AN EFFICIENT LC-MS WORKFLOW FOR IDENTIFICATION AND MONITORING OF HOST CELL PROTEINS FOR ASSISTING MONOCLONAL ANTIBODY PURIFICATION

Importance of Topic

The presence of host cell proteins HCPs in monoclonal antibody mAb products poses safety and efficacy risks in biopharmaceutical manufacturing. Detailed identification and monitoring of individual HCP impurities support purification process optimization, quality control and regulatory compliance. The development of robust LC-MS workflows enables proteome-wide coverage and complements traditional ELISA assays.

Objectives and Overview of Study

This work presents a two-step one-dimensional LC-MS platform for HCP discovery and high-throughput monitoring during mAb purification. Key goals:

• Identify low-abundance HCPs in a well-characterized NIST mAb standard and additional mAb preparations.
• Build an MS/MS spectral library for targeted HCP monitoring.
• Compare four strong cation exchange SCX protocols in their efficiency to clear specific HCPs.

Methodology and Instrumentation

Sample preparation combined proteolytic digestion with selective depletion of antibody peptides and addition of protein digest standards. Main steps:

• Denaturation reduction and alkylation of mAb using rapiGest, DTT and iodoacetamide.
• Overnight digestion with Lys-C and trypsin at 25 mg/mL protein concentration.
• Post-digest spiking of MassPREP standards ADH PHO and BSA at varied concentrations.
• Optional Protein A and SCX purification with four elution protocols on CHO-derived mAb samples.

Chromatography and mass spectrometry:

• ACQUITY UPLC I-class PLUS system with CSH C18 column 2.1×150 mm 1.7 μm.
• Gradient separations of 90 min for discovery and 30 min for monitoring assays.
• Xevo G2-XS QTof operated in data-independent MSE mode via UNIFI 1.9.4 software.
• Data processing in Progenesis QI for proteomics 4.2 with spectral library generation.

Main Results and Discussion

The discovery assay achieved a detection limit of 5 ppm identifying six HCPs in NIST mAb across triplicate injections. Two low-abundance peptides were validated by extracted ion chromatograms. High-throughput monitoring delivered robust performance with peak area RSDs under 10% for spiked protein standards. SCX protocol D demonstrated superior clearance of target HCP1 and HCP2 across multiple mAb preparations, yielding residual impurity levels of approximately 11–15 ppm.

Benefits and Practical Applications

The described LC-MS workflow offers full proteome coverage sensitivity at ppm levels orthogonal to ELISA. It supports rapid spectral library building and scalable monitoring of individual HCPs, facilitating process optimization and comparability studies in bioprocess development.

Future Trends and Applications

Further integration with automated library search, real-time data analysis, and machine learning algorithms is anticipated. Expansion to other host systems and continuous bioprocess monitoring can enhance control of impurity profiles. Advances in microfluidic LC-MS and enhanced fragmentation methods will improve throughput and depth of coverage.

Conclusion

A streamlined one-dimensional LC-MS platform combining discovery and targeted monitoring delivers sensitive reliable identification of individual HCPs. The approach enables informed selection of purification protocols and robust tracking of impurity clearance in monoclonal antibody production.

References

1. Doneanu C et al Anal Chem 2015 87 10283
2. Huang YQ et al Anal Chem 2017 89 5436