pH-gradient cation exchange purification of IgG2 disulfide isoforms

Area of Science:

• Biochemistry
• Protein Chemistry
• Biopharmaceutical Development

Background:

• Immunoglobulin (IgG) therapies are vital for treating various diseases.
• The IgG2 subclass exhibits variable disulfide bond connectivity in its hinge region, creating distinct isoforms.
• These isoforms, particularly interchain disulfide bonding variations, can influence the Fab arm structure and ultimately affect therapeutic potency.

Purpose of the Study:

• To develop and demonstrate a novel purification method for native IgG2 disulfide isoforms.
• To analyze the abundance and impact of IgG2 isoforms on therapeutic antibody function.
• To address limitations of existing purification techniques for specific IgG2 monoclonal antibodies (mAbs).

Main Methods:

• A pH-gradient cation exchange separation was developed for native purification of IgG2 disulfide isoforms.
• High-resolution denaturing reversed-phase chromatography was used for isoform content analysis.
• Collected fractions were combined to create solutions with controlled IgG2-B isoform percentages.

Main Results:

• The developed method successfully purified IgG2 disulfide isoforms for an mAb not amenable to previous salt-gradient methods.
• Isoform content was accurately determined using fluorescence detection.
• Generated solutions spanned a range of 20.3% to 80.8% IgG2-B isoform, with consistent product quality and potency assay suitability.

Conclusions:

• A novel pH-gradient cation exchange method enables native fractionation of IgG2 disulfide isoforms.
• This method is effective for IgG2 mAbs resistant to prior purification techniques.
• Understanding and controlling IgG2 isoform abundance is crucial for developing effective IgG2-based therapeutics.