Manufacturing Process And Quality Study Of Pegylated Recombinant Human Interferon Alpha 2b

Recombinant human interferonα2b(rhIFNα2b)is one of the cytokines which were the earliest to be mass-produced by genetic engineering technology,rh IFNα2b is mainly applied to treat acute and chronic viral hepatitis(e.g.hepatitis B and hepatitis C),condyloma acuminatum,hairy cell leukemia,chronic myeloid leukemia,lymphoma,AIDS-realted Kaposi’s sarcoma,malignant melanoma and other diseases.However,due to the short drug interval,many researchers are committed to the development of long-term interferon preparation.Pegylation technology is one of the effective means to deal with the short half-life of protein drugs.In this study,monomethoxy polyethylene glycol succinimidyl carbonate(m PEG-Succinimidyl Carbonate,m PEG-SC)whose molecular weight is 20KD were used to modify rh IFNα2b,the process method of pegylated recombinant human interferonα2b(PEG-rh IFNα2b)with high purity was developed,and physical and chemical properties,biological activity and stability of PEG-rh IFNα2b were studied.(1)Manufacturing and purification of pegylated recombinant human interferonα2b(PEG-rh IFNα2b).Optimization of p H,feed ratio,rh IFNα2b concentration and other parameters by DOE experimental design,establish the manufacturing process of PEG rh IFNα2b.Separate the target product by cation exchange chromatography and gel filtration chromatography.The optimal process is:under the modification conditions of p H7.0,2～8 ~oC,about 4 hours,the concentration of rh IFNα2b is 1.8mg/ml～2.7mg/ml,the feed ratio is 2.2～3.0,the single-point modification rate of PEG-rh IFNα2b was more than 44%,the reaction by-product NHS and free polyethylene glycol can be removed effectively by the established two-step purification process of PEG-IFNα2b which consisted of cation exchange chromatography and gel filtration chromatography,and purity of the gained target protein was above 95%.(2)Physicochemical properties and stability of pegylated recombinant human interferonα2b(PEG-rh IFNα2b).The molecular weight of PEG-IFNα2b is 39206.6Dalton,the isoelectric point of which is 5.8-6.0,the UV absorption wavelength of which is 280nm,the purity of which is 98.1%-98.4%by SDS-PAGE and 98.08%-99.74%by SEC-HPLC,and free polyethylene glycol was not detected,five positional isomers which have the same electrophoretic mobility of SDS-PAGE can be isolated through weak differences of charge property among them by IE-HPLC.PEG-rh IFNα2b was placed at 2～8 ~oC,25±2~oC and 37±2 ~oC,respectively,protein content,electrophoresis purity,HPLC purity,free interferon,specific activity,free PEG and other indexes were examined at different time points,three batches of PEG rh IFNα2b solution were stored inspection indexes met the requirements,the purity test was unqualified after 3months at 37~oC.(3)Biological activity of pegylated recombinant human interferonα2b(PEG-rh IFNα2b).Study on the specificity and precision of the bioassay of interferon in Pharmacopoeia of the People’s Republic of China.The results showed that the method which from Pharmacopoeia of the People’s Republic of China had a good specificity,solvent and a little of free polyethylene glycol had no influence on bioassay of pegylated recombinant human interferonα2b,good precision,RSD=14%(n=6).