Versatile LC-MS-Based Workflow with Robust 0.1 ppm Sensitivity for Identifying Residual HCPs in Biotherapeutic
Feng Yang1, Delia Li1, Regina Kufer2
1Protein Analytical Chemistry, Genentech, A Member of the Roche Group, 1 DNA Way, South San Francisco, California 94080, United States.
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.
Insights
This study introduces a novel workflow for highly sensitive detection of residual host cell proteins (HCPs) in biotherapeutics. The method enhances HCP identification sensitivity by 10- to 100-fold, crucial for drug safety and quality control.
Area of Science:
- Biopharmaceutical Analysis
- Protein Chemistry
- Analytical Chemistry
Background:
- Residual host cell proteins (HCPs) in biotherapeutics can compromise product quality, stability, and safety.
- Highly active hydrolytic enzymes, even at sub-ppm levels, pose a significant challenge for detection using conventional LC-MS/MS due to wide dynamic range differences with therapeutic proteins.
Purpose of the Study:
- To develop a highly sensitive workflow for identifying and quantifying residual host cell proteins (HCPs) in biopharmaceutical products.
- To improve the detection limits for challenging HCPs, particularly hydrolytic enzymes, at very low concentrations.
- To enhance the demonstration of HCP clearance during drug process development.
Main Methods:
- Implementation of a novel analytical workflow involving native digestion at high protein concentration and addition of sodium deoxycholate during reduction.
- Utilized solid-phase extraction with 50% MeCN elution for effective monoclonal antibody (mAb) removal prior to analysis.
- Employed a 50 cm nanoflow charged surface hybrid column to increase sample load capacity and enhance sensitivity for LC-MS/MS analysis.
Main Results:
- Achieved a 10- to 100-fold increase in sensitivity for HCP identification compared to previous methods.
- Demonstrated robust identification of HCPs as low as 0.1 ppm (34.5 to 66.2 kDa MW).
- Successfully identified over 85% of 48 UPS-1 proteins (0.10 to 1.34 ppm) in a mAb and a record 746 mouse proteins from NIST mAb in a single analysis.
Conclusions:
- The developed workflow significantly advances the capability for detecting and quantifying trace-level HCPs in biopharmaceuticals.
- This method is crucial for ensuring drug product quality and safety by effectively identifying critical impurities like low-level hydrolases.
- The enhanced sensitivity and robustness provide a valuable tool for demonstrating HCP clearance in biopharmaceutical process development.
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