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Journal of Validation Technology

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 Validation and Operation of a
Sterility Testing Isolator: A Study
Proposal | IVT
By
Tim Sandle
and Nancy Tours

Mar 8, 2013 11:26 am PST

INTRODUCTION

The sterility test is an important end-product test for medicinal products that are required to be sterile (including those that are aseptically filled
and many that are terminally sterilized, unless regulatory approval has been granted for parametric release) (1). The sterility test, as a culture
based method, is described in the harmonized pharmacopoeias: United States Pharmacopeia (USP) (<71>), European Pharmacopeia (EP)
(2.6.1), and Japanese Pharmacopeia (4.06) (2). The US Food and Drug Administration, through the Code of Federal Regulations, allows for
alternative rapid methods to be used as a replacement to the culture method. The focus of this paper is the culture-based method.

One of the concerns with the sterility test, especially when high-value products are handled, is the risk of false positives that can lead to
expensive and lengthy failure investigations (and where proving a false positive is extremely difficult, therefore, the outcome can often be batch
rejection). A false positive can be described as a contaminating microorganism that has been transferred into the test media through cross-
contamination by personnel or from the test environment; in contrast, to microbial contamination being present in the product under test.

Where the sterility test is conducted within a conventional cleanroom, the risk of cross-contamination is arguably higher than if the test is
conducted within an isolator (3). The advent of isolation technology for sterility testing, since the 1990s, has, in theory, lowered the incidence of
false positives (4). The use of isolators for sterility testing is described in USP <1208> (5).

An isolator is an arrangement of physical barriers that are integrated so that the workspace (an enclosed environment) within the isolator is
sealed from the outside environment. The barrier to the outside is measured in terms of a routine leak test and the maintenance of pressure
differential, both of which are assessed within specified limits. The isolator allows manipulations to be performed within the workspace in a way
that does not compromise the integrity of the isolator (usually through gloveports) (6).

To maintain the barrier with the external environment, isolators consist of either a flexible film for the outer wall or have a solid wall envelope. With
the exception of handling certain medicinal products, such as cytotoxic drugs, isolators used for the sterility test, when operative, are at a positive
pressure relative to the room and have a high efficiency particulate air (HEPA) filtered airflow. The pressure level of the isolators is normally
monitored by pressure alarms and readings.

As a general concept, the aims of an isolator are to:

 Provide a testing environment free from contamination, through routine sanitization using a validated cycle and confirmed by environmental
monitoring.
 Enable the isolation between the operator and the process.
 Provide a non-corrosive, drainable, and easily cleaned enclosure.
 Have doors to provide access to the equipment inside the isolator.
 Have gloveports situated to allow users access to machinery and product during testing.
 Have a mechanism by which materials can be sanitized and then be securely placed within the isolator. This can be through the use of transfer
isolators or through the use of a rapid gassing port. Some users elect to load all materials into the isolator each time and run a full sanitization
cycle.
 Provide an in-feed opening to introduce testing materials and product to the system;
 Be equipped with an exit opening to allow finished product and test materials to exit the system (7).

Isolators are most commonly sanitized using hydrogen peroxide vapor (a surface disinfectant) or peracetic acid. Isolators are said to „disinfect‟ or
to 'sanitize' rather than 'sterilize' because absolute sterility cannot be demonstrated. Sanitization, in this context, describes the reduction of a
number of microorganisms within the clean environment as demonstrated through the use of biological indicators in validation studies for different
isolator cycles.

The maintenance of isolators is important, especially in ensuring the minimization of leakage (where the leakage of air out could lead to the
ingress of air from the less-clean surrounding room environment) maintenance of isolator integrity and filter efficiency. Isolators are normally
tested biannually for HEPA filter leaks, air velocity, particle counts, induction leak tests, and differential pressure. All of these parameters must fall
within pre-defined set ranges.

This paper examines the important operational criteria for an isolator used for sterility testing. To contextualize operational criteria, the paper also
addresses aspects of validation and the maintenance program. This examination takes the form of a study proposal. The isolator system
discussed consists of two solid wall Type 1 positive pressure isolators linked by a rapid gassing port (8). The gassing port and the isolators are
attached to a gas generator. Although other isolator systems will be different, the aim of the paper is to discuss general requirements and to
provide some good practice guidance.

