Improving cultivation processes for recombinant protein production

An new cascade control system is presented that reproducibly keeps the cultivation part of recombinant protein production processes on its predetermined track. While the system directly controls carbon dioxide production mass and carbon dioxide production rates along their setpoint profiles in fed-batch cultivation, it simultaneously keeps the specific biomass growth rates and the biomass profiles on their desired paths. The control scheme was designed and tuned using a virtual plant environment based on the industrial process control system SIMATIC PCS 7 (Siemens AG). It is shown by means of validation experiments that the simulations in this straightforward approach directly reflect the experimentally observed controller behaviour. Within the virtual plant environment, it was shown that the cascade control is considerably better than previously used control approaches. The controller significantly improved the batch-to-batch reproducibility of the fermentations. Experimental tests confirmed that it is particularly suited for cultivation processes suffering from long response times and delays. The performance of the new controller is demonstrated during its application in Escherichia coli fed-batch cultivations as well as in animal cell cultures with CHO cells. The technique is a simple and reliable alternative to more sophisticate model-supported controllers.     

Protein release and foaming in Escherichia coli cultures grown in minimal medium

Protein release was studied in Escherichia coli cultivations in minimal medium under different conditions. The energy source concentration was oscillating either due to the cultivation technique or due to an applied on/off feed rate concept in fed-batch cultivations. It was found that the magnitude of protein release was dependent on the cultivation technique and the strain. The use of batch technique resulted in highest specific rate of protein release compared to fed-batch cultivations. No dependence of protein release on oscillating glucose concentration could be distinguished with oscillating periods of minutes of carbon starvation. Proteins released by cells acted as foaming agents and caused stabilisation of foam, during cultivation of Escherichia coli grown in minimal medium. Since the total cell protein was reflected in the medium the protein release is considered to be caused by cell lysis. However, only a few dominating proteins were present in the foam. The work was supported by grants from the Nordic Programme on Bioprocess Engineering under the auspices of NI, the Nordic Fund for Technology and Industrial Development; and from NUTEK, the Swedish National Board for Industrial and Technical Development.     

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

The enzyme controlled substrate delivery cultivation technology EnBase(?) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.     

Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.     

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.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol

Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.     

A sensitive monitoring system for mammalian cell cultivation processes: a PAT approach

Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 10⁵ cells ml^?1. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.     

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.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

A simple kinetic model for myeloma cell culture with consideration of lysine limitation

A simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.     

Measurement of oxygen uptake and carbon dioxide production rates ofmammalian cells using membrane mass spectrometry

A method for the measurement of oxygen uptake and carbon dioxide production rates in mammalian cell cultures using membrane mass spectrometry is described. The small stirred reactor with a volume of 15 ml and integrated pH-control permits the economical application of isotopically labelled substrates and ¹³C-labelled bicarbonate buffer. Repetitive experiments showed the reproducibility of the method. In one case bicarbonate-free HEPES buffer was used and carbon dioxide production was measured using the intensity of the peak at m/z = 44(¹²CO₂). In all other cases H¹³CO₃ ⁻ -buffer was applied and also¹²CO₂ was measured. The minimum cell density required was only 2 × 10⁴ cells ml⁻¹. In the hybridoma T-flask cultivation studied here the measured specific oxygen uptake and carbon dioxide production rates were reasonably constant during the exponential growth phase and decreased significantly afterwards. Estimated respiratory quotients were always between0.90 and 0.92 except in HEPES-buffer, where a value of 0.67 was found. In the latter case specific oxygen uptake rate was higher than in bicarbonate buffered culture, however, carbon dioxide production rate was lower, and viable cell density was lowest. The addition of phenazine methosulfate, an artificial electron acceptor, increased both rates resulting in highest viable cell density but also highest lactate production rate. Glucose and glutamine pulse-feeding increased final cell density. The method described is directly applicable for samples from batch, fed-batch and continuous cultivations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process

Various mechanistic and black-box models were applied for on-line estimations of viable cell concentrations in fed-batch cultivation processes for CHO cells. Data from six fed-batch cultivation experiments were used to identify the underlying models and further six independent data sets were used to determine the performance of the estimators. The performances were quantified by means of the root mean square error (RMSE) between the estimates and the corresponding off-line measured validation data sets. It is shown that even simple techniques based on empirical and linear model approaches provide a fairly good on-line estimation performance. Best results with respect to the validation data sets were obtained with hybrid models, multivariate linear regression technique and support vector regression. Hybrid models provide additional important information about the specific cellular growth rates during the cultivation.     

Generic model control of the specific growth rate in recombinant Escherichia coli cultivations

Generic model control is shown to be a powerful tool for keeping a microbial cultivation process close to its predetermined (optimized) control profile. This is demonstrated at the example of the green fluorescent protein expressed in genetically modified Escherichia coli host cells. It is shown that the process can be run very closely to a predefined complex profile of the specific cell growth rate mu(t). Controlling the experiments at many different growth conditions is a straightforward way of effectively collecting the data necessary for optimization of recombinant protein production systems. Although the process dynamics is rather complex, the model for the controller can be kept quite simple. The control technique, used here for specific growth rate control, is quite universal and can be applied for different biotechnological processes as well.     

Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations

The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100–19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole–Cole, and linear modeling were applied and compared in order to predict VCD. The Cole–Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole–Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole–Cole modeling.     

Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Simultaneous use of urea and potassium nitrate for Arthrospira (Spirulina) platensis cultivation

Urea has been considered as a promising alternative nitrogen source for the cultivation of Arthrospira platensis if it is possible to avoid ammonia toxicity; however, this procedure can lead to periods of nitrogen shortage. This study shows that the addition of potassium nitrate, which acts as a nitrogen reservoir, to cultivations carried out with urea in a fed-batch process can increase the maximum cell concentration (X(m) ) and also cell productivity (P(X) ). Using response surface methodology, the model indicates that the estimated optimum X(m) can be achieved with 17.3 mM potassium nitrate and 8.9 mM urea. Under this condition an X(m) of 6077 ± 199 mg/L and a P(X) of 341.5 ± 19.1 mg L(-1) day(-1) were obtained.     

Electronic nose for estimation of product concentration in mammalian cell cultivation

The off-gas composition from perfusion cultivation of a CHO-cell line producing recombinant human blood coagulation Factor VIII is monitored with an electronic nose. It is shown that the electronic nose in combination with an artificial neural network can be used for on-line estimation of the Factor VIII concentration in production-scale cultivations. The obtained prediction error (1†) for the Factor VIII concentration was 1.1 IU/ml. The potential of the electronic nose for estimation of viable cell count is outlined in laboratory-scale Factor VIII cultivations. The obtained prediction error (1†) for the viable cell count was 0.4᎒⁶ cells/ml. The results show that this non-invasive method is potentially useful for on-line bioprocess monitoring.