Versatile LC–MS-Based Workflow with Robust 0.1 ppm Sensitivity for Identifying Residual HCPs in Biotherapeutic Products

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.