Practical aspects of Microbiological Testing and
handling of OOS
Presentation By:UMESH B G
Sr.General Manager - Quality and Regulatory Affairs
Stempeutics Research Pvt Ltd, Bangalore
Agenda
• Introduction
• Why Microbiology testing
• Schedule L1 requirement
• Personnel Monitoring
• EM monitoring
• RM/PM,In process and FP testing
• MLT
• OOS w.r.t Microbiology testing
Introduction
• GMP is the basic requirement for manufacturing pharma/Biopharma
products
• GMP demand suitable contamination control procedures
• Contamination control –
• Design ,construction and operation of an environmental control
system.
• Air handling unit(AHU) ,filtering and distribution systems, building
design & construction features
• GMP main idea is to “Build in quality along the entire manufacturing process”
• All API’s & formulated products shall be manufactured in controlled area
under controlled conditions
• Poor GMP and GLP conditions at a manufacturing and testing
facility can ultimately pose a life-threatening health risk to a patient.”
Types of Contamination
•Viable & Nonviable particles
• Particles of dust, fibers, or other material are suspended in the air and may
contaminate product. These particles may, or may not, contain living
organisms (bacteria and their spores).
• The more particles in the air surrounding the product the more likely the
product will be contaminated with those particles.
Humans and bacteria
• Over 200 different species of bacteria are found associated with humans.
• Bacteria are found in the intestines, eyes, nose, mouth, hair and skin.
• Dry skin can have 1000’s of microbes / mm2 !
Staphylococcus epidermidis
Scanning EM. CDC.
Endotoxin
Endotoxin: a pyrogenic (fever inducing) substance (e.g. lipopolysaccharide)
present in the bacterial cell wall. Endotoxin reactions range from fever to death.
http://pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable – recommended conditions for inactivation are 180 0 C for 3 hours.
Spores
• What are SPORES?
• Why are they a MAJOR CHALLENGE!!!!
http://www.samedanltd.com/members/archives/PMPS/Spring2003/graphics/f1_p12.gif
http://micro.med.harvard.edu/faculty/rudner.html
Heat alone will not inactivate spores!
Viral Contamination
• Viruses are small (nm) non-living
entities that “hijack” the
machinery of a host cell
http://www.scq.ubc.ca/.../2006/08/viralreplication.jpg
Sources of Contamination
• People/Personnel
• Air/Environment
• Equipment
• Surfaces
• Others
People
•
Personnel Hygiene
•
Gowning
•
Gloves
•
Personnel.Qualification
•
Minimum number of personnel in clean areas
•
Training to all including cleaning and maintenance staff
•
Special cases
Environmental Monitoring
•
Microbiological
•
Air samples
•
Surface swabs
•
Personnel swabs
one of the most important laboratory controls is the environmental
monitoring program
Environmental Monitoring
• Elements of Environmental Monitoring program
• Sampling Methods
• Media & Incubation Conditions
• Sampling Locations
• Frequency of Sampling
• Alert & Action Limits
• Trend Analysis
• Out of Limits Investigations
• Corrective Action
Environmental Monitoring
• The selection of sampling locations depends on the room classification, design,
layout of the manufacturing process.
• Each process should be evaluated in order to identify the actual and potential
sources of contamination.
• A diagram of the sampling locations must be done as well as documenting the
procedure of collecting, incubate and analyze samples.
•
Environmental air monitoring shall be done critical areas
•
Sampling shall be done for return air and at working level
• Sampling must occur at the same location each time and at the same time of
the day.
Environmental Monitoring
•
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 2 hours and replaced for duration of activity
• Media should be capable of growing a range of bacteria and moulds
e.g. Soybean Casein Digest Agar (SCDA)
• 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
Environmental Monitoring of clean Areas
•
Surface monitoring
•
Product contact surfaces, floors, walls, and equipment should be tested
on a regular basis
•
Touch plates - used for flat surface
•
Surface Swabs - used for irregular surfaces
Environmental Monitoring of clean rooms
•
For each session - gloves should be monitored (but not immediately after
sanitizing!)
•
Periodic sampling for other locations on gown
•
Clean room operators should be regularly demonstrate that they do not
contaminate gowns during gowning up (gowning qualification)
Limit For Monitoring Clean Areas
Grade
Air sampling
Settle plates(90
mm )
(cfu/m3)
Contact plates(55
mm )
(cfu/m3)
Glove Prints
(cfu/ glove)
A
<1
<1
<1
<1
B
10
5
5
5
C
100
50
25
-
D
500
100
50
-
Environmental Monitoring:
Trending Data
•
Averages of data can be misleading and mask unacceptable localized conditions.
