Preparation And Application Research Of A New Chromatographic Meida For Purification Of Mab

Protein A chromatography plays a significant role in the purification of monoclonal antibodies(Mabs)for its high purification efficiency.However,a clean-in-place step would be integrated to minimize the impurities such as HCPs,nucleic acid and microbes.High concentration of sodium hydroxide solution has been widely used to remove tightly bound contaminants at low cost.Under such harsh condition,Protein A ligand would be damaged and the capacity of Protein A medium decreased roughly after each clean-in-place(CIP)cycle.So the genetic engineering technology was applied for improving the high capacity and alkalineresistance of Protein A ligand.In this research,a new alkaline-resisted recombinant Protein A ligand was modified and constructed,named r Protein A-ZX.And commercial r Protein A ligand was introduced as camparsion.r Protein A-ZX and commercial r Protein A were both coupled to Sepharose 4FF for for evaluating the dynamic binding capacity(DBC)and the alkali-resistance.r Protein A-ZX has higher affinity than the commercial r Protein A,but r Protein A-ZX media based on Sepharose 4FF maintained 64% of initial DBC after 40 CIP cycles using 0.5 mol/L Na OH and commercial r Protein A could retain 86% of that.From this results,r Protein A-ZX was considered to be worse alkaline-resistance than commercial r Protein A.The factor of Protein A's alkaline-resistance was investigated deeply by SDS-PAGE,enzyme-linked immunosorbent assay(ELISA)and circular dichroism spectrum(CD).Both ligands was incubating in 0.5 mol/L Na OH for 0,75,150,300,450 and 600 min,the secondary structure and concentration was characterized by CD and ELISA respectively.From the results,the variation of the content of ?-helix and concentration were similar to the change trendency of DBC in CIP cycles.From acquired datas in the former research,alkaline-resistance of ligands was considered as key point of media's performance.Futhermore,the alkaline-resistance of r Protein A-ZX media was optimized by coupling to the high cross-linked medium and the media can kept 80% of initial DBC after 40 CIP cycles,achieveing basic international level.In previous research,we found that the ligand was key factor of media's performance.According to this conclusion,r Protein A-PE was constructed on basis of r Protein A-ZX.The site-specific mutagenesis was conducted in r Protein A-ZX's sequence.The r Protein A-PE achieved high mole binding ratio for Ig G as 1:1.96.And r Protein A-PE based on Sepharose 4FF can acquire high dynamic binding capacity as 81.92 mg/m L,more than that of imported commercial product on sale.Further,the DBC and alkalineresistance of r Protein A-PE coupling to high cross-linked matrix were also investigated.The r Protein A-PE based on high cross-linked medium achieved DBC as 63.19 mg/m L and kept 95% of initial DBC after 40 CIP cycles which has reached international advanced level.