Figure 1: Photograph of the Isolator System under Evaluation (photograph Tim Sandle©).

SELECTION OF GASSING AGENT

The gassing agent used for the isolator system in this study proposal was hydrogen peroxide in the vapor state. This was based on this agent
having demonstrable and effective microcidal properties. Hydrogen peroxide has strong oxidizing properties (9). Hydrogen peroxide vapor
disinfection employs a free radical reaction mechanism to facilitate microbicidal action (10) and it is very difficult for microorganisms to develop
resistance to free radical attack (11). The vapor is typically generated by flash evaporation from 30% (wt/wt) liquid hydrogen peroxide (this is the
concentration of the hydrogen peroxide employed for the isolator system in this study proposal) and is delivered into the isolator system via a
rotating nozzle.

The mechanism of molecule distribution and gas-to-liquid phase change process is called 'micro-condensation'. Micro-condensation formation is a
simple and efficient way of delivering the agent to all exposed surfaces (12).

Hydrogen peroxide always decomposes (or disproportionates) exothermically into water and oxygen gas spontaneously through the following
equation (13): 2 H 2O2 → 2 H2O + O2.

SANITIZATION CYCLE: THE KEY STEPS

For the operation of an isolator for sterility testing a, number of steps must be followed. It is important to present these steps prior to discussing
the validation and operational requirements of the isolator.

Pre-cycle Disinfection
 Prior to final product items (and other material that is not sterilized and contained within double wrapping) being loaded into the isolator or gassing
port, some users elect to wipe items with 70% isopropyl alcohol (IPA) using a low particulate wipe (observing the required contact time for the
alcohol). Although it could be argued that such a step is not necessary, given the demonstrable kill achieved from biological indicators, some
regulatory agencies have asked for this step to be incorporated. A rationale for the inclusion of this step is that gaseous disinfection requires a
pre-cleaning step. The target of the gaseous disinfection process is surface bio-decontamination and does not match the performance of defined
penetrative sterilization processes detailed in pharmacopeiae. Surface gaseous disinfection requires a starting condition wherein a surface is
visibly clean of soiling. Visible contamination or soiling may occlude microorganisms from the disinfection process and if uncovered in-process
may present a bio-contamination risk. Any surface contamination is removed through the 70% IPA wiping step.

Gassing Port and Disinfection Cycles

The gassing port is a chamber connected to the isolators via interlocking doors; it can be opened to the room environment for loading without
compromising the integrity of the isolators. The gassing port is designed to sanitize the different sterility test loads in a faster time than would be
possible if loads were placed inside an isolator for sanitization. The gassing port is connected to a gas generator. Because hydrogen peroxide in
the passive state is very poor at diffusion, the function of the gas generator is to heat the hydrogen peroxide to produce a vapor and to provide
the generated vapor to the port. The function of the port is to actively distribute the vapor via a distribution nozzle. The function of the rotating
nozzle is to prevent hot spots arising from an uneven distribution of the gas. The dosage of the hydrogen peroxide is controlled by the user prior
to placing the chemical into the gas generator. This will provide a controlled volume of the chemical, provided the correct cycle is selected by the
user.

Sometimes the terms 'gas' and 'vapor' are used interchangeably in literature. The hydrogen peroxide used to decontaminate an isolator is a vapor
and is evaporated by a process called flash evaporation. It is a condensable vapor and not a true gas.

When loading an isolator or port, the presentation of surfaces to the sanitization process is extremely important. Material loads often rely on point
contact support, via wire racking or hangers, to assure exposure to the disinfection agent. Only those load combinations that have been validated
should be used. A sterility test load consists of the final product samples, the sterility test kit, the culture media for the test, rinsing fluids, and
environmental monitoring test plates required to conduct the sterility test.

Sanitization Cycle

The sanitization process occurs by an aqueous solution of hydrogen peroxide being evaporated in such a way as to produce the same weight
ratio in the vapor phase as in the source liquid (starting with 30% wt/wt of hydrogen peroxide, which is evaporated into the heated carrier gas
stream to produce hydrogen peroxide concentrations at a level within an expected range established at the operational qualification [OQ] and
performance qualification [PQ] stages). This vapor is transported to the chamber to be bio-decontaminated in a heated carrier gas (initially sterile
compressed air). Vapors are circulated around the chamber, then through a catalysing filer at the end of sanitization to break down the hydrogen
peroxide.

