Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

Abstract

A Chinese Hamster Ovary cell line, CHO1-15(500), producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell(-1 )h(-1)) and early decline (0.040 pg cell(-1 )h(-1)) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h(-1). Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.

Fig. 1

Chinese hamster ovary cells, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA). Cells were grown as a 1 L dispersed suspension culture using a serum free medium. (A) Cell density and cumulative product titer. (B) Cumulative volumetric cell-hours (CHvol) and cumulative product titer

Fig. 2

Cumulative protein concentration as a function of the cumulative volumetric cell-hours. The slope of the curve is the specific productivity. The specific productivity of recombinant human tPA from the CHO1-15₅₀₀ cell line is growth-phase-associated. The specific productivity changes with culture conditions. (A) High initial glucose concentration of 40 mmol L⁻¹. (B) Low initial glucose concentration of 8 mmol L⁻¹

Fig. 3

Population growth dynamics of CHO1-15₅₀₀ cells in dispersed suspension batch culture using serum free medium, from an inoculum grown in serum containing medium. The population is divided into four subpopulations on the basis of relative DNA per cell. (A) Entire viable population. The population accumulates in G1 during the lag phase and then traverses the cell cycle in partial synchrony. The population accumulates in the apoptotic subcompartment during the late decline phase. (B) Cycling fraction of the viable population (A subpopulation excluded). The cycling fraction accumulates in G1 both during the lag phase and again during the late decline phase

Fig. 4

Production of the recombinant human tPA is a linear function of the G1 cell cycle phase cell-hours, with a calculated specific productivity of 0.080 pg c-h⁻¹. This pattern suggests that tPA is produced during the G1 cell cycle phase in the CHO1-15₅₀₀ cell line. (Top left) Recombinant protein as a function of the G1 phase cell-hours. (Top right) Recombinant protein as a function of the S phase cell-hours. (Bottom left) Recombinant protein as a function of the G2/M phase cell-hours. (Bottom right) Recombinant protein as a function of the A subpopulation cell-hours

Fig. 5

Density arrested population of CHO1-15₅₀₀ cells. Cells were maintained as a confluent monolayer on a 225-cm² T-flask, using medium containing 5% FBS. The majority of the population resides in the G1 cell cycle phase. The whole population specific productivity was calculated to be 0.067 pg c-h⁻¹. The G1 associated specific productivity, calculated to be 0.081 pg c-h⁻¹, correlates well with the proportion of the population in the G1 cell cycle phase (84%)

Fig. 6

Association of the production of DHFR to the cell cycle phases. Relative DHFR associated fluorescence intensity is compared to the proportion of the population in each of the subpopulations. DHFR production correlates with the G1 and S cell cycle phases, at relative concentrations of 1 and 10, respectively. DHFR is not produced during apoptosis