Q C of Sterile Products

QUALITY CONTROL OF
STERILE PRODUCTS
4TH Prof.
Pharm.D
INTRODUCTION
 Parenteral preparations are sterile, pyrogen-
free liquids (solutions, emulsions, or
suspensions) or solid dosage forms intended
for administration by injection, infusion, or
implantation into the body.
 The word “Parenterals” has come from Greek
word:
Para= outside/besides
Enteron= intestine
2
INTRODUCTION cont’d
 Parenteral preparations may contain excipients such as solvents,

suspending agents, buffering agents, substances to make the
preparation isotonic with blood, stabilizers, or antimicrobial
preservatives.
 The addition of excipients should be kept to a minimum. When
excipients are used, they should not adversely affect the stability,
bioavailability, safety, or efficacy of the active ingredient(s), or cause
toxicity or undue local irritation.
 There must be no incompatibility between any of the components of the
dosage form.
 Water for injections is used as the vehicle for aqueous injections. It
should be freshly distilled, be free from carbon dioxide. and comply with
Test for bacterial endotoxins.
 Unless otherwise specified in the individual monograph, sodium chloride
or other suitable substance(s), may be added to an aqueous solution for
injection in order to render the preparation isotonic.
3
TYPES OF PARENTERALS
 There are four main forms of parenteral
preparations:
 injections,
 intravenous infusions (large volume parenterals),
 powders for injections, and
 implants.
 Certain injections and intravenous infusions may
be presented in the form of sterile concentrated
solutions, which must be suitably diluted before
use.
4
TYPES OF PARENTERALS cont’d
1) Injections
 Injections are sterile, pyrogen-free solutions or dispersions
(emulsions or suspensions) of one or more active ingredients in a
suitable vehicle.
 Whenever possible, an injection should be prepared using an
aqueous vehicle. If necessary, suitable non-aqueous solvents are
indicated in the individual monographs. Injections that are
dispersions should remain sufficiently stable so that, after
shaking, a homogeneous dose can be withdrawn.
 The use of single-dose injections is to be preferred and is
essential when the preparation is intended for administration by
routes where, for medical reasons, an antimicrobial preservative
is not acceptable, e.g. intracisternal, intrathecal.
5
Single-dose preparations
 Single-dose preparations should contain a sufficient
quantity of the injection readily to permit the withdrawal
and administration of the volume specified on the label.
Multidose preparations
 Multidose preparations should contain a suitable
antimicrobial preservative in appropriate concentrations,
except in cases where the preparations themselves have
adequate antimicrobial properties. The containers should
be equipped to ensure adequate protection of the contents
after partial withdrawal. In order to minimize the risk of
contamination resulting from multiple penetrations of the
closure, the contents of a multidose preparation should
normally not exceed 30 ml.
6
2) Intravenous infusions
 Intravenous infusions are sterile, pyrogen-free
aqueous solutions or emulsions with water as
continuous phase, usually prepared to be
isotonic. They are intended for administration in
large volumes (usually 100 ml or more), and
should not contain any antimicrobial
preservatives.
 On visual inspection, emulsions for intravenous
injection should show no evidence of phase
separation.
7
3) Powders for injections

 Powders for injections are solid substances (including freeze-dried materials),
distributed in their final containers and which, when shaken with the prescribed
volume of the appropriate sterile liquid, rapidly form either clear and practically
particle-free solutions or uniform suspensions. Powders for injections, after
dissolution or suspension, comply with the requirements for injections or
intravenous infusions, as appropriate.
 Uniformity of mass
 Powders for injections (single-dose use) comply with the test for Uniformity of
mass for single-dose preparations, unless otherwise specified in the individual
monograph.
 Uniformity of content
 A requirement for compliance with the test for Uniformity of content for single-
dose preparations is specified in certain individual monographs where the active
ingredient is less than 40 mg. In such cases, compliance with the test
for Uniformity of mass for single-dose preparations may not be required.
8
4) Implants
 Implants are solid preparations containing one or
more active ingredients. They are of a size and
shape suitable for parenteral implantation, and
provide release of the active ingredient(s) over
an extended period of time. They are presented
in individual sterile containers.
 All requirements for these specialized dosage
forms are given in the individual monographs.
9
Types of containers
1. Ampoules:
 They are intended for single use only, ampoules are opened by breaking the glass
at a score line on the neck. Because glass particles may become dislodged during
ampoule opening, the product must be filtered before it administered. Because of
their unsuitability for multiple-dose use, the need to filter solutions before use
and other safety considerations have markedly reduced ampoule use.
2. Vials:
 Vials are glass or plastic containers are closed with a rubber stopper and sealed
with an aluminum crimp.
 Advantages over ampoules.
 They can be designed to hold multiple doses (if prepared with a bacteriostatic agent).
 It is easier to remove the product.
 They eliminate the risk of glass particle contamination during opening.
 Drawbacks
 The rubber stopper may become cored.
 Multiple withdrawals(as with multiple-dose vials)may result in microbial
contamination.
10
Types of containers cont’d
3. Prefilled syringes:
 These designed for quickest administration and maximum
convenience. Drugs administered in an emergency
(e.g.,atropine,epinephrine) may be available for immediate
injection when packaged in prefilled syringes.
4. Infusion solutions
 Infusion solutions are divided into two categories : small
volume parenterals (SVP), those having a volume of 100
ml;and large volume parenterals (LVP), those having a volume
of 100 ml or greater. Infusion solutions are used for the
intermittent or continuous infusion of fluids or drugs.
11
QUALITY CONTROL
TESTS OF
PARENTERALS
(BRITISH PHARMACOPOEIA 2012)
12
Rabbit Pyrogen Test
 The Rabbit Pyrogen Test in an in vivo test to
detect pyrogens qualitatively.
 Rabbits have a similar pyrogen tolerance to
humans, so by observing a change in body
temperature in rabbits it is possible to make a
determination of the presence of pyrogens.
 This method can detect non-bacterial
endotoxin pyrogens as well as bacterial
endotoxins.
13
Appendix XIV D.Test for Pyrogens
(Ph. Eur. method 2.6.8)
 The test consists of measuring the rise in body temperature

evoked in rabbits by the intravenous injection of a sterile
solution of the substance to be examined.
Selection of animals
 Use healthy, adult rabbits of either sex weighing not less
than 1.5 kg, fed a complete and balanced diet not
containing antibiotics, and not showing loss of body mass
during the week preceding the test. A rabbit is not be used
in a pyrogen test:
a) if it has been used in a negative pyrogen test in the
preceding 3 days, or
b) if it has been used in the preceding 3 weeks in a pyrogen
test in which the substance under examination failed to
pass the test.
14
cont’d
 Animals' quarters: Keep the rabbits individually in a

quiet area with a uniform appropriate temperature.
Withhold food from the rabbits overnight and until the test
is completed; withhold water during the test. Carry out the
test in a quiet room where there is no risk of disturbance
exciting the animals and in which the room temperature is
within 3 °C of that of the rabbits' living quarters, or in which
the rabbits have been kept for at least 18 h before the test.
 Materials: Glassware syringes and needles. Thoroughly
wash all glassware, syringes and needles with water for
injections and heat in a hot-air oven at 250 °C for 30 min or
at 200 °C for 1 h.
15
cont’d
 Retaining boxes
 The retaining boxes for rabbits whose temperature is being
measured by an electrical device are made in such a way that the
animals are retained only by loosely fitting neck-stocks; the rest
of the body remains relatively free so that the rabbits may sit in a
normal position. They are not restrained by straps or other similar
methods which may harm the animal. The animals are put into
the boxes not less than 1 h before the first record of the
temperature and remain in them throughout the test.
 Thermometers
 Use a thermometer or electrical device which indicates the
temperature with a precision of 0.1 °C and insert into the rectum
of the rabbit to a depth of about 5 cm. The depth of insertion is
constant for any one rabbit in any one test. When an electrical
device is used it may be left in position throughout the test.
16
cont’d
Preliminary test
 After selection of the animals, one to three days
before testing the product to be examined, treat
those animals that have not been used during the
previous 2 weeks by intravenous injection of 10 mL
per kilogram of body mass of a pyrogen-free 9 g/L
solution of sodium chloride R warmed to about 38.5
°C. Record the temperatures of the animals,
beginning at least 90 min before injection and
continuing for 3 h after the injection of the solution.
Any animal showing a temperature variation greater
than 0.6 °C is not used in the main test.
17
cont’d
Main test
 Carry out the test using a group of three rabbits.
Preparation and injection of the product: Warm the
liquid to be examined to approximately 38.5 °C before the
injection. The product to be examined may be dissolved in,
or diluted with, a pyrogen-free 9 g/L solution ofsodium
chloride R or another prescribed liquid. Inject the solution
slowly into the marginal vein of the ear of each rabbit over
a period not exceeding 4 min, unless otherwise prescribed
in the monograph. The amount of the product to be
injected varies according to the product to be examined
and is prescribed in the monograph. The volume injected is
not less than 0.5 mL per kilogram and not more than 10 mL
per kilogram of body mass.
18
cont’d
 Determination of the initial and maximum