The sanitization stage (or what is sometimes called „bio-decontamination‟) functions within the chamber by depositing an even layer of hydrogen
peroxide over all surfaces (micro-condensation). According to the relevant Parenteral Drug Association (PDA) Technical Report, bio-
decontamination is “a process that is designed to remove soil (including microorganisms).” (14).

Regarding the micro-condensation process, there has been a long-standing debate as to whether „wet‟ or „dry‟ hydrogen peroxide processes are
the most effective (with the debate centred on rival technologies). Research has found that more than a few microns of molecule-monolayer
deposition (which is invisible micro-condensation) contribute little additional efficacy. The research indicates that the optimum bio-
decontamination processes are between the „wet‟ visible condensation condition (with over injection of vapor) and the invisible gas phase, a „dry‟
process that is specified to remain just below dew point. Optimum kill conditions require dew point to be reached. Achieving dew point is based on
a certain target volume of gas; it is affected by the starting temperature, relative humidity conditions, load and isolator surface temperature, and
the subsequent amount of vapor injected into the target volume (15).

Condensation formation at saturated vapor conditions, past the dew point, is the mechanism that delivers the hydrogen peroxide molecules to all
exposed surfaces. This is physical chemistry and, therefore, has physical parameters of control. Therefore, isolator and gassing port cycle times
should be established and examined for each operation to ensure that the recorded parameters fall within the ranges set during validation.

There are four key steps to ensure an optimal hydrogen peroxide vapor disinfection process:

1. Vaporization of liquid to small molecules–gas phase delivery to target volume.

2. Development of the gas concentration in the target environment to saturated vapor conditions, past dew point, and transition into liquid phase.
3. At saturated vapor conditions, the gas concentration can hold no more molecules, thus, the process of condensation formation and disinfection
agent surface deposition starts.
Micro-condensation forms on surfaces by merging molecules. Initially, nuclei form on any surface contaminants, before full condensation occurs
over the entire available surface, eventually forming a disinfectant monolayer (from gas to liquid phase).
4. Re-evaporation of the surface condensate and removal of residual gas to target endpoint concentration.

The sanitization cycle operates through the following steps:

1. The generator initially dehumidifies the ambient air (conditioning). Here, initial temperature and relative humidity is optimized before vapor
injection. Hot dry air is exchanged with the target environment to achieve required starting humidity conditions.
2. The generator then produces hydrogen peroxide vapor by passing aqueous hydrogen peroxide over a vaporizer. The gas distribution is an active
process controlled through a nozzle for a pre-determined volume of hydrogen peroxide. The gassing phase is validated to deliver a disinfection
agent dose volume that will reach required micro-condensation conditions in the target area and on target surfaces. During validation, an
expected time range for the gassing or dosage phase is determined (based on gassing pump speed); cycle times for routine operations must fall
within this range.
3. The vapor is then circulated at a programmed concentration in the air and held for a set period of time (dwell or hold phase). The dwell time is
optimized to maintain micro-condensation conditions throughout the complete dwell phase for assured disinfectant contact time.
4. After the hydrogen peroxide vapor has circulated in the enclosed space for a pre-defined period of time, it is circulated and broken down into
water and oxygen by a catalytic converter until concentrations of hydrogen peroxide vapor fall to safe levels, at 1 parts per million (ppm) (aeration
phase) (less than 1 ppm is below the occupational exposure level and deemed safe for personnel). Gas residual removal is typically achieved
with integral or supporting HEPA-catalysts and/or supporting dilution via barrier air being vented to the environment.
 Primary process variables for hydrogen peroxide vapor disinfection include:

 Starting relative humidity (RH)

 Surface and environmental temperatures
 Gas distribution for a homogeneous deposition of vapor and resulting surface condensate
 The amount of evaporated hydrogen peroxide solution dosed into the target volume (g/min) to reach saturated vapor conditions and provide a
sufficient monolayer of disinfectant
 Surface area for condensate distribution together with amount of surface absorbency (e.g., packaging).

It is important that these process variables are controlled. The establishment of these parameters is examined below.

VALIDATION OF HYDROGEN PEROXIDE CYCLES

This paper will not go into detail about the validation of the isolator and gassing port (this is a topic that requires a separate analysis);
nonetheless, the parameters set during the validation determine the operational requirements of the isolator when it is used for routine
sanitization cycles (16). Therefore, it is appropriate to discuss validation briefly.

