Critical quality attributes (CQA) are constantly monitored throughout the production process to maintain the quality of therapeutic monoclonal antibody (mAb) products. Core fucosylation of N-glycan structures is a CQA that affects the potency of the mAbs and can be analyzed at subunit level. The following subunit level analysis holds several key advantages over conventional peptide mapping or released N-glycan analyses. It reduces LC-MS run time and provides a simplified workflow. The applications, however, are limited in late stage development and QC, due to the extent of user intervention required. Here, we introduce a fully integrated, workflow-driven, LC-HRMS platform solution with automated data acquisition, processing and reporting capability for core fucosylation assessment.
Two Trastuzumab subunit samples (from different batches, 20 mg/mL) were prepared by digesting with FabRICATOR/IdeS (Genovis, Cambridge, MA, USA) and Endopeptidase S (IgGZERO)/S2 (GlycNATOR, Genovis, Cambridge, MA, USA) enzymes. The samples were analyzed by reversed-phase LC-HRMS. The data acquisition, processing, and reporting were controlled by workflow method generated in UNIFI Scientific Information System.
The enzyme mixture trims the N-glycan structures down to one GlcNAc (with or without an alpha-1,6 Fucose). The reversed-phase separation of the digest showed three ScFc fragments: aglycosylated (no glycans), afucosylated (has one GlcNAc), and fucosylated (one fucose and one GlcNAc). Fucosylated ScFc was the dominant peak and all scFc peaks were detected with less than 5 ppm mass tolerance. Aglycosylated form was observed at less than 1% (relative abundance) with a 10% RSD in repeated measurements. The data was analyzed with generic processing parameters which require minimum adjustments. This methodology can be easily introduced into late stage process development for routine fucosylation/afucosylation CQA monitoring by LC-MS.