Multi-variable operational characteristic studies of on-column oxidative protein refolding at high loading...

Area of Science:

• Biochemistry
• Protein Chemistry
• Chromatography

Background:

• Chromatographic protein refolding, particularly Size Exclusion Chromatography (SEC), offers advantages over dilution methods, including higher concentrations and simultaneous purification.
• Previous high-concentration SEC refolding studies (>1mg/mL) were limited to single-factor analyses, hindering optimization and understanding of parameter interactions.

Purpose of the Study:

• To conduct a multi-variable investigation of SEC protein refolding at high concentrations using lysozyme as a model.
• To quantify the interactions of operating parameters and optimize refolding performance.

Main Methods:

• Employed a multi-variable approach to investigate Size Exclusion Chromatography (SEC) for high-concentration protein refolding.
• Utilized lysozyme as a model protein and analyzed the impact of additives like l-arginine and redox couples.

Main Results:

• The addition of l-arginine made refolding yield independent of protein concentration and buffer pH when a redox couple was present.
• l-arginine reduced pore accessibility for small molecules, potentially preventing aggregation at higher protein concentrations.
• Achieved over 90% refolding yield for lysozyme at loading concentrations up to 40mg/mL using optimized conditions.

Conclusions:

• Multi-variable optimization of SEC protein refolding with l-arginine and a redox couple enables high yields at significantly increased protein concentrations.
• This optimized SEC method surpasses previous high-concentration refolding techniques, offering a more efficient approach for protein recovery.