12 Sterile Production

12 - Sterile
Production
Sterile Production
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12 Sterile Production
Contents:
12.A Introduction
12.A.1 Manufacturing products that can be sterilised
in the final container
12.A.2 Aseptic processing
12.A.3 Production areas/premises
12.A.4 Production equipment
12.B Air Lock Concepts

12.B.1 Personnel locks in the clean area
12.B.2 Material locks
12.C Manufacturing the solution

12.C.1 Starting materials
12.C.2 Solution batch
12.C.3 Testing the bioburden
12.C.4 Sterile filtration
12.D Washing processes

12.D.1 Stoppers
12.D.2 Particulate impurities
12.D.3 Glass containers (ampoules, bottles)
12.D.4 Transport
12.E Filling
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12.E.1 Filling equipment for solutions

12.E.2 Process for filling LVP containers in cleanliness grade C
12.E.3 Process for filling ampoules with solution in
cleanliness grade A/B
12.E.4 Filling ampoules in cleanliness grade C
and laminar flow
12.E.5 Culture medium filling (Media Fill)
12.E.6 Filling with powders
12.F Steam sterilisation

12.F.1 Sterilisers
12.F.2 Description of the procedure
12.F.3 Qualification of a steam steriliser
12.F.4 Validation of the steam sterilisation process
12.G Microbiological monitoring

12.G.1 Sources of contamination
12.G.2 Room classification
12.G.3 Monitoring program
12.G.4 Sampling
12.G.5 Sampling points
12.G.6 Measure if levels are exceeded
12.G.7 Organism identification
12.H Test for sterility

12.H.1 Parametric release
12.H.2 Sterility test
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12.H.3 Method description

12.H.4 Number of samples
12.H.5 Sample quantity
12.H.6 Reading and evaluating
12.H.7 Procedure in the event of culture medium turbidity
12.H.8 Culture media
12.H.9 Culture media controls
12.H.10 Method validation
12.I Testing for tightness and particles

12.I.1 Testing for tightness
12.I.2 Particle test
12.I.3 Sequence of operation
12.J Freeze drying

12.J.1 Description of the procedure
12.J.2 Qualification of a freeze dryer
12.J.3 Validation of the freeze drying process
12.K Dry Heat Sterilisation

12.K.1 Description of the procedure
12.K.2 Sterilisation kinetics
12.K.3 Qualification of a sterilisation tunnel
12.K.4 Validation of the sterilisation process
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12.A Introduction
Here you will find answers to the following questions:
Which requirements are sterile products expected to meet?

In which cleanliness grades do the individual products to be sterilised steps take place for
products to be sterilised in the final container or for aseptic processing?
What are the requirements for particle load and bioburden?
What problems can occur in connection with production equipment?
The absence of living microorganisms is a central requirement for a sterile medicinal product.For
category 1 preparations, the pharmacopoeia requires that sterility testing is complied with (acc. to
PhEur. 5.1.4, part 1). This is defined as a reduction in microbial count of six log factors (10-6). In
addition to the absence of microorganisms, the reduction of pyrogens and endotoxins is also
required for many products (see figure 12.A-1).
Figure 12.A-1 Definitions of pyrogens and endotoxins
Definitions
Pyrogens
Any substances (chemical, microbiological) that cause fever on parenteral

administration.
Tested using the rabbit test.
Endotoxins Components of the cell wall in gram-negative bacteria that cause fever on parenteral
administration.
Tested using the LAL test (Limulus Amebocyte Lysate test).
Parenterals (products that are administrated to the bloodstream) are also required to be "free from
visible particles". For invisible particles, the requirements apply as described in figure 12.A-2.
Figure 12.A-2 Requirement for invisible particles in preparations for infusion or injection
Requirement for invisible particles in preparations for infusion or injection
Nominal volume > 100ml
Nominal volume of 100 ml
Average number* of particles

10 m 25 P/ml

10 m, maximum 6 000 per container

25 m, maximum 3 P/ml

25 m, maximum 600 per container
* The average number is obtained from three measurements using the light blockade test procedure.
Usually, these are aqueous substances that are administrated parenterally (i.m. or i.v. as an injection
or infusion), or eye medicines (ophthalmics) for application on the eyeball. Drug substances that are
not stable in aqueous solution, or which would not survive the heat treatment for sterilisation without
becoming damaged, are often freeze-dried and resuspended with sterile solvents before application.
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Since testing for sterility (see chapter 12.H Test for sterility) always destroys the product and can
therefore only be carried out on random samples, this test does not provide absolute certainty that
each individual container in the manufactured batch is sterile. Validation of the processing steps, in
particular of sterilisation, together with detailed batch documentation of the manufacturing and
sterilisation parameters, is one method of ensuring the sterility of the whole product batch.
Due to the specific requirements for the manufacture of sterile products, the technical knowledge,
behaviour and training of the relevant personnel are key factors in reducing contamination with
microorganisms, particles and pyrogens to a minimum. Products must be manufactured according to
validated methods and procedures defined in writing. Quality aspects must not be limited to the final
manufacturing step or testing of the final product.
Sterile medicinal products are generally manufactured according to one of two different procedures:
1. Manufacturing with sterilisation in the final container (see chapter 12.A.1 Manufacturing products
that can be sterilised in the final container)
2. Aseptic processing (see chapter 12.A.2 Aseptic processing)
The crucial processing steps for achieving the ultimate aim of sterility are sterilisation and sterile
filtration (see chapter 12.C.4 Sterile filtration). Sterilisation in the final container is the preferred
method.Sterile filtration is considered if a heat sterilisation procedure would destroy the properties of
the medicinal product.
12.A.1 Manufacturing products that can be sterilised in the final container

Sterilisation in the final container should be used wherever possible. All processing steps in the runup to and environment of a sterilisation in the final container must be targeted towards the ultimate
aim of sterility by employing sterilisation methods and measures to reduce microorganisms. This
includes the need to perform the batch/filtration manufacturing operations in a closed system in
cleanliness grade D (EU-GMP-Guideline), and the filling of the final container in cleanliness grade C
under LF (Laminar Flow, class 100 US Federal Standard). Annex 1 of the EU-GMP-Guideline also
stipulates the following: "Where the product is at a high or unusual risk of microbial contamination,
(for example, because the product actively supports microbial growth or must be held for a long
period before sterilisation or is necessarily processed not mainly in closed vessels), then preparation
should be carried out in a grade C environment. Filling of products for terminal sterilisation should
be carried out in at least a grade C environment." (see figure 12.A-4).
In addition to these requirements, the microorganism and endotoxin load (see chapter 12.C.3 Testing
the bioburden) of the active and excipient ingredients must be determined before manufacturing and,
after filling from the final container, before sterilisation. The loads determined in the solution and
substances can be used to calculate the sterilisation probability of the preparation and the remaining
endotoxin load in advance, based on the efficiency of microorganism and endotoxin reduction
determined and evaluated in the qualification and process validation runs.
In special cases, particles are removed from liquids as a part of steril filtration using membrane filter
layers with a pore width of 0.22 or 0.45 .
Sterilisation in saturated steam in an autoclave at 121 C for 15 mins is the method of choice for
achieving a sterile aqueous medicinal product (PhEur.) (see chapter 12.F Steam sterilisation). An
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SAL (Sterility Assurance Level) of at least 10-6 must be achieved. This means that the probability of
finding a non-sterile object must be one in 1,000,000 units.
12.A.2 Aseptic processing

When manufacturing a sterile medicinal product that cannot be sterilised in its final container, it is
necessary to place higher demands on the personnel, the environment (see figure 12.A-3) and the
processing equipment, in order to completely exclude the possibility of microbial contamination.
Figure 12.A-3 Cleanliness grades for aseptic processing
Cleanliness grades for aseptic processing
Manufacture of the product
Cleanliness grade C
Steril filtration
Cleanliness grade B
Filling in the final container
Cleanliness grade A
Manufacture of non-filterable preparations Cleanliness grade A

A batch-specific, microbiological and particle monitoring of the environment and personnel is carried
out (see chapter 12.G Microbiological monitoring).
The suitability of all processing steps is confirmed twice yearly using a nutrition agar culture fill to
ensure <0.1% non-sterile objects (see chapter 12.E.5 Culture medium filling (Media Fill)).
12.A.3 Production areas/premises

Clean areas for the manufacturing of sterile products are graded according to the required
environmental characteristics. Each manufacturing process must fulfil the appropriate cleanliness
grade for the environment while in process, in order to minimise the risk of contamination of the
material by microorganisms or particles. To fulfil the conditions in process, the areas must be
arranged to ensure that a certain level of air purity can be achieved at rest. At rest, the technical
facility is installed and ready for operation.In process, the technical facility is being operated by staff.
Personnel must only be able to access the clean areas via air locks positioned between one cleanliness
grade and the next (see chapter 12.B Air Lock Concepts). These should correspond to the relevant
cleanliness grade and be ventilated using filters of an adequate efficiency level (see chapter 3.H
Heating Ventilation Air Conditioning (HVAC)). This means that the local separation rate of the
HEPA filter (High Efficiency Particulate Air filter) must be at least 99.97 % and must be selected and
fitted in accordance with PIC document PH 5/89 from Sept. 89 to the appropriate level for the
cleanliness grade.
Cleanliness grade A H14 99.995 % separation rate (integral value)
Cleanliness grade B H14 99.995 % separation rate (integral value)
Cleanliness grade C H13 99.95 % separation rate (integral value)
Cleanliness grade D H11 95.00 % separation rate (integral value)
Materials should enter the room via separate material locks (see chapter 12.B.2 Material locks). The
various operations such as the preparation of ingredients, formulation and filling should be carried
out in separate zones within the clean area.
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Manufacturing operations are classified into two categories:

1. Products that can be sterilised in their sealed final containers (cleanliness grade D)
2. Products for which particular stages or all stages of the manufacturing processes are carried out
under aseptic conditions (cleanliness grade C).
The cleanliness grades have different names depending on the underlying regional requirements: In
the EU-GMP-Guideline A-D, in the USA according to Federal Standard (FS) 209 E in classes 100,
10.000 and 100.000 (no longer valid since November 2001, but can still be found in practice), and
according to the ISO guideline no.14644 applicable since 2001, in ISO classes 5-8 (see figure 12.A4). For more information, see chapter 12.G.2 Room classification.
Figure 12.A-4 Cleanliness grades
Cleanliness grades
Cleanliness grade
A
(FS 209 E class
100 at rest) ISO 5
US class 100 in
operation
The local zone for operations with a high level of risk, for example in the
filling area, assembly of the filling apparatus (pump, filter, etc.), aseptic
connections (equipment, tubes, couplings) under a laminar air flow of
0.45 m/s 20%.
Microbiological limit <1 CFU/m3
In the case of faults, controlled, brief intervention by personnel from
cleanliness grade B is permitted.
SI M* 3.5
Cleanliness grade
B
(FS 209 E class
100 at rest) ISO 5
The presence of appropriately dressed personnel is permitted.

Turbulent air flow is permitted.
Microbiological limit 10 CFU/m3 (action limit)


US class 10.000 in
operation
SI M* 5.5
Cleanliness grade
C
(FS 209 E class 10
000 at rest) ISO 7
US class 100.000
in operation
SI M* 6.5
Cleanliness grade
D
(FS 209 E class
100 000 at rest)
ISO 8
US class: not
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classified
* SI M represents the classification on the basis of 0.5m particles
Limits for the microbial contamination of surfaces in these cleanliness grades are generally tested
with purchased nutrition agar contact plates with a diameter of 55 mm (corresponds to a surface area
of 23.75 cm2). You therefore need to consider the specifications per cm2 and include them in
standard operating procedures (SOPs). To be on the safe side, you can comply with the required limit
(see figure 12.A-5 and figure 12.G-3) for a contact area of > 23.75 cm2. Normally, specifications are
provided per plate with a diameter of 55 mm or per 25 cm2 (figure 12.A-5).
Figure 12.A-5 Recommended residue values for microbiological contamination (according to the EUGMP-Guideline, Annex 1)
Recommended limits for microbial contamination
(EU-GMP-Guideline, Annex 1, USP 25)
Grade
air sample
cfu/m3
settle plates
(diam. 90mm)
cfu/4 hours
contact plates
(diam. 55m)
cfu/plate
glove print
(5 fingers)
cfu/glove
<1
<3
<1
<1
3
<1
3 glove
5 clothes
10
<20
5
5
10 floor
5
10 Gloves
20 clothes
100
<100
50
25
200
100
50
For absolutely necessary interventions in cleanliness grade A, the hands covered by the gloves should
be disinfected to ensure that the limit of <1 CFU/5 fingertips is not reached. However, since
individual values of 1 and >1 cannot be excluded (<1 is an average value), in this case, the average
value of the last ten quality controls should be used. This average value must be <1 CFU/5 finger tips
(measure limit value/action limit) (see figure 12.A-5). In the presence of correct personnel behaviour
and operational cycle, a bioburden on the finger tips of >3 CFU/5 finger tips is extremely rare. If
operating procedures are not adhered to, the values are considerably higher, i.e., a value of 2 CFU/5
finger tips can be an indication of systematic failures and should be regarded as a warning sign. The
alert limit could therefore be determined at 2 CFU/5 finger tips.
Particle contamination in the air is measured at the start and end of the procedure at 3 points below
the LF at object height.
1. Start of LF in direction of transport
2. At the filling point of the objects
3. Before the sealing apparatus (stoppering or tip sealing for ampoules)
The contamination levels must be measured at the beginning and end of production.
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Figure 12.A-6 Max. permitted number of particles/m3 (greater than or equal to) according to the EUGMP-Guideline, Annex 1
Max. permitted no. of particles/m3 (equal to or above)
from the EU-GMP-Guideline, Annex 1
Cleanliness grade
At rest
In operation
0.5 m
5 m
0.5 m
5 m
3,500
3,500
3,500
350,000
2,000
350,000
2,000
3,500,000
20,000
3,500,000
20,000
not defined
not defined
This enables you to prove and document, if you are using the alert system (audio signal or red light if
the value drops below 0.35 m/s) of the LF box, that the laminarity of the air flow has been maintained
throughout the processing period.In automatic systems, selected points are recorded; usually two
points per square metre. The action limits according to the EU-GMP-Guideline, Annex 1, are shown
in figure 12.A-6.
The flow rate of the air is measured at 0.45 m/s approx. 10-30 cm below the filter surface and is
usually displayed on the digital or analogue display of the relevant LF box.The control measurement
should then be performed at the level of the opening on the object to be filled, at least at the filling
point, since in general this is where the strongest movements take place which can affect the air flow.
The lower limit of 0.35 m/s can be easily complied with if there are no serious mechanical problems.
The flow measurement must also be performed at the beginning and end of production.
12.A.4 Production equipment

Requirements for premises and equipment are to be taken from Annex 1 of the EU-GMP-Guideline.
Considering the variation in technical, physical, chemical and microbiological requirements of the
materials to be processed and the procedures used, it is understandably only possible to make very
general statements, such as "exposed surfaces should be smooth, unbroken and impervious ... airconditioned ... cleaned, disinfected and/or sterilised where appropriate ... subject to validation and
planned maintenance" (see chapter 4 Facilities and Equipment).
Production equipment must be qualified, regularly serviced and, in the event of repairs and
modifications, subject to a change control procedure. Incalculable risks in drug product safety almost
always originate from incorrect procedures in technical systems (short-term defects are particularly
dangerous) related to insufficient personnel understanding of the influence on product quality
resulting from a change in the general conditions of a sequence of operation.
Example:
A machine stop due to breakage of glassware (bottles, ampoules, etc.), switching errors and the
resulting intervention can mean that the machine is not stopped in the end position of a cycle
(compressed air release, spraying with water, pressure maintenance of pumps, time switch points of
cycles, etc.). Since nowadays, compact lines are most commonly used (washing machine, sterilisation
tunnel, filling machine in one), these faults have an impact across several levels of processing. When
eliminating faults, it must be guaranteed that objects which have not reached the end of the process
are disposed of, or that they are allowed to reach the end of the process uninfluenced.
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Examples of serious faults:

The interruption of a dosing pump movement consistently leads to dosing variations due to the
change in pressure conditions in the system cylinder - piston - suction pressure - pressure line and
switching.
In switching mechanisms employed in compact lines (for example, spraying pressure of water or
compressed air), no fault must be allowed to occur that does not lead to immediate stopping of the
machine and that can be "overridden". This may mean that objects are not adequately rinsed or that,
for example, partially filled WFI objects reach the sterilisation tunnel and are not properly sterilised.
In order to exclude problems resulting from faults as much as possible, limits must be defined in
relation to the time period of the machine cycles, pressures and temperatures of media, the revolution
speed of drives, end positions of machine levers and cams, and the mechanical process flow must be
recorded in the electronic parallel program. This ensures that when deviations occur, error displays
are generated, the machine is stopped appropriately, and both technical and pharmaceutical measures
can be implemented.
Microbiological influences are nearly always a consequence of physical or technical changes.The
deciding factor when selecting production equipment is the necessity of a requirement from a
technical and pharmaceutical perspective, together with the technical feasibility. Maximum
requirements are only reasonable in cases in which clear values must be fulfilled. For example: In the
procurement of prototype machines and filling systems that have not actually performed any
development steps, their performance can only be imagined, but not backed up with practical
experience.In this case, clear maximum requirements must be set and enforced in terms of parameters
(such as flow rates and particle counts in the air in accordance with the cleanliness grade, sterilisation
temperatures, temperature control limits and time specifications). All other requirements are
secondary and should be amicably deleted in advance between the technical and pharmaceutical
operators. "Overloading" with automatic logs, controls and networking leads to an inflation of
potential fault sources, increased costs and prolonged timeframes. The common understanding,
comprehension and processing of the requirement in the form of compromises (for example price,
capacity, performance, current costs) lead to sustainable, reasonable regulations that comply with the
demand for "state-of-the-art" and "legally conformant drug product production".
Summary
Sterile products must be free from any organisms that can survive in the product, and the levels of
particles and endotoxins must remain within the prescribed limits. Sterility is achieved either through
steam sterilisation in the final container (preferred), sterile filtration, or aseptic environmental
conditions.
Permissible limits for particles and microorganisms for production areas in grades A-D are specified
in Annex 1 of the EU-GMP-Guideline. Each individual operation is assigned to a cleanliness grade.
The main source of deviations and values that exceed the limits is machine faults.
12.B Air Lock Concepts
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How are personnel locks between cleanliness grades designed?

How are changing procedures organised?
How are material locks designed?
Air locks are rooms of a particular cleanliness grade that enable the passage of persons or materials
into an area of a lower or higher grade. Air locks must correspond to the same cleanliness grade as
the adjoining work area. They are subject to the same building and air filter requirements as all other
rooms of the same cleanliness grade (see chapter 3 Premises).
The use of different air lock concepts depends largely on the layout of the building, the machine
facilities, and the direction of any air flow channels. Regardless of the resulting design, the air lock
must always fulfil the relevant requirements in terms of minimising microbiological and particulate
contamination. The following is an explanation of the different air lock concepts.
12.B.1 Personnel locks in the clean area

A personnel lock is usually a relatively small or medium-sized room in which activities of a certain
cleanliness grade are permitted to take place. In the air lock, the cleanliness grades of adjoining areas
come into contact with each other.
The concept of a "sit-over-system" has proven easily manageable from an airflow perspective; it can
provide an air flow suitable for two cleanliness grades with use of the appropriate filters, and can
logically be integrated into activities by the persons using the air lock. Doors are only required to
maintain pressure levels in the operating areas.
Cupboards used for the storage of clean and worn clean-room clothing (for short-term absences
from the work area) should be ventilated or equipped with UV light in accordance with the
cleanliness grade. Clothing that has been worn in cleanliness grade B must be stored in a cupboard
that complies with at-rest cleanliness grade B (cleanliness grade B, ventilated), in the case of shortterm absence from the cleanliness grade B area. Clothing that has been worn in cleanliness grade C
must be stored in a cupboard that complies with at-rest cleanliness grade C (cleanliness grade C,
ventilated).
12.B.1.1 Air locks in cleanliness grade F/E

Air locks that lead to undefined areas with respect to particles (cleanliness grade F/E, for example
labs, offices), are ventilated only by an air current from E to F or into the uncontrolled area via doors
that are not locked on both sides or secured with indicators.
Required clothing: none prescribed
However, clothing regulations are recommended, for example, lab coat, shoes and possibly a head
covering in a standard colour to provide physical protection (laboratory) or to indicate that the
employee is assigned to cleanliness grade E/F (see chapter 11.B.1 Clothing).
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12.B.1.2 Air locks in cleanliness grade E/D

Starting from cleanliness grade E, the air lock to cleanliness grade D should be entered through a
door with a lock (with an emergency exit mechanism) or at least an indicator (lit "Do Not Enter"
sign), in order to prevent uncontrolled air flow through the air lock.
Layout: the air lock should be divided into two sections by a sit-over (see figure 12.B-1).
Figure 12.B-1 Air lock concept from cleanliness grade E to cleanliness grade D
Cleanliness grade E section: wardrobe cupboards for cleanliness grade E clothing/private clothing,
washing facilities, storage area for cleanliness grade D clothing.
Cleanliness grade D section: wardrobe cupboards for grade D clothing, washing facilities,
disinfectant for hand disinfection.
Ventilation: the air current in accordance with cleanliness grade D (microbiological particulates)
should flow in the direction of the sit-over diagonally from ceiling to floor towards the "unclean"
grade E side, where it is extracted. The flow conditions should be verified using smoke tubes.
Personnel: change of clothing from cleanliness grade E to D as described in figure 12.B-2.
Figure 12.B-2 Change of clothing from cleanliness grade E to cleanliness grade D
Change of clothing from cleanliness grade E to cleanliness grade D
Wait for the entry signal before opening the door.

Store clothing from cleanliness grade E in the designated cupboard.
Put on head covering.
Use washing facilities (hands)
Place grade D clothing on the sit-over and sit down.
On sitting, place shoes from grade E under the sit-over and move over to the grade D side.
Place feet in grade D shoes (temporary options: pool shoes or plastic overshoes)
If applicable, put on beard protector.
Remove grade D clothing from the packaging and first put on the jacket, followed by the
trousers.
Use the mirror to check the clothing is correctly in place: hair, ears, and beard covered,
sleeves and ankles are closed.
Wash hands in hand wash solution and dry. Disinfect hands with fast-acting disinfectant.
Open the door to cleanliness grade D (after checking the entry signal).
When leaving the grade D area, follow the above steps in reverse order (leave grade D
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clothing in cupboard in the grade D section).
12.B.1.3 Air locks in cleanliness grade D/C

Starting from cleanliness grade D, the air lock to cleanliness grade C should be entered through a
door with a lock (with an emergency exit mechanism) or at least an indicator (lit "Do Not Enter"
Layout: The air lock should be divided into two sections by a sit-over (see figure 12.B-3).
Figure 12.B-3 Air lock concept from cleanliness grade D to cleanliness grade C
Cleanliness grade D section: Wardrobe for storing grade D clothing, shelf for shoe storage, washing
facilities for hands, dryer, disinfectant dispenser.
Cleanliness grade C section: Wardrobe for grade C overalls, shelf for grade C shoes, sock dispenser,
supply of gloves and face masks next to sit-over, disinfectant dispenser.
Ventilation: The air current in accordance with cleanliness grade C should flow in the direction of
the sit-over from ceiling to floor towards the grade D area, where it is removed. Flow conditions
should be verified.
Personnel: Change of clothing from cleanliness grade D to C as described in figure 12.B-4.
Figure 12.B-4 Changing clothing between cleanliness grade D and cleanliness grade C
Changing clothing between cleanliness grade D and cleanliness grade C
Wait for the entry signal before opening the door.

Place clothing from cleanliness grade D in the cupboard, except for head covering and beard
protection.
Use washing facilities (hands)
Place grade C clothing ready on the sit-over and sit down.
On sitting, place shoes from grade D under the sit-over and move over to the grade C side.
Put on socks and then shoes for cleanliness grade C.
Put on gloves, disinfect.
Put on hood and overall for cleanliness grade C.
Put on face mask.
Disinfect gloves.
Open the door to cleanliness grade C after checking the entry signal.
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12.B.1.4 Air locks in cleanliness grade C/B

Starting from cleanliness grade D, the grade D "pre-lock" should be entered through a door that is
equipped with a lock with an emergency exit mechanism or at least an indicator (lit "Do Not Enter"
A second door shuts off this "prelock". Behind this door is the "main lock" with the two sections
cleanliness grades C and B, separated by a sit-over. The air lock is exited via a door to the grade B
work area (see figure 12.B-5).
Figure 12.B-5 Air lock concept from cleanliness grade D to cleanliness grade B
Layout: This area is divided into three sections, with a grade C/grade B sit-over in the second
section.
Cleanliness grade D section: wardrobe for storing grade D clothing, shelf for shoe storage, pool
shoes for transition to section C, washing facilities for hands, dryer, disinfectant dispenser.
Cleanliness grade C section: wardrobe for optional particle-free underclothes, shelf for storing pool
shoes, sock dispenser, washing facilities for hands, dryer, supply of gloves and face masks next to sitover, disinfectant dispenser, disinfectant spray bottle.
Cleanliness grade B section: storage of grade B shoes under the sit-over or on designated shelf or
optional overshoes, cupboard for sterilised cleanliness grade B clothing, cupboard for reusable grade
B clothing (overall, hood), safety glasses case, disinfectant dispenser, disinfectant spray bottle.
Ventilation: the air current in accordance with cleanliness grade B should flow in the direction of the
sit-over from ceiling to floor towards the grade C area, where it is removed. Flow conditions should
be verified.
Personnel: change of clothing from cleanliness grade D to B as described in figure 12.B-6.
Figure 12.B-6 Change of clothing from cleanliness grade D to cleanliness grade B
Change of clothing from cleanliness grade D to cleanliness grade B
Enter grade D pre-lock, remove shoes, put on clean room socks over normal socks, and place
feet in pool shoes.
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Wash hands - enter the main lock through the door (wait for entry signal).
Remove grade D clothing (except for head covering and beard protector) and hang in the
designated cupboard.
Wash hands, dry in dryer (class 100), and disinfect.
If required, take clean room underclothes from the storage cupboard and put on.
Go to sit-over, put on sterilised disposable gloves, and disinfect.
Prepare cleanliness grade B shoes (which are ready on the designated shelf) and climb over
the sit-over, leaving the pool shoes behind. Place feet in cleanliness grade B shoes or clogs.
Disinfect (gloved) hands.
Remove sterile packaged clothing from the cupboard or shelf (sterile packaged clothing is
sterilised in autoclaves and enters the air lock from the cleanliness grade B work area).
Open the packaging (clothing is always folded in the same way), remove head covering, and
put on (the open packaging containing the overalls can be placed on the grade B side of the
sit-over). Disinfect hands after doing up the zip and the tensioning device on the hood.
Put on face mask.
Standing with one arm stretched out, remove the overall by the collar. Lift the overall with
one hand, allow packaging to fall on the sit-over. With the other hand, open the zip on the
overall. Hold the overall tight in one hand at stomach level and let go of the collar, then hold
the hanging left and right bundles of sleeves and trouser legs to the corresponding sides of the
zip.
Divide the zip with sleeve and trouser leg bundles between both hands and pull apart. This
procedure enables you to step into the overall without the trouser legs coming into contact
with the floor or any other external influences.
Sit on the sit-over bending forwards, so as not to allow the overall to touch the sit-over, and
step into the trouser leg with one foot (clean room sock) whilst slowly releasing the bundle on
that side. Pull the foot all the way through and place in clean room shoe. Repeat the
procedure exactly with the other leg.
Stand up (the overall is now up to your hips), let go of the bundled sleeves, and place your
arms into the sleeves, all the while ensuring that the outside of the overall does not come into
contact with the body. Bend over forwards, push your hands through the bundled sleeves,
grab the neck, and pull the collar over the hood shoulder cover.
Stand up straight and pull the zip up to your throat. Use the mirror to check that clothing is
correctly positioned.
Disinfect hands.
Spray disinfection of the sit-over after disposal of the packaging from gloves and clean room
clothing.
Place a new pair of gloves over the "changing gloves" and disinfect.
12.B.2 Material locks

Material locks are rooms through which larger pieces of equipment (vessels, bins, magazines,
machine accessories, etc.) can be transferred from one cleanliness grade to another, higher,
cleanliness grade. The requirements comply with the cleanliness grade of the work area that can be
reached through the air locks. Only one door can be opened at a time. The material lock is generally a
room that separates the cleanliness grades by two doors.
Layout: the facilities usually consist only of a store for disinfectant used in spray disinfection.
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Ventilation: in single-room air locks often none, since the air is refreshed by the higher air pressure
from the clean area.
Figure 12.B-7 and figure 12.B-8 show examples of material locks from cleanliness grade D to C and
from cleanliness grade D to B.
Figure 12.B-7 Material lock from cleanliness grade D to cleanliness grade C
Figure 12.B-8 Material lock from cleanliness grade D to cleanliness grade C
In double locks, the higher grade room can be actively ventilated, and apertures (lamellae) in the door
enable the cleaner air to disperse into the pre-lock, so that a pressure difference is maintained to the
rest of the environment.
Materials: all objects that enter a higher cleanliness grade via the air lock must be cleaned and
disinfected appropriately. A residence time in the lock following disinfection must be established.
Summary
The different cleanliness grades are separated by air locks, in which personnel change into the
prescribed clean room clothing and materials are disinfected.
The personnel lock is generally divided by a sit-over bench that separates the areas corresponding to
the adjacent cleanliness grades.
Staff change clothing in accordance with a written, defined plan (standard operating procedure, SOP).
12.C Manufacturing the solution

Which requirements are starting materials expected to meet?

How can you guarantee compliance with the recipe?
In which cleanliness grades must products be manufactured?
How and with what means are microbial reduction and particle purity of a solution achieved?
How and where are work steps or controls documented?
How and by whom is the final evaluation of the solution preparation performed?
12.C.1 Starting materials

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A solution is a transparent liquid. It contains active pharmaceutical ingredients and excipients,

dissolved in a solvent. The dissolved substances can exist in several different forms:
Ion disperse: the solution contains ions as in an ionic solution.

