Growth and productivity of a recombinant Chinese hamster ovary cell line in batch culture

Abstract

A Chinese Hamster Ovary cell line (CHO1-5 500) co-transfected with h-tPA and dhfr was studied in bath culture mode. The concept of the cell-hour (the biological capacity for production) was developed, placing evaluation of cell population and protein production on the same cumulative basis, within the epigenetic time domain. The specific productivity of human tissue type plasminogen activator (h-tPA) was found to be growth-phase-associated, with maxima in the lag (0.065 pg c-h -1 and early decline (0.040 pg c-h -1) growth phases. The product titer was found to have a maximum (10.5 mg L-1) in bath culture under many different conditions. The h-tPA production was also evaluated as a function of the physiological state of the cells, by population segregation on the basis of cell cycle phase. Appearance of extracellular h-tPA was found to directly correlate to the G1 cell cycle phase (0.080 pg G1c-h-1), while DHFR content per cell was greatest during the S cell cycle phase.

Morphological type (substratum adherent, aggregate suspension, and dispersed suspension) could be induced by agitation rate and serum concentration. Aggregates, which were seen to have large void volumes stabilized by cytoplasmic bridging had a medium growth rate (umax = 0.016 h-1), but a short exponential growth phase and low batch averaged specific productivity (q1PA = 0.021 pg c-h -1). Simple dispersed suspension systems with serum-free medium (DS) attained the same product titer as microcarrier-attached systems with 10% FBS (MS), while the h-tPA concentration as a percent of total extracellular protein was greater by a factor of 8. Slow growing DS (umax = 0.012 h-1), had a higher specific productivity (qtPA = 0.045 pg c-h-1) than the MS (umax = 0.026 h-1, qtPA = 0.037 pg c-h-1).

Three metabolic phases were identified during batch culture, supporting the view that phase I gluconeogenesis and DHFR mediated glycine production are used by CHO1-5 500 cells. Production of h-tPA coincided with the first metabolic phase so that strategies to lengthen this phase should extend the productive portion of a batch culture.

A partially structured model for hybridomas was adapted to the more complex metabolism of CHOI-5 500. The model, which contained 15 equations and 24 parameters, provided a good fit to the three metabolic phases. Metabolic switches were assumed to coincide with depletion of glucose and glutamine to constant levels of 0.5 mmol L-1.