The document discusses investigations into a failed sterility test of a product. It describes the perspectives and concerns of different stakeholders when a sterility failure occurs, including the technician, lab director, quality assurance, manufacturer, and client. It then details the steps of a thorough investigation conducted by an expert to find the root cause, involving reviewing laboratory and production records, environmental monitoring data, personnel training, and sources of raw materials. The investigation found the likely cause was contamination from cardboard boxes used to package sterile containers in the laboratory rather than any issues with production, raw materials, or personnel.

1. Investigations into Sterility Failures What do you do when you find out there is a problem with a sterility test?

2. Investigations into Sterility Failures Answer: It depends on your perspective

3. Investigations into Sterility Failures A. You Cry If you’re the technician

4. Investigations into Sterility Failures The Technician I failed No one will trust me I could be fired

5. Investigations into Sterility Failures B. You Deny it If you’re the lab director

6. Investigations into Sterility Failures The Lab Director Who messed up? What about training? Will I be blamed? There goes my bonus

7. Investigations into Sterility Failures C. You get ready to reject the product If you’re QA

8. Investigations into Sterility Failures QA The FDA is coming We have a disaster Shut down the place Reject first, ask questions later

9. Investigations into Sterility Failures D. You blame someone else If you’re the manufacturer

10. Investigations into Sterility Failures The Manufacturer I can’t trust anyone Other products involved? There goes our incentive Now we have to spend money to do it right. Damn!

11. Investigations into Sterility Failures E. You Panic If you’re the client

12. Investigations into Sterility Failures The Client I’ll lose money I’ll lose credibility I might lose my business

13. Investigations into Sterility Failures How do you reconcile all these concerns? Methodically Lab Manufacturer, supplier Management

14. Investigations into Sterility Failures The Lab Technician Spore strips (media run) First time alone Identified organism Not B. stearothermophilis Retest passed

15. Investigations into Sterility Failures The Lab Director Direct inoculation (cotton) Isolated Gram negative Could not find the source Notified client Lost the account

16. Investigations into Sterility Failures QA Spore strip (Bandage) B. subtilis found Lab investigation ETO sterilization review: Excursions in cycle Material rejected

17. Investigations into Sterility Failures The Manufacturer Sterilant: propylene oxide Isolated Gram negative Lab investigation Manufacturing investigation Excipient supplier investigation: Excessive bioburden Product recalled Heads rolled (management)

18. Investigations into Sterility Failures The Client Sterile ampule sterility failure We panicked Manufacturer blamed the lab Lab blamed the manufacturer QA ready to reject Life was getting interesting

19. Investigations into Sterility Failures The Client Antibiotic in plastic ampule First failure ever (3 years) Propriobacterium acnes Lab investigation OK Recent merger

20. Investigations into Sterility Failures Product History Three year clean tract record Human-associated organism Product was inhibitory Did not make sense: validated Convinced management to wait

21. Investigations into Sterility Failures Why would I want to save the batch? Could I do anything to save the batch?

22. Investigations into Sterility Failures FDA Out-of-Specification (OOS) guidance? It essentially excludes microbiological and biological assays

23. Investigations into Sterility Failures ICH Q7A OOS guidance? States that OOS investigations are not necessary for in-process tests

24. Investigations into Sterility Failures USP <1117>? Discusses the difficulties associated with resolving a Microbial Data Deviation (MDD) Not much help

25. Investigations into Sterility Failures FDA Aseptic Processing Guidance Provides a rational, methodical, scientific approach to address the issue

26. Investigations into Sterility Failures “ Only if conclusive and documented evidence clearly shows that contamination occurred as part of testing should a new test be performed”

27. Investigations into Sterility Failures “ When available evidence is inconclusive batches should be rejected as not conforming to sterility requirements”

28. Investigations into Sterility Failures Needed a smoking gun

29. Investigations into Sterility Failures Lab? Manufacturer? Supplier? Management?

30. Investigations into Sterility Failures The initial steps Place product on hold Are other batches affected? Write an investigation protocol based on the criteria outlined in the Aseptic Processing Guideline

31. Investigations into Sterility Failures Detail exactly what should be done Seven specific areas that need to be investigated

32. Investigations into Sterility Failures 1. Identify the organism Review lab tests and deviations Review production area EM data Review lab and production personnel training and history i i

33. Investigations into Sterility Failures Review product bioburden Review production records Review manufacturing history (including media fills) i i

34. Investigations into Sterility Failures We added four more areas to investigate

35. Investigations into Sterility Failures 8. Review the API source 9. Review the excipient sources 10. Review weighing facility 11. Review contract laboratory

36. Investigations into Sterility Failures We placed the onus of the investigation on the laboratory operation, aligning it with an MDD investigation

37. Investigations into Sterility Failures We had eleven areas to find the smoking gun We told management we could do it

38. Investigations into Sterility Failures Now it was up to us (aka me) to find the source of contamination

39. Investigations into Sterility Failures Wrote the protocol Had it countersigned by senior management in my company, the manufacture and the contract laboratory We needed the full cooperation of all parties in order to thoroughly conduct the investigation

40. Investigations into Sterility Failures 1. Identification : Propriobacterium acnes - associated with human activity Confirmed by GC analysis

41. Investigations into Sterility Failures 2. Review of laboratory tests and deviations Reviewed sterility test history of the contract laboratory for 3 years Sterility failure rate and false positive rate < 0.1% Organism not found in the past

