Improved method for evaluating the dead volume and protein-protein interactions by self-interaction chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Protein Science

Background:

• Self-interaction chromatography (SIC) is a key technique for analyzing protein-protein interactions.
• Accurate determination of the second virial coefficient in SIC relies on precise evaluation of dead volume.
• Traditional methods for dead volume assessment in SIC present experimental and theoretical challenges.

Purpose of the Study:

• To develop a novel, simplified approach for evaluating dead volume in self-interaction chromatography.
• To enhance the accuracy and accessibility of quantifying protein-protein interactions using SIC.
• To validate the new method by evaluating the second virial coefficient of a monoclonal antibody.

Main Methods:

• A new method utilizing a dextran standard with a size equivalent to the protein of interest was employed to determine dead volume.
• Size exclusion chromatography was used to ascertain the effective size of the protein standard.
• The second virial coefficient was measured for a monoclonal antibody across various buffer conditions.

Main Results:

• The novel dextran standard method effectively determined dead volume, simplifying SIC procedures.
• The evaluated second virial coefficient for the monoclonal antibody showed strong agreement with membrane osmometry data.
• The new approach demonstrated good performance, particularly under conditions dominated by repulsive interactions.

Conclusions:

• The developed method offers a simpler and more accurate way to measure dead volume in self-interaction chromatography.
• This advancement facilitates the routine quantification of protein-protein interactions.
• The technique holds promise for broader application in protein interaction studies.