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6 Pharmaceutical Technology ® Trends in Manufacturing eBook May 2024 PharmTech.com Biologics good manufacturing practices (CGMP) for finished pharmaceuticals and parts 600 to 680 for additional biological product standards (3). Assurance of final product sterility also relies on procedural measures that effectively prevent contamination of biological materials, such as cleanroom technology and other GMP standards. T he compend i a l s ter i l it y tes t i s re cog n i z ed by t he i ndust r y for its ad herence to reg u lator y standards and for its reliability. Compendial test- ing involves direct inoculation of the CAR T-cell sample into two different media (4), for growth of aerobic and anaerobic microorganisms, followed by incubation at different temperatures. During incubation, v isua l examination of media is per- formed by checking turbidity. However, the final examination of the medium for evidence of micro- bial grow th occurs af ter a 14-day incubation pe- riod. This extended lead-time can be challenging for patients in urgent need of treatment once the drug product is manufactured, particularly if the final formulation cannot be cryopreserved and has a short shelf-life. The limited amount of a sample, combined wit h t he time sensitivit y and critica l need of severely sick patients, means a faster ste- rility method is required. The development of more rapid sterilit y testing met hods of fers sign if icant advantages. It can be beneficial for frozen CAR T-cell products, expedit- ing the time from product manufacture to patient administration, which is crucial in the treatment of severely sick patients in the late stages of disease. To ensure the deployment of safe and effective thera- pies to patients, the industry needs to evaluate and promote effective rapid sterility methods. Evaluating faster sterility methods Novel systems are currently under development to expedite the detection of contamination. Among these, a faster method for sterility testing was eval- uated and validated, in comparison to the standard compendial method, using the BD Bacterial Auto- mated Detection and Enumeration (BACTEC) FX40 device for early detection of potential contamina- tion. In this study, the comparability of the BACTEC method with the compendial sterilit y test was as- sessed using three batches of CAR T-cell products i ntent iona l ly cont a m i nated w it h n i ne d i f ferent microorganisms. The nine microbial reference strains (Clostridium sporogenes, Cutibacterium acnes, Staphylococcus aureus, Streptococcus pyogenes, 2/7 Brachybacte- rium nesterenkovii, Pseudomonas aeruginosa, Ba- cillus subtilis, Candida albicans, Aspergillus brasil- iensis) were inoculated into the BACTEC cultivation vials in various concentrations (100, 50, and 10 col- ony forming units [CFU]/vial), together with a fas- tidious organism supplement (FOS). Three in-house isolates (aerobic bacteria) were also included in the testing. Positive controls of microorganisms and negative controls of selected media, FOS, and CAR T-cell drug products were prepared, with testing by the compendial method performed in parallel. All BACTEC vials were cultivated in the BACTEC FX40 at 35 ± 1.5 °C up to 14 days, or until detection of microbial grow th. All samples for compendial sterilit y testing were inoculated in tr y pticase soy broth or in clear f luid thioglycolate medium and cul- tivated at 20–25 °C and 30–35 °C, respectively, for up to 14 days, or until detection of microbial growth. BACTEC method results In t he compa rabi l it y st udy (i noc u lat ion of 100 CFU), all the tested microbial strains showed pos- itive results within four days using the BACTEC FX40 met hod, a compa rable t i me f ra me to t he standard compendial method (see Table I). For the sensitivit y experiment (10 and 50 CFU), the BAC- TEC FX40 confirmed sensitivity as low as 10 CFU for all tested microorganisms, which proved supe- rior to the compendial method where no growth was obser ved in the case of Pseudomonas aerugi- nosa (see Table II). The sterilit y test using the BD BACTEC FX40 de- v ice demon st rated h ig h sen sit iv it y i n detec t i ng low levels of bacteria l and f ungal contamination in CAR T-dr ug products, wit h t he st udy showing that the time to detection was a maximum of four days. Moreover, a l l m icroorga n isms detected by the BACTEC FX40 were successfully grown in sub- sequent subcultures, validating the reliability of the method. The study confirmed the method's speci- f icit y by accurately ident i f y i ng negat ive cont rol samples, further solidif ying its reliability for rapid sterility testing. However, it's important to recognize that the sys- tem's applicability may vary depending on specific cases, and discussions with regulatory authorities are advisable. The comprehensive comparison of the BACTEC device with the classical compendial method (5) in- dicates that some environmental microorganisms t hat necessitate lower cu lt ure temperat ures ca n pose challenges for cultivation. Regulatory aspects In terms of reg ulator y compliance, there are t wo main points that should be considered. The first is to ensure the product is safe for the patient by reducing the risk of administering a contaminated product and the second is to deliver it to the patient in the required time frame.