Continuous protein refolding and purification by two-stage periodic counter-current chromatography

Area of Science:

• Biotechnology
• Protein Chemistry
• Chromatography

Background:

• Matrix-assisted refolding (MAR) offers advantages over dilution-based methods for protein recovery and buffer efficiency.
• Size exclusion chromatography (SEC) is used for MAR but suffers from dilution and long column requirements in batch mode.
• Continuous processing methods are needed to overcome limitations of batch protein refolding.

Purpose of the Study:

• To develop a continuous matrix-assisted refolding (MAR) process for L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC).
• To enhance protein refolding and purification efficiency through a novel 2-stage sequential PCC process.
• To compare the performance of the developed 2-stage process against existing batch SEC and pulse dilution methods.

Main Methods:

• A modified SEC-PCC operational scheme was developed for continuous MAR of L-asparaginase IBs.
• A 2-stage process involving sequential PCCs with different matrices was implemented for continuous refolding and purification.
• Performance metrics including volumetric productivity, specific buffer consumption, specific activity, and recovery were evaluated and compared.

Main Results:

• The initial SEC-PCC process showed a 6.8-fold increase in volumetric productivity and a 5-fold decrease in specific buffer consumption compared to batch SEC.
• The 2-stage PCC process achieved enhanced specific activity (175-190 IU/mg) and high recovery (84%) for refolded L-asparaginase.
• The 2-stage process demonstrated lower specific buffer consumption than pulse dilution and comparable levels to single-stage IMAC-PCC.

Conclusions:

• The 2-stage continuous PCC process effectively addresses challenges in protein refolding, yielding high-purity, active L-asparaginase.
• This integrated approach offers significant improvements in throughput, recovery, and operational flexibility for protein refolding.
• Seamless integration of the two stages holds potential for further increasing process throughput without compromising refolding quality.