(402c) Operational Characteristic Studies of Protein Refolding in Size Exclusion Chromatography

Proteins are one of the most important biological compounds and many of them are used as drugs beneficial to human health. Proteins produced by recombinant DNA technology using bacterial hosts often form inactive aggregates called inclusion bodies. Large amounts of proteins are trapped in these insoluble aggregates which require solubilisation and purification. The solubilisation is possible through the introduction of Denaturising and Reducing agents (D&R) to unfold the protein chain. However, the refolding of proteins into their compact structures is critical to confer biological activity on proteins allowing them to function as effective therapeutics. Due to simplicity in design and operation, batch dilution refolding is commonly practiced in laboratories and industry. However, the correct protein folding pathway often competes with misfolding and aggregation which substantially reduces refolding yields. Furthermore, the presence of aggregates in the final product as impurities has many health concerns. Therefore, though ideal at laboratory scale, this technique has serious drawbacks during scale-up due to low product concentration and purity along with large process volumes necessitating additional post-refolding cost-intensive concentration and purification steps. Chromatographic based refolding has shown to address challenges with product dilution by facilitating spatial isolation of protein and D&R molecules while simultaneously purifying the protein after refolding. Since chromatographic protein refolding allows for significantly higher protein concentrations without drastic loss of refolding yield, compared to batch dilution, a multi-variable investigation of the system performance in industrially relevant concentrations is an essential step for optimization and intensification. In this work, using Lysozyme from chicken egg white as a model protein (129 amino acids, molecular weight of 14,307gmol^-1, four disulfide bonds and theoretical isoelectric point of 11.35), the effect of operating parameters on refolding yield, protein recovery and purity were evaluated. The parameters studied were protein concentration (20-40 mgml^-1) and refolding buffer composition including its pH (8.1-9.5), ionic strength (0.2M NaCl) and Arginine presence as aggregation prevention additive (0.2M). As expected, refolding buffer pH and protein concentration played an important role in refolding yield. In the tested range, pH further away from the protein isoelectric point (8.1) and low protein concentration (20mgml^-1) resulted in higher refolding yield. However, in the presence of Arginine, system performance was different which will be discussed in detail. Addition of sodium chloride resulted in considerable protein precipitation in column and flow blockage in high protein concentration. This observation is in contrast to Lysozyme refolding in lower concentrations, where the same type and concentration of salt is recommended to decrease the self and non-specific interactions.