Optimization and robustness analysis of hybridoma cell fed-batch cultures using the overflow metabolism model

The maximization of biomass productivity in fed-batch cultures of hybridoma cells is analyzed based on the overflow metabolism model. Due to overflow metabolism, often attributed to limited oxygen capacity, lactate and ammonia are formed when the substrate concentrations (glucose and glutamine) are above a critical value, which results in a decrease in biomass productivity. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder–Mead simplex optimization algorithm. The optimal multi exponential feed rate trajectory improves the biomass productivity by 10 % as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolic state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, to model structure errors.     

Feed rate control in fed‐batch fermentations based on frequency content analysis

A new strategy for controlling substrate feed in the exponential growth phase of aerated fed‐batch fermentations is presented. The challenge in this phase is typically to maximize specific growth rate while avoiding the accumulation of overflow metabolites which can occur at high substrate feed rates. In the new strategy, regular perturbations to the feed rate are applied and the proximity to overflow metabolism is continuously assessed from the frequency spectrum of the dissolved oxygen signal. The power spectral density for the frequency of the external perturbations is used as a control variable in a controller to regulate the substrate feed. The strategy was implemented in an industrial pilot scale fermentation set up and calibrated and verified using an amylase producing Bacillus licheniformis strain. It was shown that a higher biomass yield could be obtained without excessive accumulation of harmful overflow metabolites. The general applicability of the strategy was further demonstrated by implementing the controller in another process using a Bacillus licheniformis strain currently used in industrial production processes. In addition, in this case a higher growth rate and decreased accumulation of overflow metabolites in the exponential growth phase was achieved in comparison to the reference controller. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:817–824, 2013     

Characterization of the metabolic burden on Escherichia coli DH1 cells imposed by the presence of a plasmid containing a gene therapy sequence

The presence of a plasmid, containing gene sequences for DNA immunotherapy that are not expressed in microbial culture, imposed a degradation in bioreactor performance in cultures of the host E. coli strain. Significant decreases in growth rate (24%) and biomass yield (7%) and a corresponding increase in overflow metabolism were observed in a strain containing a therapeutic sequence (a hepatitis B antigen under the control of a CMV promotor). The observed increase in overflow metabolism was incorporated into a Metabolic Flux Analysis (MFA) model (as acetate secretion). Metabolic flux analysis revealed an increase in TCA cycle flux, consistent with an increased respiration rate observed in plasmid-containing cells. These effects are thought to result from increased ATP synthesis requirements (24%) arising from the expression of the Kanr plasmid marker gene whose product accounted for 18% of the cell protein of the plasmid-containing strain. These factors will necessitate significantly higher aeration and agitation rates or lower nutrient feed rates in high-density cultures than would be expected for plasmid-free cultures.     

Optimization of biphasic growth of Saccharomyces carlsbergensis in fed-batch culture

The optimal glucose feeding policy for the fed-batch culture of Saccharomyces carlsbergensis is presented. The biphasic nature of growth results in a singular feed rate policy that is unique to this organism. When the operating cost is high, the reduction in operating time forces the cells to utilize both glucose and ethanol toward the end of fermentation time and results in a decreasing rate of glucose addition, unlike the normally observed in creasing feed rate. The optimal feeding policy depends heavily on the initial conditions and is highly sensitive to changes in kinetic parameters. A semiempirical scheme for feedback optimization is suggested for the fed-batch yeast culture.     

Approximation of continuous fermentation by semicontinuous cultures

Semicontinuous fermentations, in which a fraction of a culture is replaced with fresh media at regular intervals, have been previously used as a means of approximating continuous growth. In most cases deviations from continuous operation were erroneously estimated using Fencl's model, which is only valid when the specific growth rate is independent of the substrate concentration. An approach to modeling Semicontinuous growth that incorporates the same kinetics followed in batch and continuous growth was developed and tested for Monod's expression for the specific growth rate. A dimensionless form of the model was used to simulate Semicontinuous fermentations for comparison to continuous growth. Differences between Semicontinuous and continuous growth were found to depend on three dimensionless variables: feed concentration, replacement rate, and time between replacements. For given values of the dimensionless feed concentration and time between replacements, a range of dimensionless replacement rates can be determined over which semi-continuous cultures are approximately continuous.     

