Impact of aeration strategy on CHO cell performance during antibody production

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub‐lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air–liquid interface: bubbled direct gas sparging and a non‐bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F‐68 (PF‐68). Cells were unable to grow with direct gas sparging without PF‐68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF‐68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F‐actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub‐lethal physiological change in cells that would not be detected by the at‐line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.     

通气对乳酸发酵过程的影响

对拟干酪乳杆菌发酵产乳酸的过程进行研究,通过改变不同的通气量(不通气、0.1vvm、0.2 vvm、0.5 vvm)确定0.1vvm的通气量最有利于产生乳酸;再通过优化通气策略,在发酵0～15 h不通空气,15～50 h通0.1 vvm空气使得乳酸的产量比全程通0.1 vvm空气又提高了11.7%,同时乳酸产率也提高了16.2%。最后通过对胞内NAD~+、NADH、乳酸脱氢酶和NADH氧化酶活性、以及发酵过程氧化还原电位(Oxidation-reduction potential,ORP)变化进行分析,阐述了通气影响乳酸发酵过程的机理。     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes

An aeration strategy was proposed for foam control in an airlift reactor with double wire mesh draft tubes. The airlift reactor was employed in the cultivation of Bacillus thuringiensis for thuringiensin production. The aeration strategy involved two situations. If the foam rose and touched the foam probe, the air flow rate was dropped to a low value for a certain period. However, if the DO value was already below 10% of the saturation when the air flow rate was dropped, the conventional foam control was employed. The production of thuringiensin based on the proposed strategy was up to 70% higher than that of using the conventional cultivation method with addition of antifoam agents for foam control.     

Production of proteolytic enzyme by Rhizopus oryzae in a bubble column bioreactor

Production of proteolytic enzyme in a bubble column bioreactor was carried out using the microorganism Rhizopus oryzae. Optimization of the production conditions were done adopting the EVOP (evolutionary operation technique). Studies on two variable and three variable parameters were carried out. The variable parameters were air flow rate, aeration time and incubation period. 8 hours aeration time per day, 1.66 vvm air flow rate and 2 days incubation was found to be optimum.     

Effect of culture conditions on monoclonal antibody production from genetically modified tobacco suspension cultures

Oxygen supply and inoculum age were found to affect the production of the heavy chain monoclonal antibody (HC MAb) from genetically modified tobacco suspension cultures. The increase of oxygen supply increased both cell growth and HC MAb production. Furthermore, the increased aeration and mixing improved the production of HC MAb based on the unit amount of cells or total soluble proteins. This indicated that the increased aeration improved the production and secretion of HC MAb more than other cell components. HC MAb production and cell growth also improved when batch cultures were inoculated with actively dividing cells (5-day old) rather than the fullygrown cells (7- or 10-day old cells) that are commonly used for subcultures. The addition of glutamine to the medium also improved cell growth and HC MAb production.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

A comparison of three different mammalian cell bioreactors for the production of monoclonal antibodies

Three different commercially available stirred tank reactors for mammalian cell culturing were compared for the ability to support hybridoma cell growth and monoclonal antibody production in batch mode operation. Despite quite similar vessel geometries differences were found both in growth and production profiles in the systems. These differences can possibly be related to the different aeration modes used in the bioreactors, and the levels of shear stress created by stirrer and agitator in the tanks.     

Effect of shear rate on hybridoma cell metabolism

The effect of shear rate on cell growth and monoclonal antibody production of hybridoma cells was studied. The dependence of agitation rate on antibody production is discussed by measuring the amount of monoclonal antibody in cells cultured by a spinner vessel. The effect of shear rate is also studied by exposing a homogeneous shear flow to hybridoma cells in a cone-and-plate viscometer. The dependence of shear rate on hybridoma cells was observed and the increase of antibody production was arised from the increase of secretion from cells.     

