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

Area of Science:

• Biotechnology
• Analytical Chemistry
• Virology

Background:

• Oncolytic virus (OV) therapy is a rapidly advancing field requiring robust analytical methods for viral characterization.
• Accurate quantification of viral proteins is essential for understanding OV infection, immunogenicity, and replication stages.
• Vesicular stomatitis virus glycoprotein (VSV-GP) is a promising biotherapeutic candidate in OV therapy.

Purpose of the Study:

• To develop and validate a novel ultra-high performance liquid chromatography-multiple reaction monitoring (UPLC-MRM) assay for the absolute quantification of VSV-GP structural proteins.
• To enable precise measurement of processed GP1 and GP2 proteins and the full-length glycoprotein complex (GPC).
• To utilize the assay for tracking GPC processing in production cell lines.

Main Methods:

• Development of a UPLC-MRM assay using a Sciex 6500 triple-stage quadrupole mass spectrometer.
• Absolute quantification achieved through heavy-labeled reference standard surrogate peptides.
• Simultaneous measurement of multiple viral proteins, including processed GP1, GP2, and full-length GPC.

Main Results:

• The UPLC-MRM assay provides accurate and precise absolute quantification of VSV-GP structural proteins.
• The assay successfully quantified the ratios of processed GP1 and GP2, as well as the unprocessed GPC.
• Demonstrated the utility of the assay in tracking GPC processing within HEK-293-F cells infected with VSV-GP.

Conclusions:

• The developed UPLC-MRM assay is a robust tool for characterizing oncolytic viruses like VSV-GP.
• This analytical method is crucial for the quality control and development of viral vector-based biotherapeutics.
• Understanding GPC processing is vital for VSV-GP infectivity and therapeutic efficacy.