SUMMARY Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells, provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show the metabolic resources in CHO are >3 times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

35Keywords 36 Metabolic network, secretory pathway, biotherapeutic production, systems biotechnology 37Indeed, recent studies have incorporated portions of the secretory pathway in metabolic models of yeast 55 3-5 . Furthermore, Lund and colleagues reconstructed a genetic interaction network of the mouse secretory 56 pathway and the unfolded protein response and analyzed it in the context of CHO cells 6 . However, such a 57 network does not encompass a stoichiometric reconstruction of the biochemical reactions involved in the 58 secretory pathway nor it is coupled to existing metabolic networks of mammalian cells. 59Here we present the first genome-scale stoichiometric reconstructions and computational models of 60 mammalian metabolism coupled to protein secretion. Specifically, we constructed these for human, 61 mouse, and CHO cells, called RECON2.2s, iMM1685s, and iCHO2048s, respectively. We first derive an 62 expression for computing the energetic cost of synthesizing and secreting a product in terms of molecules 63 of ATP equivalents per protein molecule. We use this expression and analyze how the energetic burden 64 of protein secretion has led to an overall suppression of more expensive secreted host cell proteins in 65 mammalian cells. Given its dominant role in biotherapeutic production, we further focus on the 66 biosynthetic capabilities of CHO cells. We then demonstrate that product-specific secretory pathway 67 models can be built to estimate CHO cell growth rates given the specific productivity of the recombinant 68 3 product as a constraint. We identify the features of secreted proteins that have the highest impact on 69 protein cost and productivity rates. Finally, we use our model to identify proteins that compete for cell 70 resources, thereby presenting targets for cell engineering. Through this study we demonstrate that a 71 systems-view of the secretory pathway now enables the analysis of many biomolecular mechanisms 72 controlling the efficacy and cost of protein expression in mammalian cells. We envision our models as 73 valuable tools for the study of normal physiological processes and engineering cell bioprocesses in 74 biotechnology. All models and data used in this study are freely available at 75 https://github.com/LewisLabUCSD/MammalianSecretoryRecon. 76 77 RESULTS 78 A stoichiometric expression of protein secretion energetics 79In any cell, the secretory machinery is concurrently processing thousands of secreted and membrane 80 proteins, which all compete for secretory pathway resources and pose a metabolic burden. To quantify 81 this burden, we estimated the energetic cost of synthesizing and/or secreting 5,641 and 3,538 82 endogenous proteins in the CHO and human secretome and membrane proteome in terms of total 83 number of ATP equivalent molecules consumed (see Methods). These protein costs were compared to 84 the cost of five recombinant proteins commonly produced in CHO cells (Fig. 1a). To refine estimates, we 85 predicted signal peptides 7 , GPI anchor attachment signals 8 , an...