Model based design of a biochemical cultivation process

In this paper a simple design procedure is used to enhance the performance of a biotechnical cultivation process. A model supported approach is proposed. It starts with a simple classical process model obtained from literature data, which is used to design the first experiment. Then, the main procedure is an iteration of (i) improving the model making use of the deviations between the experimental data and the data predicted by the model, (ii) of designing the next experiment by determining the optimal control profiles from the current model, and (iii) of executing that designed experiment. Important for the success of the procedure is that the model development is oriented at the process performance. The procedure is demonstrated at the simple practical example of a laboratory-scale fed-batch cultivation of Escherichia coli.     

Simple adaptive pH control in bioreactors using gain-scheduling methods

A simple well-performing adaptive control technique for pH control in fermentations of recombinant protein production processes is described and its design procedure is explained. First, the entire control algorithm was simulated and parameterized. Afterwards it was tested in real cultivation processes. The results show that this simple technique leads to significant reductions in the fluctuations of the pH values in microbial cultures at a minimum of expenditures. The signal-to-noise ratio and thus the information captured by the pH signal were increased by about an order of magnitude. This leads to a substantial improvement in the noise of many other process signals that are used to monitor and control the process. For instance, respiratory off-gas data of CO₂ and its derived carbon dioxide production rate signals from the cultures carry much less noise as compared to those values obtained with conventional pH control. Detailed process analysis revealed that even very small pH jumps of 0.03 values during the fermentation were shown to result in pronounced deflections in CO₂-volume fraction of 8% (peak to peak). The proposed controller, maintaining the pH within the interval of 0.01 around the setpoint, reduces the noise considerably.     

Optimization of CSTRs in series by dynamic programming

This article concerns the development of a simple yet effective procedure for optimizing the design of a reactor system employing CSTRs in series. The basic approach used in this work was to translate the problem of reactor design to a mathematical programming model. The resulting model was then solved by dynamic programming. The procedure was tested on an IBM 3033 computer and an IBM PC-compatible machine, the CORONA PC-II microcomputer. The results of this study indicate that the optimization procedure developed is very effective.     

The effect of transcendental mediation on right hemispheric functioning.

This study reports two experiments investigating the effects of transcendental mediation on right hemispheric functioning. The task used in both experiments was the Seashore Tonal Memory Test. In the first experiment a nonmediator group and an experienced mediator group were run. The denonmeditator group and an experienced mediator group were run. The design involved three periods: a pretest, a meditation or a rest period, and then a posttest. The results showed the experienced meditators were significantly better in both pretest and posttest performance. There were no pretest--posttest differences. The second experiment was done to replicate the first experiment and to control for possible selection bias. The design was the same as the first experiment, except that an additional group of inexperienced meditators was included. The results again showed significantly superior performance for the experienced meditators compared to the nonmeditators. In addition, the experienced meditators were superior to the inexperienced meditators. There were no significant differences between the nonmeditators and the inexperienced meditators. These results support the hypothesis that meditation facilitates right hemispheric functioning. Alternative explanations, such as selection bias, are also discussed.     

Internal model control for structured rank deficient system based on full rank decomposition

In this paper, an internal model control method is first proposed for structured rank deficient systems based on full rank decomposition. The system is first converted into a column full rank system by designing a pre-compensator. Then a feedback-compensator is designed to improve the dynamic characteristics of the full rank system and decrease the controller design difficulties. Rather than performing complex designing calculations, the pre- and feedback- compensators are designed by the full rank decomposition method. Furthermore, the non-square relative gain subsystem selection criterion is used to choose the square subsystem and to realize loop pairing. Consequently, the selected square subsystem is used as an internal model to design the internal model controller. Finally, a simple process is taken as the simulation object to demonstrate the validity and feasibility of the new method. Simulations results illustrate that the proposed strategy is not only simple and easy to implement but also has a good performance even the system model is mismatched.     

Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method

Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial‐and‐error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix‐assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion‐exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α‐lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix‐assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used.     

The effect of transcendental meditation on right hemispheric functioning

This study reports two experiments investigating the effects of transcendental meditation on right hemispheric functioning. The task used in both experiments was the Seashore Tonal Memory Test. In the first experiment a nonmeditator group and an experienced meditator group were run. The design involved three periods: a pretest, a meditation or rest period, and then a posttest. The results showed the experienced meditators were significantly better in both pretest and posttest performance. There were no pretest-posttest differences. The second experiment was done to replicate the first experiment and to control for possible selection bias. The design was the same as the first experiment, except that an additional group of inexperienced meditators was included. The results again showed significantly superior performance for the experienced meditators compared to the nonmeditators. In addition, the experienced meditators were superior to the inexperienced meditators. There were no significant differences between the nonmeditators and the inexperienced meditators. These results support the hypothesis that meditation facilitates right hemispheric functioning. Alternative explanations, such as selection bias, are also discussed.The authors wish to thank Ms. Michelle Ellis and Ms. Bridget Carr for their assistance in conducting these experiments. We also wish to thank the Seattle SIMS for their cooperation in obtaining subjects.     

A balance control model of quiet upright stance based on an optimal control strategy

Models of balance control can aid in understanding the mechanisms by which humans maintain balance. A balance control model of quiet upright stance based on an optimal control strategy is presented here. In this model, the human body was represented by a simple single-segment inverted pendulum during upright stance, and the neural controller was assumed to be an optimal controller that generates ankle control torques according to a certain performance criterion. This performance criterion was defined by several physical quantities relevant to sway. In order to accurately simulate existing experimental data, an optimization procedure was used to specify the set of model parameters to minimize the scalar error between experimental and simulated sway measures. Thirty-two independent simulations were performed for both younger and older adults. The model's capabilities, in terms of reflecting sway behaviors and identifying aging effects, were then analyzed based on the simulation results. The model was able to accurately predict center-of-pressure-based sway measures, and identify potential changes in balance control mechanisms caused by aging. Correlations between sway measures and model parameters are also discussed.     

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.     

Quantitative quality control in microarray experiments and the application in data filtering,normalization and false positive rate prediction

Data preprocessing including proper normalization and adequate quality control before complex data mining is crucial for studies using the cDNA microarray technology. We have developed a simple procedure that integrates data filtering and normalization with quantitative quality control of microarray experiments. Previously we have shown that data variability in a microarray experiment can be very well captured by a quality score q(com) that is defined for every spot, and the ratio distribution depends on q(com). Utilizing this knowledge, our data-filtering scheme allows the investigator to decide on the filtering stringency according to desired data variability, and our normalization procedure corrects the q(com)-dependent dye biases in terms of both the location and the spread of the ratio distribution. In addition, we propose a statistical model for false positive rate determination based on the design and the quality of a microarray experiment. The model predicts that a lower limit of 0.5 for the replicate concordance rate is needed in order to be certain of true positives. Our work demonstrates the importance and advantages of having a quantitative quality control scheme for microarrays.     

Characterization of tissue scaffolds for time-dependent biotransport criteria – a novel computational procedure

This study aims to establish a new computational framework that allows modeling transient oxygen diffusion in tissue scaffolds more efficiently. It has been well known that the survival of cells strongly relies on continuous oxygen/nutrient supply and metabolite removal. With optimal design in scaffold architecture, its ability to sustain long distance oxygen supply could be improved considerably. In this study, finite element based homogenization procedure is first used to characterize the initial effective biotransport properties in silico. These initial properties are proper indicators to prediction of the on-going performance of tissue scaffolds over time. The transient model by adopting an edge-based smoothed finite element method with combination of mass-redistributed method is then established to more efficiently simulate the transient oxygen transfer process in tissue scaffolds. The proposed new method allows large time steps to model the oxygen diffusion process without losing numerical accuracy, thereby enhancing the computational efficiency significantly, in particular for the design optimization problems which typically require numerous analysis iterations. A number of different scaffold designs are examined either under net diffusion without cell seeding, or under cellular oxygen/nutrient uptake with or without considering cell viability. The association between the homogenized effective diffusivity of net scaffold microstructures and corresponding transient diffusion and time-dependent cellular activities is divulged. This study provides some insights into scaffold design.     