Immobilization of animal cells using photo-crosslinkable resin

A photo-crosslinkable resin, BIX12, was selected from among various photo-crosslinkable resins for the immobilization of animal cells. BIX12 had no cytotoxic effect on the growth of hybridoma cells and the production of monoclonal antibody, although other photo-crosslinkable resins had significant inhibitory effects. Using BIX12-alginate hybrid gel particles, hybridoma cells could grow in the resins and produce monoclonal antibody. For the continuous production of monoclonal antibody, perfusion culture using a fluidized-bed bioreactor with direct air bubbling was carried out. By this cultivation, monoclonal antibody could be produced stably for more than 50 d. A high viable cell density of more than 10⁷ cells/ml-gel was attained, and the antibody productivity was improved 8.5-fold compared with conventional suspension culture using a spinner flask. Anchorage-dependent cells were also immobilized in the resin particles by three immobilization procedures. Among these procedures, porous BIX12 formed by adding gelatin powder provided good support strength and allowed the cells to grow on the surface inside of the support.     

Effect of temperature on hybridoma cell cycle and MAb production

The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. (c) 1992 John Wiley & Sons, Inc.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Comparison of air-lift and stirred tank reactors for itaconic acid production by Aspergillus terreus

In a 2-l stirred tank reactor (STR), maximum production rate ofitaconic acid was 0.48g/l.h , for an agitation rate of 400 rpm andan aeration rate of 0.5 vvm. In an air-lift reactor (ALR) themaximum production rate was 0.64 g/l.h at an O supply rate of0.41 l O /l. min. Power input per unit volume which gave themaximum production rates for STR and ALR were 1180 and 542 W/m 3,respectively. If O -enriched air was used in place of air for ALR,the corre-sponding power input per unit volume was decreased to 34W/m 3 . ALR requires less power input per unit volume in comparisonwith that of STR whether therefore air or O -enriched air is used.ALR would be a suitable bioreactor for a large production of itaconicacid.     

Staphylococcal Enterotoxin B and Nuclease Production Under Controlled Dissolved Oxygen Conditions

Enterotoxin B, nuclease, and total exoprotein production by Staphylococcus aureus strain S-6 was studied in a 0.5-liter fermentor system. While these extracellular products were elaborated over a wide range of aeration rates, maximal production occurred within the very narrow range of 125 to 150 cm(3) of air per min. The levels attained at the optimal aeration rate were not increased by maintaining a constant pH, although yield of enterotoxin:cell mass was highest at a constant pH of 7.0. During the growth cycle of the cultures, when aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen (DO) levels, initially set at 100% of saturation, decreased to 5 to 10% 4 to 5 h after inoculation. The oxygen demand of the culture then maintained this level for an additional 4 to 6 h. This interval of low DO was characterized by maximal growth and exoprotein production. When the DO was controlled at a constant value throughout growth (by increasing or decreasing the airflow rate as appropriate), the culture demonstrated different optima for maximal growth and exoprotein production. A constant DO of 100% stimulated growth to extremely high densities, but the accumulation of toxin and nuclease was not observed. On the other hand, maintaining constant DO levels at 50 or 10% raised exoprotein levels higher than those achieved in a culture grown at the optimal aeration rate. Compared to the optimal aeration rate culture, the 10% DO culture yielded 20% more nuclease, 25% more toxin, and 40 to 50% more total exoprotein. These results indicate that it is the DO and not the aeration rate, per se, that is influential in controlling growth, toxin, nuclease, and total exoprotein production.     

Gas concentration and temperature gradients in a packed bed solid-state fermentor

In solid-state fermentation (SSF), interaction of heat and mass transfer with biochemical reaction (growth associated enzyme production) affects the bioreactor performance. This interaction was earlier observed to cause temperature and gaseous concentration gradients which reduced the effective bed height of the bioreactor. Since forced aeration is known to alleviate this problem, a packed column bioreactor with forced aeration was employed in the present study. Using wheat bran and Aspergillus niger CFTRI 1105, experiments were conducted for the production of the enzyme amyloglucosidase at various air flow rates. Temperatures and gas concentrations were recorded and enzyme activities estimated at different bed heights during the course of SSF. Gas concentration and temperature gradients decreased with increasing air flow rate. The packed column allowed the use of larger bed heights and yielded higher enzyme activities (6,260 Units/gDMB) than trays (345 Units/gDMB). Enzyme activity was affected more by temperature than concentration gradients, and increased with air flow rates.     

