Identification Of Internal Reference Genes For Transcriptomic Study On CHO Cells And Screening Of Potential Genes Related To Fc-fusion Protein Production

The biopharmaceutical industry has become one of the fastest-growing branches in the pharmaceutical field with an excellent curative effect,the rapid development of biotechnology,and increasing investment in research and development.The market scale in 2019 has exceeded 300 billion US dollars with the majority of antibody-related drugs(intact antibody,Fc-fusion protein,bi-specific Ab,etc.).Chinese hamster ovary cells(CHO)has been used as the major expression system due to its ideal post-translational modification on proteins.About 70%of recombinant therapeutic proteins were manufactured in CHO cells.Even the productivity of r-protein in CHO cells was increased dramatically during the past several decades,the absolute yield of Ab-based drugs still cannot meet the market demand.Therefore,improvement of mAb productivity has important practical significance.Compared with the intact antibody,Fc-fusion protein should contain different molecular mechanisms due to the lack of light chain/heavy chain interaction.To explore the mechanisms of Fc-fusion protein productivity in CHO cells,the adherent CHO-K1 cell line was firstly adapted to a serum-free suspension cell line in this study.Transfection with the plasmid containing GLP1-Fc fusion protein was next performed.5 CHO cell lines with different Qp and gene copy numbers were screened and then were analyzed with RNA-seq techniques.Candidate reference genes were selected from three sources including:(1)commonly used house-keeping genes,(2)previously identified reference genes in CHO cells producing an intact antibody,and(3)candidate genes from our transcriptomics data.Validation of the above internal reference genes was done in our experiment including 75day long-term cultivation and fed-batch cultivation processes under different conditions.geNorm,NormFinder,BestKeeper,and ΔCt programs and methods were utilized to analyze the gene expression stability and gave an overall ranking.Akr1a1,Gpx1,and Aprt in long-term cultivation and Akr1a1,Rps16 in fed-batch culture,which have not been reported previously,exhibited the highest stability of gene expression,while Pabpnl,Hirip3,and Actb in both sets of experiments together showed the weakest stability.A comparison of reference genes with different expression stability was performed.It showed that inappropriate or even opposite trends in gene expression levels can be led to by using unstable reference genes.These new reference genes should be considered for the investigations on CHO cells in related research.After the analysis of RNA-seq data,DEGs,GO function analysis of DEGs,pathways of DEGs and PPI(protein-protein interaction)were applied between high/low-producing CHO cell lines.20 potential genes related to r-protein production were obtained.With the validation of the above 20 genes by RT-qPCR reactions,12 genes showed similar trends with the RAN-seq results.These genes were then examined with RNA interference test.Two genes thbd and medag were identified to relate to the Fc-fusion protein productivity at the mRNA level and protein level.Finally,a stable cell line of the high-producing CHO-12 with an shRNA plasmid of medag was established to study the mechanisms of this gene on recombinant protein production.CHO-12-medag-2E9 with the obvious success interference on the expression of gene medag was screened.With the 3-day passage cultivation,the r-protein yield increased 13%and the doubling time decreased 1.6h(7%)which might be the reason for the higher titer of the recombinant protein.This result provides the basis for the following research on medag gene with the quite limited background.