Gillespie Paul F, Rustandi Richard R, Swartz Andrew R, Shang Liang, Raffaele Jessica, Prout Ashley, Cunningham Nicholas, Dawod Mohamed, Deng James Z, Wang Shiyi, Olson Jessica, Shieh Yvonne, Loughney John W
Analytical Research and Development.
Vaccine Process Research and Development, Merck & Co., Inc., Rahway, New Jersey, USA.
Hum Gene Ther. 2023 Jan;34(1-2):68-77. doi: 10.1089/hum.2022.147.
A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.
柯萨奇病毒A21（CVA21）的一种原型毒株正在作为一种溶瘤病毒免疫疗法进行评估。CVA21优先裂解上调细胞间黏附分子1表达的细胞，其中包括某些类型的肿瘤细胞。CVA21具有二十面体衣壳结构，由60个蛋白质亚基组成，包裹着一个病毒RNA基因组，颗粒直径为30纳米。快速且可靠的分析方法来定量CVA21的总病毒颗粒、空病毒颗粒和完整病毒颗粒，对于支持工艺开发、满足监管要求以及验证生产工艺非常重要。在本研究中，我们使用自制抗体证明了对CVA21的所有四种衣壳蛋白VP1、VP2、VP3和VP4以及空颗粒替代物VP0的检测。利用这些抗体开发了一种自动化定量毛细管蛋白质免疫印迹分析方法——Simple Western，通过VP1定量CVA21总颗粒，通过VP0定量空颗粒，通过VP0和VP4定量空颗粒与完整颗粒的相对比例，以及通过VP0和VP1定量空颗粒与总颗粒的绝对比例。最后，这种Simple Western方法作为一种高通量分析工具，用于支持CVA21细胞培养和纯化工艺优化，以便快速做出工艺决策。