A nonsingular optimization approach to the feed rate profile optimization of fedbatch cultures

A new method to calculate the optimal feed rate profile for fedbatch culture is proposed. Instead of the usual singular control approach of taking the feed rate as the control variable, the substrate concentration profile is used as the transformed control variable to avoid the computational difficulty associated with the singular control. Thus, the problem is converted into a nonsingular optimization problem of determining the optimal substrate concentration profile subject to a constraint. The equivalent feed rate profile to match the optimal substrate concentration profile is then generated. With this method the computational difficulty associated with singular controls for high-order systems is circumvented. The proposed method is illustrated by a number of examples.     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg).

A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-associated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 0.12-0.18 h-1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fed-batch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product formation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-1 culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth.     

Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition

Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. Copyright 1998 John Wiley & Sons, Inc.     

Substrate oscillations boost recombinant protein release from Escherichia coli

Intracellular production of recombinant proteins in prokaryotes necessitates subsequent disruption of cells for protein recovery. Since the cell disruption and subsequent purification steps largely contribute to the total production cost, scalable tools for protein release into the extracellular space is of utmost importance. Although there are several ways for enhancing protein release, changing culture conditions is rather a simple and scalable approach compared to, for example, molecular cell design. This contribution aimed at quantitatively studying process technological means to boost protein release of a periplasmatic recombinant protein (alkaline phosphatase) from E. coli. Quantitative analysis of protein in independent bioreactor runs could demonstrate that a defined oscillatory feeding profile was found to improve protein release, about 60 %, compared to the conventional constant feeding rate. The process technology included an oscillatory post-induction feed profile with the frequency of 4 min. The feed rate was oscillated triangularly between a maximum (1.3-fold of the maximum feed rate achieved at the end of the fed-batch phase) and a minimum (45 % of the maximum). The significant improvement indicates the potential to maximize the production rate, while this oscillatory feed profile can be easily scaled to industrial processes. Moreover, quantitative analysis of the primary metabolism revealed that the carbon dioxide yield can be used to identify the preferred feeding profile. This approach is therefore in line with the initiative of process analytical technology for science-based process understanding in process development and process control strategies.     

A first test of a new modelling approach to estimate food consumption in particle-feeding fish

Models for estimating food consumption in fish by analysing changes in stomach fullness over time are invariably based on a stomach evacuation rate obtained when the fish is fasting, on the assumption that this rate also applies to when the fish is feeding. However, this often is not the case in fish that feed on small particles. A new modelling approach was therefore tested, which is based not only on stomach fullness but also on gut contents. To eliminate errors arising from assimilation in the gut, titanium(IV) oxide (TiO₂) was used as an indigestible marker. When applied to a dataset obtained from tilapia given several equal doses of pelleted feed over a 2.5‐h period, the new approach gave a closer true consumption estimate than a conventional model. The evacuation rate proved to be a more sensitive parameter than the ingestion rate, but the former was no longer required by the new approach for estimating ingestion, thus liberating the food consumption estimate from any errors and dependencies inherent in the evacuation rate. The new approach assumes that the digesta of previous feedings can be distinguished from those of the feeding phase being analysed and therefore needs further refinement for those cases when this does not apply. Suggestions for such refinements are also given. This new approach is expected to be equally suitable for estimating consumption in stomachless fish.     

Fed-batch culture automated by uses of continuously measured cell concentration and culture volume

An automated control method of fed-batch culture in which the nutrient feed rate was determined from continuously measured cell concentration and culture broth volume was developed. Theoretical background was elucidated, from which it was found that the method is unique in that it controls specific substrate consumption rate of the microorganism. The method was experimentally applied to the fed-batch cultures of recombinant Escherichia coli HB101. It was observed that the specific substrate feed rate affects not only the specific growth rate but also the growth yield. If some conditions are satisfied, this type of automated fedbatch culture can be applied widely to any microbial systems and seems especially useful when the culture medium is composed of natural complex nutrient(s) because their concentrations are very difficult to detect and control.     

Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides

This work is related to the set-up of overflowing exponential fed-batch cultures (O-EFBC) derived from carbon limited EFBC dedicated to the production of mycosubtilin, an antifungal lipopeptide belonging to the iturin family. O-EFBC permits the continuous removal of the product from the bioreactor achieving a complete extraction of mycosubtilin. This paper also provides a dynamical Monod-based growth model of this process that is accurate enough to simulate the evolution of the specific growth rate and to correlate it to the mycosubtilin specific productivity. Two particular and dependant phenomena related to the foam overflow are taken into account by the model: the outgoing flow rate of a broth volume and the loss of biomass. Interestingly, the biomass concentration in the foam was found to be lower than the biomass concentration in the bioreactor relating this process to a recycling one. Parameters of this model are the growth yield on substrate and the maximal specific growth rate estimated from experiments led at feed rates of 0.062, 0.071 and 0.086h(-1). The model was extrapolated to five additional experiments carried out at feed rates of 0.008, 0.022, 0.040, 0.042 and 0.062h(-1) enabling the correlation of the mean specific growth rates with productivity results. Finally, a feed rate of 0.086h(-1) corresponding to a mean specific growth rate of 0.070h(-1) allowed a specific productivity of 1.27mg of mycosubtiling(-1) of dried biomassh(-1).     

流加发酵提高产胡萝卜素红酵母产量的研究

本文对产胡萝卜素的红酵母进行了间歇、恒速及变速流加发酵实验,建立了简单的数学模型控制流加。实验结果表明,变速流加可显著地提高红酵母的产量,当流加控制因子K为88×10－5,变速流水发酵比间歇发酵红酵母产量提高了56．2％。     

Growth rate control in fed-batch cultures of recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg)

Summary A recombinant Saccharomyces cerevisiae producing hepatitis B surface antigen (HBsAg) exhibited growth-assciated product formation. By controlling the medium feed rate, based on the calculated amount of medium required for 1 h, a constant specific growth rate was obtained in the range of 1.12-0.18 h^–1. In order to prolong the exponential growth phase, the medium feed rate was increased exponentially. A fedbatch cultivation method based on the production kinetics of batch culture enhanced HBsAg production ten times more than in batch culture. The reason for the increase can be explained by the fact that the production of HBsAg is expressed as an exponential function of time when the specific growth rate is controlled to a constant value in growth-associated product fromation kinetics. In the scale-up of this culture to 91, the specific growth rate could also be maintained constant and the HBsAg production trend was similar to that in a 1-l culture. However, ethanol accumulation occurred at a late stage in fed-bach culture. Ethanol produced was not reutilized and inhibited further cell growth. Offprint requests to: M. B. Gu     

Effect of cycling on final mixed culture fate

Cycling in feed substrate concentration and dilution rate is examined as a means of modifying the final fate of a mixed culture. It is shown for the case where the specific growth rate of one species is always greater than that of the second that no cycling strategy will provide the desired extinction of the faster growing species unless time delay is included in the modeling. To account for the time lag in adjusting organism metabolic activities to environmental changes, an adaptability parameter is introduced. Numerical simulations are carried out and an operating diagram indicating the conditions under which the desired extinction occurs is constructed. Cycling in feed substrate concentration and dilution rate are both found to produce the desired result.     

Run-to-run fed-batch optimization for protein production using recombinant Escherichia coli

A two-phase design approach is introduced to determine the optimal feed rate, fed glucose concentration and fermentation time to maximize protein productivity using recombinant Escherichia coli BL21 (pBAW2) strain. The first phase is applied to determine a primary S-system kinetic model using batch time-series data. Two runs were carried out in the second phase to achieve the maximum protein productivity for the fed-batch fermentation process. The computational results using the S-system kinetic model obtained from the second run are in better agreement with the experiments than those using the kinetic model obtained from batch time-series data. For cross-validation, two extra fed-batch experiments with different feed strategies were carried out for comparison with the optimal fed-batch result. From the experimental results, this approach could improve productivity by at least 3%.     