42. Investigations into Sterility Failures 3. Production area environment 3 years of EM data reviewed Organism found 18 months ago on air sampling plate Not found in sterile shroud Not associated with any sterility or environmental issues

43. Investigations into Sterility Failures Personnel Monitoring (laboratory and production) Organism not found on either the laboratory or production personnel in the past three years

44. Investigations into Sterility Failures 5. Product bioburden Past 3 years reviewed, one instance it exceeded limit of < 10 CFU/100 mL This organism was not isolated Bioburden found only once or twice a year; each time organism was Bacillus. All the other times bioburden < 10 CFU/100 mL Biofouling occurred 5 years ago, rectified with CIP 100 after media fills

45. Investigations into Sterility Failures 6. Review of Batch Records All records from the past 3 years All excursions addressed during the runs Current record was also re-reviewed and no excursions of any type were found

46. Investigations into Sterility Failures Manufacturing history No bioburden (other than the aforementioned), personnel or EM excursions in last 3 years Manufacturing area pristine, personnel well trained and man-in-the-plant for every batch Media fills (15,000 ampules each of TSB and FTM) done 2X/year, No +

47. Investigations into Sterility Failures Additional Areas

48. Investigations into Sterility Failures API source Microbial limit testing performed on every batch of API by both the overseas manufacture and the US contract laboratory

49. Investigations into Sterility Failures The microbiological history reviewed for 3 years and Propriobacterium acnes never found

50. Investigations into Sterility Failures Occasionally Bacillus found in API at levels below the specification of <15 CFU/gm and appeared infrequently in the pre-sterilization bioburden

51. Investigations into Sterility Failures 9. Excipients MLT done on every excipient lot Microbiological history reviewed for 3 years. P. acnes not found Other than WFI, excipients were corrosive or not conducive to growth of this organism

52. Investigations into Sterility Failures 10. W eighing facility (NJ) Every area thoroughly scrutinized: receiving area, holding area, weighing room, transfer area, shipping area, analytical labs Weighing room trend data < 2 CFU per settling plate site Organism not found in three years

53. Investigations into Sterility Failures 11. Contract laboratory Contract lab scrutinized: receiving area, staging area, anteroom, sterility suite, cleaning processes, HEPA filters, personnel, autoclave cycles, purchased media, sterility equipment, incubators, EM isolates

54. Investigations into Sterility Failures Anything remotely associated with the product was scrutinized Organism not isolated, 3 year data Documentation provided complete cross referencing to media prep, sterilization, growth promotion, organism maintenance, EM, cleaning and (most importantly) training

55. Investigations into Sterility Failures The bottom line: Great data, No smoking gun

56. Investigations into Sterility Failures Weakest link was the sterility test What more could I do? Management was very impatient

57. Investigations into Sterility Failures Convinced management to do a study

58. Investigations into Sterility Failures Shipped finished product from the plant to the lab for testing

59. Investigations into Sterility Failures Watched every step of the operation: Receiving and logging of FP Storing the product Preparing it for testing, staging it and transferring it into the sterility suite

60. Investigations into Sterility Failures I even gowned up with the technicians and stood behind them while they performed the sterility test

61. Investigations into Sterility Failures Everything was perfect I couldn’t find the smoking gun and was resigned to the fact that we would have to reject the batch without any definitive root cause

62. Investigations into Sterility Failures There was one glimmer of hope, however

63. Investigations into Sterility Failures During the sterility test 20 ampules were pooled into a sterile container Sterile containers sterilized by gamma radiation Shipped in a large cardboard box containing a polyethylene bag filled with 1000 sterile containers

64. Investigations into Sterility Failures Why weren’t the containers individually wrapped? Less expensive to buy them in bulk

65. Investigations into Sterility Failures The lab recently switched from individually wrapped containers to bulk packaged containers

66. Investigations into Sterility Failures Sampled the inside, outside and flaps of the cardboard box, and the polyethylene bag

67. Investigations into Sterility Failures Two samples contained Propriobacterium acnes

68. Investigations into Sterility Failures The smoking gun?

69. Investigations into Sterility Failures YES!

70. Investigations into Sterility Failures Residing in the cardboard box that contained the sample containers used to pool the product before filtration!

71. Investigations into Sterility Failures We had our evidence to invalidate the original test The lab purchased individually wrapped sample containers, invalidated the original sterility test, performed a successful retest

72. Investigations into Sterility Failures We released the product based upon the results of the retest, the completion of the investigation by my company, the contract manufacturer and the contract laboratory

73. Investigations into Sterility Failures Lessons learned: ● Don’t panic ● Let science be your guide ● Prepare a sound action plan ● Involve all stakeholders ● Keep searching for a cause ● Be brave ● Don’t give up

74. Investigations into Sterility Failures Questions?

75. Investigations into Sterility Failures Thank you Frank Settineri Veracorp LLC 973-729-8171 [email_address]

76. Investigations into Sterility Failures References 1 www.microbiologyforum.org/PMFNews/PMFNew s.12.11.0611.pdf 2 www.fda.gov/cder/guidance/3634fnl.htm OOS 3 www.fda.gov/Cber/gdlns/ichactive.pdf ICH OOS 4 USP, <1117> Best Microbiological Practice USP 29 Supplement 2, 2006 Guideline 5 www.fda.gov/cder/guidance/5882fnl.pdf Aseptic guidance