The Production of Monoclonal Antibody in Growth-Arrested Hybridomas Cultivated in Suspension and Immobilized Modes

Abstract

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.

Effects of Three‐Dimensional Culturing on Osteosarcoma Cells Grown in a Fibrous Matrix: Analyses of Cell Morphology, Cell Cycle, and Apoptosis

“…Cell cycle has been reported to be related to cell metabolism and protein expression (41,42). In particular, higher specific protein productivity has been reported for growth-arrested cells (71,(76)(77)(78)(79)(80). Further research has been conducted by incorporating cycle-dependent promoters into cells and studying their effects on the expression of recombinant genes (81)(82)(83).…”

Process parameter shifting: Part II. Biphasic cultivation—A tool for enhancing the volumetric productivity of batch processes using Epo‐Fc expressing CHO cells

“…Control of proliferation has also been pursued by a series of other research groups who described very different strategies of controlling the progress of cells throughout the cell cycle. Cell-cycle arrest in G 1 has been induced in several ways: by starvation of an essential nutrient (Seifert and Phillips, 1999), by the addition of DNA synthesis inhibitors (Al-Rubeai et al, 1992), by the addition of genotoxic agents (Gartenhaus et al, 1996), through metabolic control (Altamirano et al, 2001) and by over-expression of cell-cycle inhibitors (Kaufmann et al, 2001;Mazur et al, 1998). A simple and wellestablished method based on changes of process parameters is the concept of growth arrest by shifting the culture to low temperatures.…”

Decoupling cell growth and product formation in Chinese hamster ovary cells through metabolic control

“…The maintenance of viable cells and production capacity over a relevant period of time contrasts with the loss of viability reported when temperature shifts are applied (Jenkins and Hovey, 1993), the reduced productivity observed after shifting to low temperatures (Bloemkolk et al, 1992), the appearance of mutants escaping from growth suppression in productive process conditions (Mazur et al, 1999), or the loss in viability observed when estradiol is used to trigger IRF-1-mediated cell arrest (Carvalhal et al, 1999). On the other hand, the results obtained with a medium composition control, based on serum or phosphate reduction, for a hybridoma cell line (Seifert and Phillips, 1999), show that this type of approach can be successful, and can be applied to a perfusion culture for a period of 20 days.…”

“…This goal has been pursued by a series of investigators who described distinct strategies in this sense, which in many cases lie in controlling the progress of the cells throughout the cell cycle . Cell cycle arrest in the G1 phase has been induced by starvation of an essential nutrient (Seifert and Phillips, 1999), by the addition of DNA synthesis inhibitors (Al-Rubeai et al, 1992;Bonarius et al, 1999Hayter et al, 1992Makishima et al, 1992), by genotoxic agents (Gartenhaus et al, 1996), and by a temperature shift (Jenkins and Hovey, 1993;Kaumann et al, 1999). Also, various metabolic engineering approaches have been applied.…”