IDENTIFICATION AND QUANTIFICATION OF HOST CELL PROTEIN IMPURITIES IN HIGH PURITY MONOCLONAL ANTIBODIES DOWN TO 1 PPM: AN INTER-LABORATORY STUDY

Significance of the Topic

Biopharmaceutical products such as monoclonal antibodies may contain trace levels of host cell proteins (HCPs) that can compromise drug safety and efficacy. Controlling HCP impurities to sub-parts-per-million (ppm) levels is essential to meet regulatory expectations and ensure patient safety.

Study Objectives and Overview

This inter-laboratory investigation aims to evaluate a novel two-dimensional liquid chromatography tandem mass spectrometry method (2DLC-HDMSE) for the identification and quantification of HCPs down to 1 ppm in high-purity monoclonal antibodies. Comparative studies were conducted on a NIST reference mAb, Remicade (infliximab), and its biosimilar Inflectra to assess sensitivity and reproducibility.

Methodology

• Sample preparation: Denaturation, reduction, alkylation, and proteolytic digestion of 2.5 mg mAb using RapiGest™ SF, DTT, IAM, Lys-C and trypsin. Four protein standards spiked post-digestion for Hi3 quantification.
• 2DLC separation: First dimension: high pH reversed-phase on XBridge C18; second dimension: low pH reversed-phase on CSH C18 with online trap and stepwise elution. Ion mobility enabled MSE acquisition (HDMSE).
• Data acquisition: Data-independent MSE on SYNAPT G2-S (cycle time 0.5 s, m/z 100–1990, CE ramp 20–45 eV). IMS parameters: fixed wave velocity and wave height; ion mobility specific collision energies.
• Data processing: ProteinLynx Global Server (PLGS) 3.0.2 and Hi3 methodology for label-free quantification.

Used Instrumentation

• ACQUITY UPLC M-Class system with 2D technology.
• XBridge C18 and CSH C18 analytical columns for 2D separations.
• SYNAPT G2-S mass spectrometer with ion mobility (HDMSE).

Main Results and Discussion

• The 2DLC-HDMSE workflow achieved sensitive detection and quantification of HCPs at single-digit ppm levels, matching or exceeding traditional ELISA sensitivity.
• Reproducibility across three laboratories identified 14 common HCPs in the NIST mAb with 1–200 ppm concentrations, demonstrating robust inter-laboratory consistency.
• Quantitative profiles of Remicade and Inflectra revealed two shared HCPs, with higher levels in the biosimilar, indicating potential quality differences.

Benefits and Practical Applications of the Method

This 2DLC-HDMSE approach provides discrete identification and monitoring of individual HCPs rather than total protein burden. Its high peak capacity and orthogonal separations improve coverage of low-abundance impurities, supporting process optimization and biosimilarity assessments in biopharmaceutical development.

Future Trends and Opportunities

Advances may include higher-throughput 2DLC configurations, enhanced ion mobility resolution, integration with artificial intelligence-driven data analysis, and expansion to other biologic modalities. Combining this workflow with targeted assays could further refine impurity monitoring and accelerate regulatory approval.

Conclusion

The inter-laboratory study confirms that the 2DLC-HDMSE platform delivers reproducible, sensitive discovery and quantification of HCPs at sub-ppm levels. This method offers a powerful complement to immunoassays for ensuring biopharmaceutical quality and comparability.

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