A Workflow for Accurate and Consistent Quantitation of Host Cell Proteins by SWATH LC-MS/MS Analysis to Support Process Development.

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.