temperatures: The "initial temperature" of each rabbit is the mean
of two temperature readings recorded for that rabbit at an interval of 30 min
in the 40 min immediately preceding the injection of the product to be
examined. The "maximum temperature" of each rabbit is the highest
temperature recorded for that rabbit in the 3 h after the injection. Record
the temperature of each rabbit at intervals of not more than 30 min,
beginning at least 90 min before the injection of the product to be examined
and continuing 3 h after the injection. The difference between the maximum
temperature and the initial temperature of each rabbit is taken to be its
response. When this difference is negative, the result is counted as a zero
response.
 Rabbits showing a temperature variation greater than 0.2 °C between two
successive readings in the determination of the initial temperature are
withdrawn from the test. In any one test, only rabbits having initial
temperatures which do not differ from one another by more than 1 °C are
used. All rabbits having an initial temperature higher than 39.8 °C or less
than 38.0 °C are withdrawn from the test.
19
cont’d
 Interpretation of results: Having carried out the test

first on a group of three rabbits, repeat if necessary on
further groups of three rabbits to a total of four groups,
depending on the results obtained. If the summed response
of the first group does not exceed the figure given in the
second column of the Table 2.6.8.-1, the substance passes
the test. If the summed response exceeds the figure given
in the second column of the table but does not exceed the
figure given in the third column of the table, repeat the test
as indicated above. If the summed response exceeds the
figure given in the third column of the table, the product
fails the test.
 Rabbits used in a test for pyrogens where the mean rise in
the rabbits' temperature has exceeded 1.2 °C are
permanently excluded.
20
cont’d
21
<151> PYROGEN TEST (USP)
 The pyrogen test is designed to limit to an acceptable
level the risks of febrile reaction in the patient to the
administration, by injection, of the product concerned.
The test involves measuring the rise in temperature of
rabbits following the intravenous injection of a test
solution and is designed for products that can be
tolerated by the test rabbit in a dose not to exceed 10 mL
per kg injected intravenously within a period of not more
than 10 minutes. For products that require preliminary
preparation or are subject to special conditions of
administration, follow the additional directions given in
the individual monograph or, in the case of antibiotics or
biologics, the additional directions given in the federal
regulations .
22
<151> PYROGEN TEST cont’d
 APPARATUS AND DILUENTS

 Render the syringes, needles, and glassware free from pyrogens by heating
at 2500 for not less than 30 minutes or by any other suitable method. Treat
all diluents and solutions for washing and rinsing of devices or parenteral
injection assemblies in a manner that will assure that they are sterile and
pyrogen -free. Periodically perform control pyrogen tests on representative
portions of the diluents and solutions for washing or rinsing of the
apparatus. Where Sodium Chloride Injection is specified as a diluent, use
Injection containing 0.9 percent of NaC!.
 TEMPERATURE RECORDING
 Use an accurate temperature-sensing device such as a clinical thermometer,
or thermistor probes or similar probes that have been calibrated to assure
an accuracy of ±0.1 ° and have been tested to determine that a maximum
reading is reached in less than 5 minutes. Insert the temperature-sensing
probe into the rectum of the test rabbit to a depth of not less than 7.5 cm,
and, after a period of time not less than that previously determined as
sufficient, record the rabbit's body temperature.
23
 TEST ANIMALS
 Use healthy, mature rabbits. House the rabbits individually in an
area of uniform temperature between 20° and 23° and free from
disturbances likely to excite them. The temperature varies not
more than ±3° from the selected temperature. Before using a
rabbit for the first time in a pyrogen test, condition it not more
than seven days before use by a sham test that includes all of the
steps as directed for Procedure except injection. Do not use a
rabbit for pyrogen testing more frequently than once every 48
hours, nor prior to 2 weeks following a maximum rise of its
temperature of 0.60 or more while being subjected to the
pyrogen test, or following its having been given a test specimen
that was adjudged pyrogenic.
24
 PROCEDURE
 Perform the test in a separate area designated solely for pyrogen
testing and under environmental conditions similar to those
under which the animals are housed and free from disturbances
likely to excite them. Withhold all food from the rabbits used
during the period of the test. Access to water is allowed at all
times, but may be restricted during the test. If rectal
temperature-measuring probes remain inserted throughout the
testing period, restrain the rabbits with light-fitting neck stocks
that allow the rabbits to assume a natural resting posture. Not
more than 30 minutes prior to the injection of the test dose,
determine the "control temperature" of each rabbit: this is the
base for the determination of any temperature increase resulting
from the injection of a test solution. In anyone group of test
rabbits, use only those rabbits whose control temperatures do
not vary by more than 1 ° from each other, and do not use any
rabbit having a temperature exceeding 39.8°.
25
 Unless otherwise specified in the individual monograph, inject

into an ear vein of each of three rabbits 10 mL of the test solution
per kg of body weight, completing each injection within 10
minutes after start of administration. The test solution is either
the product, constituted if necessary as directed in the labeling,
or the material under test treated as directed in the individual
monograph and injected in the dose specified therein. For
pyrogen testing of devices or injection assemblies, use washings
or rinsings of the surfaces that come in contact with the
parenterally administered material or with the injection site or
internal tissues of the patient. Assure that all test solutions are
protected from contamination. Perform the injection after
warming the test solution to a temperature of 37 ± 2°. Record the
temperature at 30-minute intervals between 1 and 3 hours
subsequent to the injection.
26
 TEST INTERPRETATION AND CONTINUATION

 Consider any temperature decreases as zero rise. If no
rabbit shows an individual rise in temperature of 0.5° or
more above its respective control temperature, the product
meets the requirements for the absence of pyrogens. If any
rabbit shows an individual temperature rise of 0.5° or more,
continue the test using five other rabbits. If not more than
three of the eight rabbits show individual rises in
temperature of 0.5° or more and if the sum of the eight
individual maximum temperature rises does not exceed
3.3°, the material under examination meets the
requirements for the absence of pyrogens.
27
Appendix XIV E. Test for Abnormal
Toxicity (Ph. Eur. method 2.6.9)
 General test
 Inject intravenously into each of 5 healthy mice, weighing 17 g to 24
g, the quantity of the substance to be examined prescribed in the
monograph, dissolved in 0.5 mL of water for injections R or of a 9 g/L
sterile solution of sodium chloride R. Inject the solution over a period
of 15 s to 30 s, unless otherwise prescribed.
 The substance passes the test if none of the mice die within 24 h or
within such time as is specified in the individual monograph. If more
than one animal dies the preparation fails the test. If one of the
animals dies, repeat the test. The substance passes the test if none
of the animals in the 2nd group die within the time interval specified.
28
Appendix XIV E. Test for Abnormal
Toxicity (Ph. Eur. method 2.6.9)
 Immunosera (antisera) and vaccines

