A Low-temperature SPR-based Assay for Monoclonal Antibody
Galactosylation and Fucosylation Assessment Using FcγRIIA/B
Abstract
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.