Capillary gel electrophoresis for precise protein quantitation

Area of Science:

• Pharmaceutical Analysis
• Biopharmaceutical Quality Control
• Separation Science

Background:

• Capillary Gel Electrophoresis (CGE), also known as SDS-CGE, is emerging as a replacement for SDS-PAGE in the pharmaceutical industry.
• Current applications focus on identity and purity control of proteins, such as monoclonal antibodies.
• Accurate quantification with a relative standard deviation (RSD) below 2% is essential for quality control.

Purpose of the Study:

• To establish a reliable and highly precise CGE method for quantitative protein analysis.
• To optimize CGE parameters to meet the stringent precision requirements for pharmaceutical quality control.
• To compare different injection modes and investigate the use of internal standards for improved accuracy.

Main Methods:

• Optimization of CGE parameters, including sample concentration and injection volume, to achieve high signal-to-noise ratios (>70).
• Comparison of hydrodynamic injection versus electrokinetic injection for precision in long series runs (n=48).
• Investigation of internal standards for migration time and peak area normalization.

Main Results:

• Hydrodynamic injection demonstrated superior precision compared to electrokinetic injection.
• The use of an internal standard reduced migration time RSD from 0.9% to 0.2% and improved peak area RSD.
• Normalization to total area further minimized injection errors, yielding peak area ratio RSDs between 1% and 2%.

Conclusions:

• A thoroughly optimized CGE method can achieve the required precision (<2% RSD) for quantitative protein analysis in pharmaceutical settings.
• Hydrodynamic injection and internal standard normalization are key strategies for enhancing method robustness and accuracy.
• The developed CGE method is suitable for identity, purity, and quantitative control of proteins like monoclonal antibodies.