Strategies for Improved dCO₂ Removal in Large‐Scale Fed‐Batch Cultures

Abstract

Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process. A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO(2) was measured using a fiber optic based probe. dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.). Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value. A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg). Titer at day 10 of culture improved by 1.5-fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.

Decreased pCO2 accumulation by eliminating bicarbonate addition to high cell‐density cultures

“…Elevated pCO 2 has been implicated in reduced growth, metabolism, and productivity in addition to adverse effects on glycosylation (Anderson and Goochee, 1994;Aunins and Henzler, 1993;deZengotita et al, 1998deZengotita et al, , 2002Garnier et al, 1996;Gray et al, 1996;Miller, 1996, 1997;Matanguihan et al, 2001;Mostafa and Gu, 2003;Zanghi et al, 1999;Zhu et al, 2005). As bioreactor pH during perfusion cultivation is controlled at a pre-defined set point, high pCO 2 results in increased osmolality, which can also negatively impact cell growth, metabolism, and productivity (deZengotita et al,et al, 1995;Øyaas et al, 1994;Lee, 1997, 1999;Zhu et al, 2005).…”

“…There is clearly a need for bioreactor pCO 2 reduction although there have been relatively few studies addressing pCO 2 removal and control in mammalian cell bioreactors (Gray et al, 1996;Matanguihan et al, 2001;Mostafa and Gu, 2003). Stripping is an obvious CO 2 removal approach, but it has a limited impact in mammalian cell bioreactors.…”

A Framework for the Development of Integrated and Computationally Feasible Models of Large-Scale Mammalian Cell Bioreactors

“…The ratio of rates of oxygen uptake to carbon dioxide production by cells has been reported to vary within a narrow range around 1 [56]. So the oxygen uptake rate (OUR) is assumed to be 0.35 pmol·cell −1 ·h −1 , which has been reported for carbon dioxide production [57]. The overall volumetric mass transfer coefficient, k L a, is calculated using Equation (7), in which U G op,q is superficial gas velocity (m/s) for computational cell q under operating condition op [33].…”

Process analytical technology (PAT) for biopharmaceutical products: Part II. Concepts and applications

“…] bioreactor (Kirdar et al, 2008). The detrimental effects of elevated bioreactor pCO 2 and osmolality on growth of CHO cells are well-documented in the literature (deZengotita et al, 1998;Gray et al, 1996;Mostafa and Gu, 2003;Nienow, 2006;Zhu et al, 2005).…”