Seid Christopher A, Curti Elena, Jones R Mark, Hudspeth Elissa, Rezende Wanderson, Pollet Jeroen, Center Lori, Versteeg Leroy, Pritchard Sonya, Musiychuk Konstantin, Yusibov Vidadi, Hotez Peter J, Bottazzi Maria Elena
a Departments of Pediatrics and Molecular Virology and Microbiology; National School of Tropical Medicine; Baylor College of Medicine ; Houston , TX , USA.
Hum Vaccin Immunother. 2015;11(6):1474-88. doi: 10.1080/21645515.2015.1036207.
Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.
生活在世界最贫困发展中国家的4亿多人感染了钩虫，其中大多数是美洲板口线虫。由美洲板口线虫谷胱甘肽S-转移酶-1（Na-GST-1）和美洲板口线虫天冬氨酸蛋白酶-1（Na-APR-1（M74））组成的二价人钩虫疫苗目前正由萨宾疫苗研究所产品开发伙伴关系组织（Sabin PDP）进行研发。两种单价疫苗目前都处于1期试验阶段。Na-GST-1和Na-APR-1抗原均表达为重组蛋白。虽然发现Na-GST-1在毕赤酵母中能高产表达，但Na-APR-1在该宿主中的表达水平过低，不适合用于生产工艺。当评估烟草植物本氏烟草作为表达系统时，获得了可接受的溶解度、产量和稳定性水平。使用该系统观察到的Na-APR-1（M74）表达水平约为300 mg/kg。在此，我们描述了工艺开发过程中取得的成果和遇到的障碍，以及纯化的Na-APR-1（M74）蛋白和配制疫苗的特性及稳定性。还介绍了从为鉴定Na-APR-1（M74）生产工艺而进行的具有代表性的5 kg可重复性运行中获得的纯化Na-APR-1（M74）蛋白的表达、纯化和分析。该工艺已成功转移至中试工厂，并在现行药品生产质量管理规范（cGMP）下进行了50 kg规模的生产活动。50 kg的生产运行提供了足够数量的蛋白质，以支持Sabin PDP正在进行的钩虫疫苗开发项目。
