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Manufacture of sterile medicines – Advanced workshop for SFDA GMP

inspectors - Nanjing, November 2009

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Microbiological Control Tests

Mrs Robyn Isaacson



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Microbiological Testing

Objectives

• To review microbiological environmental and

quality contol testing

– Microbiological Environmental Monitoring

– Container integrity testing

– Pre-sterilization bioburden testing

– Media fill medium growth promotion testing

– Sterility Testing

– Other microbiological laboratory issues



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Environmental Monitoring

Grade Air sample (CFU/m3)

Settle plates (90mm diameter)

(CFU/4hours)

Contact plates (55mm

diameter) (CFU/plate)

Glove print (5 fingers) (CFU/glove)

A < 3 < 3 < 3 < 3

B 10 5 5 5 C 100 50 25 -

D 200 100 50 -

Table 3

– These are average values

– Individual settle plates may be exposed for less than 4 hours

• Values are for guidance only - not intended to represent specifications

• Levels (limits) of detection of microbiological contamination should be

established for alert and action purposes and for monitoring trends of air quality in the facility

Limits for Viable Particles



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Methods • Surface monitoring

– Product contact surfaces, floors, walls, and equipment should be

tested on a regular basis

– Touch plates - used for flat surfaces

• sample area of 25cm2

• medium protrudes above sides

• medium contains neutralisers

– Surface Swabs - used for irregular surfaces

• area approx 25cm2 is swabbed

• qualitative or quantitative

Surface monitoring should be performed at conclusion of aseptic processing (to

minimise risk of contaminating critical surfaces during production)

• swabs and contact plates can be used



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Methods – Active Air Monitoring

• impaction, centrifugal and membrane (or gelatin) samplers

• a certain volume of air is sampled (volume and location should be

meaningful)

• instruments should be calibrated

– Passive Air Monitoring

• Settle plates exposed for 30-60 minutes (longer may result in agar

drying out) and replaced for duration of filling

• Media should be capable of growing a range of bacteria and

moulds (e.g. Soybean Casein Digest Agar (SCDA)/Trypticase Soy

Agar (TSA)

• Should consider use of medium specific for moulds if shown to be

a problem in the environment

• Only give qualitative or semi-quantitative results

• Data generated considered in combination with active air

sampling results



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Sampling Locations – Should be based on risk of microbiolgical contamination

– Should be clustered around areas where product or components are exposed e.g.

• at filling heads on filling lines

• loading of product into lyophilizers

• stopper bowls

• where aseptic connections are made

• where there are high levels of operator activity (but without

impacting on production)

– Lower grade areas are monitored less frequently and trends

monitored



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Personnel

• For each session - gloves should be monitored (but not

immediately after sanitising!)

• Periodic sampling for other

locations on gown

• Clean room operators should

be regularly validated to

demonstrate that they do not contaminate gowns during

gowning up (gowning qualification)



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Levels and Trends • Limits in Code of GMP are for guidance only

• Manufacturers should set alert and action limits appropriate to the location

• Individual results should be considered - averaging can mask

unnacceptable localised conditions

• There should be written procedures (SOPs) for data review and action to be taken if limits are exceeded

• Trend Reports

– Short and long term reports on environmental and personnel

monitoring

– Results of EM should be included in Batch Records

– Significant changes in microbial flora should be considered



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Disinfectants • Suitablility, efficacy, limitations of disinfectants and procedures

should be assessed

– minimum contact time established

• Disinfectants in Grade A/B areas should be sterile, supplied in

sterile containers and used for a defined period

• Should be shown to be effective against facility microbial flora

• Should be sporicidal (if spores found in the environment) and for “spraying in” of components and equipment

• Disinfection SOPs should include sufficient detail to enable

reproducibility

– preparation, work sequence, contact time

• Organisms identified from adverse trends should be tested for their sensitivity to the disinfectants used



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Water • microbiological quality of water very important

• Should be an extensive, comprehensive water testing programme

• Feed water, pre-treatment, reverse osmosis (RO), deionized (DI),

purified/highly purified and water for injection (WFI) should be

tested

• Alert and Action limits set by manufacturer (with action to be taken if limits are exceeded)

