Factors affecting protein refolding yields in a fed-batch and batch-refolding system.

Area of Science:

• Biotechnology
• Protein Engineering
• Bioprocess Engineering

Background:

• Recombinant protein production in Escherichia coli often results in inclusion bodies, necessitating refolding.
• Protein aggregation during dilution of denatured forms limits yields at industrial scales.
• Optimizing refolding conditions is crucial for efficient biopharmaceutical manufacturing.

Purpose of the Study:

• To investigate the impact of five key factors on the dilution refolding of recombinant proteins in a fed-batch system.
• To identify critical parameters and their interactions influencing refolding yields.
• To compare fed-batch refolding with batch-refolding under varying mixing conditions.

Main Methods:

• A 2(5) factorial experiment was employed using lysozyme as a model system.
• Factors studied included impeller Reynolds numbers, injection rate, redox ratio, and guanidine hydrochloride (GdHCl) concentration.
• Comparative assessment of fed-batch and batch refolding modes was performed.

Main Results:

• Guanidine hydrochloride concentration, redox ratio, and injection rate were identified as the most significant factors affecting refolding yields.
• Key interactions were observed between redox ratio/GdHCl concentration and GdHCl concentration/injection rate.
• High refolding yields were achieved under specific conditions, even with rapid injection and reduced mixing.

Conclusions:

• Fed-batch refolding parameters, particularly GdHCl concentration and redox ratio, are critical for maximizing recombinant protein yields.
• Optimal refolding in batch mode occurs at high GdHCl concentrations (1.2 M) and redox ratios of unity or greater.
• Understanding these parameters enables the development of more efficient protein refolding processes.