Protein refolding methods for biomanufacturing

Abstract

Interferon alpha 2b (IFN-a2b) is a soluble cytokine and a member of the type I interferon (IFN) family of which there are fourteen members. IFN-a2b is used in the treatment of hairy cell leukaemia and both hepatitis B and C. IFN-a2b actions are mediated through specific receptor binding which initiates a series of signalling cascades leading to the transcription of genes carrying the promoters containing the interferon stimulated response element (ISRE). Due to these actions the IFNs as a whole are an ideal family of proteins to be investigated further with the possibility of the production of life saving drug products. Cobra Biomanufacturing have a keen interest in IFN-a2b especially due to its beneficial effects. This project is therefore an investigation of the IFN-a2b molecule from expression to final purified product. The refolding of the IFN-a2b was the main concern of this project due to it being the `bottleneck' for many protein based processes. The investigation of natively folded IFN-a2b led to the conclusion that IFN-a2b is a stable protein which remains folded under reducing conditions. The addition of guanidine-HCI or urea to the native protein reveals differences in the success of unfolding. These differences were also witnessed during the solubilisation of the IFNa2b inclusion bodies. The refolding is only successful when the inclusion bodies have been solubilised in guanidine-HCI. Due to the differing refolding results gained, which depended on the denaturant used to solubilise the inclusion bodies, the solubilised inclusion bodies themselves were investigated. This revealed partial structure present in the urea solubilised inclusion bodies; however this was confirmed as being not native. The implications of this for biomanufacturing are that if the starting conditions are not correct even as early as the solubilisation of the inclusion bodies then the following process will result in incorrectly folded product.