2023 FDA Science Forum
Establishment of A Feasible Method to Quantify Major Glycoforms of Human IgG1 mAb Drugs in the Production Media as a Component of Process Analytical Technology
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Contributing OfficeCenter for Drug Evaluation and Research
Abstract
Monoclonal antibody (mAb) drugs constitute a significant portion of all approved therapeutic proteins. IgG1 mAb drugs produced from CHO cells often contain three major glycoforms due to different N-glycosylation at Asn297 residue in their Fc domain: aFucosylated, Fucosylated, and High-mannose. N-glycosylation is a critical quality attribute (CQA) for many IgG1 mAb drugs as it can impact their effector function-related activities, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC), and pharmacokinetics (PK)/pharmacodynamics (PD), and thus needs to be controlled and monitored. A small fraction of IgG1 mAb is not N-glycosylated (non-glycosylated) and consequently lacks effector activity. Mass spectrometry and (U)HPLC are the current tools for analysis of these glycoforms of the purified mAbs, which is not practical for an in-process analytical characterization such as PAT (process analytical technology) during advanced manufacturing. To address the unmet analytical need, we generated two sets of mouse mAbs by immunizing mice with chemically synthesized human IgG1 Fc glycopeptides with different glycoforms-conjugated on KLH as immunogens. Selected mouse mAb-a hybridoma supernatants specifically recognized non-glycosylated human IgG1, and mouse mAb-c specifically bound to glycosylated human IgG1 and differentiated aFucosylated from Fucosylated human IgG1 upon endoglycosidase treatments. The mAb-c requires both sugar-bound Asn297 (Fuc-GlcNAc-Asn or GlcNAc-Asn) and surrounding peptide around the Asn297 for antigenic recognition and binding. Currently, we are further characterizing the purified mouse mAbs (mAb-a and mAb-c) using differently glycosylated human IgG1 mAb drugs, and establishing a Biolayer Interferometry (BLI) method to quantify the major glycoforms of human IgG1 mAb drugs in the production media or unprocessed bulk after treatments with different endoglycosidases: Endo-S, Endo-M, Endo-H, and Endo-F3 that show distinct specificities for their glycoforms. This technology can be potentially developed into a component of PAT. This OPQ-mission relevant project will facilitate development of advanced/continuous manufacturing of IgG1 mAb drugs through identification of glycan-related critical material attributes (CMA) and critical process parameters (CPP).
