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Secretory pathway differences between HEK293 and CHO enable rescue of difficult to express human proteins

ABSTRACT: Biologics have emerged as leading blockbuster therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here we identify bottlenecks limiting expression of challenging recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293. Surprisingly, one third of the challenging human proteins displayed an improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, a key set of genes with extreme expression variation was identified. Among these, ATF4 and SRP9 were validated as general productivity boosters in CHO. Further, we found that more heavily glycosylated products benefit more from the elevated activities of the N- and O-glycosyltransferases found in HEK293 cells. Collectively, our results demonstrate the utilization of HEK293 cells for expression rescue of human proteins and suggest a strategy for identification of secretory pathway components improving yield and quality in HEK293 and CHO.

ORGANISM(S): Homo sapiens Cricetulus griseus

PROVIDER: GSE157729 | GEO | 2022/06/22

REPOSITORIES: GEO

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Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in the last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets to improve production and quality of RP. Some of these transcriptomic and proteomic profiles have been acquired under low temperature, butyrate addition, hyperosomotic pressure, protein degradation phenotypes, cellular engineering, production stability and productive phenotypes. In case of productive phenotypes, several whole cell proteomic studies have been previously reported, where cell populations secreting fusion proteins, monoclonal antibodies (mAb) and antibody fragments, at different specific productivity (Qp), have been compared. However, because of this information has been obtained from whole cell homogenates, highlighted categories represent abundant proteins and have limited coverage of proteome from some organelles such as the classical secretory pathway. Due to the central role of this pathway in protein and lipid metabolism, organelle biogenesis, cell cycle, apoptosis and proliferation, and to be recognized as a bottleneck for protein secretion in mammalian cells, its characterization through subcellular proteomics represents a promising strategy for identification of key targets. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the Qp of HEK293 and CHO cells producing different RP. Recently, proteomic reports of subcellular compartments from a variety of cells have been extensively used to study the protein composition of organelles, protein dynamics and discovering of novel proteins involved in the secretion pathway. Thus, this approach was applied to the secretory pathway of CHO cells to identify novel proteins linked to Qp in the present study. Differential proteomics from all remaining compartments was also carried out, giving new lights over other cellular mechanisms associated to the higher producer phenotype. The novel differentially expressed proteins (DEP) found will improve the engineering strategies of CHO cells that will have a positive impact on titer and quality of RP. Cell lines CHO DP-12 clone #1933 CRL-12444 and clone #1934 CRL-12445, which secrete a mAb against human interleukin 8 (IL-8), were used as cell models of recombinant CHO cell lines producing the same RP at different Qp. Our goal is that the processing of raw proteomics data and the identification and classification of DEP belonging to the classical secretory pathway will allow to propose a model by which the increase in the productivity of CHO cells has been associated with the deregulation of proteins from this pathway that show differential expression among the clones under study. This strategy of subcellular proteomics allowed us to identify a large number of new targets and cell pathways with respect to previous whole cell proteomic studies, which can improve the understanding of the molecular mechanisms behind RP production and provide the basis to design new sub-lines with stronger capabilities.

Project description:Ammonium is a waste product that inhibits cell growth, recombinant protein production, and protein glycosylation in mammalian cell culture. Recent studies have demonstrated that ammonium adversely affects glycosylation-related gene expression in Chinese hamster ovary (CHO) cells. However, since the CHO cell line species has not been fully sequenced, a glycosylation transcriptome analysis is not possible in this cell line. Therefore, to further understand the effects of ammonium on glycosylation-related gene expression, NS0 cells, a mouse myeloma cell line, were cultured under elevated ammonium. NS0 cells are similar to CHO cells, in that the NS0 cells are anchorage-independent and commonly used to commercially produce recombinant proteins. Additionally, DNA microarrays containing all known mouse glycosylation-related genes were available to be used to examine gene expression. NS0 cells were cultured under normal (control), elevated ammonium, elevated salt, and elevated ammonium with proline. It was observed that the control and treatments culture growth rates were not significantly different; however, the final cell densities were significantly different. The DNA microarray data was analyzed using a Welch ANOVA test with a Benjamini and Hochberg false discovery rate correction for the multiple comparisons of the glycosylation-genes. No significant difference in gene expression levels between the four conditions examined were observed. The results of this study demonstrated that NS0 cells, at the gene expression level, are insensitive to ammonium. Thus, the decreased glycosylation observed in NS0 cell cultures at elevated ammonium is likely due to changes in synthesis and degradation enzyme activity. In contrast, CHO cells have decreased glycosylation levels due to decreased sialytransferase gene expression and not increased degradation enzyme activity. Therefore, even though NS0 and CHO cells are both commonly used recombinant hosts for glycoprotein synthesis, it appears that NS0 and CHO cells had different control mechanisms respect to glycosylation-related gene expression under elevated ammonium.

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Secretory pathway differences between HEK293 and CHO enable rescue of difficult to express human proteins

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