 Unless otherwise prescribed, inject intraperitoneally 1 human dose but not more
than 1.0 mL into each of 5 healthy mice, weighing 17 g to 24 g. The human dose
is that stated on the label of the preparation to be examined or on the
accompanying leaflet. Observe the animals for 7 days.
 The preparation passes the test if none of the animals shows signs of ill health. If
more than one animal dies, the preparation fails the test. If one of the animals
dies or shows signs of ill health, repeat the test. The preparation passes the test
if none of the animals in the 2nd group die or shows signs of ill health in the time
interval specified.
 The test must also be carried out on 2 healthy guinea-pigs weighing 250 g to 400
g. Inject intraperitoneally into each animal 1 human dose but not more than 5.0
mL. The human dose is that stated on the label of the preparation to be
examined or on the accompanying leaflet. Observe the animals for 7 days.
 The preparation passes the test if none of the animals shows signs of ill health. If
more than one animal dies the preparation fails the test. If one of the animals
dies or shows signs of ill health, repeat the test. The preparation passes the test
if none of the animals in the 2nd group die or shows signs of ill health in the time
interval specified.
29
Particulate matter
 Matter of biological or non-biological origin and
with observable length, width, and thickness,
e.g., bacteria, fungi, dust, dirt, fibers, plastic,
rubber, lint etc. It may be any matter, mixed
accidentally during manufacturing in the
parenteral product which does not belong to the
product. Particulate matter may be tiny pieces of
lint, glass, dust, rubber, metal fibers, hair,
microbes or unidentified and can make the
product impure, unclean or unfit for use.
30
Sources Of Particulate Matter
 Particulate contamination particularly of cellulose fibers, dust, cotton fibers, hair,
dandruff and loose skin from human origin as well as microbial contamination may
arise from the following main sources.
 1. Material arising from the drug: undissolved substances and trace contaminants etc.
 2. Material arising from vehicle or added substances: These may include those
material not filtered out during a clarification process before to filling the final
container.
 3. Materials present in the final container: Material already present in container and
which were not removed by rinsing prior to filling
 4. Materials falling by chance into the final container during the filling process
 5. The container or closures which may be deposited in the product during
sterilization, e.g. carbon black, whiting, zinc oxide and clay
 6. Packaging components: Including glass, plastic, rubber, I/V administration sets, etc.
 7. Environmental contaminants: Including air, work tops, insects’ parts
 8. Processing equipments: Including glass, stainless steel, rubber, or filter fiber, etc.
 9. Personnel: Including skin, hair, and clothing etc.
31
Particle size
 Particles present in injectable are non-reactive, apyrogenic,
sterilized. However, by virtue of their size may biologically
hazardous. The particulate matter may be capable of
blocking the blood vessels with severe results on induction
into body with injection.
 A person with 20/20 vision under inspection conditions is
able to detect particles of size range 40 – 50 μm. However,
it is universally accepted that the particles size of 50 μm is
detected visually by an unaided eye.
 Particle size greater than 7 μm diameter is considered to be
more threatening. Pulmonary capillary are approximately 7
μm in diameter, thus particle of this much size entrapped in
vascular bed resulting in multiple pulmonary infarction.
32
Appendix XIII A. Particulate
Contamination: Sub-visible Particles
 Particulate contamination of injections and infusions

consists of extraneous, mobile undissolved particles,
other than gas bubbles, unintentionally present in the
solutions.
 For the determination of particulate contamination 2
procedures, Method 1 (Light Obscuration Particle Count
Test) and Method 2 (Microscopic Particle Count Test), are
specified hereinafter. When examining injections and
infusions for sub-visible particles, Method 1 is preferably
applied. However, it may be necessary to test some
preparations by the light obscuration particle count test
followed by the microscopic particle count test to reach a
conclusion on conformance to the requirements. 33
Appendix XIII A. Particulate Contamination: Sub-
visible Particles
(Ph. Eur. method 2.9.19) Cont’d
 Not all parenteral preparations can be examined for sub-visible particles by

one or both of these methods. When Method 1 is not applicable, e.g. in case
of preparations having reduced clarity or increased viscosity, the test is
carried out according to Method 2. Emulsions, colloids, and liposomal
preparations are examples. Similarly, products that produce air or gas
bubbles when drawn into the sensor may also require microscopic particle
count testing. If the viscosity of the preparation to be tested is sufficiently
high so as to preclude its examination by either test method, a quantitative
dilution with an appropriate diluent may be made to decrease viscosity, as
necessary, to allow the analysis to be performed.
 The results obtained in examining a discrete unit or group of units for
particulate contamination cannot be extrapolated with certainty to other
units that remain untested. Thus, statistically sound sampling plans must be
developed if valid inferences are to be drawn from observed data to
characterise the level of particulate contamination in a large group of units.
34
visible Particles
 Method 1. Light obscuration particle

count test
 Use a suitable apparatus based on the principle of
light blockage which allows an automatic
determination of the size of particles and the
number of particles according to size.
 The apparatus is calibrated using suitable certified
reference materials consisting of dispersions of
spherical particles of known sizes between 10 µm
and 25 µm. These standard particles are dispersed
in particle-free water R. Care must be taken to avoid
aggregation of particles during dispersion.
35
visible Particles
 General precautions
 The test is carried out under conditions limiting particulate contamination,
preferably in a laminar-flow cabinet.
 Very carefully wash the glassware and filtration equipment used, except for the
membrane filters, with a warm detergent solution and rinse with abundant
amounts of water to remove all traces of detergent. Immediately before use,
rinse the equipment from top to bottom, outside and then inside, with particle-
free water R.
 Take care not to introduce air bubbles into the preparation to be examined,
especially when fractions of the preparation are being transferred to the
container in which the determination is to be carried out.
 In order to check that the environment is suitable for the test, that the glassware
is properly cleaned and that the water to be used is particle-free, the following
test is carried out:
 determine the particulate contamination of 5 samples of particle-free water R,
each of 5 mL, according to the method described below. If the number of
particles of 10 µm or greater size exceeds 25 for the combined 25 mL, the
precautions taken for the test are not sufficient. The preparatory steps must be
repeated until the environment, glassware and water are suitable for the test.
36
visible Particles
 Method
 Mix the contents of the sample by slowly inverting the container 20
times successively. If necessary, cautiously remove the sealing closure.
Clean the outer surfaces of the container opening using a jet of particle-
free water Rand remove the closure, avoiding any contamination of the
contents. Eliminate gas bubbles by appropriate measures such as
allowing to stand for 2 min or sonicating.
 For large-volume parenterals, single units are tested. For small-volume
parenterals less than 25 mL in volume, the contents of 10 or more units
are combined in a cleaned container to obtain a volume of not less than
25 mL; where justified and authorised, the test solution may be
prepared by mixing the contents of a suitable number of vials and
diluting to 25 mL with particle-free water R or with an appropriate
solvent without contamination of particles when particle-free water R is
not suitable. Small-volume parenterals having a volume of 25 mL or
more may be tested individually.
37
visible Particles
 Powders for parenteral administration are reconstituted

with particle-free water R or with an appropriate solvent
without contamination of particles when particle-free water
R is not suitable.
 The number of test specimens must be adequate to provide
a statistically sound assessment. For large-volume
parenterals or for small-volume parenterals having a
volume of 25 mL or more, fewer than 10 units may be
tested, based on an appropriate sampling plan.
 Remove 4 portions, each of not less than 5 mL, and count
the number of particles equal to or greater than 10 µm and
25 µm. Disregard the result obtained for the first portion,
and calculate the mean number of particles for the
preparation to be examined.
38
visible Particles
 Evaluation
 For preparations supplied in containers with a nominal volume of more than 100 mL, apply the
criteria of test 1.A.
 For preparations supplied in containers with a nominal volume of less than 100 mL, apply the
criteria of test 1.B.
 ♦For preparations supplied in containers with a nominal volume of 100 mL, apply the criteria of
test 1.B.♦
 If the average number of particles exceeds the limits, test the preparation by the microscopic
particle count test.
 Test 1.A – Solutions for infusion or solutions for injection supplied in containers with a nominal
content of more than 100 mL
 The preparation complies with the test if the average number of particles present in the units
tested does not exceed 25 per millilitre equal to or greater than 10 µm and does not exceed 3 per
millilitre equal to or greater than 25 µm.
 Test 1.B – Solutions for infusion or solutions for injection supplied in containers with a nominal
content of less than 100 mL
 The preparation complies with the test if the average number of particles present in the units
tested does not exceed 6000 per container equal to or greater than 10 µm and does not exceed
600 per container equal to or greater than 25 µm.
39
visible Particles
 Method 2. Microscopic particle count

