Sterility testing of Pharmaceutical products

a detailed overview of Sterility testing of Pharmaceutical products according to the IP, BP, USP

1. UNIT III 3.3 Sterility testing of products Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

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3. 3 INTRODUCTION Sterility testing Sterility testing examines samples of the final product for the presence of microorganisms Should be applied to all products that are designated as sterile A satisfactory result only indicates that no contaminating microorganism has been found in the sample examined in the conditions of the test

4. 4 Culture media   Fluid thioglycollate medium For the culture of aerobic and anaerobic bacteria pH after sterilization 6.9 to 7.3 To be incubated at 30–35 °C Soya-bean casein digest medium Suitable for the culture of both fungi and aerobic bacteria pH after sterilization 7.1 to 7.5 To be incubated at 20–25 °C Sterility check of media (Negative control) Incubate portions of the media for 14 days. No growth of should occur

5. 5 Growth promotion test of aerobes, anaerobes and fungi (Positive control) Inoculate each media using test organism Incubate under the specified conditions The media are suitable if a clearly visible growth of the microorganisms occurs Aerobic bacteria Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276 Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134 Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275 Anaerobic bacterium Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437, NBRC 14293

6. 6 Fungi Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594 Aspergillus brasiliensis ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455 ATCC - American Type culture collection, USA CIP - Collection de l'Institut Pasteur, France NCTC - National collection of type cultures, England NCIMB - National Collection of Industrial and Marine Bacteria, National Collections of Industrial, Food and Marine Bacteria, UK ,Scotland NBRC – National Biological Resource Centre, Japan IMI- CABI GRC (Strain numbers: IMI): The Genetic Resource Collection, CABI Bioscience UK Centre UK

7. 7 Sterility testing methods 1. Direct inoculation methods Involves introducing test samples directly into nutrient media 2. Membrane filtration method • Involves filtration of fluids through a sterile membrane filter • Microorganism present being retained on the surface of the filter • Portions of the filter are transferred to suitable culture media

8. 8 Sterility testing by direct inoculation method Growth - contaminated

9. 9 Sterility testing of pharmaceutical products Direct inoculation method   If the product to be examined has antimicrobial activity: Carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium Oily liquids Use media to which have been added a suitable emulsifying agent at a concentration shown to be appropriate in the method suitability of the test, for example, polysorbate 80 at a concentration of 10 g/L

10. 10 Ointments and creams Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as peptone(1 g/L). Transfer the diluted product to a medium not containing an emulsifying agent Incubate the inoculated media for not less than 14 days Observe the cultures several times during the incubation period Shake cultures containing oily products gently each day When fluid thioglycollate medium is used for the detection of anaerobic microorganisms keep shaking or mixing to a minimum in order to maintain anaerobic conditions Examination of the media for macroscopic evidence of microbial growth

11. 11 If no evidence of microbial growth is found, the product to be examined complies with the test for sterility If evidence of microbial growth is found the product to be examined does not comply with the test for sterility The test may be considered invalid if A review of the testing procedure used during the test in question reveals a fault The data of the microbiological monitoring of the sterility testing facility show a fault Microbial growth is found in the negative controls

12. 12 Sterility testing of pharmaceutical products Membrane filtration method Membrane filtration is used for • Filterable aqueous preparations, • Alcoholic or oily preparations • Preparations miscible with or soluble in aqueous or oily solvents (Provided these solvents do not have an antimicrobial effect in the conditions of the test) Growth - contaminated

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14. 14 Controls used during sterility testing Positive controls • To show that microorganisms will actually grow under the conditions of the test • The media is inoculated with test organism and incubated along with test samples Negative control • To ascertain the sterility of the media • Media without sample or test organism should be incubated

15. 15 Summary • Sterility testing examines samples of the final product for the presence of microorganisms and applied to all products that are designated as sterile • Media used - Fluid thioglycollate medium (aerobic and anaerobic) Soya-bean casein digest medium (for fungi) • Methods – direct inoculation and membrane filtration • Positive control – To show that microorganisms will actually grow under the conditions of the test • Negative control -To ascertain the sterility of the media

16. 16 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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