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A Low-temperature SPR-based Assay for Monoclonal Antibody Galactosylation and Fucosylation Assessment Using FcγRIIA/B
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  • Gregory De Crescenzo,
  • Jimmy Gaudreault,
  • Catherine Forest-Nault,
  • Michel Gilbert,
  • Yves Durocher,
  • Olivier Henry
Gregory De Crescenzo
Polytechnique Montreal Departement du Genie Chimique

Corresponding Author:[email protected]

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Jimmy Gaudreault
Polytechnique Montreal Departement du Genie Chimique
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Catherine Forest-Nault
Polytechnique Montreal Departement du Genie Chimique
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Michel Gilbert
National Research Council Canada Human Health Therapeutics Research Centre
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Yves Durocher
National Research Council Canada Montreal - Decelles
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Olivier Henry
Polytechnique Montreal Departement du Genie Chimique
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Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multi-billion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγR), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labelling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5 oC and 25 oC. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviours, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve (AUC) of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (less than 5 minutes per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.
Submitted to Biotechnology and Bioengineering
29 Jan 2024Submission Checks Completed
29 Jan 2024Assigned to Editor
29 Jan 2024Review(s) Completed, Editorial Evaluation Pending
29 Jan 2024Reviewer(s) Assigned
31 Jan 2024Editorial Decision: Accept
14 Feb 2024Published in Biotechnology and Bioengineering. https://doi.org/10.1002/bit.28673