Abstract
The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity.
Highlights
Glycosylation of therapeutic proteins is crucial for their biological activity as has been previously identified [1]
Preparation and analysis of molecular integrity of different glycan variants Starting from one single batch of IgG1, different glycan variants were produced by in vitro glycoengineering (IVGE) as described in the Materials and Methods section
Four glycan variants were produced from the starting material: A hypo-galactosylated variant, i.e. IgG1 that comprises predominantly G0F Fc glycan species, a hyper-galactosylated variant, a mono-sialylated variant and a di-sialylated variant
Summary
Glycosylation of therapeutic proteins is crucial for their biological activity as has been previously identified [1]. Variability in glycan patterns based on manufacturing variability was described for marketed antibody products [4, 5]. This variability might be even more pronounced during development of monoclonal antibodies based on multiple changes implemented during process optimization. The impact of non-fucosylated complex type Fc glycans on the effector function of monoclonal antibodies has been shown in different publications [6,7,8,9]. Several reports conclude that different galactosylation levels do not influence ADCC activity [10,11,12]. Positive correlation between galactosylation and FcγRIIIa binding has been observed in multiple studies [13, 14]
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