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The emerging role of systems biology for engineering protein production in CHO cells.

ABSTRACT: To meet the ever-growing demand for effective, safe, and affordable protein therapeutics, decades of intense efforts have aimed to maximize the quantity and quality of recombinant proteins produced in CHO cells. Bioprocessing innovations and cell engineering efforts have improved product titer; however, uncharacterized cellular processes and gene regulatory mechanisms still hinder cell growth, specific productivity, and protein quality. Herein, we summarize recent advances in systems biology and data-driven approaches aiming to unravel how molecular pathways, cellular processes, and extrinsic factors (e.g. media supplementation) influence recombinant protein production. In particular, as the available omics data for CHO cells continue to grow, predictive models and screens will be increasingly used to unravel the biological drivers of protein production, which can be used with emerging genome editing technologies to rationally engineer cells to further control the quantity, quality and affordability of many biologic drugs.

SUBMITTER: Kuo CC

PROVIDER: S-EPMC5988649 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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The emerging role of systems biology for engineering protein production in CHO cells.

Kuo Chih-Chung CC Chiang Austin Wt AW Shamie Isaac I Samoudi Mojtaba M Gutierrez Jahir M JM Lewis Nathan E NE

Current opinion in biotechnology 20171207

To meet the ever-growing demand for effective, safe, and affordable protein therapeutics, decades of intense efforts have aimed to maximize the quantity and quality of recombinant proteins produced in CHO cells. Bioprocessing innovations and cell engineering efforts have improved product titer; however, uncharacterized cellular processes and gene regulatory mechanisms still hinder cell growth, specific productivity, and protein quality. Herein, we summarize recent advances in systems biology and ...[more]

PMID: 29223005

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Project description:BackgroundSialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell-cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties.MethodsThe soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA).ResultsA range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format.ConclusionStable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme.

Project description:Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development.

Dataset Information

The emerging role of systems biology for engineering protein production in CHO cells.

Publications

The emerging role of systems biology for engineering protein production in CHO cells.

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