Key LC-MS Publications for Analysis of Host Cell Proteins in Biopharmaceuticals

Significance of the Topic

Accurate identification and quantitation of residual host cell proteins (HCPs) in biopharmaceuticals are critical for ensuring product safety, efficacy and regulatory compliance.

Objectives and Study Overview

This summary reviews key publications and technical notes that describe mass spectrometry–based strategies for HCP profiling in monoclonal antibody (mAb) products.

Methodology

Approaches include:

• Non‐denaturing tryptic digestion with and without removal of undigested antibody prior to LC‐MS/MS analysis.
• Data‐independent acquisition (DIA) used for broad peptide detection, followed by parallel reaction monitoring (PRM) for confirmation and quantitation.
• Data‐dependent acquisition (DDA) workflows with inclusion/exclusion lists to enhance coverage of low‐abundance HCPs.
• Label‐free quantitation of HCPs in cell culture supernatants using GO annotation for functional classification.

Used Instrumentation

Major instruments reported:

• Thermo Scientific Q Exactive™ Plus Hybrid Quadrupole‐Orbitrap™ mass spectrometer.
• Thermo Scientific LTQ Orbitrap™ mass spectrometer.
• Orbitrap Fusion™ Lumos Tribrid™ mass spectrometer.
• High‐resolution accurate‐mass (HRAM) Orbitrap platform for improved dynamic range.

Main Results and Discussion

Key findings include:

• Sensitivity down to 0.5 ppm spiked protein in 12.5 mg/mL mAb preparations using non‐denaturing digest protocols.
• DIA workflows detected more peptide features than two‐dimensional HPLC‐MS but no additional HCP species.
• Use of drug product exclusion lists in DDA increased HCP coverage despite potential peptide overlap with the therapeutic.
• Label‐free quantitation identified dynamic changes in HCP profiles during fed‐batch culture of CHO cells.

Practical Benefits and Applications

These methods enable:

• Comprehensive monitoring of critical quality attributes (CQAs), including charge variants and aggregation.
• Enhanced detection of low‐abundance impurities to support downstream purification strategies.
• Streamlined workflows for comparability studies and biosimilar development.

Future Trends and Applications

The field is moving toward integrated high‐throughput platforms combining DIA and PRM approaches, improved sample‐preparation techniques to enrich low‐level impurities, and universal LC‐MS assays capable of five‐order dynamic range for trace‐level protein impurity analysis.

Conclusion

Mass spectrometry–based HCP analysis has matured into robust workflows that balance sensitivity, specificity and throughput. Continued innovations in instrumentation and data acquisition strategies will further enhance biopharmaceutical quality control.

References

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8. Houel S., Huguet R., Abbatiello S., Sarracino D., Josephs J. ASMS Poster 2018.
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