Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein separation

Area of Science:

• Chromatography
• Analytical Chemistry
• Biochemistry

Background:

• Intact protein separation is crucial in biopharmaceutical analysis.
• Existing chromatographic methods may lack the selectivity and sensitivity required for complex protein mixtures.
• Development of novel stationary phases is essential for advancing protein separation technologies.

Purpose of the Study:

• To develop and evaluate a novel mixed-mode stationary phase (C8PN) for intact protein separation.
• To characterize the chromatographic properties of the C8PN phase, including reversed-phase and charged-repulsion interactions.
• To assess the performance of C8PN compared to commercial stationary phases and its application potential.

Main Methods:

• Synthesis and characterization of the C8PN mixed-mode stationary phase.
• Evaluation of chromatographic properties using probe compounds.
• Separation of a standard protein mixture using reversed-phase mode.
• Investigation of mobile phase composition (acetonitrile content) and buffer salt effects.
• Application to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone.

Main Results:

• The C8PN phase demonstrated effective reversed-phase and positively charged repulsion interactions.
• C8PN exhibited different selectivity for proteins compared to a commercial C4 stationary phase.
• Protein retention was highly sensitive to acetonitrile content, as indicated by retention equation parameters.
• Increased buffer salt concentration enhanced protein retention, confirming charged-repulsion effects.
• Successful separation of oxidized- and reduced- forms of Recombinant Human Growth Hormone was achieved.

Conclusions:

• The developed C8PN mixed-mode stationary phase offers unique selectivity for intact protein separation.
• The phase shows significant potential for analyzing complex protein samples, including post-translational modifications.
• This advancement contributes to improved methods for protein characterization in biopharmaceutical research and development.