Temperature: A simple parameter for process optimization in fed-batch cultures of recombinant Chinese hamster ovary cells

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.

Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 98%

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

Trummer

Fauland

Seidinger

et al. 2006

“…In contrast to other environmental factors such as DOT and pH, temperature has been studied intensively (Bloemkolk et al, 1992;Chuppa et al, 1997;Fogolin et al, 2004;Fox et al, 2004;Furukawa and Ohsuye, 1998;Kaufmann et al, 1999;Moore et al, 1997;Rodriguez et al, 2005;Roessler et al, 1996;Sureshkumar and Mutharasan, 1991;Yoon et al, 2003aYoon et al, ,b, 2005. In our experiments, process temperature exerted a significant effect on cell growth.…”

Section: Effect Of Temperaturementioning

confidence: 97%

“…The dissolved oxygen tension (DOT) in the culture medium has also been shown to affect productivity, cell metabolism and glycosylation (Kunkel et al, 1998(Kunkel et al, , 2000Kurano et al, 1990;Lin et al, 1993;Link et al, 2004;Ozturk and Palsson, 1991). However, compared to pH and DOT the influence of temperature has been studied even more intensively (Andersen et al, 2000;Bloemkolk et al, 1992;Chuppa et al, 1997;Fogolin et al, 2004;Fox et al, 2004;Furukawa and Ohsuye, 1998;Kaufmann et al, 1999;Moore et al, 1997;Rodriguez et al, 2005;Roessler et al, 1996;Sureshkumar and Mutharasan, 1991;Yoon et al, 2003aYoon et al, , 2003bYoon et al, , 2005. Low temperature cultivation has been shown to significantly increase specific recombinant production rates and to arrest growth, while maintaining or improving product quality and reducing cell metabolism.…”

Section: Introductionmentioning

confidence: 96%

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo‐Fc expressing CHO cells cultivated in controlled batch bioreactors

Trummer

Fauland

Seidinger

et al. 2006

191

149

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

“…Large-scale production can be achieved by modifying the culture medium (Xie and Wang, 1994), extending culture time (Rossler et al, 1997;Ryu and Lee, 1992), increasing culture volume, enhancing the expression of target protein (Cherlet and Marc, 2000;Lamotte et al, 1999), and recycling or retaining the cultured cells in a bioreactor under a perfusion culture mode (Chotigeat et al, 1994;Woodside et al, 1998). Numerous approaches have been developed for mass production on the basis of batch, fed-batch and perfusion cultures (Van Der Velden De Groot, 1995;Woodside et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Erythropoietin production from CHO cells grown by continuous culture in a fluidized‐bed bioreactor

Wang

Yang

Huzel

et al. 2001

A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days. The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27. The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate. The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture. The EPO accumulated to a total production of 28,000 kUnits over the whole culture period. The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up. The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask. The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period. The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor. These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks.