Imaged capillary isoelectric focusing in native condition: A novel and successful example

Area of Science:

• Biochemistry
• Analytical Chemistry
• Protein Science

Background:

• Imaged capillary isoelectric focusing (icIEF) is crucial for protein charge variant analysis.
• Denaturants in icIEF matrices can hinder separation for certain proteins, like aggregation-prone antibodies.
• There is a need for icIEF methods that maintain protein integrity.

Purpose of the Study:

• To develop a novel icIEF matrix that avoids denaturants.
• To improve assay robustness, peak resolution, and accuracy for challenging proteins.
• To enable native protein charge variant analysis using icIEF.

Main Methods:

• Development of a new matrix formula: non-detergent sulfobetaine and taurine (NDSB-T).
• Application of NDSB-T in imaged capillary isoelectric focusing (icIEF).
• Evaluation of NDSB-T's performance with an aggregation-prone antibody.

Main Results:

• The NDSB-T matrix significantly improved assay robustness and peak resolution for the target antibody.
• Assay throughput increased over 10-fold with enhanced accuracy.
• NDSB-T enabled separation and quantification of protein charge species in their native state, avoiding denaturation artifacts.

Conclusions:

• NDSB-T is a denaturant-free matrix effective for proteins incompatible with standard icIEF conditions.
• This novel formulation enhances icIEF assay performance and accuracy.
• NDSB-T facilitates native protein analysis, potentially expanding icIEF applications.