Rapid Sterility Test Method For Cellular Therapy Product

无菌检测是细胞治疗产品生产过程和产品放行检测的重要组成部分。FDA特别建议按照21 CFR 610.12进行无菌检测。此外，由于他们担心抗生素可能会干扰无菌测试的准确评估，FDA要求根据USP“<71>无菌测试”对所有含有抗生素的样品进行初步的抑菌和抑菌测试，包括在含有抗生素的培养基中生长的细胞。CFR和USP标准中描述的无菌检测方法是在25年前开发的，并且是劳动密集型的，需要培养14天。自从这些方法发表以来，已经开发出更灵敏和快速的方法来检测各种体液中的微生物生长。这些现代方法包括用于检测血液和其他通常无菌液体中微生物生长的自动化系统。尽管许多实验室使用这些自动化系统来评估细胞治疗产品的无菌性，但FDA尚未批准这一应用，因为没有公布的数据表明这些新方法与CFR和USP方法进行了正式比较。由于这些原因，我们开发了一种验证方案，将CFR和USP方法与两种自动培养方法（生物梅里埃BacT/Alert系统和Becton Dickinson Bactec系统）进行比较。细胞产品将在选定的细菌和真菌中播种，然后用每种方法进行测试。将评估检测阳性培养物的灵敏度和时间。验证方案已经提交给FDA征求他们的意见，我们预计这项研究将在2002年秋季开始。作为FDA提交的支持数据，我们已经启动了CFR和USP方法与生物梅里埃自动化BacT/Alert系统（目前在NIH微生物实验室使用）的初步比较。在8月至9月期间制备的所有细胞治疗产品（N=187）在两种系统中进行了测试。共有7种产品在一个或两个系统中检测到生长：3种产品仅在BacT/Alert系统中检测到生长，2种产品仅在CFR/USP方法中检测到生长，2种产品在两个系统中检测到生长。这些阳性培养的临床意义将被评估。该初步研究还将用于定义(1)CFR/USP和BacT/Alert方法检测阳性培养的时间；(2)假阳性培养信号的发生率（例如，系统呈现阳性[浑浊；自动化系统的阳性信号]，但系统中没有恢复任何生物体）。

Sterility testing is an essential part of in-process and release testing for cellular therapy products. The FDA specifically recommends that sterility testing be performed as outlined in 21 CFR 610.12. Furthermore, because they are concerned that antibiotics may interfere with the accurate assessment of sterility testing, the FDA requires preliminary bacteriostasis and fungistasis testing according to the USP "<71> Sterility Test" on all samples containing antibiotics, including cells grown in antibiotic-containing media. The methods for sterility testing described in the CFR and USP standards were developed more than 25 years ago, and are labor-intensive and require incubation for 14 days. Since the time these methods were published, more sensitive and rapid methods have been developed for detection of microbial growth in various body fluids. These modern methods include automated systems for the detection of microbial growth in blood and other normally sterile fluids. Despite the fact many laboratories use these automated systems to assess sterility of cellular therapy products, the FDA has not sanctioned this application because there are no published data from any formal comparison of these newer methods with the CFR and USP methods. For these reasons, we have developed a validation protocol comparing the CFR and USP methods with two automated culture methods: the bioMerieux BacT/Alert system and the Becton Dickinson Bactec system. Cell products will be seeded with selected bacteria and fungi and then tested with each method. The test sensitivity and time to detect a positive culture will be assessed. The validation protocol has been submitted to the FDA for their comments and we anticipate this study will be initiated in the Fall, 2002. As supporting data for this FDA submission, we have initiated a preliminary comparison of the CFR and USP methods with the automated bioMerieux BacT/Alert system (currently used in the NIH Microbiology Laboratory). All cellular therapy products (N=187) that have been prepared during August-September were tested in both systems. A total of 7 products had growth detected in one or both systems: 3 products in the BacT/Alert system only, 2 products in the CFR/USP method only, and 2 products in both systems. The clinical significance of these positive cultures will be assessed. This preliminary study will also be used to define (1) the time to detection of a positive culture in the CFR/USP and BacT/Alert methods; and (2) the incidence of false-positive culture signals (e.g., the system appears positive [cloudy; positive signal by the automated system] but no organisms are recovered in the system).