Basu Rajeswari, Dambra Richard, Jiang Di, Schätzlein Sophia A, Njiyang Shu, Ashour Joseph, Chiramel Abhilash I, Vigil Adam, Papov Vladimir V
Materials and Analytical Sciences, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut, USA.
Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, Connecticut, USA.
Microbiol Spectr. 2024 Aug 6;12(8):e0365123. doi: 10.1128/spectrum.03651-23. Epub 2024 Jun 25.
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.
溶瘤病毒（OV）治疗领域发展迅速，因此需要开发新的、改进的分析方法，以在生产和研发过程中对病毒进行表征。病毒蛋白的准确监测和绝对定量对于OV产品表征至关重要，有助于理解病毒复制的感染、免疫原性和发育阶段。像多反应监测（MRM）这样的靶向质谱方法提供了一种强大的方式，可直接检测和定量由替代肽代表的特定靶向蛋白。我们通过将超高效液相色谱（UPLC）与Sciex 6500三重四极杆质谱仪相结合，利用了MRM的强大功能，开发了一种可准确、绝对定量用于新型生物治疗的假型水疱性口炎病毒（VSV）结构蛋白的分析方法（以下称为VSV-GP，以将其与天然VSV区分开来）。新的UPLC-MRM方法通过使用重标记参考标准替代肽实现绝对定量。当以已知的精确量添加到标准品和样品中时，参考标准可对样品制备和/或仪器性能期间的任何小扰动进行归一化并加以考虑，从而实现准确、精确的定量。由于MRM的多重性质，所有靶向蛋白可同时进行定量。优化后的分析方法得到了改进，可在同时测量包膜蛋白GP复合物（GPC；全长GPC）的任何剩余或未加工形式的同时，定量加工后的GP1和GP2蛋白的比例。
近年来，溶瘤病毒治疗的发展势头迅猛。水疱性口炎病毒糖蛋白（VSV-GP）是溶瘤病毒治疗平台中新兴的一种新型生物治疗药物。能够准确、精确地定量病毒蛋白的新型分析方法对于病毒载体作为生物治疗药物的成功开发至关重要。我们开发了一种基于超高效液相色谱多反应监测的分析方法，用于定量VSV-GP不同结构蛋白的绝对浓度。GP复合物（GPC）的完全加工是病毒感染性的先决条件。该分析方法扩展了定量全长GPC的潜力，有助于了解GPC的加工过程（同时分别对GP1和GP2进行定量）。我们使用该分析方法追踪感染VSV-GP的HEK-293-F生产细胞系中GPC的加工过程。