Molecular disperse: the solution contains molecules, as in a non-ionic solution (particle size
10-8 cm).
An important feature of a solution is that the dissolved substances are returned to their initial state
when the solvent is removed.
The EU-GMP-Guideline does not define any values for microbial purity, but requires that a solution
should "contain the minimum of microbial impurities". The USP requires that the necessary
precautionary measures are implemented to reduce the bioburden to an acceptably low level before
sterilisation. For solids, a value of 100 CFU/g is standard practice for solvent production with
subsequent steril filtration and sterilisation. In aseptic processing, sterilised or sterile filtered starting
materials must be used.
The most common solvents used in pharmaceutical manufacturing are:
Purified water and water for injection (WFI)

Alcohol
Oils
Mixtures of various solvents
Purified water is used in cleaning as rinse water for containers and equipment after use, and as rinsing
water (particle filtration 2 m < 10 m) for immediate containers, immersion basin water, and splash
water during the washing process. There then follows the final rinse with WFI (see chapter 5.A Water
types).
Active pharmaceutical ingredients can be materials from natural sources or substances manufactured
by gene technology or synthetic methods.
Excipients include all components of the preparation that are necessary in order to deliver the active
pharmaceutical ingredient to the patient in a tolerable manner, in accordance with its dosage form.
In accordance with the EU-GMP-Guideline, active pharmaceutical ingredients, solvents, and
excipients are all classified as starting materials.
12.C.1.1 Rooms used for weighing

In general, materials should be weighed in clean areas, to prevent changes to their initial purity. They
should be weighed in completely separate areas according to their biological activities (separate fume
cupboards, air flow pattern and accumulation of dust) (see chapter 11.G Weigh-in). Grade D, C or A
is required, depending on the starting purity (<100 CFU/g to sterile). Sterile substances are weighed
in sterilised containers (such as beakers) under grade A (clean bench) conditions, and transferred to
solution containers under cleanliness grade A. Here, the background environment must comply with
cleanliness grade B.
Due to particle shedding (from solids) in the air, and other possible effects on people, handling of
these substances is subject to general safety regulations. When weighing sterile substances, personnel
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clothing complies with cleanliness grade B, and the clothing also provides personal protection. In
cleanliness grade D, a dust mask/safety spectacles may also be necessary to meet safety requirements.
"Weighing boxes" are also available, which have a specific exhaust mechanism attached to the
balance and negative pressure in relation to the surrounding cleanliness grade. These can be useful to
prevent cross-contamination.
The substances to be used are specified in the processing instructions.
Starting materials are labelled with a designation and a company-specific ID number, which details
the required specification of the substance, and usually the current test results regarding approval for
use in drug product manufacturing. Only released substances should be used for further processing
(see chapter 14.J Batch release ).
12.C.1.2 Processing instructions (manufacturing instructions)

Approved manufacturing instructions are required for all products and each batch siye to be
manufactured. These are used to develop processing instructions that contain detailed handling steps
in accordance with the available equipment, and processing steps (see chapter 15.C Batch
documentation). IT-based documentation systems and a change management procedure (see chapter
19.C Change control) are designed to ensure that correct current specifications are always compiled.
Documentation in accordance with GMP requires that each individual step in the manufacturing
process is recorded in writing, if possible at the time of the activity, and is evaluated and initialled by
the responsible persons, for example in a doer/checker procedure. The "Doer" adds their initials to
confirm the handling, and the "Checker" checks and confirms the whole protocol based on
specifications and the weighing record, for example, checking the printed balance quantities and the
results of identity checks.
During the course of solution preparation, the equipment used means that a large number of
processing substeps take place in different locations, using different equipment, performed by
different people and at different times. It is therefore practical to identify these individual activities
numerically and keep the documentation (for example, record of a sterilisation process of filters in
autoclaves, doer/checker assessment) at the location in which the activity is executed. Only the
numerical ID of the activity is then entered in the processing instructions, together with the date and
initials of the employee responsible.
It can also be helpful to structurally divide the processing instructions into different sections and,
once they are completed and approved, send the completed sections (each with separate page
numbering) to the different locations involved in the procedure, or even generate them on a computer
system in each location. Once each activity is completed in the separate locations (e.g. weighing,
machine preparation, washing of containers, in-process checks), the completed sections are combined
and form the processing protocol. A procedure of this type ensures that the relevant part of the
processing instructions is always available at the correct location and at the right time.
12.C.1.3 Weighing of starting materials

The header section of each printed section of the processing instructions includes the date of printing,
the name of the preparation, the number of the applicable manufacturing instructions, the batch
number of the batch, the batch number of the filling, and company-specific ID numbers for
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pharmaceutical tracking based on the use of components and the final container. These ID numbers
are also used for business monitoring and accounting.
An example of the first line of a weighing record, starting with the first substance in the order in
which they are used in the batch, is shown in figure 12.C-1.
Figure 12.C-1 Example weighing record
Example weighing record
Item no. Name Article no. Release/experiment no. Required amount Actual initial weight Doer
(100%)
0001
xyz
111111
1234567890
1000.000 g
.... . ... g
.......
A sample should be taken from each individual pack of a substance and checked for identity. The
result must be attached to the weighing record before the substance is further processed. If several
packs of the same substance are weighed in initial weighing, all the results must be documented. The
test method is selected by the responsible unit, as long as it is validated or is a standard
pharmacopoeia procedure.
The substance can be weighed in containers made from various materials (stainless steel, plastic,
glass, plastic bags, etc.) that have been cleaned using a validated procedure, or materials of a known
cleanliness grade (plastic bags). All weighed substances are labelled to indicate their application
(substance name, quantity, for preparation XY, batch Z, etc.). The individual containers should be
sealed and secured, and all containers for one batch should be stored together.
12.C.2 Solution batch

In accordance with the processing instructions for the batch (execution in cleanliness grade D or C), a
visual control should be performed for each step to ensure that the cleaning and sterilisation steps for
the devices and filters have been correctly performed and the labelling is correct, and the person
responsible for the control then enters their name in the designated field. The solvents are generally
placed in the reaction vessels in advance. This is performed in the form of a weight or volume
measurement.
Each operation (in accordance with the specified SOP) is confirmed by the employee's initials in the
processing instructions immediately after it is performed, and any printouts from balances or
measuring instruments are attached to the processing instructions.
The addition of previously weighed substances to the batch, and the compliance with specified
parameters, methods, and timescales (for example temperature, stirring duration, stirrer revolutions,
pressures, reaction times, sampling and measured values (such as pH or density), should be checked,
monitored, and confirmed by two members of staff (doer/checker principle). Figure 12.C-2 shows an
example of how the documentation can be structured.
Figure 12.C-2 Division of processing instructions into Action, Check and IPC
Division of processing instructions into Action, Check and IPC (independent of processor)
Activity _xyz___ acc. to SOP__xxx_________________________________________
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Name__Doer_______Comments____________________________________________
Activity _xyz_____ acc. to SOP_xxx____Target:_____ Actual:______ Result________
Name__Checker___ Comments___________________________________________
IPC Process laboratory:
For activity _xyz____ Target value:__0000____ Actual value:_____ Result__________
Name__Doer_______ ___Checker____
One exception is the bulk production of WFI, which is used in the manufacturing of parenterals and
for the final rinsing of equipment, containers, and final containers. In this case, compliance with the
specification (see chapter 5.A Water types) must be proven by monitoring at the WFI manufacturing
plant and point of withdrawal. The test for the presence of endotoxins in the WFI in the reaction
vessel, which provides a relatively quick result (residue limit for WFI <0.25 EU/ml), also provides a
partial conclusion to be drawn regarding the purity of the vessel.
Further subsequent steps in the manufacturing process are listed and processed in the same way. At
the end of the process, the target value for the batch according to the recipe and actual value are
compared, and expressed as a percentage yield. The yield must lie within a previously specified
target range. If the value is not within this range, a plausible explanation for the deviation must be
provided (see figure 12.C-3).
Figure 12.C-3 Yield outside the specified range
Yield outside the specified range
Example 1:
The target yield specifies a range of 85 to 95% and incorrect operation of the filter ventilation results
in a solution loss of over 10%, as the ventilation cannot be completely closed (sealing error). This
gives rise to the following questions: Were staff insufficiently trained? Was an incorrect gasket
installed? Were there errors in assembly? Was an incorrect device used?
Example 2:
Filtration was stopped because an unusual decrease in the filtration rate was established (filter
blockade due to starting material load with particulate impurity). This gives rise to the following
questions: Was the specification of the starting material inadequate? Were analysis results incorrect?
Was the starting material manufactured incorrectly?
Example 3:
The operational cycles have run perfectly. The implementations and the use of substance quantities
are at the upper limits, and hence > 95%. This leads to the following questions: Why don't processes
always run in this way? Were there errors in validation? Is the process under control?
Solids should be added to the reaction vessel with the minimum possible generation of dust (extra
exhaust in order to exclude cross-contamination). The technology of absorption of solids should
always be preferred if the flow properties of the substance and the diameter of the pipes in the
container permit.
When transporting solutions through pipes, due to possible sealing problems and particle
shedding, no pumps should be used, but filtered nitrogen or compressed air should be used instead.
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SOPs for solution manufacturing should not only describe consecutive operations and activities, but
should also require checks of the previous steps for equipment and accessories. Any anomalies and
irregularities must be recorded and discussed.
In solution preparation, the reliability of the preparation, labelling, and the sound functioning of all
the necessary equipment and tools are particularly important. In the case of malfunctions,
replacement devices must be available that have been cleaned and sterilised. Experience shows that
the time required for a failure of this type in the process is around three hours. If this happens, the
process flow of the scheduled batch does not correspond to the planned time schedule, and therefore
requires a "procedural deviation" report (see chapter 11.K Deviations).
If expected values from the manufacturing instructions (processing instructions) are not met or, for
example, there is a loss of yield, this leads to an OOS result (see chapter 14.H Out-of-specification
results). This may be associated with special checks to provide evidence that the quality of the
preparation has not been impaired. It also initiates the long-term elimination of the cause of the
failure and training of personnel.
Attention to detail in the initial phase of a manufacturing process can therefore have a decisive
influence on the reliability of a process flow. Of course, this means that the responsible managers
must always be available at these times and contribute towards the problem solution. Figure 12.C-4
lists some examples of weak points that can lead to OOS results.
Figure 12.C-4 Weak points that can lead to OOS results
Weak points that can lead to OOS results
Failure
Cause
Stiffness/blockage of plug and

screw connectors and valves
Damaged screw thread, damaged or ageing seals, not changed

regularly
Leaks in piping, equipment, and

containers
Assembly errors: Incorrect, missing seal, incorrect mounting

direction
Irregular filtration times, low

measurement recordings
Incorrect pore size of the filter, possible substance properties,

defective pressure readings, recording equipment not monitored
after the start phase.
Sampling is a special case. Here the problem is not only that, if insufficient care and attention is paid
to all points (see chapter 14.A Sampling), an incorrect result is generated and then corrected on
repetition, but that the significance of any results is affected. This means that for each subsequent
sample taken from another batch, doubts may arise as to the accuracy of the found values. Any
measures that are implemented are then incorrect; laboratory data is cast into doubt and time is spent
searching for errors that do not exist. The method description for sampling, and compliance with the
method are therefore of the utmost importance. For example, the sequence of valve operations should
be described. If valves are opened to take samples, a defined quantity must be collected as a
preliminary run (in a separate container). Subsequently - without moving the valve, let alone closing
it and reopening it - the actual sample quantity is filled in the sample container. This prevents the
rinsing of dead spaces in the piping/valve and the generation of false results.
Figure 12.C-5 to figure 12.C-7 list the processes involved in solution preparation in chronological
order for products with and without final sterilisation.
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Figure 12.C-5 Practical process flow for preparation of solutions (cleanliness grade D) that can be
sterilised in the final container
Practical process flow for preparation of solutions (cleanliness grade D) that can be sterilised in
the final container
Check that all required equipment (measuring and test devices), containers, and starting
materials are available and suitable.
Provision of solvent, check of all IPC laboratory data
Add starting materials while ensuring the sequence and completeness are adhered to, the
prescribed temperature is regulated, and the labelling or naming is validated.
Solution duration and complete dissolution/IPC laboratory data
If required, add corrective substances for (pH, density, content, etc.)
IPC lab data
Filtration using monitored 0.22 m product/filtration equipment (or 0.45m in special cases)
at the prescribed temperature in a suitable, cleaned and sterilised storage container/IPC lab
data integrity test of filters
Yield calculation
Figure 12.C-6 Preparation of a product that can be sterilised in the final container (cleanliness grade
D)
Figure 12.C-7 Practical process flow for preparation of solutions that cannot be sterilised in the final
container (cleanliness grade C)
Practical process flow for preparation of solutions that cannot be sterilised in the final
container (cleanliness grade C)
In particular, check the package integrity of sterilised accessories.

All surfaces that come into contact with the product must be handled in a non-contact
manner. Around these surfaces, only use sterilised utensils (for example, tweezers, etc.) for
sealants. In the case of uncontrolled contact, device components must be replaced.
Filter integrity test in cleanliness grade D before sterilisation of the product filter with 0.22
m pore size
Cleanliness grade C
Check that all required equipment (measuring and test devices), containers, and starting
materials are available and suitable.
Provision of solvent, check of all IPC laboratory data
Add starting materials while ensuring the sequence and completeness are adhered to, the
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prescribed temperature is regulated, and the labelling or lettering is validated.

Solution duration and complete dissolution/IPC laboratory data
If required, add corrective substances (for pH, density, content, etc.)
IPC lab data - bioburden
Cleanliness grade B
Filtration from the container in cleanliness grade C, through the tested, 0.22 m pore size
filter in cleanliness grade B, into the storage container (coupling procedures under LF,
cleanliness grade A).
After disconnecting the piping, filter integrity test (ventilation, medium and product filter)
Yield calculation
12.C.3 Testing the bioburden

Since, when manufacturing a solution, all surfaces, starting materials, surrounding air and other
media (nitrogen, compressed air), as well as all personnel can be a source of microbiological
contamination, the microbiological load of the product must be known before application of standard
sterilisation methods. This enables you to ensure process reliability. An SAL (Sterility Assurance
Level) of 10-6 is required, which means that the sterilisation conditions achieve a microbial reduction
level in which only a maximum of one in one million objects may be unsterile.
For products sterilisable in the final container, the microbial count is determined using membrane
filtration with the contents of a primary container, or 50 ml per container as a double determination.
The limit is usually 100 CFU/ml (CFU = colony-forming unit), as is the case for purified water.
There is no binding specification.
Aseptically processed products are tested in bulk. In this case, the residue limit is 10 CFU/100 ml (in
accordance with CPM/QWP/486/95).
The use of rinsing or buffer solution during or after sample filtration (to eliminate the influence of
starting materials on microbial growth) should be validated and incorporated in an SOP for the
bioburden test. This should describe, for example, that after filtration of the sample solution for
bioburden testing via a membrane filter with a pore size of 0.45 m, the filter should be rinsed, for
example, with 3 x 100 ml pH 7.0 sodium chloride peptone buffer solution. The rinsing procedure
should also be validated for each specific product. The membrane filter should then be removed from
the filter stand (obviously all carried out in cleanliness grade A, Clean Bench) and placed on an Agar
culture medium for incubation. Casein soy peptone agar is used as a culture medium (for bacteria)
and Sabouraud agar is for moulds.
If the limit is exceeded, further filtration should be performed. Since the result is not available until
several days later, the process reliability can be calculated based on the batch quantity, filter surface,
original microbial count, and validated separation rate (performed by the filter manufacturer for
various germ species) (SAL 10-6) (see chapter 12.F Steam sterilisation).
12.C.4 Sterile filtration

Sterile filtration is the process of separating all microorganisms, apart from viruses, from a liquid. A
sterile filter must block all microorganisms in a flow of liquid without affecting product quality.
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12.C.4.1 History
Since the beginning of the industrial manufacture of medicinal products around 1900, three major
methods of filtration have been pursued: porcelain, cellulose, asbestos filters, and since 1930,
membrane filters.
After porcelain filters, cellulose asbestos filters (Seitz EK) were most common until the mid 1970s.
Cooperation between filter manufacturers and pharmaceutical manufacturers enabled all
disadvantages of the first generation of membrane filters, compared to cellulose-asbestos and
porcelain filters, to be eliminated. Until the late 1960s, a pore size of 0.45 m qualified as a
"sterilisation filter".
These filters were qualified using a microorganism (Serratia marcescens) measuring 0.6 x 1 m,
which was at that time a standard bacteria in water and membrane analytics. As evidence showed that
this microorganism occasionally penetrated the membrane with a test level of 104 to 106 per cm2, a
pore size of 0.22 m, for the effective retention of the 0.3 x 0.6 to 0.8 m Pseudomonas
diminuta/Brevundimonas diminuta (American Type Culture Collection (ATCC) as culture No.
19146), became the standard for the qualification of sterilisation filters.
12.C.4.2 Mode of operation

In general, it is assumed that the filtration effect is similar to the function of a sieve. Depending on
their size, microorganisms are not let through the pores, but instead collect on the surface. This is the
most important reason, but not the only reason why filtration works. This applies for geometric solid
particles. Smaller particles and microorganisms are retained in the pores due to adsorption forces,
depending on the pressure differential, flow rate, number of particles, surface tension, and degree of
ionisation. The pressure differential is particularly important, since this must not lead to a
deformation of the microorganisms. If the microorganism becomes deformed, it can become capable
of passing through the pores and may be pushed through.
For the probability of bioburden reduction, the following factors must be taken into account:
The greater the number of particles (including microorganisms), the greater the number of
particles that are not retained. This means that the bioburden has a significant influence on the
effectiveness of the filtration. The bioburden should therefore obviously be kept as low as
possible. Germ species differentiation during monitoring is important in order to determine
the size of the organisms. The data from routine monitoring (see chapter 12.G
Microbiological monitoring) provides a clear picture of which organisms are present, which
filtration pressure is required, and whether a slippage of microorganisms appears possible.
The lower the pressure differential, the greater the probability of segregation of
microorganisms.
The longer the residence time of an organism in a pore, the more reliable its retention in the
filter.
12.C.4.3 Materials, designs and properties

A sterile filter is defined as a filter capable of retaining 107 CFU of Brevundimonas diminuta ATCC
19146/cm2 of filter surface under specified conditions. Since the classification of a sterile filter is
defined by the pore size (integrity test value), this has replaced the bacterial retention rate in
production processes. A filter is classified as a "sterile filter" with a pore size of 0.22 m. Filters with
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greater pore widths are simply membrane filters of different sizes (for example, 0.5 m/1.0 m/1.2
m etc.). Due to the influences on the effectiveness of the filter as described above (see chapter
12.C.4.2 Mode of operation), the appropriate filter should be selected depending on the product.
Materials used include cellulose ester, nylon, polyester, polytetrafluoroethylene, polyvinylfluoride,
polycarbonate, polypropylene, polysulfone, and polyethersulfone. In addition, hydrophobic or
hydrophilic membranes are used.
Similar to the diversity of technical possibilities, filter housing, and mounts, the design of the filters
are also varied: Disc filters, candle filters, and cartridges. Cartridges are supplied by the manufacturer
and are membrane filter layers (0.2 m, 0.45 m, 1.2 m etc.) that are pre-shrink-wrapped in a
polypropylene housing, which are installed and used in piping following sterilisation. With disc
filters, the users themselves have to fit membrane filter layers. This also applies for candle filters that
have to be fitted in a filter housing (for example, those made by Sartorius and Pall).
It is important to note the possible release of extractables from the filter material, which could reach
the product. Analytical methods should be available and the appropriate studies performed depending
on the application of the filter.
It is also important to monitor the possible adsorption of active pharmaceutical ingredients from the
membrane material, which may potentially cause changes to the product.
12.C.4.4 Filter integrity test

The filter manufacturers carry out a challenge test to prove the classification as a sterile filter, and,
depending on the agreement, supply a certificate, an FDA number, and detailed documentation
together with the filter.
The user is also obliged to validate sterile filtration with the product. The validation can be processed
together with the filter manufacturer.
For integrity testing, manual or automatic tests are permitted. These tests must be validated. Filter
manufacturers have also often developed their own test devices for their filters.
There are several tests that can be used to check the properties and correct functioning of membrane
filters:
Bubble point test

Diffusive flow/forward flow test
Pressure hold/decay test
The commonly used pressure hold/decay test is an indirect method of the forward flow test. This test
provides a quantitative measurement of the sum of a test gas diffused through the membrane and the
flow through each open pore.
Test equipment arrangement:
Pressure-gas - Valve (for separation) - Pressure/time recorder (mbar/s) - Filter -Room atmosphere.
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In this method, the filter housing is placed under a certain pressure and then separated from the
pressurisation system.
The compressed gas flowing through the membrane is quantitatively measured as a pressure drop in
mbar/180 s. The pressure hold/decay test measures the diffused gas (air or nitrogen) through all moist
pores and the gas flow through larger, non-moist pores, when a particular gas pressure is applied to a
moist filter. The pressure drop is measured on the inflow side after disconnection of the gas supply.
The pressure drop is a function of the gas flow through the filter and the volume of the filter system
on the inflow side. The test also checks the integrity of the whole assembly, including seals. It is
therefore important to eliminate faulty seals in order to conclude that there is definitely a problem
with filter integrity.
If the pressure change is lower than the permitted maximum value, the filter has passed the physical
integrity test. If the pressure drop reaches or exceeds the maximum value, the filter is defective.
Investigations into the preparation processes of the filter must be introduced. The solution filtered
through this filter must undergo refiltration (or another measure).
12.C.4.5 Executing sterile filtration

When performing sterile filtration of a solution, it is recommended to use a checklist (or a list
included in the SOP) of all required components of the equipment and accessories, in order to ensure
that no steps are omitted. All openings on equipment and accessories must be covered with
sterilisation paper and secured with bio-indicator strips. All components must be labelled with the
batch no. and drug product. The pore width and type of all filters must also be specified.
All components and devices (see figure 12.C-8) have been cleaned and sterilised in accordance with
the SOP.
Figure 12.C-8 Components and devices for sterile filtration
Equipment for sterile filtration in cleanliness grade B:
Suitable pipeline/connector hose + seal from the container to the filter

Seal + filter containing membrane filter and seal
If applicable, on/off valve and spare gaskets
Storage/filling container
Sterilised tweezers and gloves
Disinfectant and disinfectant cloths
The assembly process must be performed from the cleaner grade B to grade C.
All accessory components removed from sterilisation devices (for example, pass-through autoclave
from cleanliness grade D/C to cleanliness grade B) must be checked to ensure the integrity of the
sterilisation seals and packaging, and transported together to the place of use.
When working under LF, the following must always be noted:
The necessary interventions under LF are only permitted following disinfection of gloved
hands.
The work should always be performed from underneath the open surfaces exposed to the
airflow
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Whenever possible, use tweezers or other utensils (use small tools and instruments so as not
to disturb the LF).
The filter assembly is put together as described in figure 12.C-9.

Figure 12.C-9 Filter assembly
Filter assembly
After sterilisation, connect the filter to the support nozzle of the container under LF,
cleanliness grade A
Before placing the components under the LF, disinfect the objects at the points at which they
were touched during transport (handling)
Loosen the bio-indicator strips, remove, and check for any colour change
Prepare the seal for mounting (open the packaging of the seal)
Lift the sterilisation paper from the connection of the container
Place the seal (removed from the opened sterile packaging) on the support nozzle
Place the filter on the seal - close with a safety clamp (tri-clamp)
If using glass apparatus, the connection can also be made using glass olives and tube material.
The sterile connection from the filter to the storage container has now been established. The container
for the sterile filtered solution is either sterilised together with an assembled air filter, or this filter
must be mounted, as described for the sterile filter in figure 12.C-9.
In CIP/SIP facilities, cleaning and sterilisation of equipment is easier than described in figure 12.C-9,
but still requires assembly steps for which the same level of care must be observed.
The sterile filtration process is the same as described in figure 12.C-10.
Figure 12.C-10 Sterile filtration
Sterile filtration
The product pipe for the unfiltered product can be assembled on the inflow side of the sterile
filter in cleanliness grade B, but should also be performed under LF.
Starting from cleanliness grade B, the piping to grade C can now be set up (according to the
dimensions of the wall inlet) and connected to the batch container.
By overlaying with filtered nitrogen or compressed air on the container, the solution is
compressed into the sterile filter (cleanliness grade B).
The ventilation valve on the "unclean" side of the filter housing (for flooding the housing) can
be used here, for example, to take a sample for the bioburden test.
Once the ventilation valve is closed, the pressure in the filter is increased and filtration into
the storage container (fill container) begins.
Observation of the filtration pressure and the filtration time enable early recognition of any
deviations and failures in the process.
The use of nitrogen as a compressed gas creates a nitrogen atmosphere in the storage container as a
result of the nitrogen dissolved in the solution. If the sterile solution is left to stand for a longer time,
it should be actively overlaid with nitrogen. Standing times before filling must be validated and
limited.
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The sterile filter is subsequently removed from the storage container (see figure 12.C-11).
Figure 12.C-11 Removal of the sterile filter from the storage container
Removal of the sterile filter from the storage container
At the end of filtration, close the valve on the container.

Lift the filter, including seal, from the container supports.
Cover the surface with sterilised film.
If you are using glass apparatus with olives and glass valves, follow a similar procedure as
appropriate. The filter is removed from the piping to grade C and undergoes an integrity test (see
chapter 12.C.4.4 Filter integrity test).
Summary
This chapter describes the systematic sequence of the necessary process steps and presents answers to
the questions posed at the beginning.
The main behaviour and activities of personnel in connection with the planned and prescribed activity
are described (doer/checker interface).
The chapter also describes the procedure for handling any deviations from values and prescribed
processes (OOS).
12.D Washing processes

What are the main container materials used in primary packaging?

Which agents are used to wash final containers?
What particle and microbial content requirements are the cleaning agents expected to meet,
and how are these achieved?
Which temperatures and quantities of cleaning agent are required and which functional steps
are necessary in automatic washing?
Which changes to the surface should be noted?
Which washing procedures are common practice?
12.D.1 Stoppers
12.D.1.1 Material
Stoppers for sterile pharmaceutical products must fulfil the requirements listed in figure 12.D-1.
Figure 12.D-1 Requirements of stoppers
Requirements of stoppers
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Of a suitable hardness, dimensions, and shape for the purpose

Pre-cleaned by the stopper manufacturer to minimise the presence of fill material residue,
colour pigments, and plasticisers
Must not absorb any of the "packed" pharmaceutical product
Must not release any substances into the pharmaceutical product
Smooth surfaces and clean cut edges
Minimum friability
Stoppers for pharmaceutical purposes are usually made from rubber (vulcanised natural caoutchouc).
Due to its high elasticity, the material provides a very good seal against the surface of the bottle neck.
The high durability against wear also enables multiple piercing of the stopper without fragments
breaking off. The material can be sterilised in super-heated steam, is free from toxic ingredients, and
can be cleaned with relative ease. The material can be coloured and produced in varying degrees of
hardness. Natural rubber and its synthetic derivatives - butyl or chlorobutyl rubber - are sufficient to
meet the relevant application requirements in 90 % of cases.
Any applications for which the requirements are not fulfilled with these elastomers either have very
specific requirements (for example, high temperature tolerance, resistance to mineral oil, etc.), or are
subject to maximum requirements for safety reasons (for example, seals for test substances, absolute
physiological safety, or similar). These problems can be solved by using Teflon-clad stoppers or
silicone rubber. The term "silicone" is not applied uniformly. Sometimes it is used to refer to all
silicon organic compounds, while sometimes it is used as a more precise definition for certain types
of Si bonds. These are synthetically produced polymer compounds in which hydrocarbon residues
(usually methyl groups) form long chain, sheet, or spatial structures with silicon atoms, bound by
oxygen bridges. The compounds are heat-tolerant, hydrophobic, isolating, not very volatile, not
flammable, and barely change viscosity in the case of temperature fluctuations. Depending on the
length of the molecular chains and the degree of interlinking, materials are produced in varying states
of liquid "silicone oils" or solid "silicone rubber". This material is most commonly used for tubes and
gaskets. Silicone rubber is mostly obtained from a reaction between silicon and methyl chloride
(Rochow process). The resulting chlorosilane is then fractionated, followed by hydrolysis,
condensation, or polymerisation. The beneficial properties of silicon compounds are summarised in
figure 12.D-2.
Figure 12.D-2 Properties of silicone compounds
Properties of silicone compounds
Heat and cold resistance

High tensile strength
Flexibility
Physiological indifference
Resistant to ageing
Long lifespan
Hydrophobic behaviour
Surface activity
Smooth surface
Low proportion of volatile substances
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12.D.1.2 Manufacture
Quality and purity are the priority aims of manufacturers who guarantee "pharmaceutical quality".
This includes clean room conditions and compliance with standards and internal regulations.
All materials are tested and each of the production steps roling, mixing, shaping, and vulcanising are
monitored according to precise specifications. All shaped (punched) stoppers undergo a final
inspection. The stoppers are cleaned. Each manufacturer has their own procedure. Since shaping the
stoppers in metal trays requires the use of separating media (usually silicone oils), manufacturers
must ensure that all traces of any separating medium are removed. The final state of the stoppers after
cleaning depends on the cleaning procedure and the cycles used.
12.D.2 Particulate impurities

Attempts to keep stoppers 100 % particle-free by washing are illusionary, due to the elastic nature of
the material and its affinity towards particles from the shaping environment. Stoppers can only
accurately be described as low in particles. The determination of the number and size of particles on a
stopper is a very time consuming and complicated process. The total displacement of particles from
the stopper in a washing fluid for particle measurement is necessarily performed as a separate
procedure. Once no further particles are displaced from the stopper into a washing fluid specifically
designed for this purpose, it is unlikely that the product itself will displace any further particles from
a stopper. Of course, this must be proven in comparative studies.
Only the lower part (approx. one third of the stopper surface) of the stopper used in the final
container is moistened by the solution, and the stopper can only shed particles in this area.
If the stoppers are sterilised in a steam steriliser (sterilisation bag), the particle status of the stoppers
changes once again. The influence of the transport system on the filling machine is also significant
for the stoppers (particle emission due to shear forces in the propellant vessel).
It is important that the particle load of the individual stopper must only cause a slight change to the
particle content of the pharmaceutical solution in the range of 10 m/ml and 25 m/ml (see figure
12.D-3).
Figure 12.D-3 Particle load limits
Particle load limits
Small-volume parenteral,
for example 20 ml vial (SVP)
Large-volume parenteral,
for example 100 ml infusion bottle (LVP)
Maximum permitted particle count: Maximum permitted particle count:

6000 P 10 m
600 P 25 m
25 P/ml 10 m
(corresponds to 2500 P/100 ml container)
3 P/ml 25 m
(corresponds to 300 P/100 ml container)
(The Japanese pharmacopoeia requires 2 P/ml 25 m.)
The theoretical causes of particle load in a solution are as follows:

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a) Particles from the solution

b) Particles from the laminar flow (LF) air
c) Particles from the internal surface of the final container, which were not removed in the washing
procedure (can be determined with the use of suitable laboratory procedures).
d) Particles from the surface of the stopper that comes into contact with the product
Re. a): Particles from the solution (filtered over 0.2 m membrane filter and 8 to 30 m needle filter)
can be easily measured and are generally not significant. These filtrations minimise a particle count
of over 10 m to far below the permitted particle count.
Re. b): Particle load from the controlled laminar flow (cleanliness grade 100) can be eliminated,
because no particles larger than 5 m are present in the air.
Therefore, only c) and d) are likely influencing variables.
If you want to evaluate the influence of the stopper washing procedure, this must be compared to the
particulate quality in the final container for (c) (unless the cleaning is just performed "as well as
possible"). During the filling process, final sterilisation, transport, etc., these particles are also shed
into the product.
The required quality of the particulate purity of the Final Rinse (WFI 25 P/ml 10 m and 3 P/ml
25 m) provides a good basis for the calculation.
Residual moisture (WFI) in containers of sizes between 2 and 250 ml after blowing out with particlefree air is present in quantities ranging between 10 and 150 mg, which corresponds to a particle load
of 5 particles 10 m and 0.6 particles 25 m per object. This is therefore negligible compared to
the permitted level of 6,000 particles 10 m and 600 particles 25 m per container and for 100 ml
infusion bottles, 2,500 particles 10 m and 300 particles 25 m per bottle.
Re. d): The nature of an elastomer and its manufacturing process means that its surface is subject to
higher particle load than, for example, a glass surface. It can be assumed that approx. 80 % of the
particle load of a product is caused by the stopper sealing system (teflon-clad stoppers show better
results).
12.D.2.1 Stopper washing

The washing process must be executed in accordance with a valid SOP and must be incorporated as a
work step in the manufacturing instructions.
Reasons for washing stoppers in pharmaceutical operations include:
The (particulate and microbiological) cleanliness of the stoppers is ensured by your own
validated processes.
The time from cleaning to use can be planned, to guarantee microbiological quality.
The unification of the surface properties enables bottles to be processed on sealing machines
(this is often the main problem with faster filling machines). It is important to keep a detailed
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record of the delivery status of the stoppers by implementing a goods in check and
monitoring.
To remove the particles from the surface without affecting the properties of the stopper, the
conditions listed in figure 12.D-4 must be fulfilled.
Figure 12.D-4 Stopper washing
Stopper washing
Washing fluid made from purified water (if necessary with tenside for improved surface
moistening)
Temperature of the washing fluid as high as possible (minimum 70 C)
Determine the percentage quantity of the washing fluid/number of stoppers
Continual separation of particles from the washing fluid by filters (pore size 2 m) using a
circulation pump
Stoppers moved with minimum friction (if possible, flotation) of the stoppers to prevent any
surface changes
Apply as many boundary layer influences as possible (air/liquid/steam) to the surface of the
stopper, which increases the cleaning effect
Rinse phases and final rinse >80 C for microbial reduction
Sterilisation or steam overlay for microbial reduction
These essential cleaning steps are executed with the help of washing machines that control the use of
media and include programs to regulate the chronological sequences. Washing machines for this
purpose are supplied by several manufacturers. Separate wash programs can be defined and validated
depending on the type and quantity of the stoppers (freeze-drying stoppers, injection stoppers,
infusion stoppers, etc.). The washed stoppers should be removed from the washing machines into
washed and, if necessary, sterilised containers. Stoppers that need to be sterilised in autoclaves should
be placed and heat-sealed in particle-free sterilisation bags. Since these washing machines are very
expensive and can be laborious to operate, and the quality of the washed stoppers has to be constantly
monitored, as an alternative, "ready-to-use" stoppers can be purchased from the manufacturer
(accompanied by the appropriate certificate).
12.D.3 Glass containers (ampoules, bottles)
12.D.3.1 Types of glass

Glass containers for pharmaceutical contents are manufactured in glass of varying composition, and
are classified into hydrolytic (water resistance) classes 1, 2 and 3. This classification is necessary,
since the pharmacopoeias of different countries require that injectible products must only be filled in
containers whose hydrolytic surface resistance meets the requirements of glass type 1 (hydrolytic
class 1). Glass containers in accordance with hydrolytic class 1 are also known as borosilicate glass,
and hydrolytic classes 2 and 3 are known as soda glass. To achieve the hydrolytic classification, the
glass must contain a specific mixture of components, which indicates the hardness of the glass as well
as the behaviour of the surface in contact with the filled product. It has been determined that the
temperatures required for processing glass to make containers cause changes to the internal surface
that can have a negative effect on the filled product. This mainly refers to the vaporisation of Na2O
that collects on the internal surface. In aqueous solutions, this can lead to changes in the pH value and
other interactions. The internal surface of the glass therefore has to be processed. This improves the
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hydrolytic class of the internal surface, for example, from class 2 to class 1. To improve the
hydrolytic class, the glass is treated with ammonium chloride (or ammonium sulphate). During the
expansion process at approx. 580 C, the Na2O residues form a chemical compound with the coating
material, which gives the surface a slightly milky appearance. This coating can be easily removed in
the initial washing phases during normal cleaning procedures.
12.D.3.2 Manufacture
Two different procedures are used for manufacturing containers:
Deformation of glass pipes for ampoules of between 1 and 50 ml and bottles between 5 and
30 ml.
Production from molten glass by glass blowing, for bottles between 50 and 1000 ml.
Manufacturers of glass containers for the pharmaceutical industry have fulfilled the basic regulations
of the GMP requirements by setting up clean rooms after the tempering oven (580 C) for packaging
with plastic trays and cartons in shrink film. It is essential to use a clean room or an LF box for the
steps Wrapping in film, Heat sealing and subsequent shrinking, to keep the levels of organisms and
particles as low as possible. The different types of packaging are commonly known as safe-pack,
hygiene-pack or clean-pack.
12.D.3.3 Washing
The washing processes for ampoules and bottles take place upstream of the filling process in
compact lines. These systems consist of a combination of washing machine, sterilisation tunnel, and
filling machine. The washing machines are designed as rotary or bar-based systems. The media used
are filtered compressed air (0.1 m), purified water or a WFI connection for supplying the circulatory
water (7 m/2 m filtration). WFI is prescribed for the final rinse (with a particle filter 20 to 30 m
against particle emission through valve seals and abrasion from pressure pumps). The cleaning
procedure comprises the steps listed in figure 12.D-5
Figure 12.D-5 Cleaning procedure for containers
Cleaning procedure for containers
Ultrasound
Blow out with compressed air (0.1 m particle filtration) or outlet (for infusion bottles with
large bottle necks)
Spray inside and out with purified water via nozzles (using approx. one third of the object
volume per spray cycle)
Blow out with filtered compressed air
Spray with filtered, purified water
Spray with filtered compressed air
Spray with WFI (final rinse, 20 to 30 m particle filtration)
Blow out with filtered compressed air
The use of ultrasound in the first stage of the cleaning procedure (the object is filled and transported
immersed in water) has been demonstrated to contribute the mechanical detachment of impurities due
to a "cavitation" effect (implosion of hollow spaces in the water created by the ultrasound).
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When spraying with filtered, purified water, you should ensure that the water can also drain away
within the available time phase, as otherwise, only rinse water that contains particles circulates in the
object and the particles are not rinsed off in the water.
It is important to use a quantity of rinse water corresponding to approx. 1/3 to 1/2 of the object
volume, to ensure that the boundary layer effects of air/fluid can be allowed to act on the internal
surface. The number of spray processes depends on the requirements of the pharmaceutical
manufacturer.
Validation
All washing processes must be validated.
Since the overall washing effect results from the parameters listed in figure 12.D-6, all these
parameters must be determined and validated.
Figure 12.D-6 Validation parameters for washing containers
Validation parameters for washing containers
Purity of the purified water

Residence time of the object in the water bath with ultrasound
Temperature of the water bath
Quantity of splash water
Spray pressure of the water in object (1 to 3 bar)
Run-off time/remaining amount of water after blowing out with compressed air (approx. 1.5
bar)
Number of cycles
The effectiveness of the procedure can be determined by spiking with endotoxins followed by
endotoxin reduction, and by deliberate impurification of objects with standard particles (for
example, 20 m standard particles as used for testing particle measuring devices). It is important to
remember that the endotoxin reduction will be disproportionately more difficult, since quantities of
endotoxin applied to the glass surface structures adhere much more strongly to the surface. A 2-log
reduction is normal. When rinsing particles from the surface, the quantities of rinsing agent are less
significant. The impact velocity of the rinsing agent and the frequency of repetition of the process are
more important factors.
For example: If 99 % of the particles present are rinsed out in one rinse procedure, three rinse
procedures will cause a 6 log reduction. This means that more than three cycles will not make a
significant difference to particle reduction, but will in the case of endotoxin reduction. In the latter
process, the spray pressures and hence also the "penetration depth" into the surface structures are
more important. However, these functions are limited by the design of the washing machine, its time
settings (residence time of objects in the spray phase), and the output of washed objects per hour.
The final rinse with WFI has a particularly important effect. This stage must ensure that the internal
surface quality of the objects matches with the specification of the WFI. Blowing the objects out with
filtered compressed air ensures that only a small amount of residual WFI remains in the container (10
to 150 mg), and that any substances contained in this water can be precipitated on the internal surface
during the sterilisation process in the hot air tunnel.
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12.D.3.4 Ready to fill

In general, these are ampoules or radiation-sterilised plastic containers in sterile packaging which,
after passing through an air lock into cleanliness grade B, can be passed to filling machines with LF
or clean benches where they are filled with solution. This method of production is suitable if
investments for larger machine systems are not feasible and qualification, process validation, and
monitoring are too complex given a small number of items.
12.D.4 Transport
Stoppers are supplied by the manufacturers in plastic bags as a mass product with several hundred
and up to a thousand items per bag. After loading into the washing machine, washing and sterilisation
in the washing machine, the stoppers are automatically or semi-automatically ejected into stainless
steel transport containers, which are then transported to the capping machine. Here they are mounted
onto the pipe connections of the capping machine under LF. The objects can also be output in
sterilised transport bags, which are subsequently heat-sealed and later emptied into the transport
systems (propellant vessels) of the capping machine.
Stoppers that are supplied by the manufacturer in sterilisation bags "ready to use" must be sterilised
in the steam steriliser, removed under grade B conditions, and then loaded into the transport system
of the capping machine.
Stoppers that are used for sterilisable final containers, and therefore do not have to be sterile to begin
with, should be stored in a cool place until needed. The microbiological load over the planned storage
period must be validated.
During processing of the washing process, dry heat sterilisation, and filling stages, glass containers
are transported through the compact lines on transport racks, conveyor belts, and helical conveyors.
At the end of the process, the objects are transferred to stainless steel or plastic magazines. In these
magazines, they can be sterilised and forwarded for visual control.
Summary
The above section describes the manufacture, material properties, and delivery packaging of the final
containers and their treatment in the washing process. The processes and performance of the washing
machines are described, together with the microbiological and particulate values that should be
achieved in the washed containers.
12.E Filling
What clean room conditions are required for filling in an open container?
What is the difference between products sterilised in the final container and products filled in
aseptic conditions?
What are the tasks of the various system components in the filling system?
What are the different types of filling systems and how are they evaluated?
Which steps are required to execute and control a filling process?
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Which errors may occur?

