LC-MS-based host cell protein (HCP) identification and monitoring during biopharmaceutical downstream process development

Importance of the topic

Host cell proteins (HCPs) are process-related impurities released during cell culture and purification of monoclonal antibodies (mAbs). Even trace levels of these proteins can pose immunogenic risks, affect product potency, or catalyze unwanted degradation reactions. Regulatory agencies require rigorous monitoring of HCP levels to ensure safety and efficacy of biopharmaceuticals. LC-MS/MS offers an orthogonal method to conventional ELISA, providing both qualitative identification and quantitative measurement of individual HCPs independent of their immunogenicity.

Study goals and overview

This application note demonstrates a streamlined LC-MS based workflow for HCP identification and relative quantitation during mAb downstream process development. Three intermediate purification pools (Protein A, anion exchange, cation exchange) from a platform mAb process were analyzed. Objectives included:

• Assess HCP clearance across purification steps.
• Identify high-risk HCPs that co-purify with mAb.
• Validate a non-denaturing digestion approach to improve detection sensitivity.

Methodology and instrumentation

Sample preparation employed an optimized non-denaturing trypsin protocol: residual undigested mAb was removed by heat precipitation, enriching low-abundance HCP peptides. Four stable reference proteins were spiked in for peptide-based quantitation. Peptides were separated on a Vanquish Flex UHPLC system with an Accucore C18 column at 60 °C and a 0.3 mL/min gradient. MS analysis used a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer in data-dependent Top10 mode with 70 000 resolution (MS1) and 35 000 resolution (MS2). Data processing and HCP database searching were performed in Thermo Scientific BioPharma Finder 4.1, leveraging its new host cell protein analysis workflow, decoy database searching, and Top3 quantitation algorithm.

Key results and discussion

Non-denaturing digestion reduced mAb peptide interference, enhancing detection of low-level HCPs down to ~1 ppm. Total detected HCPs and cumulative HCP levels (ppm) were:

• Protein A pool: 676 HCPs, 49 473 ppm
• Anion exchange pool: 111 HCPs, 1 253 ppm
• Cation exchange pool: 7 HCPs, 90 ppm

Stepwise purification achieved effective HCP removal. The HCP workflow enabled visualization of sequence coverage, peptide identification, and quantitation trends. Three previously reported high-risk HCPs (phospholipase B-like, peroxiredoxin-1, peroxiredoxin-2) were tracked:

• Phospholipase B-like: 561 ppm → 135 ppm → 63 ppm
• Peroxiredoxin-1: 596 ppm → 88 ppm → 57 ppm
• Peroxiredoxin-2: 57 ppm → 13 ppm → 1 ppm

System reproducibility was demonstrated by low technical and biological CVs across replicates and chromatographic stability over 135 min runs.

Benefits and practical applications

• Provides individual HCP identification and relative quantitation throughout purification.
• Supports selection and monitoring of high-risk HCPs to guide process optimization.
• Offers a single software platform for peptide mapping and HCP analysis, facilitating data review and regulatory compliance.

Future trends and opportunities

Integration of LC-MS HCP workflows with PAT (process analytical technology) and continuous manufacturing could enable real-time impurity monitoring. Advances in data-independent acquisition (DIA) and machine learning–based spectral interpretation may further increase HCP coverage and quantitation accuracy. Customized HCP databases and improved sample preparation methods will continue to enhance sensitivity for trace impurity detection.

Conclusion

The optimized LC-MS/MS workflow with non-denaturing digestion and high-resolution mass spectrometry effectively identified and quantified HCPs during mAb purification. The approach delivered high sensitivity down to single-digit ppm levels, robust tracking of high-risk HCPs, and comprehensive data analysis in a unified software environment, offering a powerful tool for downstream process development and quality control.

References

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