Transmissible spongiform encephalopathy agent clearance by the immunoaffinity and anion-exchange chromatography steps of the REFACTO® manufacturing process
Transmissible spongiform encephalopathy agent clearance by the immunoaffinity and anion-exchange chromatography steps of the REFACTO® manufacturing process
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DOI：
10.1111/j.1365-2516.2007.01441.x
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发表时间：
2007-09-01
期刊：
影响因子：
3.9
通讯作者：
Kelley, B.
中科院分区：
文献类型：
--
作者：
Booth, J.;Vicik, S.;Kelley, B.
REFACTO（R）（morphocog alfa）是一种批准用于治疗血友病A的重组因子VIII，通过哺乳动物细胞培养过程生产，该过程在细胞培养基中包括治疗级人血清白蛋白（HSA）。虽然迄今为止尚未出现因临床使用HSA而导致传染性海绵状脑病（TSE）的病例，但Wyeth进行了一项研究，以证明如果TSE因子作为HSA中的污染物存在，则REFACTO生产工艺具有显著的去除TSE因子的能力。评价了免疫亲和（8A4琼脂糖）和阴离子交换（Q琼脂糖）色谱步骤对仓鼠TSE因子（菌株263 K）的清除。本药物非临床研究质量管理规范研究使用适当合格的实验室规模色谱系统进行。将来自TSE感染仓鼠的过滤脑匀浆加入两种色谱柱的上样液中，并使用经验证的蛋白质印迹定量方法测定上样液和产品合并液中TSE因子的浓度。重复色谱运行一致，分别通过5.2 log减少证明。这些数据提供了高度的保证，即在REFACTO细胞培养工艺中使用的HSA发生TSE污染的不太可能事件中，纯化步骤有可能将感染因子清除至极低水平，从而显著降低接受REFACTO的患者的风险。
REFACTO (R) (moroctocog alfa), a recombinant factor VIII approved for the treatment of haemophilia A, is produced by a mammalian cell-culture process that includes therapeutic-grade human serum albumin (HSA) in the cell-culture medium. While to date there have been no cases of transmissible spongiform encephalopathy (TSE) resulting from the clinical use of HSA, Wyeth conducted a study to demonstrate that the REFACTO manufacturing process has significant capacity to remove a TSE agent if it were present as a contaminant in the HSA. The immunoaffinity (8A4 Sepharose) and anion-exchange (Q Sepharose) chromatography steps were evaluated for the clearance of the hamster TSE agent, strain 263K. This Good Laboratory Practice study was performed using appropriately qualified, laboratory-scale chromatography systems. Filtered brain homogenate from TSE-infected hamsters was added to loads of both chromatographic columns, and the concentration of TSE agent in the loads and product pools were determined using a validated western blot quantitation method. Replicate chromatography runs were consistent, as demonstrated by the 5.2 log reduction respectively. These data provide a high degree of assurance that in the unlikely event of a TSE contamination of the HSA used in the REFACTO cell-culture process, the purification steps have the potential to remove the infectious agent to extremely low levels, thereby significantly reducing the risk to patients receiving REFACTO.