Applying UHPLC HRAM MS-MS/MS method to assess host cell protein clearance during the purification process development of therapeutic mAbs

Significance of the Topic

Host cell proteins (HCPs) represent critical impurities in monoclonal antibody production. Their precise identification and quantification are essential to ensure product safety, efficacy and regulatory compliance. Traditional ELISA methods provide total HCP measurements but lack the sensitivity and molecular detail needed for in-depth process development. Implementing an ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution accurate mass (HRAM) MS/MS platform addresses these gaps by enabling comprehensive, site-specific HCP profiling even at sub-ppm levels.

Objectives and Study Overview

This work presents the development of a robust UHPLC–HRAM MS–MS/MS workflow to monitor individual HCP clearance across multiple downstream purification steps for a therapeutic trastuzumab product. Two in-house samples (Protein A and polishing pools) and a well-characterized NISTmAb reference were analyzed in triplicate to assess method performance, sensitivity and quantitative precision. The study also evaluated the ability of a POROS Caprylate mixed-mode resin to remove high-risk and difficult-to-remove HCPs during polishing.

Methodology

Samples were digested under non-denaturing conditions with trypsin and separated on a 25 cm C18 UHPLC column (2.1 × 250 mm, 2.2 μm) at 60 °C and 300 μL/min over a 90 min linear gradient. Data-dependent acquisition (DDA) was performed on an Orbitrap Ascend BioPharma Tribrid instrument, capturing one full MS scan (120k resolution) followed by 15 MS/MS scans (30k resolution). A dual-search strategy combining Sequest HT and CHIMERYS improved protein identification coverage.

Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system
• Acclaim VANQUISH C18 column (2.1 × 250 mm, 2.2 μm)
• Orbitrap Ascend BioPharma Tribrid mass spectrometer
• Thermo Scientific Proteome Discoverer 3.1 software

Main Results and Discussion

The optimized method identified 385 HCPs in the Protein A pool and 91 in the polish pool for trastuzumab, and 235 HCPs from the NISTmAb reference, spanning a dynamic range down to 0.007 ppm. Quantitative repeatability was excellent, with 80 % of HCPs showing ≤ 15 % CV across replicates. Application of POROS Caprylate resin achieved > 96 % reduction of key high-risk and difficult-to-remove HCPs in the polishing step.

Benefits and Practical Applications

• High sensitivity for sub-ppm HCP detection
• Comprehensive profiling of individual process-related impurities
• Quantitative precision suitable for process development and quality control
• Rapid assessment of novel polishing resins and purification strategies

Future Trends and Opportunities

Integration of data-independent acquisition (DIA), machine-learning-driven data processing and real-time process analytical technologies will further enhance HCP coverage and throughput. Expansion of spectral libraries and streamlined workflows can support high-throughput screening of resin chemistries and continuous bioprocessing platforms.

Conclusion

A platform UHPLC–HRAM MS–MS/MS method was established, delivering robust identification and quantification of HCPs across key purification steps. The approach demonstrated high sensitivity, reproducibility and practical utility in evaluating resin performance and ensuring product purity.

References

1. Beaumal C, et al. Proteomics. 2023; doi:10.1002/pmic.202300172
2. Huang H, et al. Analytical Chemistry. 2017;89(10):5436–5444
3. Thermo Fisher Scientific. POROS Caprylate Mixed Mode Resin, Catalog A51049
4. Biophorum. Host cell protein searchable database
5. Singh S, et al. Biotechnology Progress. 2019;1–12