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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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Sensitive RP-LC Method Enabling PTM-Specific Quality Control and MS-Compatible Characterization of Fc-Containing

Jin Xu1,2,3, Anqi Zhou1,2,3, Mengjiao Xu1,2,3

  • 1State Key Laboratory of Macromolecular Drugs and Large-Scale Preparation, School of Pharmaceutical Sciences, Cixi Biomedical Research Institute, Wenzhou Medical University, Wenzhou 325035, China.

Journal of the American Society for Mass Spectrometry
|May 6, 2026
PubMed
Summary
This summary is machine-generated.

A new reverse-phase liquid chromatography (RP-LC) method rapidly profiles post-translational modifications (PTMs) in Fc-containing GLP-1 therapeutics. This advanced analytical technique enhances quality control and process analytical technology for biologics development.

Keywords:
GLP-1-Fc therapeuticsaccuratecomprehensive (RAC) platformrapidreverse-phase chromatography

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Area of Science:

  • Biopharmaceutical Analysis
  • Analytical Chemistry
  • Protein Chemistry

Background:

  • Fc-containing GLP-1 therapeutics display complex post-translational modification (PTM) heterogeneity.
  • Existing analytical methods struggle with comprehensive PTM profiling for quality control (QC) and process analytical technology (PAT).

Purpose of the Study:

  • To develop and validate a rapid, accurate, and comprehensive (RAC) RP-LC method for PTM-specific profiling of Fc-GLP-1 biologics.
  • To enable advanced QC and support PAT implementation for biosimilar and next-generation therapeutic development.

Main Methods:

  • Developed an optimized RP-LC method using dulaglutide (IgG4-Fc) as a model, focusing on mobile-phase additives, acid concentration, and shallow gradients.
  • Incorporated difluoroacetic acid (DFA) for mass spectrometry (MS) compatibility, enabling intact-mass characterization of PTM variants.
  • Validated the method's applicability across different Fc subtypes (IgG4 and IgG2) using supaglutide.

Main Results:

  • Successfully resolved critical PTM variants including hydroxylation, N-terminal truncation, disulfide reduction, and glycosylation within 40 minutes.
  • Identified additional PTMs, such as site-specific O-glycosylation and process-dependent truncations, through MS compatibility.
  • Demonstrated direct quantification of impurities and detection of process-induced variants across biosimilar clones, with broad applicability across Fc subtypes.

Conclusions:

  • The developed MS-compatible RP-LC platform offers a robust solution for PTM-specific QC of Fc-GLP-1 therapeutics.
  • This method accelerates biosimilar development and facilitates the implementation of PAT for next-generation biologics.
  • The RAC approach provides comprehensive characterization crucial for biopharmaceutical quality assurance.