How is it possible to prove the success of the aseptic filling method?
What needs to be taken into account when filling with powders?
12.E.1 Filling equipment for solutions

The solution exists as a batch in the storage container (filling container) and has been filtered and
released for further processing. The containers to be filled are cleaned, sterilised, and depyrogenated,
and are transported to the filling machine by conveyor belts or manually from magazines. The
containers are filled with solutions by machines specifically designed for this purpose, which are
available from several manufacturers. Filling systems have undergone considerable changes in recent
decades, particularly in terms of their electronic controls. The main differences when compared with
single and double-figure ampoule and bottle filling machines with dosage pumps are as follows:
1. Linear filling machines or rotary systems with several filling points and weighing cubicles
2. Filling machines with time-pressure filling systems and turbines
There are advantages and disadvantages to all machines, which differ according to fill quantity per
object and solution properties of the solution, and a system must be selected based on these properties
(see figure 12.E-1).
Figure 12.E-1 Advantages and disadvantages of different types of filling machine
Advantages and disadvantages of different types of filling machine
Linear filling
machines
Routine technology
Space requirement
Rotary systems
High capacity, long fill time for

individual objects
Complicated mechanics and pump control
Time-pressure
filling
system
No pump, good cleaning
Dosage problems for different size containers

and preparations, capacity/h
Weighing
cubicle
systems
No pump, safe dosage
Complicated transport system, capacity/h
Other important points are: Viscosity, density and solid matter content of the solution. These
parameters influence the flow rate in piping, pumps, and hollow needles for filling objects, and thus
affect the time required to fill each object. These parameters are used to calculate the filling capacity
of a machine.
12.E.1.1 System structure

A dosage filling system (piston dosing), consisting of a solution container, pump and final container
must include the following components:
Storage vessel (or container), containing the solution to be filled

Level vessel in which smaller quantities of the solution are kept for the pump system
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Solution distributor (feed to individual pumps)

Valve mechanism for controlling the aspiration/pressure phase of the pumps
Piston dosage pump (or pump with integrated aspiration or pressure configuration)
Hollow needle for filling the final container
Connecting pipes (stainless steel) or tubes (for example, silicone, teflon, etc.) for liquid flow
in all system components.
Self-priming pumps are used in all cases apart from the time-pressure filling system, which does not
use any pumps.
The function of each system component is described in the following:
Storage vessel
Stores the solution in the tank and maintains temperature at room temperature (occasionally at higher
temperatures during filling). Transports the solution due to constant gas pressure (filtered nitrogen), if
necessary via organism-particle filters, into the level vessel of the filling machine.
Level vessel
The level vessel is made from stainless steel or glass and has a valve mechanism that guarantees a
maximum and minimum level of liquid in the vessel with no pressure. The difference in the liquid
level compared to the aspirating piston dosage pumps should be no more than minus 60 cm. The
aspiration pressure of the pumps can be increased or decreased by setting the level vessel at different
heights (can also be positioned using the pumps).
Solution distributor
These are small containers (0.5 to 10 l), from which the connected pump pipes or tubes suck the
quantity of solution directly into the pump. Due to the negative pressure that forms in the solution
distributors, they extract solution from the level vessel and refill themselves in time for the next work
cycle. A solution distributor is therefore not required if the level of liquid in a storage vessel is
approximately equal to or greater than that in the pumps. In general, however, this does not apply,
since the level of liquid in the storage vessel is usually drastically reduced during the filling process.
This means the height difference becomes too great for suction to be possible.
Valve mechanism (cursor)

The aim of the valve mechanism is as follows: To establish a suction channel from the solution feed
to the pump cylinder on retraction of the piston in a synchronised cycle. Then, at the peak of the
movement, to shut off the suction channel and open an outflow channel to the filling needle. On
forward propulsion of the piston by the cylinder, the previously extracted quantity of solution is
ejected towards the filling needle. At the end of the piston movement, the outflow channel is closed
and the suction channel is quickly reopened (back suction).
Piston dosage pump

On the intake stroke of the piston movement in the cylinder, the piston dosage pump takes up a
certain quantity into the cylinder space created and, after the cursor has switched position, ejects it
via the (adjustable) set route. The primary aim is to always eject the same set quantity. A metering
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accuracy of 0.5 % should be achieved in order to fulfil the requirements of the pharmacopoeias
(USP, JP, EP). The material for the piston dosage pumps can be selected from stainless steel,
ceramic, glass, or combinations with seals. The selection of the material depends on the dosage
volume and the long-term stability. An important factor is whether the pumps in an installed state can
be cleaned (CIP) and sterilised or steam-treated within the machine (SIP). For pumps made from
stainless steel and ceramic, this procedure is the state of the art. In cases in which pumps and pipes
have to be dismantled and taken apart after use, the individual components are cleaned in accordance
with an SOP and sterilised before re-use (steam sterilisation or dry heat sterilisation for glass
accessories). CIP/SIP treatment (cleaning in place/sterilisation in place) of pumps, pipes, and filling
needles almost always includes simultaneous cleaning of the solution feed from the storage vessel,
since this is the point at which the WFI and steam for cleaning are connected and supplied. The
CIP/SIP procedure is then carried out controlled by a program (see chapter 4.I CIP (Cleaning in
Place)).
Hollow needle (filling needle)

Hollow needles for filling containers are selected in different diameters according to the solutions to
be filled (viscosity, foam characteristics, flow rate and surface tension), and depending on the
container opening. In general, these are pipe sections with a diameter of 2 to 10 mm. When the
hollow needle is inserted into the container, the solution should be squeezed into the container
(ampoule/bottle) in the pressure phase of the pump piston. Turbulence in the existing solution in the
container should be minimised in order to avoid foam formation. Foam means that when the bubbles
burst, droplets of liquid land around the neck of the bottle, where they are pressed on the lateral seal
surface of the stopper. This may result in crystal deposits, which can enter the solution as
crystallisation inducer.
Foam formation in the filling phase of an ampoule is even more serious, since the chimney effect of
the rising air carries foam bubbles through the glass ampoule, and they precipitate around the tip seal.
This effect also occurs after the end of the filling phase, when a larger foam layer remains on the
surface of the fluid. The rotation phase at the tip sealing station causes the foam bubbles to burst, and
the liquid droplets slide into the glass ampoule where they dry out or even become charred. In the
subsequent steam sterilisation process, these cracked solution ingredients are then rinsed in the
solution and lead to a directly visible and detectable reject. It is even worse if these dried substances
are not removed until after the optical control. Suitable measures must be introduced to prevent these
problems as far as possible. A deciding factor can be to reduce the rate at which the solution flows
from the needle into the container. To reduce this rate, it is necessary to employ a combination lower
filling pressure and the largest possible diameter of filling pipe. It can be advantageous to start the
first phase of filling quickly and to fill more slowly towards the end of the filling quantity
(mechanical-technical filling characteristics of movement control of the filling pumps).
Connecting lines/tubes
Connecting lines (pipes) for transporting the solution should always be used if there is no movement
involved and the machine can be cleaned in its installed state. Tubes must be used at points in which
constant synchronised motion is required (for example, the lifting and lowering of a filling needle
attached to a tube), and at points in which the filling equipment has to be disassembled or partially
disassembled for cleaning and sterilisation. From a technical flow perspective, tubes have to fulfil the
same task as fixed connecting lines (pipes). This means that the composition of tube walls must be a
stable shape, and changes in the volume of the tube under pressure must be kept to a minimum (there
must be a minimal "breathing" effect).
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Time-pressure filling system

This type of dosage of solutions into a container requires an exact, very fast measurement method and
calculation of the opening time phase of the cut-off mechanism against which the solution flows. The
cut-off mechanism can be a method for squashing a silicone tube, a membrane valve, or a mechanical
cursor. The required accuracy is the most important factor in the selection of a cut-off mechanism,
which means that the options of membrane valve or mechanical cursors are less frequently
considered. Due to the required dose accuracy, time-pressure filling systems are only useful for
amounts greater than 50 ml. It is important to remember that the preliminary pressure (nitrogen or
compressed air) created to transport the solution through the opened cut-off mechanism and the
hollow needle into the container must be sufficient to force an excess of solution (volume) through
the hollow needle within the residence time of the container under the hollow needle. Switching the
cut-off mechanism limits the quantity of solution. The opening time of the cut-off mechanism is
therefore dependent on the current density (according to the solution temperature) of the transported
solution. This must be controlled in a computer-controlled pressure/density/temperature relationship.
If processing several different solutions (preparations) and container sizes, the applicability of this
system is limited. If filtration (of particles and organisms) or different-sized containers (tanks) and
different lengths of piping are then also used, problems will undoubtedly arise. The advantage of this
system is the complete absence of pumps and hence the associated problems
Weighing cubicle
The principle is the initial weighing into the container and the switching of the filling process by the
weighing cubicle. The dosage is measured for each container and maintained by constant monitoring.
This controls the filling system. However, the system involves expensive transport systems to the
weighing cubicle and zeroing and weighing each container takes time, which means that capacity is
limited. All other problems of a filling system, such as cleaning, partial dismantling, and sterilisation,
also remain. No pumps are used. Other tasks that are usually fulfilled by a pump are performed by
exerting a preliminary pressure on the solution in the supply tank to the on/off valve, which is
controlled by the weighing cubicle.
Filling turbines
The fill solution flows through the turbines within the predefined pressure range, for example 1 to
2 bar, and the revolutions (converted to volume) are "opened" and "closed off" according to the dose.
In my experience, the technology is not yet sophisticated enough in the range from 5 to 20 ml.
12.E.2 Process for filling LVP containers in cleanliness grade C

Figure 12.E-2 shows the individual steps required for filling in a facility for LVP (Large Volume
Parenterals) in cleanliness grade C. Dosage controls should be performed hourly, and half-hourly for
high capacity machines. An additional dosage control should also be performed for every anomaly.
Figure 12.E-2 Process for filling LVPs solution in cleanliness grade C
Process for filling LVPs solution in cleanliness grade C
Check the bottle washing machine (acc. to checklist) and hot air tunnel, if applicable
Check pressure differentials between cleanliness grade C/cleanliness grade D and the
immediate containers (release, batch, hydrolytic class, etc.)
Check that the reservoir/holding tank is assigned to the filling system (batch, date,
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preparation, final container)

Check the LF equipment (particle and flow rate measurement), documentation
Check the stoppers to be used (type, release, cleaning and sterilisation processes)
Check and calibration of the weighing apparatus for dosage control
Place the stoppers in the clean propellant vessel of the capping machine
Attach the cleaned and sterilised pumps, terminal filters and tube connections to the level
vessel
Connect the solution feed, if necessary with a 0.2 m/0.45 m filter from the holding
tank/reservoir to the level vessel of the filling machine
Start the pump function for self-priming and pumping out in a sample vessel for IPC of the
integrity of the solution
Check the immersion depth of the filling needle in the object
Check the dosage amount
Check that the capping machine is functioning correctly (crimping)
Check the dosage measurement and torque control of the sealing cap
Enter documentation in the dosage control sheet
Fine dosage adjustment
Enter documentation in the dosage control sheet
Start of filling, dosage control and documentation after release by the IPC lab
Test the sealing cap, documentation
End of filling
Check the LF equipment (particle and flow rate measurement), documentation
Compare number of items/supplied quantity of solution, consumption of bottles and sealing
caps
Disassemble pumps, tubes and level vessel for cleaning and sterilisation, for CIP/SIP, switch
connections for WFI, steam and compressed air feed
Clean and disinfect machine surfaces
When using CIP/SIP systems, the first activity is usually to attach several pipe and tube connections
to the pump system, since this is how the WFI, steam and compressed air are supplied. Figure 12.E-3
lists possible faults and information on their cause and possible prevention.
Figure 12.E-3 Possible faults when filling infusion bottles / vials
Possible faults when filling infusion bottles/vials
Possible faults
Cause/prevention
Solution flowing into the

bottle sputters at the end of
the filling needle
Solution forms too much

foam on the surface.
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Pump fill pressure too high (machine speed)

-> Reduce machine speed
Cross-section of the filling needle too small
-> Use needle with a larger cross-section
Drop height of the solution into the bottle is too high
-> Lower the filling needle into the bottle
Filling needle too close to the bottom of the bottle
-> Soften the impact of the solution by lifting the filling needle
Temperature of the solution is too high
-> maximum 30 C
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Filling needle drips
Pore size of the needle filtration sieve is too small

-> Choose larger pores or mesh size
Reverse suction effect of the pump is too small
-> Increase time for pump reverse suction (reverse suction route
in the needle 1 to 3 cm)
Temporary dosage
fluctuations
Suction conditions have altered due to stiffness of pump and

tailings of solutions
-> Ensure the pump and valve control are running smoothly and
check the fill level control in the level vessel
Dosage changes
Change in machine speed and thus suction conditions

-> Reset the pump dosage
Stoppering is too difficult

(incomplete)
Fit between the bottle neck diameter/stopper diameter is too

narrow
-> Measure the containers and stoppers
Adhesive power between the stopper material and glass is too
high
-> Increase the moisture level of the stoppers and bottles (if
necessary, use siliconisation)
Stoppering is too easy

(stoppers are ejected by air
bubbles)
Fit between the bottle diameter and the stopper is too wide and
too moist.
-> Measure the containers and stoppers, use drier stoppers and
bottles (if necessary, increase the blowing out of the bottles with
compressed air and ensure that the bottle opening does not
become wet from the solution)
Crimping incomplete, not

tight enough
Too little side material, too little downwards pressure on the cap
-> Increase pressure of the plunger
Incorrect roller alignment of the crimping head
Crimping deforms the

aluminium cap
Pressure on the cap is too high, material overhang on the bottle

neck
-> Reduce the pressure on the plunger, check the roller position
of the crimping head
12.E.3 Process for filling ampoules with solution in cleanliness grade A/B
Figure 12.E-4 describes the filling process in cleanliness grade A within a grade B environment as it
is prescribed for aseptic manufacturing.
Figure 12.E-4 Filling process in cleanliness grade A/B
Filling process in cleanliness grade A/B
Check the ampoules supplied in hygienic packaging (release, batch, quantity, size, etc.)
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Check the washing machines and the hot air sterilisation tunnel according to the checklist
(media pressures, temperatures, capacity specification, etc.)
Start the equipment (normally located in cleanliness grade D)
Check the pressure differential between cleanliness grade B/grade C/grade D
Assemble the filling equipment (pumps, hollow needles, tubes, filters for nitrogen gas and
solution distributor
Check (in operation) the LF in cleanliness grade A, particle measurement, flow rate,
microbial count determination using the air sampler method, CFU determination of the
machine surface in contact procedures
Check (in operation) the environment in cleanliness grade B using the air sampler method and
for contact procedures, check equipment surface and floor, as well as particle measurement.
The filling container/reservoir is usually in cleanliness grade B close to the filling machine or,
less commonly, in cleanliness grade C in the case of ongoing sterile filtration into cleanliness
grade B in a level vessel.
Control and calibration of weighing apparatus
Aspiration of the solution into the solution distributor by starting the filling pumps or by
using a vacuum pump for smaller fill volumes and relatively long pipelines with a relatively
large tube diameter
Set the filling speed
Pump solution from the hollow needles as a preliminary run into a sample vessel for IPC and
for general dosage settings
Control the immersion depth and centring of the hollow needles in the ampoules
Dosage measurement, documentation in the dosage log, and balance printout
Fine adjustment of dosage, documentation in the dosage log, and balance printout
Control CFU determination, personnel fingerprints in grade B
Adjust the flame settings in the tip sealing station
Start filling following release by IPC lab
Check the ampoule seal, glass ampoule shape and purity
Check dosage, document in the dosage log and balance printout
Send filled and sealed ampoules to IPC lab
Remove filled and sealed ampoules in steel magazines, attach a label with a consecutive
number, preparation, batch, etc.
End of filling
Check the environment:

CFU determination by way of contact procedure - grade A machine surface in LF
CFU determination by way of contact procedure - grade B surfaces (tables, balances, exterior
of filling machine)
CFU determination by way of contact procedure - grade B floor
Personnel controls:
CFU determination by way of contact procedure - cleanliness grade B fingerprints
Disassemble pumps, tubes, solution distributors, etc.
Clean the filling machine, disinfect surfaces, disinfect floor
12.E.4 Filling ampoules in cleanliness grade C and laminar flow

This work area is used for solutions that are sterilised in the sealed ampoule. All steps described for
filling ampoules in a grade A/grade B environment (see figure 12.E-4) must be executed, with the
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exception of microbiological tests, since there are no batch-specific tests for cleanliness grade C. In
general, these are monitored weekly or at other regular intervals as a part of quality control. Different
limits also apply (see chapter 12.G Microbiological monitoring).
Figure 12.E-5 shows the possible faults that can occur when filling ampoules.
Figure 12.E-5 Possible faults when filling ampoules
Possible faults when filling ampoules
Possible faults
Ampoule neck becomes wet from
solution
Cause/prevention
Bubbles or foam form on the solution

surface in the ampoule
Drop formation on the filling needle
Ampoule lance has incorrect shape:

Bubble head
Incorrect positioning (centring) of the filling needle

> Check the centring and alignment of the filling
needle bar
Filling needle is bent
> Straighten the needle
Pump fill pressure is too high
> Reduce machine speed
Cross-section of the filling needle is too small
> Choose a larger cross-section
Drop height of the solution into the ampoule is too
high, insufficient low level filling
> Reduce immersion depth of the needle
Filling needle is too close to bottom of ampoule
> Increase distance between filling needle and the
bottom of the ampoule.
Reverse suction effect of the pump is too low.
> Change the control of the reverse suction effect.
Pressure tubing to filling needle is too long
("breathing")
> Shorten the tube, use harder material if necessary
Pump is leaking
> Replace the pump
Temperature of the solution is too high
> Reduce solution temperature
Solution wets the lance part and evaporates
> Check the centring and shape of the filling needle
Tip sealing flame is too hot
> Reduce gas supply
Solution is too cool (slight vacuum effect)
> Bring solution to room temperature
12.E.5 Culture medium filling (Media Fill)
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After the three media fills for validation, a nutrition agar fill should be performed at regular intervals,
since this is the only way to definitely prove the aseptic filling technique. This control is necessary
due to the large number of process steps involved in an aseptic operation and possible deviations
from the ideal requirements in terms of personnel, activities, environment, media and equipment.
Routine monitoring of normal production provides only a snapshot of the operating status.
The 2004 FDA guideline on aseptic technique (chapter D.10 Guidance for Industry Sterile Drug
Products Produced by Aseptic Processing - Current Good Manufacturing Practice) states that after
validation, a media fill should be performed twice yearly.
Figure 12.E-6 Warning and action limit depending on the number of filled containers
No. of filled containers
Alert limit
Action limit1
3,000
Not applicable
4,750
6,300
7,760
9,160
10,520
11,850
13,150
14,440
15,710
10
16,970
11
1 Media fill failed.

The culture medium should enable growth of the widest possible spectrum of organisms. The
medium, for example, casein soya bean digest broth, should have a low selectivity and be
recommended by a pharmacopoeia (for example, USP). For culture medium filling (media fill), all
the same activities are required as for grade A ampoule production in a grade B environment (see
figure 12.E-4), but under worst-case conditions. This means the slowest fill speed (containers and
solution are exposed to environmental conditions for longer), planned interventions (multiple), as
they can occur in the case of faults in normal production, and maximum presence and activity, for
example, of technical personnel. A minimum number of 3000 filled containers is required. For a
confidence level of 95%, none of the containers must be contaminated. It is therefore common
practice to fill a minimum of 4750 containers. The alert threshold limit is then 1 contaminated
container, with an action threshold limit of two contaminated containers (see figure 12.E-6).
If you consider that a modern system can fill 120 to 300 ampoules per minute, this would mean a
time period of just 10 to 25 minutes. This is a short time period for assessment. In addition, it is
difficult or impossible to allow filling cycles and tip sealing processes to run as slowly as possible.
These operations can then not be correctly executed and, due to the properties of the culture medium
solution, lead immediately to filling errors, breakage of glass containers, and insufficient tip sealing.
In practice, therefore, a slow fill speed is selected at which all functions can still be guaranteed, and
the planned interventions and number of items correspond to a runtime of approximately one hour.
Added to the set-up and set-down times for the media fill, this requires a time period of over two
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hours. The work required for the subsequent microbiological processing, such as incubation and
quality control (visual control) then remains reasonable and provides a realistic process simulation.
Figure 12.E-7 Execution of a media fill
Execution of a media fill
Pump two large quantities of solution (approx. 500 ml) into sterilised glass containers (for
example, beakers) for incubation and inoculation with organisms to test for growth. This can
be used to ensure that the equipment is sterile and the personnel performed the assembly
procedures properly.
Control CFU determination, personnel fingerprints in grade B
Take fingerprints from all employees in cleanliness grade B and in the planned interventions
and samples (must be described in detail in the SOP for media fill).
Stop the machine, remove 10 to 20 objects before, at, and after the filling point, loosen and
tighten the hollow needle, restart the filling machine, set the dosage, and after filling and
capping the first subsequent ampoules, remove them as a sample and perform an additional
incubation and assessment. All this should also be included in an SOP to be created by the
unit.
The culture medium solution for the medium fill is treated in exactly the same way as the product, i.e.
it is generally sterile-filtered. It may be necessary to heat the culture medium solution, as otherwise
the filters can quickly become blocked. Connections to the filling equipment tubes should be made
under LF.
The ampoules are filled as described in figure 12.E-4 Ampoule filling in cleanliness grade A/B.
Additional and variant steps are shown in figure 12.E-7.
The manufacturing instructions for the media fill include the time specifications and conditions for
incubation of the filled objects (for example, normal or upside-down), and the results protocols of the
incubated objects in the individual magazines.
If non-sterile objects are identified, you can narrow down the time the sample was filled, and note
any peculiarities that occurred within this period (intervention? dosage control? dosage change?
unplanned stop due to washing machine or sterilisation tunnel fault? etc.). The results of analysis of
the non-sterile objects for microbial species are documented in the manufacturing instructions.
Microbiological results and assessment

Calculation of the proportion of non-sterile objects following incubation of the filled containers (see
figure 12.E-6): Target <0.1 %.
CFU determination at the monitoring points in the grade B filling room (surfaces, floor, air).
CFU determination at the monitoring points (see chapter 12.G Microbiological monitoring) of
personnel and material locks in grades C and B (air and surfaces).
Yield calculation, assessment and release by management.
12.E.6 Filling with powders

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Sterile powders are normally obtained from sterile filtered solution by spray drying or crystallisation,
milled, micronised (if applicable), mixed with lubricants and additives and, if necessary, sterilised
with dry heat or gas. The properties of the powder, such as fluidity, bulk density, dust production
(abrasion), sensitivity to moisture, and the type of containers to be filled, are determining factors for
the construction of the filling machine used.
Two basic variants are possible:
Machines that use a feed shoe to mechanically fill a mold with a certain quantity of powder
and eject or blow this out into the specified container.
Machines that transport powder to the container in the weighing cubicle via vibrating rails
until the planned preset dose is reached, then automatically stop the conveying and transport
the next container for filling.
The requirements for a machine for powder filling are just as demanding as for a filling machine for
solutions. Particular attention must be paid to the areas of cleaning, assembly and disassembly of
components, and the flow profiles of air due to particle load from the contents.
It should also be possible to perform a culture medium fill instead of, or in combination with, the
powder to provide evidence of aseptic manufacturing methods.
12.E.6.1 System layout of the filling equipment

A system for filling containers with sterile powder usually consists of the following components,
which are used for the functions described below:
Reservoir
Storage of the powder during filling. Feeding powder into the filling system through the vibration
facility on the reservoir or helical conveyor.
Coupling system, helical conveyor, vibration piping

The system must enable containers to be connected, and in some cases replaced, under LF (possibly
outside of the machine LF).
Feed shoe for molds - vibrating bars

The feed shoe must fill a space in the mold with powder in accordance with the required dose. A
vibrating bar or a vibrating pipe transports powder to the designated container through adjustable
vibration (time and amplitude) and gradients. The vibration time is controlled by the dosage balance.
Aspiration system for developing dust

Apart from the usual grade A environmental conditions used for aseptic filling, the build up of dust
cannot be avoided when filling containers with powder, and it is not possible to comply with the
acceptable particle count for ISO class 5 (previously class 100 209E) "in operation" at any point in
the LF. This means that an aspiration system for dust is required at transition points between the feed
shoe and the mold or the vibration pipes. This aspiration may have a slight influence on the laminar
stream in the LF, but it limits the particle load at these points. A transportable suction system
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(vacuum cleaner for the ISO class 5 clean room area ) must be available in case of faults in the
production process.
Weighing system - vibration conveyor

Start the vibration conveyor after weighing and zeroing the empty container. Switch off depending on
the set dosage.
12.E.6.2 Practical process using a glass bottle as an example

The process should be performed in accordance with a valid SOP. The filling takes place in grade A
conditions within a grade B environment.
Check the bottle washing machine (acc. to checklist) and hot air sterilisation tunnel.
Check pressure differentials between cleanliness grade C/cleanliness grade B and the
immediate containers (release, batch, hydrolytic class, etc.)
Check the reservoir(s) (batch, date, preparation, assignment to filling equipment and
immediate container, and sterilisation seal).
Check the LF equipment (particle and flow rate measurement), documentation.
Check that the aspiration system of the filling machine is functioning correctly.
Check the seals for the immediate containers (type, release, cleaning and sterilisation
processes or ready-to-use documentation).
Check the calibration of the balance equipment for dosage and for a system check.
Stock the capping system with seals.
Assemble the cleaned and sterilised product-stirring filling mechanism
Connect the reservoir under LF to the coupling system
Control (in operation) of the LF grade A, particle measurement, flow rate, CFU determination
using the air sampler method (see chapter 12.G Microbiological monitoring), CFU
determination of machine surface by way of the contact procedure.
Control (in operation) of the grade B environment using the air sampler method and contact
procedure on equipment surfaces and floors, and particle measurement of the air.
Start the conveying and filling mechanisms
Check the dosage amount
Check that the capping unit is functioning correctly
Set the dosage
Control, CFU determination, personnel fingerprints in grade B
Start filling following release by IPC
Check the dosage and documentation (automated using the weighing system when using
vibration conveyor and weighing cubicle)
Output of filled and sealed containers in subsets (magazines) with label containing
consecutive number, preparation, batch, etc.
End of filling
Microbiological environmental monitoring of the following positions:

o in grade A, machine surface in LF
o in grade B, working surfaces (e.g. exterior of filling machine, tables, etc.) and floors,
fingerprint of personnel
Disassemble the feed hopper
Clean the filling machine, disinfect surfaces, disinfect floor surfaces.
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Figure 12.E-8 lists the potential faults, together with possible causes and prevention or testing
methods.
Figure 12.E-8 Possible faults when filling with powder
Possible faults when filling with powder
Possible faults
Cause/prevention
Tailings of powder from

the reservoir faulty
Machine switches off due

to insufficient powder feed
Fill level in reservoir is too high, causing too much weight

and reduced vibration of the coupling system > Reduce
the fill level
Faulty flow properties of the powder, humidity is too
high? > Test
Change in particle size? > Test
Adjust pipe diameter or insufficient column width of the
reservoir to the coupling system
> Adjust the settings
Fill speed of the machine is too high
> Reduce or adjust speed
Summary
This section describes the main functions of a filling system and explains the necessary system
components and their technical/physical tasks. Possible faults are discussed together with their causes
and prevention. The execution of the operating steps for filling containers in grade C and grade A/B
environments in a systematic process is described.
The section also describes the process of a nutrition agar fill, as well as the background behind the
procedures and further microbiological processing.
Special systems are required for filling containers with powder. Particular attention has to be paid to
particle load in the air resulting from the powder product.
12.F Steam sterilisation

Which parameters need to be monitored for steam sterilisation?

