Research Of Modified Purification Process Of Recombinant Human Serum Albumin(rHSA)

Plasma human serum albumin (pHSA ) has been used clinically to treat many kinds of disease for many years.Today blood is polluted more and more seriously, so it becomes necessary to develope a substitute of HSA by DNA technology. Recombinant human serum albumin (rHSA ) derived from Pichia Pastoris by PCR technology contains different impurities from that of plasma HSA. Because HSA has been used in high dosage, so extremely pure product is required in large-scale production. For rHSA, the removal of heterologous impurities becomes crucial fact.In this paper, we developed a new purification process using cation exchangers of HSL(high salt ligand) as first step. The culture cell supernatant(CCS) can be loaded directly into column without diluted. This cation exchanger shorten run time and its special ligand has higher selectivity to rHSA of CCS. Analysis methods of impurities such as pigments,host saccharide,host protein,residue DNA etal, are set-up. A detail study removing impurities by process has been done. Research shows the appearance of product purified by this process is light-yellow, corlor ratio (by A350/280) is lower than 0.01.The content of host protein in final product is less than 0.1ppm, residue DNA is lower than 100ng/dose(under the sensitive baseline).We also studied strictly on secreted polysaccharide form Pichia Pastoris and trace saccharide in final product. The results show there isn't any mannan which cause allergic reaction in clinically. The product produced by modified process is better than that of original process.Compared with pHSA, we detect rHSA. It has immune reaction with mono antibody of HSA. Its purity of HPLC is above 99%. The results of reduced and non-reduced SDS-PAGE show single band. Its C-terminal and N-terminal sequence are identical with that of pHSA. CD spectrum and peptide mapping of rHSA are almost similar to that of pHSA. Its molecule weight is 66330 by reduced SDS-PAGE and is 66530 by ES-MS which is near to the theoretic value.