Practical aspects of Microbiological Testing and handling of OOS

1. 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

2. 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

3. 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.”

4. 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. Viable & Nonviable particles Types of Contamination

5. 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.

6. 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.

7. What are SPORES? Why are they a MAJOR CHALLENGE!!!! Spores 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!

8. 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

9. Sources of Contamination • People/Personnel • Air/Environment • Equipment • Surfaces • Others

10. People • Personnel Hygiene • Gowning • Gloves • Personnel.Qualification • Minimum number of personnel in clean areas • Training to all including cleaning and maintenance staff • Special cases

11. Environmental Monitoring • Microbiological • Air samples • Surface swabs • Personnel swabs one of the most important laboratory controls is the environmental monitoring program

12. 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

13. 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

14. 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

15. 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 Areas

16. 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) Environmental Monitoring of clean rooms

17. Limit For Monitoring Clean Areas

18. 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

19. Environmental Monitoring Recommended Frequency of Monitoring:-

20. Raw or Packaging Materials In-process and Finished Products Bioassays Utilities Microbiology Testing

21. Sterility Testing Bacterial Endotoxin Test Mycoplasma Testing MLT Microbiology Testing

22. 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

23. 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

24. 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

25. 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

26. 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 Testing

27. Sterility Test IP/EP/USP

28. Sterility Test IP/EP/USP

29. 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

30. 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

31. 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

32. Bacterial Endotoxin

33. 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 Mycoplasma Testing

34. 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 Microbial Limit Test

35. 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 Bioburden testing

36. Potable water Purified water, Highly purified water Water for injection Pure Steam Compressed Air/Nitrogen/CO2 Utilities

37. 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) Pharmaceutical Water

38. 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

39. 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 Bioassay

40. 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 handling in Microbiology Testing

41. 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

42. 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

43. 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

44. 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

45. 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

46. 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-get-to 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

47. 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

48. 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

49. 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

50. 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