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Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.

ABSTRACT: Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.

SUBMITTER: Hansen HG

PROVIDER: S-EPMC4676018 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.

Hansen Henning Gram HG Nilsson Claes Nymand CN Lund Anne Mathilde AM Kol Stefan S Grav Lise Marie LM Lundqvist Magnus M Rockberg Johan J Lee Gyun Min GM Andersen Mikael Rørdam MR Kildegaard Helene Faustrup HF

Scientific reports 20151211

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete i ...[more]

PMID: 26657798

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Project description:Biopharmaceuticals are medical compounds derived from biological sources and are often manufactured by living cells, primarily Chinese hamster ovary (CHO) cells. CHO cells display variation among cell clones, leading to growth and productivity differences that influence the product's quantity and quality. The biological and environmental factors behind these differences are not fully understood. To identify metabolites with a consistent relationship to productivity or cell death over time, we analyzed the extracellular metabolome of 11 CHO clones with different growth and productivity characteristics over 14 days. However, in bioreactor processes, metabolic profiles and process variables are both strongly time-dependent, confounding the metabolite-process variable relationship. To address this, we customized an existing hierarchical approach for handling time dependency to highlight metabolites with a consistent correlation to a process variable over a selected timeframe. We benchmarked this new method against conventional orthogonal partial least squares (OPLS) models. Our hierarchical method highlighted several metabolites consistently related to productivity or cell death that the conventional method missed. These metabolites were biologically relevant; most were known already, but some that had not been reported in CHO literature before, such as 3-methoxytyrosine and succinyladenosine, had ties to cell death in studies with other cell types. The metabolites showed an inverse relationship with the response variables: those positively correlated with productivity were typically negatively correlated with the death rate, or vice versa. For both productivity and cell death, the citrate cycle and adjacent pathways (pyruvate, glyoxylate, pantothenate) were among the most important. In summary, we have proposed a new method to analyze time-dependent omics data in bioprocess production. This approach allowed us to identify metabolites tied to cell death and productivity that were not detected with traditional models.

Project description:Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Dataset Information

Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.

Publications

Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.

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