Reverse-Phase Ultra-Performance Chromatography Method for Oncolytic Coxsackievirus Viral Protein Separation and Empty to Full Capsid Quantification

Hum Gene Ther. 2022 Jul;33(13-14):765-775. doi: 10.1089/hum.2022.013. Epub 2022 Jun 1.

Abstract

Oncolytic virus immunotherapy is emerging as a novel therapeutic approach for cancer treatment. Immunotherapy clinical drug candidate V937 is currently in phase I/II clinical trials and consists of a proprietary formulation of Coxsackievirus A21 (CVA21), which specifically infects and lyses cells with overexpressed ICAM-1 receptors in a range of tumors. Mature Coxsackievirus virions, consisting of four structural virion proteins, (VPs) VP1, VP2, VP3, and VP4, and the RNA genome, are the only viral particles capable of being infectious. In addition to mature virions, empty procapsids with VPs, VP0, VP1, and VP3, and other virus particles are produced in V937 production cell culture. Viral protein VP0 is cleaved into VP2 and VP4 after RNA genome encapsidation to form mature virions. Clearance of viral particles containing VP0, and quantification of viral protein distribution are important in V937 downstream processing. Existing analytical methods for the characterization of viral proteins and particles may lack sensitivity or are low throughput. We developed a sensitive and robust reverse-phase ultra-performance chromatography method to separate, identify, and quantify all five CVA21 VPs. Quantification of virus capsid concentration and empty/full capsid ratio was achieved with good linearity, accuracy, and precision. ClinicalTrials.gov ID: NCT04521621 and NCT04152863.

Keywords: LC/MS; RP-UPLC; V937; capsid empty/full ratio; capsid quantification; oncolytic Coxsackievirus; therapeutic viral vector; virion protein analysis.

MeSH terms

  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Capsid* / metabolism
  • Chromatography
  • Oncolytic Viruses* / genetics
  • Oncolytic Viruses* / metabolism
  • RNA, Viral / analysis
  • RNA, Viral / metabolism
  • Viral Proteins

Substances

  • Capsid Proteins
  • RNA, Viral
  • Viral Proteins

Associated data

  • ClinicalTrials.gov/NCT04521621
  • ClinicalTrials.gov/NCT04152863