Operation of Sterility Testing

Isolators and validation issues

http://www.pharmamicroresources.com/

https://www.pharmig.org.uk

Introduction

Sterility testing concerns

Introduction to isolators

Isolator decontamination cycles

Biological indicators

Validation of isolator cycles

Key operational decisions

Maintaining isolators

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Major concern with the sterility test is the risk of false positives.

These can lead to expensive and lengthy failure investigations.

Proving a false positive is extremely difficult.

A false positive is a microorganism that has been transferred through cross-contamination by personnel or from the test environment.

This differs 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.

Isolators

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.

• 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.

• May have doors to provide access to the equipment inside the

isolator.

• Have gloveports situated to allow users access to machinery

and product during testing or half-suits.

• Have a mechanism by which materials can be sanitized in place

or moved into the isolator via the use of transfer isolators or

through the use of a rapid gassing port.

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

isolators used for the sterility

test are at a positive pressure

relative to the room and have a

high efficiency particulate air

(HEPA) filtered airflow.

The maintenance of isolators is important, especially

in ensuring the minimization of leakage.

Isolators are normally tested biannually for HEPA

filter issues.

Regular checks for leakage and pressure differential,

often with alarms in place.

• Isolators are most commonly sanitized using hydrogen

peroxide vapor (a surface disinfectant) or peracetic acid.

• Isolators are said to ‘disinfect’, ‘biodecontaminate’ or

to 'sanitize' rather than 'sterilize’.

• 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.

Biodecontamination # 1

Biodecontamination # 2

• Hydrogen peroxide vapor disinfection employs a free

radical reaction mechanism to facilitate microbicidal

action.

• Hydrogen peroxide always decomposes (or

disproportionates) exothermically into water and

oxygen gas spontaneously.

Biodecontamination # 3

VHP

• Reliant on a high vapour phase concentration - achieved by initial dehumidification of the air.

• The process uses 30-35% hydrogen peroxide liquid which is flash evaporated to achieve the

required vapour phase concentration.

• The process is always stopped before the dew point is reached - a “Dry” process.

Biodecontamination # 3

HPV

• The process also flash evaporates 30-35% hydrogen peroxide to create a vapour.

• No initial dehumidification.

• The process is brought beyond the dew point to saturated vapour conditions.

• The process relies on the formation of micro-condensation on surfaces - a “Wet” process.

• Reliant upon ambient temperature and humidity.

iHP

• Less common, produced by ultrasonics.

QUALIFYING AN ISOLATOR

FOR STERILITY TESTING

A CASE STUDY

This presentation examines the important operational criteria for an isolator used for sterility

testing.

Looking at aspects of validation and the maintenance program.

This examination takes the form of a study proposal.

The isolator system consists of two solid wall Type 1 positive pressure isolators linked by a rapid

gassing port.

The gassing port and the isolators are attached to a gas generator.

Images of the isolator examined – taken by Tim Sandle

Introducing isolator

decontamination cycles

ISOLATOR CYCLE: STEP 1

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, items should be disinfected.

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 pharmacopeia.

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.

ISOLATOR CYCLE: STEP 2 (A)

Gassing Port and Disinfection Cycles

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.

ISOLATOR CYCLE: STEP 2 (b)

Gassing Port and Disinfection Cycles

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.

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.

ISOLATOR CYCLE: STEP 3 (a)

Sanitization Cycle #1

The sanitization process occurs by an aqueous solution of

hydrogen peroxide being evaporated to produce the same

weight ratio in the vapor phase as in the source liquid.

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.

ISOLATOR CYCLE: STEP 3 (b)

Sanitization Cycle #1

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.

In this case study – a micro-condensation process.

Optimum kill conditions require dew point to be reached,

which is affected by various parameters.

ISOLATOR CYCLE: STEP 3 (c)

Sanitization Cycle #2

There are some 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.

1. With this case study: at saturated vapor conditions, the gas concentration can hold no more molecules, thus, the process of condensation formation and disinfection agent surface deposition starts.

3. Re-evaporation of the surface condensate and removal of residual gas to target endpoint concentration.

ISOLATOR CYCLE: STEP 3 (d)

Sanitization Cycle #3

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.

2. The generator then produces hydrogen peroxide vapor by passing aqueous hydrogen peroxide over a vaporizer.

1. The gassing phase is validated to deliver a disinfection agent dose volume that will reach required in the target area and on target surfaces.

3. The vapor is circulated at a programmed concentration in the air and held for a set period of time (dwell phase).

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 to safe levels, at 1 parts per million (ppm).

ISOLATOR Cycle

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)

Surface area for condensate distribution together with amount of surface absorbency (e.g., packaging).

It is important that these process variables are controlled

Validation of isolator cycles

Biological indicators

With this case study, the gassing cycles were validated and developed

using biological indicators.

