Chin Pei-Ju, Lambert Christophe, Beurdelay Pascale, Charlebois Robert L, Colinet Anne-Sophie, Eloit Marc, Gilchrist Shanaz, Gilleece Maria, Hess Matthew, Leimbach Andreas, de Man Tom J B, Vandeputte Olivier, Walas Dawid, Wang Weihong, Khan Arifa S
Office of Vaccines Research and Review, Center for Biologics Evaluation and Review, U.S. Food and Drug Administration, Silver Spring, MD, 20993, USA.
GSK, Rue de l'Institut 89, Rixensart, Belgium.
NPJ Vaccines. 2025 Mar 29;10(1):61. doi: 10.1038/s41541-025-01104-1.
The safety of all biological products includes demonstrating the absence of adventitious viruses by testing various types of samples at different stages of the manufacturing process. Seven laboratories evaluated short-read high-throughput sequencing (HTS) for sensitivity and breadth of adventitious virus detection using viruses with distinct physicochemical and genome properties. These five viruses are currently designated as CBER NGS Virus Reagents and include: Epstein-Barr virus (EBV; or human herpes virus 4), feline leukemia virus (FeLV), respiratory syncytial virus (RSV), mammalian orthoreovirus type 1 (Reo1), and porcine circovirus type 1 (PCV1). To evaluate adventitious virus detection in a biological material with a high production virus titer and low cellular background, the 5 viruses were mixed and different copies of the viral genomes spiked into 1 - 5 × 10 genome copies per mL (GC/mL) of purified adenovirus 5. Independent protocols were used by each laboratory for the entire HTS workflow. All laboratories detected 10 GC/mL of the five viruses by both targeted and non-targeted bioinformatic analyses. Additionally, the limit of detection of squirrel monkey retrovirus and porcine endogenous retrovirus, which pre-existed in EBV and PCV1 virus stocks, respectively, was evaluated. The five laboratories that tested 10 GC/mL, detected all 5 viruses with the targeted analysis, and Reo1 and EBV with the non-targeted analysis. It was noted that some laboratories achieved a better sensitivity for detection of the five viruses ( ≤10 GC/mL). This study presents an approach for HTS validation for viral safety testing of vaccines and other biologics using a panel of reference viruses. The results highlight that optimization of steps in the HTS workflow can improve the limit of detection for adventitious viruses.
所有生物制品的安全性包括在生产过程的不同阶段对各类样品进行检测，以证明不存在外源病毒。七个实验室评估了短读长高通量测序（HTS）检测外源病毒的灵敏度和广度，所使用的病毒具有不同的物理化学和基因组特性。这五种病毒目前被指定为CBER NGS病毒试剂，包括：爱泼斯坦-巴尔病毒（EBV；或人类疱疹病毒4型）、猫白血病病毒（FeLV）、呼吸道合胞病毒（RSV）、1型哺乳动物正呼肠孤病毒（Reo1）和1型猪圆环病毒（PCV1）。为了评估在高生产病毒滴度和低细胞背景的生物材料中检测外源病毒的情况，将这五种病毒混合，并将不同拷贝数的病毒基因组掺入到每毫升1 - 5×10个基因组拷贝（GC/mL）的纯化腺病毒5中。每个实验室在整个HTS工作流程中使用独立的方案。所有实验室通过靶向和非靶向生物信息学分析均检测到了浓度为10 GC/mL的这五种病毒。此外，还评估了分别预先存在于EBV和PCV1病毒储备中的松鼠猴逆转录病毒和猪内源性逆转录病毒的检测限。测试浓度为10 GC/mL的五个实验室通过靶向分析检测到了所有五种病毒，通过非靶向分析检测到了Reo1和EBV。值得注意的是，一些实验室在检测这五种病毒（≤10 GC/mL）时具有更高的灵敏度。本研究提出了一种使用一组参考病毒对疫苗和其他生物制品进行病毒安全性检测的HTS验证方法。结果表明，HTS工作流程中步骤的优化可以提高外源病毒的检测限。
