2023 FDA Science Forum
The Big Protein Project: Development and Assessment of Modern Protein Glycosylation Characterization Techniques
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Contributing OfficeCenter for Drug Evaluation and Research
Abstract
Analytical characterization of the structure and chemistry of monoclonal antibodies (mAb) in a formulation is critical for the purpose of quality assurance. In particular, glycosylation – the enzymatic attachment of oligosaccharides to the protein backbone - is a critical quality attribute (CQA) that must be well characterized and controlled before regulatory approval because different glycans can impact the safety and efficacy of mAbs and other therapeutic proteins. Glycans can differ in monosaccharide composition, branching, and connectivity which can make analysis challenging. The conventional workflow for N-glycan analysis is to enzymatically release them from the protein then label the reducing end aldehyde with a fluorophore, a time- and resource-intensive process.
Glycans can then be separated by hydrophilic interaction chromatography and detected and quantified with fluorescent detection (HILIC-FLD). The FDA’s Office of Testing and Research (OTR) initiated the Big Protein Project (BPP) to develop a panel of parallel analytical procedures to characterize therapeutic protein drug products that can be done more efficiently than historical methods without sacrificing specificity or sensitivity. For glycan characterization, the BPP focused on high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques using state-of-the-art instrumentation along with conventional HLIC-FLD quantitation.
In this study, analytical characterization was performed on multiple lots of rituximab from two sources, one approved and one not approved for the US marketplace by the FDA. Multiple orthogonal analytical techniques were performed including analysis of released glycans by HILIC-FLD, liquid chromatography (LC)-MS-based multi-attribute method (MAM), intact-mass LC-MS, and NMR. Individual glycoforms detected by MS and HILIC-FLD were compared directly while NMR results quantified classes of CQAs (afucosylation, galactosylation and high-mannose). While each method presented advantages and disadvantages, there was concurrence between methods in the identified lot-to-lot and manufacturer-based differences. Results herein demonstrate how agreement can be found between conventional and state-of-the-art analytical methods; these high-resolution methods can provide increased confidence in comparative analytical studies and provide flexibility in method selection based on the needs and expertise of individual researchers.
