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.

Fig 1. Cell pool generation and cell line development for PYC2 engineering.

(A-D) Schematic diagram of two-phase selection of pMPYC transfected CHO cells with varying concentration of MTX and puromycin. (B-D) Cell density, viability and lactate production profiles for stable pool 1B, pool 1C and pool 2D in fed-batch culture mode as a function of time. (E-F) Flow diagram depicts the steps followed to obtain a clonal CHO cell line from the heterogeneous population of a stable pool. Flow-cytometry used to gate the healthy cells population and sort the single CHO cell in a 384 well plate. Live cell imager is used to monitor the clonality and growth of the colony/cell in the consecutive days. Single clones are expanded from 384 well plates to T25 flask.

Fig 2. A comparative shake flask fed-batch study of the screened clones expressing PYC2 gene.

Graph representing culture profile: (A) Cell density (B) cell viability (C) lactate profile (D) glucose consumption profile for PYC2-expressing cells in shake-flask culture.

Fig 3. Gene copy number and mRNA expression analysis of the PYC2 expressing clones.

(A) Fold expression of the PYC2 m-RNA relative to the house keeping β-actin gene (cDNA used as a template). (B) Calculation of gene copies relative to house-keeping β-actin gene for selected PYC2 clone#12 (genomic DNA used as a template).

Fig 4. Culture performance of the clone#12 expressing PYC2 and parental CHO cell in shake flask fed-batch culture.

Graph representing culture profile: (A) Cell density,(B) cell viability, (C) Glutamine (D) lactate and (E) glucose consumption profiles.

Fig 5. Culture performance of the clone 12 expressing PYC2 and parental CHO cell in culture medium MamPF77, CDM4perMAB and Dynamis in batch mode.

Graph representing culture profile: (A and D) Cell density, (B and E) cell viability, (C and F) lactate profile.

Fig 6. Amino acid analysis of spent medium.

Concentration of the glutamic acid, aspartic acid and alanine profile of parental CHO cells and clone#12 grown in a fed -batch culture at different time interval. (A) Glutamic acid (B) Aspartic acid (C) Alanine.

Fig 7. Comparative fed batch study of mAb expression in CHO-PYC2 clone 12 and CHO-S control cells.

(A) Cell density, (B) Cell viability, (C) Lactate profile (D) Specific productivity and (E) Titer profile.

Fig 8. Comparative glycoform analysis of mAb secreted from the clone expressing PYC2 gene and a set of control without PYC2 over-expression.

Relative abundance of glycan composition of a mAb.