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Updated: Aug 6, 2026
Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Published on: January 8, 2014
Aglycosylated Immunoglobulin G1 Fc Stabilized Through Disulfide Bond Addition Exhibits Compositional Homogeneity and
Anjali Shenoy1, Daniel J Falconer2, Adam W Barb1,3,4
1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Background:
The interaction between human immunoglobulin G (IgG)1 Fc and the Fc gamma receptor (FcγR) IIIa/CD16a elicits protective immune responses. Antibody N-glycosylation stabilizes the FcγR-binding interface and is thus essential for interaction with wildtype IgG1 Fc. Furthermore, the N-glycan introduces substantial compositional and functional heterogeneity, with distinct glycoforms providing different affinities and discrete responses in vivo. Accordingly, various engineering endeavors to improve antibody binding strive to boost the therapeutic efficacy of monoclonal antibodies but do not directly address compositional heterogeneity.
Objective:
Here, we describe a previously unexplored approach to engineer IgG1 Fc. We eliminated carbohydrate heterogeneity by removing the N-glycan but stabilizing the FcγR-binding interface with disulfide bonds.
Conclusions:
These newly generated Fc domains served as a starting point for protein engineering through yeast surface display to enhance receptor-binding affinity. We recovered Fc variants from this approach that demonstrated FcγRIIIa binding affinities comparable to the starting sequence and thus serve as a proof-of-principle for this strategy.
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