With this study proposal, the gassing cycles were validated and developed using biological indicators. The indicators used were Geobacillus
stearothermophilus spores dried onto stainless steel discs. The carrier used must be inert, promote the even dispersion of spore suspension, and
have a limited effect on the resistance of the microorganism. The metal discs were placed in Tyvek pouches (polyethylene fibre) to create the
biological indicators used to validate the gassing cycle. This met the PDA recommendation that the microorganism used as the biological
indicator should have a higher population and resistance than the bioburden on the item to be decontaminated (the isolator system, contained
within a cleanroom, was located in an environment where the bioburden challenge is low as shown by environmental monitoring data).
Furthermore, the microorganism must geminate under defined conditions in a standard culture media and have a low pathogenicity to humans.

The Geobacillus stearothermophilus used was traceable to the American Type Culture Collection (ATCC) with either equivalent strains ATCC
7953 or 12980 used. The requirement of the biological indicator is to demonstrate adequate sanitization of the isolator through achieving a six-
log10reduction. For this, a population of >5x10 5 is considered to be of sufficient number. Scientific data indicates that there is a relationship
between kill time and surface temperature and that faster kills are seen near the dew point of gassing vapors. The rate at which spores are killed
is logarithmic (17).

Figure 2: An Operator Accessing the Sterility Testing Isolator (photograph ©Tim Sandle).

Hydrogen peroxide is a surface disinfectant; therefore, it is important that the biological indicators are presented to ensure maximum surface area
exposure (the envelope was folded back so that the biological indicator was not lying flat on a surface; Kapton tape was also used). After the
exposure of each biological indicator, the discs were transferred into tryptone soya broth (equivalent to the soya-bean casein digest medium
described in the pharmacopoeias) and incubated at 55-60oC for a minimum of 14 days. The temperature range was suitable for the recovery of
thermophilic bacterium. The incubation time was longer than recommended in the pharmacopoeias. This was to allow for sufficient time for any
stressed or sub-lethally damaged microbial cells to recover. Positive and negative controls were performed for each cycle.

Development of Sterility Test Load Patterns

 An important part of the OQ and PQ stages was the development of load patterns for sterility testing. For this:

 The different sterility test load combinations were mapped out. These were evaluated for the smallest and largest configurations by assessing the
different load types (this was assessed by calculating the surface area and by an assessment of the degree of absorbent material). OQ data
demonstrated that the maximum load was the worst-case.
 Once assessed, the maximum load was evaluated against biological indicators using different cycle parameters. A cycle that achieved total
biological indicator kill was chosen as a base cycle and performed on three occasions.
 From the satisfactory outcome of the maximum load validation, it was reasoned that all intermediate loads would also be satisfactory based on
the load of the largest surface area being considered as the „worst-case‟ and the demonstrated reproducibility and repeatability of the successive
validation cycles. Routine cycle parameters were then established by incorporating an „overkill‟ for gassing agent volume and dwell time to ensure
a robust cycle.
 During performance qualification each different load type was assessed using biological indicators and the established routine cycle.
 Duplicate biological indicators were used at each location due to the theoretical risk of „rogue‟ biological indicators (multiple layers of spores or
debris on the carrier so that some spores are not exposed to the surface disinfectant) could give a false positive result.
 The following strategy was applied:
o Both duplicates indicate no growth–test pass.
o Both duplicates indicate growth–test failure. Cycle parameters to be reviewed.
o One duplicate indicating growth and one no growth–raise out-of-specification investigation. Repeat in triplicate: all replicates must indicate no
growth for cycle acceptance.

Once established, the load patterns became the standardized load patterns for sterility testing. No significant changes were permissible without
undertaking a new load qualification.

Requalification of Isolator Loads

An important consideration is the frequency of isolator load requalification and, when undertaken, the types of load patterns required (as a
minimum this is ordinarily the largest size for each load type). Requalification is to verify that the isolator system and gassing port continue to
operate as expected; that is to demonstrate that the gassing system continues to kill a known population of resistant spores (a „kill‟ or „no kill‟
test). In relation to this study proposal, the decision taken was to re-qualify the entire isolator system using biological indicators on an annual
basis.