test
 Use a suitable binocular microscope, filter assembly for retaining particulate
contamination and membrane filter for examination.
 The microscope is equipped with an ocular micrometer calibrated with an
objective micrometer, a mechanical stage capable of holding and traversing
the entire filtration area of the membrane filter, 2 suitable illuminators to
provide episcopic illumination in addition to oblique illumination, and is
adjusted to 100 ± 10 magnifications.
 The ocular micrometer is a circular diameter graticule (see Figure 2.9.19.-1.)
and consists of a large circle divided by crosshairs into quadrants,
transparent and black reference circles 10 µm and 25 µm in diameter at 100
magnifications, and a linear scale graduated in 10 µm increments. It is
calibrated using a stage micrometer that is certified by either a domestic or
international standard institution. A relative error of the linear scale of the
graticule within ± 2 per cent is acceptable. The large circle is designated the
graticule field of view (GFOV).
40
visible Particles
 2 illuminators are required. One is an episcopic

brightfield illuminator internal to the microscope,
the other is an external, focusable auxiliary
illuminator adjustable to give reflected oblique
illumination at an angle of 10-20°.
 The filter assembly for retaining particulate
contamination consists of a filter holder made of
glass or other suitable material, and is equipped with
a vacuum source and a suitable membrane filter.
 The membrane filter is of suitable size, black or dark
grey in colour, non-gridded or gridded, and 1.0 µm or
finer in nominal pore size.
41
visible Particles
42
visible Particles
 General precautions
 The test is carried out under conditions limiting particulate contamination,
preferably in a laminar-flow cabinet.
 Very carefully wash the glassware and filter assembly used, except for the
membrane filter, with a warm detergent solution and rinse with abundant
amounts of water to remove all traces of detergent. Immediately before
use, rinse both sides of the membrane filter and the equipment from top to
bottom, outside and then inside, with particle-free water R.
 In order to check that the environment is suitable for the test, that the
glassware and the membrane filter are properly cleaned and that the water
to be used is particle-free, the following test is carried out: determine the
particulate contamination of a 50 mL volume of particle-free water
R according to the method described below. If more than 20 particles 10 µm
or larger in size or if more than 5 particles 25 µm or larger in size are present
within the filtration area, the precautions taken for the test are not
sufficient. The preparatory steps must be repeated until the environment,
glassware, membrane filter and water are suitable for the test.
43
visible Particles
 Method
 Mix the contents of the samples by slowly inverting the container 20
times successively. If necessary, cautiously remove the sealing closure.
Clean the outer surfaces of the container opening using a jet of particle-
free water Rand remove the closure, avoiding any contamination of the
contents.
 For large-volume parenterals, single units are tested. For small-volume
parenterals less than 25 mL in volume, the contents of 10 or more units
are combined in a cleaned container; where justified and authorised, the
test solution may be prepared by mixing the contents of a suitable
number of vials and diluting to 25 mL with particle-free water R or with
an appropriate solvent without contamination of particles when particle-
free water R is not suitable. Small-volume parenterals having a volume
of 25 mL or more may be tested individually.
 Powders for parenteral administration are constituted with particle-free
water R or with an appropriate solvent without contamination of
particles when particle-free water R is not suitable.
44
visible Particles
 The number of test specimens must be adequate to provide a statistically sound
assessment. For large-volume parenterals or for small-volume parenterals
having a volume of 25 mL or more, fewer than 10 units may be tested, based on
an appropriate sampling plan.
 Wet the inside of the filter holder fitted with the membrane filter with several
millilitres of particle-free water R. Transfer to the filtration funnel the total
volume of a solution pool or of a single unit, and apply vacuum. If needed, add
stepwise a portion of the solution until the entire volume is filtered. After the last
addition of solution, begin rinsing the inner walls of the filter holder by using a
jet of particle-free water R. Maintain the vacuum until the surface of the
membrane filter is free from liquid. Place the filter in a Petri dish and allow the
filter to air-dry with the cover slightly ajar. After the filter has been dried, place
the Petri dish on the stage of the microscope, scan the entire membrane filter
under the reflected light from the illuminating device, and count the number of
particles that are equal to or greater than 10 µm and the number of particles that
are equal to or greater than 25 µm. Alternatively, partial filter count and
determination of the total filter count by calculation is allowed. Calculate the
mean number of particles for the preparation to be examined.
45
visible Particles
 The particle sizing process with the use of the circular diameter graticule is
carried out by transforming mentally the image of each particle into a circle
and then comparing it to the 10 µm and 25 µm graticule reference circles.
Thereby the particles are not moved from their initial locations within the
graticule field of view and are not superimposed on the reference circles for
comparison. The inner diameter of the transparent graticule reference
circles is used to size white and transparent particles, while dark particles
are sized by using the outer diameter of the black opaque graticule
reference circles.
 In performing the microscopic particle count test do not attempt to size or
enumerate amorphous, semi-liquid, or otherwise morphologically indistinct
materials that have the appearance of a stain or discoloration on the
membrane filter. These materials show little or no surface relief and present
a gelatinous or film-like appearance. In such cases the interpretation of
enumeration may be aided by testing a sample of the solution by the light
obscuration particle count test.
46
visible Particles
 Evaluation
 For preparations supplied in containers with a nominal volume of more than 100 mL,
apply the criteria of test 2.A.
 For preparations supplied in containers with a nominal volume of less than 100 mL,
apply the criteria of test 2.B.
 ♦For preparations supplied in containers with a nominal volume of 100 mL, apply the
criteria of test 2.B.
 Test 2.A – Solutions for infusion or solutions for injection supplied in containers with a
nominal content of more than 100 mL
 The preparation complies with the test if the average number of particles present in
the units tested does not exceed 12 per millilitre equal to or greater than 10 µm and
does not exceed 2 per millilitre equal to or greater than 25 µm.
 Test 2.B – Solutions for infusion or solutions for injection supplied in containers with a
nominalcontent of less than 100 mL
 The preparation complies with the test if the average number of particles present in
the units tested does not exceed 3000 per container equal to or greater than 10 µm
and does not exceed 300 per container equal to or greater than 25 µm.
47
Appendix XIII B. Particulate
Contamination: Visible Particles
 Particulate contamination of injections and infusions consists of extraneous, mobile
undissolved particles, other than gas bubbles, unintentionally present in the
solutions.
 The test is intended to provide a simple procedure for the visual assessment of the
quality of parenteral solutions as regards visible particles. Other validated methods
may be used.
 Apparatus
 The apparatus (see Figure 2.9.20.-1) consists of a viewing station comprising:
 — a matt black panel of appropriate size held in a vertical position,
 — a non-glare white panel of appropriate size held in a vertical position next to the
black panel,
 — an adjustable lampholder fitted with a suitable, shaded, white-light source and
with a suitable light diffuser (a viewing illuminator containing two 13 W fluorescent
tubes, each 525 mm in length, is suitable). The intensity of illumination at the viewing
point is maintained between 2000 lux and 3750 lux, although higher values are
preferable for coloured glass and plastic containers.
48
Contamination: Visible Particles (Ph. Eur.
method 2.9.20)
49
Contamination: Visible Particles (Ph. Eur.
method 2.9.20)
 Method
 Remove any adherent labels from the
container and wash and dry the outside.
Gently swirl or invert the container, ensuring
that air bubbles are not introduced, and
observe for about 5 s in front of the white
panel. Repeat the procedure in front of the
black panel. Record the presence of any
particles.
50
Leakage test
 Leakage test is employed to test the package
integrity.
 Package integrity reflects its ability to keep the
product in and to keep potential contamination
out.
 It is because leakage occurs when a discontinuity
exists in the wall of a package that can allow the
passage of gas under pressure or concentration
differential existing across the wall.
 Leakage differs from permeation, which is the
flow of matter through the barrier itself.
51
Leakage test cont’d
Followings are the leak test methods.

