Increasing sensitivity of high throughput host cell protein analysis on a novel high- resolution accurate mass platform

Importance of the topic

Recombinant biotherapeutics rely on non-human host cell lines for production. Trace amounts of host cell proteins (HCPs) can compromise drug safety and efficacy. Standard techniques like ELISA assess total HCP content but cannot characterize individual impurities. High-resolution mass spectrometry has become essential for quantifying and identifying HCPs at sub-ppm levels. Advances in proteomic workflows are needed to address the dynamic range challenge between abundant therapeutic proteins and low-level impurities.

Objectives and overview of the study

The study evaluates the Thermo Scientific Orbitrap Astral mass spectrometer for high-throughput, sensitive detection of low-ppm HCPs in biotherapeutics. Key aims include:

• Reducing the dynamic range between monoclonal antibody (mAb) peptides and HCP peptides
• Increasing throughput using a 20.2-minute gradient (60 samples per day method)
• Demonstrating identification of over 130 HCPs at sub-ppm levels

Methodology and instrumentation

Proteolysis: Native NISTmAb samples were digested using SMART Digest immobilized trypsin under non-denaturing conditions (37 °C, 3 h) and compared to denaturing digestion (70 °C, 30 min), followed by precipitation and reduction with DTT.
Chromatography: Peptides (200–500 ng) were separated on a 150 µm × 15 cm PepMap Neo column with a 20.2 min gradient on a Vanquish Neo UHPLC in Trap & Elute mode.
Mass spectrometry: The Orbitrap Astral operated in DIA mode with 3 Th isolation windows and MS2 acquisition speeds up to 200 Hz.
Data analysis: Proteome Discoverer 3.1 with the Chimerys algorithm searched a UniProt Mus musculus database. Filtering at <1% peptide and protein FDR and requiring two unique peptides per HCP ensured high confidence.

Main results and discussion

• Non-denaturing proteolysis reduced mAb peptide signals in key retention regions, lowering dynamic range and improving HCP detection limits.
• Over 130 HCPs were identified at <1% FDR with at least two unique peptides in three replicate injections using a 20.2 min gradient.
• The workflow achieved ~5× increased throughput compared to standard methods while maintaining comparable HCP coverage to recent studies.
• High-quality MS2 spectra across a concentration range from sub-ppb to >100 ppm supported robust identification.

Practical benefits and applications of the method

• Enables detailed characterization of individual HCP impurities for biotherapeutic development and quality control.
• Delivers rapid turnaround and high throughput (60 samples per day) without loss of sensitivity.
• Facilitates regulatory compliance by identifying trace impurities that may impact safety or efficacy.

Future trends and potential applications

Combining ultra-fast HRAM DIA scans with advanced chromatographic strategies and AI-driven data analysis will further enhance sensitivity and throughput. Applications may include real-time HCP monitoring during purification, extended profiling of protein degradants, and tailored workflows for diverse biologic modalities such as ADCs and fusion proteins.

Conclusion

This work demonstrates the exceptional performance of the Orbitrap Astral mass spectrometer for rapid, sensitive analysis of trace-level host cell proteins in biotherapeutics. The integration of non-denaturing digestion, fast chromatography, high-speed DIA, and advanced data analysis enables comprehensive HCP profiling at sub-ppm levels in high-throughput settings.

References

• Huang L. et al., Anal. Chem. 2017, 89 (10), 5436–5444.
• Claydon A. et al., Thermo Scientific App. Note no. 73412, 2020.
• Beaumal C. et al., Proteomics. 2023 May 6, doi:10.1002/pmic.202300172.
• Johnson B. et al., Anal. Chem. 2020, 92 (15), 10478–10484.