Proteoform-Resolved FcɤRIIIa Binding Assay for Fab Glycosylated Monoclonal Antibodies Achieved by Affinity Chromatography Mass Spectrometry of Fc Moieties.

Steffen Lippold,Simone Nicolardi,Manfred Wuhrer,David Falck

doi:10.3389/fchem.2019.00698

Steffen Lippold, Simone Nicolardi + Show 2 more

Open Access

https://doi.org/10.3389/fchem.2019.00698

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Abstract

Fcɤ receptors (FcɤR) mediate key functions in immunological responses. For instance, FcɤRIIIa is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). FcɤRIIIa interacts with the fragment crystallizable (Fc) of immunoglobulin G (IgG). This interaction is known to be highly dependent on IgG Fc glycosylation. Thus, the impact of glycosylation features on this interaction has been investigated in several studies by numerous analytical and biochemical techniques. FcɤRIIIa affinity chromatography (AC) hyphenated to mass spectrometry (MS) is a powerful tool to address co-occurring Fc glycosylation heterogeneity of monoclonal antibodies (mAbs). However, MS analysis of mAbs at the intact level may provide limited proteoform resolution, for example, when additional heterogeneity is present, such as antigen-binding fragment (Fab) glycosylation. Therefore, we investigated middle-up approaches to remove the Fab and performed AC-MS on the IgG Fc to evaluate its utility for FcɤRIIIa affinity assessment compared to intact IgG analysis. We found the protease Kgp to be particularly suitable for a middle-up FcɤRIIIa AC-MS workflow as demonstrated for the Fab glycosylated cetuximab. The complexity of the mass spectra of Kgp digested cetuximab was significantly reduced compared to the intact level while affinity was fully retained. This enabled a reliable assignment and relative quantitation of Fc glycoforms in FcɤRIIIa AC-MS. In conclusion, our workflow allows a functional separation of differentially glycosylated IgG Fc. Consequently, applicability of FcɤRIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.

Highlights

The fragment crystallizable (Fc) of antibodies mediates immunological responses, for example through binding to Fc receptors (Nimmerjahn and Ravetch, 2008; Pincetic et al, 2014)
Fc fragments consisting of paired polypeptide chains were observed rather than single Fc/2 chains which is attributable to non-covalent interactions of the Fc polypeptides (Bern et al, 2018)
In line with our observations for Kgp in FcÈRIIIa affinity chromatography (AC)-mass spectrometry (MS), binding of papain generated Fc was comparable to intact immunoglobulin G (IgG)

Summary

Introduction

The fragment crystallizable (Fc) of antibodies mediates immunological responses, for example through binding to Fc receptors (Nimmerjahn and Ravetch, 2008; Pincetic et al, 2014). Fucosylation of Fc glycans drastically decreases FcÈRIIIa affinity which is attributable to an unique glycan-glycan interaction (Ferrara et al, 2011) Other glycosylation features such as galactosylation were shown to affect the Fc-FcÈRIIIa interaction (Thomann et al, 2015; Dekkers et al, 2017). They are used in the treatment of various diseases, such as cancers or autoimmune diseases (Chan and Carter, 2010; Weiner et al, 2010). Glycoengineering for enhanced FcÈRIIIa affinity and ADCC has been therapeutically exploited (Jefferis, 2009; Beck and Reichert, 2012)

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