Model-based optimization of viral capsid protein production in fed-batch culture of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

An optimized fed-batch cultivation process for the production of the polyoma virus capsid protein VP1 in recombinant Escherichia coli BL21 bacteria is presented. The optimization procedure maximizing the amount of desired protein is based on a mathematical model. The model distinguishes an initial cell growth phase from a protein production phase initiated by inducer injection. A new approach to model the target protein formation rate was elaborated, where product formation is primarily dependent on the specific biomass growth rate. Lower growth rates led to higher specific protein concentrations. The model was identified from a series of fed-batch experiments designed for parameter identification purposes and possesses good prediction quality. Then the model was used to determine optimal open-loop control profiles by manipulating the substrate feed rates in both phases as well as the induction time. Feed-rate optimization has been solved using Pontryagin's maximum principle. The solution was validated experimentally. A significant improvement of the process performance index was achieved.     

Increasing batch-to-batch reproducibility of CHO cultures by robust open-loop control

In order to guarantee the quality of recombinant therapeutic proteins produced in mammalian cell systems, the straightforward approach in industry is to run the processes as reproducible as possible. It is first shown that considerable distortions in the currently operated processes appear when the initial cell density deviates from its nominal value. Small deviations in the initial cell mass may lead to severe deviations from the desired biomass trajectory. Next, it is shown how to design a fed-batch production process in such a way that it is robust with respect to variations in the viable cell density. A simple open loop strategy is proposed for that purpose. Here we show for the first time at animal cell cultures (CHO cells) that by means of an appropriate glutamine feed rate profile F(t), which keeps the specific growth rate of the cells on a predefined value below its maximal value while maintaining the viabilities on a high level, the diverging viable cell count profiles change over into a robust converging set of profiles. The CHO cells used to validate the procedure could be focused to any specific growth rates below μ_max.     

Improving the batch-to-batch reproducibility of microbial cultures during recombinant protein production by regulation of the total carbon dioxide production

Batch-to-batch reproducibility of fermentation processes performed during the manufacturing processes of biologics can be increased by operating the cultures at feed rate profiles that are robust against typically arising disturbances. Remaining randomly appearing deviations from the desired path should be suppressed automatically by manipulating the feed rate. With respect to the cells' physiology it is best guiding the cultivations along an optimal profile of the specific biomass growth rate mu(t). However, there are two problems that speak for further investigations: Upon severe disturbances that may happen during the fermentation, the biomass concentration X may significantly deviate from its desired value, then a fixed mu-profile leads to a diminished batch-to-batch reproducibility. Second, the specific growth rate cannot easily be estimated online to a favourably high accuracy, hence it is difficult to determine the deviations in mu from the desired profile. The alternative discussed here solves both problems by keeping the process at the corresponding total cumulative carbon dioxide production-profile: it is robust against distortions in X and the controlled variable can accurately be measured online during cultivations of all relevant sizes. As compared to the fermentation practice currently used in industry, the experimental results, presented at the example of a recombinant protein production with Escherichia coli cells, show that CPR-based corrections lead to a considerably improved batch-to-batch reproducibility.     

Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.     

Process design for recombinant protein production based on the promoter,P<Subscript><Emphasis Type="Italic">malK</Emphasis></Subscript>

P_malK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate (cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: (1) catabolite derepression achieved from low glucose concentration and (2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg^–1 h^–1 and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.     

On the optimal control of fed-batch reactors with substrate-inhibited kinetics

The optimal feed rate profiles, for fed-batch fermentation that maximizes the biomass production and accounts for time, are analyzed. The solution can be found only if the final arc of the optimal control is a batch arc, since in this case the final concentrations of substrate and biomass can be determined by ulterior conditions on the mass balance and on the final growth rate of biomass and thus it is possible to solve the resulting time optimal problem by using Green's theorem. This evidences the "turnpike property" of the solution, which tries to spend the maximum time on or at least near the singular arc along which the substrate concentration is maintained constant. The optimality of the final batch arc is related to the time operational cost in the performance index. The sequence of the control depends on the initial conditions for which six different regions, with the respective patterns, have been identified, in case the performance index allows the control sequence to have a final batch.     

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.     

Effects of post-induction feed strategies on secretory production of recombinant streptokinase in Escherichia coli

The effects of post-induction nutrient feed rates, on recombinant streptokinase production in fed-batch processes, were investigated using various feed profiles. Recombinant streptokinase was produced using a secretory expression system and was induced by a temperature up-shift, using a temperature-sensitive λ_PL promoter. The specific growth rates decreased sharply upon induction of recombinant protein expression, thus necessitating a variable feed strategy in the post-induction phase. The various feed profiles employed in the post-induction phase included constant feed rates, linearly increasing feed rate and exponentially varying feed rates. Significant differences were obtained in the specific activity of streptokinase produced in these fed-batch processes. A maximum activity per unit biomass of 4.96 × 10⁶ and 4.43 × 10⁶ IU/g DCW was achieved for exponentially decreasing feed and linearly increasing feed, respectively. The decrease in specific growth rate during the post-induction phase was also less pronounced in these cases in comparison to other fed-batch experiments. It was observed that streptokinase productivity is governed by the nutrient feed rate per unit biomass at a critical juncture after induction. The highest activity per unit biomass was obtained when the nutrient feed rate per unit biomass was around 0.7–0.8 g glucose (g DCW)⁻¹ h⁻¹, between 2 and 4 h after induction.     

