Absolute quantification of viral proteins from pseudotyped VSV-GP using UPLC-MRM

The rapidly developing fieldof oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the characterization of the virus during production and development. Accurate monitoring and absolute quantificationof viral proteins are 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) offera robust way to directly detect and quantify specifictargeted 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 quantifiesthe structural proteins of a pseudotyped vesicular stomatitis virus (VSV) intended for use as a new biotherapeutic (designated hereafter as VSV-GP to differentiateit from native VSV). The new UPLC-MRM method provides absolute quantificationwith 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 quantifiedat 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 GP complex (GPC; full-length GPC).