1. Wyeth-Ayerst Pharmaceuticals1 CLEANING VALIDATION Microbial Monitoring Anthony M. Cundell Ph. D. Associate Director, QA Microbiological Development & Statistics Wyeth-Ayerst Pharmaceuticals Pearl River, New York

2. Wyeth-Ayerst Pharmaceuticals2 CLEANING VALIDATION What are the the current Good Manufacturing Practices requirements for cleaning validation?

3. Wyeth-Ayerst Pharmaceuticals3 CLEANING VALIDATION 21 CFR 211.67 Equipment cleaning and maintenance a) Equipment and utensils shall be cleaned, maintained and sanitized at appropriate intervals to prevent contamination that would alter the safety, identity, strength, quality or purity of the drug product.

4. Wyeth-Ayerst Pharmaceuticals4 CLEANING VALIDATION 21 CFR 211.113 Control of microbiological contamination (a) Appropriate written procedures, designed to prevent objectionable microorganisms in drug products not required to be sterile, shall be established and followed.

5. Wyeth-Ayerst Pharmaceuticals5 CLEANING VALIDATION 21 CFR 211.113 Control of microbiological contamination (b) Appropriate written procedures, designed to prevent objectionable microorganisms in drug products required to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.

6. Wyeth-Ayerst Pharmaceuticals6 CLEANING VALIDATION What is the accepted industry practice for microbial monitoring during cleaning validation?

7. Wyeth-Ayerst Pharmaceuticals7 CLEANING VALIDATION FDA Mid-Atlantic Region Inspection Guide on Cleaning Validation (July, 1992) does not address microbial monitoring.

8. Wyeth-Ayerst Pharmaceuticals8 CLEANING VALIDATION FDA Guide to Inspection of validation of Cleaning processes (July, 1993) states that microbiological aspects of equipment cleaning should be considered. There should be some evidence that routine cleaning and storage does not allow microbial proliferation.

9. Wyeth-Ayerst Pharmaceuticals9 CLEANING VALIDATION PhRMA report on microbiological monitoring in nonsterile pharmaceutical manufacturing areas (March, 1997) recommended that depending on the product, e.g. inhalation products,oral aqueous liquids, vaginal creams, etc, cleaning validation should include microbial sampling to ensure microbiological quality.

10. Wyeth-Ayerst Pharmaceuticals10 CLEANING VALIDATION PhRMA Guidelines for the Validation of Cleaning Procedures for Bulk Pharmaceutical Chemicals published in September, 1997 declared that microbial issues was not within the scope of the report.

11. Wyeth-Ayerst Pharmaceuticals11 CLEANING VALIDATION Microbial contamination can be prevented by: n Selection of suitable equipment n Sound cleaning programs n Cleaning equipment directly after use n Dry storage of equipment

12. Wyeth-Ayerst Pharmaceuticals12 CLEANING VALIDATION Microbial Monitoring Methods n Swab Method n Surface Rinse Method n RODAC Plate Method n Limulus Amoebocyte Lysate Method n ATP Bioluminescence Method

13. Wyeth-Ayerst Pharmaceuticals13 CLEANING VALIDATION What are the Advantages & Disadvantages of the the Available Microbial Monitoring Methods?

14. Wyeth-Ayerst Pharmaceuticals14 CLEANING VALIDATION Swab Method n Advantages: Most common method used with selective media to isolate directly different microbial populations. n Disadvantages: Recovery may not be reproducible & quantitative.

15. Wyeth-Ayerst Pharmaceuticals15 CLEANING VALIDATION Surface Rinse Method n Advantages: Higher counts obtained than swab method & better overall assessment possible. n Disadvantages: Entire surface evaluated, microbial population must be detached & membrane filtration necessary to obtain countable numbers.

16. Wyeth-Ayerst Pharmaceuticals16 CLEANING VALIDATION RODAC Plate Method n Advantages: Direct growth on media in contact plate is convenient, neutralizers may be included in media & different media may be used. n Disadvantages: Only applicable to surfaces that are smooth & have low counts.

17. Wyeth-Ayerst Pharmaceuticals17 CLEANING VALIDATION Limulus Amoebocyte Lysate Method n Advantages: Rapid, sensitive, quantitative measure of bacterial endotoxin levels. n Disadvantages: Indirect measurement of high numbers of gram-negative bacteria only.

18. Wyeth-Ayerst Pharmaceuticals18 CLEANING VALIDATION Bioluminescence Method n Advantages: Rapid, highly accurate & reliable method. n Disadvantages: Suitable for microbial counts in the range of 10 4 to 10 8 organisms as insufficiently sensitive for low microbial counts.

19. Wyeth-Ayerst Pharmaceuticals19 CLEANING VALIDATION Microbial requirements for non- sterile pharmaceutical drug products: Control of the total bioburden, elimination of USP indicator and objectionable microorganisms.

20. Wyeth-Ayerst Pharmaceuticals20 CLEANING VALIDATION Microbial Limit for a oral solid dosage form, i.e., Tablet or capsule. Total Aerobic Microbial Count NMT 1000 cfu/g Total Combined Yeast & Mold Count NMT100 cfu/g Absence of USP Indicator organisms, i.e., E. coli, S. aureus and Salmonella spp.

21. Wyeth-Ayerst Pharmaceuticals21 CLEANING VALIDATION Case history: What should be the acceptance criteria for a blender used to process a wet granulation tablet?

