Kielpinski G, Prinzi S, Duguid J, du Moulin G
Department of Quality Systems, Genzyme Biosurgery, Cambridge, Massachusetts 02139, USA.
Cytotherapy. 2005;7(6):531-41. doi: 10.1080/14653240500361079.
In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system.
For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days.
The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method.
Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.
2004年2月，美国食品药品监督管理局（FDA）批准了我们的生物制品许可证补充申请，允许用于软骨细胞（Carticel），即自体培养软骨细胞，使用BacT/ALERT微生物检测系统替代药典规定的无菌检查用于产品放行。本文提供了我们的审批过程路线图。该批准代表了超过4年的开发和验证研究，比较了Steritest紧凑型系统和BacT/ALERT微生物检测系统。
在本研究中，从一个经过鉴定的细胞库中制备新鲜培养的软骨细胞。微生物分离株取自美国典型培养物保藏中心（ATCC）菌株或内部污染物。对于每种测试条件，将软骨细胞悬液和测试微生物接种到需氧培养基（SA标准成人培养瓶、FA FAN、胰蛋白胨大豆肉汤）和厌氧培养基（SN标准成人培养瓶、FN FAN、硫乙醇酸盐流体培养基）中，并使用Steritest紧凑型系统（美国马萨诸塞州贝德福德的密理博公司）和BacT/ALERT微生物检测系统（美国北卡罗来纳州达勒姆的生物梅里埃公司）进行无菌测试。阴性对照瓶接种软骨细胞但不接种微生物。所有瓶子孵育14天并每天进行读数。通过目视检查Steritest罐或通过BacT/ALERT系统检测阳性来确定细菌生长情况。14天后，使用革兰氏染色和划线平板法确认阳性瓶和阴性瓶。
在每项验证研究中，总结了Steritest紧凑型系统或BacT/ALERT系统检测到的每种微生物的阳性情况。与药典方法相比，将孵育温度从35摄氏度降至32摄氏度的研究产生的数据改善了自动化方法的检测时间。其他改进包括使用FAN需氧和厌氧培养基来吸收制备的软骨细胞样品培养基中所含的庆大霉素。单独的软骨细胞在药典方法或自动化方法中均未产生阳性结果。
验证研究的数据支持使用BacT/ALERT微生物检测系统替代软骨细胞的无菌检查。