Effect of different inoculum levels of heterogeneous mixed culture in acidogenic fermentation

Summary Acidogenesis during fermentation by a heterogeneous mixed population inoculated at different initial biomass levels was studied. Butyric and lactic acid production and biomass formation were measured. Acidogenesis appeared to be a two step process, lactic acid being produced first, followed by butyric. Fermentation started more rapidly with higher levels of initial biomass, after which the fermentation rate remained unaffected indicating a change in the fermentation profile     

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.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations

The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated.     

红曲霉菌液相培养高产色素低产桔霉素的代谢调控研究

从8株固态发酵高产红曲色素菌株中筛选出一株适合于液相培养工艺的高产红曲色素菌株FL1,并对其液相培养过程特性及高产色素低产桔霉素的代谢调控策略进行了研究.发酵过程动力学研究结果表明,红曲色素与桔霉素作为次级代谢产物,其合成与红曲霉菌生长的关系属于非生长偶联型.本文首次采用连续补料的Fed-Batch培养技术,用于红曲霉...     

Two-dimensional fluorescence spectroscopy: a novel approach for controlling fed-batch cultivations

In industrial fed-batch cultivations it is often necessary to control substrate concentrations at a low level to prevent the production of overflow metabolites and thus optimize the biomass yield. A new method for on-line monitoring and fed-batch control based on fluorescence measurements has been developed. Via instantaneous in situ measurements and multivariate data analysis a chemometric model has been established, which enables the rapid detection of ethanol production at aerobic Saccharomyces cerevisiae fed-batch cultivations. The glucose feed rate is controlled by predicting the metabolic state directly from the fluorescence intensities. Thus, ethanol production could be avoided completely while increasing the biomass yield accordingly. The robust instrumentation is suitable for industrial applications.     

The quorum-sensing effect of aerobic granules on bacterial adhesion, biofilm formation, and sludge granulation

Quorum sensing (QS) through signal chemical molecules is known to be essential to bacterial adhesion and biofilm formation. In this study, the QS ability of aerobic granules—a special form of biofilms used for biological wastewater treatment—was investigated and compared with that of conventional activated sludge flocs. A novel sectional membrane bioreactor was used together with a flow-cell to evaluate the possible influence of signal chemicals produced by the source sludge on the growth mode of bacterial cells. The results demonstrate the apparent production of QS chemicals from granules and its impact on initial cell attachment and granule formation. When granules were used as the signal-producing biomass, the attached-growth mode was dominant for the free cells, and the biofilm formation rate in the flow-cell was about ten times faster than in cases which used activated sludge as the signal source biomass. In addition, the intracellular extract from mature granules significantly accelerated the sludge granulation process. It is argued that the production and expression of QS signal chemicals from granules and granule precursors might have induced the gene expression of bacteria in suspension for attached growth rather than suspended growth, leading to granule formation and its stable structure.     

Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate

The regulation of benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate was investigated in batch and continuous culture. In batch culture, R. pickettii PKO1 achieved a maximum specific growth rate with benzene of 0.18 h⁻¹, while succinate allowed much faster growth (μ_max = 0.5 h⁻¹). Under carbon excess conditions succinate repressed benzene consumption resulting in diauxic growth whereas under carbon-limited conditions in the chemostat both substrates were used simultaneously. Moreover, the effect of succinate on the adaptation towards growth with benzene was investigated in carbon-limited continuous culture at a dilution rate of 0.1 h⁻¹ by changing the inflowing carbon substrate from succinate to different mixtures of benzene and succinate. The adaptation process towards utilisation of benzene was rather complex. Three to seven hours after the medium shift biomass production from benzene started. Higher proportions of succinate in the mixture had a positive effect on both the onset of biomass production and on the time required for induction of benzene utilisation. Strikingly, after the initial increase in biomass and benzene-catabolising activities, the culture collapsed regularly and wash-out of biomass was observed. After a transient phase of low biomass concentrations growth on benzene resumed so that finally rather stable and high biomass concentrations were reached. The decrease in biomass and degradative activities cannot be explained so far, but the possibilities of either intoxication of the cells by benzene itself, or of inhibition by degradation intermediates were ruled out.     

Hybrid modeling as a QbD/PAT tool in process development: an industrial <Emphasis Type="Italic">E. coli</Emphasis> case study

Process understanding is emphasized in the process analytical technology initiative and the quality by design paradigm to be essential for manufacturing of biopharmaceutical products with consistent high quality. A typical approach to developing a process understanding is applying a combination of design of experiments with statistical data analysis. Hybrid semi-parametric modeling is investigated as an alternative method to pure statistical data analysis. The hybrid model framework provides flexibility to select model complexity based on available data and knowledge. Here, a parametric dynamic bioreactor model is integrated with a nonparametric artificial neural network that describes biomass and product formation rates as function of varied fed-batch fermentation conditions for high cell density heterologous protein production with E. coli. Our model can accurately describe biomass growth and product formation across variations in induction temperature, pH and feed rates. The model indicates that while product expression rate is a function of early induction phase conditions, it is negatively impacted as productivity increases. This could correspond with physiological changes due to cytoplasmic product accumulation. Due to the dynamic nature of the model, rational process timing decisions can be made and the impact of temporal variations in process parameters on product formation and process performance can be assessed, which is central for process understanding.