Purification And Stability Of Recombinant Thrombopoietin Mimetic Peptide-human Serum Albumin Fusion Protein

Recombinant human thrombopoietin(rhTPO)failed on clinical trial in patients for treating thrombocytopenia in the United States due to its high risk of development neutralizing antibodies against native TPO induced by this drug,which lead to further reductions in platelet counts.Previous work in our lab showed that dimer of TMP-HSA(dTMP-HSA)has been successfully expressed in CHO expression system by a DNA recombinant technology to fuse HSA to a tandem thrombopoietin mimetic peptide(dTMP)without homologous sequence with native TPO.The dTMP-HSA fusion protein overcomes the shortcomings of rhTPO,and effectively extends the plasma half-life of small molecules dTMP.Furthermore,large-scale expression of dTMP-HSA fusion protein was produced in WAVE reactor.It is urgent to establish an efficient and economical downstream purification process for obtaining a large amount of dTMP-HSA fusion protein from the culture supernatant of WAVE reactor.In present work,the purification process of dTMP-HSA fusion protein expressed in CHO system in WAVE reactor was established.After purification,the desired protein with purity of not less than 93% was obtained,the total recovery of dTMP-HSA was 18%.The purified protein was further identified and its quality control was involved.The results showed that the percentage of peptide coverage of dTMP-HSA fusion was more than 97%,the molecular weight for TMP-HSA was 71.50 kDa,the isoelectric point was 5.54,and the maximum ultraviolet absorption peak was 280 nm ± 2nm.Western blot results showed that dTMP-HSA fusion protein was positive towards anti-HSA antibody and anti-dTMP antibody.Host DNA residues were no more than 100 pg per dosage,and the host protein residues were lower than 0.05% of the total protein.The effects of pH value and some drug adjuvants on the stability of dTMP-HSA were also studied.The storage conditions for desired protein were determined,and acceleration experiment suggested that dTMP-HSA should be stable at 4? for 694 days or 45? for 45 days.These results provided useful cues for the pharmaceutical development of dTMP-HSA.