CE methods for charge variant analysis of mAbs and complex format biotherapeutics

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biopharmaceutical Analysis

Background:

• Charge heterogeneity in monoclonal antibodies (mAbs) impacts therapeutic efficacy and safety.
• Analyzing complex mAb formats like bispecifics presents unique analytical challenges.
• Existing methods may lack the resolution or scope for comprehensive charge heterogeneity assessment.

Purpose of the Study:

• To develop and optimize capillary electrophoresis (CE)-based methods for analyzing charge heterogeneity in therapeutic mAbs and complex formats.
• To enhance the resolution and analytical scope of capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF).

Main Methods:

• Development of a novel buffer system and optimization of the background electrolyte (BGE) for CZE, including a dynamic coating agent and polymeric additive.
• Optimization of iCIEF using a specific ampholyte combination for a linear pH gradient and investigation of non-denaturing stabilizers.
• Increased BGE pH in CZE to improve separation of high pI and complex mAb variants.

Main Results:

• Optimized CZE method demonstrated enhanced resolution for high pI and complex format mAbs.
• Optimized iCIEF method provided a linear pH gradient suitable for broad mAb analysis.
• Non-detergent sulfobetaine 195 identified as an effective alternative to denaturing stabilizers in iCIEF.

Conclusions:

• The developed CZE and iCIEF methods offer a robust framework for comprehensive charge heterogeneity analysis of therapeutic mAbs.
• These optimized techniques improve the characterization of critical quality attributes for biopharmaceutical development.
• The study provides a foundation for routine charge heterogeneity assessment of diverse mAb formats.