二代测序技术检测生物制品中外源病毒的技术方法和应用进展

生物制品中外源病毒污染一直是监管机构对其安全性评价的重要内容，目前主要采用的技术包括体外细胞培养、接种动物、种属特异性检查等传统方法，但是传统方法普遍存在灵敏度低、检测范围窄等缺点，因此有些病毒可能无法检出。二代测序法(Next Generation Sequencing，NGS)曾被用于发现已获批上市的疫苗中存在外源病毒污染，说明该法具有全面检测病毒的潜力。使用NGS法检测外源病毒，能从源头上保障生物制品的安全。然而，生物制品中的病毒含量可能很低，甚至还可能和已知病毒的基因序列存在较大差异，NGS法能否全面准确地检出病毒尚不清楚；而且，NGS是一项复杂的技术， 在操作流程和数据解读方法等方面尚无任何指导原则。本文首先回顾了世界范围内的主要药品生产企业和监管机构对NGS技术检测生物制品中外源病毒的观点，接着详细讨论了该方法的技术流程和将方法标准化时面临的挑战，最后陈述了部分NGS技术检测生物制品中外源病毒的应用实例，为利用NGS技术检测生物制品中外源病毒方法的建立、标准化和验证提供启示。

The Techniques and Progress of Adventitious Virus Detection by Next Generation Sequencing

The contamination of adventitious viruses in biological products has always been a major safety concern for regulatory agencies. So far, the recommended methods have included in vitro cell culture, in vivo animal inoculation and species specific testing. However, several drawbacks of the routine methods, such as low sensitivity and limited detection capacity, have led to detection failure of some viruses. Next generation sequencing (NGS) has been applied to detecting a case of adventitious virus contamination of a licensed vaccine, demonstrating the potential of this method for universal virus detection. The application of NGS into the safety evaluation of biological products can offer fundamental safety protection. However, the viral content of biologics may be low and some novel viruses may have low genomic similarities compared with known viruses. Whether or not NGS can broadly and precisely detect the viruses mentioned above is not clear. Besides, NGS is a complex technique and no guidelines exist for operational procedures and data interpretation methods. The current review initially summarized the perspectives of manufactures and regulatory agencies worldwide on NGS for adventitious virus detection, then discussed in detail the methodologies and the challenges faced with in standardizing andvalidating the techniques. Finally, some applications of NGS for biologicals were illustrated, providing insights for the establishment, standardization and validation of NGS for adventitious virus testing.