22. Wyeth-Ayerst Pharmaceuticals22 CLEANING VALIDATION Approach: The microbial limit in terms of cfu per 25 cm 2 can be determined from a knowledge of the internal surface area of the blender, the quantity of granulation processed and the Microbial Limit for the product (oral solid dosage form)

23. Wyeth-Ayerst Pharmaceuticals23 CLEANING VALIDATION Example: A Gemco 20 blender has an internal surface area of 46,128 cm 2 & a capacity of 220 kg of granulation. With a surface bioburden of 100 cfu/ 25cm 2 there is potential to transfer <1 cfu/g to the granulation. cf. Microbial Limit for an oral solid dosage form NMT 1000 cfu/g.

24. Wyeth-Ayerst Pharmaceuticals24 CLEANING VALIDATION Example: A Gemco 75 blender has an internal surface area of 100,944 cm. sq. & a capacity of 500 kg of granulation. With a surface bioburden of 100 cfu/25cm 2 there is potential to transfer <1 cfu/g to the granulation. c.f. Microbial Limit for an oral solid dosage form NMT 1000 cfu/g.

25. Wyeth-Ayerst Pharmaceuticals25 CLEANING VALIDATION Example: A Littleford blender has an internal surface area of 22,718 cm. sq. & a capacity of 100 kg of granulation. With a surface bioburden of 100 cfu/25 cm 2 there is potential to transfer <1 cfu/g to the granulation. c.f. Microbial Limit for an oral solid dosage form NMT 1000 cfu/g.

26. Wyeth-Ayerst Pharmaceuticals26 CLEANING VALIDATION Recommended Acceptance Criteria for Blenders: NMT 100 cfu per 25 cm 2 & Absence of USP Indicator Organisms. Recommended Monitoring Method: Swab Method

27. Wyeth-Ayerst Pharmaceuticals27 CLEANING VALIDATION Microbial requirements for sterile pharmaceutical drug products: Control of total bioburden to maintain the Sterility Assurance Level & elimination of endotoxin to prevent pyrogenic reactions.

28. Wyeth-Ayerst Pharmaceuticals28 CLEANING VALIDATION Case history: What should be the acceptance criteria for a pre- sterile bulk solution tank used to aseptically fill a parenteral product?

29. Wyeth-Ayerst Pharmaceuticals29 CLEANING VALIDATION Approach: The microbial limit in terms of cfu per 25 cm 2 can be determined from a knowledge of the internal surface area of the bulk tank, the volume of the bulk solution processed and the square footage of the cartridge filter used to sterile filter the product.

30. Wyeth-Ayerst Pharmaceuticals30 CLEANING VALIDATION Calculations: Challenge cfu per sq. cm of Filter = Fill Volume(Liters) X Bioburden (cfu/l)/Surface Area of the Filter (sq.cm) Conversion factor: 10 sq. ft. = 9290 sq. cm

31. Wyeth-Ayerst Pharmaceuticals31 CLEANING VALIDATION Example: A 1000 Liter bulk tank with a bioburden of 10 cfu per 100 mL will represent a total bacterial challenge of 10 5 to a 10 sq. ft. cartridge filter, i.e., 10 cfu per cm 2 of filter surface, which well below the filter retention rating of 10 7 per sq. cm.

32. Wyeth-Ayerst Pharmaceuticals32 CLEANING VALIDATION Acceptance criteria (Sterile products): Based on pre-sterile bulk solution bioburden limit, i.e. NMT 10 cfu/100 mL (aseptically- filled products), heat resistance of bacterial spores (terminally- sterilized products or equipment) & endotoxin contribution.

33. Wyeth-Ayerst Pharmaceuticals33 CLEANING VALIDATION Example: 125 Liter pre-sterile filtration bulk tanks (Precision Stainless, Inc.) with a product contact surface area of 2356 sq. cm. With a surface bioburden of 33 cfu/25 cm 2 there is potential to transfer 4 X 10 3 organisms or 1 cfu/100 mL to the bulk solution.

34. Wyeth-Ayerst Pharmaceuticals34 CLEANING VALIDATION Endotoxin considerations: Endotoxin limits for parenteral products are set using the maximum human dose for the individual products. Since a bacterial cell weights 10 -13 grams, 10 5 cells in the bulk solution represents 10 -8 gram of cellular material (10 ng).

35. Wyeth-Ayerst Pharmaceuticals35 CLEANING VALIDATION Endotoxin considerations: If endotoxin represents 10% of the cell weight then a bulk solution of 125 Liters will contain less 0.00001 ng/mL of endotoxin.

36. Wyeth-Ayerst Pharmaceuticals36 CLEANING VALIDATION Example: 125 Liter pre-sterile filtration bulk tanks (Precision Stainless, Inc.) with a product contact surface area of 2356 sq. cm. With a surface endotoxin level of 0.5 EU/25 cm 2 there is potential to transfer up to 20 EU or 0.002 EU/mL to the bulk solution.

37. Wyeth-Ayerst Pharmaceuticals37 CLEANING VALIDATION Recommended Acceptance Criteria for pre-sterile filtration bulk tanks NMT 33 cfu per 25 cm 2 & NMT 0.2 EU per sq. cm Recommended Monitoring Method: RODAC Plate Method &/or Rinse sampling.

38. Wyeth-Ayerst Pharmaceuticals38 CLEANING VALIDATION Conclusions: Based on the desired Microbial Limit for a non-sterile product or a pre-sterile filtration bulk solution, given a certain equipment surface to material quantity ratio, it is possible to set appropriate surface bioburden limits for cleaning validations.