Kee Gaik Sui, Pujar Narahari S, Titchener-Hooker Nigel J
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
Biotechnol Prog. 2008 May-Jun;24(3):623-31. doi: 10.1021/bp070472i. Epub 2008 Apr 23.
Virus-like particles (VLPs) are expressed intracellularly in Saccharomyces cerevisiae and the recovery process involves the use of a detergent, which facilitates the release of VLP from host cell components. The detergent-mediated liberation of VLPs is a critical step in primary recovery and is responsible for setting the backdrop for subsequent purification in terms of product yield and characteristics of the process stream. In this paper the use of Triton X-100 detergent for the recovery of lipid envelope VLPs, using the hepatitis B surface antigen (HBsAg) as the VLP model, was investigated. To develop a framework that can be adopted in process design for future generation VLP vaccine candidates, the impact of Triton X-100 was characterized via different response factors: (i) recovery and activity of the HBsAg; (ii) level of protein and lipid contamination from the host cell; and (iii) indirect impact on the performance of an ultrafiltration step following primary recovery. Our studies identified that an increase in detergent concentration favors recovery of HBsAg only to a specific threshold, 0.5% v/v Triton X-100. Further increase in detergent results in delipidation of HBsAg leading to loss in antigenic activity. The level of contamination due to host protein and lipid co-liberation is in proportion with the amount of detergent employed. Greater membrane resistance during ultrafiltration was observed for samples generated using higher concentrations of detergent due to the increase in membrane fouling by the contaminants. Based on this study, Triton X-100 concentrations in the range of 0.2-0.5% v/v appears to be most suitable for recovery of native HBsAg. Choosing between 0.2-0.5% v/v would involve identifying a suitable tradeoff between desired product yield and the level of contamination that can be tolerated by downstream operations.
病毒样颗粒（VLPs）在酿酒酵母细胞内表达，回收过程需要使用去污剂，以促进VLPs从宿主细胞成分中释放出来。去污剂介导的VLPs释放是初次回收的关键步骤，在产品产量和工艺流特性方面为后续纯化奠定了基础。本文以乙型肝炎表面抗原（HBsAg）作为VLPs模型，研究了使用Triton X-100去污剂回收脂质包膜VLPs的情况。为了建立一个可用于下一代VLP候选疫苗工艺设计的框架，通过不同的响应因素对Triton X-100的影响进行了表征：（i）HBsAg的回收率和活性；（ii）宿主细胞中蛋白质和脂质的污染水平；（iii）对初次回收后超滤步骤性能的间接影响。我们的研究发现，去污剂浓度的增加仅在特定阈值（0.5% v/v Triton X-100）之前有利于HBsAg的回收。去污剂浓度进一步增加会导致HBsAg的脱脂，从而导致抗原活性丧失。宿主蛋白和脂质共同释放引起的污染水平与所用去污剂的量成正比。由于污染物导致的膜污染增加，使用较高浓度去污剂产生的样品在超滤过程中观察到更大的膜阻力。基于这项研究，0.2 - 0.5% v/v范围内的Triton X-100浓度似乎最适合回收天然HBsAg。在0.2 - 0.5% v/v之间进行选择需要在期望的产品产量和下游操作可容忍的污染水平之间找到合适的平衡点。
