(393e) Study of Protein Refolding in High Concentrations | AIChE

(393e) Study of Protein Refolding in High Concentrations

Authors 

Saremirad, P. - Presenter, Western University



Inclusion Bodies (IBs) production is a widely used method in biopharmaceuticals due to very high expression levels. Large amounts of proteins are trapped in these insoluble aggregates which require solubilisation and purification. IBs are usually solubilized using high concentrations of suitable chaotropic salts and reducing agents 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. The simplest refolding method is batch dilution in which the denaturized protein is diluted in a suitable refolding buffer. However, the correct protein folding pathway often competes with misfolding and aggregation which substantially reduces refolding yields. Furthermore, presences of solubilized aggregates impurities in most biopharmaceutical processes are not acceptable as they may cause health problems in patients. Therefore, batch dilution has serious drawbacks during scale-up due to low product concentration and purity along with large process volumes necessitating additional concentration and purification steps. On-column chromatographic based refolding has shown to address challenges with product dilution by facilitating isolation of protein and chaotropic and reducing 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, it is essential to investigate the system performance in industrially relevant concentrations. In this work, using Lysozyme from chicken egg white as a model protein, the effect of 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) on refolding yield, protein recovery and purity were evaluated and will be discussed in detail.