• WHO recommendations (next slide)

• For purified/highly purified water and WFI, limits defined in

pharmacopoeia

– purified <100CFU/mL

– Highly purified and WFI 10CFU/100mL (but is usually kept at high

temperatures)



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Sampling Location Target Alert Action

Raw water 200 300 500

Post multimedia filter 100 300 500

Post softener 100 300 500

Post activated carbon filter 50 300 500

Feed to RO 20 200 500

RO permeate 10 50 100

Point of use 1 10 100

Suggested Microbial Limits (CFU/mL)

for facility water



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Water • Water should also be tested for presence of coliforms and/or

pseudomonads if appropriate (may cause biofilm)

• Water used for parenterals should be tested for pyrogens

– limit is not more than 0.25 EU/mL

• Water should be tested using R2A agar (low nutrient for the

recovery of water borne organisms) incubated for at least 5 days at 30-35°C

• Sampling procedures should follow those used in production

Compressed Air/Nitrogen/CO2 • Should be tested for non-viables and viables

• Pressure reduction orifices should be used to provide a steady

stream of air, validation of media should be ensured with

consideration of validation



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Container Integrity Testing

• Integrity of container/closure system

– is intitally validated by filling container with sterile growth medium then inserting container in broth containing 106

CFU/mL of suitable microorganism

– containers sealed under a vacuum should be periodically tested to demonstrate that vacuum is maintained over shelf

life

– procedures in place to detect faulty containers during manufacture

– operators involved in visual inspection should have frequent

breaks and regular eye-sight tests



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Bioburden/IPC Testing

• Should be written procedures for pre-sterilization

bioburden, in-process control and raw material

testing

• method should be validated for the recovery of low numbers of organisms

• use of anaerobic medium should be considered if

shown to be present in environment

• target, alert and action limits should be

documented and include action taken if limits

exceeded



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Growth Promotion Testing

• Media used for microbiolgical testing should be

tested for its ability to support microbial growth – media used for media fills should be able to support the

growth of a wide range of microorganisms (bacteria and

moulds)

– Soybean Casein Digest Medium is usually used. An anaerobic medium may also be substituted occasionally if environmental

monitoring indicates presence

– After the media fill has been completed, it is important to demonstrate that the media would have been able to support

the growth of organisms if they had been present

– containers with media should be inoculated with 10-100 CFU of organims such as Bacillus subtilis, Staphylococcus aureus,

Candida albicans, Aspergillus niger. Environmental isolates

should also be included



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Growth Promotion Testing

Media – The inoculated media should be capable of showing growth

within 3 days of incubation

– Media used in environmental monitoring should also be tested

for its growth promoting properties. Validation of recovery of organisms under test conditions should be carried out to

demonstrate neutralization of disinfectant residuals (media

should contain neutralisers).

– Media purchased externally should also be tested

– Media used for media fills and environmental monitoring

should be pre-incubated to demonstrate “sterility” prior to use

– Media should have a validated shelf life



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Sterility Testing

• Sterility test is a quality control test used as part

of product release for product required to be

sterile – Has significant statistical limitations - will really only detect

gross contamination

• Sampling – No of containers and volume to be tested defined in

Pharmacopoeia

– Samples from aseptically manufactured product should be taken from beginning, middle and end of batch fill and also

after interventions and stoppages

– Samples from terminally sterilized product should be taken

from previously identified cool spots within load

– Sampling should be sufficient to allow for retests if needed



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Sterility Testing

• Facilities

– Sterility testing should be carried out under the same

conditions as aseptic manufacture

• In a Grade A laminar air flow cabinet in a Grade B

background (may also be carried out in an isolator)

• Air supply through HEPA filters, pressures should be

monitored and alarmed

• Access to area should be through airlocks

• Operators should be appropriately gowned is sterile

garments

• Operators should be appropriately trained and validated

• Appropriate cleaning, sanitisation and disinfection procedures should be in place

