Improving the refolding process of rhBMP-2 and development of a novel control strategy for refolding processes

Abstract

Escherichia coli is a very important host cell for the production of biopharmaceuticals. Over the last decades a great amount of knowledge about its biological features has been developed, which makes E.coli an attractive host cell. Proteins are often developed in E.coli in the form of inclusion bodies. Production of inclusion bodies is considered as a promising production strategy because it generates a very pure protein source and high output levels. However, the production of inclusion bodies requires an additional refolding step, which still remains a bottle neck in the downstream process. Previous studies have shown that the redox system is especially important for the refolding of disulphide-bond containing proteins. The system should work under controlled conditions in order to trigger constant outcomes. A relevant control method for refolding is to control the redox potential via dissolved oxygen. In this work we aim at improving the refolding process for the disulphide-bond containing rhBMP-2 (recombinant human bone morphogenetic protein-2), which is an important bone growth factor. Different protein concentrations, redox systems and CHAPS concentrations have been investigated in an univariate study. Best operation conditions were identified at 15mM L-cysteine, at a protein concentration of 80-g/ml and at 20mM CHAPS (51%, 5d). Interestingly, a correlation between a break-down of redox potential and stop of refolding was observed. Based on this observation, a new simultaneous control strategy for redox potential and dissolved oxygen (dO2) was developed. Different redox potential and dO2 set-points were analysed. We found that the set point values -250mV and 10% dO2 caused the most promising results (66%, 44h). Moreover, dO2 and redox controlled and uncontrolled runs were compared, suggesting that under controlled conditions (66%, 44h) higher yields and faster kinetics can be achieved (uncontrolled 51%, 120h). Based on these results, controlled processes for refolding should be promoted. Reproducibility is still immature with this control strategy, so repetitions are needed and conducting the control strategy with other proteins maybe achieve even more efficient outcomes.