Konstantin Konstantinov

Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells

Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0 degrees C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 x 10(6) cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 328-338, 1997.

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

The accurate, on-line measurement of cell concentration in animal-cell cultures is an on-going problem in bioprocess engineering, and the development of new monitoring techniques is an area of intensive and fruitful research. This article summarizes the recent advances, trends and problems in this field and focuses, in particular, on optical sensors, including the latest laser and infrared probes. Alternative methods, such as multiple-extinction fluorimetry, real-time imaging and particle-size analysis, are also discussed. Although many of these techniques are still at an experimental stage, we believe that some of them have been developed sufficiently that we advocate their routine use in bioprocess monitoring and control.

Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production

Abstract The efficiency of the fed-batch cultivation of recombinant Escherichia coli AT2471 for phenylalanine production is highly dependent on the distribution of the carbon flow between the main process products — biomass, phenylalanine, acetic acid and carbon dioxide. In order to enhance the process performance, the effects of several factors, namely glucose feeding, tyrosine feeding and oxygen supply, were investigated experimentally. As a result, a set of control strategies was developed, designed to tolerate phenylalanine synthesis at the expense of the remaining products. The DO was controlled to prevent acetic acid excretion due to oxygen limitation. The total amount of tyrosine fed was used to provide an optimal balance between biomass synthesis and that of phenylalanine. Special algorithms for control of the glucose feed rate were applied to eliminate the threat of acetic acid excretion due to overfeeding, and at the same time, to reduce excessive CO 2 evolution caused by unnecessarily severe glucose limitation. The joint application of these strategies resulted in greatly improved efficiency in the phenylalanine production process: the final phenylalanine concentration reached 46 g/ l , the yield was above 17%, and the productivity-0.85 g/ l ·h. In combination, these data exceed the results reported by others, and are much higher than those obtained by use before the implementation of the proposed complex of techniques.

The business impact of an integrated continuous biomanufacturing platform for recombinant protein production

The biotechnology industry primarily uses batch technologies to manufacture recombinant proteins. The natural evolution of other industries has shown that transitioning from batch to continuous processing can yield significant benefits. A quantitative understanding of these benefits is critical to guide the implementation of continuous processing. In this manuscript, we use process economic modeling and Monte Carlo simulations to evaluate an integrated continuous biomanufacturing (ICB) platform and conduct risk-based valuation to generate a probabilistic range of net-present values (NPVs). For a specific ten-year product portfolio, the ICB platform reduces average cost by 55% compared to conventional batch processing, considering both capital and operating expenses. The model predicts that these savings can further increase by an additional 25% in situations with higher-than-expected product demand showing the upward potential of the ICB platform. The ICB platform achieves these savings and corresponding flexibility mainly due to process intensification in both upstream and downstream unit operations. This study demonstrates the promise of continuous bioprocessing while also establishing a novel framework to quantify financial benefits of other platform process technologies.

Dielectric measurement to monitor the growth and the physiological states of biological cells

Measurement of capacitance, also referred to as dielectric permittivity, is a new method of estimating the concentration of cells, monitoring the growth and detecting the physiological changes during the cultivation of organisms in various bioprocess. Several types of biological cells were studied, namely; Saccharomyces cerevisiae, Escherichia coli, Perilla frutescens (plant cells) and AFP-27 hybridoma cells. Generally, a linear correlation between cell capacitance (C) and other biomass measurement technique such as optical density (OD) and dry weight (DW) was obtained using the different types of cell suspension. Therefore, this method could be used to monitor the growth of the organism during the active growth. It could be conveniently used to make a rapid estimate of the cell concentration such as in plant cell suspension culture. The capacitance sensor could also be designed to be installed and autoclaved in-situ in a bioreactor and used for on-line monitoring of cell growth. On the other hand, distinct deviations in the capacitance value were observed in relation with the growth stage of the organism. This was observed in all the organisms studied but the type of deviation depends on the physiology of the organism. This variation in cell capacitance showed the possibility of using this method as a means to indicate changes in the physiological state of cells during cultivation. This capability would be very useful in designing control strategies that would depend on the physiological states in the bioprocess.

