Versatile LC–MS-Based Workflow with Robust 0.1 ppm Sensitivity for Identifying Residual HCPs in Biotherapeutic Products
Feng Yang, Delia Li, Regina Kufer, Lance Cadang, Jennifer Zhang, Lu Dai, Jia Guo, Stefanie Wohlrab, Midori Greenwood‐Goodwin, Amy Shen, Dana Duan, Hong Li, Inn H. Yuk
Abstract
Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying >85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.