Namila Fnu, Zhou Tianyi, Wang Lu, Jin Mi
Drug Substance Development, Spark Therapeutics, Inc., Philadelphia, USA.
Biotechnol J. 2025 Jan;20(1):e202400636. doi: 10.1002/biot.202400636.
Adeno-associated virus (AAV) vectors have become a leading platform for gene delivery. A major portion of gene therapy currently in clinical trials are AAV-based for a wide range of diseases. A commonly used method for AAV production is by mammalian or insect cell culture, with or without added viruses to introduce needed genetic elements for AAV production. There are potential risks of virus contamination from the production cell line, process residuals, or adventitious contamination in the production of biotherapeutics, including AAV-based gene therapy products; therefore, it is imperative to demonstrate that the drug substance manufacturing process has sufficient capability to clear process-related or adventitious viruses. In the AAV-based gene therapy manufacturing process, cell lysis, affinity chromatography, and ion exchange chromatography steps are often effective to inactivate or remove viruses. To increase the viral clearance robustness, virus filtration (VF) is increasingly recommended by regulatory agencies for gene therapy products as a dedicated viral clearance step in the downstream purification process. In the current study, two commercially available virus filters were evaluated in the context of AAV manufacturing. The filter throughput and process yield were assessed under different operational modes. Virus clearance performance was evaluated by spiking in Adenovirus type 5 (Adv-5) and Simian virus 40 (SV-40). The viral filters assessed in this study demonstrated manufacturable throughputs, acceptable process yields, and robust virus clearance capabilities for viruses greater than 40 nm.
腺相关病毒（AAV）载体已成为基因递送的主要平台。目前正在进行临床试验的基因治疗中，很大一部分是基于AAV用于治疗多种疾病。一种常用的AAV生产方法是通过哺乳动物或昆虫细胞培养，添加或不添加病毒以引入AAV生产所需的遗传元件。在生物治疗产品（包括基于AAV的基因治疗产品）的生产过程中，存在来自生产细胞系、工艺残留或偶然污染的病毒污染潜在风险；因此，必须证明原料药生产工艺有足够能力清除与工艺相关或偶然的病毒。在基于AAV的基因治疗生产工艺中，细胞裂解、亲和色谱和离子交换色谱步骤通常对灭活或去除病毒有效。为提高病毒清除的稳健性，监管机构越来越推荐将病毒过滤（VF）作为基因治疗产品下游纯化过程中的专门病毒清除步骤。在本研究中，在AAV生产背景下评估了两种市售病毒过滤器。在不同操作模式下评估了过滤器通量和工艺收率。通过加入5型腺病毒（Adv-5）和猿猴病毒40（SV-40）评估病毒清除性能。本研究中评估的病毒过滤器对大于40nm的病毒显示出可制造的通量、可接受的工艺收率和强大的病毒清除能力。
