Gillespie Paul F, Wang Yanjie, Yin Kuo, Groegler Emily, Cunningham Nicholas, Stiving Alyssa Q, Raffaele Jessica, Marusa Natalia, Tubbs Christopher M, Loughney John W, Winters Michael A, Rustandi Richard R
Analytical Research & Development, Merck & Co., Inc., Rahway, NJ 07065, USA.
Process Research & Development, Merck & Co., Inc., West Point, PA 19486, USA.
Vaccines (Basel). 2024 Dec 5;12(12):1373. doi: 10.3390/vaccines12121373.
BACKGROUND/OBJECTIVES: Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions.
Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE.
RESULTS/CONCLUSIONS: Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.
背景/目的：宿主细胞蛋白（HCP）含量是生物制品和疫苗产品的一个主要属性，在产品获批上市之前必须进行广泛的特性鉴定。酶联免疫吸附测定（ELISA）和质谱分析（MS）是用于生物制品和疫苗产品中宿主细胞蛋白定量分析的传统方法。这两种技术通常都非常繁琐、 labor-intensive，并且难以转移到其他实验室。此外，ELISA方法需要二维SDS-PAGE和二维蛋白质免疫印迹抗体试剂验证来确定试剂覆盖范围。这种试剂覆盖范围提供了一个目前被接受的相当薄弱的环节，因为蛋白质免疫印迹是在变性条件下进行的，而ELISA是在天然条件下进行的。Simple Western™是一种相对较新的、自动化的、基于毛细管蛋白质免疫印迹的技术，可实现蛋白质的分离、印迹和检测。但是，与传统蛋白质免疫印迹不同，Simple Western™是定量的，能够对生物制品和疫苗样品中的HCP含量进行定量。抗体试剂验证要直接得多，因为试剂覆盖范围可以在二维方法和Simple Western™之间直接关联，因为它们都是在变性和还原条件下进行的。
本文描述了一种毛细管蛋白质免疫印迹方法的开发，用于定量使用Vero细胞系生产的针对2019冠状病毒病（COVID-19）的一种研究性活病毒候选疫苗（V590）所产生的样品中的HCP含量。使用三种方法评估COVID-19疫苗样品中的HCP含量：新的毛细管蛋白质免疫印迹法、金标准ELISA法和SDS-PAGE法。
结果/结论：在毛细管蛋白质免疫印迹法和SDS-PAGE法之间的HCP含量数据中观察到高度一致性，而ELISA的HCP数据是异常值，这表明毛细管蛋白质免疫印迹法所产生的HCP浓度更接近真实浓度。这是首次报道使用毛细管蛋白质免疫印迹技术分析疫苗样品中的HCP。