What is the procedure for process evaluation when applying the F0 concept?
What are the most important points of an SOP for performing steam sterilisation?
Steam sterilisation is the method of choice for manufacturing sterile products or sterilising equipment
and containers. Alternatives such as sterile filtration are only considered if steam sterilisation is not
possible, for example because the product is destroyed at high temperatures. Aseptic processing, in
which no sterilisation of the product is possible, is described in chapter 12.A.2 Aseptic processing.
Exposure to ethylene oxide gas and irradiation with gamma rays are only permitted for a limited
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application area. Since ethylene oxide is a carcinogen, this procedure is only used in certain cases
(for example, for sterilising substances in the form of powder or crystals, and for thermo-unstable
devices in the medical area). Problems with this procedure are the long desorption times and possible
residual amounts in the material. Handling the gas and destroying it after use (gas washing with
reaction agents) are time-consuming and should be avoided if possible by using other methods of
sterilisation.
Sterilisation by radiation using gamma systems (high-activity cobalt radiation sources) and electron
accelerators are used in the manufacture of dressing material and disposal medical products, such as
prefilled syringes, and therefore primarily for surface sterilisation. Since this involves working with
sources of radiation, a high level of compliance with radiation regulations is required, although this is
compensated for by high production rates and hence efficiency.
12.F.1 Sterilisers
Steam sterilisers (autoclaves) usually consist of a pressure chamber surrounded by a heating mantle,
and peripheral measuring and control devices.
The technical regulations for the sterilisation temperature, pressure, and the sterilisation time, and the
measurement of the sterilisation temperature of 121 C via chamber sensors are defined in the
qualification documentation and the technical descriptions for the steam steriliser. The temperatures,
timescales, and pressure conditions are documented on a multi-channel recorder of the steam
steriliser.
In addition to these "conventional" steam sterilisers, sterilisers with turbulent steam-air mixture and
hot water sprinkler sterilisers are also used. Depending on the task for which they are designed
(device sterilisation, laundry sterilisation, sterilisation of objects filled with solution), steam sterilisers
are equipped with programs for drying, self-cooling, or active cooling using cool water or air. A
"vacuum test" step in the program must display the seal tightness of the whole system, in order to
ensure the ventilation of the chamber and hence the sterilised devices/accessories at the end of
sterilisation via a sterile filter (0.2 m).
12.F.2 Description of the procedure

The steam sterilisation process has to fulfil three conditions: The steam must be saturated, be under
positive pressure, and be kept at a temperature of at least 121 C. It must be possible to maintain
these conditions for any chosen length of time. In order to fulfil the conditions, "chamber autoclaves"
(steam sterilisers) are used. The steam sterilisation procedure consists of the phases described in
figure 12.F-1.
Figure 12.F-1 Phases in steam sterilisation
Phases in steam sterilisation
Vacuum for deaeration

Supply of saturated steam in several cycles
Sterilisation
Cooling
Drying
Ventilation
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The sterilisation process using a steam autoclave is described using a time-temperature curve (see
figure 12.F-2).
Figure 12.F-2 Chronological procedure for autoclave sterilisation
12.F.2.1 Sterilisation
The aqueous solutions, immediate containers or accessories that are to be sterilised must come into
contact with a saturated steam phase or a self-generated steam phase (for solutions). Saturated steam
refers to steam that is in a state of equilibrium with its liquid, which means that if liquid condensate is
present, the steam is definitely saturated. Saturated steam has a particular pressure at each
temperature. This physical relationship means that in the case of defects in measuring technology, it
is possible to evaluate the sterilisation conditions on the basis of the pressure course or temperature
course.
In order to establish the steam/steam air or hot water sprinkling conditions, any air present in the
autoclave at the beginning of the process must be removed by suction. To do this, a circulation pump
is used to create a vacuum of <100 mbar (abs). The chamber is then filled with saturated steam via
the heating mantle, followed by aspiration once more (in three to ten cycles), which takes into
account the different designs and uses of the steam steriliser and the type of product undergoing
sterilisation. Subsequently, saturated steam is supplied to a positive pressure of approx. 1.1 bar. The
resulting temperature corresponds to the steam pressure-temperature curve.
120 C = 1.9854 bar

121 C = 2.0492 bar
122 C = 2.1145 bar
123 C = 2.1816 bar
124 C = 2.2504 bar
Based on requirements of the various pharmacopaeiae (not lower than 121 C) and according to USP
(1.0 C deviation in the empty chamber), this results in a pressure regulation range of 65.3 mbar
(from 121 C to 122 C).
121.1 C = 2.0556 bar

121.5 C = 2.0811 bar
122.0 C = 2.1145 bar
122.5 C = 2.1470 bar
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The different loads of an autoclave (equipment, containers of varying sizes and mass) mean that
different amounts of heat (in the form of saturated steam) are transferred to the sterilisation goods
over differing lengths of time, and the resulting condensate is continually removed by condensate
separators, the temperatures in the chamber and in the objects can change so quickly that controlling
the steam feed using heat sensors becomes too slow. It is therefore necessary to control the steam
feeding based on the pressure in the chamber. This provides immediate regulation and can be set to
an accuracy of 0.1 C = approx. 7 mbar.
It is then possible to measure the temperature in reference objects, in the chamber, and in the
condensate outlet, the start of the sterilisation time from 121 C, the end of the cooling phase etc.,
with double Pt100 heat sensors. A temperature measurement is then sent to the measuring
equipment and the recorder on the autoclave. The parallel measurement is used as a safety or
comparison measurement (or for monitoring purposes) and leads to a measured value printer with F0
calculation. A prerequisite condition for the start of the time period for the sterilisation phase is a
temperature of at least 121 C at the coldest reference point in the sterilisation goods (for example,
inside a bottle or in a filter, valve, or tube).
Figure 12.F-3 provides an overview of the specifications for sterilisation time in the various
pharmacopoeias.
Figure 12.F-3 Pharmacopoeia specifications for sterilisation time
Pharmacopoeia specifications for sterilisation time
European pharmacopoeia,
JP, BP
at a minimum of 121 C for 15 min
DAB, EP
121 C for 15 min with 2 C and 10 kPa measured in the coldest

point of the autoclave
USP
Using a temperature not less than 121 C ..... the F0 concept may be
appropriate........
12.F.2.2 Drying
After the sterilisation phase, there follows a drying phase at approx. 70 C (temperature of the
steriliser mantle) under a vacuum.
In the sterilisation of closed containers (ampoules, bottles), the high pressure built up inside the
container (approx. 4 bar due to the expansion of air in the empty space above the solution) has to be
accounted for since, as the steam pressure in the chamber is reduced in the cooling and drying phase,
a support pressure of up to 2.5 bar is built up using compressed air. This prevents bottles and
ampoules from exploding and seals from deformation due to an excessive pressure differential. In the
case of open piston stoppers in prefilled syringes, it is particularly important to control the support
pressure to minimise any movement of the piston stopper in the glass object during the sterilisation
and cooling phase.
The necessary duration of the drying phase for the relevant load and features of the sterilisation goods
(for example, membrane filter layers require very long drying times) must be established in the
performance qualification (PQ) and described in an SOP. Figure 12.F-3 shows the requirements of
the pharmacopoeias for sterilisation time.
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12.F.2.3 Sterilisation kinetics

The inactivation of microorganisms follows the kinetics of a first-order reaction, which means that
the number of microorganisms inactivated within a given time is always proportional to the number
of existing microorganisms that are still alive (see figure 12.F-4).
Figure 12.F-4 Microbial reduction and survival rate
Microbial reduction and survival rate
log Nu = - U/D + log No
Nu = Microbial count after U minutes
No = Original microbial count
D = Decimal reduction time for the relevant temperature
Definition of the symbols used to describe sterilisation kinetics:

n: Number of organisms, expressed as CFU (colony-forming units), also known as microbial count.
D value: If microorganisms are subjected to heat with a higher moisture content (at a constant
temperature), the microbial count decreases in relation to time. The D value is the decimal mortality
rate in minutes at a given temperature, or the time in minutes that is required to kill 90 % of the
spores or vegetative cells of a specified microorganism at a given temperature. The D value always
refers to one temperature and one microbial species, for example, D121 C Bacillus subt. Figure 12.F-5
shows an example of how to calculate the D value.
Figure 12.F-5 Calculating the D value
Calculating the D value
Sterilisation conditions (moist heat) Microbial reduction D value
100 min at 100 C
104 -> 103
100 min
10 min at 110 C
104 -> 103
10 min
1 min at 120 C
104 -> 103
1 min
Z value: value in degrees Celsius that causes a change in the decimal D value (or: the change in
temperature that causes the D value to change by a power of ten). The Z value is also defined as the
relative resistance of a given microorganism against different temperatures. The Z value for Bacillus
stearothermophilis is 10 C.
F0 121 C: The total amount of heat that acts on the sterilisation goods during a sterilisation process,
adjusted according to the relevant temperature (here 121 C) in min, or the temperature course of the
whole sterilisation curve including the heating and cooling phase. Since the calculation is of
American origin, the specification is 250 Fahrenheit, which corresponds to 121.1 degrees Celsius.
For example: The F0 value specifies the sterilisation time (min) at 121 C or the corresponding
sterilisation time (min) at 121 C for sterilisation temperatures above or below 121 C. The calculable
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F0 value can be used to compare sterilisation procedures performed at temperatures above or below
121 C in terms of the microbial killing effect of the standard autoclave at 121 C.
At a constant temperature, the following applies:
t = sterilisation time (min)

T = sterilisation temperature(C)
z = 10 C.
For a fluctuating temperature, the following applies:
For example:
F0 value of the procedure for 60 min at 105 C F0 value of the procedure for 20 min at 120 C
F0 = 60 x 10 ( 105 - 121)/10
F0 = 20 x 10 (120 - 121)/10
F0 = 60 x 10 -16/10
F0 = 20 x 10 -1/10
F0 = 60 x 10 -1.6
F0 = 20 x 10 -0.1
F0 = 1.507
F0 = 15.88
With reference to the calculation basis of 121.1 C, which is also the basis for all automatic programs
for F0 calculation, the following example uses realistic setting values based on 121.5 C and 20 min
sterilisation time:
F0 value of the procedure for an actual 21.5 min sterilisation time at 121.8 C.
F0 = 21.5 x 10 (121.8 - 121.1)/10
F0 = 21.5 x 10 (0.7)/10
F0 = 21.5 x 10 0.07
F0 = 25.26
12.F.3 Qualification of a steam steriliser

The operation of a steam steriliser must ensure that the objects or solutions treated in the system
achieve the required result - i.e. they are free from living microorganisms. This can be ensured by
technical design and qualification.
The requirements are described in a user requirement (see chapter 6.D Design qualification (DQ)).
The aim of qualification is to provide evidence that the steam steriliser meets the defined
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requirements in terms of design, function and operability. Inspection by the authorities concentrates
in particular on the points named in figure 12.F-6.
Figure 12.F-6 Test points for inspections
Test points for inspections
Materials
Pipe connections (welding seams), aseptic connections
Electrical components
MSR components
Function
Monitoring
Documentation
When selecting a design (steam sterilisers are not constructed according to customer requirements,
but are, at most, modified by manufacturers of these devices), it is important to be clear about the
requirements and transfer these to a technical specification. A qualification plan must be created in
which the qualification process is described (see chapter 6 Qualification).
The qualification activities must be monitored and checked by an independent department or person
who is responsible for ensuring that they are processed correctly, and attests to this with a signature.
The operator is responsible and must be sure that the qualification process has been performed
correctly. To realise this responsibility properly, the responsibilities must be defined in the
qualification plan (see figure 5.D-19).
System functions and process steps can be classified as critical or uncritical using a risk analysis. A
risk analysis can form a very useful basis for qualification and validation plans (see chapter 10.D
Methodologies to be Used to Facilitate Risk Management).
12.F.3.1 Installation qualification

The execution of the tests is described in detail (see figure 12.F-7), to enable exact replication of the
same tests in the future. Measured values must be recorded in the test protocol and documents
(printouts, diagrams, etc.) must be included as attachments.
Figure 12.F-7 Tests in installation qualification
Tests in installation qualification
Bring the steam steriliser into cleanliness grade D

Seal the steriliser for grade C
Connect the pipes for steam and compressed air
Seal the bottom drainage
Connect the electricity
Valve control functions
etc.
Calibration of measuring points

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As a part of preparation for operational qualification, an initial calibration of the measuring

equipment should be performed before start-up of the steam steriliser. The measuring points defined
in the IQ, the type of measurement, operating range, adjustment thresholds, calibration intervals and
accuracy categories must all be included in the calibration for process validation (see figure 5.D-16).
Quality-relevant measuring points in steam sterilisers are as follows:
Temperature (chamber, reference object, condensate)

Time
Pressure (positive pressure and vacuum).
Measured calibration values must be compared with reference measuring systems from a higher
class. To perform a reliable calibration, for example, a three-point calibration is performed. For a
steam steriliser, these are room temperature 20 to 25 C, a drying temperature of approx. 70 C, and
the sterilisation temperature of 121 C. These three measured values are compared to the reference
equipment (in this case, a tested Hg thermometer with 1/10 C increments, tempered in an oil
thermostat).
For values outside the acceptable measured value tolerance, the measuring chain must be adjusted
and recalibrated. The operational qualification cannot take place until a successful initial calibration
has been performed. In general, this results in 10 to 15 temperature measuring points for calibration.
Heat sensors
Pressure and vacuum resistant control heat sensors are used (type Pt 100) with feeds that are fed into
the steam sterilisation chamber, where they are positioned according to a distribution plan. The feeds
are connected to a measuring point recorder of the appropriate quality grade.
If using tracers (for example, made by Ball), the cables become unnecessary, since these devices
store temperature values and can be imported to a PC before and after they are used. The calculation
program can be used to calculate and print F0 values, curves, numerical values, etc., as required. In
both procedures, the control heat sensors must be calibrated. In general, twelve control heat sensors
are tested in accordance with an SOP, before and after the validation measurement at 121 C (not a 3point measurement) in the oil bath thermostat. The number of heat sensors depends on the trays in the
steam steriliser, but in order to provide a representative heat distribution test, must cover the whole
space divided into parts of approximately the same volume (see chapter 12.F.4.2 Loading
configurations). In productive operation of When operating a steam steriliser in production, three heat
sensors are absolutely necessary:
1. A "product sensor" at the coldest point
2. A free chamber sensor
3. A heat sensor in the draining condensate
Modern sterilisers also use as many as twelve heat sensors in production operations. The maximum
permitted deviation is 0.5 C. A correction factor is determined from the average values before and
after as follows:
Before validation, a temperature of 121.0 C is set in an oil bath (Hg thermometer stem corrections
must be taken into account). The heat sensor to be used is inserted into the oil bath and shows, for
example, a temperature of 121.2 C. The determined deviation is +0.2 C. This sensor is then used to
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measure the temperature in the steam steriliser. After this measurement, the deviation in the oil bath
is determined once again. This results in, for example, 121.4 C. The deviation is therefore +0.4 C.
The resulting correction factor is +0.6 C divided by 2 = +0.3 C.
All calculations for this sensor are performed with a correction of -0.3 C, because the measured
values are changed by +0.3 C during the sterilisation phase. The data is documented in a calibration
record.
Chamber pressure transmitters

The chamber pressure transmitters are tested against a control device at pressures of 1.1 and 2.1 bar
(abs). They must meet the requirement of 0.05 bar pressure differential. The data is documented in a
calibration record.
12.F.3.2 Operational qualification

After the DQ and IQ, the equipment and function of the steam steriliser are tested using the defined
process parameters. This test is the operational qualification (OQ) for the device. In the operational
qualification, all the operating states of the steam steriliser are simulated to their limits (worst case),
and the results documented (see figure 12.F-8).
Figure 12.F-8 Test points in operational qualification (extract)
Test points in operational qualification (extract)
Does an alarm sound if the temperature drops below the sterilisation temperature?
Is the sterilisation time interrupted?
Is there an automatic restart once the sterilisation temperature is reached?
Is the process documented on the system measuring devices?
Is a failure message/analysis documented (for example, drop in steam pressure)?
Testing heat distribution

During operational qualification, the three empty chamber sterilisation runs in a full cycle must show
an even heat distribution in the empty chamber. In addition, there must be no cold spots (average
value of the temperatures from all control sensors (see chapter 12.F.4.2 Loading configurations)
<1 C must be determined. The coldest spot is established as follows: One individual measured value
is -1.4 C below the average value of all the heat sensors used, for example, 122.4 C (measured
value = 121 C). This means that a cold spot would exist in this position, as the permitted deviation
limit is only 1 C.
This test proves that the construction of the steam steriliser, the distribution of its steam inlets and
distance of the storage surfaces from the "cold" doors (not insulated by the mantle) do not form
condensation surfaces for the steam, which may affect the temperature. Since all common steam
sterilisers are constructed in a way to specifically prevent these influences, deviations with cold spots
when examining empty chambers tend to be theoretical.
Function tests
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In addition to calibrations, the function tests listed in figure 12.F-9 are also performed. These tests
should be repeated in process validation, since some time may have passed since the OQ and changes
may have occurred.
Figure 12.F-9 Function tests
Function tests
Recorder feed of the external
device
Function check of the steam

steriliser - time measurement
Tightness test
Check paper feed with markings on the recorder paper and

time measurement
Documentation: markings on the recorder chart with time
measurement
The built-in recorder in the steam steriliser is tested using
markings on the recorder paper and time measurements.
Maximum deviation 1 min/h
(corresponds to 60 1 mm/h paper feed).
Documentation: Markings on the recorder chart and time
measurement
Start the vacuum test program
Evacuate to 65 mbar (abs), duration 10 min
Maximum pressure increase 15 mbar (to 80 mbar abs)
Documentation: Recorder printout
Inspection of media quality

During qualification, the quality of the pure steam and the air used to ventilate the autoclave is also
tested. This test is also repeated during process validation, as the steam system must have undergone
a separate qualification and process validation. The data from these studies can be used. However,
current values must be available from monitoring or must be determined during process validation of
the steriliser. The requirements for pure steam are summarised in figure 12.F-10.
Figure 12.F-10 Requirements for pure steam
Requirements for pure steam
Full analysis of the pure steam condensate Corresponds to WFI
Particle measurement
25 P/ml 10 m
3 P/ml 25 m
CFU determination
10 CFU/100 ml
Endotoxin determination
< 0.25 EU/ml
Before entering the chamber, the steam is filtered via 13 m and 0.1 m particle filters (for example
by Pall, Composit 0.1 m). Air is guided over vent filters, before it enters the chamber. A retention
rate of 0.2 m is achieved, for example with the Pall filter MCS 4440V002. Filter integrity tests are
executed and documented before and after the validation activities.
Following successful completion of qualification, the qualification report (consisting of an
introduction, results and summary/evaluation) is created and released by the person responsible for
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validation and/or head of production or quality control, and by quality management (see chapter
6.C.3 Qualification report).
12.F.4 Validation of the steam sterilisation process

The first step after qualification is to compile the validation protocol based on internal company
guidelines. The validation protocol formulates the aim to provide evidence that a reproducible
sterilisation process is achieved in the steam steriliser under overkill conditions.
Figure 12.F-11 Extract from a validation protocol
Extract from a validation protocol
Responsibilities
Person responsible for validation
Head of Production, head of quality control
Validation manager
Project manager
Validation team
Staff of the unit (users)

QA staff
Responsible for implementation
Device: steam steriliser, manufacturer XYZ no.12345 building 000

Process parameter temperature 121C (worst case), set value 122.5 C
Time 20 min (worst case) to 23 min sterilisation
Pressure = saturated steam curve approx. 2.1 bar (abs)
Validation runs: 3 runs with each of the min and max worst case loading conditions
Reproducibility: evaluation of recorder for range of process values
Overkill conditions: by using bio-indicators and adjusted, shortened sterilisation time (partial cycle),
to provide evidence that the process parameters for the worst case of 121 C and 20 min overkill
conditions (F0 12 min/D121 C = 1 min) are achieved.
A prerequisite for executing the validation is the employment of qualified personnel, and proof that
the systems and test equipment are qualified for the intended purpose, and the measuring equipment
is calibrated.
12.F.4.1 Description of equipment and process

The steam steriliser supplied by XYZ is installed in building 000 in cleanliness grade XY. In most
cases, autoclaves are used as pass-through autoclaves with two doors leading from one cleanliness
grade to another cleanliness grade. The steam steriliser is used to sterilise final containers and
accessories (for example cylindrical ampoules, rubber stoppers, and crimping caps in sterile bags or
sterilisation boxes). The sterilisation is performed in accordance with the relevant SOP (see figure
12.F-12 and figure 12.F-13).
Figure 12.F-12 SOP for sterilisation in the steam steriliser (autoclave sterilisation)
SOP for sterilisation in the steam steriliser (autoclave sterilisation)
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Author:
Checked:
QA approval:
Released by head of production:
Signatures:.......................Valid from:
Contents:
1. Aim/Purpose
2. Responsibilities
3. Scope
4. Description of procedure execution
5. Documentation
6. Other relevant SOPs
7. Attachments
1. Aim/Purpose
This SOP describes the use of the steam steriliser XYZ, inventory no. 12345 for the sterilisation of
sterile goods in accordance with the equipment manufacturer's operating instructions.
2. Responsibilities
The procedure described in the SOP is the responsibility of employees after instruction, as well as the
departmental and company management.
3. Scope
Organisational unit: XYZ and subunit: XYZ.
Location of the steam steriliser

Cleanliness grade
Media (pure steam, compressed air, drinking water, purified water/WFI)

Process
Start up/ start the steam steriliser by switching on the electricity

Open the media valves
Prepare the steriliser for standard sterilisation with a short empty chamber sterilisation
(approx. 5 min.)
Label the steriliser: sterilisation goods for preparation and batch, date, name of the operator,
start time and predicted end of sterilisation
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Load the sterilisation goods according to the loading configuration (marked with sterilisation
indicators) and position the heat sensors
Select program: sterilisation time/drying time
Start of sterilisation - control of documentation equipment (recorder functions)
Monitor of the measuring equipment vacuum-steam intervals (-0.8 bar to >0.3 bar),
temperature rise to ~122 C (temperature in the condensate). Start the sterilisation process
(time) by the coldest heat sensor >121 C.
Sterilisation time >15 min.
End of steam phase, pressure drop to <0.3 bar.
Monitor the initiation of the vacuum pump and reduction in chamber pressure to <-0.8 bar,
start the drying phase (depending on the program, >3 hours)
When drying phase is complete, ventilation to 0 bar.
End of program
Remove the sterilisation goods and check the sterile packaging is undamaged and the indicator turn
over (marks the goods as sterilised), if applicable. In all other cases, the sterilised products must be
assigned a "sterilised" label.
5. Documentation
Check and initial the time-temperature-pressure protocol of the recorder

If applicable, checking and initialling of the recorder protocol (for example, numerical
documentation) by the employee who performs the sterilisation (doer).
Evaluation of the recorder protocol of the steam steriliser according to SOP XYZ
(temperature, time course and pressure) by the tester (checker)
Enter results in the batch production instructions an record

For example: Recorder evaluation, SOP XYZ
Tightness test SOP XYZ
Use of filter SOP XYZ
Sterilisation of filters and tubes, SOP XYZ
etc.
7. Attachments
Loading configuration
Program course for device and valve function
The steam steriliser consists of the central pressure chamber with two doors surrounded by a heating
mantle, and peripheral measuring and control devices. When attaching control measuring equipment
to the steam steriliser and bioindicators in the immediate vicinity, the whole space in which the goods
to be sterilised are positioned should be taken into account. This usually means that several storage
levels are set up in the steam steriliser, so that a number from 10 to 12 positions must be measured in
addition to the actual chamber or condensate sensors. For the validation of steam sterilisers for final
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containers (for the sterilisation of filled ampoules and bottles), the procedure is the same as the
validation protocol above, but the temperature is always measured in the objects (bottle or ampoule
or reference object). This includes the bioindicators. This ensures that the different heat-up times and
cooling phases of small and large volumes are taken into account. This must include the very long
heat-up times of filter housing, steel mantle tubes and media fill solutions in open steel pressure
containers.
The sterilisation temperature of 121 C to 122.5 C is determined using the chamber sensor and the
sterilisation time is selected using a timer switch. The sterilisation is regulated based on pressure in
accordance with the saturated steam curve. The temperature, time and pressure are recorded using a
multi-channel recorder. More detailed technical information (for example, the electrical technology
description of the control loop) is available in the qualification documentation.
The three validation runs for each loading configuration follow the usual sequence of operation for a
steam sterilisation process (see figure 12.F-13).
Figure 12.F-13 Sequence of operation
Sequence of operation
Program selection
Vacuum test
Self-cooling
Drying
Pre-heating phase
Opening the steam feeding

Steam flows into the heating mantle and heats up the steriliser in advance (all
parts of the system must be at operating temperature). During operation, a
"short sterilisation" of approx. 5 min is carried out, and an equipment check is
performed at the same time.
The pre-heating phase is complete when the heating mantle has a steam
pressure of approx. 0.3 bar.
Load the steriliser

Close and seal the steriliser (manual or automatic, depending on the model)
Start
Ventilation/prevacuum
Evacuation of the pressure chamber to 150 mbar
Sterilisation
Steam flows at set intervals via the heating mantle into the pressure chamber
until the sterilisation temperature is reached. Automatic start and processing of
the preset sterilisation time.
Pressure release by After the sterilisation phase, in the program step "pressure release", the
self-cooling
autoclave naturally becomes depressurised as the steam feeding is stopped and
due to natural cooling.
Drying
The steam is released from the chamber and the chamber is evacuated ( 150
mbar). The drying phase runs for the set time at a chamber temperature of
approx. 70 C (indirect heating via the heating mantle).
Final ventilation
The final ventilation is performed via two consecutive sterilised vent filters with
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a pore size of 0.2 m.

In the program for performing the vacuum test (leak test), the chamber is evacuated to a pressure of
65 mbar (abs). The pressure subsequently must not rise to more than 80 mbar (abs) within 10
minutes.
12.F.4.2 Loading configurations

During validation, all loading configurations that are possible during routine operation are tested, as
the success of sterilisation greatly depends on loading. The different loading configurations and the
relevant positions of the heat sensors and bioindicators is shown below.
X = external heat sensor (Pt100), tracer
0 = Bioindicators
K = Chamber sensor
I = Object to be sterilised (filters, tubes, accessories)
Empty steam steriliser

View from front
View from above 4th layer (top) D
3rd layer C
2nd layer B
1st layer (bottom) A
Steam steriliser with minimum loading

View from front
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3rd layer C
2nd layer B
Steam steriliser with maximum loading

View from front
3rd layer C
2nd layer B
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12.F.4.3 Bioindicators
Bioindicators provide an additional check of the success of sterilisation. These are marketed
ampoules or spore strips with defined germ solutions (microbial count and type) (see figure 12.F-14).
These are positioned as described in chapter 12.F.4.2 Loading configurations. After use, the
bioindicators must be incubated for 3 to 5 days at 56 2 C (ampoules are directly incubated, spore
strips are placed in a culture broth). No microbial growth must be visible on any ampoules or spore
strips (see chapter 12.H.6 Reading and evaluating).
Figure 12.F-14 Bioindicators
Bioindicators
For example, Attest ampoules supplied by For example, spore strips supplied by
BAG
3M
Test organism: Bacillus stearothermophilus
Bacillus stearothermophilus
Batch:
see certificate
see certificate
D121 value
see certificate
see certificate
Insemination:
see certificate
see certificate
Expiration
date:
see certificate
see certificate
12.F.4.4 Determining the sterilisation time

To check the microbiological effectiveness, only bioindicators with an F0 value of <10 min are
available. These indicators cannot be used for evidence of an overkill procedure (12 min). The
routine sterilisation time is therefore reduced to a partial cycle.
Using bioindicators with a known insemination of Bacillus stearothermophilus, together with the
choice of a suitable reduced sterilisation time (partial cycle), three validation runs are performed for
each loading configuration. For revalidation the recognised procedure is to use one run with
minimum load plus three runs with maximum load. Spore strips with a sufficient insemination and D
value can be used in a full cycle (for example, insemination 2.58 x 106 CFU, D value 2.1 = F0 of
13.56 under conventional conditions T = 121.1 C, D121 C = 1 min). The principles listed in figure
12.F-15 to figure 12.F-17 are used to calculate the sterilisation times.
Figure 12.F-15 Determining the minimum sterilisation time
Determining the minimum sterilisation time
F = n x D121
D121 = Decimal reduction time (TReference = 121.1 C)
F = Effectiveness of the sterilisation process
n = Original microbial count (log 10)
Figure 12.F-16 Determining the maximum sterilisation time
Determining the maximum sterilisation time
F0 Overkill /F0 Part = tTot /t Part
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F0 Part = Effectiveness of the partial cycle (see bioindicator)

tTot = Total sterilisation time
tPart = Maximum partial cycle time
F0 Overkill = 12 min
Figure 12.F-17 Sterilisation times for equivalent temperatures
Sterilisation times for equivalent temperatures
tE = Sterilisation time for equivalent temperature

tR = Sterilisation time at reference temperature (for example, F0)
TE = Equivalent temperature
TR = Reference temperature (121.1 C)
12.F.4.5 Executing the validation

The particular configuration (size, internal construction of the chamber, trays, inlets and outlets) is
used to determine the minimum and maximum (worst case) loads based on the routine loading
configuration with sufficient steam penetration. Whether sufficient steam has reached the sterilisation
goods can be judged by the killing of the bioindicators: If no moisture is present, the required killing
rates are not reached.
The steriliser temperature is between the permitted limits of 121 C to 122.5 C. The D value and the
insemination of the bioindicator used (for example, Attest ampoules) together with the sterilisation
temperature, are taken into account to determine the required sterilisation times (see figure 12.F-17).
Some companies stop the sterilisation process after these calculations and once company-defined F0
values have been reached (to protect the active pharmaceutical ingredient being sterilised). The great
advantage of calculating and determining F0 values is the provision of definitive data regarding the
sterilisation robustness if the sterilisation time is slightly longer or shorter than the prescribed time for
technical reasons. Since the realistic sterilisation temperatures only become evident during the actual
sterilisation process, times must be prescribed for the temperatures 121 C/122 C and 123 C. Once
these temperatures and times are achieved, the operating personnel must terminate the sterilisation
phase. The time of the partial cycle is measured when the coldest measuring point of the loading
configuration (see chapter 12.F.3.2 Operational qualification) has reached 121 C. Testing the heat
distribution in the empty chamber during operational qualification does not usually reveal the coldest
spot, due to the appropriate design of the steam steriliser. Deviations from temperature courses
during the sterilisation process are only caused by the sterilisation goods, and evaluation and control
of the sterilisation time for the sterilisation goods are therefore required, which should be established
in process validation (operational qualification).
The documentation includes the following documents:
Time-temperature-pressure record of the sensors installed in the steam steriliser
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Time-temperature record of the control heat sensor

Results of the evaluation of bioindicators following incubation
Certificates of the bioindicators
Once the sterilisation process is complete, the data is evaluated. In terms of temperature, the
evaluation is based on measured data once the stable plateau phase is reached. The temperature-time
curves (see figure 12.F-2) should be evaluated in the final third of the partial cycle time.
Assessment of saturated steam conditions (temperature, pressure)

Requirement: pressure-temperature combination in accordance with the saturated steam curve
Assessment of the required heat penetration (temperature, time in the objects)
Required temperature 121 C, sterilisation time within the corresponding min, max partial
cycle time
Assessment of the measurement results from the device measuring equipment compared to
the control measuring equipment (evaluation of heat-up time)
Assessment of effectiveness using physical data (F0)
Assessment of bioindicators for sterility (in parallel with positive controls)
Assessment of the sterilisation process (according to PDA monograph no.1, relating to
calculation principles for microbial reduction)
The microbiological effectiveness is calculated according to the formula in figure 12.F-18.

Figure 12.F-18 Calculation of the microbiological effectiveness of the partial cycle (F)
Calculation of the microbiological effectiveness of the partial cycle (F)
F = D x (log10 A - log10 (2.303 x log10 n/q))
A = Insemination of bioindicator
n = Total no. of bioindicators used
q = Bioindicators with no growth (negative result)
Testing of ventilation piping

Replacement of the vent filters (after 6 or 12 months) means that the ventilation valve and the pipes
need to be sterilised. Attaching a spore strip in the ventilation piping provides evidence of a 12-log
microbial reduction in full-cycle sterilisation. After incubation of the bioindicator, no growth must
appear.
Reproducibility
The reproducibility is checked, for example, under the following conditions:
Temperature: 121 C (lower permitted limit)

Pressure: 2.1 bar (abs)
Time: 21.5 min
The following requirements must be fulfilled:
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The assessment should be carried out using measuring data determined after 121 C has been
reached.
Time, t: 20 min and 23 min
Temperature, T: 121 C to 122.5 C
Pressure, p: Saturated steam conditions (T, p)
Assessment of the evaluation and control of the recorder chart (in accordance with company
SOP).
Documentation: Time-temperature-pressure records from routine sterilisations.
Completion of validation
The results of the tests must be recorded in a validation report. When the validation process is
complete and all results are positive, the steam steriliser can be released in the described scope for
one year. After this time, revalidation is required. Requalifications must be performed if technical
changes are made to the steam steriliser (Change Control).
Despite successful validation of a sterilisation process and a the understanding and proof of microbial
reduction theories parametric release is not common practice (see chapter 12.H.1 Parametric
release). The deciding factor is that in the case of technical deviations, no biological verification
(sterile test) is possible and the batch would be lost.
Summary
Due to the importance of steam sterilisation in the production of sterile products, the qualification and
process validation should be planned and executed based on a background knowledge of sterilisation
kinetics. It is important to pay particular attention to the accuracy and reliability of the temperature
measuring equipment and pressure display and regulation. Monitoring and documentation of the
sterilisation conditions by personnel and appropriate measuring point recorders help to ensure reliable
processing.
The loading configurations selected in process validation must always be adhered to in operational
processing. If the loading configuration of an autoclave is changed, a change control procedure and
repeated process validation are required.
The bioburden before sterilisation should be established. Limits on the sterilisation time beyond the
relevant legal regulations are defined by the manufacturer during the development and introduction of
a product.
Monitoring with bioindicators should be carried out based on process validation.
12.G Microbiological monitoring

Which microbial contamination risks must be anticipated during the manufacture of sterile
preparations?
What level should be set for individual clean areas?
Which methods should be used for the environmental monitoring?
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At what frequencies should the investigations be carried out?