Modeling of ferrous iron oxidation by a Leptospirillum ferrooxidans-dominated chemostat culture

The objective of this study was to evaluate a direct classical bioengineering approach to model data generated from continuous bio-oxidation of Fe(2+) by a Leptospirillum ferrooxidans-dominated culture fed with either 9 g or 18 g Fe(2+) L(-1) under chemostat conditions (dilution rates were between 0.051 and 0.094 h(-1)). The basic Monod and Pirt equations have successfully been integrated in an overall mass balance procedure, which has not been previously presented in this detail for Fe(2+) oxidation. To ensure chemostat conditions, it was found that the range of the dilution rates had to be limited. A too long retention time might cause starvation or non-negligible death rate whereas, a too short retention time may cause a significant alteration in solution chemistry and culture composition. Modeling of the experimental data suggested that the kinetic- and yield parameters changed with the overall solution composition. However, for respective feed solutions only minor changes of ionic strength and chemical speciation can be expected within the studied range of dilution rates, which was confirmed by thermodynamic calculations and conductivity measurements. The presented model also suggests that the apparent Fe(3+) inhibition on specific Fe(2+) utilization rate was a direct consequence of the declining biomass yield on Fe(2+) due to growth uncoupled Fe(2+) oxidation when the dilution rate was decreased. The model suggested that the maintenance activities contributed up to 90% of the maximum specific Fe(2+) utilization rate, which appears close to the critical dilution rate. Biotechnol. Bioeng. 2008;99: 378-389. (c) 2007 Wiley Periodicals, Inc.     

Respirometric evaluation and modeling of glucose utilization by Escherichia coli under aerobic and mesophilic cultivation conditions

The study presents a mechanistic model for the evaluation of glucose utilization by Escherichia coli under aerobic and mesophilic growth conditions. In the first step, the experimental data was derived from batch respirometric experiments conducted at 37 degrees C, using two different initial substrate to microorganism (S(0)/X(0)) ratios of 15.0 and 1.3 mgCOD/mgSS. Acetate generation, glycogen formation and oxygen uptake rate profile were monitored together with glucose uptake and biomass increase throughout the experiments. The oxygen uptake rate (OUR) exhibited a typical profile accounting for growth on glucose, acetate and glycogen. No acetate formation (overflow) was detected at low initial S(0)/X(0) ratio. In the second step, the effect of culture history developed under long-term growth limiting conditions on the kinetics of glucose utilization by the same culture was evaluated in a sequencing batch reactor (SBR). The system was operated at cyclic steady state with a constant mean cell residence time of 5 days. The kinetic response of E.coli culture was followed by similar measurements within a complete cycle. Model calibration for the SBR system showed that E. coli culture regulated its growth metabolism by decreasing the maximum growth rate (lower microH) together with an increase of substrate affinity (lower K(S)) as compared to uncontrolled growth conditions. The continuous low rate operation of SBR system induced a significant biochemical substrate storage capability as glycogen in parallel to growth, which persisted throughout the operation. The acetate overflow was observed again as an important mechanism to be accounted for in the evaluation of process kinetics.     

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed‐batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top‐down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1023–1033, 2013     

Bioreactor hydrodynamic effect on Escherichia coli physiology: experimental results and stochastic simulations

A microorganism circulating in a bioreactor can be submitted to hydrodynamic conditions inducing a significant effect on its physiology. The mixing time exhibited by the stirred bioreactor and the circulation of microorganisms are both involved in this reacting system. The mixing component determines the intensity of the concentration gradient and the circulation component determines the way in which the microorganism is exposed to this gradient. These two components linked to the experimental evaluation of microbial physiology can be analysed by a structured stochastic model in the case of a partitioned or “scale-down” reactor (SDR). A stochastic model indeed enables to simulate the mixing process as well as the circulation of microorganisms in SDRs. The superimposition of mixing and circulation processes determines the concentration profile experienced by a microorganism in the reactor. In the present case, the glucose concentration experienced by Escherichia coli has been modelled during a fed-batch culture. In this context, the use of a stochastic hydrodynamic model has permitted to point out an interesting feed pulse retardant effect in the SDRs. Nevertheless, the metabolic response of E. coli is not easy to interpret because of the possible simultaneous developments of overflow metabolism and mixed acid fermentation induced by the strong glucose concentration in the reactor.