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.     

Quality control for quantitative PCR based on amplification compatibility test

Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however there is currently no method to allow for reliable control of the amplification process without directly modifying the sample mix. Herein we present a statistical approach based on multivariate analysis of the amplification response data generated in real-time. The amplification trajectory in its most resolved and dynamic phase is fitted with a suitable model. Two parameters of this model, related to amplification efficiency, are then used for calculation of the Z-score statistics. Each studied sample is compared to a predefined reference set of reactions, typically calibration reactions. A probabilistic decision for each individual Z-score is then used to identify the majority of inhibited reactions in our experiments. We compare this approach to univariate methods using only the sample specific amplification efficiency as reporter of the compatibility. We demonstrate improved identification performance using the multivariate approach compared to the univariate approach. Finally we stress that the performance of the amplification compatibility test as a quality control procedure depends on the quality of the reference set.     

Incorporating prior parameter uncertainty in the design of sampling schedules for pharmacokinetic parameter estimation experiments

An experiment design procedure is proposed for nonlinear parameter estimation studies that formally incorporates prior parameter uncertainty. The design criterion derives from information theory considerations and involves an asymptotic interpretation of the expected posterior information provided by an experiment. A pharmacokinetic sample schedule design problem is used to illustrate and evaluate this information theoretic design strategy. The model considered is commonly used to describe the plasma concentration of a drug following its oral administration. The limitations and advantages of the proposed design procedure are discussed in relation to other previously reported design techniques for incorporating parameter uncertainty.     

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.     

Ab initio construction of protein tertiary structures using a hierarchical approach

We present a hierarchical method to predict protein tertiary structure models from sequence. We start with complete enumeration of conformations using a simple tetrahedral lattice model. We then build conformations with increasing detail, and at each step select a subset of conformations using empirical energy functions with increasing complexity. After enumeration on lattice, we select a subset of low energy conformations using a statistical residue-residue contact energy function, and generate all-atom models using predicted secondary structure. A combined knowledge-based atomic level energy function is then used to select subsets of the all-atom models. The final predictions are generated using a consensus distance geometry procedure. We test the feasibility of the procedure on a set of 12 small proteins covering a wide range of protein topologies. A rigorous double-blind test of our method was made under the auspices of the CASP3 experiment, where we did ab initio structure predictions for 12 proteins using this approach. The performance of our methodology at CASP3 is reasonably good and completely consistent with our initial tests.     

Effects of auditory frequency-shifts on zero and below-zero SCR habituation

The purpose of the present experiment was to test a procedure for the measurement of effects of zero- and below-zero habituation (BZH) on responding to frequency shifts in auditory stimuli. The present procedure avoided some of the drawbacks of other procedures, that is, long duration, ambiguity in the definition of BZH, and inadequate control procedures. Two groups received 18 stimulus presentations each; group 1 received first a tone of 1000 Hz 12 times, then 1400 Hz (test stimulus 1) 3 times, and 1850 Hz (test stimulus 2) 3 times. Group 2 received the same stimuli, but 3, 12, and 3 times, respectively. The procedure had the advantages of short duration of the experiment, zero habituation and BZH were operationally defined (as 3 and 12 stimulus presentations, respectively), and there were adequate control conditions since a control group that did not receive stimulus change on the relevant trial was employed. The results showed no effects of stimulus shifts on responding to the test stimuli. Group 2 responded significantly less than group 1 across trials, though, and this may be explained by an inhibitory process elicited by changes in weak stimulation during the habituation process.