Validation Of Rapid Sterility Test Method For Cellular T

细胞 T 快速无菌测试方法的验证

• 批准号: 7006564
• 负责人: Patrick R Murray
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7006564
• 关键词: antiseptic sterilization; biological products; diagnostic tests; evaluation /testing; human tissue; microorganism growth; tissue /cell culture

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 (CFR/USP). For these reasons, we have developed a validation protocol comparing the CFR/USP method with two automated culture methods: the bioMerieux BacT/Alert (BTA) system and the Becton Dickinson (Bactec) system. Six different cell products from the Department of Transfusion Medicine (DTM) were seeded with selected bacteria (8 strains) and fungi (2 strains) (10 colony forming units and 50 CFUs) and then tested in triplicate with each method. The test sensitivity and time to detect a positive culture were assessed. With each bacterium and fungus, the automated systems were found to be more rapid and sensitive for detection of contaminated cell products. Growth of strict aerobic and anaerobic organisms grew preferentially in aerobic and anaerobic blood culture broths, respectively; therefore, each cell product would have to be cultured in the two broth formulations. Results: Positive cultures were detected in a mean (range) of 72% (7-100%) of the cultures for CFR/USP, 82% (0-100%) for BTA, and 93% (57-100%) for Bactec. For nine of the 10 organisms tested, overall detection rates for BTA and Bactec were equivalent to or higher than CFR/USP. Of the six product media tested, detection of organisms was impaired only by the medium containing multiple antibiotics: this occurred in all three systems. Both BTA and Bactec had shorter times to detection than the CFR/USP method, with overall means (ranges) of 87 (24-264 ) hours for CFR/USP, 24 (12-54) hours for BTA, and 33 (12-80) hours for Bactec. Detection occurred coonsistently within 7 days for both BTA and Bactec, but not for CFR/USP.

无菌检查是细胞治疗产品过程中和放行检查的重要组成部分。FDA特别建议按照21 CFR 610.12所述进行无菌试验。此外，由于他们担心抗生素可能干扰无菌试验的准确评估，FDA要求根据USP“<71>无菌试验”对所有含抗生素的样品（包括在含抗生素培养基中生长的细胞）进行初步抑菌和抑真菌试验。CFR和USP标准中描述的无菌测试方法是25年前开发的，属于劳动密集型，需要孵育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正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尚未批准此应用，因为这些新方法与CFR和USP方法（CFR/USP）的任何正式比较都没有发表数据。出于这些原因，我们制定了一项验证方案，将CFR/USP方法与两种自动培养方法进行比较：bioMerieux BacT/Alert（BTA）系统和Becton Dickinson（Bactec）系统。 用选定的细菌（8株）和真菌（2株）（10个菌落形成单位和50 CFU）接种来自输血医学科（DTM）的6种不同细胞产品，然后用每种方法重复检测3次。评估了检测灵敏度和检测阳性培养物的时间。对于每种细菌和真菌，发现自动化系统在检测污染的细胞产物方面更快速和灵敏。严格需氧和厌氧微生物的生长分别优先在需氧和厌氧血液培养肉汤中生长;因此，每种细胞产物必须在两种肉汤制剂中培养。 结果如下：CFR/USP、BTA和Bactec的阳性培养物检出率的平均值（范围）分别为72%（7-100%）、82%（0-100%）和93%（57-100%）。对于检测的10种微生物中的9种，BTA和Bactec的总体检出率等于或高于CFR/USP。在供试的6种产品培养基中，仅含多种抗生素的培养基会影响微生物的检出：这在所有3种系统中均发生。BTA和Bactec的检测时间均短于CFR/USP方法，CFR/USP的总体平均值（范围）为87（24-264）小时，BTA为24（12-54）小时，Bactec为33（12-80）小时。BTA和Bactec在7天内检出一致，但CFR/USP未检出。