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

The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral protein...

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Published inMicrobiology spectrum Vol. 12; no. 8; p. e0365123
Main Authors Basu, Rajeswari, Dambra, Richard, Jiang, Di, Schätzlein, Sophia A., Njiyang, Shu, Ashour, Joseph, Chiramel, Abhilash I., Vigil, Adam, Papov, Vladimir V.
Format Journal Article
LanguageEnglish
Published United States American Society for Microbiology 06.08.2024
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Abstract The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
AbstractList The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC).The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC).The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.IMPORTANCEThe development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC).IMPORTANCEThe development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC). The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
ABSTRACT The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC).IMPORTANCEThe development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (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 an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the 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.
The rapidly developing field of 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 quantification of 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) offer 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 (VSV) 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 GP complex (GPC; full-length GPC).
Author Jiang, Di
Dambra, Richard
Papov, Vladimir V.
Chiramel, Abhilash I.
Schätzlein, Sophia A.
Vigil, Adam
Njiyang, Shu
Ashour, Joseph
Basu, Rajeswari
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Keywords GPC tracking
oncolytic virus
absolute quantification
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Present address: Denali Therapeutics Inc., Development Sciences, South San Francisco, California, USA
All authors were employed by Boehringer Ingelheim Pharmaceuticals Inc. (BIPI) at the time of the experiments.
Present address: Regeneron Pharmaceuticals, Tarrytown, New York, USA
The authors declare no conflict of interest.
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Snippet The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new...
The rapidly developing field of oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the characterization of...
ABSTRACT The rapidly developing field of oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the...
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SubjectTerms absolute quantification
Genomics and Proteomics
GPC tracking
oncolytic virus
Research Article
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Title Absolute quantification of viral proteins from pseudotyped VSV-GP using UPLC-MRM
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