Which points should be investigated?
How should one proceed if the level is exceeded?
How can one identify isolates?
It has become generally accepted that the manufacture of pharmaceutical preparations must take
place under controlled microbiological conditions. Microbiological monitoring is therefore
indispensable. The aim of the monitoring is to detect deviations from the validated state. Monitoring
is designed to demonstrate that the process is under control.
12.G.1 Sources of contamination

Because the medicinal products come into contact either with air in the room or, under laminar flow
units, with suitably purified air, air is often viewed as the main source of contamination. This is also
reflected in the GMP guidelines which in places include extremely detailed requirements for the air
system. For the manufacture of sterile products, the EU-GMP-Guideline (Annex 1: Manufacture of
Sterile Medicinal Products) states that the air microbial count should be determined during
operation in clean areas. The results should be taken into account when releasing a batch.
Surfaces are also relevant as possible sources of contamination. In the first instance, one must
naturally view parts that come into contact with the product as particularly critical for product
contamination, as organisms can be directly transferred here. However, there is no such thing as a
non-critical point, as even here we must anticipate contamination via the air. For these reasons, all
areas should be examined, not only those parts of the machine that come into contact with the
product.
If the room fittings are defective (unsealed ceilings and windows, poorly equipped air lock and
overpressure systems, etc.), massive invasions of organisms must be anticipated. Even the smallest
scratches and cracks in the wall or floor covering give organisms a good chance of surviving
disinfection measures and potentially developing into colonies. Particular attention must therefore be
paid to the coating of floors and walls, as the paint and wall or floor covering will determine the
success or otherwise of disinfection measures (see chapter 3.E Construction elements). The EUGMP-Guideline gives very detailed specifications for structural fittings.
Because people are involved in nearly all production steps, particular attention must be paid to
personnel hygiene. We must assume that personnel represent a main source of contamination. This
is understandable when one considers the results of relevant investigations. A human being carries
numerous organisms; not only aerobes, but also nearly as many anaerobes. Organisms are transferred
to the product directly if, for example, an employee comes into contact with the medicinal product or
indirectly, if the organism is first released into the air and then lands on the medicinal product.
Due to their nature, the usual measures for microbial prevention (sterilisation, disinfection) are not
suitable for people, or only to a limited degree. One is generally limited to a hand disinfection here.
Alongside this, suitable protective clothing in particular has an extremely important effect on
organism release from people. Annex 1 of the EU-GMP-Guideline therefore contains very detailed
specifications for protective clothing for cleanliness grades A to D. As well as wearing the clothes
correctly, it is important to change them regularly as organisms penetrate through the material after a
certain gestation period. It is particularly important to change gloves regularly to catch even the
smallest amount of unnoticed damage (see chapter 11.B Personnel hygiene).
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Due to the risk of the medicinal product being contaminated by the environment, monitoring must
include the following areas:
Air
Surfaces
Personnel
12.G.2 Room classification

If you wish to monitor the quality of a room, you need standards. Annex 1 of the EU-GMPGuideline and the Microbiological Evaluation of Clean Rooms and Other Controlled Environment
chapter of the USP recommend limits against which to evaluate the quality of the room. However,
these are only guidelines. Alert and action levels (see chapter 12.G.3 Monitoring program) must be
determined for the specific unit (figure 12.G-1).
Figure 12.G-1 Room grades in accordance with the EU-GMP-Guideline (Annex 1)
Area
Required for
Critical
area A
Sterile preparations - sterilised in sealed final containers
Filling of products if the operation presents an unusual risk
Sterile preparations - aseptic preparations
Critical
area B
Aseptic preparation
Aseptic filling
Transfer of partially sealed containers that are used for freeze drying
Preparation of ointments, creams, suspensions and emulsions
Filling of ointments, creams, suspensions and emulsions
Operations with a high level of risk: filling area, stopper bowl, open ampoules
and vials
Container sealing systems for aseptically filled vials that are not fully sealed
until the aluminium cap is crimped on.
Background environment for an area with cleanliness grade A

Transfer of partially sealed containers that are used for freeze drying, only in
sealed transfer baskets
Controlled Sterile preparations - sterilised in sealed final containers

area C
Preparation of solutions if the operation presents an unusual risk.
Filling of products
Background environment for a zone with cleanliness grade A for filling products
if the operation presents an unusual risk
Preparation of ointments, creams, suspensions and emulsions
Filling of ointments, creams, suspensions and emulsions
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Preparation of solutions to be filtered

Background environment for blow/fill/seal machine (protective clothing A, A/B)
Carrying out less critical stages in the manufacture of sterile products
Area with Sterile preparations - sterilised in sealed final containers

requirement
D
Preparation of solutions and components for subsequent filling
Background environment for blow/fill/seal machine
Handling of components after washing

Carrying out less critical stages in the manufacture of sterile products
Background environment for an isolator
In order to determine useful levels, it is first necessary to divide the manufacturing areas into
different room grades according to the type of product and production stage. Stricter room
requirements are selected for preparations with high cleanliness requirements. We base our division
of manufacturing areas on the room grades in Annex 1 of the EU-GMP-Guidelines (see chapter 3.D
Room classes).
It is possible to compare the EU Guidelines (Annex 1: Manufacture of Sterile Medicinal Products),
the FDA Aseptic Guidance and US Guidelines (USP) in terms of the particle limitation in the bodies
of rules (see figure 12.G-2).
12.G.3 Monitoring program

Implementation of monitoring must be set down in writing. It is advisable to draw up the monitoring
program in the form of a standard operating procedure. This standard operating procedure should
include:
Level
Methods/equipment
Frequencies
Measures in the event of deviations
Sampling (responsibilities)
Processing (responsibilities)
Sampling points
Documentation
The monitoring SOP should also define where the documents (raw data, reports, deviation
documentation, etc.) are stored. This is particularly important for any audits, as it is necessary to
make documents available within a short space of time. Other details (e.g. incubation, culture media,
etc.) should be described in the relevant laboratory SOPs.
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12.G.3.1 Limits (level)

As mentioned above, alert and action levels should be determined for the specific unit. The EUGMP-Guideline also requires that procedures for countermeasures are specified for the event that
these limits are exceeded.
By alert level, we mean the limit above which there may be possible problems. Corrective measures
are not necessarily required. If action levels are exceeded, one must assume that the process may be
out of control. Investigations must be carried out to check this. Corrective measures may be required
and their success should be checked (see chapter 12.G.6 Measure if levels are exceeded).
Inspectors expect levels to be determined in consideration of previous monitoring results and the
room qualification. If you determine the level "by feeling", you lay yourself open to the accusation of
making it too easy by setting the level too high. This is particularly the case for alert levels.
Figure 12.G-2 Room classification according to the FDA Aseptic Guidance, USP and EU-GMPGuideline
FDA Aseptic Guidance
USP
EU-GMP-Guideline
Operation room
Operation room
Clean
ISO
0.5 m
Area
Designati particles/
Classificati on
m3
on
Clas Particles equal to and

s
larger
nam than 0.5 m
e
U.S.
m3
Customa
ry
100
3,520
M 100
3.5
1,000
35,200
No equivalent
10,000
352,000
M 10,000
5.5
3,530
Clas Quiet room

s
Max. permissible number of

particles/m
(equal to or greater than)
0.5 m 5 m 0.5 m 5 m
ft3
100
3,500
1(e)
3,500
1(e)
353,000 10,00 B
0
C
100,000
Operation
room
3,520,00 M 100,000 3,530,0 100,0

0
6.5
00
00
3,500
1(e)
350,000 2,000
350,000 2,000 -
3,500,0 20,00
00
0
D
3,500,0 20,00 Not

00
0
determined
It is relatively common to use the average of the results from a relatively long period of time +2 s
(alert level) and +3 s (action level) as the basis for calculating the level (s = standard deviation). This
procedure is even suggested by the German supervisory authorities. However, with this type of
calculation, values are based on a normal distribution and outliers and level exceedances are
overevaluated. Results from environmental monitoring usually follow a Poisson or negative
exponential distribution. This is allowed for in the following formulae:
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Figure 12.G-3 Alert level (AL)
C = average value of all measurements

Figure 12.G-4 Action level (L)
AL = alert level, C = average value of all measurements

It is advisable to use the following procedure to implement these requirements for determining alert
and action levels for daily monitoring:
Action level If applicable, the level reported to the FDA must be taken into consideration
(otherwise changes must be notified) and a regulation must also be established for handling
the average values specified in Annex 1: Manufacture of Sterile Medicinal Products. To
interpret the term "average value", we can use the recommendation from the FIP for air
investigations (the average value should be smaller than 1 and be calculated from at least ten
measurements) made in 1990.
Alert level: It is advisable to calculate the level for the individual unit using the formulae
stated above. However, infinitely high values are not acceptable.
Below are suggestions for various objects of investigation (air, surfaces, personnel) in the different
cleanliness grades. The alert levels represent an upper limit.
Air
Annex 1 of the EU-GMP-Guideline recommends limits for microbiological contamination in order to
assess the quality of the room for the manufacture of sterile products. Chapter <1116> and the FDA
Aseptic Guidance also contain appropriate suggestions. In figure 12.G-5, there is an overview of the
requirements and a suggestion for implementation. The action levels were based on the values from
the bodies of rules.
Figure 12.G-5 Suggestions for requirements for microbiological quality of air
Grade
FDA Aseptic
EU-GMPProposal for
(operation
Guideline
Guideline
implementation
room)
USP
2004
Recommended
Action
Determination
1987
Alert level
Action
limits**
level
level
100 (ISO 5)
Quantitative
1* CFU/m3 <3 CFU/

m3
<3 CFU/ m3 <1 CFU/ m3
0 (1) CFU/
m3
Settle plates
1* CFU/4 h -
<1 CFU/4h
Quantitative
7 CFU/m3
Settle plates
3 CFU/4 h -
1,000 (ISO 6)
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10,000 (ISO 7)
Quantitative
10 CFU/m3 -
<20 CFU/
m3
10 CFU/ m3
3 CFU/m3
7 CFU/m3
Settle plates
5 CFU/4 h -
5 CFU/4 h
Quantitative
100
CFU/m3
<100
CFU/m3
<100 CFU/m3
50 CFU/m3
100 CFU/m3
Settle plates
50 CFU/4 h -
50 CFU/4 h
100,000 (ISO 8)
<80
CFU/m3
D (particle count not determined)

Quantitative
<200 CFU/m3
200 CFU/m3 400 CFU/m3
Settle plates
100 CFU/4 h
* Air monitoring samples of critical areas should normally yield no microbiological contaminants, **
Averages
Surfaces (rooms/technical equipment)

In Annex 1 of the EU-GMP-Guideline, there are general limits for each cleanliness grade A to D, but
no information about whether these are for personnel or surfaces and no differentiation according to
location (table, wall, floor, etc.). As there is additional data available, these are certainly meant to be
requirements for surfaces. We can assume that these are essentially surfaces close to the product and
not floors (microbiological checks are made here to verify the disinfection procedures and not as part
of the production monitoring). The USP and FDA Aseptic Guidance (for product contact only) also
contain relevant suggestions. In figure 12.G-6, there is an overview of the requirements and a
suggestion for implementation.
Figure 12.G-6 Suggestions for requirements for microbiological quality of surfaces
Grade
FDA Aseptic
EU-GMPProposal for
(operation room)
Guideline
Guideline
implementation
USP
2004
Recommended
Action
Determination
1987
Alert level
Action level
limits**
level
100 (ISO 5)
Product contact
0*CFU/
25 cm2
< 1 CFU/
25 cm2
0 (1) CFU/
25 cm2
Surfaces
3 CFU/25
cm2
2 CFU/
25 cm2
3 CFU/
25 cm2
Floor
3 CFU/25
cm2
3 CFU/25
cm2
5 CFU/
25 cm2
Surfaces
5 CFU/
25 cm2
5 CFU/
25 cm2
2 CFU/
25 cm2
5 CFU/
25 cm2
Floor
10 CFU/
25 cm2
5 CFU/
25 cm2
10 CFU/
25 cm2
25 CFU/
25 CFU/
50 CFU/
10,000 (ISO 7)
100,000 (ISO 8)
Surfaces
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25 cm2
Floor
25 cm
25 cm
100 CFU/
25 cm2
200 CFU/
25 cm2
D (particle count not determined)

Surfaces
50 CFU/
25 cm2
Floor
* Critical surfaces that come into contact with the sterile product should remain sterile throughout an
operation, ** Averages
Personnel
As already mentioned, Annex 1 to the EU-GMP-Guideline contains recommended limits for
monitoring personnel in cleanliness grades A and B. Chapter <1116> also contains relevant
suggestions. The FDA Aseptic Guidance points out that production personnel should wear noncontaminated gloves and clothing in the sterile room during intervention. In figure 12.G-7, there is an
overview of the requirements and a suggestion for implementation.
Figure 12.G-7 Suggestions for microbiological requirements for examination of personnel
Grade
FDA Aseptic
EU-GMPProposal for
(operation room)
Guideline
Guideline
implementation
USP
2004
Recommended
Determination
1987
Alert level Action level
Action
limits**
level
100 (ISO 5)
Gloves
0*CFU/25 cm2 -
3 CFU/25
cm2
< 1 CFU/
5 fingers
Clothing
0*CFU/25 cm2 -
5 CFU/25
cm2
Hood
0*CFU/25 cm2 -
5 CFU/25
cm2
Gloves
Clothing
Hood
0 (1) CFU/
25 cm2
10 CFU/25 5 CFU/
cm2
5 fingers
2 CFU/
25 cm2
3 CFU/
25 cm2
10 CFU/25 cm2
5 CFU/
25 cm2
10 CFU/
25 cm2
20 CFU/25 cm2
5 CFU/
25 cm2
10 CFU/
25 cm2
10,000 (ISO 7)
* An ongoing goal for manufacturing personnel in the aseptic processing room is to maintain
contamination-free gloves and gowns throughout operation, ** Averages
12.G.3.2 Methods and equipment

This item in the monitoring program must clearly determine the method with which the investigation
is to be carried out. Details for sampling should be regulated in laboratory SOPs.
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Microbiological testing of air

Airborne organisms do not generally levitate but are bound to particles. However, all attempts to
establish a reproducible relation between organisms and particles have failed to date. This means one
must carry out microbiological investigations in order to make any conclusions about the air
microbial count. There are both qualitative and quantitative methods available for checking the air
microbial count (see figure 12.G-8).
Qualitative procedure
Settle plates
Quantitative procedures
Filtration
Impaction
Impingement
For the settle plate procedure, petri dishes containing a suitably solid medium are left open in a
room for a certain time (30 minutes to 2.5 hours; EU-GMP-Guideline Annex 1: 4 hours). The
organisms that settle on the agar surface in this time can be counted following incubation. Even
though the EU-GMP-Guideline contains levels for this method, quantitative conclusions cannot be
drawn, as they depend on numerous coincidences. This method should therefore only be used in
conjunction with quantitative methods. This is also referred to in the FDA Aseptic Guidance.
Figure 12.G-8 Methods for air testing
The FDA Aseptic Guidance also states that, as part of the method validation with settle plates, no
drying out must be allowed to occur, as this can influence the recovery of organisms. For these types
of conformance tests, it is certainly not sufficient to use the USP reference strains, otherwise isolates
from the unit being checked would also be required. This requirement to prevent settle plates drying
out should be checked for the selected settle plates. There are obviously differences between the
cultural medium plates of different manufacturers. A fill quantity of 30 ml/plate is generally more
favourable than 20 ml. Such tests can be carried out by weighing. This indicates that, to all intents
and purposes, plates that are available on the market show an extraordinarily low degree of drying
(<2 %), even with the exposure time of four hours suggested by the FDA when stating the levels.
Whether this would be accepted by the FDA as a prevention of drying out is questionable.
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With the filtration method, the air is sucked through membrane filters (generally gelatine filters).
Microorganisms are caught by these and can then be cultured. A main advantage of the filtration
method is the possibility of taking isokinetic measurements (suction rate > flow rate) that are needed
to be able to measure under LF.
The impaction procedure (air striking solid culture media) includes the Anderson sampler, popular
in the USA, the Reuter centrifugal sampler (RCS) and the slit sampler (STA method). With these
methods, the air is sucked in, accelerated and blown onto solid culture media. Due to their mass, the
particles and any organisms bound to them remain on the culture medium. This technique is only able
to determine a certain range of particle sizes. Whereas the Anderson sampler covers a very broad
range of particle sizes with its cascade arrangement of six sieve plates, the RCS unit and the slit
sampler aim to cover the range that is typical for the air (this varies, however, depending on the
cleanliness grade of the room).
In the USA, the STA method is favoured by the FDA. In the Microbiological Evaluation of Clean
Rooms and other Controlled Environment chapter of the USP, air levels are therefore also set with
reference to the STA (Using a Slit-to-Agar Sampler or Equivalent). The use of equivalent samplers is
expressly permitted.
With the slit sampler, the air to be tested is blown onto a rotating agar plate. This makes it possible
to detect contamination in relation to time. According to the manufacturer, all particles greater than
0.5 m are detected. Bacteria are sometimes smaller than this, but because they cling to particles they
are reliably detected with this particle separation. The manufacturer states an exposure time of up to
90 minutes. This is in line with the requirements of FDA investigators to cover as long a period of
time as possible. A test volume of 55 l/min is possible, which corresponds to 3.3 m3/h (maximum
total testable volume: approx. 5 m3). However, special plates with a diameter of 15 cm are required.
These are then analysed with a graded template for recognising temporal context. In order to save
material, it has been suggested on various occasions that a plate can be used several times. This is of
course possible, but the possibility of detecting temporal context is then lost.
The RCS method is very practical. The battery-operated unit works according to the impaction
principle and allows a qualitative separation of airborne microorganisms from a sample volume of
between 10 and 1000 litres. The flow of air enters the rotor from the front, is rotated by the fan blade
and organisms in the air are separated onto the airborne organism indicator by centrifugal force. The
air outlet is directed towards the rear, parallel to the unit, to prevent turbulence in the intake area. The
sample volume at a speed of 6,100 rpm is approximately 50 l/min, whereby lighter or smaller
particles and those that are somewhat further from the agar surface have enough time to settle in the
gap between the inner part of the rotor and the agar surface. Because a sufficiently high suction rate is
achieved and a measurement of 1 m3 is possible, the unit can be used under LF.
Finally, we should look at the impingement procedure (the collection of organisms in a liquid).
With this method, a defined volume of air is sucked through a liquid or directly through a culture
broth. Following sufficient mixing, the microorganisms present in the air are retained in the liquid,
which can then be microbiologically tested for microbial content. Although the impingement
procedure is often viewed as the "standard procedure" in scientific investigations, it is seldom used
for routine testing as it is extremely expensive.
The question is, which method should one routinely use? Numerous comparative studies have shown
that the results are extraordinarily dependent on the method. For this reason, the method used should
always be stated for results and requirements. It is important that routine air testing is always carried
out with the same method. This is the only way to detect changes in the air quality.
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12.G.3.3 Microbiological testing of surfaces and personnel

If possible, a sample should only be taken from surfaces that come into contact with the product after
production has finished, so as not to endanger the product. This is also the case for personnel, so that
gloves are not contaminated.
The following methods are usually used today to test the microbial count on surfaces and personnel
(see figure 12.G-9):
Contact plate method with solid culture media

Swabs
The following method is also used for personnel monitoring:
Hand washing method (rinsing procedure)
Figure 12.G-9 Methods for surface/personnel testing
Contact plate method with solid culture media

The contact plates have a convex agar surface (25 cm2 total area) that allows them to be pressed
directly onto the surface to be tested. Agar films make it possible to test even very curved surfaces
(e.g. in vessels). Agar films also have a convex agar surface (approx. 25 cm2 total area), so that they
can also be pressed directly onto the surface to be tested.
When using contact plates of agar films, you must make sure that there is no condensate (the
colonies could run together). They must also be checked macroscopically for any contamination.
Only plates with an expiry date that is after the anticipated incubation period may be used. Once the
sample has been taken, it is extremely important to clean the agar residue from the contact surface
with sterile-filtered 70 % alcohol. This is the only way to ensure that agar residue does not improve
microbial growth of possible contaminants on the tested surface.
When personnel are tested, gloves must not be disinfected immediately before samples are taken. The
result would be massively distorted and is not therefore usable. This is also referred to in the FDA
Aseptic Guidance.
Because the contact pressure and the contact duration should have a significant influence on the
microbial yield, bioMrieux (bioMrieux Deutschland GmbH, Postfach 1204, D-72602 Nrtingen,
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Germany) have brought an aid for contact plates onto the market which standardises these two
parameters (uniform pressure of 500 50 g over a period of 10 1 seconds).
Swabs
A swab consists of a rod to which a cotton wool pad or alginate pad is affixed. The surface can be
wiped with this pad.
A damp swab (swab dampened with sterile physiological NaCl solution) is used to test dry surfaces.
The sample material collected is then wiped onto casein soya bean digest agar. The top of the pad can
then be cast into casein soya bean digest agar and both plates incubated.
A dry swab is used to test damp places (e.g. remains of cleaning water on the floor). The sample
material collect is then wiped onto casein soya bean digest agar. The top of the pad is then put into
casein soya bean digest broth for concentration for evidence of pseudomonas aeruginosa. The plate
and test tube are then incubated.
The alginate swab gives the best microbial yield. It should be used especially when only small
microbial counts are expected. The pad on the alginate swab is first soaked in a sodium-citrate buffer.
The solution or suspension is used to carry out a blend test in casein soya bean digest agar. The
remaining pad is cast into casein soya bean digest agar and both plates are incubated.
Hand washing method (rinsing procedure)

The FIP (Fdration Internationale Pharmaceutique) describes this interesting method. Both hands
are washed in 1 litre of sterile saline solution, with the addition of a disinfectant neutralising
substance if necessary. The microbial count is measured by filtering portions of the wash fluid.
It is certainly expedient to add 0.1 % of Tween 80 to the wash fluid to ensure better cleaning. The
microbial count on the hands can be determined by filtering the wash fluid and incubating the filter.
The FIP suggests 30 CFUs as the level for both hands. However, this is high for a gloved hand and
not acceptable to an inspector. If you wish to use this method, you should carry out a comparative
study of hand contact with an acceptable result and the hand washing method level.
In literature, this hand washing method appears to be similar to the rinsing procedure of horizontal
surfaces with subsequent filtration. In my opinion, this procedure is not particularly suitable, as it is
virtually impossible not to create puddles, which entail their own problems with water organisms (in
particular pseudomona aeruginosa).
Culture media
According to suggestions in the USP, it is advisable to use casein soya bean digest agar. As this
medium is prescribed for the total microbial count determination of final products, it is assuredly
well suited for this purpose. Inactivating agents should be added to the medium so that any growthneutralising substances picked up are inactivated. This is naturally always the case for surface testing,
as disinfectant residues must be expected on the tested surfaces, as well as for contact by a gloved
hand, for example. An inactivating additive can also be useful for air testing, for example, if
antibiotic dust is to be expected.
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If you purchase ready-to-use contact plates, you will get at least polysorbate and lecithin as
neutralisers (e.g. contact plates from BBL), plus perhaps L-Histidine (e.g. Count-Tact plates from
bioMerieux, GK-HYCON organism indicator from Biotest and contact plates from Heipha). If you
are working with swabs, you should be aware that disinfectant residue can be picked up during the
swabbing procedure, which can make it necessary to add suitable substances to the medium. In figure
12.G-10, there is a selection of neutraliser combinations recommended by the German Society for
Hygiene and Microbiology (Deutschen Gesellschaft fr Hygiene und Mikrobiologie - DGHM).
Figure 12.G-10 Neutraliser combinations
Recommended inactivation mixtures
Polysorbate 80 30 g/l, lecithin 3 g/l, L-cysteine 1 g/l, a. bidest. 1000 ml

Polysorbate 80 30 g/l, saponine 30 g/l, L-histidine 1 g/l, L-cysteine 1 g/l,
a. bidest. 1000 ml
Polysorbate 80 30 g/l, lecithin 3 g/l, L-histidine 1 g/l, sodium thiosulphate 5 g/l,
a. bidest. 1000 ml
Polysorbate 80 30 g/l, lecithin 0,3 g/l, L-histidine 0.1 g/l, a. bidest. 1000 ml
B-cyclodextrin 10 mM, a. bidest. 1000 ml
Polysorbate 80 30 g/l, lecithin 3 g/l, sodium thiosulphate 5 g/l, CSL 30 g/l,
a. bidest. 1000 ml
Particular attention must be paid to avoiding bringing organisms into the sterile room on the exterior
of the test material. It is often attempted to disinfect the exterior by spraying with a disinfectant. This
should be done with care so as not to influence the growth promotion of the medium with disinfectant
residue. This procedure must be validated. It is preferable to use double-wrapped sterilised plates or
strips.
As with all microbiological testing, the culture medium should be checked for growth promotion. For
this, the agar strips or plates must be inoculated with up to 100 CFU of the usual test strain of USP
or Ph. Eur. 5 (see figure 12.G-11 and chapter 12.H Test for sterility).
Figure 12.G-11 Reference organisms and strains for checking growth promotion
Organism group
Organism
Strain, e.g.
Aerobes
B. subtilis
Staphylococcus aureus
Pseudomonas aeruginosa
Isolate from environmental monitoring
ATCC 6633
ATCC 6538
ATCC 9027
Anaerobes
Clostridium sporogenes
ATCC 11437
ATCC 19404
Fungi
Candida albicans
Aspergillus niger
ATCC 10231
ATCC 16404
Incubation
The FDA Aseptic Guidance states the following as incubation conditions:
Aerobes: 48-72 hours at 30-35 C

Yeast and mildew: 5-7 days at 20-25 C.
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If the FDA suggestion is followed, it means that two samples must be taken (one sample for 4872 hours at 30-35 C and one for 5-7 days at 20-25 C). By way of compromise, a single sample can
be taken and first incubated for 5(-7) days at 20-25 C and then (after an interim reading) 48(-72)
hours at 30-35 C. Both suggestions entail a relatively high amount of effort for carrying out the test
and even more documentation (two readings). If previous incubation has only ever been at one
temperature, incubation at two temperatures means an interruption in the trend recording.
If you decide to incubate at only one temperature (in accordance with the FIP suggestion, for
example: 5 days at 30 2 C), the equivalence of the procedure must be proved in extensive tests.
This excludes all the problems mentioned above, but appropriate validation studies must be carried
out to also include isolates. This type of procedure is acceptable, but the FDA expressly states that,
for environmental monitoring, microbiological test methods other than the traditional ones (e.g. USP)
may be used if it has been proven that these methods provide equal or better results. If only one
incubation temperature has been used previously, this procedure is to be recommended, in spite of the
validation requirements, as the trend recording can be continued.
The question of the need for anaerobic incubation of the monitoring samples comes up again and
again. If we consider that anaerobic organisms occur in humans in the same quantities as aerobic
organisms, this need seems to be understandable In the context of media fills (see chapter 12.E.5
Culture medium filling (Media Fill) and chapter 12.H.9 Culture media controls), FDA investigators
ask for information about anaerobes. If this is not available, they request media fills with
thioglycolate medium. By comparing the level exceedances in the tests of samples with aerobic and
anaerobic incubation, we were able to determine that anaerobic incubation only rarely provided
additional information.
During media fills, we therefore carry out anaerobic incubation on a second sample in some places in
order to show that we do not have any problems with anaerobes and that culture medium filling using
thioglycolate medium is not necessary.
If solid culture media (agar plates, contact plates, agar strips, etc.) are to be incubated in anaerobic
conditions, this is carried out in large quantities in an anaerobic jar. If only a small number is to be
incubated, this can also be done with anaerobic bags (e.g. GENbag gas generators by bioMrieux,
Nrtingen). It is important that the anaerobic ratios can be actually achieved on the agar strips, in the
plate, etc. Agar strips must not be adhered under any circumstances. If cams are present, hooks must
be placed in the plates to enable gas exchange.
Method validation
Methods for microbiological monitoring must be validated. This is referred to in the FDA Aseptic
Guidance. Inspectors, in particular those from the FDA, will also want to see the relevant
documentation. Unfortunately, it is not always so easy to carry out suitable tests, as often the
necessary technical equipment (such as an aerosol chamber for producing a microbe cloud) is not
available. However, it is possible to carry out the expected validation up to a certain degree and to
submit this if necessary.
Figure 12.G-12 The recovery rate of various surface methods and the influence of various factors
(Seyfarth, H.: Microbiological monitoring of surfaces/personnel; In: Gute Hygiene Praxis; Pharma
Technologie Journal 1080, 116-154 (2000)
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In surface testing methods, it is primarily the recovery rate that is questioned. This means that the
surfaces must be artificially contaminated and then tested to see how many organisms can be detected
with the selected method. Here, we will generally attempt to answer this question with data from
literature. In figure 12.G-12, there is a summary of relevant tests carried out by the author. The source
quoted also contains the associated statistical data.
For air testing methods, the FDA Aseptic Guidance requires that active collectors are calibrated and,
when using settle plates, that the exposure conditions are optimised to the unit with relevant testing.
In practice, the relevant data on the air volume tested and on the air velocity (important when using
the equipment under LF) should be requested from the equipment manufacturer. In the laboratory,
this data should be checked regularly (six-monthly) if possible. If this is not possible, the equipment
must be sent in and must have the relevant test certificates. Every batch of agar strips, contact plates,
etc., must be tested for growth promotion (see Culture media page 14). If culture media are not
covered at the end of the incubation, it must be demonstrated that organism growth would not have
been possible by dripping test organisms onto them (maximum 100 CFUs, e.g. of staphylococcus
aureus ATCC 6538).
It is also important to validate the air lock process. For this, inoculation with organisms (complete
range of organisms, see Culture media page 14) after the air lock should show that nothing is growing
on the medium in the H2O2, formalin, etc. in the material lock and thus that the growth promoting
properties are still present to a sufficient degree.
12.G.4 Sampling
It is not sufficient to describe sampling procedures, etc. here (see chapter 14.A Sampling). It is
particularly important to determine the responsibilities for sampling and, if necessary, also for
transportation from the unit to the processing laboratory. This is the only way to ensure that the
monitoring runs smoothly.
It is similar for the processing of the samples, in that the responsibilities must be clearly defined. It
must be ensured that no samples are left unprocessed (see chapter 14.A.2 Sampling plan
(instructions)).
12.G.4.1 Frequencies
For a monitoring program to be compiled, the testing frequencies must be considered. There are
guidelines for this in both the European and the American directives. Whereas in Europe, testing at
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the end of a process is sufficient, the FDA Aseptic Guidance requires that monitoring takes place
during the manufacturing and filling activities. If the operation has several shifts, the daily
monitoring should also cover every shift. This is interpreted generally to mean testing during
manufacturing, spread out over the process. FDA investigators also require concentration on the
actual filling area.
Air
The supplement to the EU-GMP-Guideline for the manufacture of sterile medicinal products contains
specific information about testing frequencies. Microbiological monitoring is required during
production for aseptic processes, in the same way as for FDA production. FDA investigators expect
general measurements spread out over the production process. By contrast, the supplement to the EUGMP-Guideline for the manufacture of sterile medicinal products is satisfied with monitoring based
on the processing step: air during or at the end of production. From now on, FDA investigators
require concentration on the actual filling area, whereby they would like a long period of time
covered for air. In figure 12.G-13, there are frequencies for sterile areas that have been accepted by
the FDA.
Figure 12.G-13 Frequencies for air testing in sterile areas
Frequencies for air testing in sterile areas
Sampling
points
Frequency
Critical area A EU production: for products to be manufactured aseptically, per batch after the
critical processing step. A single measurement if several batches are manufactured
per day.
US production: at the start of a batch, then every 2 hours (min. 2)
Critical area B EU production: after the critical processing step and during use if there is no
increased risk caused by the measurement.
US production: at the start, in the middle and at the end of production of a batch
Controlled
area C
operation room - daily to quarterly (depending on the production frequency or room

usage)
Area with
requirements
D
Operation room - monthly to quarterly
Surfaces
The supplement to the EU-GMP-Guideline for the manufacture of sterile medicinal products contains
information about testing frequencies for surfaces too. Microbiological monitoring is required during
production for aseptic processes, in the same way as for FDA production. FDA investigators expect
general measurements spread out over the production process. By contrast, the supplement to the EUGMP-Guideline for the manufacture of sterile medicinal products requires that the sample is taken at
the end of a critical processing step so that the drug product is not endangered. In figure 12.G-14,
there are frequencies for sterile areas that have been accepted by the FDA.
Figure 12.G-14 Frequencies for surface testing in sterile areas
Frequencies for surface testing in sterile areas
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Sampling
points
Frequency
Critical area A
EU production: for aseptically produced products, per batch after the critical
processing step.
US production: several times on a day of production
EU production: for aseptically produced products, per batch after the critical
processing step.
Weekly to quarterly (according to production frequency and room usage)
Additionally for US production: several times on a day of production
EU production: after the critical processing step and during use if there is no
EU production: after the critical processing step and during use if there is no
EU production: weekly to monthly (according to production frequency and

room usage)
Surfaces
Table
Wall
Floor
Surfaces
Work surface
/machine
Wall
Floor
Critical area B
Surfaces
Work surface
/machine
Wall
Floor
Controlled area C
Area with requirements D

Surfaces
Table
Sterile Production
Monthly to six-monthly (depending on threat to product)
Page 84 of 140
Wall
Floor
Personnel
The supplement to the EU-GMP-Guideline for the manufacture of sterile medicinal products also
contains information about testing frequencies for microbiological checks of personnel.
Microbiological monitoring is required during production for this, in the same way as for FDA
production. The sample should again be taken at the end of the process in order not to endanger the
drug products. In figure 12.G-15, there are frequencies for sterile areas that have been accepted by the
FDA.
Figure 12.G-15 Frequencies for personnel testing
Frequencies for personnel testing
Sampling location
Frequency
Critical area A
EU production:
Hand
Batch-related after the critical processing step
Forearm
Monthly after air lock

(each member of staff at least 1x annually)
Hood
(around the breathing mask)

USA production:
on production days, all personnel are fully tested (hand, forearm, hood) at the end of a shift
Critical area B
EU production:
Hand
Batch-related after the critical processing step
Forearm

