Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate
Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate
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DOI：
10.1016/j.pep.2019.04.001
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发表时间：
2019-08-01
影响因子：
1.6
通讯作者：
King, C. Richter
中科院分区：
文献类型：
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作者：
Lee, Shwu-Maan;Plieskatt, Jordan;King, C. Richter
为了控制并最终消除疟疾，长期以来一直在寻求开发阻断传播的疫苗。然而，在临床试验中评估的抗原很少，这通常是由于抗原的表达和纯化的产量和质量的限制。 Pfs230 是配子细胞的表面抗原，最近作为使用铜绿假单胞菌外蛋白 A 载体蛋白的结合疫苗进入了临床评估。在这里，我们继续在杆状病毒系统中开发 Pfs230 候选药物 Pfs230C1 (aa 443-731) 的基础上，通过系统的工艺开发努力来提高产量和纯度。首先评估了包括 High Five、Sf9 和 Super Sf9 在内的各种昆虫细胞以及三种昆虫细胞培养基的抗原质量和数量。在 Sf9 细胞的选择中，完整的 Pfs230C1 被表达并在 48 小时收获用于下游发育。下游工艺利用固定金属亲和柱 (IMAC)、离子交换 (IEX) 膜 (Mustang S)、最后 IEX 层析 (DEAE) 产生纯 Pfs230C1 蛋白。整个过程在 20 L 规模下重复 3 次。为了支持 Pfs230C1 最终的化学制造和控制 (CMC)，开发了包括单克隆抗体在内的分析工具来表征 Pfs230C1 的身份、完整性和纯度。这些分析工具与优化的流程相结合，在实施时考虑了现行良好生产规范 (cGMP)，以实现 I 期临床试验的最终目标。
In an effort to control and eventually eliminate malaria, the development of transmission-blocking vaccines has long been sought. However, few antigens have been evaluated in clinical trials, often due to limitations in the expression and purification of the antigen in sufficient yield and quality. Pfs230, a surface antigen of gameto-cytes, has recently advanced to clinical evaluation as a conjugate vaccine using the Pseudomonas aeruginosa exoprotein A carrier protein. Here we continue to build upon prior work of developing a Pfs230 candidate in the baculovirus system, Pfs230C1 (aa 443-731), through systematic process development efforts to improve yield and purity.Various insect cells including High Five, Sf9 and Super Sf9 were first evaluated for quality and quantity of antigen, along with three insect cell media. In the selection of Sf9 cells, an intact Pfs230C1 was expressed and harvested at 48 h for downstream development. A downstream process, utilizing immobilized metal affinity column (IMAC), followed by ion exchange (IEX) membranes (Mustang S) and finally IEX chromatography (DEAE) yielded a pure Pfs230C1 protein. The complete process was repeated three times at the 20 L scale.To support the eventual chemistry manufacturing and controls (CMC) of Pfs230C1, analytical tools, including monoclonal antibodies, were developed to characterize the identity, integrity, and purity of Pfs230C1. These analytical tools, taken in combination with the optimized process, were implemented with Current Good Manufacturing Practices (cGMP) in mind with the ultimate objective of Phase I clinical trials.