Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve.

Area of Science:

• Biopharmaceutical analysis
• Protein characterization
• Analytical chemistry

Background:

• Capillary electrophoresis with SDS (CE(SDS)) is widely used for analyzing antibody (mAb) size heterogeneity.
• Accurate identification of protein fragments in CE(SDS) remains a challenge.
• Previous 2D CE systems offered online mass spectrometry but required improvements in stability and handling.

Purpose of the Study:

• To develop a more robust and user-friendly 2D CE system for mAb impurity characterization.
• To enhance the identification of protein fragments using capillary electrophoresis coupled with mass spectrometry.
• To simplify the decomplexation strategy in 2D CE-SDS-CZE-MS.

Main Methods:

• Introduction of a novel 8-port valve with four sample loops for improved separation.
• Optimization of a simplified decomplexation strategy using solvent and cationic surfactant injection.
• Testing different solvent and surfactant volumes to maximize removal efficiency.
• 2D measurements of reduced NIST mAb light and heavy chains using the new system.

Main Results:

• The novel 8-port valve and optimized strategy significantly increased system robustness.
• The simplified decomplexation strategy enabled efficient analysis without additional detectors.
• Successful 2D measurements demonstrated the system's capability for analyzing mAb fragments.
• The improved system facilitates routine application for protein impurity characterization.

Conclusions:

• The developed 8-port valve system and optimized decomplexation strategy enhance the robustness of CE(SDS)-CZE-MS.
• This advancement simplifies the analysis of antibody size heterogeneity and protein fragments.
• The novel setup represents a significant step towards routine biopharmaceutical impurity characterization using 2D CE-MS.