Glucose feeding strategy accounting for the decreasing oxidative capacity of recombinant Escherichia coli in fed-batch cultivation for phenylalanine production

Abstract The glucose feed rate is known to be a key factor in the control of various fed-batch processes. It plays a special role in the cultivation of Escherichia coli strains because of their inherent ability to readily excrete inhibitory metabolites, particularly acetic acid, when an inappropriate feeding strategy is adopted. Here we present the results of our study on the peculiarities of acetic acid excretion by recombinant Escherichia coli AT2471 in fed-batch cultivation for phenylalanine production. In addition to confirming that under aerobic conditions acetic acid excretion is a function of the glucose feeding strategy, we found that the excretion is closely related to the decreasing oxidative capacity of the cell population. This is clearly expressed by a declining dynamical profile of the so called “critical specific glucose uptake rate”, which discriminates the process field into distinct regions. We have concluded that the actual specific feed rate should always be dynamically adjusted below the critical limits, otherwise the oxidative capabilities of the population will be exceeded and acetate will be excreted. To provide the required time-course of the specific glucose feed rate, a closed-loop control algorithm was developed which utilizes a useful empirical relationship. Its application resulted in a suitable profile of the specific glucose feed rate, prevention of acetic acid excretion and improved phenylalanine synthesis, the final concentration of which reached 46 g/l.

On-line monitoring of representative structural variables in fed-batch cultivation of recombinant Escherichia coli for phenylalanine production

During cultivation, the cell population often undergoes severe physiological transformations which can be interpreted as structural alterations in the process. In such cases, adequate control can be achieved only after accounting for these phenomena. A natural basis for accomplishing this task (often called diagnostics) is offered by pattern recognition theory. Practically, its application requires selection and calculation of a set of variables, jointly capable of describing the structural state of the process. This set can be used consequently to monitor the process structural alterations, allowing the control system to react in a timely and suitable manner. Because of the well-known characteristics of biological processes, the problem of selection of representative structural variables is far from being trivial, and requires special attention. Here we present our experience in the definition and calculation of such variables in the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production.

An Expert Approach for Control of Fermentation Processes as Variable Structure Plants

Abstract Structural variability is a fundamental property of biological systems. Fermentation processes, in particular, are often accompanied by physiological phenomena which have the characteristics of structural alterations. The conventional control approach does not have the competence to handle such processes, because it is based on the general assumption for a single-structure plant. In order to overcome this limitation, we consider under some conditions fermentation processes as variable structure plants and propose a two-level hierarchical scheme for their control. At the higher hierarchical level, which performs the organizing functions and operates in the structural space of the plant, the current plant structure is recognized as an element of a finite set of structures defined on the basis of ‘deep’ expert knowledge of the cell physiology and/or ‘shallow’ expert knowledge of the behavioral characteristics of the microbial population. The recognition is based on fuzzy decomposition of the structural space of the plant into several subspaces, mapped isomorphically into a respective set of rational control strategies. A set of expert rules is used for the actual synthesis of the recognition procedure, which is finally tuned by supervised learning. The adopted methodology at this hierarchical level provides the system with a kind of intelligence. To the lower hierarchical level, two distinct functions are assigned. The first one performs the global task of control of the plant structure and operates in its structural space. The second function has a local character and consists of control of the plant in a particular subspace of constant structure. It works in the state space of the plant. Under supervision of the higher level, the control strategy relevant to the current plant structure is picked out from a predefined pool and the corresponding control action is calculated. Extensive use of expert knowledge in the conceptual formulation of proper control strategies is assumed. Compared with the conventional single-structure approach, the variable structure concept results in rational and physiologically motivated control of fermentation processes. Instead of the development of a single but complicated process model and control structure, construction of several simple control structures, often in a ‘model-less’ form, is required. This method naturally incorporates and theoretizes the widely accepted policy of artificial induction of structural transformations in the plant, which is essential for enhanced productivity in many modern fermentation technologies. The proposed approach has been realized as a real-time software system, provided with enough flexibility to be used with different fermentation processes and equipment. This system is orientated to IBM personal computers and works under the QNX multi-tasking real-time operating system.

Knowledge-based systems, artificial neural networks and pattern recognition: applications to biotechnological processes.

Recent years have witnessed the increasing application of artificial intelligence techniques, specifically, knowledge-based systems, artificial neural networks, and pattern recognition, to biotechnological processes. Although progress has been made in simple control applications, more work is needed to establish the advantages of these techniques for industrial process control, for diagnosis/monitoring, and to upgrade the information content of historical data.

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.