Hood
(around the breathing mask)

USA production:
on production days, all personnel are fully tested (hand, forearm, hood) at the end of a shift
12.G.5 Sampling points
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The critical points must be considered in the first instance when determining the sampling points. The
results of the initial monitoring when setting up a sterile room must also be taking into consideration
when determining the sampling points. For example, in the filling station, testing is certainly
expected near to the filling needle. If other problem areas come to light later (for example, during
investigations), these must also be included in the routine monitoring.
However, you should beware of testing as many points as possible. This would only produce a vast
quantity of data (and cost) without any additional gain of information. In the very compact facilities
that are usual nowadays, one or two sampling points in the filling machine is sufficient - and likewise
in the surrounding area. The PDA Technical Report 13 Revised Fundamentals of an Environmental
Monitoring Program, which was mentioned earlier, contains a table of examples of sampling points
(see figure 12.G-16).
Figure 12.G-16 Examples of sampling points (PDA 2001)
Examples of sampling points (PDA 2001)
System
Point
Air (filling line)
Near to the open and/or filled container
Room air
Near to the working area
Surface (room)
Floor
Door handle
Walls
Curtains
Surface (equipment)
Filling line
Control panel
Stopper bowl
Personnel on the filling line
Fingerprint (as a minimum)
Laminar air flow

(e.g. cover)
Near the area with high activity
The sampling points must be clearly determined (e.g. not only "on the table", but "30 cm at the centre
of the table"). By way of description, it is also useful to have a plan of the sterile room with sampling
points plotted. This facilitates discussions in the event of an inspection.
12.G.6 Measure if levels are exceeded

The measures for this should only be referred to in general terms in the monitoring SOP. Details
should be set down in a separate SOP. It is not simply a case of surveying and logging the results. If
necessary, corrective measures must be implemented and appropriately documented. First, an
investigation plan (testing plan) must be drawn up and then, following successful testing, an
investigation report must be compiled.
Investigation plan
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The following must be checked as part of this investigation:
Results
Extent of the problem
Influence on the products
Are quarantine measures required
Follow-up testing
And the following must be implemented:
Investigations to find the cause and

Investigation of measures to show their success
It is important to first ask whether this is a question of a deviation in production hygiene or possibly
an incorrect laboratory result.
If there is no error in analysis of the microbiology, the question of the influence on the product
quality is of vital importance. The product should be quarantined if the deviation is relevant to the
product. There is generally no relevance to the product if there are deviations in the personnel lock,
for example. If the deviation is relevant to the product, investigations must be carried out to
determine whether the product quality is actually affected.
In PDA Technical Report 13 Revised Fundamentals of an Environmental Monitoring Program
(2001), there is a useful summary of corrective measures in the event of deviations (see figure 12.G17).
Figure 12.G-17 Typical corrective measures if levels are exceeded (PDA 2001)
Typical corrective measures if levels are exceeded (PDA 2001)
System
Room air/
HVAC
Corrective measures
Review level of personnel activity

Review/perform air flow patterns/smoke tests
Review aseptic technique of personnel
Review gowning requirements for area
Inspect air filters for leaks and pressure differential across filter
Review room disinfection/sanitisation procedures, sanitisation intervals and
disinfectant efficacy
Check area pressure differentials, particularly with respect to the last
sanitisation
Evaluate mechanical equipment in area as possible source of contamination
Evaluate integrity of the room (e.g. peeling paint, cracks in ceiling, walls, and
floors)
Review risk to product
Perform investigation for possible sources of contamination

Evaluate sanitisation/disinfection practice
Review cleaning records
Review possible unusual events during manufacturing operation
Examine areas during usage
Verify that controls were not circumvented
Facility
surfaces
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Personnel
gowning
Review risk to product

Determine sensitivity of isolate to disinfectants being used
Review isolates for occurrence in other types of tests
Evaluate possible operator impact upon product

Examine areas during usage
Review sterility test data
Review other environmental monitoring data for area
Review preparation and expiry dates for disinfectants used on gloves
Identify all morphologically unique isolates (human vs. environmental)
Evaluate training of operator
Interview operator for potential causes
Retrain/requalify operator
If necessary, additional testing must be carried out to find the source of the organisms. If the action
level is exceeded, the organisms must always be identified. This will often provide information about
the source of the organisms. Once the measures have been carried out, appropriate testing must
demonstrate that they were successful and that the area or process is under control again.
Investigation report
Once the investigations are complete, an investigation report must be drawn up, documenting the
following:
Investigations
Measures
Review by management
Distribution to affected personnel
Any future corrective measures
12.G.7 Organism identification

Organisms discovered must be identified as part of the monitoring, at least if the levels are exceeded.
The hope is that this will provide information about the source. Unfortunately, conventional methods
for identifying organisms are not always enough to detect clear contamination contexts. This is
expressly stated in the new, harmonised sterile testing chapter of the Ph. Eur. and the FDA Aseptic
Guidance also contains reference to this. Because most microbiological laboratories do not have the
facility for molecular isolate typing, one generally has to be satisfied with conventional methods. But
even with these results, it is possible to draw certain conclusions about the possible source of the
organisms based on the type. Relevant specialist literature is also helpful, such as Mitscherlich, E.
and E.H. Marth, Microbial Survival in the Environment, Springer-Verlag, Berlin, Heidelberg, New
York, Tokyo (1984). There is also help on the Internet at the address
http://141.150.157.117:8080/prokPUB/index.htm.
In environmental monitoring, one must anticipate very varied proportions of the different organism
groups:
Gram-positive rods (floor, dust): 10-20 %

Gram-positive cocci (human and animal origin): 80-85 %
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Gram-negative rods (water, floor): 5-10 %

Yeast and mildew: 2-5 %
The following section presents some possibilities for the examination procedure to identify isolates
(bacteria) in environmental monitoring. It is extremely important to note that this type of work is
covered by the infection protection legislation and must therefore be notified to the responsible
authorities (epidemic unit of the responsible regional government) and approved by them.
Normally, not all colonies that have exceeded a level are identified and one must be satisfied with the
identification of all morphologically different colonies. It is extremely important to run one, or often
even two, passages on blood agar to get a single colony. The organisms are revitalised by this and
acquire the potential of carrying out their typical biochemical reactions. They should be cultured in a
fractionated smear so that pure cultures can be obtained for the subsequent work. The individual
colonies are checked first (colour, size, shape, hemolysis, any odour, metallic gloss, etc.) and the
results are logged. The subsequent procedure is determined with a gram preparation (figure 12.G-18
).
Figure 12.G-18 Examination procedure to identify isolates from environmental monitoring - growth
of pure cultures and gram staining
The following diagrams show examination procedures for gram-negative rods (figure 12.G-19),
gram-positive rods (figure 12.G-20), gram-negative cocci (figure 12.G-21) and gram-positive cocci
(figure 12.G-22). The bioMrieux api identification system is stated as an example. The other
systems available on the market can of course also be used here.
Figure 12.G-19 Examination procedure to identify isolates from environmental monitoring identification of gram-negative rods
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Figure 12.G-20 Examination procedure to identify isolates from environmental monitoring identification of gram-positive rods
Figure 12.G-21 Examination procedure to identify isolates from environmental monitoring identification of gram-negative cocci
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Figure 12.G-22 Examination procedure to identify isolates from environmental monitoring identification of gram-positive cocci
Of course, it is not sufficient to only consider bacteria. If problems with fungi occur, a distinction
must first be made between yeast and mildew. If a more precise identification is required, one must
generally turn to specialists as, for mildew at least, there are no reliable identification systems on the
market.
As every organism found cannot generally be identified for reasons of capacity, one must decide
when it is desirable to identify the organisms and to what extent. According to the FDA Aseptic
Guidance, organisms should be identified to species level as far as possible and - if appropriate - to
genus level.
If the action level is exceeded in cleanliness grade A and B, organisms should certainly be identified
to species level if possible. If the alert level is exceeded then identification to genus level is usually
sufficient. If there is a suspicion that special organisms are present, e.g. spore formers or
pseudomonads, these should also be identified.
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In cleanliness grade C, identification to genus level is generally sufficient if the action level is
exceeded. If there is a suspicion that special organisms are present, e.g. spore formers or
pseudomonads, these should also be identified here.
For isolates from grade D, colonies that occur particularly often should be identified. Gram staining
is usually sufficient.
Summary
When manufacturing and filling sterile drug products, all possible sources of microbial contamination
from the environment must be controlled. This primarily concerns air, surfaces and personnel.
A monitoring program must be established for this, in which levels, sampling points, frequencies,
methods/equipment and measures in the event of deviations, amongst other things, must be regulated.
These individual points are explored and suggestions are made for practical implementation.
12.H Test for sterility

Which drug products must be tested for sterility?

What is the procedure for parametric release?
How is a sterility test carried out?
Which investigations must be carried out alongside the test?
What is the procedure if there is substance turbidity in the culture medium?
What needs to be considered when validating a sterile test method?
In the European pharmacopoeia (Ph. Eur. 4), there are various monographs concerning the
requirement for sterility (see figure 12.H-1).
It is not specified that a test for this must be carried out. It is only asked that, if the test is carried out,
the substances/preparations/products must conform to the test for sterility. This is expressly referred
to in the "Guidelines for Using the Test for Sterility" ("A manufacturer is neither oblieged to carry
out such tests..."). This pharmacopoeia method is the arbitration method that is to be implemented in
the event of disputes ("The purpose of the test for sterility, as that of all pharmacopoeial tests, is to
provide an independent control analyst with the means of verifying that a particular material meets
the requirements of the European Pharmacopoeia") and that serves as a standard for changes to the
method (the manufacturer is not precluded from "using modifications of or alternatives to the stated
method provided they are satisfied that, if tested by the official method, the material in question
would comply with the requirements of the European Pharmacopoeia").
Figure 12.H-1 Drug products for which the pharmacopoeia requires sterility
Drug products for which the pharmacopoeia requires sterility
Drug product group
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Requirement
Page 92 of 140
Parenterals
Injection preparations
Infusion preparations
Concentrates for the manufacture
of injection preparations
Concentrates for the manufacture
of infusion preparations
Powders for the manufacture of
injection preparations
Powders for the manufacture of
infusion preparations
Implants
Ophthalmics
Radioactive medicinal products for

parenteral use
Vaccines for humans
Must comply with the "test for sterility".

If the result of the test cannot be waited for, the
parametric release method should be chosen.
Regulation in the individual monographs
Bacterial vaccines
Bacterial toxoids
Viral vaccines
Vaccines for veterinary use
Eye drops
Eye baths
Powders for eye drops
Powders for eye baths
Semi-solid preparations for ocular
use
Eye inserts
Protein solutions
If specified in the monograph, must comply with the

"test for sterility".
Bacterial vaccines
Bacterial toxoids
Viral vaccines
Vector vaccines
Immunsera for humans
Immunsera for veterinary use
Must comply with the "test for sterility" (>100 ml,

membrane filtration if possible)
Intramammary preparations ad us. vet.
Sutures for veterinary use
Must comply with the "test for sterility" for "catgut and
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other surgical sutures".

Designated drug products (e.g. ointments) If the preparation is designated as "sterile", it must
comply with the "test for sterility".
12.H.1 Parametric release

Although the introduction to the "Sterility test chapter" 2.6.1 in the pharmacopoeia states that sterility
testing must be carried out on substances, preparations or products for which sterility is specified, this
does not in any way mean that testing must actually be carried out - as already mentioned. Because of
the statistical uncertainty of the "sterility testing", parametric release is preferable for products
sterilised in the final container. The pharmacopoeia specifically agrees to this (see figure 12.H-2).
Figure 12.H-2 Statement from the European pharmacopoeia about parametric release
Statement from the European pharmacopoeia about parametric release
"In the case of terminally sterilised products, physical proofs, biological based and automatically
documented, showing correct treatment throughout the batch during sterilisation are of greater
assurance than the sterility test. The circumstances in which parametric release may be considered
appropriate are described under Methods of preparation of sterile products (5.1.1).
"When a fully validated terminal sterilisation method by steam, dry heat or ionising radiation is used,
parametric release, that is the release of a batch of sterilised items based on process data rather than
on the basis of submitting a sample of the items to sterility testing, may be carried out, subject to the
approval of the competent authority."
However, chapter 5.1.1 does not contain any further details about parametric release other than the
instruction that the process must be validated. For this, we can turn to the PDA Technical Report No.
30, which describes parametric release for products sterilised with steam in the final container (see
figure 12.H-3).
Figure 12.H-3 Parametric release according to PDA Technical Report No. 30
Parametric release according to PDA Technical Report No. 30
Parameter
Requirement
Minimum Sterilisation
Efficacy
The sterilisation process validated to achieve a SAL of 10-6
Critical Process
Parameters
Critical process parameters are defined and limits are set which must
be met prior to release of the product
Sterilisation
procedure
Procedures are established that ensure the entire lot of finished

product to be released was subjected to the sterilisation process
Procedures are established that ensure critical process parameters are
met for every sterilisation load
Procedures are established that ensure critical process parameters are
met in every part of the load
Bioindicator
Verification
Sterile Production
All bioindicators should be specified according to a written

procedure.
Page 94 of 140
Presterilisation
Bioburden
the organism's name

the supplier
the spore population on the carrier
expiration date
storage conditions
resistance
Where overkill cycles are not employed, the indigenous bioburden

must be characterised in unsterilised product for at least 3 lots
initially, and at predefined intervals for an extended period thereafter.
Bioburden alert and action level must be established
Maximum allowable manufacturing time limits between initial
product compounding and sterilisation of the last filled container
must be established to ensure presterilisation bioburden does not
increase beyond established levels.
Container/Closure
Integrity
Integrity for each container/closure system has to be demonstrated

over the shelf life of the product
Process Validation
Installation Qualification (IQ)/Operational Qualification (OQ)

Temperature mapping of the empty chamber
Pressure
Cooling water - if used
Validation of electronic data processing system
Performance Qualification (PQ)
Temperature distribution
Requalification and Change Control Programme
Requalification of the sterilisation process
A defined preventive maintenance programme
Change control procedure
Process Parameters
and Records
Product Bioburden Control

Sampling procedures
Test procedures
Nutrient media,
Types of tests
Frequency of testing
Screening for aerobes
Where appropriate, screening for anaerobes
Where appropriate, screening for heat-resistant spore-forming
bacteria
Critical Process Parameters
Dwell time
Minimum temperature
Chamber pressure
Minimum limits for F0 for processes monitored by integrator

Physical/chemical indicators
Process parameters
and records (cont.)
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Product Release
Procedure
Records (all critical process parameters must be recorded)
Batch Records Review

Evaluation of the validation status of the process
However, it is not so easy to switch from the sterility test to parametric release; the authorities
responsible must agree. This is particularly problematic if the product has been registered in several
countries and every authority with which the sterility test is recorded for the drug product concerned
must be notified and give their consent.
There are now papers relating to this on the "official side". Annex 17: Parametric release to the EUGMP-Guideline, is closely related to a relevant EMEA Note for Guidance. The Pharmaceutical
Inspection Convention (PIC) has also issued a relevant guideline. These papers go further than the
PDA paper: as well as parametric release of sterile drug products, it is also generally discussed for
other tests.
12.H.2 Sterility test

If the sterility test is selected for products sterilised in the final container, or if parametric release is
not possible, e.g. for aseptically filled products, it is advisable to carry out the sterility test in
accordance with the specifications in the pharmacopoeia. A different procedure would also be
possible here (page 1), but it may result in endless discussions in the event of an inspection.
The following section covers the most important points of the test. The sterility test included in Ph.
Eur. 4.6 is the result of an international harmonisation to the end that a test exists that is recognised in
Europe as well as in the USA and Japan. In addition to a simple explanation of the test, the most
important changes in comparison to the previous test should be highlighted. As not all points could be
harmonised, the points that will be handled differently in the future in the USA and Japan than in
Europe will also be mentioned.
12.H.2.1 Environmental conditions

The sterility test must be carried out under aseptic conditions in order to prevent secondary
contamination. In order to achieve this, the test environment must be adapted to the test procedure.
However, the measures implemented must not affect any existing contaminants.
In the non-harmonised, non-binding Guidelines for Using the Test for Sterility, for example, the
European pharmacopoeia mentions the use of a laminar flow bench that should be present in a class B
clean room (according to the requirements of the EU-GMP-Guideline for the manufacture of aseptic
products) or the use of an isolator. There is a number of points that speak in favour of an isolator:
High level of result reliability due to maximum product protection (reduction of the secondary
contamination rate)
FDA investigators recommend the use of isolators for the sterility test.
Tried and tested technique in North America (in <71> Sterility Tests, the USP expressly
refers to the use of isolators for the sterility test).
High degree of flexibility for personnel deployment because the air lock procedure is not
required.
Low space requirements (normal sterile test isolator approx. 1 x 2 x 2 m)
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No requirement for an expensive HVAC system for the sterile test rooms and their
maintenance (legislators do not require any special environmental conditions for isolators. In
Ph. Eur. there are no background requirements for isolators, USP Sterility Testing Validation of Isolator Systems: Isolators for sterility testing need not be installed in a
classified clean room)
No costs for sterile clothing (purchase, care, laundry and sterilisation)
Minimal routine monitoring in the isolator, no monitoring of the environment or personnel.
However, it must be made clear that the cost for a sterile test isolator with validation will cost around
400,000 euros. This cost quickly proves its worth though, when we consider that "false-positive"
results can be avoided. This usually means that batches that are actually sterile are not quarantined
because of the impracticality of carrying out follow-up tests (see chapter 12.H.6 Reading and
evaluating).
12.H.2.2 Environmental monitoring

The work conditions specified for carrying out the tests must be regularly monitored with suitable
environmental checks in the working area and using checks of the aseptic work methods.
Monitoring of the hygiene status in sterile test rooms

In figure 12.H-4 and figure 12.H-5, there is a suggestion for the implementation of the requirement
for environmental checks in the sterile test room which is based on the limits and frequencies for
monitoring production.
The usual methods for aseptic processes are used for monitoring a clean room (chapter 12.G
Microbiological monitoring). There are no far-reaching requirements as in aseptic processes.
Figure 12.H-4 Limits for environmental monitoring in the sterile test room
Limits for environmental monitoring in the sterile test room
Measurement
Alert limit
Action limit
Air particles
0.5 m
max. 3,500/m3
5 m
max. 0/m3
Under LF
0 (1) CFU/m3
Outside LF
3 CFU/m3
7 CFU/m3
Table/wall under LF
2 CFU/25 cm2
3 CFU/25 cm2
Surface/wall outside LF
2 CFU/25 cm2
5 CFU/25 cm2
Floor
5 CFU/25 cm2
10 CFU/25 cm2
Forearm
0 (1) CFU/25 cm2
Hand
0 (1) CFU/25 cm2
Hood exterior
0 (1) CFU/25 cm2
Under LF
Air microbial count
Surfaces
Personnel
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Page 97 of 140
Figure 12.H-5 Suggestion for environmental monitoring in the sterile test room - frequencies
Suggestion for environmental monitoring in the sterile test room - frequencies
Techniques
Frequency
Locations
Implementation
Air particles
Leak test
Six-monthly
Every LF bench
Particle
counter
Continuous
Every LF bench
Climate workshop
Air microbial count

Air under LF
EU
After the test
Under LF on the table
Laboratory
personnel
FDA
During the test
Laboratory
personnel
Before the test starts According to the plan
Laboratory
personnel
Under LF
Before and after the Under LF on the table

test
Laboratory
personnel
Outside LF
After the test
Laboratory
personnel
Personnel
After the start of the

test
Air
outside LF
Surfaces
Right and left work hand

Right and left forearm around the
wrist
Hood exterior around the
respiratory protection
Laboratory
personnel
Once every six months, a second set of plates or agar strips is also applied to check
for anaerobes
Monitoring of the hygiene status in isolators

A main advantage of using sterile test isolators is that the tests are carried out in a sterilised
environment free from personnel. This reduces the contamination risk to zero. However, it is a
mistake to think that you can get by without monitoring. This is clearly stated in Annex 1 to the EUGMP-Guideline: ("Monitoring should be carried out routinely and should include leak testing of the
isolator and glove/sleeve system.").
The question of which limits to set then arises. It seems logical to apply the normal air limits for
room grade A, but it has become accepted to require 0 CFU/m3 air in the sterile test isolator. This is
also in line with the suggestions in the FDA Aseptic Guidance, where no microorganisms are
tolerated in the air for aseptic processes (Samples from Class 100 ... environments should normally
yield no microbiological contaminants). 0 CFU/25 cm3 must be achieved for surfaces as this is of
course a true, validated sterilisation (see figure 12.H-6).
Figure 12.H-6 Limits for environmental monitoring in the isolator
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Limits for environmental monitoring in the isolator

Measurement
Alert limit
Air particles
0.5 m
5 m
Air microbial count
0 CFU/m3
Surfaces
0 CFU/25 cm2
Action limit
max. 3,500/m3
max. 0/m3
There is however uncertainty about the scope of the test for checking isolators. A suggestion is made
in figure 12.H-7.
Figure 12.H-7 Suggestion for environmental monitoring in the isolator - frequencies
Suggestion for environmental monitoring in the isolator - frequencies
Test object
Frequency
Air
Active collectors
Start and end of the test
Open bottles
Test duration
Surfaces
RODAC contact plate
End of test
Each glove
Fingers
End of test
Glove
End of test
Cuff
End of test
The monitoring methodology is particularly problematic. One must anticipate residual H2O2 in the
room, which is emitted from the damp agar surfaces and which prevents any microorganisms present
from growing. Proving growth promotion using control organisms (positive controls) takes on
particular significance here. For this, max. 100 CFU Staphylococcus aureus ATCC 6538 is dripped
onto the plates or strips with no growth following incubation. This demonstrates that no organisms
would have been able to grow.
For air testing, it is possible to try to come to a satisfactory solution using multiple extractions (e.g. 2
x 500 l with RCS). For the RCS unit, there are now agar strips on the market with a suitable
neutralisation additive which alleviate the problems of proving air microbial counts. For surface
contact testing, Becton-Dickinson have brought a suitable contact plate onto the market which
contains D/E neutralising agar. Plates with a suitable neutralising additive should also be used for a
collector with solid culture medium petri dishes.
Aseptic working methods

The aseptic working method testing required by the pharmacopoeia (negative controls) must be
regularly monitored using proven sterile preparations. These controls must be carried out at the same
time as the testing, as the result must be used when making a decision about any follow-up tests (see
chapter 12.H.6 Reading and evaluating). If different methods are implemented (membrane
filter/direct inoculation of the culture medium) and drug products are tested that require different
preparation types (e.g. aqueous ampoules, vials with lyophilisates, etc.), the test must be carried out
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for the methods and the preparation types. If several people carry out the test, negative controls are
also required for each person.
12.H.3 Method description

The pharmacopoeia contains method descriptions for the membrane filtration and direct inoculation
methods. Membrane filtration is stipulated as the method of choice (The technique of membrane
filtration is used whenever the nature of the product permits ...)
The membrane filter method is described for the following dosage methods:
Aqueous solutions
Soluble powders
Oils and oily solutions
Ointments and creams
The direct inoculation method is described for the following dosage forms:
Oily liquids
Ointments and creams
Catgut and other surgical suture materials for veterinary use
There are a few small modifications here that may be extremely useful. For aqueous solutions, for
example, a solution quantity is not stated, but validation is suggested. The wash cycles are limited to
5 x 200 ml, unless a validation study shows that this is not sufficient to wash out antimicrobial
substances. (Japan requires 5 times 100 ml per filter for this.)
In addition, there are detailed descriptions for testing parenterals, ophthalmics and other preparations
not intended for injection. These have been adopted virtually unchanged. There are only two changes.
The instructions "The total volume filtered through a membrane must not exceed 1000 ml, except in
justifiable and authorised cases" (for membrane filtration) and "If the filling volume of the sample
container is more than 100 ml, the membrane filter method should be used, except in justifiable and
authorised cases" (for the direct inoculation method) were difficult to justify objectively and have
been removed and not replaced.
12.H.3.1 Incubation
The harmonised text stipulates a minimum of 14 days' incubation. Liquid thioglycolate medium (for
determining bacterial contamination) 30-35 C and soya-bean casein digest medium (for proving
fungal contamination) 20-25 C. This means that the previous option in the USP for "seven days'
incubation for products sterilised in the final container" can no longer be used.
12.H.4 Number of samples

The numbers of samples were agreed as part of the harmonisation, but only USP and JP want to
include these numbers in the binding part. In Ph. Eur., they are in the non-binding "Guidelines for
Using the Test for Sterility" as before (figure 12.H-8). It is certainly a good idea to abide by these
suggestions. Otherwise the table is unchanged; only regulations for testing bulk materials have been
added.
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When determining sample numbers, the minimum test quantities must also be taken into
consideration (see figure 12.H-8), as the number of samples must be doubled if the entire content of a
container is to be tested.
Figure 12.H-8 Number of samples for sterility testing
Minimum number sterility testing
Number of items in the
batch
Minimum number of items to be tested for each medium, unless

otherwise justified and authorised*
Parenteral preparations
100 containers
> 100 but 500
> 500
10 % or 4 containers whichever is the greater

10 containers
2 % or 20 containers ** whichever is less
Ophthalmic and other non-injectable preparations

200 containers
> 200 containers
5 % or 2 containers whichever is the greater

10 containers
If the product is presented in the form of single-dose containers, apply the scheme shown above for
preparations for parenteral use.
Catgut and other surgical suture materials for veterinary use
2 % or 5 packages whichever is the greater, up to a maximum total of 20
packages
Bulk solid products
< 4 containers
> 4 containers but 50
containers
> 50 containers
Each container
20 %t or 4 containers whichever is the greater
2% of 10 containers whichever is the greater
Pharmacy bulk packages of antibiotics (greater than 5 g)

6 containers
* If the contents of one container are enough to inoculate the two media, this column gives the
number of single containers needed for both the media together.
** JP and USP will indicate 10 containers for large-volume parenterals
12.H.5 Sample quantity

The required test quantity is contained in a table in the main part of the pharmacopoeia (and is
therefore binding). As part of the harmonisation, some of the quantities to be tested have been
changed (which may mean revalidation of the methods used). An overview of the requirements is
given in figure 12.H-9 and figure 12.H-10, which also shows the previously required quantities, so
that it is easy to see whether validation is required for drug products that have already been
introduced.
Figure 12.H-9 Sample quantities for the sterility test - parenterals
Sample quantities for the sterility test - parenterals
Quantity per
container
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Minimum quantity to be used for each medium unless otherwise justified

and authorised
Page 101 of 140
Ph. Eur. 4
Ph. Eur. 5
Liquids
< 1 ml
The whole contents of each container The whole contents of each container
1-40 ml
Half the contents of each container
Half the contents of each container but

not less than 1 ml
> 40 ml 100 ml 20 ml
20 ml
>100 ml
10 percent of the contents of the container

but not less than 20 ml
20 ml
Antibiotic liquids
<1 ml
1 ml
Half the contents of each container but 1 ml

not more than 20 ml
Solids
<50 mg
50 mg
but <300 mg
Half the contents of each container

not less than 50 mg
300 mg - 5 mg
150 mg
150 mg
>5g
150 mg
500 mg
Figure 12.H-10 Sample quantities for the sterility test - not intended for injection
Sample quantities for the sterility test - not intended for injection
Quantity per
container
Minimum quantity to be used for each medium unless otherwise justified and
authorised
Ph. Eur. 4
Ph. Eur. Suppl. 4.6
Ophthalmic and other non-injectable preparations

Aqueous solutions
< 1 ml
The whole contents of each container to The whole contents of each container
provide not less than 2.5 ml
1-40 ml
The whole contents of each container to Half the contents of each container but
not less than 1 ml
> 40 ml and
100 ml
The whole contents of each container to 20 ml

>100 ml
The whole contents of each container to 10 % of the contents of the container but
not less than 20 ml
Antibiotic liquids
<1 ml
The whole contents of each container
The whole contents of each container
1 ml

not more than 20 ml
1 ml
Other preparations soluble in water or in isopropyl myristate

The whole contents of each container to The whole contents of each container to
provide not less than 0.25 g
provide not less than 200 mg
Insoluble preparations, creams and ointments to be suspended or emulsified
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The whole contents of each container to The whole contents of each container to
provide not less than 0.25 g
provide not less than 200 mg
Catgut and other surgical sutures for veterinary use
3 sections of a strand (each 30 cm long) 3 sections of a strand (each 30 cm long)
12.H.6 Reading and evaluating

Several times during and after the incubation time, the cultures are checked macroscopically for
visible growth of microorganisms. If there is growth, the test can be interrupted and then evaluated.
If no growth is determined, the tested product complies with the sterility test. However, if growth is
detectable, the product does not satisfy the requirements, unless the test can be proven to be invalid
for reasons that are not related to the product itself. The test can only be viewed as invalid and
repeated if one or more of the conditions summarised in figure 12.H-11 are fulfilled.
Point four in figure 12.H-11 ("after determination of the identity of the microorganisms isolated from
the test, the growth of this species or these species may be ascribed unequivocally to faults with
respect to the material and/or the technique used in conducting the sterility test") is only sufficient
grounds for a repetition if the organism species can be clearly assigned. A similar species with
conventional identification methods is not sufficient in this case. This is expressly stated in the nonharmonised, non-binding "Guidelines for Using the Test for Sterility". If the identification of the
microorganisms is to be used as the only criterion for declaring the sterility test invalid, more
sensitive techniques must be applied as microbiological/biochemical methods, e.g. molecular typing
with RNA/DNA homologies.
Other reasons for the sterility test being invalidated are conceivable, for example, if a test was carried
out in the isolator and there were problems with the integrity of the system. This instruction was
contained until now in the USP.
Figure 12.H-11 Invalidity declaration of the sterility test
Invalidity declaration of the sterility test
The test can only be viewed as invalid if one or more of the conditions are fulfilled
the data of the microbiological monitoring of the sterility testing facility show a fault
a review of the testing procedure used during the test in question reveals a fault
microbial growth is found in the negative controls
after determination of the identity of the microorganisms isolated from the test, the growth of
this species or these species may be ascribed unequivocally to faults with respect to the
material and/or technique used in conducting the sterility test procedure.
12.H.7 Procedure in the event of culture medium turbidity

If the material to be tested clouds the culture medium so that the existence or otherwise of microbial
growth is difficult to determine at the end of the incubation time, suitable quantities of the culture
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medium must be transferred into fresh containers with the same culture medium. The main restraint
when carrying out a test, as stated by both the USP and Ph. Eur., was how to proceed in the event of
culture medium turbidity. The Ph. Eur. required: The incubation of the original container and the
container with the transferred culture medium to be continued for a total at least 14 + 7 days,
calculated from the original inoculation.
The USP required sub-cultures to be created between the third and seventh day and the sub-cultures
to be incubated for 11 to 7 days, so that they had a total incubation time of 14 days. Although this
incubation time is pleasantly short in comparison to the Ph. Eur., this procedure is somewhat
problematic. Firstly, when the incubation time was determined by the USP for the original test, it was
assumed that organisms require 14 days to multiply to a quantity visible in a liquid medium with the
direct inoculation method. If there is turbidity (which generally occurs with the direct inoculation
method), it is now assumed that after the third day there is a sufficient quantity of organisms for
enough organism material to be detected when transferred into a new culture medium container. This
is an unfortunate misconception. This long incubation time of 14 days is not necessary because the
organisms grow so slowly but because the organisms often have a very long lag phase in which they
do not grow. A problematic organism for aseptic filling is Propionibacterium acnes, for example,
which in our experience often does not develop into a visible organism concentration in the culture
medium until after the twelfth day. These organisms are certainly not detected with the procedure
suggested by the USP.
This major difference has now been rectified. The harmonised text now states that 14 days after the
start of incubation, portions (each no less than 1 ml - JP requires transfer suitable portions and omit
here) of the culture medium should be transferred to fresh containers with the same culture medium.
The incubation of the original container and the freshly inoculated container should then continue for
at least four further days so that there is a total incubation time of 18 days.
12.H.8 Culture media

The two pharmacopoeias name the following as culture media:
Fluid thioglycolate medium

This is primarily used to detect anaerobic bacteria, but can also be used to detect aerobic
bacteria.
Soya-bean casein digest medium
This is mainly suitable for detecting aerobic bacteria and fungi.
Other culture media can be used on the condition that there is evidence of its growth promoting
properties and validation tests have been carried out. It is not advisable to make use of this opening
clause, as neither Japan nor the USA will adopt it.
Very detailed information is available about storing culture media:

This must be stored in sterile, air-tight containers between 2 and 25 C. If more than the upper
third of the medium turns pink, it can be made useable again by heating until the pink colour
disappears then rapidly cooling it. The medium may only be stored in the long term with the
relevant validation (JP and USP will not adopt this clause). The question of what "long term"
means is unanswered - but certainly longer than 1 month. If the medium is stored in unsealed
containers, it may be used for a month with the prerequisite that the growth promoting
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properties have been tested within two weeks of the medium being used and the colour
indicator does not cover more than the upper third.
Soya-bean casein digest medium
This must be stored in sterile, well-sealed containers between 2 and 25 C, unless the medium
is to be used immediately. The medium may only be stored in the long term following the
relevant validation.
12.H.9 Culture media controls

Every batch of the culture media used must comply with the tests listed below, which can be carried
out before or at the same time as the test of the product being investigated:
Sterility: Samples of the culture media are incubated for 14 days according to their intended
use, i.e. thioglycate medium is generally incubated at 30-35 C and soya-bean casein digest
medium at 20-25 C. No microbial growth must be detected.
The European pharmacopoeia stipulates that every batch manufactured must be tested on ready-touse medium. This requirement is not so high in the JP and USP. The two pharmacopoeias wish to
point out that, in suitable cases, periodic testing of the various manufacturing batches manufactured
from the same batch of dehydrated medium is acceptable.
Because there is always the risk that a medium is over-sterilised and thus that growth promotion
properties are reduced, for example due to caramelisation of the sugars, this option must not be used
under any circumstances for tests for Japan or the USA. This would be problematic for the USA at
least, because the problem of over-sterilisation is expressly referred to in the FDA-Guide to
Inspection of Microbiological Pharmaceutical Quality Control Laboratories.
In order to test the growth promotion properties, samples of the culture media are inoculated with a
small quantity (10 to 100 CFU) of microorganisms and incubated. The microorganisms must create a
visible turbidity within the specified time.
The following microorganisms are specified:
Fluid thioglycolate medium: Clostridium sporogenes, Pseudomonas aeruginosa,

FDA investigator also expect wild strains to be used (organisms that have been found as part
of the monitoring in the units).
Soya-bean casein digest medium: Aspergillus niger, Bacillus subtilis, Candida albicans
Incubation is for up to three days for bacteria and five days for fungi. Reference strains are
given in Fig. 12.H-12. The number of passages from the original culture is limited to 5, as
before.
Figure 12.H-12 Reference organisms for the sterility test for method validation and culture
media controls
Reference organisms for the sterility test for method validation and culture media
controls
Organism
Strains
Newly recorded
ATCC 6538, CIP 4.83,
Aerobic bacteria
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NCTC 10788, NCIMB 9518

Bacillus subtilis
ATCC 6633, CIP 52.62,

NCIMB 8054
Pseudomonas
aeruginosa
ATCC 9027, NCIMB 8626,

CIP 82.118
ATCC 19404, CIP 79.3, NCTC 532
ATCC 11437
Candida albicans
ATCC 10231, IP 48.72
NCPF 3179,
ATCC 2091, IP1180.79
Aspergillus niger
ATCC 16404
IP 1431.83, IMI 149007
Anaerobic bacteria
Fungi
12.H.10 Method validation

All methods must be validated for the specific substance and/or drug product using all the
microorganisms named in figure 12.H-13. The strains stated in figure 12.H-12 must be used. If use
has been made of the previous option of using one type of aerobe, one type of anaerobe and one type
of fungus, this will mean revalidation in all cases. FDA investigators sometimes expect validation
with organisms that have been found in the unit
Figure 12.H-13 Microorganisms for method validation
Medium
Microorganisms
Pseudomonas aeruginosa
Soya-bean casein digest medium Bacillus subtilis

Aspergillus niger
Candida albicans.
For the validation, the lowest number of organisms possible (maximum 100) are added to the last
portion of the rinsing fluid for membrane filtration or to the drug product/culture medium mixture for
the direct inoculation method and incubated for up to five days (previously three days for bacteria) at
30-35 C (liquid thioglycolate medium) or 20-25 C (soya-bean casein digest medium). After
incubation, growth must be detectable comparable to the accompanying control containers. The
method can then be viewed as valid. If organism inhibition is observed, this must be eliminated, for
example by neutralisation, dilution, increase of the rinsing volume, etc. Especially for preparations
with a high content of antimicrobial substances, it can be useful to test the growth promotion again
following the incubation.
The question of when a validation must be carried out is clearly regulated:
If the sterility test is carried out with a new product

If there is a change in the experimental conditions of the test, which does not make clear when
one must assume changes in the experimental implementation (this should not understood in
too narrow a sense, in my opinion).
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Another important question is how often and with which product batches this type of validation must
be carried out. There is no direct information about this in the sterility test regulations in the
pharmacopoeias. However, in the Validation of Microbial Recovery From Pharmacopeial Articles
chapter of the USP, it states that three independent validations should be carried out. The FDA
explains that the validation must be carried out with three different batches in order to rule out any
influences of drug product formulation, etc. ("The three tests should also be performed on three
different lots to demonstrate that the test is not affected by within specification variations of different
lots of the formulation"). Although this only relates to the validation of microbial count
determinations, it is a good idea to use it for the validation of sterility test batches.
There are always discussions about whether validations are transferrable to other active substance
concentrations. The European Pharmacopoeia states that validations must be carried out if the
sterility test is performed for a new product or if there is a change in the experimental conditions.
This means that a validation must be carried out for every new drug product and in the event of
changes in the experimental implementation of the test. The only conceivable exception to this is if a
drug product with an identical composition has several different dosages of the active substance. In
this case, the validation of the highest and lowest concentration is transferable to the dosages in
between on the condition that the two validated methods are identical.
Summary
The pharmacopoeia requires sterility for an extensive range of drug products. However, this does not
mean that sterility must be tested. Parametric release is also possible for preparations sterilised in the
final container.
If it is decided that sterility testing is to be carried out, the guidelines in the pharmacopoeia must be
followed. Investigations must be carried out at the same time as the test as well as the pure
performance of the test.
It is important that a validation specific to the substance/drug product is carried out before the sterile
test is carried out.
12.I Testing for tightness and particles

What are the common procedures for testing tightness?

