| Objective:To study the optimal reaction conditions of modifying recombinant human interferona-2b(rhIFNα-2b) by 20kD Methoxyl PEG Succinimidyl Ester(mPEG-NHS) and the method for the purification of mono-PEG- rhIFNα-2b that meet the requirements of biotechnological products. Then investigate it's physicochemical properties, pharmacokinetics, immunogenicity and pharmacodynamics.Methods: Selected the optimal modification conditions by analyzing scan maps of SDS-PAGE R250 staining and purifid the reaction mixture by ion-exchange and size exclusion chromatography. Different methods were used to study on the identification, molecular weight, concentration, UV spectrum, purity and biological titer of PEG-rhIFNα-2b. Inject mouse with a single dose of PEG-rhIFNα-2b and rhIFNα-2b respectively and determine the concentrations of samples in sera by ELISA and analyze the pharmacokinetic parameter. Immunize mouse with rhIFNα-2b and PEG-rhIFNα-2b according to daily and clinical administration with ip. administration, then determine the serum combining and neutralizing antibody by ELISA and cytopathogenic effect inhibition assay (VSV-WISH). HepG2.2.15 cell line was used as cell model. MTT assay was adopted to evaluate the toxicity of PEG-rhIFNα-2b and rhIFNα-2b on cell line. High, medium, low doses of PEG-rhIFNα-2b and rhIFNα-2b were added to the cell every other day for 8 days, ELISA was adopted to detect the level of HBsAg in supernatant. Same doses of PEG- rhIFNα-2b and rhIFNα-2b were added to the cell according to the frequency of clinical administration for one week, that is PEG-rhIFNα-2b was added for 1 time while rhIFNα-2b was added for 3 times, ELISA to determine the secretion of HBsAg. condition:pH9.0,3:1 for the mass ration of PEG to rhIFNa-2b,4℃for reaction of 2h, and the final yield is 24.72% after the two steps of purification. Determination of physicochemical properties showed that PEG modification don't change the epitopes of rhIFNα-2b, the molecular weight of PEG-rhIFNa-2b is 44.06kD, The concentration of PEG-rhIFNα-2b is about 100μg/mL, the UV absorption peak of PEG-rhIFNα-2b is 279.5nm while that of rhIFNα-2b is 280nm, the the purity of the PEG-rhIFNα-2b met the specification of biotechnological products, the activity of PEG-rhIFNα-2b retained 11.8% of rhIFNα-2b. Pharmacokinetic results displayed that after rhIFNα-2b modified by PEG, the half-life extended from 1.670h to 7.670h, an increase of nearly 5-fold.AUC increased from 700.173ng·h/mL to 4709.97ng·h/mL, an increase of 6.7-fold, and the CL decreased from 0.357 mL/h·kg to 0.053 mL/h·kg, a decrease of 6.73-fold. The combining antibody titer and the neutralizing antibody titer are 7517.72 and 77.63 induced by rhIFNα-2b, while the titers are 694.62 and 26.98 induced by PEG-rhIFNα-2b according to daily administration. The two types of antibody titers are 5538.51 and 44.99 induced by rhIFNα-2b, while those are 311.61 and 12.96 induced by PEG-rhIFNα-2b according to clinical administration. Pharmacodynamics results demonstrated that TC50 (PEG-rhIFNα-2b)>20000IU/mL, TC50(rhIFNα-2b)>20000IU/mL. C50(PEG-rhIFNα-2b)=559.88IU/mL, IC50(rhIFNα-2b) =478.71IU/mL. The inhiting ratio of PEG-rhIFNα-2b was a bit lower than that of rhIFNα-2b with the same doses. The inhibition of the both have no significant differences according to the frequency of clinical administration.Conclusion:Established a stable modification process of PEG-rhIFNα-2b with high percentage and a purification process of PEG-rhIFNα-2b with high purity. Established methods to research part of physicochemical properties in quality standard according to reporting requirements of new biologicals. The results shows that the modification of rhIFNα-2b with PEG can significantly prolong the half-life, decreased the immunogenicity and don't affect anti-HBV effect in vitro. This study provided a comprehensive evaluation on influence of rhIFNα-2b modified by PEG separately from physicochemical properties, pharmacokinetics and pharmacodynamics of PEG-rhIFNα-2b, and it would make preparations for the research and industrialization of PEG-rhIFNα-2b.
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