OTHER OPERATIONAL DECISIONS

Further decisions, based on the validation outcomes and the process cycles selected, are required for the daily operation of an isolator system.
These include setting the frequency of sanitizing the entire system and the policy for glove changes. These aspects are discussed below.

Frequency of Sanitization of the Isolators

The frequency of sanitizing the entire isolator system needs to be established. This was a separate activity to the gassing of each load required
for the sterility test in the gassing port. In part, this decision was based on environmental monitoring data. For this study protocol, the two isolators
and the gassing port were sanitized empty once per week. The purpose of this was to minimize the risk of microorganisms survi ving within the
isolator chamber.

Over time, the established frequency can be reviewed and increased if necessary. Circumstances that would trigger this would include the
environmental monitoring data from sterility testing indicating an upward trend or following a sterility test failure. Equally, it could be that, after a
considerable period of satisfactory data, the frequency could be extended. Additional supporting data can be provided from conducting post-
sanitization microbial monitoring.

Routine Cleaning and Disinfection Agents

There will be occasions when the inside of the isolator should be cleaned and disinfected, such as following a spillage incident. It may also be that
the user elects to do this prior to operating the cycle for the sanitization of the entire system (as described above).

For this, the cleaning and disinfection agents should be carefully selected. For cleaning, the detergent selected should be a sterile neutral
detergent because any residues are less likely to react with the disinfectant. The disinfectant of choice, given that most isolators are constructed
from stainless steel, is 70% IPA. This is because other disinfectants could damage the stainless steel inner chamber of the isolator (by causing
scratching, rouging, and etc.). Seventy percent IPA may not be appropriate for cleaning perspex visors; sterile filtered water can then be used to
remove any debris. Where a more efficacious disinfectant is needed, such as a sporicide, then due to many sporicides being aggressive, 70%
IPA should be used to remove any residues of the sporicide post-application.

Frequency of Glove Changes

Isolator gloves are prone to leakage, and this represents a weak point with any isolator system. Some users test all gloves for leakage before and
after each test. One problem here is that leak-testing devices are not available for all types of isolators, and those that are available can be
unreliable. Where leak testing is difficult to perform, some users adopt a policy to change the gloves on a regular basis (such as weekly) with a
visual inspection before and after each test. Additional supporting data for a glove change policy can be provided from post-sterility test and post-
sanitization microbial finger plate monitoring. The use of a water intrusion test for gloves that have been changed can provide further data as to
the frequency that leaks occur.

Requirements for Environmental Monitoring

Environmental monitoring should be conducted for each sterility test. The testing isolator environment should be monitored every time a sterility
test is performed. The typical environmental samples taken are: air-samples (using a volumetric sampler) and settle plates (during the test); and
finger plates, swabs, or contact plates (immediately post-test). Alert and action levels are based on those applicable to an International
 Organization for Standardization (ISO) 14644 class 5/European Union (EU) good manufacturing practice (GMP) Grade A environment, which are
typically 1 colony forming unit (cfu). Static state particulate monitoring is performed weekly as part of an on-going check.

Environmental monitoring data is used to show the level of contamination inside the isolator, and, if a final product fails a sterility test, the data will
be used to help determine the validity of the test. All out-of-limits results should be investigated.

The culture media (sometimes called „recovery media‟) used should be subjected to inhibition studies to determine that hydrogen peroxide does
not have an inhibitory effect upon the growth promoting properties of the culture medium.

The room in which the isolator system is housed will also be subject to viable and particulate monitoring at an interval determined by risk
assessment. Upward trends and individual out-of-limits should be investigated, and, where contamination occurs within the isolator, a crosscheck
should be made to the microorganisms recovered within the room because any connection may indicate contamination ingress.

Isolator Housing Room

Many isolators are held within a cleanroom or a controlled environment. The isolator described in this study protocol was held within an EU GMP
Grade D/ISO 14644 class 9 („in operation‟) cleanroom.

Where a cleanroom is used, the cleanroom will have to meet various operational parameters itself. These parameters will include:

 Positive pressure differential to the outside corridor

 Controlled temperature, given that this can affect isolator gassing parameters (in this study proposal the temperature was set at 21 4 oC) (similarly,
humidity control is important; in this study proposal, the room had a maximum 70% relative humidity)
 High efficiency particulate air (HEPA) (with filters of 99.99% efficiency)
 Controlled air-changes (for this study proposal, these were set at 25 per hour in the main isolator room)
 Particles monitored in the dynamic state
 Room subject to viable environmental monitoring
 Room falls under a routine maintenance plan including classification.

