Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids using Capillary Western.

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 (ICAM-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. Here, 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 via VP1, empty particles via VP0, relative ratio of empty to full particles via VP0 and VP4, and the absolute ratio of empty to total particles via 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.