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Metabolic engineering of CHO cells for the development of a robust protein production platform.

ABSTRACT: Chinese hamster ovary (CHO) cells are the most preferred mammalian host used for the bio-pharmaceutical production. A major challenge in metabolic engineering is to balance the flux of the tuned heterogonous metabolic pathway and achieve efficient metabolic response in a mammalian cellular system. Pyruvate carboxylase is an important network element for the cytoplasmic and mitochondrial metabolic pathway and efficiently contributes in enhancing the energy metabolism. The lactate accumulation in cell culture can be reduced by re-wiring of the pyruvate flux in engineered cells. In the present work, we over-expressed the yeast cytosolic pyruvate carboxylase (PYC2) enzyme in CHO cells to augment pyruvate flux towards the TCA cycle. The dual selection strategy is adopted for the screening and isolation of CHO clones containing varying number of PYC2 gene load and studied their cellular kinetics. The enhanced PYC2 expression has led to enhanced pyruvate flux which, thus, allowed reduced lactate accumulation up to 4 folds and significant increase in the cell density and culture longevity. With this result, engineered cells have shown a significant enhanced antibody expression up to 70% with improved product quality (~3 fold) as compared to the parental cells. The PYC2 engineering allowed overall improved cell performance with various advantages over parent cells in terms of pyruvate, glucose, lactate and cellular energy metabolism. This study provides a potential expression platform for a bio-therapeutic protein production in a controlled culture environment.

SUBMITTER: Gupta SK

PROVIDER: S-EPMC5538670 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Metabolic engineering of CHO cells for the development of a robust protein production platform.

Gupta Sanjeev Kumar SK Srivastava Santosh K SK Sharma Ankit A Nalage Vaibhav H H VHH Salvi Darshita D Kushwaha Hiralal H Chitnis Nikhil B NB Shukla Pratyoosh P

PloS one 20170801 8

Chinese hamster ovary (CHO) cells are the most preferred mammalian host used for the bio-pharmaceutical production. A major challenge in metabolic engineering is to balance the flux of the tuned heterogonous metabolic pathway and achieve efficient metabolic response in a mammalian cellular system. Pyruvate carboxylase is an important network element for the cytoplasmic and mitochondrial metabolic pathway and efficiently contributes in enhancing the energy metabolism. The lactate accumulation in ...[more]

PMID: 28763459

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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

Metabolic engineering of CHO cells for the development of a robust protein production platform.

Publications

Metabolic engineering of CHO cells for the development of a robust protein production platform.

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