Analytical characterization of IgG Fc subclass variants through high-resolution separation combined with multiple...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• The increasing use of monoclonal antibodies and IVIg necessitates deeper understanding of human IgG subclasses.
• Effective methods for detailed characterization of IgG subclasses are crucial for pharmaceutical development and research.

Purpose of the Study:

• To develop and validate a comprehensive LC-MS approach for deep characterization of IgG Fc/2 sequence variants and glycans.
• To identify and quantify sequence variants across all human IgG subclasses in complex biological matrices.

Main Methods:

• High-resolution HPLC coupled with intact liquid chromatography-mass spectrometry (LC-MS) for separation and analysis.
• Peptide mapping following sample fractionation to identify amino acid sequence variants.
• Application of the developed method to Intravenous Immunoglobulin (IVIg) for validation.

Main Results:

• Successfully identified and relatively quantified 19 amino acid sequence variants across IgG1, IgG2, IgG3, and IgG4 subclasses.
• Obtained unique glycan information for each IgG subclass, notably for IgG3, which was previously challenging.
• Demonstrated the method's efficacy in characterizing complex samples like IVIg.

Conclusions:

• The developed LC-MS approach provides a powerful tool for in-depth characterization of IgG Fc/2 regions in complex samples.
• The findings offer valuable insights into human IgG subclass heterogeneity and glycan profiles, particularly for IVIg.
• This methodology supports advancements in antibody-based therapeutics and understanding of immune system function.