Enhanced HCP quantitation LC-MS/MS workflow solution using a MS sample prep method

Significance of the topic

Host cell proteins (HCPs) represent critical impurities in biotherapeutic production that may affect product safety and efficacy. Conventional ELISA methods provide aggregate HCP measurements but can miss non-immunogenic species. High-resolution LC-MS/MS with targeted quantitation enables identification and precise measurement of individual HCPs, offering an orthogonal and complementary approach to ensure comprehensive impurity control.

Study objectives and overview

This study aimed to develop an enhanced HCP quantitation workflow by integrating protein A–based depletion, Thermo Scientific EasyPep sample preparation, and heavy-labeled AQUA peptide standards. Two monoclonal antibodies (rituximab and trastuzumab) and a reference NISTmAb were analyzed to compare the performance of the novel workflow against conventional native digestion methods.

Methodology and instrumentation

Sample preparation involved:

• Affinity purification of antibodies followed by protein A MagBead depletion.
• EasyPep digestion with an added semi-tryptic trypsin elution step to recover low-abundance HCPs.
• Native tryptic digestion controls performed with and without prior depletion.

Targeted quantitation employed a 65-peptide HeavyPeptide AQUA panel covering 28 high-risk CHO HCPs. Linearity and limit of quantitation (LLOQ) were assessed via six-point dilution series (0.03–200 fmol).

Instrumentation:

• Thermo Scientific Dionex UltiMate 3000 RSLCnano System.
• Thermo Scientific Q Exactive Plus mass spectrometer.
• Data analysis with Thermo Scientific Proteome Discoverer 3.0 and Skyline software.

Key findings and discussion

The protein A–depleted EasyPep workflow outperformed native digestion approaches:

• Trastuzumab samples: 1575 HCPs detected with depleted EasyPep vs. 859 native and 563 native-depleted.
• NISTmAb: 807 HCPs identified by depleted EasyPep, compared with 235 by native digestion.
• Rituximab: 13 HCPs detected in depleted EasyPep vs. 3 in native digestion.
• PRM quantitation displayed excellent linearity (R² ≥ 0.95) for the majority of heavy peptides and an LLOQ of 0.03 fmol.

Combining protein A depletion with a targeted heavy peptide panel and semi-tryptic elution mitigated dynamic range challenges and improved recovery of low-abundance HCPs.

Benefits and practical applications

Key advantages of the enhanced workflow include:

• Improved sensitivity and depth of HCP coverage compared to conventional methods.
• Specific quantitation of individual HCP species not detected by ELISA.
• Robust monitoring of HCP profiles throughout downstream processing for quality control.

Future trends and potential applications

Emerging opportunities include:

• Automation of sample preparation for higher throughput and reproducibility.
• Expansion of heavy peptide panels to encompass additional risk-associated HCPs.
• Integration with advanced mass analyzers or ion mobility separation to further enhance detection depth.
• Implementation within real-time process analytical technology (PAT) frameworks to support continuous biomanufacturing.

Conclusion

The protein A–depleted EasyPep LC-MS/MS workflow significantly enhances HCP identification and quantitation in monoclonal antibody samples. By leveraging targeted heavy peptides and optimized depletion protocols, the method delivers superior sensitivity, linearity, and practical utility for biotherapeutic quality control.

Reference

• Jones et al. (2021) “High-risk” host cell proteins (HCPs): A multi-company collaborative view. Biotechnol Bioeng. 118(8):2870–2885.