Bioprocess Biosyst Eng (2004) 26: 347351 DOI 10.

1007/s00449-004-0383-z

M IN I R E V I E W

Carl-Fredrik Mandenius

Recent developments in the monitoring, modeling and control of biological production systems

Received: 5 May 2003 / Accepted: 13 July 2004 / Published online: 5 August 2004 Springer-Verlag 2004

Abstract Current trends in the development of methods for monitoring, modeling and controlling biological production systems are reviewed from a bioengineering perspective. The ability to measure intracellular conditions in bioprocesses using genomics and other bioinformatics tools is addressed. Devices provided via micromachining techniques and new real-time optical technology are other novel methods that may facilitate biosystem engineering. Mathematical modeling of data obtained from bioinformatics or real-time monitoring methods are necessary in order to handle the dense ows of data that are generated. Furthermore, control methods must be able to cope with these data ows in ecient ways that can be implemented in plant-wide computer communication systems. Keywords Genomics and proteomics Chemometrics Multivariate process control In situ optical sensors Miniaturized devices Control models

For all life science-oriented applications, the description of the biological system is obtained by observing it using a variety of information methods 1, 2. These are adapted from general chemical, physical or biological analytical methods. Once recorded, the information obtained from these methods is processed into a form that the observer can interpret in order to draw conclusions about the state of biological molecules, metabolism or physiology, either at a research laboratory or in a production plant. The intended use for this information: for decision-making, to draw conclusions about the biological state for automatic control actions, or to initiate specic activities in experimentation, production, regulatory work or elsewhere, for instance, is the decisive factor when choosing the approach used to acquire the information [3] (Fig. 1).

Biological systems
Deeper insights into cell metabolism and physiology call for better and more precise analytical tools for monitoring the progression of growth and dierentiation of cells. The timescales for the biological events that occur in the system set specic requirements on the measurements, limitating the analytical methods that can be used (Fig. 2). If these limits could be extended, in both timescale directions, new valuable information would in many cases be unravelled. When cells are reengineered genetically or metabolically for heterologous protein or gene vector production, the processing of the genetic information in the cell from the gene sequences to the expressed products needs to be resolved at a scale of detail that allows us to estimate the activity of the cells global control systems and their molecular responses. This makes more sophisticated analysis necessary. During biological production processes, there is also the need to detect very minute quantities of compounds, mostly biological molecules (1) in order to meet regulatory safety requirements, and (2) to allow the early

Introduction
With the scientic and industrial development of modern biotechnology, better methods of measurement, monitoring and modeling have become necessary, in order to generate more detailed information about the biosystems so that they can be better understood and controlled. To large extent, the focus so far has been on biotechnological production processes, but recent work in other elds of life science that have also benetted from the genomics research has widened the scope of possible applications.
Mini-review for the proceedings of the M3C conference C.-F. Mandenius Division of Biotechnology, Linko ping University, 581 83 Linko ping, Sweden E-mail: cfm@ifm.liu.se

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Fig. 1 Biological systems are complex measurement environments that require advanced information systems (sensors and other instrumentation combined with ecient evaluation methods) to generate data for automatically- or manually-controlled systems

detection of adverse intracellular states, as revealed by specic signalling molecules like ppGpp [4]. Another example is the need for better infectant control, which is dicult for bacterial systems and even more demanding for cell lines of mammalian origin. Important steps toward a better understanding of microbial physiology during bioprocessing have been taken by using gene-chip technology. This has already resulted in valuable insight into intracellular stress response interrelationships in popular host cell organisms such as E. coli [5] and Bacillus sp. [6]. Recent observations of intercellular communication during high density cultivationso-called quorum sensingunder production conditions have spread new light on the interplay between intracellular and extracellular events in bioreactor cultures [7].