•
Alert and action levels should be set for each sample site
•
Individual sample results should be evaluated against the action and alert levels
Environmental Monitoring
Recommended Frequency of Monitoring:SAMPLING
ACTIVE, PASSIVE,
SURFACE &
PERSONNEL
SAMPLING LOCATIONS
GRADE
SAMPLING FREQUENCY
LAFS in Both Vial & PFS Filling area
LAFS in Cooling zone ( for both Vial Filling &
PFS)
A
LAFS in Vial & PFS filtration area
Every Operation or Weekly
Twice during non
manufacturing days.
LAF in Vial Sealing area
ACTIVE, PASSIVE,
SURFACE &
PERSONNEL
Vial & PFS Filling area
(Man material movement, chances of product
exposure to environment)
PASSIVE & ACTIVE
Vial & PFS filtration areas
Vial & PFS Cooling zone areas
B
(Critical areas)
B
(Non Critical areasclosed systems)
Corridor (near entry to filling room)
Every Day during Batch
manufacturing and
weekly twice during non
manufacturing days.
Every Day during Batch
manufacturing and
weekly once during non
manufacturing days
Vial sealing area
PASSIVE
Vial & PFS Bulk manufacture
C
Every Day during Batch
manufacturing and
weekly once during non
manufacturing days
Vial & PFS Component preparation room
Vial & PFS Washing areas
Change rooms
PASSIVE
Change rooms
D
Weekly once or APP
Microbiology Testing
•
Raw or Packaging Materials
•
In-process and Finished Products
•
Bioassays
•
Utilities
Microbiology Testing
•
Sterility Testing
•
Bacterial Endotoxin Test
•
Mycoplasma Testing
•
MLT
Sterility Testing
•
Sterility test is a quality control test used for raw material analysis and 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
Sterility Testing
• Methods are defined in Pharmacopoeia
• Membrane filtration is the preferred method if product is filterable
• Direction inoculation is alternative
• Media types
• Soybean Casein Digest medium (SCD), 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.
• Growth should be evident after 3 days (bacteria), 5 days (moulds)
• media may be purchased or made in-house using validated sterilization
procedures
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/, 30-35°C for FTM
• Test containers should be inspected at intervals
Sterility Testing
• Negative Controls
• 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
• 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.
• Growth should be evident after 3 days (bacteria), 5 days (moulds)
Sterility Testing
• Results
• Any growth should be identified (Genotypic)
• Automated/Semi-automated systems used for identification should be
periodically verified using reference strains
• 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
Sterility Test IP/EP/USP
Parameters IP
EP
USP
Remarks
Sampling
Table 1 (IP2010)
P. No 57 of IP
(10%,5 and 2%)
Table 2.6.1.2 of EP
Table 3 of EP
Facilities
Grade A laminar air flow or an
isolator
Grade A laminar air flow
located within a class B clean
room or an isolator
Same as EP
Nil
Methods
Membrane Filtration
Direct Inoculation
Same as IP
Same as IP & EP
Nil
Media
FTM
SCDM
Same as IP
Same as IP & EP
Incubation
FTM at 300 to 350
SCDM 200 to 250 Incubate for
14 days
Same as IP
Same as IP & EP
Nil
Interpretation
No evidence of microbial
growth sterility test complies
( macroscopically)
Same as IP
Same as IP & EP
Nil
Controls
Positive Control
Negative Control
Positive test controls
Same as IP
Same as IP & EP
Nil
Nil
Alternative
Thiglycollate
medium
(IP and USP)
Sterility Test IP/EP/USP
Parameters
IP
EP
USP
Remarks
Re-testing
When it is clearly shown test is invalid
Same as IP
Same as IP
Nil
In valid Test
•Microbial growth in Negative control
•ENM of testing facility is faulty
•Testing procedure reveals a fault
•After identifying the microorganism
isolated from the test, the growth of
these species may be ascribed
unequivocally to faults with respect to
the material and/ technique used
microbial growth
Same as IP
USP
Nil
Repeat testing
Same number of unit as in the original
test
If no evidence of microbial growth
found in repeat test, preparation
complies for sterility test
Same as IP
USP
Nil
Bacterial 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 Testing
Bacterial Endotoxin
• Types of LAL test
• Gel Clot ( Limit Test)
• Gel Clot ( SemiQuantitative test)
• Turbidimetric Kinetic Method
• Chromogenic Kinetic Method
• Turbidimetric End point Method
• Kinetic End point Method
• Equipment used in test must be Endotoxin free
• Validation of accuracy and reliability of the method for each product is
essential
Bacterial Endotoxin
• Gel Clot Method
• Original method
• The official “referee test”
• The specimen is incubated with LAL of a known sensitivity. Formation of a
gel clot is positive for endotoxin.