 A) VISUAL INSPECTION
 Visual inspection is the easiest leak test method to
perform. But this method is least sensitive. The method is
used for the evaluation of large volume parenterals. To
increase the sensitivity of the method, the visual inspection
of the sample container may be coupled with the
application of vacuum to make leakage more readily
observable. This method is simple and inexpensive.
However, the method is insensitive, operator dependent,
and qualitative.
 Sometimes, the method is used in combination with
pressure and /or temperature cycling to accelerate leakage
to improve sensitivity.
52
 B) BUBBLE TEST
 The test package is submerged in liquids. A differential pressure
is applied on the container. The container is observed for bubbles.
Sometimes, surfactant added liquid is used for immersion of test
package. Any leakage is evident after the application of
differential pressure as the generation of foaming in immersion
liquid. The method is simple and inexpensive. The location of the
leaks can be observed in this method. However, it is relatively
insensitive and the findings are operator dependent and are
qualitative. The optimized conditions can be achieved using a
surfactant immersion fluid along with the dark background and
High intensity lighting. Generation of a differential positive
pressure of 3 psi inside the vial and observation of any leakage
using magnifying glass within a maximum test time of 15
minutes.
53
 C) DYETESTS
 The test container is immersed in a dye bath. Vacuum and
pressure is applied for some time. The container is removed
from the dye bath and washed. The container is then
inspected for the presence of dye either visually or by
means of UV spectroscopy. The dye used may be of blue,
green, yellowish-green color. The dye test can be optimized
by use of a surfactant and or a low viscosity fluid in the dye
solution to increase the capillary migration through the
pores. The dye test is widely accepted in industry and is
approved in drug use. The test is inexpensive and is requires
no special equipment required for visual dye detection.
However, the test is qualitative, destructive and slow. The
test is used for ampoules and vials.
54
 D) VACUUM IONIZATIONTEST
 Vacuum ionization test is useful for testing leakage in the
vials or bottled sealed under vacuum. This test is used for
online testing of the lyophilized products. High voltage,
high frequency field is applied to vials which to cause
residual gas, if present to glow.
 Glow intensity is the function of headspace vacuum level.
The blue glow is the indicative of vacuum while the purple
glow indicative of no vacuum. The sensitivity of the method
is not documented. This test is on-line, rapid and is non
destructive test. However, the proteins present in the test
sample may be decomposed. This method is used for the
lyophilized vials of biopharmaceuticals.
55
Appendix XVI A. Sterility
A. Test for Sterility1
 The test is applied to substances, preparations or articles
which, according to the Pharmacopoeia, are required to be
sterile. However, a satisfactory result only indicates that no
contaminating micro-organism has been found in the sample
examined in the conditions of the test.
 Precautions against microbial contamination
 The test for sterility is carried out under aseptic conditions.
In order to achieve such conditions, the test environment
has to be adapted to the way in which the sterility test is
performed. The precautions taken to avoid contamination
are such that they do not affect any micro-organisms which
are to be revealed in the test. The working conditions in
which the tests are performed are monitored regularly by
appropriate sampling of the working area and by carrying
out appropriate controls.
56
A. Test for Sterility
(Ph. Eur. method 2.6.1) CONT’D
 Culture media and incubation
temperatures
 The following culture media have been found to
be suitable for the test for sterility.
 Fluid thioglycollate medium is primarily
intended for the culture of anaerobic bacteria;
however, it will also detect aerobic bacteria.
 Soya-bean casein digest medium is suitable for
the culture of both fungi and aerobic bacteria.
57
 Fluid thioglycollate medium is to be incubated at
30-35 °C.
 For products containing a mercurial preservative
that cannot be tested by the membrane-
filtration method, fluid thioglycollate medium
incubated at 20-25 °C may be used instead of
soya-bean casein digest medium provided that it
has been validated as described in growth
promotion test.
 Where prescribed or justified and authorised, the
alternative thioglycollate medium may be used.
58
 Soya-bean casein digest medium is to be incubated at 20-25 °C.
 The media used comply with the following tests, carried out before
or in parallel with the test on the product to be examined.
 Sterility
 Incubate portions of the media for 14 days. No growth of micro-
organisms occurs.
 Growth promotion test of aerobes, anaerobes
and fungi
 Test each batch of ready-prepared medium and each batch of
medium prepared either from dehydrated medium or from
ingredients. Suitable strains of micro-organisms are indicated in
Table 2.6.1.-1.
59
60
 Inoculate portions of fluid thioglycollate medium with a small number (not more
than 100 CFU) of the following micro-organisms, using a separate portion of
medium for each of the following species of micro-organism:
 Clostridium sporogenes,
 Pseudomonas aeruginosa,
 Staphylococcus aureus.
 Inoculate portions of soya-bean casein digest medium with a small number (not
more than 100 CFU) of the following micro-organisms, using a separate portion
of medium for each of the following species of micro-organism:
 Aspergillus brasiliensis,
 Bacillus subtilis,
 Candida albicans.
 Incubate for not more than 3 days in the case of bacteria and not more than 5
days in the case of fungi.
 Seed lot culture maintenance techniques (seed-lot systems) are used so that the
viable micro-organisms used for inoculation are not more than 5 passages
removed from the original master seed-lot.
 The media are suitable if a clearly visible growth of the micro-organisms occurs.
61
 Method suitability test
 Carry out a test as described below under Test for sterility of the
product to be examined using exactly the same methods except
for the following modifications.
 Membrane filtration
 After transferring the contents of the container or containers to
be tested to the membrane add an inoculum of a small number
of viable micro-organisms (not more than 100 CFU) to the final
portion of sterile diluent used to rinse the filter.
 Direct inoculation
 After transferring the content of the container or containers to
be tested (for catgut and other surgical sutures for veterinary
use: strands) to the culture medium add an inoculum of a small
number of viable micro-organisms (not more than 100 CFU) to
the medium.
62
 In both cases use the same micro-organisms as those described above under
Growth promotion test of aerobes, anaerobes and fungi. Perform a growth
promotion test as a positive control. Incubate all the containers containing
medium for not more than 5 days.
 If clearly visible growth of micro-organisms is obtained after the incubation,
visually comparable to that in the control vessel without product, either the
product possesses no antimicrobial activity under the conditions of the test or
such activity has been satisfactorily eliminated. The test for sterility may then be
carried out without further modification.
 If clearly visible growth is not obtained in the presence of the product to be
tested, visually comparable to that in the control vessels without product, the
product possesses antimicrobial activity that has not been satisfactorily
eliminated under the conditions of the test. Modify the conditions in order to
eliminate the antimicrobial activity and repeat the method suitability test.
 This method suitability test is performed:
a) when the test for sterility has to be carried out on a new product;
b) whenever there is a change in the experimental conditions of the test.
 The method suitability test may be performed simultaneously with the test for
sterility of the product to be examined.
63
 Test for sterility of the product to be examined
 The test may be carried out using the technique of membrane filtration
or by direct inoculation of the culture media with the product to be
examined. Appropriate negative controls are included. The technique of
membrane filtration is used whenever the nature of the product
permits, that is, for filterable aqueous preparations, for alcoholic or oily
preparations and for preparations miscible with or soluble in aqueous or
oily solvents provided these solvents do not have an antimicrobial effect
in the conditions of the test.
 Membrane filtration
 Use membrane filters having a nominal pore size not greater than 0.45
µm whose effectiveness to retain micro-organisms has been
established. Cellulose nitrate filters, for example, are used for aqueous,
oily and weakly alcoholic solutions and cellulose acetate filters, for
example, for strongly alcoholic solutions. Specially adapted filters may
be needed for certain products, e.g. for antibiotics.
64
 The technique described below assumes that membranes about
50 mm in diameter will be used. If filters of a different diameter
are used the volumes of the dilutions and the washings should be
adjusted accordingly. The filtration apparatus and membrane are
sterilised by appropriate means. The apparatus is designed so
that the solution to be examined can be introduced and filtered
under aseptic conditions; it permits the aseptic removal of the
membrane for transfer to the medium or it is suitable for carrying
out the incubation after adding the medium to the apparatus
itself.
 Aqueous solutions If appropriate, transfer a small quantity of a
suitable, sterile diluent such as a 1 g/L neutral solution of meat or
casein peptone pH 7.1 ± 0.2 onto the membrane in the apparatus
and filter. The diluent may contain suitable neutralising
substances and/or appropriate inactivating substances for
example in the case of antibiotics.
65
 Transfer the contents of the container or containers to be tested
to the membrane or membranes, if necessary after diluting to
the volume used in the method suitability test with the chosen
sterile diluent but in any case using not less than the quantities of
the product to be examined prescribed in Table 2.6.1.-2. Filter
immediately. If the product has antimicrobial properties, wash
the membrane not less than 3 times by filtering through it each
time the volume of the chosen sterile diluent used in the method
suitability test. Do not exceed a washing cycle of 5 times 100 mL
per filter, even if during the method suitability test it has been
demonstrated that such a cycle does not fully eliminate the
antimicrobial activity. Transfer the whole membrane to the
culture medium or cut it aseptically into 2 equal parts and
transfer one half to each of 2 suitable media. Use the same
volume of each medium as in the method suitability test.
Alternatively, transfer the medium onto the membrane in the
apparatus. Incubate the media for not less than 14 days.
66
67
 Soluble solids Use for each medium not less than the quantity prescribed
in Table 2.6.1.-2 of the product dissolved in a suitable solvent such as the solvent
provided with the preparation, water for injections, saline or a 1 g/L neutral
solution of meat or casein peptone and proceed with the test as described above
for aqueous solutions using a membrane appropriate to the chosen solvent.
 Oils and oily solutions Use for each medium not less than the quantity
of the product prescribed in Table 2.6.1.-2. Oils and oily solutions of sufficiently
low viscosity may be filtered without dilution through a dry membrane. Viscous
oils may be diluted as necessary with a suitable sterile diluent such as isopropyl
myristate shown not to have antimicrobial activity in the conditions of the test.
Allow the oil to penetrate the membrane by its own weight then filter, applying
the pressure or suction gradually. Wash the membrane at least 3 times by
filtering through it each time about 100 mL of a suitable sterile solution such as 1
g/L neutral meat or casein peptone containing a suitable emulsifying agent at a
concentration shown to be appropriate in the method suitability test, for
example polysorbate 80 at a concentration of 10 g/L. Transfer the membrane or
membranes to the culture medium or media or vice versa as described above for
aqueous solutions, and incubate at the same temperatures and for the same
times.
68
 Direct inoculation of the culture