表达抗-CD25单克隆抗体的GS-NS0骨髓瘤细胞无血清培养及代谢特性

抗-CD25单克隆抗体作为免疫抑制剂拥有广阔的市场前景和巨大的经济价值。本实验以表达抗?CD25单克隆抗体的GS-NS0细胞为研究对象,开发了支持其大规模培养和抗体表达的无血清低蛋白培养基,批培养最大活细胞密度和最大抗体浓度分别达3×106cells/mL和300mg/L以上,比商业无血清培养基(Excell 620+0.2% primatone)分别提高了100%和46%。通过批培养实验,研究了细胞的生长、葡萄糖和氨基酸代谢、以及产物表达特点,并揭示了批培养过程中初始葡萄糖浓度对GS-NS0细胞生长与代谢的影响规律。为优化GS-NS0细胞培养过程和抗CD25单抗成功迈向产业化提供了重要的科学依据。     

<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis>: effect of oxygen on growth and acetic acid production

The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1). In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.     

Effect of aeration on the production of carotenoid pigments by Rhodotorula rubra-lactobacillus casei subsp. casei co-cultures in whey ultrafiltrate

Under intensive aeration (1.3 l/l min) the associated growth of Rhodotorula rubra GED2 and Lactobacillus casei subsp. casei in cheese whey ultrafiltrate (55 g lactose/l) proceeded effectively for both cultures with production of maximum carotenoids (12.4 mg/l culture fluid). For maximum amount of carotenoids synthesized in the cell, the yeast required more intensive aeration than the aeration needed for synthesis of maximum concentration of dry cells. Maximum concentration of carotenoids in the cell (0.49 mg/g dry cells) was registered with air flow rate at 1.3 l/l min, and of dry cells (27.0 g/l) at 1.0 l/l min. An important characteristic of carotenogenesis by Rhodotorula rubra GED2 + Lactobacillus casei subsp. casei was established--the intensive aeration (above 1.0 l/l min) stimulated beta-carotene synthesis (60% of total carotenoids).     

Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.     

Synthesis of staphylococcal enterotoxin A and nuclease under controlled fermentor conditions.

The production of enterotoxin A and nuclease by Staphylococcus aureus strain 100 was studied in a 1.0-liter fermentor. The effects of the gas flow rate, pH, and dissolved oxygen were evaluated. Toxin and nuclease secretion occurred under all conditions which permitted growth of the organism. Final yields of toxin and nuclease in cultures grown at constant air flow rates, ranging from 50 to 500 cm3 per min, were higher at successively higher flow rates. An optimum flow rate for either toxin or nuclease production was not observed. When the aeration rate alone or aeration rate and pH were held constant, the dissolved oxygen levels in the culture decreased from the initial 100% level to 0 to 5% 3 to 4 h after inoculation. The O2 demand of the culture then maintained this level for an additional 4 to 5 h. This low dissolved oxygen interval was characterized by rapid growth and extracellular protein production. Controlling the dissolved oxygen at a constant level throughout growth did not increase the final levels of toxin and nuclease above those achieved at the respective constant pH values. Growth under the influence of a constant aeration rate of 500 cm3 per min and a constant pH of 6.5 and 7.0 yielded the highest titers of nuclease (1,550 units/ml) and toxin (10.5 mug/ml) obtained in any of the fermentations conducted in this study. Sparging fermentor cultures with pure oxygen at a rate of 100 cm3 per min yielded growth and extracellular protein levels similar to those achieved at the sparge rate of 500 cm3 of air per min. Controlling the dissolved oxygen at 100% of pure oxygen saturation appeared to inhibit the culture, as the final cultural turbidity as well as the levels of toxin and nuclease were reduced. These data indicate that enterotoxin and nuclease secretions are closely associated with the growth of strain 100. Analyses of the production rates of these components indicated that early log phase was the most efficient production interval in the growth cycle and that this efficiency was increased by pH control at 6.7 to 6.8 and dissolved oxygen control at 10% of air saturation.