• Gel clot ( quantitative method) quantifies bacterial endotoxins in the test
solution by titration to an end point
Bacterial Endotoxin
Gel Clot
Chromogenic
Endpoint
Chromogenic
Kinetic
Turbidimetric
Semiquantitative
Quantitative
Quantitative
Quantitative
Simple Least
expensive,
Requires 37°C bath
Requires
spectrophotometer
or plate reader
Requires
Requires
incubating plate or tube incubating plate or
reader
tube reader
Manually read and
recorded
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Can be automated,
allows electronic
data storage
Sensitive down
to 0.03 EU/ml
Sensitive down
to 0.1 EU/ml
Sensitive down
to .005 EU/ml
Sensitive down
to .001 EU/ml *
* (Sensitivities vary by reagent manufacturer, instrumentation and testing conditions)
Mycoplasma Testing
•
Mycoplasma Testing
• Therapeutic products for both human and animal use are required to
undergo mycoplasma testing throughout various steps in the production
process
• Methods
•
Culture Method
•
Indicator Cell culture method
•
Neuclic Amplification Methods
Microbial Limit Test
• Microbial Limit Test ( Test for specified Microorganism)
• The tests designed primarily to determine whether a substance or
•
•
Total Aerobic Microbial Count (TAMC Or TABC)
•
•
preparation complies with an established specification of microbiological
quality
Total viable aerobic count
TSA
Total Yeast and Mould Count (TYMC or Total Fungal count)
•
SDA
• TVAC= TAMC + TYMC
•
Report – ml or gm
Bioburden testing
• Bioburden 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
organismsm
• 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
Utilities
• Potable water
• Purified water,
•
Highly purified water
• Water for injection
• Pure Steam
• Compressed Air/Nitrogen/CO2
Pharmaceutical Water
• For purified/highly purified water and highly purified water , limits defined in
pharmacopoeia
• Purified <100CFU/mL
• Highly purified and WFI 10CFU/100mL (but is usually kept at high
temperatures)
• Pure steam – WFI standard
• WFI, Pure steam and Highly Purified water (BET <0.25 IU per ml)
•
Alert and Action limits set by manufacturer (with action to be taken if limits are
exceeded)
Bioassay
• Microbiological assay for antibiotics
• The potency of an antibiotic is estimated by comparing the inhibition of
growth of sensitive microorganism produced by known concentrations of
the antibiotic to be examined and a reference substance
• Activity has been precisely determined with reference to the
corresponding international standard or international reference
preparation
•
Methods
•
•
•
Diffusion
Turbidimetric
Calculate the potency using appropriate statistical methods
Bioassay
• Bioassays for Biologics
• Used to assess the potency of proteins, antibodies or hormones by
comparing their effects on a culture of living cells or a test organism to those
of a control preparation.
• The concentration or potency of a substance is determined by measurement
of the biological response that it produces.
• Types of Bioassays
•
In-Vivo Bioassays
•
•
Animal based bioassays
In-Vitro Bioassays
•
•
•
Cell based potency assays
Antiviral cell based assays
Cell proliferation assays
OOS handling in Microbiology Testing
• Investigation into a laboratory result that did not meet normally expected
criteria
• Test Results Vs Performance standard
• Affects
•
•
•
•
Personnel Monitoring
EM monitoring
RM/PM testing
Product testing
OOS
• Test Results not meeting specifications is not a failure
• Due to
•
•
•
•
•
Operator Error
Out of Calibration
Not adhering to procedure
Un assignable Reasons
Method Not Validated
OOS
• What happens when a sterility test failure Occurs?
• How should one investigate a sterility test failure?
