A workflow for accurate and consistent quantitation of host cell proteins by SWATH LC-MS/MS analysis to support
Jia Guo1, Regina Kufer2, Midori Greenwood-Goodwin3
1Protein Analytical Chemistry, Genentech, A Member of the Roche Group, South San Francisco, CA, United States.
Journal of Pharmaceutical Sciences
|November 17, 2024
Summary
Accurate quantification of residual host cell proteins (HCPs) is crucial for drug safety. This study enhances SWATH MS/MS methods for precise HCP analysis, improving drug product quality and reliability.
Area of Science:
- Biopharmaceutical analysis
- Mass spectrometry
- Protein quantification
Background:
- Residual host cell proteins (HCPs) can compromise drug product quality, stability, efficacy, and safety.
- Accurate quantification of HCPs at low levels (≥ 1 ppm) is essential for biopharmaceutical development.
- Existing methods like data-dependent acquisition (DDA) and targeted approaches have limitations for HCP analysis.
Purpose of the Study:
- To enhance the accuracy and reliability of SWATH MS/MS-based methods for quantifying residual host cell proteins (HCPs).
- To develop a robust method for consistent HCP quantification, overcoming biases associated with reference protein selection.
- To support biopharmaceutical process development through improved HCP analysis.
Main Methods:
- Utilized data-independent sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS/MS.
- Optimized reference protein selection based on ranking values to minimize quantitative bias.
- Incorporated sodium deoxycholate (SDC) during digestion to improve protein detection and quantitation consistency.
- Employed standard addition combined with SWATH quantitation.
- Validated results using targeted MRM-MS/MS and ELISA.
- Applied an automated Spectronaut data analysis workflow.
Main Results:
- Achieved enhanced accuracy in SWATH-based HCP quantitation relative to spiked-in reference proteins.
- Demonstrated improved protein detection and quantitation consistency with the addition of SDC.
- Showcased good accuracy and reproducibility in measuring HCP levels using SWATH with standard addition.
- Confirmed the reliability of the method through validation with targeted MRM-MS/MS and ELISA.
- Successfully generated quantitative HCP results for various in-process pools using an automated workflow.
- Enabled measurement of specific HCPs like Peroxiredoxin-1 with high throughput and consistency.
Conclusions:
- The enhanced SWATH MS/MS method provides accurate and reproducible quantification of residual HCPs.
- Optimized reference protein selection and SDC addition significantly improve quantitation reliability.
- The automated workflow facilitates efficient HCP analysis, supporting biopharmaceutical process development.
- This approach is valuable for ensuring drug product quality, safety, and efficacy by controlling HCP levels.