• Environmental monitoring should be conducted



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Sterility Testing

• Methods are defined in Pharmacopoeia – membrane filtration is the preferred method if product is

filterable

– direction innoculation is alternative

• Media types – Soybean Casein Digest medium (SCD), (also knows as

Trypticase Soy Broth(TSB)) and Fluid Thioglycollate medium

(FTM) is usually used (to detect aerobic and anaerobic organisms)

– validation studies should demonstrate that the media are

capable of supporting growth of a range of low numbers of

organisms in the presence of product. May need to incorporate inactivators

• growth should be evident after 3 days (bacteria), 5 days (moulds)

– media may be purchased or made in-house using validated

sterilization procedures



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Sterility Testing

• Media – should be tested for growth promoting qualities prior to use

(low number of organisms)

– should have batch number and shelf life assigned

• Incubation Period – At least 14 days incubation

– 20-25°C for SCD/TSB, 30-35°C for FTM

– Test containers should be inspected at intervals

– temperatures should be monitored and temperature

monitoring devices should be calibrated

– if product produces suspension, flocculation or deposit in

media, suitable portions (2-5%) should be transferred to fresh media, after 14 days, and incubated for a futher 7 days



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Sterility Testing • Negative Contols

– media should be incubated for 14 days prior to use, either a portion or 100% of batch (may be done concurrently with test)

– negative product controls - items similar in type and

packaging to actual product under test should be included in each test session

• facilitate interpretation of test results

• negative control contamination rate should be calculated and

recorded

• Positive Test Controls – bactiostasis/fungistasis test

• should demonstrate that media are capable of supporting growth

of a range of low numbers of organisms in the presence of

product. May need to incorporate inactivators

– growth should be evident after 3 days (bacteria), 5 days

(moulds)



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Sterility Testing

• Positive Controls • should be performed on all new products and when any changes

are made.

• Should be repeated annually

– Stasis test recommended particularly for product with

antibiotics or preservatives or slow release tested by direct innoculation

• demonstrates that media can support growth at the end of the

incubation period and has not been affected by product

• Results – Any growth should be identified (genotypic)

– Automated/Semi-automated systems used for identification should be periodically verified using reference strains



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Sterility Testing

• Interpretation and Repeat Tests

– No contaminated units should be found

– A test may only be repeated when it can be

demonstrated that the test was invalid for causes

unrelated to the product being examined

– European Pharmacopoeia criteria

• (a) the data of the micro monitoring of the sterility test

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 negative controls

• (d) after determination of the identity of the

microorganisms isolated from the test, the growth of this species or these species may be ascribed unequivocally to

faults with respect to the material and/or technique used in conducting the sterility test procedure



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Sterility Testing

• Interpretation and Repeat Testing • When conditions (a), (b) or (c) apply the test should be aborted

• If a stasis test performed at the end of the test shows no

growth of challenge organisms, this also invalidates the test

• For conditions (d) to apply must demonstrate that the

orgamisms isolated from the sterility test is identical to an isolate from materials (e.g. media) and/or the environment

– must use genotypic identification methods

– Repeat test is carried out with same number of samples

as first test

– Any contamination detected in repeat test, product does

not comply



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Other Microbiological Laboratory Issues

Reference Culture Collections • Reference cultures may be used for

• Quality contol of media

• Test method validation

• Control of test reagents

• Must remain genetically stable to retain characteristics for

which they have been selected.

• Cultures of microorganisms tend to undergo

variation/genetic change that can affect characteristics of a

culture - unsuitable for further use.

• Probability of variation/genetic change increases with

frequency of repeated subculture of reference culture –

working culture must be no more than 5 generations

removed from original source culture.



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Reference Cultures (2)

• Laboratory must have a system for preserving and

maintaining reference cultures with their original

characteristics.

• Laboratory should:

– maintain suitable reference cultures for QC of culture media

and test reagents and for test method validation;

– ensure reference cultures are traceable to a recognised culture

collection eg. ATCC, NCTC;

– ensure reference cultures are uniquely identified within

laboratory, with traceability to recognised culture collection.



