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.
Summary
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.
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.
