Open AccessRapid screening of IgG quality attributes – effects on Fc receptor binding
Author(s) -
Geuijen Karin P. M.,
OppersTiemissen Cindy,
Egging David F.,
Simons Peter J.,
Boon Louis,
Schasfoort Richard B. M.,
Eppink Michel H. M.
Publication year - 2017
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.12283
Subject(s) - deamidation , neonatal fc receptor , receptor , antibody , chemistry , immunoglobulin fc fragments , fragment crystallizable region , biochemistry , immunoglobulin g , monoclonal antibody , in vivo , binding site , fc receptor , receptor–ligand kinetics , microbiology and biotechnology , biology , immunology , enzyme
The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (Fcγ RI , Fcγ RII a, Fcγ RII b, Fcγ RIII a, and Fcγ RIII b) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo . Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (Fcγ RII a, Fcγ RII b, Fcγ RIII a, and Fcγ RIII b), while Fcγ RI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to Fcγ RIII a and Fcγ RIII b. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.