Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems
Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems
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DOI：
10.1016/j.jchromb.2006.05.006
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发表时间：
2006-09-14
影响因子：
3
通讯作者：
Rito-Palomares, Marco
中科院分区：
文献类型：
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作者：
Benavides, Jorge;Mena, Jimmy A.;Rito-Palomares, Marco
病毒样颗粒具有广泛的应用，包括疫苗接种、基因治疗，甚至作为纳米材料。它们的成功利用取决于选择性和可扩展的产品回收和纯化方法的可用性，这些方法与上游操作有效集成。在这项工作中，开发了一种基于水性两相系统（ATPS）的策略，用于回收由昆虫细胞杆状病毒表达系统产生的双层轮状病毒样颗粒（dlRLP）。评估聚乙二醇 (PEG) 分子量、PEG 和盐浓度以及体积比（Vr，顶部相体积/底部相体积），以确定 dlRLP 和污染物浓缩到相反相的条件。由 PEG 400-磷酸盐组成的两级 ATPS，其 Vr 为 13.0，连接线长度 (TLL) 为 35% (w/w)，pH 7.0，为处理破碎细胞的高浓缩粗提物提供了最佳条件（dIRLP 浓度为 5 μg/mL）。在这种条件下，细胞内 dIRLP 积聚在顶部阶段（回收率 90%），而细胞碎片保留在界面中。此外，使用 Vr 为 1.0、pH 为 7.0 的 ATPS、PEG 3350（10.1%，w/w）和磷酸盐（10.9%，w/w）时，来自培养物上清液的 dlRLP 优先在界面中积累（回收率为 82%）。 ATPS 后，培养物上清液中 dlRLP 的纯度提高了 55 倍。考虑到细胞内和细胞外 dIRLP 的纯化，ATPS 的使用导致回收过程产生的 dIRLP 纯度在 6% 至 11% 之间，总产品收率为 85% (w/w)。总体而言，本研究提出的策略比恢复 dIRLP 的传统方法更简单，并且代表了轮状病毒感染疫苗生产过程的可扩展且经济可行的替代方案，具有广泛的通用商业应用范围。 (c) 2006 Elsevier B.V. 保留所有权利。
Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dIRLP concentration of 5 mu g/mL). In such conditions intracellular dIRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dIRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dIRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dIRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application. (c) 2006 Elsevier B.V. All rights reserved.