High-level Expression And Bioactivity Of HSA/dTMP Fusion Proteins In Mammalian Cells

In our previous studies,a novel fusion protein that composed of tandem dimer of TMP(dTMP)genetically fused at the N-terminus of human serum albumin(HSA)was successfully constructed and expressed in Chinese hamster ovary(CHO)cells and methanol yeast,respectively.In vitro bioactivity assays showed that the bioactivity of dTMP-HSA fusion protein expressed by CHO was about 50 times higher than that of product expressed by yeast.However,the extremely low transient expression level of dTMP-HSA fusion protein in CHO cells,less than 1 mg/L,severely limits the following pharmacological and pharmacodynamic studies.In addition,how to accurately quantify the noval fusion protein is also a problem required to be solved at once,because there is no anti-TMP antibody or ELISA test kit for dTMP-HSA in market.Moreover,it has been reported that fusion orientation could remarkably influence the bioactivity of HSA fusion proteins.When dTMP is genetically fused at the C-terminus of HSA,the bioactivity of HSA-dTMP fusion protein is unknown.This study focuses on above problems.We first prepared rabbit anti-TMP polyclonal antibody with high titer,and on this basis,a novel sandwich ELISA for HSA/dTMP fusion proteins was established.The precision and specificity of the method accorded with the demand of general ELISA.Next,after the optimization of host cells,expression vector,serum free medium,key parameters of transfection,and culture conditions post transfection,we established an efficient transient expression system for HSA/dTMP fusion proteins.In follow-up experiments,CHO-S cells were transfected using 10 L Wave Bioreactors under optimized TGE conditions.As a result,the optimal process obtained above was scalable and the final concentration of dTMP-HSA in batch culture was 87.4 mg/L.After purification,both in vivo and in vitro bioactivity assays showed that,the bioactivity of dTMP-HSA was half of that of HSA-dTMP.In addition,HSA successfully improved the pharmacokinetic profile of dTMP,and both HSA-dTMP and dTMP-HSA fusion proteins prepared in this study displayed prolonged circulating half-life of over 16 h in mice.In order to achieve industrial applications of HSA/dTMP fusion proteins,we employed dihydrofolate reductase(DHFR)mediated gene amplification system to establish high-level stable CHO cell lines.In batch culture of recombinant cell line 2A5H8,dTMP-HSA fusion protein yields up to 400 mg/L were achieved.In batch culture of recombinant cell line 2A6G8,HSA-dTMP fusion protein yields up to 190 mg/L were achieved.And,after 75 days of continuous culture,yields of the two recombinant CHO cell lines did not decrease obviously.This result laid the foundation for the realization of the industrialization application of HSA/dTMP fusion proteins.