Native fluorescence detection with a laser driven light source for protein analysis in capillary electrophoresis

Area of Science:

• Analytical Chemistry
• Biochemistry
• Biotechnology

Background:

• Ultraviolet (UV) absorbance detection in capillary electrophoresis (CE) is common but suffers from low concentration sensitivity and baseline fluctuations.
• Alternative detection methods are needed to improve sensitivity and reduce noise for protein analysis.

Purpose of the Study:

• To develop and characterize a native fluorescence detection (NFD) scheme for protein analysis using a commercial CE platform (PA 800 Plus).
• To compare the performance of CE-NFD with traditional UV absorbance detection in SDS-CGE and cIEF for protein analysis.

Main Methods:

• Implemented a laser-driven light source for excitation in a native fluorescence detection (NFD) setup on a commercial CE system.
• Characterized the CE-NFD system using tryptophan and a reduced immunoglobulin G (IgG) standard.
• Compared CE-NFD performance against UV absorbance detection in sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (cIEF).

Main Results:

• CE-NFD achieved a 14.6-fold higher signal-to-noise ratio for non-reduced IgG in SDS-CGE compared to UV detection at 214 nm.
• CE-NFD required approximately 170 times less sample mass for comparable profile quality in cIEF analysis of NISTmAb (Humanized IgG1κ) versus UV detection at 280 nm.
• CE-NFD eliminated baseline anomalies and reduced interference from other absorbing species observed with UV detection.

Conclusions:

• Native fluorescence detection (NFD) significantly enhances sensitivity and reduces baseline noise in capillary electrophoresis (CE) for protein analysis.
• CE-NFD offers a practical and powerful alternative to UV absorbance detection for protein characterization in the biopharmaceutical industry.
• The developed CE-NFD method provides improved performance for SDS-CGE and cIEF applications.