medium
 Transfer the quantity of the preparation to be examined
prescribed in Table 2.6.1.-2 directly into the culture medium so
that the volume of the product is not more than 10 per cent of
the volume of the medium, unless otherwise prescribed.
 If the product to be examined has antimicrobial activity, carry out
the test after neutralising this with a suitable neutralising
substance or by dilution in a sufficient quantity of culture
medium. When it is necessary to use a large volume of the
product it may be preferable to use a concentrated culture
medium prepared in such a way that it takes account of the
subsequent dilution. Where appropriate, the concentrated
medium may be added directly to the product in its container.
69
 Oily liquids Use media to which have been added a suitable
emulsifying agent at a concentration shown to be appropriate in the
method suitability test, for example polysorbate 80 at a
concentration of 10 g/L.
 Incubate the inoculated media for not less than 14 days. Observe the
cultures several times during the incubation period. Shake cultures
containing oily products gently each day. However when fluid
thioglycollate medium is used for the detection of anaerobic micro-
organisms keep shaking or mixing to a minimum in order to
maintain anaerobic conditions.
70
 Observation and
interpretation of results
 At intervals during the incubation period and at its conclusion, examine
the media for macroscopic evidence of microbial growth. If the material
being tested renders the medium turbid so that the presence or absence
of microbial growth cannot be readily determined by visual
examination, 14 days after the beginning of incubation transfer portions
(each not less than 1 mL) of the medium to fresh vessels of the same
medium and then incubate the original and transfer vessels for not less
than 4 days.
 If no evidence of microbial growth is found, the product to be examined
complies with the test for sterility. If evidence of microbial growth is
found the product to be examined does not comply with the test for
sterility, unless it can be clearly demonstrated that the test was invalid
for causes unrelated to the product to be examined.
71
 The test may be considered invalid only if one or more of the following
conditions are fulfilled:
a) the data of the microbiological monitoring of the sterility testing
facility show a fault;
b) a review of the testing procedure used during the test in question
reveals a fault;
c) microbial growth is found in the negative controls;
d) after determination of the identity of the micro-organisms 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 procedure.
 If the test is declared to be invalid it is repeated with the same number
of units as in the original test.
 If no evidence of microbial growth is found in the repeat test the product
examined complies with the test for sterility. If microbial growth is found
in the repeat test the product examined does not comply with the test
for sterility.
72
 Application of the test to parenteral preparations,
ophthalmic and other non-injectable preparations
required to comply with the test for sterility
 When using the technique of membrane filtration, use, whenever
possible, the whole contents of the container, but not less than
the quantities indicated in Table 2.6.1.-2, diluting where
necessary to about 100 mL with a suitable sterile solution, such
as 1 g/L neutral meat or casein peptone.
 When using the technique of direct inoculation of media, use the
quantities shown in Table 2.6.1.-2, unless otherwise justified and
authorised. The tests for bacterial and fungal sterility are carried
out on the same sample of the product to be examined. When
the volume or the quantity in a single container is insufficient to
carry out the tests, the contents of 2 or more containers are used
to inoculate the different media.
73
 Minimum number of items to be tested
 The minimum number of items to be tested in relation to
the size of the batch is given in Table 2.6.1.-3.

74
LAL Bacterial Endotoxins Test
 The LAL (limulus amebocyte lysate) Assay is an in
vitro assay used to detect the presence and
concentration of bacterial endotoxins in drugs and
biological products.
 Endotoxins, which are a type of pyrogen, are
lipopolysaccharides present in the cell walls of gram-
negative bacteria.
 Limulus amebocyte lysate (LAL) is an aqueous extract
of blood cells (amoebocytes) from the horseshoe
crab, Limulus polyphemus.
 LAL reacts with bacterial
endotoxin or lipopolysaccharide (LPS).
 This reaction is the basis of the LAL test, which is used
for the detection and quantification of bacterial
endotoxins.
75
LAL Bacterial Endotoxins Test cont’d
 The use of LAL for endotoxin detection was derived from

Bang’s observation that the infection of Limulus
polyphemus, the horseshoe crab, induced by GNB, results in
extensive intravascular clotting and death.
 Later on, Levin and Bang demonstrated that the
extracellular coagulation of Limulus hemolymph (blood) was
caused by an reaction between endoxin and a coagulative
protein in amebocytes, circulating in Limulus hemolymph.
 In a subsequent study, Levin et al. developed a sensitive
assay for endotoxin in human plasma using the material
lysed from Limulus amebocytes.
 Young, Levin and Prendergast then isolated, purified and
described the LAL coagulative protein and proved that the
reaction between lysate and endotoxin is of enzymatic
nature.
76
LAL Bacterial Endotoxins Test cont’d
Advantages of the LAL test:

 Small amount of the tested sample is required for the LAL test.
 Several samples could be tested daily.
 Only one trained worker is needed to carry on the tests.
 LAL test is more economical, despite higher initial expenses.
 A lower level of endotoxins could be detected than in rabbit pyrogen
test.
 The testing method is highly standardised.
 Data for the final evaluation of the test could be obtained relatively
quickly.
Disadvantages of the LAL test:
 High emphasis on the precise implementation of the test procedure.
 Slight disturbances could influence the outcome of the test.
77
Appendix XIV C. Test for Bacterial
Endotoxins (LAL Test)
 The test for bacterial endotoxins (BET) is used to detect or quantify endotoxins from
gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus). There are 3 techniques for this test: the gel-
clot technique, which is based on gel formation; the turbidimetric technique, based
on the development of turbidity after cleavage of an endogenous substrate; and the
chromogenic technique, based on the development of colour after cleavage of a
synthetic peptide-chromogen complex.
 The following 6 methods are described in the present chapter:
 Method A. Gel-clot method: limit test
 Method B. Gel-clot method: quantitative test
 Method C. Turbidimetric kinetic method
 Method D. Chromogenic kinetic method
 Method E. Chromogenic end-point method
 Method F. Turbidimetric end-point method
 Proceed by any of the 6 methods for the test. In the event of doubt or dispute, the
final decision is made based upon method A unless otherwise indicated in the
monograph.
 The test is carried out in a manner that avoids endotoxin contamination.
78
Appendix XIV C. Test for Bacterial Endotoxins
(LAL Test)
(Ph. Eur. method 2.6.14) cont’d
 1. Apparatus
 Depyrogenate all glassware and other heat-stable apparatus in a hot-air oven
using a validated process. A commonly used minimum time and temperature is
30 minutes at 250 °C. If employing plastic apparatus, such as microtitre plates
and pipette tips for automatic pipetters, use apparatus shown to be free of
detectable endotoxin and which does not interfere in the test.
 2. Reagents, test solutions
 (1) Amoebocyte lysate is a lyophilised product obtained from amoebocyte
lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).
This reagent refers only to a product manufactured in accordance with the
regulations of the competent authority.
 (2) Lysate solution: Dissolve amoebocyte lysate in water for BET or in a
buffer, as recommended by the lysate manufacturer, by gentle stirring. Store the
reconstituted lysate, refrigerated or frozen, as indicated by the manufacturer.
 (3) Water for BET (water for bacterial endotoxins test) Water for
injections R or water produced by other procedures that shows no reaction with
the lysate employed at the detection limit of the reagent.
79
(LAL Test)
 3. Preparation of the standard endotoxin stock

solution
 The standard endotoxin stock solution is prepared from an endotoxin reference
standard that has been calibrated against the International Standard.
 Endotoxin is expressed in International Units (IU).
 NOTE: one International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.).
 Follow the specifications in the package leaflet and on the label for preparation and
storage of the standard endotoxin stock solution.
 4. Preparation of the standard endotoxin solutions

