Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591-6707, USA.
Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591-6707, USA.
J Pharm Biomed Anal. 2024 May 15;242:116009. doi: 10.1016/j.jpba.2024.116009. Epub 2024 Feb 6.
Many methods using liquid chromatography-mass spectrometry (LC-MS) have been established for identifying residual host cell proteins (HCPs) to aid in the process development and quality control of therapeutic proteins. However, the use of MS-based techniques for adeno-associated virus (AAV) is still in its infancy, with few methods reported and minimal information available on potentially problematic HCPs. In this study, we developed a highly sensitive and effective differential digestion method to profile residual HCPs in AAV. Unlike direct digestion, which completely digests both AAV and HCPs, our differential digestion method takes advantage of AAV's unique characteristics to maintain the integrity of AAV while preferentially digesting HCPs under denaturing and reducing conditions. This differential digestion method requires only several micrograms of sample and significantly enhances the identification of HCPs. Furthermore, this method can be applied to all five different AAV serotypes for comprehensive HCP profiling. Our work fills a gap in AAV HCP analysis by providing a sensitive and robust strategy for detecting, monitoring, and measuring HCPs.
许多使用液相色谱-质谱联用技术（LC-MS）的方法已经被建立，用于鉴定残留的宿主细胞蛋白（HCPs），以辅助治疗蛋白的工艺开发和质量控制。然而，基于 MS 的腺相关病毒（AAV）技术的应用仍处于起步阶段，报道的方法很少，关于潜在问题 HCPs 的信息也很少。在这项研究中，我们开发了一种高度敏感和有效的差异消化方法来分析 AAV 中的残留 HCPs。与直接消化不同，直接消化完全消化 AAV 和 HCPs，我们的差异消化方法利用 AAV 的独特特性，在变性和还原条件下优先消化 HCPs ，同时保持 AAV 的完整性。这种差异消化方法仅需要几微克的样品，并且显著增强了 HCPs 的鉴定。此外，这种方法可以应用于所有五种不同的 AAV 血清型，用于全面的 HCP 分析。我们的工作通过提供一种灵敏而强大的检测、监测和测量 HCPs 的策略，填补了 AAV HCP 分析中的空白。