Advances in biological measurement technology

Today there are many inventive examples of methods carried out on the micro-scale that allow nano-volume handling of nucleic acids, proteins or other cellular materials. Although these methods contribute to many elds of molecular science, the micro-devices are
Fig. 2 Event timescales and detection limits are decisive to the information that can be obtained from observing a system using a particular technique. The value of the technique is related to the justin-time concept, which requires in-depth understanding of the nature of the biological system

particularly useful for analyzing biological systems in bioprocess engineering applications in order to capture information on transient changes related to stressed production situations. Micro-dimensional designs for DNA arrays that favor analytical convenience and high-speed, and are therefore highly suited to process development, or even production, seem highly desirable. Designs that utilize electronics for detection in combination with miniaturization are being developed in several labs that are investigating cultivation problems [8, 9]. Interesting demonstrations of how the microsystems can be extended to encompass the entire uidic path from pretreatment to nal detection of complex biological samples have been shown [10, 11]. This would be of great value not only during clinical analysis but also when analyzing crude samples from bioreactors thereby providing shorter delays as well as high-throughput options. A further development of the micro-device array concept to include specic protein ligands (protein microarrays) based on sensitive labeling methods or mass detection, for example with surface plasmon resonance, should probably be realized when suitable antibody fragment libraries become available. The readiness of biotech companies to develop novel sensor devices for bioanalysis is apparent within companies like Ciphergen, Agilent, Biacore and Amersham Biosciences. Table 1 lists a few of these devices based on miniaturization technologies that are already on the market and that have great potential for bioprocess analysis. Progress has been made with conventional analytical techniques as well. Fibre optics make it possible to apply established optical methods on-line, if sterilizable bre probes are tted close to or inside the bioreactor. These probes also facilitate in situ spectral scanning in the bioreactor. When using multi-wavelength uorescence,

349 Table 1 Commercial micro-devices for biotechnological applications Technique SELDI ProteinChip PicoLabChip Biacore Nanochip Genechip GyroLab Principle Surface enhanced laser desorption/ionization technology applied on protein arrays Lab-on-a-chip technology, integrated multifunction uidic systems Surface plasmon resonance Electronic microarray that preconcentrates sample Hybridization uorescence detection Lab-on-a-chip technology, CD design, MALDI MS detection Vendor Ciphergen Biosystems Inc, CA Agilent Technologies Inc. Biacore Intrl AB, SE Nanogen Inc., San Diego, CA Aymetrix Inc. CA Gyros AB, SE Application Separation, detection and analysis of proteins at the femtomole level Complex samples, DNA/RNA, proteins Biomolecular interaction RNA/DNA detection DNA/RNA Proteins

monitoring of components in the culture medium can be performed by applying multivariate analysis methods to the dense information ow obtained when scans are compared over a whole cultivation run, as shown with yeast and Bacillus cultivations [12, 13]. Cell properties can be analyzed in real-time using in situ microscopy in combination with advanced image analysis [14]. Image analysis, and in particular the ability to link it to data in order to evaluate production states on-line, provides a valuable tool that has the potential to be integrated into the production plant environment. Another in situ technique that can be applied to bioreactor monitoring in real-time is radio-frequency impedance measurement. The technique quanties cell viability based on physical principles that still are under debate. Applications to E. coli, yeast cells and mammalian cells are reported in the literature [15].

spectrum where selected wavelength numbers are correlated with one or several analytes in the medium by multi-linear least square regression models. Studies in bacterial and mammalian cell cultures have shown that these methods can easily be adapted to real-time in situ monitoring under a number of conditions that are realistical for use in industry [16,17]. However, calibration of the models is time-consuming, and recalibration may be necessary, especially when medium conditions change during a batch. Analytical methods based on ow cytometry signals from cell cultures continue to improve steadily, in particular through the use of powerful computer imaging [18]. The study of necrosis and apoptosis and their roles in cell culture processing may benet considerably from these methods, hopefully contributing to suppress these phenomena in production processes [19].

Data evaluation in biological measurement technology

Data obtained from several of the measurement and monitoring methods used in biotechnology can be interpreted more eectively by applying advanced data processing and data analysis. Mathematical methods, including multivariate analysis and chemometrics as well as statistical methods, are already valuable tools for exploiting the massive volumes of data obtained from microarrays, scanning spectrometers, and multi-sensor devices. An advantage of these methods is that sampling the data ow with one analytical procedure normally allows the construction of a model that can handle several analytical variables simultaneously, for example medium concentrations of biomass, nutrient components, or excreted by-products. Furthermore, many of the methods are suitable for non-invasive real-time monitoring, as with mass spectrometers and electronic noses, or in situ probes, as with spectroscopic methods with bre optics. However, to use these methods, it is necessary to build robust and reliable calibration models that are precise enough for the measurement purpose, which requires skill. Examples of successful applications are the use of infrared spectroscopy in the near- or mid-regions of the

