High-sensitivity Host Cell Protein (HCP) analysis on a new Orbitrap Excedion Pro BioPharma hybrid mass spectrometer with optimized sample preparation

Importance of the Topic

Host cell proteins (HCPs) are residual impurities in biotherapeutic manufacturing that can compromise product safety, efficacy and stability. Conventional ELISA assays quantify total HCP levels but cannot discriminate individual proteins or cover the required dynamic range. High-resolution LC-MS methods address these limitations by enabling sensitive, specific profiling of HCPs across orders of magnitude in abundance, supporting advanced risk assessment and process optimization.

Study Objectives and Overview

This study evaluates the performance of the new Thermo Scientific Orbitrap Excedion Pro BioPharma hybrid mass spectrometer for high-sensitivity HCP profiling. Two biotherapeutic models are investigated: NISTmAb under native tryptic digestion and trastuzumab using both native digestion and Protein A-depleted EasyPep workflows. The aim is to benchmark sensitivity, reproducibility and dynamic range, and to compare sample preparation strategies for comprehensive HCP identification.

Methodology

• Sample Preparation: NISTmAb and trastuzumab were processed by native trypsin digestion or Protein A bead-depleted EasyPep digestion, followed by quantification of digested peptides.
• Chromatography and Acquisition: Peptides were separated on a Thermo Scientific Acclaim Vanquish C18 column (2.1×250 mm) with a 135 min gradient at 300 µL/min. Data were acquired in data-dependent mode on the Orbitrap Excedion Pro BioPharma at 120 000 resolution for MS1 and 30 000 for MS2, using HCD fragmentation.
• Data Analysis: MS2 spectra were searched against UniProt Mus musculus and Cricetulus griseus databases using Proteome Discoverer 3.2 with CHIMERYS and Sequest HT algorithms. Results were filtered at <1% peptide and protein FDR and ≥2 unique peptides per protein.

Used Instrumentation

• Orbitrap Excedion Pro BioPharma hybrid mass spectrometer
• Thermo Scientific Acclaim Vanquish C18 UHPLC column (2.1×250 mm)
• Thermo Scientific Proteome Discoverer 3.2 with CHIMERYS and Sequest HT

Key Results and Discussion

• NISTmAb Profile: Native digestion yielded 163 HCPs spanning concentrations from 169 ppm to 0.007 ppm, with excellent reproducibility (CV 1.08%). Comparison with prior FAIMS-based data confirmed 128 overlapping HCPs and revealed 31 novel identifications.
• Trastuzumab Comparison: Protein A-depleted EasyPep digestion detected 1 550 HCPs versus 874 by native digestion. Of these, 826 were common, 670 unique to EasyPep and 48 unique to native digestion, highlighting superior sensitivity of the depleted workflow for low-abundance HCPs.
• Reproducibility and Dynamic Range: Both workflows demonstrated consistent high-quality spectra for low-level HCPs. High-abundance HCP profiles were similar, confirming methodological comparability.

Benefits and Practical Application

The optimized LC-MS workflow on the Orbitrap Excedion Pro BioPharma enables comprehensive and sensitive HCP profiling, facilitating targeted monitoring of individual impurities during biotherapeutic development and quality control. Enhanced detection of low-abundance HCPs supports improved risk assessment and process optimisation.

Future Trends and Potential Applications

• Integration of ion mobility or FAIMS to further extend dynamic range.
• AI-driven spectrum interpretation for accelerated data analysis.
• Enhanced workflows for multiplexed quantitation across multiple mAb products.
• Automation and real-time monitoring in continuous bioprocessing.

Conclusion

The new Orbitrap Excedion Pro BioPharma mass spectrometer, combined with native and Protein A-depleted EasyPep digestion strategies, delivers ultrahigh-sensitivity, reproducible HCP profiling across a wide dynamic range. The Protein A-depleted workflow outperforms native digestion for low-abundance HCP detection, offering a robust platform for advanced biopharmaceutical quality control.

References

1. Huang X. et al. A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies. Anal Chem. 2017;89(10):5436–5444.
2. Jae C. et al. EasyPep Sample Enrichment and Quantitation of Host Cell Proteins. Thermo Fisher Scientific Poster PO-220; 2024.
3. Beaumal C. et al. Comprehensive HCP Profiling by Proteomics. Proteomics. 2023;doi:10.1002/pmic.202300172.