Guo Jia, Kufer Regina, Greenwood-Goodwin Midori, Wohlrab Stefanie, Woodruff Fem, Li Delia, Reckermann Katharina, Youn Jonghae, Kandula Dilip Kumar Reddy, Xiong Lei, Yang Feng
Protein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, United States.
Pharma Technical Development Analytics, Roche Diagnostics GmbH, Penzberg, Germany.
J Pharm Sci. 2025 Feb;114(2):1002-1009. doi: 10.1016/j.xphs.2024.11.007. Epub 2024 Nov 16.
Residual host cell proteins (HCPs) in drug products may impact product quality, stability, efficacy and safety. To support consistent and accurate quantitative analysis for low levels of HCPs (≥ 1 ppm), the data-independent sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS/MS-based method provides unique advantages over data dependent acquisition (DDA) or targeted methods for HCP identification and quantification. However, SWATH MS/MS-based methods can generate biased quantitative results that are highly dependent on the selected reference protein. In this study, we enhanced the accuracy of SWATH-based HCP quantitation relative to a spiked-in reference protein by selecting appropriate reference proteins based on their ranking values. We developed a reliable SWATH-based method for quantifying specific HCPs by adding sodium deoxycholate (SDC) during digestion to enhance both protein detection and quantitation consistency. By combining SWATH-based quantitation with standard addition, we showed its use in measuring HCP levels with good accuracy and reproducibility, confirmed by both targeted MRM-MS/MS and ELISA. Additionally, we demonstrated an automated Spectronaut data analysis workflow can efficiently generate SWATH quantitative results for HCPs in different in-process pools. Using SWATH-based quantitation, we were able to measure specific HCPs (e.g. Peroxiredoxin-1) and support process development with good throughput and quantitation consistency.
药品中的残留宿主细胞蛋白（HCPs）可能会影响产品质量、稳定性、功效和安全性。为了支持对低水平HCPs（≥1 ppm）进行一致且准确的定量分析，基于数据非依赖型全理论碎片离子谱（SWATH）的串联质谱方法相较于依赖数据采集（DDA）或靶向方法在HCP鉴定和定量方面具有独特优势。然而，基于SWATH串联质谱的方法可能会产生高度依赖所选参考蛋白的有偏差的定量结果。在本研究中，我们通过根据其排名值选择合适的参考蛋白，提高了基于SWATH的HCP定量相对于掺入的参考蛋白的准确性。我们开发了一种可靠的基于SWATH的方法，通过在消化过程中添加脱氧胆酸钠（SDC）来增强蛋白质检测和定量一致性，从而对特定的HCPs进行定量。通过将基于SWATH的定量与标准加入法相结合，我们展示了其在准确测量HCP水平方面的应用，这通过靶向MRM-串联质谱和酶联免疫吸附测定（ELISA）得到了证实。此外，我们证明了自动化的Spectronaut数据分析工作流程可以有效地生成不同生产过程中间产物池中HCPs的SWATH定量结果。使用基于SWATH的定量方法，我们能够测量特定的HCPs（如过氧化物还原酶-1），并以良好的通量和定量一致性支持工艺开发。