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Reference Cultures (3)

• Lab should have documented procedures:

– that maintain hierarchical control of reference cultures (ie.

master, stock & working cultures);

– for purchase, preservation, maintenance, identification and

frequency of subculturing of reference cultures;

– that prevent use of working cultures as replacements for

depleted stock and/or master cultures.

• Maintain records for each reference culture:

– identity, source and history and date of receipt of master culture;

– resuscitation, preservation, maintenance and storage conditions for master, stock and working cultures;

– results of purity and identification tests for master and/or stock cultures; and

– dates of preparation of stock and working cultures.



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QC of Culture Media

• Media other than sterility testing media and media fill media

must be subject to quality contol

• quantitative or semi-quantitative method/s to assess growth

promotion/fertility

• use of positive and negative controls for selective and/or

dirrerential culture media

• different levels of QC required dependent on whether

culture is

– manufactured in house (every batch should be tested)

– purchased ready to use (supplier tests media with testing periodically verified in house)



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QC of Culture Media (2)

• Laboratory should:

– have documented procedures for preparation, QC, release and

storage of culture media;

– have validated shelf life of culture media under normal storage

conditions;

– maintain records of preparation and QC of individual batches

of culture media;

– ensure that records of microbiological QC performance testing

are traceable to batch preparation records; and

– that microbiological QC performance test results are assessed against acceptance/rejection criteria.



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Sterilization processes for Culture Media

• sterilzation process for culture media should be validated

and monitored using same procedures as for sterilization of

product

Equipment Calibration and Checks

• Laboratory equipment (e.g. pipettes, balances, incubators,

refrigerators, thermometers, autoclaves, laminar flow

workstations etc) should be calibrated and recalibrated and

routinely monitored (where appropriate)

Personnel

• Should be appropriately trained and authorized to perform

testing



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Testing of Biological Indicators

• if tested in-house the method should include a heat-shock

step (this verifies that the indicators do actually contain

spores and not vegetative organisms)

• BIs should occasionally be tested in house to verify the

suppliers count

Endotoxin Testing

• Parenteral products should be free from endotoxin

• Endotoxin is a lipopolysaccharide present in the cell wall of

gram negative bacteria which can cause fever if introduced

into the body

• Raw materials, WFI used in manufacture and some finished

product must be tested for endotoxin



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Endotoxin Testing (2)

• LAL (Limulus Amebocyte Lysate) test is used for detecting

endotoxin (previously a rabbit test)

– based on clotting reaction of horseshoe crab blood to

endotoxin

• Types of LAL test

– Gel Clot

– Turbidimetric

– Colorimetric

• Equipment used in test must be endotoxin free

• Validation of accuracy and reliability of the method for each

product is essential



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Gel Clot Method • Original method

• The official “referee test”

• The specimen is incubated

with LAL of a known senstivity. Formation of a gel

clot is positive for endotoxin.




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Turbidimetric Method

• A kinetic method

• The specimen is incubated with LAL and either the rate of increase in turbidity or the time taken to reach a particular turbidity is measured spectrophotometrically and compared to a standard curve.




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Colorimetric Method

• Endotoxin catalyzes the activation of a proenzyme in LAL which will cleave a colorless substrate to produce a colored endproduct which can be measured spectrophotmetrically and compared to a standard curve.

• Can be kinetic or endpoint




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* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)

Sensitive down

to .001 EU/ml *

Sensitive down

to .005 EU/ml

Sensitive down

to 0.1 EU/ml

Sensitive down

to 0.03 EU/ml

Can be automated,

allows electronic

data storage

Can be automated,

allows electronic

data storage

Can be automated,

allows electronic

data storage

Manually read and recorded

Requires

incubating plate or

tube reader

Requires

incubating plate or

tube reader

Requires

spectrophotometer

or plate reader

Simple Least expensive,

Requires 37 degree bath

Quantitative Quantitative Quantitative Semi-

quantitative

Turbidimetric Chromogenic

Kinetic

Chromogenic

Endpoint

Gel Clot




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