Which procedure is suitable for which container/product?
What are the weaknesses of the different procedures?
What are the state-of-the-art procedures for particle detection?
What is the capacity of the procedures?
Why are certain pre- and post controls essential?
How is it possible to compare and evaluate the procedure and the visual control?
12.I.1 Testing for tightness
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To prevent microorganisms and external atmospheric oxygen from penetrating filled immediate
containers such as ampoules and bottles, these must not have any cracks or fissures. The containers
are examined for these faults by a visual optical control, and faulty containers are rejected. Since this
method cannot guarantee a 100% certainty, various methods are used to test the containers for
tightness.
Depending on the product, the size of the container and the visual or automatic electronic inspection,
different methods have become established.
Dye bath (usually blue) for ampoules and bottles (old procedure, used for small batches or for
solutions with low conductivity)
Water bath for freeze-dried drug products
Steam overlay for oily solutions in ampoules and bottles
Crack test in a high-frequency range for electric current (for solutions with a conductivity >5
S/cm)
Weight difference compared to an average calculated from samples
The test for tightness should ideally be performed as the final step in the operational cycle before the
containers are sorted into transport packaging, since all steps up to this point are associated with a
risk of cracks and fissures.
For tests that involve a dye bath, water bath or steam overlay, this is obviously not possible. The
high-frequency crack test for electric current has therefore become very popular. Regardless of which
method is used to detect a leak, it is important to investigate whether the cause is systematic or a oneoff event. The appropriate measures to prevent cracks in glass objects or leaks in plastic objects must
be complied with (see figure 12.I-1).
Figure 12.I-1 Measures for the prevention of cracks
Measures for the prevention of cracks
Check transport routes in the manufacturing process and queuing and regulatory switches to
ensure that the containers are not compressed
Keep speed differences between two objects to a minimum - avoid impacts between objects
Avoid edges on machine guideways, which can cause pressure points on containers
Insert containers into transport packaging "softly" by hand
12.I.1.1 Testing for tightness using a dye bath

Following steam sterilisation in the chamber autoclave, the dye bath test is carried out in the
chamber (if technically possible) or in a separate, conventional, modified pressure vessel in upright
magazines.
The containers are immersed in the dye bath and subjected to negative pressure. If the container
contains a crack air escapes and, following overlay with positive pressure, the dye solution passes
through the crack into the container. The dyed contents can be recognised by visual control and the
container rejected.
For small numbers of containers, the dye bath is a relatively cheap method and does not require too
much technical apparatus. In contrast, the method has the disadvantages listed in figure 12.I-2.
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Figure 12.I-2 Disadvantages of the dye bath test

Disadvantages of the dye bath test
The dye bath solution must be controlled (dye content), maintained, and later disposed of in
an environmentally appropriate manner. In addition, rinse and cleaning procedures, as well as
detection methods have to be developed that reliably identify any residue.
The crack in the container must be sufficiently large to cause discolouration that can be
identified visually or by photometric methods.
If brown glass is used, the detection of discolouration becomes even more problematic.
The test requires approx. 1 hour if performed in an autoclave.
If performed in a modified pressure vessel, the time required for the evacuation and pressure
phases (approx. 30 mins each) and the loading, removal and space requirements can disrupt
the sequence of operation.
The optical control for discolouration along the longitudinal axis of the container means that a
particle control cannot be performed simultaneously.
Insufficient reliability of detection means that there is a possible risk of microbial or chemical
contamination by the dye solution.
12.I.1.2 Testing for tightness in the water bath (for freeze-dried drug products)
Similar to the dye bath test, the water bath test is performed in a modified pressurised container.
Purified water is used.
Figure 12.I-3 Testing in the water bath
Testing in the water bath
Advantages:
Cheap method with respect to apparatus

required
Reliable visual detection during particle
inspection, since lyophilisates (cakes) become
fluidised or deformed when they come into
contact with water or steam phases.
Reliable automatic detection due to highfrequency crack test, because differences are
clear compared to intact objects.
Disadvantages:
Containers that are initially not filled

and are subjected to freeze drying
may be evaluated as defective and
must be tested.
12.I.1.3 Testing for tightness in steam

Containers that are filled with oily solutions are subjected to evacuation and subsequent vapour phase
in the autoclave, so that steam can penetrate into the container. This results in a visible turbidity of
the oil phase, or in the case of larger cracks, a visible oil/water phase in the bottom of the container.
Both can be detected visually and electronically.
12.I.1.4 High-frequency crack test

The measuring principle is based on measuring current flow when a high-frequency high voltage
(500 Hz, 16-30 kV) is applied to the glass container (ampoules, vials). The current flow measured for
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an intact container is used as a control variable (baseline) for the container. A constant low electrical
discharge between the electrodes and the test object can be detected.
Figure 12.I-4 shows the test set-up. According to Ohm's law, the current I flows as follows: I1 = U /
Z1 + R + Z2
In a leaky container, one of the electrical impedances is decreased, which leads to a higher current
flow (up to a lightning surge) and considerably exceeds the baseline signal.
The difference of current flow between the baseline signal and a higher current flow is represented on
diodes, and is used as a criterion for detection in accordance with Ohm's law I = U/R + Z. Under
constant current, a higher current flows for a defective container.
Figure 12.I-4 Electrical circuit diagram
The principles described above mean that insufficiently conductive liquids (for example, WFI) are
not suitable for this test. One possible workaround is to enrich the cooling water in the autoclave or
the immersion bath with an electrolyte (for example, NaCl), since then the contents of the container
become conductive in the case of leakage.
Sensitivity of the method

The principle of the test also means that each drug product and each test point, such as floor to
middle, and middle to the tip or neck of the container, has to be assigned a specific setting for
sensitivity and high voltage (see figure 12.I-5).
Figure 12.I-5 Structure of the function test
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Over many years of experience and cooperation with pharmaceutical companies, machine
manufacturers have developed electrode position configurations/high voltage values and sensitivity
settings for 1-100 ml glass containers. This takes into account the solution properties such as
conductivity, container surface moisture, and technical properties of the container. Rotation of the
containers during the process under the electrodes is standard practice for identifying any cracks that
may be in geometrically inaccessible positions.
Test objects for function tests

Testing the functional reliability of a crack test machine would require containers with cracks of a
defined size. Since it is not possible to manufacture containers with cracks of a defined size, this was
attempted using ampoule models made of Teflon with inbuilt resistors. This method has proved
largely impractical, since the min/max settings of the test parameters necessarily cause electrical arcs,
which very quickly destroy the test object. It has therefore become common practice to use containers
as test objects (for testing functional reliability before the start of production), which have been
identified as faulty in previous production processes. Containers with cracks that have been
technically induced in the lab (by shocking the hot ampoule tip with water and hence forming microcracks) and have been microscopically evaluated can also be used.
All test objects only have limited suitability:
Run off and drying of the solution on the crack causes the crack to close.
Cracks are enlarged by electrical arcs.
Thin glass areas (tips of balloons on ampoules or point-cut countersinks) may shatter.
This means that a constant supply of containers with faults must be available for use in functional
testing.
Assessment of the method

When testing containers using solutions with a conductivity of considerably more than 5 S/cm, a
reproducibility of >96 % is expected. The method also enables you to simultaneously identify
containers with thin glass areas that are later in danger of breakage during transport.
12.I.1.5 Testing for tightness by weighing

Weighing a large number of visually inspected containers and calculating an average enables the
individual weight of a container to be compared and a calculated plus-minus weight difference can be
used to determine whether there is a significant increase or decrease in total weight. This would
detect any leakage in the container. A more exact inspection of the container is absolutely necessary.
This procedure is only practical in transport or packaging facilities in which weighing equipment can
be integrated into the system.
12.I.2 Particle test

Particle contamination refers to foreign, mobile, undissolved particles that are unintentionally present
in the solution, with the exception of gas bubbles (Pharm Eur.). The test can either be performed
visually, using a semi-automated procedure, or by a validated electronic procedure that would be
regularly controlled.
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Annex 1 of the EU-GMP-Guideline, point 90, requires that: "Filled containers of parenteral products
should be inspected individually for extraneous contamination or other defects. When inspection is
done visually, it should be under suitable and controlled conditions of illumination and background".
The European pharmacopoeia, section 2.9.20 Particle contamination - visible particles (fig. 2.9.20-1),
describes that a vertical mat black plate of a suitable size should be aligned next to a suitably sized
vertical non-reflective white plate. A source of white light should be used that generates 2,000 and
3,750 Lux. After the content of the container has been lightly whirled, the container should be turned
so that no air bubbles remain in the liquid. The liquid is then inspected for 5 seconds in front of the
white plate. The inspection is repeated in front of the black plate. Any presence of particles is
recorded.
To satisfy the requirement for inspection for "other" defects, the use of magnifying glasses (1.5x)
considerably improves the detection of cracks, external impairments and damage to seals or tips (of
ampoules). This also applies for the inspection of the external shape of the container, such as length,
diameter, colour, defective material (inclusions in the glass, misshapes from glass forming at the
manufacturer). This also enables particles to be detected more easily.
In order to be able to detect particles in a solution, the particle must visibly stand out from the
solution, or at least, the interface between the solution and the particle must be visible under light.
This requires an adequate light source and a coloured background on the object to be inspected. It
must be possible to comply with the normal viewing distance of approx. 25 cm.
Particles and defects are identified by:
Different colours
Different transparency
Solid shapes
Varying density and hence light reflection at the interfaces
Varying density and hence light penetration
Cosmetic, external faults on the container.
This results in different options for visually and electronically recognising particles.
12.I.2.1 Visual inspection

An important task of visual inspection is to identify the type and material of the particle from its
visual impression, and to identify the cause and place of contamination (see figure 12.I-6).
Figure 12.I-6 Particles and their causes
Type and material of the particle
Cause and place of contamination
Metal, ceramic, glass
Pump abrasion
Rubber particles
Seals of filter housing
Fibres, particles from environment and improper handling Filter assembly

Adhesive particles of stopper material and fibres from the Rubber stoppers
environment
Semi-melted fibres and particles and glass chips from
glass manufacturer
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Containers
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Particles from conveyors for stoppers and sealing

components
Filling machine
Glass splinters
From damage and breakage on the filling

machine
Sintered or cracked ingredients of the solution,

crystallisation
Sealing processing step
A visual assessment and classification of the particle is not always sufficient, in which case an
analysis of the isolated particle must be carried out. It has been shown that with sufficient experience
and exact knowledge of the production steps, the particles can be classified and assigned by the visual
identification alone. Material inspection is only required in rare cases. In general, staff require
extensive training before they are able to identify particles, if these reach a size of approx. 80 mm. It
is important to take into account any magnification caused by the shape and diameter of the
container. During the training program, the control employee should be introduced to all recognised
types of fault.
Figure 12.I-7 Frequency of particles in solutions
Frequency of particles in solutions
Ampoules
High
Fibres
Glass splinters
Black particles
Brown particles
Colourless particles
Suspended particles
(amorphous)
Glass bottles
Low
Fibres
Glass splinters
Particles of stopper
material
Coloured particles
Metallic shiny
particles
Suspended particles
(amorphous)
Plastic containers (clear)
Fibres
Colourless particles
Brown-ish particles
Metallic shiny
particles
Coloured particles
Suspended particles
(amorphous)
In all containers, the dosage (fill level) and the presence of cracks should also be checked at the same
time, and any conspicuousness evaluated as a type of fault.
The visual inspection is reliant on staff management, expectations, clear instructions and controlled
handling:
Handle container with as little movement as possible - avoid shaking

Do not begin to turn the object in the hand until the start of the observation in the visual field any "small particles" deposited on the floor of the container (in some cases below the
visibility level) should be detected.
Stick to the observation system from bottom to top of the container.
Good training and constant visual inspection activity should lead to an "automated" (almost
subconscious) ability of the member of staff to reject faulty objects. The member of staff should be
made aware of their responsibility by emphasising the importance of the visual inspection as the last
possibility for detecting faults. This motivation is particularly important if fewer faulty objects occur
and a sense of doubt ("there are no faults here anyway. ...") leads to negligence. A failure rate of
<0.5% should be aimed for as a good production result.
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Visual optical controls are based on the laws of probability. If a presence (detection) or absence (nondetection) is possible, this is a chance result. The frequency of an event (e.g. detection), for example
"70x detected in 100 observations", is expressed as a fraction 70/100 = 0.7. This is described as the
relative frequency of the event. With a sufficient number of observations, the relative frequency can
be expressed as a numerical value for the probability (P, statistical probability).
12.I.2.2 Visual control with semi-automated equipment

Bottles
These are control machines in which the containers are fed individually via a conveyor belt. The
container is held in mechanical supports, and in front of the observer, is turned upside down and
rotated around its own axis at least once. The characteristics of a particle can be observed (reflection,
flotation, falling speed, colour, etc.).
Within the required 5 seconds observation time, the solution contents are checked for particles and
the external integrity of the container is checked. Objects classified as good are subsequently
subjected to tightness weighing or a high-frequency crack test.
Ampoules/vials
The ampoules/vials are loaded from their delivery containers into a loading receptacle (with glass
bottom) of the machine. The objects undergo a preliminary inspection from underneath through the
glass bottom. The ampoules are checked to ensure that the lance part is the correct shape and that no
impurities have resulted from the tip sealing process. Vials are checked to ensure that the crimping
cap is the correct shape and that the cap is the correct colour.
The machine mechanics automatically load the containers (4 to 6 at a time) into a rotation mechanism
and turn them. When the rotation stops, the control employee inspects the containers. The solution in
the container continues to rotate and any particles that are also rotating can be identified. Defective
containers can be rejected at the touch of a button.
For containers containing powders or lyophilisates, the rotation speed must be regulated (1-2 x per
observation time) so that faults (such as black particles, glass splinters) are not "washed away", but
instead are made clear.
12.I.2.3 Electronic control for visible particles

There are three main electronic control procedures in use that have been developed by different
machine manufacturers:
Rotation of the container and measurement of the shadow of moving particles using
illumination once they have stopped moving.
Rotation of the container and measurement of the reflection of light scattering by cameras,
once the particles have stopped moving.
Camera systems for image comparison against an imported ideal image of the container.
In principle, all electronic procedures perform a 2-station and/or multiple control (measurement).
The advantage of all three procedures is the elimination of human error such as:
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Decreasing visual acuity

Decreasing stamina
Temporary alertness
Physical and psychological changes throughout the day
General daily form throughout the week
and a possible higher inspection ability and sensitivity settings for different particle sizes.
The disadvantages are:
High purchase price

Complex validation activities and specific long-term technical support
Relatively high mistaken evaluation as poor due to misinterpretation of physical states that are
thought to be particles
Impossibility of detecting certain faults that are outside the detection spectrum (preliminary
inspection of tips or subsequent control necessary)
In addition, none of the three systems is capable of interpreting faults. These must always be
classified by personnel after the control.
Figure 12.I-8 Electronic control for visible particles
Electronic control for visible particles
Advantages:
Disadvantages:
Shadow formation
All particles (colourless,

smooth, metallic, amorphous)
form a shadow.
Air bubbles do not form a
shadow.
A preliminary inspection for external faults is

required.
Only solutions that do not have any stria (from
concentration differences in the solution of the
container caused by sterilisation) can be inspected.
Solutions must be homogenised before inspection
(pre-rotation stations or shaking equipment), if they
contain >10% solid components.
Particles floating in the meniscus are not detected.
Reflection measurement
Concentration differences
(stria) in the solution do not
matter.
All light-reflecting particles
are detected.
Inspection for external faults is required.

The reflection caused by air bubbles must be
suppressed by polarisation filters.
Black/coloured, non-smooth particles are not detected
as they have no or only weak reflection.
Particles rotating in the meniscus are not detected.
Ideal image comparison using cameras
Technical measurement of
shape
Reflections appear as crack or
breakage
Comparison of the ideal
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A good external cleanliness grade must be achieved

(preliminary washing)
Each image deviation leads to rejection (high
proportion of faultless objects rejected).
Page 115 of 140
solution image to the real

image
Figure 12.I-8 compares the three methods against each other.

When a visible or detected fault occurs, the container should be separated and added to the rejects.
The type of fault and number of affected objects must be documented in the manufacturing
instructions.
Evaluation of the inspection procedure

Since the pharmacopoeias stipulate the requirement "free from visible particles", the aim is to achieve
at least the same level of effectiveness in electronic control as in the visual control. This means that
an evaluation method is required for comparing the automated inspection against the visual
inspection. Electronic controls are set up with the effectiveness of the visual inspection as a
benchmark.
Evaluation methods for the inspection ability of personnel

The visual inspection is subject to the regulations in the applicable SOP, for example, in terms of
illumination, background and observation time. The effectiveness of the member of staff must be
determined based on the detection of a particle in solution according to the laws of probability. This
can be established by mixing test objects (if possible containing all potential faults, for example see
figure 12.I-6), which are found by experienced members of staff, at up to 10 to 20% within several
hundred to thousands of numbered objects of the same container size. This sample is controlled by
seven to ten experienced members of staff ten times. The frequency with which an object is detected
is assigned to the object as a quality number (QN) from 0-1.0. This means that an object with a QN =
0.7 is detected as faulty 7 times in ten controls. Objects classified in this way are divided into three
areas based on their quality number.
The effectiveness of the control is determined using the objects from the reject zone QN 0.7 - 1.0.
The percentage of these objects found by members of staff or in electronic procedures is specified.
Figure 12.I-9 Object classification based on quality number
Object classification based on quality number
QN = 0 - 0.3
No faults
(accept zone)
QN = 0.4 - 0.6
Acceptable
(grey zone)
QN = 0.7 - 1.0
Faulty
(reject zone) RZ
Determining effectiveness (RZE)

When executed in practice, however, several hundred objects with the QN 0-0.6 should be used, in
which 20% with a QN of 0.7-1.0 are distributed, in order to achieve a random distribution in the
sample and to prevent the "memory" effect of control personnel.
For each QN range, replacement objects should be kept on standby in order to keep the sample range
within the evaluation range even if an object is lost (for example, broken). The sample should be
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assessed by several members of staff at regular intervals in order to evaluate the QN of the individual
objects. The QN of individual objects can decrease, for example, to < 0.7 due to the breakdown of
particles over time, or can increase to > 0.7 due to the release of particles originally adhering to the
glass wall of the container. When the QN is confirmed, a new expiration date should be determined.
Figure 12.I-10 Determining effectiveness (RZE)
Determining effectiveness (RZE)
This example is based on three objects (for example, ampoules) with a QN of 0.7-1.0 (faulty, reject
zone RZ)
The objects are mixed into a batch (containing at least 5 times as many objects) of objects with QN
0-0.6 (no faults to acceptable, accept and grey zone).
In the subsequent 10x control, each object can theoretically be detected a maximum of 10 times
(corresponding to RZN 10), which means a total RZN of 30 for all three objects.
In practice, however, the three objects are detected as faulty (RZR) 7 times, 8 times and 9 times,
which amounts to a total of RZR 24 (Reject Zone Reject).
Therefore:
RZE = RZR/RZN
RZE = 24/30 x 100
RZE = 80%
(The member of staff has been proven capable of detecting faulty objects with a QN of 0.7-1.0 with a
probability of 80%).
Aptitude test for staff

Members of staff entrusted with visual inspection activities should achieve a reject zone efficacy
(RZE) of > 70%. If this value is not achieved, this person is not permitted to carry out any visual
control activities. The test should be repeated every two years, just like an eyesight test at the
ophthalmologists.
12.I.3 Sequence of operation

After sterilisation, the containers of a sterilisation batch are submitted to the Optical Control (several
sterilisation batches may result from one filling batch), clearly separated and with the prescribed
labelling (product name, batch, colour indicator proof, date, time and no. of items), with all signatures
and recorder charts in accordance with the manufacturing instructions. There the documents and
objects are checked for completeness. The primary aim is to avoid mistakes and mix-ups.
Figure 12.I-11 SOP for execution of visual control
SOP for execution of visual optical control
Author:
Checker:
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QA approval:
Released by head of production:
Signatures: ........................................... Valid from:...................
Table of contents:
1. Aim/Purpose
2. Responsibilities
3. Scope
5. Documentation
1. Aim/Purpose
This SOP describes the operation for the visual optical control of containers by appropriate and
trained staff.
2. Responsibilities
The procedure described in the SOP is the responsibility of suitable members of staff (positive
aptitude test) following instruction, as well as the departmental and company management.
3. Scope
Organisational unit: XYZ and subunit: xyz
Location of manual or semi-automated work stations

Cleanliness grade
Media (electricity supply, purified water)
Process
Receipt (in the appropriate sequence according to the manufacturing steps from start to end of
the batch, in the order of sterilisation batches) of the production batch by the responsible
member of staff and check for conformance with the drug product name, batch number, type
of container, article number, sterilisation batch number, number of items, no. of transport
containers (pallets, magazines, boxes, etc.), number of labels text, and indicator turnover.
Assignment of the batch for checking to particular work stations (type, station number).
Product name, batch, article number, fill date entered in the work station log book.
Occupation time of the work station (date from ......... to.............)
Number of controlled containers, number:......Good:.......Poor:..............
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Sample withdrawal:..............
Type of loading in transport container for packaging: ............. Number:..............
Name of members of staff involved:.................
Type of reject:......(glass, fibre, particles, etc.) Item:.......... Percent:...........
Notes/special features:...................
Start-up/start of illumination by switching on the electricity

Labelling of the work station with drug product, batch and date
Control of first objects and sample withdrawal for chemical analysis for release
Sampling of the first sterilisation batch in accordance with SOP QA in random distribution
during visual control
Observation in accordance to the description of visual control
(see chapter 12.I.2 Particle test)
Test for tightness (see chapter 12.I.1 Testing for tightness):
Number:..........Good:.............Poor:......................
5. Documentation
Control and signature of executing member of staff (doer)

Evaluation of figures in accordance with SOP XYZ by the tester (checker)
Entry of results into batch production record

For example, yield calculations, SOP XYZ
Staff test, SOP XYZ
Container cleaning in washing machines, SOP XYZ
Monitoring of quality of visual optical control, SOP XYZ
In the first step, containers are removed as samples for release analytics. In accordance with the
visual control procedure specified in the manufacturing instructions for the product, the processing
step is preceded by the identification and labelling of machines and workplaces. Entries are made in
the log book and checklists for operational tests are worked through. After the control equipment is
released (for example, confirming the absence of extraneous objects), and the designation on the
delivery container (closed pallets, transport carts, magazines, etc.) is checked, the containers are
forwarded to the visual control system strictly in the sequence of filling. During this process, samples
are taken for the sterility test in accordance with the sterilisation batch (biological batch). All samples
taken must be documented in the batch production record. Figure 12.I-11 shows an example of an
SOP for executing visual optical control.
During the control

When faulty containers are identified, these are rejected, counted, and classified by type of fault, or
further analysed. Faulty containers (particles, dosage, external faults, etc.) should be aligned in
chronological order of filling and filling personnel should be informed immediately in order to
introduce appropriate measures as soon as possible. After the tightness test and the inclusion of
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visually controlled objects into the transport packaging, these are labelled (product name, batch, date,
no. of items, sequence no., etc.). The numbers of items are entered as yield and rejects in the batch
production record. After evaluation of the yield (target range/deviation/discrepancies), the batch
production record is completed by the signature of the head of production.
Monitoring the quality of Optical Controls

At regular intervals, randomised samples are taken from electronically or visually controlled
containers of a batch and reviewed by independent, suitably qualified staff.
Summary
Tightness testing is designed to detect cracks or leaky seals. Methods available include a dye bath,
water bath (for freeze-dried products), and the high-frequency crack test (for conductive liquids).
An optical control method or a combination of methods must be chosen for each product from the
procedures described for particle control, based on the physical reasons for detecting particles
electronically or visually. The weaknesses of each method in the detection of particular particles must
be taken into account before manufacturing begins. Appropriate practical measures must be taken to
prevent this type of error from occurring.
Due to the highly specific nature of requirements, the qualification and validation of electronic
particle controls in the container requires very close cooperation with machine manufacturers.
12.J Freeze drying

How is freeze drying performed?

What accompanying process parameters have to be measured?
What are the quality-determining physical principles/processes?
Where must the technical requirements of a freeze-drying system be described?
The process of freeze drying can be used to very gently obtain dry substances from aqueous
solutions. Freeze drying is primarily used to preserve the stability of sensitive substances such as
hormones, antibiotics, enyzmes, protein preparations, blood preparations, serum, vaccines, etc. Many
medicinal substances decompose in water over time. In order to keep them sterile and free of
particles, they are dissolved and sterile-filtered. Containers filled under aseptic conditions are
subjected to a freeze drying procedure, whereby the water is extracted (dried) from the frozen
solution. This results in a stable, sterile powder or solid "cake". The containers are then aseptically
sealed, for example ampoules undergo tip sealing at the sealing station of an ampoule filling machine,
or injection vials are sealed with freeze-drying stoppers in the freeze dryer. The resulting readily
soluble powder (lyophilisate) is not dissolved in sterilised water for injection until immediately
before the medicinal product is used. The solution formed can then be injected.
12.J.1 Description of the procedure

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The characteristic feature of freeze drying is that the water is removed from a frozen drug product
without defrosting, i.e. through sublimation.
The usual liquid/gaseous phase transition that takes place in a conventional drying process is replaced
by the phase transitions liquid/solid (freezing) and solid/gaseous (otherwise known as sublimation or
freeze drying).
After freeze drying, the original properties of a drug product are retained in an optimal way. Freeze
drying is the method of choice for the preservation of sensitive substances, particularly if they are of
biological origin.
Execution
First, the solution is frozen in the container (ampoule, bottle) and is then subjected to a vacuum,
taking care to ensure that no defrosting occurs. In the vacuum, steam molecules escape from the
frozen, aqueous substance until a partial pressure of steam corresponding to its temperature (steam
saturation pressure) develops above the frozen substance (see figure 12.J-1).
Figure 12.J-1 Steam saturation pressure (source: Leybold Ltd.)
For drying, appropriate pumps (vacuum pumps) are used to constantly extract condensable gases and
steam using the "steam pump" (condenser). Steam molecules escape from the frozen product
sublimation until the product is dry (minimum residual water content). After freeze drying, the
ampoules must therefore be sealed in a room under adjusted dry conditions (< 20% rel. humidity).
Bottles enter the freeze dryer with a special freeze-drying stopper already in place, and following
drying, are sealed in the facility by pressing in the stopper.
The method of freezing and the cooling temperature are determined by the properties of the product,
the size and shape of the containers, and the most important factor of the eutectic temperature
(solidification temperature of solutions). This temperature also affects the subsequent appearance of
the "cake".
The freezing temperature must be adjusted to suit the product - for example, it can be -15 C for
one product or -50 C for another product. Systems should be specifically selected for this reason, or
the freezing process should be performed outside the freeze drying system in special freezing
facilities (such as a rotation method). Different methods of freezing are shown in figure 12.J-2.
Figure 12.J-2 Overview of the major methods of freezing
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Layers of the drug product should not be higher than 2 cm, in order not to inhibit the transport of
steam through the product. The ideal temperature range for freeze drying is -20 C to -40 C, since
small ice crystals are formed in this range, and hence the steam is quickly transported through the
drying product during the drying process. It should be noticed that at low substance concentrations no
"cakes" of uniform appearance can be formed in the solution to be dried. For caking to be achieved,
the following measures are possible:
Add "additives" such as dextrin to form a matrix on which the drug substance can be evenly
distributed. This point has to be considered at the development stage of the drug product.
Increase the concentration of solid matter content, so that the "cake" is not too severely
deformed on drying (breakages, clumping), and to ensure it has a uniform appearance after
drying.
Select the container according to the ratio of wet surface/volume of solution/solid matter
content. This is also a task for the development phase of the drug product (figure 12.J-3).
Figure 12.J-3 Temperature-time course of freeze drying
The duration of freeze drying depends on the temperature in the ice-containing part of the drug
product - the higher this temperature, the faster the process. It is therefore of utmost importance to
control the temperature in the ice-containing part of the drug product to be dried. The ice nucleus of
the drug product must be prevented from thawing during heating at all costs. Monitoring the
electrical conductance in the frozen product is used to help regulate the system. When the frozen
solution is warmed up, there is a sharp decrease in electrical resistance when the eutectic temperature
is reached, which is interpreted as a sign that the ice in the drug product is beginning to melt. The
measurement of electrical resistance can therefore provide information about the behaviour of the
drug product during the freezing and drying process. The drying process is described in figure 12.J-4.
Figure 12.J-4 Drying process
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12.J.1.1 System components

Freeze-drying chamber
The freeze-drying chamber is where the drug product is placed, and consists of trays that can be
cooled or heated. Multiple trays can be aligned, for example, over each other, and can be moved
together using a hydraulic mechanism to close bottles with freeze-drying stoppers. Freeze dryers are
supplied by many manufacturers in a wide variety of designs to accommodate the diversity of
products and containers used figure 12.J-5).
Figure 12.J-5 Principles of a freeze dryer
The cooling and heating system, using silicon oil as a heat transfer medium, must enable even
changes of temperature across the trays and continuous temperature regulation. The low temperature
level of the trays during freezing is determined by the requirements of the drug product. The distance
between the trays depends on the size of the containers for the drug product. The total tray surface
area, the maximum loading with containers and the maximum thickness of layer of the solution to be
dried result in the basic capacity for the amount of ice collecting in the steam pump (condenser).
Steam pump (condenser, cryopump)

The action of the steam pump is based on a surface area of very low temperature (lower than the
temperature in the drug product) on which the steam condenses and becomes ice, and thus occupies a
very small space compared to its gas volume. The steam, which, according to gas laws, will spread to
occupy the entire remaining space (chamber and condenser), is then extracted from the chamber
space. This means that on condensation, the gas proportion becomes reduced, and the gas is
transported from the chamber to the condenser. This is only possible, however, if obstacles caused by
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molecule collisions with water molecules are prevented, by the extraction of non-condensable gases
by vacuum pumps in the initial phase.
The construction of the condenser is a very important factor for its effectiveness. Its lowest possible
end temperature determines the partial pressure of steam that the "steam pump" can achieve. The
intermediate valve of the chamber/condenser transition must have a large transition diameter, and
must be designed to deal with the large volumes of gas. At minus 40 C, ten kilograms of ice
represents a gas volume of approx. 100,000 m3, which has to pass through the narrow transition to the
condenser within a few hours. The condensation heat produced during condensation is equivalent to
the amount of heat passed to the frozen drug product during freeze drying by the heating of the trays
to compensate for the sublimation coldness.
Since most drug products have a very low residual moisture, the end temperature of the ice condenser
must therefore be particularly low (see figure 12.J-1).
To pump the last remaining steam (usually a very small amount) from the drug product, vacuum
pumps are used that have an end vacuum of up to approx. 10-5 mbar. The interaction between the
steam pump and the vacuum pumps for pressure regulation is controlled by programmable automatic
programs. It must also be possible to control the freeze dryer manually, although this is not ideal as it
consumes energy and time, and is personnel-intensive, since control points must be monitored every
two hours.
Ideally, the pressure in the drying chamber should be set at a level so that the heat transfer from
convection caused by the molecules in the drying chamber contributes significantly to heat transport.
When selecting a system, it is also important to consider the maximum condenser capacity
(condenser capacity per 24 h, or total ice capacity before thawing).
Measurements
For the optimum freeze drying process for a particular drug product, you need the following
information:
The required temperature in the ice nucleus of the drug product

The maximum temperature in the layer that is already dry
The approximate residual moisture at which the drying process is considered complete (taking
into account the desorption isotherm of the drug product)
Pressure measurement (chamber/condenser).
This results in the following measuring elements:
Heat sensors
At least two product sensors (thermocouples)
Condenser temperature sensor (Pt 100)
Sterilisation temperature sensor (Pt 100)
Tray temperature sensor (Pt 100)
Conductivity measuring point
Pressure measuring equipment
Vacuum measuring tube (principle of heat transfer, available from several manufacturers)
12.J.2 Qualification of a freeze dryer

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The use of a freeze dryer must guarantee that any aseptically filled objects handled in the system
achieve the required result - namely the gentle removal of water content (except for a defined
permitted residual moisture) - without microbiological contamination. This requirement is ensured
through the technical design and qualification.
The following points need to be considered in the qualification of a freeze dryer:
Materials
Pipe connections
Electrical components
MSR components
Function
Monitoring
Documentation
Technical sequence of loading and unloading
Transport from the filling machine to the freeze dryer in accordance with the cleanliness
grade
Uniform temperature transmission and thus freeze drying conditions throughout the contact
areas of container/transport vessel/tray.
12.J.2.1 Installation qualification (IQ)

The execution of the tests is described in detail to ensure that the same tests can be accurately
replicated in the future. Measured values must be recorded in the test record and documents
(printouts, diagrams, etc.) must be included as attachments. Figure 12.J-6 shows an example of some
test points.
Figure 12.J-6 Tests in installation qualification
Tests in installation qualification
Introduce the freeze dryer into cleanliness grade E, D or C

Sealing of the freeze dryer towards cleanliness grade B
Pipe connections for steam, nitrogen, compressed air, hot and cold water
Connection for floor drainage
Electrical connections
Functional control of the mechanical components (valves, vacuum system, etc.)

