2D-CEX-FcRn-MS to Study Structure/Function Relation of mAb Charge Variants

Area of Science:

• Biochemistry and Biophysics
• Analytical Chemistry
• Protein Therapeutics

Background:

• Understanding the relationship between monoclonal antibody (mAb) structure and function is crucial for therapeutic development.
• Assessing charge variants and their impact on neonatal crystallizable fragment receptor (FcRn) binding is essential for mAb efficacy.
• Existing workflows for mAb characterization are often time-consuming and resource-intensive.

Purpose of the Study:

• To develop and validate an automated two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) platform for simultaneous structural and functional analysis of mAb charge variants.
• To elucidate the impact of specific modifications, particularly oxidation, on mAb binding affinity to FcRn.
• To improve the efficiency and reduce the resource requirements of mAb characterization compared to established methods.

Main Methods:

• Utilized a 2D-LC-MS system employing cation-exchange chromatography (CEX) in the first dimension and FcRn affinity chromatography in the second dimension.
• Employed a multiple heart-cutting valve for automated collection and transfer of charge variants between dimensions.
• Incorporated post-column denaturing solution addition to enhance MS sensitivity and utilized multidimensional LC-MS (mD-LC-MS) for detailed structural analysis.

Main Results:

• Successfully resolved and characterized mAb charge variants induced by forced degradation.
• Identified heavy chain (HC) M₂₅₃ and M₄₂₉ oxidation as primary drivers of reduced FcRn binding affinity, with increased oxidation leading to decreased binding.
• Demonstrated a 7-fold improvement in throughput and a significant reduction in material requirements compared to traditional offline methods, with good correlation to ELISA binding data.

Conclusions:

• The automated 2D-LC-MS platform provides a streamlined and efficient approach for multiattribute analysis of mAbs, integrating structural and functional assessments.
• This method enables precise ranking of charge variants based on FcRn binding affinity, highlighting the critical role of oxidation.
• The developed platform accelerates the development of safer and more effective antibody therapeutics by providing rapid, comprehensive mAb characterization.