The indicators used were Geobacillus stearothermophilus spores dried

onto stainless steel discs.

ATCC 7953 or 12980.

The microorganism used as the biological indicator should have a

higher population and resistance than the bioburden on the item to

be decontaminated

Metal discs placed in Tyvek pouches (polyethylene fibre)

The microorganism must geminate under defined conditions in a

standard culture media.

Biological indicators

Hydrogen peroxide is a surface disinfectant; therefore, it is important that the biological indicators are presented to ensure maximum surface area exposure.

Need to determine number and location.

To place in the isolator the envelope must be folded back so that the biological indicator does not lying flat on a surface.

Kapton tape can be used.

After the exposure of each biological indicator, the discs must be transferred into tryptone soya broth and incubated at 55-60 C for a minimum of 7 days.

Temperature range suitable for the recovery of thermophilic bacteria.

Developing load patterns #1

An important part of the OQ and PQ stages is with 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.

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.

Developing load patterns #2

Duplicate biological indicators were used at each location due to the theoretical

risk of ‘rogue’ biological indicators.

The following strategy was applied:

Both duplicates indicate no growth–test pass.

Both duplicates indicate growth–test failure. Here cycle parameters to be

reviewed.

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.

When to requalify 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

Frequency of decontamination

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 surviving within the isolator chamber.

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

There will be occasions when the inside of the isolator should be

cleaned and disinfected, such as following a spillage incident.

The cleaning and disinfection agents should be carefully selected.

The disinfectant of choice, given that most isolators are

constructed from stainless steel, is 70% IPA or hydrogen peroxide.

This is because many other disinfectants, such as chlorine

based ones, 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.

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.

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: air-samples (using a volumetric sampler) and

settle plates (during the test); and finger plates, swabs, or contact

plates (immediately post-test).

Action levels are based on EU GMP Grade A environment,.

Static state particulate monitoring is performed regularly.

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.

The culture media used should be subjected to inhibition studies.

The room in which the isolator system is housed will also be subject to

viable and particulate monitoring.

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 and humidity.

High efficiency particulate air (HEPA) (with filters of 99.995% efficiency)

Controlled air-changes

Particles monitored in the dynamic state

Room subject to viable environmental monitoring room falls under a routine maintenance

plan including classification.

Maintaining sterility testing

isolators

In order for an isolator to function as required, a number of physical attributes

and tests must be performed and measured, such as:

Volume of hydrogen peroxide used

Gas concentration alert level

Alarm status during the cycle

Gas injection phase time

Humidity at the start of gassing.

Selected parameters should also be trended.

Useful reference point is: ISO 14644 Part 7

Cycles: temperature and humidity

Case study:

Gas injection temperature:

Alarms set at 41oC to 50oC (determined during OQ)

This temperature range is important in order to determine that the gas entering the chamber is within the correct range to create a vapor of the required level of saturation.

Temperature inside port:

15–30oC (operation of the gassing port during the gassing cycle).

This is part of the conditioning phase. The risk of the air temperature being affected by cold external air upon the isolator is minimal

Humidity:

15-70% RH

The gassing port is designed to condition the air prior to its passing through the isolator system or from the commencement of gassing.

Gassing time Time is an important parameter.

If the time falls outside the set range, then the gassing cycle will probably be ineffective.

The cycle parameters of peroxide injection rates and phase times were initially developed to

ascertain the required gas concentration profile during the qualification.

Gassing dwell time is set during the cycle development stage of OQ for each different cycle

Positive pressure

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.

Leak rate test of the isolator

From a starting pressure of 150 Pa, a timed sequence of monitoring leakage over a 90 second

period is undertaken. By the end of the 90 seconds the pressure must not fall by more than 25 Pa.

From ISO 14644-7

Air and airflow

Maintaining the Grade A zone

HEPA filtered H14 standard

Although unidirectional airflow is not required for a sterility testing isolator, there is a theoretical argument that the airflow should demonstrably remove contamination.

Air speed should be 0.45 meters per second (ms-1) at the filter face (±20%)

A set number of air changes is necessary for the dilution and removal of any potential contaminants from the isolator environment.

Particle levels:

0.5 µm = 3, 520 counts per cubic meter 5.0 µm = 20 counts per cubic metre

This presentation examined a case study for an isolator system used

for sterility testing drawing upon personal experience.

The study focused on 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.

Further reading:

Sandle, T. and Tours, N. (2013). Validation and Operation of a

Sterility Testing Isolator: a Study Proposal, Journal of Validation

Technology, Vol. 19, No.1

Summary

Sterility testing concerns

Introduction to isolators

Isolator decontamination cycles

Biological indicators

Validation of isolator cycles

Key operational decisions

Maintaining isolators

THANK YOU

Dr. Tim Sandle

Pharmaceutical Microbiology:

http://www.pharmamicroresources.com/

Pharmig: https://www.pharmig.org.uk