Effect of sample loop dimension on lysozyme refolding in size-exclusion chromatography

Area of Science:

• Biotechnology
• Protein Chemistry
• Chromatography

Background:

• Misfolded protein aggregates, particularly inclusion bodies, significantly limit protein refolding yields.
• Size-exclusion chromatography (SEC) offers simultaneous refolding and separation, making it a promising buffer exchange method for enhancing protein refolding.
• Optimal operating parameters for SEC-based protein refolding remain incompletely understood.

Purpose of the Study:

• To investigate the impact of sample loop dimensions (diameter and length) on aggregate formation and renaturation yield during SEC refolding.
• To identify critical process parameters influencing protein aggregate formation and refolding efficiency.

Main Methods:

• Size-exclusion chromatography (SEC) was employed for protein refolding.
• Experiments varied sample loop dimensions (contraction, expansion, control) and evaluated aggregate formation and renaturation yield.
• The influence of injection volume and protein concentration was also assessed.

Main Results:

• Aggregate formation patterns showed minimal differences between contraction and control sample loops.
• Expansion cases exhibited an additional peak, suggesting altered aggregate behavior.
• Aggregate formation was independent of sample loop dimensions but significantly influenced by injection volume and protein concentration.

Conclusions:

• Reducing aggregate formation and enhancing renaturation yield in SEC refolding is primarily dependent on optimizing sample volume and concentration.
• A strategy involving large sample volumes and low protein concentrations is recommended for improved refolding outcomes.
• These findings provide insights for developing more effective protein refolding strategies.