Study Of Recombinant Human Serum Albumin-Interferon-α 2b Fusion Protein

Objective:To prepare a potent, long-lasting recombinant human serum albumin-Interferon-α2b fusion protein (rHSA/IFNα 2b) expressed in Pichia pastoris by albumin fusion technology. This fusion protin is suitale for treat some kinds of tumors, chronic hepatitis B and C infections.Methods:1.rHSA/IFNα 2b was expressed in the Pichia pastoris GS115,including cloning cDNA of HSA and IFNα 2b, constricting the recombinant expression vector,tranformating and screening the high-level expression strain.2.Optimization of fermentation conditions of rHSA/IFNα 2b was expressed in the Pichia pastoris GS115.3.Optimization the separation and purification methods of rHSA/IFNα 2b expressed in the Pichia pastoris GS115.4.Purified rHSA/IFNα 2b was identificated and characterized by SDS-PAGE, IEF, protein hybridization, terminal amino acid sequence, MS, peptide mapping, CD and in vitro biological activity analysis.5.Pharmacokinetic and pharmacodynamic studies of rHSA/IFN a 2b were conducted in cynomolgus monkeys. Concentration changes of rHSA/IFNα 2b and 2',5'-OAS in serum was monitored and compared with normalIFNα 2b.Immunogenicity of repeated administration of rHSA/IFNα 2b was also studied.Results:1. cDNA of HAS and IFNα 2b was cloned by RT-PCR,then cloned to expression vector pGENYD2 and get the recombinant expression vector.This vector was transformed to pichia pastoris GS115 and intergrated to yeast chromosone.The high-level expression strain was screened by flask test and rHSA/IFNα 2b was screted to supernatant. rHSA/IFNα 2b concentration in broth was about 0.05g/L in flask which was detected by SDS-PAGE.2.High-density fermentation process in automatic fermenter was studied according to fermentation conditions optimized in flask test. The optimized condition was 0.5% methonal, 0.1% tween-80,22℃ additive of organic culture media,and the sutibale fermentation time was 96 hours. rHSA/IFNα 2b concentration in broth could reach to 0.432g/L under these fermentation conditions.3.Fermentation broth of rHSA/IFNα 2b was concentrated by ultrafitration and purified by dye-affinity chromatography,hydrophobic interaction chromatography, ion-exchange chromatography and gel-fitration chromatography combinational. Purity of the prepared rHSA/IFNα 2b was greater than 98% and the yield of protein recovery was greater than 20%.4.MW of rHSA/ IFNα 2b is 86KD when detected by reduced SDS-PAGE. Its pI was 5.3 and showed a single band on 1EF gel. Molecular weight determined by MALDI-TOF was 86004.3±29.2. Amino-terminal and carboxyl-terminal amino acid sequences were identical to predicted sequence. Its specific activity in vitro was greater than 5.0×10~5 IU/mg fusion protein, retaining about 1.4% of that of unmodified rIFNα on a molar basis.5.The results of pharmacokinetic and pharmacodynamic studies proved that the half-life of rHSA/IFNα 2b was prolonged to 96 hours in vivo after subcanuous adminstration,improved 19.6 times compare to normal IFNα 2b.6.The results of pharmacodynamic studies indicated that 2',5'-OAS expression remarkably increased and was related to the dose of rHSA/IFNα 2b.Furthermore,it had been maintained on the 14th day after the injection, compared to that of IFNα 2b (only 2 days).7. Anti-rHSA/IFNα 2b antibody was induced after 2 weeks administration and neutral antibody wasinduced after 8 weeks administration.Conclusions:1 .rHSA/IFNα 2b fusion protein was expressed high-yield level in pichia system. Purified rHSA/IFNα 2b was identificated and characterized by physical, chemical and biological method.All the results showed that correct and biological active rHSA/IFNα 2b was prepared from pichia system. Protein recovery yield was greater than 20% and purification process was feasible. rHSA/IFNα 2b prepared from pichia system can be used for clinical trial.2.Pharmacokinetic and pharmacodynamic studies proved that the half-life of rHSA/IFNα 2b was prolonged and was more potent than normal IFNα 2b.Unfortunately, immunogenicity was also proved in cynomolgus monkeys.3.All the results indicated that rHSA/IFNα 2b expressed in pichia has longer half-life and more potent than normal IFNα in animal body,and longer half-life means reduced dosing frequency which will improve patient compliance, improving the response to treatment.