Comprehensive Characterization of IgG2 Disulfide Isoforms Using Native Cation Exchange Chromatography-Mass...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Immunoglobulin G2 (IgG2) antibodies possess complex disulfide bond connectivity in their hinge region, leading to three distinct isoforms (A, A/B, B).
• Conventional structural elucidation methods for IgG2 isoforms, such as reversed-phase chromatography or capillary electrophoresis, often require denaturing conditions, limiting comprehensive analysis.
• Accurate characterization of IgG2 disulfide isoforms is crucial for understanding antibody structure-function relationships and ensuring therapeutic efficacy.

Purpose of the Study:

• To develop and validate a native cation exchange chromatography-mass spectrometry (CEX-MS) method for the separation and characterization of IgG2 disulfide isoforms.
• To apply this method to analyze IgG2 F(ab')2 fragments, enabling high-quality mass spectrometry (MS) analysis while mitigating Fc-related heterogeneity.
• To investigate IgG2 disulfide bond rearrangements and elucidate the structural basis of an IgG2 mutant with enhanced agonistic activity.

Main Methods:

• Development of a native CEX-MS method utilizing volatile salts for IgG2 disulfide isoform separation.
• Application of a middle-up strategy focusing on F(ab')2 fragments to localize and analyze disulfide bond rearrangements.
• Implementation of redox treatment and site-directed mutagenesis to determine the elution order of IgG2 disulfide isoforms.
• Optimization of nonreduced peptide mapping with an isotope envelope confidence score to enhance peptide identification and analyze interchain disulfide patterns.

Main Results:

• Successful separation and characterization of IgG2 disulfide isoforms using native CEX-MS.
• Demonstration of the method's ability to analyze IgG2 F(ab')2 fragments, yielding optimal chromatographic separation and high-quality MS spectra.
• Elucidation of the elution order of IgG2 disulfide isoforms through redox treatment and mutagenesis.
• Characterization of an IgG2 mutant exhibiting a pseudoisoform B structure stabilized by noncovalent interactions, linked to agonistic activity.
• Development of an optimized peptide mapping strategy that enhances the identification of complex hinge-related peptides.

Conclusions:

• Native CEX-MS is a powerful tool for the structural characterization of IgG2 disulfide isoforms, overcoming limitations of denaturing methods.
• The middle-up strategy focusing on F(ab')2 fragments is effective for analyzing IgG2 hinge region heterogeneity.
• The developed methods provide new insights into IgG2 structure, including the characterization of functionally relevant mutants and complex disulfide linkages.