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An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry
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High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for

Iain D G Campuzano1, Emma M Pelegri-O'Day1, Nithya Srinivasan1

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|October 7, 2022
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Summary

A novel high-throughput solid-phase extraction mass spectrometry (HT-SPE-MS) method analyzes 96 samples in under 45 minutes. This accelerates biotherapeutic characterization, including monoclonal antibodies and nucleic acids, significantly faster than traditional methods.

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Area of Science:

  • Biopharmaceutical Analysis
  • Mass Spectrometry
  • Analytical Chemistry

Background:

  • Reversed-phase liquid chromatography-mass spectrometry (rpLC-MS) is crucial for pharmaceutical research but faces throughput limitations.
  • Increasing complexity of biotherapeutics like monoclonal antibodies (mAbs) and bispecific antibodies (BsAbs) necessitates faster analytical methods.
  • Emerging modalities like bispecific T-cell engagers and nucleic acid therapeutics require robust MS characterization.

Purpose of the Study:

  • To develop a modality and target agnostic high-throughput mass spectrometry (HT-MS) method.
  • To significantly reduce analysis time for large sample sets compared to traditional rpLC-MS.
  • To enable comprehensive characterization of diverse biotherapeutic species.

Main Methods:

  • Implementation of a high-throughput solid-phase extraction coupled with mass spectrometry (HT-SPE-MS) workflow.
  • Analysis of a 96-well plate using the developed HT-SPE-MS method.
  • Comparison of run times against traditional reversed-phase liquid chromatography-mass spectrometry (rpLC-MS) methods.

Main Results:

  • The HT-SPE-MS method achieved analysis of a 96-well plate in 41.4 minutes, a substantial improvement over the 14.4 hours required by traditional rpLC-MS.
  • The method accurately determined molecular weights for monodispersed and polydispersed biotherapeutics and membrane proteins.
  • Characterization capabilities include quantification of glycosylation, glycation, formylation, detection of chain mispairing, and accurate drug-to-antibody ratio (DAR) determination.

Conclusions:

  • The developed HT-SPE-MS method offers a significant advancement in analytical throughput for biopharmaceutical research.
  • This agnostic approach supports the characterization of a wide range of therapeutic modalities, accelerating drug development.
  • The method's speed and comprehensive analytical power are essential for handling evolving and expanding biotherapeutic pipelines.