Abstract
The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure–function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.
Highlights
Glyco-engineering of the antibody fragment crystallizable (Fc) has improved the immune effector function of direct-targeting therapeutic antibodies (Ab) for almost a decade
With the developed approach described, we demonstrate the applicability of the hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) technique to monitor relevant structural changes using enzymatically altered Fc glycosylations, created by the previously introduced, in vitro glyco-engineering (IVGE) technology [23]
We recently presented a targeted Hydrogen/deuterium exchange (H/DX)-MS approach to monitor minor structural alterations, wherein reliable assignments of H/DX differences resulting from methionine oxidation were demonstrated
Summary
Glyco-engineering of the antibody fragment crystallizable (Fc) has improved the immune effector function of direct-targeting therapeutic antibodies (Ab) for almost a decade. Mogamulizumab (Poteligeo® , Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) is a humanized anti-CC motif chemokine receptor 4 (CCR4) immunoglobulin G1 (IgG1) derived from Chinese hamster ovary (CHO) cells [1]. It was followed by Roche’s anti-CD20 obinutuzumab (Gazyva® ) in 2013. An enhanced antibody-dependent cellular cytotoxicity (ADCC) of up to 100-fold compared to its fucosylated counterpart, was the rationale for this Fc engineering milestone [2].
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