Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel® disk carriers was determined. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clinical and early clinical trials. It was originally operated with a perfusion of 2.6vvd during production phase in order to supply the high cell density (2.5×107cellml−1 of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a reduction of the medium perfusion rate by −25% and −50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clinical trials with an improved second-generation process. With a −25% reduction of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a −30% loss of productivity was obtained when the medium perfusion rate was further reduced to −50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in reference conditions. Finally, a reduction of −25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the molecule, while reducing costs of media, material and manpower of the production process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the production phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufacturing operation

Endothermic microbial growth. A calorimetric investigation of an extreme case of entropy-driven microbial growth

Life is almost always associated with the generation of heat. Thus far, all chemotrophic life forms that have been studied in calorimeters were found to be exothermic. Certain literature reports have even cast doubt on the existence of endothermic growth, even though thermodynamic principles do not rule it out. The present report describes the first experiments demonstrating the actual existence of chemotrophic life forms that take up heat rather than produce i

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I2281

The influence of residual ethanol on metabolism of food grade Gluconacetobacter xylinus I2281 was investigated during controlled cultivations on 35g/l glucose and 5g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidation of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35g/l glucose and 5g/l acetate. It was found that acetyl CoA was activated by the acetyl coenzyme A synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calculation showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis liked to ethanol catabolism. This calculation showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethano

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

In the present study, the optimal medium perfusion rate to be used for the continuous culture of a recombinant CHO cell line in a packed-bed bioreactor made of Fibra-Cel disk carriers was detd. A first-generation process had originally been designed with a high perfusion rate, in order to rapidly produce material for pre-clin. and early clin. trials. It was originally operated with a perfusion of 2.6 vvd during prodn. phase in order to supply the high cell d. (.apprx.2.5*107 cell ml-1 of packed-bed) with sufficient fresh medium. In order to improve the economics of this process, a redn. of the medium perfusion rate by -25% and -50% was investigated at small-scale. The best option was then implemented at pilot scale in order to further produce material for clin. trials with an improved second-generation process. With a -25% redn. of the perfusion rate, the volumetric productivity was maintained compared to the first-generation process, but a -30% loss of productivity was obtained when the medium perfusion rate was further reduced to -50% of its original level. The protein quality under reduced perfusion rate conditions was analyzed for purity, N-glycan sialylation level, abundance of dimers or aggregates, and showed that the quality of the final drug substance was comparable to that obtained in ref. conditions. Finally, a redn. of -25% medium perfusion was implemented at pilot scale in the second-generation process, which enabled to maintain the same productivity and the same quality of the mol., while reducing costs of media, material and manpower of the prodn. process. For industrial applications, it is recommended to test whether and how far the perfusion rate can be decreased during the prodn. phase - provided that the product is not sensitive to residence time - with the benefits of reduced cost of goods and to simplify manufg. operations. [on SciFinder (R)

Optimal Control of Fed-Batch Fermenters

Optimal control of fed-batch fermenters S. Valentinotti† C. Cannizzaro‡ M.Rhiel‡ U. Holmberg† U. von Stockar‡ D. Bonvin† †Institut d’Automatique, EPFL, 1015 Lausanne, Switzerland ‡Institut de Genie Chimique, EPFL, 1015 Lausanne, Switzerland Fermentors are often run in a fed-batch manner to avoid the formation of overflow metabolites. At a high growth rate, the most efficient metabolic pathway(s) of certain microorganisms become saturated resulting in overflow metabolite production. These byproducts are undesirable since their accumulation in the reactor may be inhibitory and the productivity of biomass and growth-associated products is reduced. The ideal way to run such fed-batch fermentation is to grow the cells in the reactor at the critical growth rate, i.e., the point at which overflow metabolite production begins. However, since this value changes from run to run, or even during a given fermentation, its identification is not trivial. A simple way to overcome this difficulty is to maintain a very small, but constant overflow metabolite concentration in the reactor, ensuring that most of the substrate is consumed efficiently. However due to exponential cell growth, standard controllers can maintain a constant concentration only for a limited time period. In this work an adaptive control strategy to maintain a constant overflow metabolite concentration in fed-batch fermentation is presented. The proposed approach requires the knowledge of only two system parameters: the yield coefficient, expressing the relation between overflow metabolite and substrate, and the instantaneous concentration of the overflow metabolite. Baker’s yeast fed-batch experiments were performed with the ob jective of maximizing biomass productivity and minimizing ethanol production. Mid-infrared spectroscopy was used to measure the ethanol concentration that was provided on-line to the controller. The results from numerous experiments have demonstrated the effectiveness of the proposed control strategy. The specific growth rate was maintained constant, at a value close to the critical point, until oxygen transfer limitation occurred. Then, the controller automatically reduced the feed rate to prevent excess ethanol production. The biomass increased from 0.5 to 65 grams per liter during the exponential growth phase. Simulation results based on this control strategy show its applicability to other overflow metabolite organisms, such as Escherichia coli

Influence of residual ethanol concentration on the growth of Gluconacetobacter xylinus I 2281

The influence of residual ethanol on metab. of food grade Gluconacetobacter xylinum I 2281 was investigated during controlled cultivations on 35 g/l glucose and 5 g/l ethanol. Bacterial growth was strongly reduced in the presence of ethanol, which is unusual for acetic acid bacteria. Biomass accumulated only after complete oxidn. of ethanol to acetate and carbon dioxide. In contrast, bacterial growth initiated without delay on 35 g/l glucose and 5 g/l acetate. It was found that acetyl CoA was activated by the acetyl CoA synthetase (Acs) pathway in parallel with the phosphotransacetylase (Pta)-acetate kinase (Ack) pathway. The presence of ethanol in the culture medium strongly reduced Pta activity while Acs and Ack remained active. A carbon balance calcn. showed that the overall catabolism could be divided into two independent parts: upper glycolysis linked to glucose catabolism and lower glycolysis linked to ethanol catabolism. This calcn. showed that the carbon flux through the tricarboxylic cycle is lower on ethanol than on acetate. This corroborated the diminution of carbon flux through the Pta-Ack pathway due to the inhibition of Pta activity on ethanol. [on SciFinder (R)