Electrophoretic evidence for the presence of structural isoforms specific for the IgG2 isotype

Abstract

Recombinant monoclonal antibodies of therapeutic interest were analyzed by a nonreduced CE-SDS (nrCE-SDS) method developed for the evaluation of size-based variants. We found that immunoglobulins analyzed by this technique exhibited different behavior depending on their subclasses. Under nrCE-SDS conditions, IgG1 molecules were separated in a well-resolved, single peak, whereas IgG2 molecules were consistently separated as a doublet. Investigation of these isoforms showed that they were structurally different, and that the difference was not caused by cell culture condition, glycosylation structure, or recombinant expression system. Commercially available IgG2 affinity-purified from human plasma also showed the presence of structural isoforms. The structural isoforms remained present under pH- and temperature-stressed conditions. Application of a mild cysteine/cystine redox potential converted the main peak doublet into a single peak, indicating that these isoforms were disulfide bond-related species. Bioactivity measured before and after application of a redox potential gave similar values, indicating that the structural isoforms have comparable potency. The nrCE-SDS technique described here demonstrated a unique capability to resolve IgGs, leading to the discovery of novel structural isoforms specific to the IgG2 isotype.

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

“…27 When wild-type IgG2 antibodies were analyzed by CGE, two peaks were found, peak #1 was identified as the IgG2-A and IgG2-A/B isomer and peak #2 was identified as the IgG2-B isomer, which was supported by Lys-C peptide mapping and historical data collected from orthogonal techniques including reverse-phase chromatography and cation exchange chromatography (data not shown).…”

Stability of Protein Pharmaceuticals: An Update

“…The preferred method for detecting and quantifying these disulfide species appears to be capillary electrophoresis (CE) in the presence of SDS (253)(254)(255), although RP-HPLC and LC-MS has also been reported to resolve them as well (128). There appears to be a functional difference associated with placement of the disulfides in IgG2s (256,257).…”

Development, validation, and implementation of capillary gel electrophoresis as a replacement for SDS‐PAGE for purity analysis of IgG2 mAbs

“…This technique is optimized for detection of fragments and it is capable of resolving aglycosylated heavy chain from glycosylated heavy chain, allowing reproducible quantitation for purity and glycosylation occupancy [10]. A recent report has also shown the applicability of CGE for monitoring disulfide isomer heterogeneity [19].…”