Heterogeneity of recombinant antibodies: linking structure to function

Area of Science:

• Biochemistry
• Protein Chemistry
• Immunology

Background:

• Recombinant IgG1 antibodies are critical therapeutics but exhibit structural heterogeneity.
• This heterogeneity arises from variations in conserved and unique structural features.
• Common sources include Fc glycosylation, C-terminal Lys processing, methionine oxidation, and hinge-region cleavage.

Purpose of the Study:

• To identify and characterize structural variations in recombinant IgG1 antibodies.
• To investigate the impact of specific modifications on antibody structure and potential function.

Main Methods:

• Utilized cation exchange chromatography and hydrophobic interaction chromatography to detect aspartate isomerization.
• Employed Ellman's reagent to detect unpaired cysteines.
• Applied papain digestion followed by hydrophobic interaction chromatography to resolve thiol-containing Fab fragments.

Main Results:

• Detected aspartate isomerization to iso-aspartate in trastuzumab and omalizumab.
• Identified unpaired cysteines in omalizumab.
• Resolved thiol-containing Fab fragments, indicating structural heterogeneity.

Conclusions:

• Structural variations in recombinant IgG1 antibodies are prevalent and arise from multiple sources.
• Specific modifications like aspartate isomerization and unpaired cysteines can be detected using chromatographic and chemical methods.
• Chromatographic resolution of structural variants suggests potential impacts on antibody potency and efficacy.