The scope of the IQ includes defining measuring points, the type of measurement, operating range,
adjustment points, calibration intervals and levels of accuracy. The quality-relevant measuring points
of freeze dryers are as follows:
Temperature (condenser, trays, product)

Conductivity
Time
Pressure (vacuum, excess atmospheric pressure)
Measured calibration values must be compared with reference systems from a higher class. If
measured values are outside the acceptable measured value tolerance, the measurement chain must be
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adjusted and recalibrated (see chapter 4.G Calibration). The operational qualification cannot take
place until a successful initial calibration has been performed.
Heat sensors
Pressure and vacuum resistant control heat sensors are used, which must be suitable for use in the
fine vacuum phase and resistant to steam sterilisation pressures. The feeds are connected to a
measuring point recorder of the appropriate quality grade. The thermocouples should be calibrated at
0 C.
Vacuum measurement/pressure
Measuring tubes for fine vacuum measurement in the range 10-3 mbar (hPa) according to the
principle of heat transfer are recommended by manufacturers for pressure regulation in different
variations and measuring ranges. These can be used to indirectly determine the product temperature
according to the steam-pressure curve over ice. The calibration should be performed in several steps
from <10-4 mbar to 1000 mbar. The excess pressure in the sterilisation phase (which should also be
considered as a cleaning step) with super-heated saturated steam is measured using pressure
transmitters, which are tested against a control device at pressures of 1.1 and 2.1 bar (abs). As this is
a process for sterilising surfaces that do not come into contact with the product, the temperature
should only be measured in a few points (coldest spot - condense water drainage, chamber
condenser).
12.J.2.2 Operational qualification (OQ)

In the operational qualification, all the operating states of the freeze dryer are simulated to their limits
(worst case), and the results are documented. Critical test points are listed in figure 12.J-7.
Figure 12.J-7 Test points in operational qualification (extract)
Is the intended end vacuum of the vacuum pumps reached?

Is the intended heat transmitter temperature achieved by the cooling machine?
Is the freezing temperature automatically regulated within the required limits?
If the pressure is higher than the specified range, is the product cooled, and if the pressure is
lower, is the heating started?
If the pressures and temperatures are outside the specified range, are they automatically
displayed as errors and corrected?
Does the thaw water drain away completely in the condenser?
Are the mechanical components functioning correctly? (chamber seal, hydraulic mechanism
of the stopper sealing system, intermediate valve, level switching of the cooling aggregates,
etc.)
Is the whole system tight and is the final vacuum reached at the end temperature of the
condenser? (approx. -85 C for two-phase cooling technology)?
The test to check the uniform heat distribution on the plates and heat transfer to the objects is
particularly important for the formation and evenness of the "cakes" in all containers. This test also
proves that all objects are subject to the same temperatures in the freezing and drying phases. Suitable
substances for test runs are, for example, dextran or mannitol (5 %) (figure 12.J-8).
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Figure 12.J-8 Function tests

Function tests
Recorder feed of the external device
Check that the freeze drying time

measurement is functioning
correctly.
Check paper feed with markings on the recorder paper

and time measurement
Documentation: Markings on the recorder chart with
time measurement
Recorders integrated in the freeze dryer are assigned
markings on the recorder paper and tested by time
recording.
Maximum deviation: 1 mm/h
Documentation: Markings on the recorder chart and
time measurement
0 C comparison
Heat sensor comparison at 0.1 C
Tightness test
Evacuate the system to 10-3 mbar with the

corresponding configuration of vacuum pumps.
After approx. ten hours, increase the pressure from 10-2
to no more than 10-1 mbar.
Media quality
During qualification, the quality of the pure steam and the nitrogen used to ventilate the freeze dryer
is also tested. The warm potable water used for thawing the condenser is also analysed. The data from
these investigations can be used, as long as current values are available from monitoring or are
determined during process validation of the freeze drying process (see chapter 12.J.3 Validation of
the freeze drying process).
Before entering the system, the steam is filtered by particle filters with a pore size of 13 m and
0.2 m, warm water (for thawing) is filtered using a pore size of 8 m and 0.2 m. The system is
ventilated via a drying agent (silica gel) with a vent filter (0.2 m pore size) in class B. This filter is
subsequently subjected to an integrity test.
Once the qualification is successfully completed, the qualification report is compiled and approved
(see chapter 6.C.3 Qualification report).
12.J.3 Validation of the freeze drying process

The validation protocol describes the structure, monitoring, and documentation of the validation
activities (see chapter 7.H Validation protocol and report). Figure 12.J-9 shows an extract from a
validation protocol for freeze drying. The following is an example for the description of devices and
process and for the execution of validation.
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12.J.3.1 Description of equipment and process

The freeze dryer supplied by XYZ is installed in building 000 in cleanliness grade XY. In freeze
dryers for aseptically processed solutions, the loading openings of the drying chamber are in
cleanliness grade B. The rest of the machine is located in a lower grade, which facilitates technical
monitoring.
Figure 12.J-9 Extract from a validation protocol
Responsibilities
Person responsible for validation
Head of Production, Head of Quality Control
Validation manager
Project manager
Validation team
Staff from the unit (users)

QA staff
Device: Freeze dryer, manufactured by XYZ, no. 12345...... Building 000

Process parameter freezing temperature: -20 C to - 40 C
Tray temperature: -40 C
Freezing time: 6 hours
Condenser temperature: -50 C to - 85 C
Pressure regulation (drying phase), depending on the drug product: 10-1 to 10-2 mbar
Temperature increase: 3 C/h (main drying)
Product target temperature 0 C: Temperature maintenance until sol reflux from tray, constant
0 C
Begin final drying: Set pressure range to <10-2, product temperature to final temperature
(depending on the drug product, +10 to +35 C) and maintain for between 4 and X hours.
Pressure increase measurement: Pressure increase close to 0.
End of drying: Ventilation via sterilised membrane filter, pore size 0.2 m.
The freeze drying process is carried out in accordance with a company SOP. The general structure of
this type of SOP is described in figure 12.F-12.
The freeze drying process is executed using pressure and temperature regulation and is recorded
using multichannel recorders. More detailed technical information is available in the qualification
documentation (electrotechnical descriptions of the control loops).
The three validation runs for each loading configuration and drug-product-specific requirements
(eutectic temperature, permitted residual moisture, appearance of the lyophilisate) follow the defined
sequence of operation as described in figure 12.J-10.
Figure 12.J-10 Sequence of operation
Program selection
Automatic/manual start
Freezing
Cool trays (approx. -40 C)
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Load trays
Close system
Main drying
Condenser cooling (approx. -50 C)

Switch on vacuum pump
Open the intermediate valve
Pressure range regulation
Tray temperature regulation
Tray temperature>0 C
Final drying
Pressure range regulation

Tray temperature regulation up to the set end temperature
> +10 C to .............
Pressure increase
measurement
Pressure increase in the chamber after shutting off the ice condenser using
the intermediate valve.
Target value: close to 0
Ventilation
Final ventilation using drying agent via sterilised membrane filter with a
pore size of 0.2 m.
12.J.3.2 Executing the validation

The scope of the validation includes checking all loading configurations that are possible in routine
operation according to the specified construction of the machine. In particular, the intensive contact
with the cooling surfaces (trays) is tested at all points in order to ensure even freezing and hence
formation of uniform "ice bodies" in each container.
The temperature differentials of the cooling and heating medium at the entrance to and exit from the
trays should be minimal. This is achieved by uniform loading of the trays and sufficient flow of
medium.
The low temperature to be reached by the trays must be adapted according to the requirements of the
drug product. The electrical resistance in the drug product should be recorded as a measure of
crystallisation or recrystallisation processes, and incorporated in the operating program as a control
variable.
Switch on the register and control devices

Set the working pressure, for example to 0.2 Torr (2.7 x 10-1 mbar) for a product temperature
of approx. - 25 C. This corresponds to a steam saturation pressure of 0.5 Torr (7 x 101
mbar).
Start the circulation of the heat transfer medium and temperature controller (target values are
taken from the program control for heating and cooling).
Condenser cooling (program control)
Vacuum pump/s (program control)
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Vacuum control via valve regulation at switching points specified in the program. Pump valve
closes = Pressure increases until it reaches the upper switching point -> Pump valve opens =
Pressure decreases to the lower switching point = Pump valve closes, etc.
View the objects visible in the observation window to check the appearance of the
lyophilisate.
Maintain time schedules and temperature increase steps (deviations from temperature and
pressure courses due to technical faults are detected by the program control and product
protection is initiated by tray cooling that starts automatically).
Pressure increase is measured once the end temperature is reached.
System is ventilated.
Determination of water content of the freeze-dried drug product

Time-temperature-pressure protocol of the sensors installed in the freeze dryer.
Evaluation of the time - pressure - temperature course data in accordance with company SOP,
and checking of the recorder charts.
To confirm the aseptic working conditions, after the containers are filled with nutrition agar, these
are placed in the freeze dryer. All processes such as transport to the freeze dryer, placement in the
freeze-drying chamber, withdrawal following a short standing time, are executed, except for freezing.
The system is evacuated at room temperature to approx. 100 mbar (to simulate gas transport). The
system is then ventilated via a 0.2 m membrane filter, the removed objects are sealed, and incubated
as normal.
The system is cleaned by wiping the surfaces of the freeze-drying chamber with disinfectant and
subsequent sterilisation of the chamber and the condenser in accordance with a company SOP.
The results of the tests must be compiled in a validation report. After successful execution and
positive assessment of all results, the freeze dryer can be released.
Summary:
In freeze drying, products that are unstable in water are frozen and water is extracted from them by
means of sublimation in a vacuum, until a specified product-specific residual moisture is reached.
Because the products are usually sterile products that cannot be subjected to heat sterilisation, this
procedure must be carried out under aseptic conditions. It is important to ensure that a uniform cake
is formed that can easily be re-dissolved.
The freeze-drying process is controlled and monitored by measuring the temperature, conductivity,
time and pressure using calibrated measuring devices. In validation, the individual phases, from
freezing, drying and pressure increase, through to ventilation, are inspected. The aseptic conditions
are confirmed by a simulation of the process (without freezing).
12.K Dry Heat Sterilisation

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Which requirements are sterilisation tunnels expected to meet?

Which factors determine the temperature range of a sterilisation tunnel?
How can the process be evaluated?
Which parameters must be documented using the system measuring devices?
How is a depyrogenation process assessed?
Since the 1970s, an increasing number of compact lines has been developed and used for the mass
production of ampoules and bottles in the pharmaceutical industry. These consist of a combination of
washing machine/sterilisation tunnel/filling machine in continual operation. In the past, most
sterilisation tunnels were tunnels with infrared quartz tubes. In these devices, the temperature was
regulated by switching the individual quartz tubes on and off, which caused particles to be emitted on
the tubes and on the internal surfaces of the tunnel. This also meant that the air flow in the tunnel was
no longer stable, which has a negative effect on the pressure conditions at the transition to the filling
equipment's cleanliness grade. This was one reason why these devices were superseded by laminar
flow hot air tunnels, which are equipped with terminal particle filters in the hot air and offer an
improved, controlled air flow pattern. Laminar flow units also offer secure connections at the
entrance and exit of the conveyor belt.
The requirements of the pharmacopoeias for the parameters time/temperature are shown in figure
12.K-1.
Figure 12.K-1 Pharmacopoeia requirements for dry heat sterilisation
Pharmacopoeia requirements for dry heat sterilisation
USP (USA) 250 C 15 C and 3-log reduction of USP units of bacterial endotoxin
JP (Japan)
160-170 C (120 mins),

170-180 C (60 mins) and
180-190 C (30 mins)
EP
(Europe)
At least 160 C for two hours

Evidence of the SAL of 10 -6 for the sterilisation and for 3-log reduction of endotoxins
at > 220 C
It must be possible to maintain these conditions for any chosen length of time. To meet these
requirements, sterilisation tunnels are used. These transport the objects on speed-regulated conveyor
belts through the hot air zone and thus fulfil the temperature/time conditions of the empty glass
container. The phases of dry heat sterilisation in the sterilisation tunnel are listed in figure 12.K-2.
Figure 12.K-2 Phases of dry heat sterilisation in the sterilisation tunnel
Phases of dry heat sterilisation in the sterilisation tunnel
Conveying (or manual placement) of the washed containers with a known residual moisture
into the "infeed LF".
Transport in the hot air LF of the sterilisation tunnel at the fixed maximum transport speed of
the conveyor belt (results in the minimum sterilisation time in the hot air part).
Entry into the LF cool air stream and cooling of the objects to under 30 C.
Transition to the LF of the filling machine (for example, cleanliness grade C, grade B, grade
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A).
The sterilisation process in a sterilisation tunnel can be described using a time-temperature curve (see
figure 12.K-3).
Figure 12.K-3 Temperature course in the hot air part
12.K.1 Description of the procedure

The hot air flow must come into contact with the inside and outside of the empty glass containers to
be sterilised (ampoules, bottles), or the objects must be brought to the necessary temperature for
sterilisation and depyrogenation within the required time by the radiating heat from neighbouring
objects. The sterilisation time/depyrogenation time does not begin until the defined temperature is
reched within the container. In closed containers, it can take a long time to achieve the required
internal temperatures (often several hours). Hot air sterilisation tunnels are usually not suitable for
this type of object. In this case, sterilisation or depyrogenation is performed in hot air sterilisation
chambers.
When sterilising empty, open glass containers in compact lines with enclosed hot air sterilisation
tunnels, much higher temperatures than those given in figure 12.K-1 are often used. The temperature
can be >250 C, mainly to avoid having to provide evidence of the 3-log reduction in endotoxins
using bioindicators (spores).
Above 180 C, the D values of these bioindicators are well below 1 min (at 160 C 5 mins, at 200 C
under 1 sec, for example ATCC 9372). In the continual process, the temperature in the container also
rises constantly without a plateau phase until it reaches the maximum temperature. It is therefore not
possible to remove the bioindicator at the appropriate time (or position in the tunnel) without
affecting the temperature.
Depyrogenation
Containers for parenterals must be pyrogen-free if they are filled aseptically. From volumes of 15 ml
and above, injectable drug products for use in humans must be tested for the presence of endotoxins
or pyrogens. In practice, following dry heat sterilisation, the remaining residual quantities in the
empty container are below the limit of detection of 0.03 EU/ml. Based on the known effectiveness of
the washing process, the initial endotoxin load established by monitoring, and the residual moisture
in the final rinse that contains < 0.25 EU/ml, the quantities of endotoxin to be destroyed in dry heat
sterilisation are very low. The risk that endotoxins will enter from the solution is several times
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greater, due to the WFI and active ingredients or components (with the permitted endotoxin content)
that they contain. To assess the overall situation, the endotoxin limits for parenterals in accordance
with the USP should be observed.
To achieve the deactivation of pyrogens by 3 log takes 1.5 mins at 250 C. For this reason, in
practice, the sterilisation tunnel should aim to achieve object temperatures of > 250 C and residence
times of > 3 mins above this temperature.
12.K.2 Sterilisation kinetics

The same rules apply as described in chapter 12.F Steam sterilisation, but different variables are used
for the D and Z values.
n Number of germs, expressed as CFU (colony-forming units), also known as microbial count.
The D value is the decimal mortality rate in minutes at a given temperature, or the time in minutes
that is required to kill 90% of the spores or vegetative cells of a specified microorganism at a given
temperature. The D value always refers to a specific temperature and germ species, for example D170
C.
The Z value for hot air is the Z value of a given microorganism defined against different
temperatures and is 20 C (see figure 12.K-4).
FH 170 C The total amount of heat that acts on the sterilisation goods during a sterilisation process,
converted to the relevant temperature (here 170 C) in minutes or the temperature course of the whole
sterilisation curve.
Figure 12.K-4 Z value (hot air)
At a constant temperature, therefore:
FH = t x 10 (T - 170 )/Z
t = Sterilisation time (mins)
T = Sterilisation temperature (C)
Z = 20 C
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The following example is based on real-life settings on the basis of 255 C and 3.5 mins. FH value of
the process for an actual 3-min. sterilisation time for the container at 251 C.
FH = 3 x 10 (251 - 170 )/20

FH = 3 x 10 (81)/20
FH = 3 x 10 4.05
FH = 33660
Calculation of the F value (total lethality)
According to the formula F = n x D, at D 180 C = 30 secs, Z = 20 C, an assumed original germ count

of approx. 103 and expected contamination probability of 10 -6 results in:
F180 C = 9 x 30 secs = 270 s = 4.5 mins
Based on existing experimental data, the F value for an equivalent procedure can be calculated
according to the following formula:
FZ 180 C /FT = 10(T - 180 )/Z; FT = FZ 180 C /10(T - 180 )/Z
Inserting the values F 180 C = 4.5 mins and Z = 20 C into the above equation results in:
F 200 C = 4.5/10 (200 - 180)/20

F 200 C = 4.5/1020/20 = 4.5/10 = 0.45 mins.
For an equivalent procedure at 200 C, this results in an equivalent value of 0.45 mins.
12.K.3 Qualification of a sterilisation tunnel

The operation of a sterilisation tunnel must ensure that the objects or solutions treated in the system
achieve the required end result - i.e. they are free from living microorganisms. Furthermore, it should
also be possible to perform depyrogenation of the objects by selecting a suitable temperature. The
fulfilment of this task can be ensured by the technical layout and the qualification (see figure 12.K-5).
Figure 12.K-5 Test points in qualification
Test points in qualification
Materials
Filter classifications
MSR components
Function
Monitoring (temperature/belt speed)
Documentation
When selecting the design of tunnel, it is essential to decide whether you only want to use the tunnel
for sterilisation, or also for depyrogenation. This affects the temperature of the tunnel in operation
and also the design, which later will not permit any temperature increase.
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12.K.3.1 Installation qualification

The execution of the tests is described in detail to ensure that the same tests can be accurately
replicated in the future. Measured values must be recorded in test records and documents (printouts,
diagrams, etc.) must be included as attachments.

The scope of installation qualification includes performing an initial calibration of the measuring
equipment before start-up of the sterilisation tunnel. The measuring points within IQ, their
measurement type, operating range, adjustment points, calibration intervals and degree of accuracy
must all be incorporated in the initial calibration. Quality-relevant measuring points in the
sterilisation tunnel are as follows:
Temperature (sterilisation air, cool air)

Belt speed
Time
Air velocity (LF hot air, LF inflow, LF cool air)
Pressure differentials (LF air currents/exhaust)
Particle measurement in the LF in accordance with class 100
Measured values for calibration must be compared with reference systems from a higher class. For
values outside the acceptable measured value tolerance, the measuring chain must be adjusted and
recalibrated. The operational qualification cannot take place until a successful initial calibration has
been performed.
12.K.3.2 Operational qualification

In the operational qualification, all the operating states of the sterilisation tunnel are simulated to their
limits (worst case), and the results documented (see also figure 12.K-8 and figure 12.K-6).
Figure 12.K-6 Test points in operational qualification (extract)
Is an alarm activated if the temperature falls below the sterilisation/depyrogenation

temperature?
Is an alarm activated if the speed of the conveyor belt is exceeded?
Does an alarm sound if the LF air velocity drops below the defined setting?
Are the measured values documented?
Does the LF meet the requirements of cleanliness grade 100 at operating temperature?
Testing heat distribution

During operational qualification, a heat distribution test must be performed on the empty conveyor
belt. To do this, three measuring points are fixed on the conveyor belt across the direction of transport
(see figure 12.K-7).
The temperature difference at the heat registering points between one side of the tunnel and the other
should not exceed 5 C.
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Figure 12.K-7 Alignment of thermocouples for measuring heat distribution
Figure 12.K-8 Function tests

Function tests
Recorder feed of the external
device
Function check of the

sterilisation tunnel - time
measurement
Check paper with markings on the recorder paper and time

measurement.
Documentation: Markings on the recorder paper with time
measurement.
The built-in recorder in the sterilisation tunnel is tested
using markings on the recorder paper and time
measurements.
Maximum deviation 1 min/h (corresponds to 60 1 mm/h
paper feed).
Documentation: Markings on the recorder paper and time
measurement.
12.K.4 Validation of the sterilisation process

The validation protocol (see chapter 7 Process Validation) states the objective to provide evidence
that a reproducible process can lead to a 3-log reduction in bacterial endotoxin content.
In accordance with the EP, when using hot air at >220 C for sterilisation, the evidence of a 3-log
reduction in endotoxins is acceptable instead of evidence from bioindicators. The validation protocol
is used to describe, structure, monitor and document the validation activities (figure 12.K-9).
Figure 12.K-9 Extract from a validation protocol
Responsibilities
Person responsible for validation Head of Production, Head of Quality Control
Validation manager
Project manager
Validation team
Staff from the unit (users)

QA staff
Device: Hot air sterilisation tunnel made by XYZ, no. 1234, Building 000
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Process parameters:
Temperature 250 C (worst case),
set value 285 C, time >2 minutes.
Validation runs: Three runs with worst-case loading configurations:
a) Start phase for first objects
b) End phase for last objects
Reproducibility:
Evaluation of recorder for range of process values
Endotoxin reduction: 3 log at a minimum of 1000 EU insemination/object
Prerequisite for executing the validation is the employment of qualified personnel, and proof that the
systems and test equipment are qualified for the intended purpose, and the measuring equipment is
calibrated.
12.K.4.1 Description of the device

Example
The hot air sterilisation tunnel made by XYZ is installed in building 000 in cleanliness grade XY.
The sterilisation tunnel is filled with washed containers (ampoules/bottles) from a washing machine
automatically or by hand. A test point for rejecting objects with more than approx. 100 mg residual
moisture in the container is located upstream of the entrance to the sterilisation tunnel.
The sterilisation tunnel consists of the inlet LF box, a hot air part, the cool air LF, and the passage
sector to cleanliness grade C, A or B (see figure 12.K-10).
Figure 12.K-10 Temperature and flow conditions in the sterilisation tunnel
The heating register is usually located on the side of the hot air part. This aspirates air from the room
atmosphere via a prefilter, heats it, and passes it on through a HEPA filter. The partial amount of air
that is exhausted or pushed from the hot air part into the inlet LF and the cold air stream is replaced
by atmospheric air from the room via the prefilter.
At an air velocity of 0.7-1 m/s, when the air makes contact with the containers and the conveyor belt,
the air decelerates to approx. 0.4 m/s. The residual moisture from the containers also contributes to
this effect by forming a steam cushion against the air current of the hot air part. To minimise the loss
of hot air to the LF units and to maintain the stability of the flow conditions in the sterilisation tunnel,
the isolation between inlet LF/hot air part/cool air LF/cleanliness grade filling must be adjusted to
permit the minimum clearance above the containers at the passages.
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When equipping a sterilisation tunnel with control sensors calibrated in an oil bath at 140 C (three
Fe-Co thermocouples), and running parallel test containers loaded with endotoxin immediately
adjacent to the sensors, the whole width of the conveyor belt should be covered.
12.K.4.2 Preparation of the endotoxin test objects

Depyrogenated containers are used as validation samples for measuring endotoxin reduction. A
container (ampoule or bottle) of approx. 0.5 ml, is filled with endotoxin solution corresponding to
between 4000 and 6000 EU, distributed, and dried for 24 hours under a vacuum over silica gel at
room temperature. Three out of twelve samples prepared in this way are subsequently tested for
endotoxin recovery (usually approx. 50-100%) and used as a basis for calculating the endotoxin
reduction after depyrogenation of the other samples. To interpret the results, all individual values are
compared to the lowest and highest recovery rates and calculated. This results in the log reduction in
values from ... to ....
12.K.4.3 Description of the process

The three validation runs follow the normal process flow of continual dry heat sterilisation.
Figure 12.K-11 Sequence of operation
Program selection
Type/size of container,
hot air temperature
Conveyor belt started

LF equipment started
Heating to sterilisation temperature
Conveyor belt loaded with containers
Sterilisation/depyrogenation
Containers transported through the sterilisation tunnel
12.K.4.4 Position of the heat sensors

The internal surface temperature of the containers is measured by fixing the thermocouple at the neck
of the ampoule or bottle to the floor of the container under tension, and the thermocouple junction
makes contact with the floor of the glass. The containers loaded with endotoxin are positioned
immediately adjacent to the containers equipped with the thermocouples and transported through the
sterilisation tunnel.
12.K.4.5 Determining the endotoxin reduction

The success of sterilisation is tested using the evidence of a 3-log reduction in gram-negative
bacteria endotoxins (lipopolysaccharides) . To do this, the containers to be sterilised are loaded with
endotoxin, subjected to processing in the sterilisation tunnel, and the decrease in the amount of
endotoxin is determined.
Determining the sterilisation time

To check the microbiological effectiveness, the duration of the container temperature 250 C > 2
minutes is determined based on recordings of the thermocouple temperatures.
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12.K.4.6 Executing the validation

The particular configuration (size, internal construction of the tunnel and inlets and outlets) is used to
determine the minimum and maximum (worst case) loads based on the routine loading
configurations. The sterilisation temperature is above 250 C. Since the actual sterilisation
temperatures are not recorded until the sterilisation process itself, for temperatures >250 C, set
temperature values above 250 C are specified in order to correspond to the USP 265 C.
Time-temperature record of the measurement sensors installed in the sterilisation tunnel

Time-temperature record of the control heat sensors
Results of the evaluation of endotoxin reduction
Certificates of the endotoxins and LAL used
Once the sterilisation process is complete, the data is evaluated. The evaluation in terms of
temperature is based on measured data after the temperature of 250 C is reached, taking into account
the correction factors of the thermocouples.
Requirement: Temperature is 250 C >2 minutes (temperature, time in the objects).
Assessment of the measurement results from the device measuring equipment compared to
the control measuring equipment. Determination of the difference between the set temperature
and the actual container temperature.
Assessment of effectiveness based on physical data (FH)
Assessment of the sterilisation process (in accordance with PDA monograph no. 3)
Reproducibility
The reproducibility of the following setting conditions is tested:
Temperature: 250 C (lower permitted limit)

Time: 2 mins
Endotoxin reduction: 3 log
The following requirements must be fulfilled:
The assessment should be carried out using measured data determined after the required
temperature was reached.
Time: 2 mins
Temperature: 250 C - 280 C
Assessment of the evaluation (in accordance with company SOP) and control of the recorder chart.
Documentation: Time-temperature record from routine sterilisations.
When the validation process has been successfully completed and all results are positive, the
sterilisation tunnel can be released in the described scope for one year. After this time,
requalification/revalidation is required.
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Summary
A hot air tunnel consists of an inlet section, a hot air part, and an outlet part, which is connected to
the filling mechanism. Depending on the temperature of the hot air, the tunnel can be used for
depyrogenation as well as sterilisation. To test the effectiveness of depyrogenation in the validation
process, ampoules are spiked with endotoxins and tested to ensure that a 3-log reduction in
endotoxins has taken place. Dry heat sterilisation is usually used to prepare empty containers for
filling in compact lines.
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12 Sterile Production

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12 Sterile Production

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12 - Sterile

12.B Air Lock Concepts

12.C Manufacturing the solution

12.D Washing processes

12.E.1 Filling equipment for solutions

12.F Steam sterilisation

12.G Microbiological monitoring

12.H Test for sterility

12.H.3 Method description

12.I Testing for tightness and particles

12.J Freeze drying

12.K Dry Heat Sterilisation

Which requirements are sterile products expected to meet?

Any substances (chemical, microbiological) that cause fever on parenteral

Nominal volume of 100 ml

Average number* of particles

Average number* of particles

Average number* of particles

Average number* of particles

12.A.1 Manufacturing products that can be sterilised in the final container

12.A.2 Aseptic processing

Filling in the final container

Manufacture of non-filterable preparations Cleanliness grade A

12.A.3 Production areas/premises

Manufacturing operations are classified into two categories:

The presence of appropriately dressed personnel is permitted.

The presence of appropriately dressed personnel is permitted.

The presence of appropriately dressed personnel is permitted.

12.A.4 Production equipment

Examples of serious faults:

12.B Air Lock Concepts

Here you will find answers to the following questions:

How are personnel locks between cleanliness grades designed?

12.B.1 Personnel locks in the clean area

12.B.1.1 Air locks in cleanliness grade F/E

12.B.1.2 Air locks in cleanliness grade E/D

Wait for the entry signal before opening the door.

clothing in cupboard in the grade D section).

12.B.1.3 Air locks in cleanliness grade D/C

Wait for the entry signal before opening the door.

12.B.1.4 Air locks in cleanliness grade C/B

12.B.2 Material locks

Figure 12.B-8 Material lock from cleanliness grade D to cleanliness grade C

12.C Manufacturing the solution

Which requirements are starting materials expected to meet?

12.C.1 Starting materials

A solution is a transparent liquid. It contains active pharmaceutical ingredients and excipients,

Ion disperse: the solution contains ions as in an ionic solution.

Purified water and water for injection (WFI)

12.C.1.1 Rooms used for weighing

12.C.1.2 Processing instructions (manufacturing instructions)

12.C.1.3 Weighing of starting materials

12.C.2 Solution batch

Stiffness/blockage of plug and

Damaged screw thread, damaged or ageing seals, not changed

Leaks in piping, equipment, and

Assembly errors: Incorrect, missing seal, incorrect mounting

Irregular filtration times, low

Incorrect pore size of the filter, possible substance properties,

In particular, check the package integrity of sterilised accessories.

prescribed temperature is regulated, and the labelling or lettering is validated.

12.C.3 Testing the bioburden

12.C.4 Sterile filtration

12.C.4.2 Mode of operation