 After vigorously mixing the standard endotoxin stock solution, prepare appropriate
serial dilutions of this solution using water for BET.
 Use the solutions as soon as possible to avoid loss of activity by adsorption.
80
(LAL Test)
 5. Preparation of the test solutions

 Prepare the test solutions by dissolving or diluting active substances or
medicinal products using water for BET. Some substances or
preparations may be more appropriately dissolved or diluted in other
aqueous solutions. If necessary, adjust the pH of the test solution (or
dilution thereof) so that the pH of the mixture of the lysate and test
solution falls within the pH range specified by the lysate manufacturer,
usually 6.0 to 8.0.
 6. Determination of the Maximum Valid
Dilution
 The Maximum Valid Dilution (MVD) is the maximum allowable dilution
of a sample at which the endotoxin limit can be determined. Determine
the MVD using the following formulae:
81
(LAL Test)
 Endotoxin limit :
 The endotoxin limit for active substances administered parenterally, defined
on the basis of dose, is equal to:
K
M
 K= threshold pyrogenic dose of endotoxin per kilogram of body
mass,
 M=maximum recommended bolus dose of product per kilogram of
body mass.
 When the product is to be injected at frequent intervals or infused
continuously, M is the maximum total dose administered in a single hour
period.
 The endotoxin limit for active substances administered parenterally is
specified in units such as IU/mL, IU/mg, IU/Unit of biological activity, etc., in
monographs.
82
(LAL Test)
 Concentration of test solution:

 — mg/mL if the endotoxin limit is specified by
mass (IU/mg),
 — Units/mL if the endotoxin limit is specified by
unit of biological activity (IU/Unit),
 — mL/mL if the endotoxin limit is specified by
volume (IU/mL).
 λ=the labelled lysate sensitivity in the gel-clot
technique (IU/mL) or the lowest concentration
used in the standard curve of the turbidimetric or
chromogenic techniques.
83
(LAL Test)
 7. Gel-clot technique
(Methods A and B)
 The gel-clot technique allows detection or
quantification of endotoxins and is based on clotting
of the lysate in the presence of endotoxins. The
minimum concentration of endotoxins required to
cause the lysate to clot under standard conditions is
the labelled lysate sensitivity. To ensure both the
precision and validity of the test, confirm the
labelled lysate sensitivity and perform the test for
interfering factors as described under 1. Preparatory
testing.
84
(LAL Test)
 1. Preparatory testing
 (i) Confirmation of the labelled lysate sensitivity
 Confirm in 4 replicates the labelled sensitivity λ, expressed in IU/mL, of the
lysate solution prior to use in the test. Confirmation of the lysate sensitivity is
carried out when a new lot of lysate is used or when there is any change in the
test conditions which may affect the outcome of the test.
 Prepare standard solutions of at least 4 concentrations equivalent to 2λ, λ, 0.5λ
and 0.25λ by diluting the standard endotoxin stock solution with water for BET.
 Mix a volume of the lysate solution with an equal volume of 1 of the standard
solutions (such as 0.1 mL aliquots) in each tube. When single test vials or
ampoules containing lyophilised lysate are employed, add solutions of standards
directly to the vial or ampoule. Incubate the reaction mixture for a constant
period according to the recommendations of the lysate manufacturer (usually at
37 ± 1 °C for 60 ± 2 min), avoiding vibration. Test the integrity of the gel: for
tubes, take each tube in turn directly from the incubator and invert it through
approximately 180° in one smooth motion. If a firm gel has formed that remains
in place upon inversion, record the result as positive. A result is negative if an
intact gel is not formed.
85
(LAL Test)
 The test is considered valid when the lowest concentration of the

standard solutions shows a negative result in all replicate tests.
 The end-point is the lowest concentration in the series of decreasing
concentrations of standard endotoxin that clots the lysate. Determine
the geometric mean end-point concentration by calculting the mean of
the logarithms of the end-point concentrations of the 4 dilution series,
take the antilogarithm of this value, as indicated by the following
expression:
 Σe = sum of the log end-point concentrations of the dilution series used,

 f = number of replicates.
 The geometric mean end-point concentration is the measured
sensitivity of the lysate solution (IU/mL). If this is not less than 0.5λ and
not more than 2λ, the labelled sensitivity is confirmed and is used in the
tests performed with this lysate.
86
(LAL Test)
 (ii)Test for interfering factors

 Prepare solutions A, B, C and D as shown in Table 2.6.14.-1, and use
the test solutions at a dilution less than the MVD, not containing any
detectable endotoxins, operating as described under 1. Preparatory
testing, (i) Confirmation of the labelled lysate sensitivity.
 The geometric mean end-point concentrations of solutions B and C
are determined using the expression described in 1. Preparatory
testing, (i) Confirmation of the labelled lysate sensitivity.
 The test for interfering factors must be repeated when any changes
are made to the experimental conditions that are likely to influence
the result of the test.
 The test is considered valid when all replicates of solutions A and D
show no reaction and the result of solution C confirms the labelled
lysate sensitivity.
87
Appendix XIV C. Test for Bacterial Endotoxins (LAL Test)
88
(LAL Test)
 If the sensitivity of the lysate determined with solution B is not less

than 0.5λ and not greater than 2λ, the test solution does not contain
interfering factors under the experimental conditions used.
Otherwise, the test solution interferes with the test.
 If the preparation being examined interferes with the test at a
dilution less than the MVD, repeat the test for interfering factors
using a greater dilution, not exceeding the MVD. The use of a more
sensitive lysate permits a greater dilution of the preparation being
examined and this may contribute to the elimination of interference.
 Interference may be overcome by suitable validated treatment, such
as filtration, neutralisation, dialysis or heat treatment. To establish
that the treatment chosen effectively eliminates interference
without loss of endotoxins, repeat the test for interfering factors
using the preparation being examined to which the standard
endotoxin has been added and which has then been submitted to
the chosen treatment.
89
(LAL Test)
 2. Limit test (Method A)

 (i) Procedure
 Prepare solutions A, B, C and D as shown in Table 2.6.14.-2, and perform the test on
these solutions following the procedure described under 1. Preparatory testing, (i)
Confirmation of the labelled lysate sensitivity.

