Absolute Quantification of Viral Proteins from Pseudotyped Vesicular Stomatitis Virus (VSV-GP) using Ultra High-Performance Liquid Chromatography- Multiple Reaction Monitoring (UPLC-MRM)

The rapidly developing field of oncolytic virus (OV) therapy necessitates development of new and improved analytical approaches for characterization of the virus during production and development. Accurate monitoring and absolute quantification of viral proteins is crucial for OV product characterization and can facilitate the understanding of infection, immunogenicity, and development stages of viral replication. Targeted mass spectrometry methods, like multiple reaction monitoring (MRM), offers a robust way to directly detect and quantify specific targeted proteins represented by surrogate peptides. We have leveraged the power of MRM by combining ultra-high performance liquid chromatography (UPLC) with a Sciex 6500 triple stage quadrupole mass spectrometer to develop an assay that accurately and absolutely quantifies the structural proteins of a pseudotyped vesicular stomatitis virus intended for use as a new biotherapeutic (designated hereafter as VSV-GP) to differentiate it from native VSV. The new UPLC-MRM method provides absolute quantification with the use of heavy labeled reference standard surrogate peptides. When added in known exact amounts to standards and samples, the reference standards normalize and account for any small perturbations during sample preparation and/or instrument performance, resulting in accurate and precise quantification. Because of the multiplexed nature of MRM all targeted proteins are quantified at the same time. The optimized assay has been enhanced to quantify the ratios of the processed GP1 and GP2 proteins while simultaneously measuring any remaining or unprocessed form of the envelope protein GPC (full-length GPC). IMPORTANCEDevelopment of oncolytic viral therapy has gained considerable momentum in the recent years. VSV-GP is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral proteins are a necessity for the successful development of viral vector as a biotherapeutic. We developed a UPLC-MRM based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GPC is a pre-requisite for infectivity of the virus. The assay extends the potential for quantifying full-length GPC, which provides an understanding of the processing of GPC (along with the quantification of GP1 and GP2 separately). We used this assay in tracking GPC processing in HEK-293-F production cell lines infected with VSV-GP.