Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany.
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; Institute of Bioprocess Engineering and Pharmaceutical Technology, Technische Hochschule Mittelhessen, Wiesenstrasse 14, 35390 Giessen, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Jun 1;1117:103-117. doi: 10.1016/j.jchromb.2019.03.037. Epub 2019 Apr 1.
Options for hydrophobic-interaction chromatography (HIC) for purification of cell culture-derived influenza A and B virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing virus purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived viruses and viral vectors.
已使用半高通量 96 孔板格式评估了用于纯化细胞培养衍生的甲型和乙型流感病毒的疏水相互作用色谱 (HIC) 的选择。在制备规模上进行的后续实验用于表征动态结合容量、病毒血凝素蛋白 (HA 蛋白) 回收率以及流感病毒 (IV) 株对产量和污染水平的影响。实现了高达 96%的病毒回收率，残留 DNA 水平约为 1.3%。为了达到用于人类的流感疫苗制造所需的 DNA 污染水平，在最终设置中使用了包括澄清、失活、浓缩、基于柱的阴离子交换色谱 (AEC) 和 HIC 的纯化工艺。使用强季铵配体的 AEC 被用作通过吸附进行 DNA 耗竭的正交方法。随后，使用带有聚丙烯二醇作为功能基团的 HIC 可逆结合病毒颗粒以进行捕获并去除残留的污染 DNA 和蛋白质（流穿物）。两步色谱过程既不需要缓冲液交换步骤也不需要核酸酶处理，对 IV A/PR/8/34（H1N1）的总病毒颗粒收率为 92%。蛋白质和 DNA 污染水平可分别降低至 42%和至少 1.0%。每个单价剂量的总蛋白为 17.2μg，DNA 为 2.0ng，该纯度水平符合欧洲药典对细胞培养衍生的人用疫苗的限制。总体而言，所提出的下游工艺代表了现有病毒纯化方案的有价值替代方案。此外，它仅利用市售材料，是一种简单且经济的用于生产细胞培养衍生病毒和病毒载体的工艺。
