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.
Abstract:
Fc-containing GLP-1 therapeutics exhibit complex post-translational modification (PTM) heterogeneity, necessitating advanced analytical methods for quality control (QC) and process analytical technology (PAT). We developed a reverse-phase liquid chromatography (RP-LC) method for the PTM-specific profiling of these biologics. Using dulaglutide (IgG4-Fc) as a model, critical parameters─including mobile-phase additives, acid concentration, and shallow gradients─were optimized to resolve PTM variants (e.g., hydroxylation, N-terminal truncation, disulfide reduction, glycosylation) within 40 min. Mass spectrometry (MS) compatibility was enabled by adopting difluoroacetic acid (DFA) as an alternative ion-pairing reagent to support intact-mass characterization of variants. This enabled the identification of additional PTMs not readily resolved under the initial RP-LC conditions, including site-specific HyK-Gal-Glc O-glycosylation and process-dependent truncations. The method also allowed for the direct quantification of critical impurities and the detection of process-induced variants across biosimilar clones. The method was further demonstrated on the IgG2-subtype GLP-1-Fc-fusion (supaglutide), showing applicability across the two Fc subtypes examined without additional optimization. This robust, MS-compatible RP-LC platform provides a rapid, accurate, and comprehensive (RAC) means of conducting PTM-specific QC for Fc-GLP-1 therapeutics, supporting PAT implementation and accelerating biosimilar and next-generation drug development.
