Hagen A J, Oliver C N, Sitrin R D
BioProcess R&D Department, Merck Research Labs, West Point, Pennsylvania 19486, USA.
Biotechnol Prog. 1996 May-Jun;12(3):406-12. doi: 10.1021/bp950081g.
Poly(ethylene glycol) precipitation has been successfully used to concentrate and purify hepatitis A virus from crude lysate preparations for production of VAQTA, a highly purified, formalin-inactivated hepatitis A vaccine. Initial results showed that nucleic acids present in the starting material were problematic for the performance of the poly(ethylene glycol) precipitation step. Extensive experiments were carried out to identify processing conditions suitable for vaccine manufacture which would enhance product yield and improve purity. Results of these studies indicated that the earlier practice of concentrating crude virus-containing lysate using semipermeable membranes led to aggregation of high molecular weight nucleic acids. This aggregated material coprecipitated with the virus during the subsequent poly(ethylene glycol) precipitation step; variable amounts of nucleic acids led to inconsistent virus recovery and product purity. Nuclease treatment of the crude lysate preparations decreased the molecular size of the nucleic acids and significantly reduced their coprecipitation with the virus. Further experiments demonstrated that optimal placement of the nuclease treatment was at the lysate stage followed by a capture step using anion exchange chromatography. These steps combined with optimization of the virus concentration, ionic strength, and pH of the poly(ethylene glycol) precipitation led to effective and selective concentration of the virus which significantly enhanced process reproducibility and control.
聚乙二醇沉淀法已成功用于从粗裂解物制剂中浓缩和纯化甲型肝炎病毒，以生产VAQTA，一种高度纯化的、福尔马林灭活的甲型肝炎疫苗。初步结果表明，起始原料中存在的核酸对聚乙二醇沉淀步骤的性能有问题。进行了广泛的实验，以确定适合疫苗生产的加工条件，这将提高产品产量并提高纯度。这些研究结果表明，早期使用半透膜浓缩含粗病毒裂解物的做法导致高分子量核酸聚集。这种聚集的物质在随后的聚乙二醇沉淀步骤中与病毒共沉淀；不同量的核酸导致病毒回收率和产品纯度不一致。对粗裂解物制剂进行核酸酶处理可降低核酸的分子大小，并显著减少其与病毒的共沉淀。进一步的实验表明，核酸酶处理的最佳位置是在裂解物阶段，随后是使用阴离子交换色谱的捕获步骤。这些步骤与聚乙二醇沉淀的病毒浓度、离子强度和pH值的优化相结合，导致病毒的有效和选择性浓缩，显著提高了工艺的可重复性和可控性。
