Performance Characteristics of Commercially Available Gels for Protein Analysis by Capillary Gel Electrophoresis with UV Detection

Importance of the Topic

This study evaluates commercially available separation matrices for capillary gel electrophoresis (CGE) with UV detection, a key technique for protein size analysis and quality control in biotechnology. CGE offers automated handling, high reproducibility and resolution, making it essential for therapeutic protein characterization such as monoclonal antibodies.

Aims and Study Overview

The comparison focuses on two dextran-based gels, Beckman Coulter’s SDS Gel Buffer and Advanced Analytical’s Protigel, using an Agilent 7100 CE system. Two sets of samples were analyzed: a protein size standard with BSA for molecular weight determination, and a reduced antibody standard for quantification of IgG subunits. Additional impurity detection experiments tested sensitivity to low molecular weight proteins.

Methodology

The study employed two protocols:

• Beckman Coulter SDS Gel Buffer: 50 μm i.d. capillary, 33 cm total length, preconditioning with NaOH, HCl, water, and gel buffer; injection at –5 kV; separation at –16.5 kV with pressure support; detection at 220 nm.
• Advanced Analytical Protigel A and P: 75 μm i.d. capillary, identical length; preconditioning with Protigel solution and voltage equilibration; injection at –5 kV; separation at –10 kV with pressure support; detection at 200 nm.

Used Instrumentation

• Agilent 7100 Capillary Electrophoresis system
• 50 μm and 75 μm bare fused silica capillaries
• UV detector set at 200–220 nm

Main Results and Discussion

Both gels achieved high precision: relative migration time repeatability below 0.2% RSD and BSA molecular weight accuracy within ±10%. The Beckman Coulter gel provided slightly better resolution of heavy chain variants, while both gels showed comparable peak area repeatability (≤2% RSD). Impurity detection down to 0.1% β-lactoglobulin spike in non-reduced IgG was successful with both matrices.

Benefits and Practical Applications

The evaluated gels are well suited for routine CGE analysis in quality control environments. Their high reproducibility and resolution support accurate molecular weight determination, antibody subunit quantification, and low-level impurity detection, essential for biopharmaceutical development and manufacturing.

Future Trends and Applications

Advancements may include integration of mass spectrometry detection, development of novel polymer matrices for enhanced resolution, and microchip-based CGE platforms for faster analysis. These trends will expand CGE applications in proteomics, biomarker discovery, and process monitoring.

Conclusion

The comparison demonstrates that both commercial gels deliver reliable and precise performance for protein analysis by CGE with UV detection on an Agilent 7100 system. Users can select either matrix based on resolution needs, while achieving consistent results across capillary and gel batches.