Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding

An automated glucose feeding strategy that avoids acetate accumulation in cultivations of Escherichia coli is discussed. We have previously described how a probing technique makes it possible to detect and avoid overflow metabolism using a dissolved oxygen sensor. In this article these ideas are extended with a safety net that guarantees that aerobic conditions are maintained. The method is generally applicable, as no strain-specific information is needed and the only sensor required is a standard dissolved oxygen probe. It also gives the highest feed rate possible with respect to limitations from overflow metabolism and oxygen transfer, thus maximizing bioreactor productivity. The strategy was implemented on three different laboratory-scale platforms and fed-batch cultivations under different operating conditions were performed with three recombinant strains, E. coli K-12 UL635, E. coli BL21(DE3), and E. coli K-12 UL634. In spite of disturbances from antifoam and induction of recombinant protein production, the method reproducibly gave low concentrations of acetate and glucose. The ability to obtain favorable cultivation conditions independently of strain and operating conditions makes the presented strategy a useful tool, especially in situations where it is important to get good results on the first attempt.     

Optimal Bioreactor Operational Policies for the Enzymatic Hydrolysis of Sugarcane Bagasse

The consolidation of the industrial production of second-generation (2G) bioethanol relies on the improvement of the economics of the process. Within this general scope, this paper addresses one aspect that impacts the costs of the biochemical route for producing 2G bioethanol: defining optimal operational policies for the reactor running the enzymatic hydrolysis of the C6 biomass fraction. The use of fed-batch reactors is one common choice for this process, aiming at maximum yields and productivities. The optimization problem for fed-batch reactors usually consists in determining substrate feeding profiles, in order to maximize some performance index. In the present control problem, the performance index and the system dynamics are both linear with respect to the control variable (the trajectory of substrate feed flow). Simple Michaelis–Menten pseudo-homogeneous kinetic models with product inhibition were used in the dynamic modeling of a fed-bath reactor, and two feeding policies were implemented and validated in bench-scale reactors processing pre-treated sugarcane bagasse. The first approach applied classical optimal control theory. The second policy was defined with the purpose of sustaining high rates of glucose production, adding enzyme (Accellerase® 1500) and substrate simultaneously during the reaction course. A methodology is described, which used economical criteria for comparing the performance of the reactor operating in successive batches and in fed-batch modes. Fed-batch mode was less sensitive to enzyme prices than successive batches. Process intensification in the fed-batch reactor led to glucose final concentrations around 200 g/L.     

Model-based optimization of a sequencing batch reactor for biological nitrogen removal

An optimal operating mode for a sequencing batch reactor was determined via a model-based optimization. Synthetic wastewater containing mainly organic matter (as glucose) and nitrogen (as ammonium chloride) was treated without any addition of an external carbon source to accomplish denitrification step. A simplified model was used to describe process dynamics, comprised of six ordinary differential equations and an empirical correlation for oxygen consumption rate. Batch cycle time was the chosen objective function to be minimized for a fixed volume of waste to be treated. Furthermore, as SBR operation is divided in two major phases - aerobic and anoxic, to achieve total pollutants removal within minimum time, these phases can be repeatedly alternated. To ensure availability of organic matter necessary for denitrification, these two phases were combined with feed steps. Different feed strategies were tested using one, two or three feed steps. A successive quadratic programming algorithm was used, and maximum values for final COD, nitrate and ammonium concentrations, as well as maximum feed pump flow rate were some the process constraints. One step feed strategy was indicated by the optimization leading to a batch cycle time of 5h.     

Controlled pilot development unit-scale fed-batch cultivation of yeast on spruce hydrolysates

Yeast production on hydrolysate is a likely process solution in large-scale ethanol production from lignocellulose. The hydrolysate will be available on site, and the yeast has furthermore been shown to acquire an increased inhibitor tolerance when cultivated on hydrolysate. However, due to over-flow metabolism and inhibition, efficient yeast production on hydrolysate can only be achieved by well-controlled substrate addition. In the present work, a method was developed for controlled addition of hydrolysate to PDU (process development unit)-scale aerobic fed-batch cultivations of Saccharomyces cerevisiae TMB 3000. A feed rate control strategy, which maintains the ethanol concentration at a low constant level, was adapted to process-like conditions. The ethanol concentration was obtained from on-line measurements of the ethanol mole fraction in the exhaust gas. A computer model of the system was developed to optimize control performance. Productivities, biomass yields, and byproduct formation were evaluated. The feed rate control worked satisfactorily and maintained the ethanol concentration close to the setpoint during the cultivations. Biomass yields of 0.45 g/g were obtained on added hexoses during cultivation on hydrolysate and of 0.49 g/g during cultivation on a synthetic medium with glucose as the carbon source. Exponential growth was achieved with a specific growth rate of 0.18 h-1 during cultivation on hydrolysate and 0.22 h-1 during cultivation on glucose.     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.     

基于差分进化算法的酿酒酵母分批补料培养在线自适应控制

酿酒酵母分批补料培养中,葡萄糖添加过量会导致乙醇大量积累,破坏细胞结构及功能,降低葡萄糖利用效率;葡萄糖添加不足会限制细胞生长。为解决这一矛盾,提出了一种基于差分进化算法的在线自适应控制策略,并利用计算机仿真方法对该策略、传统的间歇流加、分段恒速流加及PID控制策略的控制性能进行了研究和比较。结果表明,在该控制策略下,发酵液中的乙醇浓度能够被稳定地控制在1g/L的低水平,而细胞浓度却达到34.45g/L的高水平,比采用间歇流加、分段恒速流加及PID控制策略的批次分别提高了243%、18%和29%。由此可知,该自适应控制策略能够将葡萄糖流加速率控制在适宜水平,避免乙醇过量积累的同时保证细胞的快速增殖。     

High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii

The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring.     