MAINTAINING THE STERILITY TEST ISOLATOR

In order for an isolator to function as required, a number of physical attributes and tests must be performed and measured at periodic intervals.
The types of parameters to measure the appropriate limits and the frequencies of testing will vary according to the particular isolator and will fall in
line with different organisation policies. The important aspects are that many of these attributes are defined upfront and justified.

For example, it is particularly important that, following completion of each cycle of the gassing port, the critical parameters are examined. These
will be used to verify that the gassing cycle was satisfactory and, by implication, the result of the sterility test is valid. The parameters
recommended to be examined are:

 Volume of hydrogen peroxide used

 Gas concentration alert level
 Alarm status during the cycle
 Gas injection phase time (refer to Table II)
 Humidity at the start of gassing.

Selected parameters should also be trended in order to determine if the gassing port is operating as expected and as a means to determine on-
going performance.

Many of the important aspects have been are summarized in the tables below. Some of these are based on the recommendations stated in
international cleanroom standard ISO 14644 Part 7 (18). Whilst these are appropriate to the isolator and gassing port assessed as part of this
study proposal, they nonetheless provide a benchmark for other isolator users to compare (19).

Tables have been constructed for:

 The isolator
 The gassing port
 The gas generator.
Table I: Physical and Operational Aspects for the Isolator.

Parameter Limit/Requirement Justification Frequency of Testing

Pressure Positive Pressure 20–250 Pascals (Pa)) Positive pressure relative to the isolator
room is required to prevent the ingress of
contamination from the outside environment
into the enclosed clean zone.
The minimum specification of +20 Pa is the
lowest setting to prevent this occurrence and
to leave some safety margin.
The maximum specification of 250 Pa
relates to the integrity of the isolator
chamber.
Monitored each working week
prior to the isolator system
being sanitized. Given that the
likelihood of failure is low,
based on past trends, this
interval was considered
sufficient.
In addition, the pressure is
checked prior to each test.
Pressure gauges are calibrated
biannually. This frequency was
based on the recommendation
of the isolator manufacturer.

Visor Perspex Aids operator comfort through glove ports. Assessed as part of leak
testing.

Leak Rate Test From a starting pressure of 150 Pa, a timed sequence of Recommended by ISO 14644 Part 7. Monitored each working week
monitoring leakage over a 90 second period is undertaken. prior to the isolator system
By the end of the 90 seconds the pressure must not fall by being sanitized. Given that the
more than 25 Pa. likelihood of failure is low,
based on past trends, this
interval was considered
sufficient.

Airflow Airflow pattern Although laminarity is not required for a
sterility testing isolator, there is a theoretical
argument that the airflow should
demonstrably remove contamination, at
faster rate, away from the critical zone.
This was assessed as a one-off
airflow visualization (smoke)
study. Such a study would be
repeated in the event of a
HEPA filter change.

Air-speed 0.45 meters per second (ms-1) at the filter face (±20%) Required by ISO 14644 Part 7 and EU GMP Assessed biannually.
for the effective removal of any
contamination.
In addition, the air-speed is
displayed on the isolator panel.
An alarm is set to sound in the
event of a low air-speed. The
alarm is set at 0.35 ms-1.

Air-supply HEPA filtered with >99.995% retention total (>99.975% Required by ISO 14644 Part 7. HEPA filter will theoretically
retention local) filter all but 0.01% of 0.3 mm
particle size (defined as the
HEPA filter based on European norm: EN
most penetrating particle size).
H14 standard 1822: 2009

Assessed biannually for HEPA

filter integrity as recommended
by the manufacturer.

Air Changes >20 air-changes per hour A set number of air changes above 20 is Recommended by Midcalf et
necessary for the dilution and removal of any al, 2004: 27.
potential contaminants from the isolator
environment.
In practice, the isolator
achieved considerably more
than 20 air changes per hour.
Parameter Limit/Requirement Justification Frequency of Testing

Particle Counts 0.5 mm = 3, 520 counts per cubic meter EU GMP limit. This is tighter than the ISO Assessed biannually as per ISO
14644 Part 1 specification. However, ISO 14644.
14644 Part 1 methodology and frequency of
5.0 mm = 20 counts per cubic metre.
testing is employed.
Static state monitoring is
performed weekly during
normal operations as part of an
on-going check.