• Many facilities have no real idea how to begin an investigation
• Many facilities don’t have a plan, i.e., a specific SOP which describes how a sterility
test failure should be investigated
• They use the QC- OOS SOP which describes what to do, but is chemistry test oriented
• Typically only negative findings are documented to any great extent
• Often the scope (breadth & depth) of the investigation isn’t sufficient to detect the
root cause
• Documentation doesn’t reflect efforts expended
• Assumptions are made that preclude finding the root cause of the sterility test failure
OOS
• Investigation of sterility positives
• Difficult to support invalidation of a positive sterility test
• One must have conclusive and documented evidence that clearly shows that
the contamination occurred due to the testing that was performed
OOS
• Investigation of Sterility Positives –
• Key Elements of the Investigation
• Identification (speciation) of the organism isolated from the sterility test
[a strain level identification is desirable of such investigations]
• Review and confirm
•
•
•
•
•
•
Record of laboratory tests and deviations
Monitoring of production area environment
Personnel Monitoring
Product Pre-sterilization bioburden
Production Record Review
Manufacturing History
OOS
• Investigation Approach
• Need to have an open mind!!! Don’t jump to conclusions. Consider all evidence.
• Need to document everything that was reviewed [good, as well as bad]
• Manufacturing Investigation
• Validation Investigation :- production sterilization processes & sterility test isolator
• Microbiology Investigation
OOS
Extraordinary Environmental Monitoring
• Definition: Additional environmental monitoring performed during sterility test
failure investigations.
•
Samples are typically taken using swabs, because irregular surfaces and hard-to-getto sites [nooks & crannies] need to be sampled.
• Samples are taken at non-routine sites which may not have been cleaned & sanitized
effectively. Increased sampling frequency is required.
• Most cases one needs to perform aggressive sampling to have a chance to find the
source of the sterility test contaminant
• The rate of growth of sterility test contaminants may be very slow and some types of
microorganisms won’t ever be seen during routine EM, e.g., Propionibacterium acnes
[microaerophillic or anaerobic] and Cladosporium species [dematiaceous mold]
OOS
EM data TRENDS that could contribute to a Batch Sterility
•
•
•
•
•
Increased numbers of viable microorganisms in critical areas – one doesn’t have to
exceed Alert or Action Levels to have a batch failure
New or unusual isolates in the facility
Increase in baseline microbial “Load” over time
Increase in bioburden of raw materials
Presence of a microorganism resistant to disinfectant used in the facility
OOS
Root Cause vs. Contamination Source
•
•
•
•
Just because you find the sources of the microbial contaminant, that doesn’t mean
that you have also found the root cause
Contamination may be transferred to filling machines or sterility test isolators/hoods
in more than one way
Don’t assume that you have found the only root cause, if the investigation is
incomplete
Don’t terminate the investigation prematurely
OOS
Recommendations
•
Make no assumptions and keep an open mind
•
Document everything!!!!!
•
Obtain the best possible identification for the sterility test failure isolates – the
goal is a strain level ID
•
Perform Aggressive Extraordinary Environmental Monitoring to find the source of
the sterility test failure isolates
OOS
Test Results:• Suggested procedures to be followed for resolution of out-of-specification
microbiology test results:-
•
Confirm the correct microbiological test method was used for testing
•
Confirm the analyst is qualified to perform the test method
•
Confirm calculations (if applicable) are correct
•
Confirm all negative controls for media, diluents, and test equipment were negative
•
Confirm growth promotion testing for all media were satisfactory
•
Confirm environmental samples taken during testing were satisfactory
•
Determine if the sample was taken aseptically by a qualified individual
OOS
Test Results
•
Confirm incubators, hoods and isolation chambers and other laboratory systems
(where applicable) were in calibration and functioning properly
•
Determine if other samples tested in the same time frame using the same lots of
media, diluents and testing equipment were satisfactory
•
Review historical data to determine if similar microbiological problems have been
previously reported
OOS
Test for outliers:•
The test for outliers is used when a result from a data pool with mainly
homogenous results differs so greatly from the others
•
The “outlier” may result from a laboratory or production error that was not
identified and is thus not representative.
•
Keep in mind: “Outliers” are only accepted as such if they can be proved
statistically (e.g. > triple standard deviation) and only for biological and not for
chemical tests!
Summary
•
•
Microbiology testing is critical
Approved specification must be in place
•
GTP and STP need to be in place
•
Testing method must be Validated.
•
Lab person - Personnel Qualification must.
•
All activities and reports need to be recorded
•
GLP plays major role in deciding the Quality of Test reports