Identification of Host Cell Proteins (HPCs) in Monoclonal Antibodies At Sub-ppm Levels Using the SYNAPT XS Mass Spectrometer

Significance of the Topic

Detection of residual host cell proteins (HCPs) in monoclonal antibody (mAb) therapeutics is critical for ensuring product safety, stability and efficacy. Regulatory agencies mandate rigorous identification and quantification of HCPs at sub-ppm levels to minimize immunogenic risks and maintain drug quality.

Objectives and Study Overview

This application note presents a highly sensitive HCP discovery assay that leverages HDMSE data-independent acquisition on the SYNAPT XS mass spectrometer. The goal is to identify and quantify HCPs in a highly purified mAb to levels as low as 100 ppb, demonstrating improved coverage compared to traditional MSE methods.

Methodology and Used Instrumentation

Sample Preparation– An adapted protocol based on mAb precipitation allows selective digestion of low-abundance HCPs while preserving the bulk mAb intact. Proteins are digested overnight with trypsin in native conformation, followed by denaturation, reduction and alkylation post-digestion. The major mAb component is precipitated and removed by centrifugation, enriching HCP peptides.

Chromatography– Ultra-high-performance liquid chromatography using an ACQUITY PREMIER CSH C18 column (2.1 × 150 mm, 1.7 µm) at 60 °C with a flow rate of 50 µL/min. Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B). A standard gradient over 50 µL injection volumes with MeOH washes for system cleaning.

Mass Spectrometry– SYNAPT XS QTof system operating in ESI+ with HDMSE acquisition. Key parameters:

• Capillary voltage: 2.5 kV; cone voltage: 40 V.
• Source temperature: 120 °C; desolvation temperature: 300 °C.
• Ion mobility separation time: 15–20 ms; transfer CE ramp: 0–85 V synchronized to drift times.
• Mass range: m/z 50–2000; scan rate: 1 s.
• Software: MassLynx 4.2 for acquisition; Progenesis QI for Proteomics v4.2 for data processing.

Main Results and Discussion

By comparing MSE and HDMSE modes in triplicate analyses of the NIST mAb, the HDMSE method identified 48 HCPs versus only five abundant proteins by MSE. Detection sensitivity improved from 10 ppm (MSE) to 100 ppb (HDMSE). A Venn diagram comparison with previous studies highlighted 14 common HCPs and 20 unique to this workflow, 14 of which were measured below 1 ppm, demonstrating the assay’s enhanced depth of coverage. The use of fast ion mobility separation reduces spectral complexity and enhances peptide fragmentation quality, yielding more confident identifications.

Practical Benefits and Applications

Implementing HDMSE on the SYNAPT XS platform allows biopharmaceutical laboratories to achieve comprehensive, proteome-wide HCP profiling at sub-ppm levels. This assay supports process development, quality control and regulatory submissions by offering:

• Improved detection of low-abundance impurities.
• Reduced risk of immunogenic or stability issues.
• Compatibility with established sample preparation protocols.

Future Trends and Opportunities

Advances in ion mobility and data-independent acquisition will continue to enhance sensitivity and throughput. Combining orthogonal sample preparation strategies (e.g., HILIC fractionation, molecular-weight enrichment) can further extend HCP coverage. Integration with automated platforms and machine-learning-driven data analysis promises faster and more reproducible impurity profiling.

Conclusion

The HDMSE-based discovery assay on the SYNAPT XS mass spectrometer significantly advances HCP detection in monoclonal antibodies, achieving sub-ppm sensitivity and expanding the repertoire of identifiable impurities. This approach offers a robust tool for comprehensive quality assessment in biotherapeutic development.

References

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3. Chen W et al. Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry. In: Technologies for Therapeutic Monoclonal Antibody Characterization, Vol. 3. ACS Symposium Series. 2015:357–393.
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