Modeling in high level data interpretation

Biochemical engineering science has provided numerous mathematical models that describe the relationships of cells in the bioreactor environment. Most of the models are unstructured, but an increasing number of structured models are appearing. Tying these models to advanced measurement methods in order to control the biological system in the bioreactor gives varying results, depending on how well the models describe those parts of the biosystem that are of relevance. This makes model-related research based on classical as well as unconventional methods very tempting. The detailed modeling of specic organisms under various cultivation regimes and with pertinent assumptions of metabolic conditions is a necessary development, but must also be done in a realistic way in terms of experimental eort, as is further discussed by other authors in this issue of the journal. Control models that take these considerations into account can, for example, be created by dividing the process into one part that contains linear relations between substrates and metabolites, and another part which describes the cell growth, as has been demonstrated for yeast fed-batch fermentation [20].

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To model biological processes from rst engineering principles is a challenge, especially when new genomics and proteomics data are taken into account. Models for optimization, monitoring and control can be improved signicantly by using on-line data from the process: so-called data-driven modeling [21]. This can be based on either multivariate correlation models for monitoring, time series models, or models that combine the rst engineering principles with corrective data. These approaches have been shown to be useful in practical applications, such as for correlation models in yeast fedbatch or continuous cultivations, and for predictive time series models in wastewater treatment [21, 22]. Modeling based on principal component analysis (PCA) has been applied for a variety of purposes. Examples of its use range from studies into the optimization of the composition of organic phases in biotransformation [23], to the interpretation of multivariate data from spectroscopic instruments such as multiwavelength uorimetry [24, 13], near-infrared spectroscopy [17] and pyrolysis mass spectrometry [25]. A new approach intending to expand the range beyond that normally attained by multivariate and neural network (ANN) models is being persued by using more structured model systems like those provided by evolutionary algorithms. Results with the lamentous fungus S. erythraea indicate a good ability to model growth and product kinetics using such methods, which could impact on the above-described control models [26]. More experimental investigations of the dierent modeling approaches are important in order to test their robustness in real applications. Evaluating the modeling methods with unusual organisms as micro-algae in photo-bioreactors is an example of an investigation that broadens the experience and applicability of general growth and product models [27].

Better control of bioproduction by improved monitoring and modeling

The link between data communications in large networks is often the critical factor when converting many of the ne academic research results into practical industrial engineering. The investment in time and eort
Fig. 3 Modeling is a key tool for linking the monitoring and control of biological systems

required to achieve plant-wide computer systems for coping with large data ows from plant bioreactors and purication units can easily become too big for many biotech companies. Reports describing large-scale architectures achieved for such systems are few and farbetween [28]. Furthermore, to realize bioprocess control with dierent instrumentation and dierent control methods, complex network communication systems are required [29]. Monitoring and control methodology in such systems has only been demonstrated to a limited extent; for example with trajectory-based control of recombinant processes [30]. The integration of heuristic knowledge from the plant environment into models may be performed, as suggested by Lu bbert, using hybrid supervisor systems [31]. This expands our ability to represent the production process by incorporating additional modeling techniques into classical models. For example, other techniques may be included on top of a backbone of basic mass balances, such as ANNs with fuzzy reasoning. Alternatively, the modeling tools could more clearly link to knowledge about the physiology of the cells in a way that takes advantage of the latest insights into the cell signalling systems via mRNA and signal molecule data. This could allow us to measure just-in-time, using methods such as ngerprinting from mass spectrometry. For example, a much better prediction of the physiological states of a production culture could be reached using pyrolysis mass spectrometry (PyMS), which would lead to better monitoring and control [32]. In conclusion, the eld of monitoring, modeling and control of biological processes is steadily developing. Genomics and micro-machining techniques, as well as new optical techniques, present a more detailed and highly resolved picture of the biosystems. Mathematical models are becoming more important. Access to more computational power has improved the chances of being able to make use of these dierent methods in daily practice. Future development will most probably be typied by further progress in these directions. Integration of modeling at dierent stages in data processing and control of the biosystems will consequently play a key role, with signicant potential (Fig. 3).

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