Site-Specific Pegylation Of Recombinant Interferon-α2b

Recombinant interferon alpha-2(IFN-α2) has proven useful for treating a varietyof human viral diseases and cancers because of its antiviral, immunoregulatory andantiproliferative activities. Unfortunately, IFN-α2has a short circulating half-life invivo，which necessitates daily or three weekly administration to patients in order tomaintain an effective blood concentration. However, the PEGylation of IFN-α2canimprove physicochemical and biological characteristics, and extend plasmacirculating half-life. We introduce a “free” cysteine residue, that is, a cysteine residuenot involved in a disulfide bond, into the recombinant human IFN-α2b (rhIFN-α2b)using gene recombination technology, followed covalent modification of the addedcysteine residue with cysteine-specific maleimide-PEG, then we create themonoPEGylated recombinant human IFN-α2b without structural isomers.The recombinant human IFN-α2b was expressed in the form of inclusion bodiesin E.coli W3110. The yield and purity of protein was increased by optimizing washingconditions of the inclusion bodies and its in vitro solubilization and refoldingconditions. Besides, we also optimized several conditions of the PEGylation ofrecombinant human IFN-α2b, such as reducing agent, pH, reaction time, reactiontemperature and the concentration of rhIFN-α2b. Through optimizing these conditions,we improved the yield of monoPEGylated rhIFN-α2b.Currently, purification methods of the PEGylated protein are dominated by ionexchange and size exclution chromatography. When the PEG molecular was bondedto protein, they could partially shield the surface charge of the protein. On the basis ofcharge difference, we studied the purification of the PEGylated reaction mixture usingcation ion exchange chromatography.