 Prepare solution A and solution B (positive product control) using a dilution not
greater than the MVD and treatments as described in 1. Preparatory testing, (ii) Test
for interfering factors. Solutions B and C (positive controls) contain the standard
endotoxin at a concentration corresponding to twice the labelled lysate sensitivity.
Solution D (negative control) consists of water for BET.
90
(LAL Test)
 (ii) Interpretation
 The test is considered valid when both replicates of solution B and C are
positive and those of solution D are negative.
 When a negative result is found for both replicates of solution A, the
preparation being examined complies with the test.
 When a positive result is found for both replicates of solution A, the
preparation being examined does not comply with the test.
 When a positive result is found for one replicate of solution A and a
negative result is found for the other, repeat the test. In the repeat test,
the preparation being examined complies with the test if a negative
result is found for both replicates of solution A. The preparation does not
comply with the test if a positive result is found for one or both
replicates of solution A.
 However, if the preparation does not comply with the test at a dilution
less than the MVD, the test may be repeated using a greater dilution,
not exceeding the MVD.
91
(LAL Test)
 3. Quantitative test (Method B)

 (i) Procedure
 The test quantifies bacterial endotoxins in the test solution by titration
to an end-point. Prepare solutions A, B, C and D as shown in Table
2.6.14.-3, and test these solutions according to the procedure described
under 1. Preparatory testing, (i) Confirmation of the labelled lysate
sensitivity.
 (ii) Calculation and interpretation
 The test is considered valid when the following 3 conditions are met:
 (a) both replicates of solution D (negative control) are negative,
 (b) both replicates of solution B (positive product control) are positive,
 (c) the geometric mean end-point concentration of solution C is in the
range of 0.5λ to 2λ.
92
(LAL Test)
 To determine the endotoxin concentration of solution A, calculate

the end-point concentration for each replicate, by multiplying each
end-point dilution factor by λ.
 The endotoxin concentration in the test solution is the end-point
concentration of the replicates. If the test is conducted with a
diluted test solution, calculate the concentration of endotoxin in the
original solution by multiplying the result by the dilution factor.
 If none of the dilutions of the test solution is positive in a valid test,
report the endotoxin concentration as less than λ (or, if a diluted
sample was tested, report as less than the lowest dilution factor of
the sample × λ). If all dilutions are positive, the endotoxin
concentration is reported as equal to or greater than the largest
dilution factor multiplied by λ (e.g. in Table 2.6.14.-3, the initial
dilution factor × 8 × λ).
 The preparation being examined meets the requirements of the test
if the endotoxin concentration in both replicates is less than that
specified in the monograph.
93
94
(LAL Test)
 8. Photometric quantitative techniques

 (Methods C, D, E and F)
 1.Turbidimetric technique (Methods C and F)

 This technique is a photometric test to measure the
increase in turbidity. Based on the test principle employed,
this technique may be classified as being either the end-
point-turbidimetric test or the kinetic-turbidimetric test.
 The end-point-turbidimetric test (Method F) is based on the
quantitative relationship between the endotoxin
concentration and the turbidity (absorbance or
transmission) of the reaction mixture at the end of an
incubation period.
95
(LAL Test)
 The kinetic-turbidimetric test (Method C) is a

method to measure either the time (onset
time) needed for the reaction mixture to
reach a predetermined absorbance or
transmission, or the rate of turbidity
development.
 The test is carried out at the incubation
temperature recommended by the lysate
manufacturer (usually 37 ± 1 °C).
96
(LAL Test)
 2. Chromogenic technique
(Methods D and E)
 This technique is used to measure the chromophore released from a
suitable chromogenic peptide by the reaction of endotoxins with the
lysate. Depending on the test principle employed, this technique may be
classified as being either the end-point-chromogenic test or the kinetic-
chromogenic test.
 The end-point-chromogenic test (Method E) is based on the
quantitative relationship between the endotoxin concentration and the
quantity of chromophore released at the end of an incubation period.
 The kinetic-chromogenic test (Method D) measures either the time
(onset time) needed for the reaction mixture to reach a predetermined
absorbance, or the rate of colour development.
 The test is carried out at the incubation temperature recommended by
the lysate manufacturer (usually 37 ± 1 °C).
97
(LAL Test)
 3. Preparatory testing
 To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory
tests are conducted to show that the criteria for the standard curve are satisfied and that the test
solution does not interfere with the test.
 Validation of the test method is required when any changes are made to the experimental
conditions that are likely to influence the result of the test.
 (i) Assurance of criteria for the standard curve
 The test must be carried out for each lot of lysate reagent.
 Using the standard endotoxin solution, prepare at least 3 endotoxin concentrations within the
range indicated by the lysate manufacturer to generate the standard curve. Perform the test using
at least 3 replicates of each standard endotoxin solution as recommended by the lysate
manufacturer (volume ratios, incubation time, temperature, pH, etc.).
 If the desired range is greater than 2 log in the kinetic methods, additional standards must be
included to bracket each log increase in the range of the standard curve.
 The absolute value of the correlation coefficient, | r |, must be greater than or equal to 0.980, for
the range of endotoxin concentrations set up.
98
(LAL Test)
 (ii)Test for interfering factors

 Select an endotoxin concentration at or near
the middle of the endotoxin standard curve.
 Prepare solutions A, B, C and D as shown in
Table 2.6.14.-4. Perform the test on at least 2
replicates of these solutions as recommended
by the lysate manufacturer (volume of test
solution and lysate solution, volume ratio of
test solution to lysate solution, incubation
time, etc.).
99
100
(LAL Test)
 The test is considered valid when the following conditions

are met:
 — the absolute value of the correlation coefficient of the
standard curve generated using solution C is greater than or
equal to 0.980;
 — the result with solution D does not exceed the limit of
the blank value required in the description of the lysate
reagent employed, or it is less than the endotoxin detection
limit of the lysate reagent employed.
 Calculate the mean recovery of the added endotoxin by
subtracting the mean endotoxin concentration in the
solution (if any) (solution A, Table 2.6.14.-4) from that in the
solution containing the added endotoxin (solution B, Table
2.6.14.-4).
101
(LAL Test)
 The test solution is considered free of interfering factors if under

the conditions of the test, the measured concentration of the
endotoxin added to the test solution is within 50-200 per cent of
the known added endotoxin concentration, after subtraction of
any endotoxin detected in the solution without added endotoxin.
 When the endotoxin recovery is out of the specified range, the
test solution is considered to contain interfering factors. Repeat
the test using a greater dilution, not exceeding the MVD.
Furthermore, interference of the test solution or diluted test
solution not to exceed the MVD may be eliminated by suitable
validated treatment, such as filtration, neutralisation, dialysis or
heat treatment. To establish that the treatment chosen
effectively eliminates interference without loss of endotoxins,
repeat the test for interfering factors using the preparation being
examined to which the standard endotoxin has been added and
which has then been submitted to the chosen treatment.
102
(LAL Test)
 4.Test
 (i) Procedure
 Follow the procedure described in 3. Preparatory testing, (ii) Test for interfering factors.
 (ii) Calculation
 Calculate the endotoxin concentration of each replicate of solution A using the standard curve
generated by the positive control solution C.
 The test is considered valid when the following 3 requirements are met:
 (1) the results obtained with solution C comply with the requirements for validation defined under 3.
Preparatory testing, (i) Assurance of criteria for the standard curve,
 (2) the endotoxin recovery, calculated from the endotoxin concentration found in solution B after
subtracting the endotoxin concentration found in solution A, is within the range of 50-200 per cent,
 (3) the result obtained with solution D (negative control) does not exceed the limit of the blank value
required in the description of the lysate employed, or it is less than the endotoxin detection limit of the
lysate reagent employed.
 (iii) Interpretation
 The preparation being examined complies with the test if the mean endotoxin concentration of the
replicates of solution A, after correction for dilution and concentration, is less than the endotoxin limit
for the product.
103

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QUALITY CONTROL OF

 Parenteral preparations may contain excipients such as solvents,

3) Powders for injections

 The test consists of measuring the rise in body temperature

 Animals' quarters: Keep the rabbits individually in a

 Determination of the initial and maximum

 Interpretation of results: Having carried out the test

 APPARATUS AND DILUENTS

 Unless otherwise specified in the individual monograph, inject

 TEST INTERPRETATION AND CONTINUATION

 Immunosera (antisera) and vaccines

 Particulate contamination of injections and infusions

 Not all parenteral preparations can be examined for sub-visible particles by

 Method 1. Light obscuration particle

 Powders for parenteral administration are reconstituted

 Method 2. Microscopic particle count

 2 illuminators are required. One is an episcopic

Followings are the leak test methods.

 Direct inoculation of the culture

 The use of LAL for endotoxin detection was derived from

Advantages of the LAL test:

 3. Preparation of the standard endotoxin stock

 4. Preparation of the standard endotoxin solutions

 5. Preparation of the test solutions

 Concentration of test solution:

 The test is considered valid when the lowest concentration of the

 Σe = sum of the log end-point concentrations of the dilution series used,

 (ii)Test for interfering factors

 If the sensitivity of the lysate determined with solution B is not less

 2. Limit test (Method A)

 3. Quantitative test (Method B)

 To determine the endotoxin concentration of solution A, calculate

 8. Photometric quantitative techniques

 1.Turbidimetric technique (Methods C and F)

 The kinetic-turbidimetric test (Method C) is a

 (ii)Test for interfering factors

 The test is considered valid when the following conditions

 The test solution is considered free of interfering factors if under

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