Optimization of PEGylation condition of recombinant Interferon beta-1a

Abstract

Interferon beta-1a is a single-chain glycosylated protein with a molecular weight of 22.5 kDa. Injection of this drug slows down the progression of disease in relapsing Multiple Sclerosis patients. Due to its high clearance, it needs to be used 3-4 days a week, which may cause many side effects. PEGylation is the covalent conjugation of active polyethylene glycol (PEG) to a specific site of the protein, so by PEGylation which will result in the size increase, it is possible to decrease the injection frequency. In this study, N-terminal PEGylation of IFNβ-1a with 20 kDa mPEG-aldehyde was studied and the results were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aim:The aim of this study is the optimization of the PEGylation condition of IFNβ-1a including reaction temperature, duration of incubation, reaction pH, and PEG-to-protein molar ratio.Methods:N-terminal PEGylation of IFNβ-1a was done by mPEG-aldehyde. PEGylation profile was studied on 10% gels using SDS-PAGE and followed by silver staining. Results:IFNβ-1a has successfully PEGylated at the N-terminal site with linear mPEG-aldehyde. According to the obtained results, the optimum condition for its PEGylation includes PEG-to-protein molar ratio of 4, reaction pH of 4, and reaction incubation time of 10 hours at 15 °C. Conclusion:The aim of this thesis was the optimization of the affecting parameters in N-terminal PEGylation of IFNβ-1a. This study is planned to be scaled up later, so the impact of affecting parameters has considered from the economical view too. Considering all parameters, limitation of IFNβ-1a source, and based on the achieved results, the optimum condition for PEGylation of IFNβ-1a with 20 kDa linear mPEG-aldehyde includes PEG-to-protein molar ratio of 4, reaction pH of 4, and reaction incubation time of 10 hours in 15 °C.