Over-expression of a Codon Optimized Yeast Cytosolic Pyruvate Carboxylase (PYC2) in CHO Cells for an Augmented Lactate Metabolism.

Sanjeev K Gupta,Ankit Sharma,Hiralal Kushwaha,Pratyoosh Shukla

doi:10.3389/fphar.2017.00463

Sanjeev K Gupta, Ankit Sharma + Show 2 more

Open Access

https://doi.org/10.3389/fphar.2017.00463

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Abstract

Monoclonal antibodies are the most demanding biotherapeutic drugs now a days used for the cure of various critical illnesses. Chinese hamster ovary (CHO) cells are one of the main hosts used for the large scale production of these antibodies. However, the cell line and production processes are the key factors to determine the cost and affordability of these antibodies. The metabolic waste lactic acid and ammonium are accumulated during a cell culture process and adversely affects productivity as well as product quality. To control the lactate metabolism of mAb (IgG1-kappa) producing CHO clones, we super-transfected the cells with a mammalian construct bearing codon optimized yeast cytosolic pyruvate carboxylase (PYC2) and a strong fusion promoter for optimal expression of PYC2 enzyme. A pool study was also performed for the assessment of cell’s performance, post-translational modification of a mAb and its expression in a CHO clone. The current study resulted an improved mAb titer up to 5%, galactosylation up to 2.5-folds, mannosylation up to twofold and marginal improved main and basic peaks in the charge variant profile at the cell pool stage. Such, approach may be suitable for the implementation in CHO cells producing recombinant protein for a better process control for the production of biotherapeutics.

Highlights

Continuous cell lines (CCLs) involve in the accumulation of wasteful byproducts in the form of lactic acid and ammonium which are produced from glucose and glutamine metabolism
The codon optimized yeast pyruvate carboxylase (PYC2) gene was used for the cloning and super-transfection of Chinese hamster ovary (CHO)-monoclonal antibody (mAb) clone, which triggers the cytosolic pyruvate toward the TCA cycle of the cell’s metabolic pathway
The current study reveals that an optimal expression of the pyruvate carboxylase 2 (PYC2) gene in CHO cells enhances its metabolic activity, which in turn directly or indirectly affects the overall cell’s performance, including titer and glycosylation composition of a therapeutic mAb

Summary

Introduction

Continuous cell lines (CCLs) involve in the accumulation of wasteful byproducts in the form of lactic acid and ammonium which are produced from glucose and glutamine metabolism. Metabolic engineering has been practiced to lower the accumulation of these wasteful byproducts in the culture which results in enhanced product titers as well as product quality which is possible by genetic manipulation of the host cells (Gupta and Shukla, 2016b). In a fed-batch culture, the extended cell viability is generally linked to the extent of byproducts ammonia and lactate accumulation in the culture medium, which can be harmful to. Long-term Chinese hamster ovary (CHO) culture shows a desynchronized metabolism of glucose associated with high lactate accumulation, that can lead to medium acidification followed by undesired increased osmolality due to the addition of alkali for the maintenance of culture pH (Omasa et al, 1991; Ozturk et al, 1992; Lao and Toth, 1997; Cruz et al, 2000). Glycosylation pattern of monoclonal antibodies and other glycoproteins can be altered by the ammonia accumulation (Borys et al, 1994; Andersen and Goochee, 1995; Gawlitzek et al, 2000; Yang and Butler, 2002; Chen and Harcum, 2006)

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