Validated method for a

Sanitization Validated decontamination cycle Run weekly for isolator
minimum six log system. This interval is
reduction of Geobacillus considered to be suitable to
prevent bioburden build up.
stearothermophilus spores
using hydrogen peroxide Effectiveness is assessed
through a review of
vapor. environmental monitoring data.

Sanitizing Agent Hydrogen peroxide liquid (30% w/w) Manufacturer’s certificate of analysis N/A
reviewed.

Gloves: Leakage Low level of leakage Recommended by USP<1208> Tested as part of the leak test
of the isolator system (each
working week).

Gloves are changed each week.

Following a change of gloves a
water test is conducted to
assess leaks and replacement
gloves are re-tested as part of
the isolator leakage test.

Gloves are additionally

visually inspected prior to each
sterility test being performed.
At the end of the sterility test,
finger plates are taken to assess
microbial growth.

In the event of gloves requiring

changing, the isolator will be
re-sanitized as a precautionary
measure.

Gloves: Design Neoprene (polychloroprene) Manufacturer’s recommendation. Purchased from an approved

supplier.
Gloves are especially designed for isolator
use.

Table II: Physical and Operational Aspects for the Gassing Port.

Parameter Limit/Requirement Justification Frequency of Testing

Gas Injection Alarms set at 41oC to 50oC
Temperature (determined during OQ)
The gassing nozzle fitted in the port
has a temperature alarm. The port is
This temperature range is important in order to Monitored during each cycle.
determine that the gas entering the chamber is
within the correct range to create a vapor of the
Verified during six-month servicing and
required level of saturation.
calibration.
Parameter Limit/Requirement
designed to abort the cycle should
the temperature go outside of the set
(validated) range.
Temperature Inside 15–30oC (operation of the gassing
Port port during the gassing cycle).
Temperature control is important
because hydrogen peroxide vapor is
evaporated by flash evaporation.
Delivery losses can theoretically
occur due to a reduction of the
temperature of the high
concentration vapor stream resulting
in condensation before the vapor
reaches target areas.
Relative Humidity 15–70%
Air-supply for the HEPA filtered with >99.95%
Chamber retention (total)/ >99.75% retention
(local).
H13 standard.
Particle Counts 0.5 mm = three 520 counts per
cubic meter
5.0 mm = 20 counts per cubic meter
Total Gassing Time The gassing time is dependent upon
the volume of hydrogen peroxide
and the pump speed in relation to
different cycles. The cycles are:
 Port
 Port and one isolator
 Entire system (port plus both
isolators).
Justification Frequency of Testing
Manufacturer’s operational setting. Temperature is measured at the start of gassing.
The gassing port is designed to heat air and to Assessed biannually as part of routine service
pass warm air through the entire isolator system. and calibration.
This is part of the conditioning phase. The risk
of the air temperature being affected by cold
external air upon the isolator is minimal because
the isolator room is set to operate at 21 ±2 oC.
The biological indicator studies conducted
during OQ included the measurement of the
temperature at each location; each temperature
fell within the operational range of the gassing
port.
The gassing port is programmed not to function
unless temperature and humidity are
satisfactory.
Manufacturer’s operational setting. Relative humidity is measured at the start of the
gassing cycle.
The gassing port is designed to condition the air
prior to its passing through the isolator system Assessed every six months as part of routine
or from the commencement of gassing. Relative service.
humidity depends not only on temperature but
also on the pressure of the system of interest.
The biological indicator studies conducted
during OQ included the measurement of the
humidity.
The gassing port is programmed not to function
unless temperature and humidity are
satisfactory.
Required by ISO 14644 Part 7<. HEPA filter will theoretically filter all but
0.03% of 0.3 mm particle size (defined as the
most penetrating particle size).
European norm: EN 1822: 2009
Assessed every six months for HEPA filter
integrity as recommended by the manufacturer.
EU GMP limit. This is tighter than the ISO Biannually as per ISO 14644.
14644 Part 1 specification. However, ISO
14644 Part 1 methodology and frequency of
Static state monitoring is performed weekly
testing is employed.
during normal operations as part of an on-going
check.
Time is an important parameter. If the time falls Reviewed after each operation. The gassing
outside the set range, then the gassing cycle will time must fall within the range established by
probably be ineffective. Problems with the the manufacturer.
gassing time may indicate that the peristaltic
pump needs changing.
This parameter should be trended.
The cycle parameters of peroxide injection rates
and phase times were initially developed to
ascertain the required gas concentration profile
during the qualification.
The gassing times must not fall outside the
Parameter Limit/Requirement Justification Frequency of Testing

