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 APIs & 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 1000s 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)

<1

<1

<1

<1

B C D

10 100 500

5 50 100

5 25 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 LAFS in Both Vial & PFS Filling area LAFS in Cooling zone ( for both Vial Filling & PFS) LAFS in Vial & PFS filtration area LAF in Vial Sealing area ACTIVE, PASSIVE, SURFACE & PERSONNEL Vial & PFS Filling area (Man material movement, chances of product exposure to environment) B (Critical areas) Every Day during Batch manufacturing and weekly twice during non manufacturing days. Every Operation or Weekly Twice during non manufacturing days. GRADE SAMPLING FREQUENCY

PASSIVE & ACTIVE

Vial & PFS filtration areas

Vial & PFS Cooling zone areas Corridor (near entry to filling room) Vial sealing area

B (Non Critical areasclosed systems)

Every Day during Batch manufacturing and weekly once during non manufacturing days

PASSIVE

Vial & PFS Bulk manufacture C Vial & PFS Component preparation room Vial & PFS Washing areas Change rooms Every Day during Batch manufacturing and weekly once during non manufacturing days

PASSIVE

Change rooms

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 20-25C for SCD/, 30-35C 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
Sampling Table 1 (IP2010) P. No 57 of IP (10%,5 and 2%) Grade A laminar air flow or an isolator Membrane Filtration Direct Inoculation FTM SCDM

EP
Table 2.6.1.2 of EP

USP
Table 3 of EP

Remarks
Nil

Facilities

Grade A laminar air flow located within a class B clean room or an isolator Same as IP Same as IP

Same as EP

Nil

Methods Media

Same as IP & EP Same as IP & EP

Nil Alternative Thiglycollate medium (IP and USP) Nil

Incubation

FTM at 300 to 350 SCDM 200 to 250 Incubate for 14 days

Same as IP

Same as IP & EP

Interpretation

No evidence of microbial growth sterility test complies ( macroscopically)

Positive Control Negative Control Positive test controls

Same as IP

Same as IP & EP

Nil

Controls

Same as IP

Same as IP & EP

Nil

Sterility Test IP/EP/USP

Parameters
Re-testing In valid Test

IP
When it is clearly shown test is invalid 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 number of unit as in the original test If no evidence of microbial growth found in repeat test, preparation complies for sterility test

EP
Same as IP Same as IP

USP
Same as IP USP

Remarks
Nil Nil

Repeat testing

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

Semiquantitative Simple Least expensive, Requires 37C bath Manually read and recorded

Chromogenic Endpoint
Quantitative Requires spectrophotometer or plate reader Can be automated, allows electronic data storage

Chromogenic Kinetic
Quantitative

Turbidimetric
Quantitative

Requires Requires incubating plate or tube incubating plate or reader tube reader 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
preparation complies with an established specification of microbiological quality Total viable aerobic count

Total Aerobic Microbial Count (TAMC Or TABC)

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 dont 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 isnt sufficient to detect the root cause Documentation doesnt 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!!! Dont 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 wont 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 doesnt 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 doesnt 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 Dont assume that you have found the only root cause, if the investigation is incomplete Dont 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