title: Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.
abstract: Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71>) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex(®) Detection System [RMDS]), and 2) by CO(2) monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iAST and iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1CFU than the compendial methods (p<0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p<0.05). RMDS detected all microorganisms significantly faster than the compendial method (p<0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p<0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
authors.affiliation: Division of Biological Standards and Quality Control, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and Research, FDA, Rockville, MD, USA.
authors.forename: Seema
authors.initials: S
authors.lastname: Parveen
authors.forename: Simleen
authors.initials: S
authors.lastname: Kaur
authors.forename: Selwyn A Wilson
authors.initials: SA
authors.lastname: David
authors.forename: James L
authors.initials: JL
authors.lastname: Kenney
authors.forename: William M
authors.initials: WM
authors.lastname: McCormick
authors.forename: Rajesh K
authors.initials: RK
authors.lastname: Gupta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001419:Bacteria / Q000254:growth & development / Q000378:metabolism; D001688:Biological Products / Q000592:standards*; D002245:Carbon Dioxide / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004340:Drug Contamination; D005658:Fungi / Q000254:growth & development / Q000378:metabolism; D008828:Microbiological Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013678:Technology, Pharmaceutical / Q000379:methods*; D014612:Vaccines / Q000592:standards*
chemical_list: D001688:Biological Products; D014612:Vaccines; D002245:Carbon Dioxide; D000255:Adenosine Triphosphate