limits set. To do so would create a risk that a

blockage, in relation to pump delivery, has
occurred or that the incorrect volume of
hydrogen peroxide has been used. In this event,
the cycle must be rejected, an investigation
raized, and the cycle repeated. In the event of
adverse trends, the isolator should not be used
until the investigation is complete.

Gassing Dwell Time Gassing dwell time is set during the This was set during the OQ based on biological Reviewed after each operation. This is a set
cycle development stage of OQ for indicator studies. An overkill time was added condition and time outside of the parameter
each different cycle. (double the time taken to achieve kill during the would lead to a cycle failure. The gassing dwell
OQ). time must fall within the range established by
the manufacturer.

Gas Concentration Minimum concentration set during This level indicates whether the correct Reviewed after each operation. A level outside
Alert Level PQ for each different cycle. concentration of vapor is being achieved in the of the established range does not necessarily
port. constitute a cycle failure. However, a result
outside of the range should be investigated.
Either the gassing or the dwell phase peak
concentration can be selected as the parameter
to measure.

Post-cycle hydrogen Level should be less than 1 ppm. This is a safety function (based on workplace Reviewed after each operation and prior to
peroxide levels exposure levels) and, additionally, a check opening the isolator/gassing port interlocking
against introducing a high level of hydrogen doors.
peroxide into the isolator.

Table III: Physical and Operational Aspects for the Gas Generator.

Parameter Limit/Requirement Justification Frequency of Testing

Temperature 50–75oC (operation of the gas Manufacturer’s operational setting. Temperature is measured at the start of gassing.
generator)
The gas generator is designed to condition and Assessed every six months as part of routine
heat air and to pass warm air into the gassing service.
port. This is assessed as part of the conditioning
phase. To leave the unit, the air must be about
50oC. The unit will not proceed to gassing until
the heaters have achieved the required
temperature during the pre-gassing phase.

The risk of the air temperature being affected by

cold external air upon the isolator is minimal as
the isolator room is set to operate at 21 ±4 oC.

The biological indicator studies conducted

during OQ included the measurement of the
temperature at each location; each temperature
fell within the operational range of the gassing
port.

The gas generator is programmed not to

function unless temperature and humidity are
satisfactory.
 Parameter Limit/Requirement Justification Frequency of Testing

Relative Humidity Not greater than 75%
(If conditions are too dry, this can
theoretically cause a problem
sincereaching dew point is critical
under saturated vapor conditions via
the surface micro-condensate. A
relative humidity of 75% or below
is recommended by the
manufacturer).
Manufacturer’s operational setting. Relative humidity is measured at the start of the
gassing cycle.
The gassing port is designed to condition the air
prior to passing through the isolator system or Assessed every six months as part of routine
from the commencement of gassing. For this, service.
the room humidity must not exceed 85%.
The gassing port is programmed not to function
unless temperature and humidity are
satisfactory.

Air-supply into the HEPA filtered with >99.995% Required by ISO14644 Part 7. HEPA filter will theoretically filter all but
Gassing Port retention total (>99.975% retention 0.01% of 0.3 mm particle size (defined as the
local). most penetrating particle size).

H14 standard. Assessed every six months for HEPA filter

BS EN1822
integrity as recommended by the manufacturer.

SUMMARY

This paper has examined a study proposal for an isolator system used for sterility testing drawing upon the experiences of the authors. The study
has focused upon the development of cycles with reference made to validation parameters and to the critical physical parameters that require
monitoring for routine sanitization cycles and maintenance of the isolator in a state that ensures it is free from contamination. Given that there are
different isolator systems available, and that this paper is based on a possible study proposal, not all of the parameters described will be
applicable to each system. However, many of the processes and parameters will be of relevance and can allow other users to benchmark their
own systems and aid these users in developing an operational rationale.

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