SFDA GMP Inspectors Workshop on Sterile Medicine Manufacturing

Manufacture of sterile medicines Advanced workshop for SFDA GMP
inspectors - Nanjing, November 2009

1
Microbiological Control Tests
Mrs Robyn Isaacson

2
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


3
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

4
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 25cm
2
medium protrudes above sides
medium contains neutralisers

Surface Swabs - used for irregular surfaces
area approx 25cm
2
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

5
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


6
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


7
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)

8
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


9
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

10
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)

11
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

12
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 R
2
A agar (low nutrient for the
recovery of water borne organisms) incubated for at least 5 days
at 30-35C
Sampling procedures should follow those used in production

Compressed Air/Nitrogen/CO
2

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

13
Container Integrity Testing
Integrity of container/closure system
is intitally validated by filling container with sterile growth
medium then inserting container in broth containing 10
6
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


14
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

15
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

16
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

17
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


18
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


19
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


20
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
20-25C for SCD/TSB, 30-35C 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


21
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)


22
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

23
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

24
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

25
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.

26
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.


27
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.


28
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)


29
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.


30
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

31
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


32
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

33
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.


34
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.


35
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

36
* (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

37
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Manufacture of sterile medicines Advanced workshop for SFDA GMP

inspectors - Nanjing, November 2009

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