Online detection of feed demand in high cell density cultures of Escherichia coli by measurement of changes in dissolved oxygen transients in complex media

A starvation-based dissolved oxygen (DO) transient controller was developed to supply growth-limiting substrate to high cell density fed-batch cultures of recombinant Escherichia coli. The algorithm adjusted a preexisting feed rate in proportion to the culture's oxygen demand, which was estimated from transients in the DO concentration after short periods of feed interruption. In this manner, the addition of glucose feed was precisely controlled at a rate that did not exceed the acetate production threshold, thus preventing acetate accumulation. In comparison to exponential feed algorithms commonly used in industry, the implementation of the new feeding strategy increased the final cell density from 32 to 44 g (dry cell weight).L(-1), with less than 16 mM acetate accumulated, producing an ideal culture for subsequent induction. Despite a constant starvation level and relatively low levels of acetate, experimental cultivations still tended to produce acetate towards the end of the process. The use of a simple Monod model provided an explanation as to why this may occur in high cell density cultivations and suggests how it may be overcome.     

Benchmarking of commercially available CHO cell culture media for antibody production

In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.     

Feeding strategies for <Emphasis Type="Italic">E. coli</Emphasis> fermentations demanding an enriched environment

The addition of a carbon nutrient feed to a fed-batch cultivation is often not enough to obtain satisfactory growth and/or production. In some cases, an additional feed with for example supplementary amino acids or complex media is required. This work presents the development of feeding strategies where more than one feed is required and the knowledge of the growth requirements is low. Simulations and cultivations with E. coli are shown using the proposed feed controllers which are based on a probing control concept. The strategies work well and they can be used to shorten the process development phase considerably.     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

Adapted ultra scale-down approach for predicting the centrifugal separation behavior of high cell density cultures

The work presented here describes an ultra scale-down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5- to 10-fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous-flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to approximately 2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell-cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot-scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large-scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale-up, are discussed.     

The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell densityEscherichia coli fermentations

Summary Two kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period.     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

On‐Line Control of Glucose Concentration in High‐Yielding Mammalian Cell Cultures Enabled Through Oxygen Transfer Rate Measurements

Glucose control is vital to ensure consistent growth and protein production in mammalian cell cultures. The typical fed‐batch glucose control strategy involving bolus glucose additions based on infrequent off‐line daily samples results in cells experiencing significant glucose concentration fluctuations that can influence product quality and growth. This study proposes an on‐line method to control and manipulate glucose utilizing readily available process measurements. The method generates a correlation between the cumulative oxygen transfer rate and the cumulative glucose consumed. This correlation generates an on‐line prediction of glucose that has been successfully incorporated into a control algorithm manipulating the glucose feed‐rate. This advanced process control (APC) strategy enables the glucose concentration to be maintained at an adjustable set‐point and has been found to significantly reduce the deviation in glucose concentration in comparison to conventional operation. This method has been validated to produce various therapeutic proteins across cell lines with different glucose consumption demands and is successfully demonstrated on micro (15 mL), laboratory (7 L), and pilot (50 L) scale systems. This novel APC strategy is simple to implement and offers the potential to significantly enhance the glucose control strategy for scales spanning micro‐scale systems through to full scale industrial bioreactors.     

Effective extracellular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis

An automated two-component substrate feeding strategy with a pH-stat modal fed-batch culture using a high pH limit was developed to effectively porduce esterase from a hyperprotein exreting Bacillus brevis HPD31 harboring a plasmid pHSC131 which carries a Bacillus stearothermo philus esterase gene. First, the effect of single- and multi-substrate feedings on the growth and activity of the excreted esterase was investigated. Then a two-component (polypepton + glucose) feeding using different feed rates was studied. Highest activity of the excreted esterase (34 U/mL) was obtained when the concentrations of poly-pepton and glucose in the nutrient feed solution were 250 and 41.60 g/L respectively. The absence and excessive amount of glucose in the nutrient feed solution was ineffective for the exracellular esterase formation because without glucose the increase in cell concentration was minimum while excessive amount of glucose favored cell growth at the expense of the esterase production. It is believed that the mechanism of enzyme excretion is growth dependent and that a higher cell growth of the host is in effect unfavorable for the enzyme production. The feed rate, automatically controlled by the direct signal of the pH change, at 0.30 mL/pulse was found optimum for the esterase production while lower (0.15 mL/pulse) and higher (0.67 mL/pulse) feed rates did not produce good results. The activity of the excreted esterase was increased more than eight times from 4 U/mL obtained in the conventional batch culture to 34 U/mL obtained in this study. The esterase productivity was likewise increased more than threefold. (c) 1992 John Wiley & Sons, Inc.     

Modelling the effects of glucose feeding on a recombinant E. coli fermentation

A simple unstructured model is described and compared with experimental data for fed-batch fermentations. The process studied is typical of the most common industrial recombinant fermentation in which temperature induced E. coli produces high yields of a heterologous protein (4?g met-asp-Bovine Somatotropin/l) using a complex peptone feed and a glucose feed. The model accurately predicts the effect of glucose feeding and shows that there is an optimal glucose feedrate to maximize productivity. At higher glucose feedrates, acetate levels become inhibitory and at lower levels glucose starvation occurs. The model is novel since it combines terms for acetate production and inhibition, effect of peptone feeding and temperature induction into a single relatively simple unstructured model. However the present model is unable to predict the effect of induction at different cell densities and reasons for this are suggested.     

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.