SEC coupled with in-line multiple detectors for the characterization of an oncolytic Coxsackievirus

James Z Deng; Richard R Rustandi; Andrew Swartz; Yvonne Shieh; Jack B Baker; Josef Vlasak; Shiyi Wang; John W Loughney

doi:10.1016/j.omto.2021.12.009

SEC coupled with in-line multiple detectors for the characterization of an oncolytic Coxsackievirus

Mol Ther Oncolytics. 2021 Dec 11:24:139-147. doi: 10.1016/j.omto.2021.12.009. eCollection 2022 Mar 17.

Authors

James Z Deng¹, Richard R Rustandi¹, Andrew Swartz², Yvonne Shieh², Jack B Baker¹, Josef Vlasak¹, Shiyi Wang¹, John W Loughney¹

Affiliations

¹ Analytical Research & Development, Merck & Co., Kenilworth, NJ 07033, USA.
² Process Research & Development, Merck & Co., Kenilworth, NJ 07033, USA.

Abstract

V937 is an oncolytic virus immunotherapy clinical drug candidate consisting of a proprietary formulation of Coxsackievirus A21 (CVA21). V937 specifically binds to and lyses cells with over-expressed ICAM-1 receptors in a range of tumor cell types and is currently in phase I and II clinical trials. Infectious V937 particles consist of a ∼30 nm icosahedral capsid assembled from four structural viral proteins that encapsidate a viral RNA genome. Rapid and robust analytical methods to quantify and characterize CVA21 virus particles are important to support the process development, regulatory requirements, and validation of new manufacturing platforms. Herein, we describe a size-exclusion chromatography (SEC) method that was developed to characterize the V937 drug substance and process intermediates. Using a 4-in-1 combination of multi-detectors (UV, refractive index, dynamic and static light scattering), we demonstrate the use of SEC for the quantification of the virus particle count, the determination of virus size (molecular weight and hydrodynamic diameter), and the characterization of virus purity by assessing empty-to-full capsid ratios. Through a SEC analysis of stressed V937 samples, we propose CVA21 thermal degradation pathways that result in genome release and particle aggregation.

Keywords: Oncolytic Coxsackievirus A21 virus; aggregation; capsid particle concentration; dynamic light scattering; empty and full capsid; immunotherapy; multi-angle light scattering; particle size; size-exclusion chromatography; virus capsid empty/full ratio; virus molecular weight.