Control of fed-batch fermentations.

The “Pharmaceutical Current Good Manufacturing Practices (CGMPs) for the 21st Century—A Risk Based Approach” initiative announced by the FDA in August 2002 to improve and modernize pharmaceutical manufacturing facilitated adoption of process analytical technology (PAT) by the pharmaceutical industry. The potential for improved operational control and compliance resulting from continuous real-time quality assurance was highlighted as a likely benefit that would result from PAT implementation. A considerable amount of work has been done on this topic by academic and industrial contributors in the last decade. In this paper, we will start with a brief overview of evolution of PAT concepts and a review of their application in the wider pharmaceutical industry. The rest of the paper focuses on PAT applications for biotech processes with emphasis on developments in the last five years. It is our observation that while significant advances have been accomplished with regard to our ability to analyze/monitor key process and quality attributes in the biotech industry, much more needs to be done with regard to utilizing the collected data for subsequent control of the process, to achieve optimum yield and product quality. The latter is necessary to achieve the benefits that will result from PAT implementation.

Process analytical technology (PAT) for biopharmaceutical products.

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

Advances in animal cell recombinant protein production: GS-NS0 expression system

In this work, radio-frequency (RF) impedance is reviewed as a method for monitoring and controlling cell culture manufacturing processes. It is clear from the many publications cited that RF Impedance is regarded as an accurate and reliable method for measuring the live cell bio-volume both on-line and off-line and the technology is also sutable for animal cells in suspension, attached to micro-carriers or immobilized in fixed beds. In cGMP production, RF Impedance is being used in three main areas. Firstly, it is being used as a control instrument for maintaining consistent perfusion culture allowing the bioreactor to operate under optimum conditions for maximum production of recombinant proteins. In the second application it has not replaced traditional off-line live cell counting techniques but it is being used as an additional monitoring tool to check product conformance. Finally, RF Impedance is being used to monitor the concentration of live cells immobilized on micro-carriers or packed beds in cGMP processes where traditional off-line live cell counting methods are inaccurate or impossible to perform.

On-line Measurements and Control of Viable Cell Density in Cell Culture Manufacturing Processes using Radio-frequency Impedance

Productivity in a CHO perfusion culture reactor was maximized when pCO2 was maintained in the range of 30–76 mm Hg. Higher levels of pCO2 (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO2 production rates for CHO cells in perfusion culture at 5.55×10-17 mol cell-1 sec-1 and 5.36×10-17 mol cell-1 sec-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (kLa+kAA) for oxygen and carbon dioxide were 0.07264 min-1 and 0.002962 min-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×107 cells/mL, the low CO2 removal efficiency would limit culture density to only 2.4×106 cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO2 transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO2 levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO2) for a culture at 107 cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.

CO(2) in large-scale and high-density CHO cell perfusion culture.

https://www.cell.com/trends/biotechnology/pdf/0167-7799(94)90049-3.pdf

Real-time biomass-concentration monitoring in animal-cell cultures

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors.

In recent years, the development of advanced systems for bioprocess monitoring and control has become an area of intensive research. Along with traditional techniques, there are several new approaches which are increasingly being applied to bioprocess operations. Among these, of special note is expert system technology, which provides possibilities for the design of efficient bioprocess control systems with new functional capabilities. This technology has been successfully applied to variety of microbial processes at laboratory and industrial scale. The present paper analyzes the possibility for application of expert systems to animal cell cultures processes whose high complexity is well suited to expert control. The discussion focuses on the organization and the functionality of the intelligent control systems, and covers some practical aspects of their design.

Expert systems in the control of animal cell culture processes: Potentials, functions, and perspectives

Feeding strategies based on glucose limitation are becoming increasingly popular in mammalian cell cultivation. While until recently this approach has been limited to small-scale experiments, nowadays there is a considerable interest in its large-scale application. In order to investigate the overall effect of glucose limitation, we have applied this strategy to the industrial scale perfused cultivation of NSO myeloma cell line for antibody production. Glucose-limited and non-limited experiments were conducted in a 200 L sparged bioreactor, equipped with an external cell retention device. The experiments continued for several months at high cell density (20-60x106 cells/mL), which enabled the reliable monitoring of physiological phenomena with large time constants. A comprehensive set of process variables representing cell physiology and process efficiency were analyzed both in terms of statics and dynamics. Glucose limitation provides several important advantages, including control of the glucose metabolism, reduced lactate concentration, better medium utilization, and simple perfusion control. Apart from these advantages, however, glucose limitation provoked phenomena with profound effect on cell physiology and process efficiency. Based on our experimental data, it was concluded that glucose limited feeding has large potential for industrial application, but should be used cautiously, after careful evaluation of its long-term effect on the cell culture.

Advantages and disadvantages of glucose limitation in perfused mammalian cell cultures

Feeding strategies based on glucose limitation are becoming increasingly popular in mammalian cell cultivation. While until recently this approach has been limited to small-scale experiments, nowadays tbere is a considerable interest in its large-scale application. In order to investigate tbe overall effect of glucose limitation, we have applied this strategy to tbe industrial scale perfused cultivation of NSO myeloma cell line for antibody production. Glucose-limited and non-limited experiments were conducted in a 200 L sparged bioreactor, equipped witb an external cell retention device. The experiments continued for several months at high cell density (20-60xl06 cellslmL), which enabled tbe reliable monitoring of physiological phenomena with large time constants. A comprehensive set of process variables representing cell physiology and process efficiency were analyzed botb in terms of statics and dynamics. Glucose limitation provides several important advantages, including control of tbe glucose metabolism, reduced lactate concentration, better medium utilization, and simple perfusion control. Apart from these advantages, however, glucose limitation provoked phenomena with profound effect on cell physiology and process efficiency. Based on our experimental data, it was concluded tbat glucose limited feeding has large potential for industrial application, but should be used cautiously, after careful evaluation of its long-term effect on the cell culture.

Konstantin B. Konstantinov

Papers

Real-time biomass-concentration monitoring in animal-cell cultures

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors.

Expert systems in the control of animal cell culture processes: Potentials, functions, and perspectives

Advantages and disadvantages of glucose limitation in perfused mammalian cell cultures

ADVANTAGES AND DISADVANTAGES OF GLUCOSE LIMITATION IN PERFUSED MAMMALIAN CELL CULTURES: AlUIlysis of a Large-Scale, High-Density Myeloma Cultivation