Studies on generalized kinetic model and Pareto optimization of a product-driven self-cycling bioprocess

The aim of this study is the optimization of a product-driven self-cycling bioprocess and presentation of a way to determine the best possible decision variables out of a set of alternatives based on the designed model. Initially, a product-driven generalized kinetic model, which allows a flexible choice of the most appropriate kinetics is designed and analysed. The optimization problem is given as the bi-objective one, where maximization of biomass productivity and minimization of unproductive loss of substrate are the objective functions. Then, the Pareto fronts are calculated for exemplary kinetics. It is found that in the designed bioprocess, a decrease of emptying/refilling fraction and an increase of substrate feeding concentration cause an increase of the biomass productivity. An increase of emptying/refilling fraction and a decrease of substrate feeding concentration cause a decrease of unproductive loss of substrate. The preferred solutions are calculated using the minimum distance from an ideal solution method, while giving proposals of their modifications derived from a decision maker’s reactions to the generated solutions.     

The use of windows of operation as a bioprocess design tool

Bioprocess design problems are frequently multivariate and complex. However, they may be visualised by a graphical representation of the design constraints and correlations governing both the process and system under consideration, namely windows of operation. Windows of operation exist at all stages of process design and find use both in the identification of key constraints from limited information, and also, with more detailed knowledge, the sensitivity of a process to design or operating changes. In this way windows of operation may be used to help understand and optimise a bioprocess design. In this paper the formulation, development and application of windows of operation is discussed for a range of biological processes including fermentation, protein recovery and biotransformation.UCL is the Biotechnology and Biological Sciences Research Council's Interdisciplinary Research Centre for Biochemical Engineering and the Council's support is gratefully acknowledged.     

Development of a versatile computer integrated control system for bioprocess controls

A general approach is described for the implementation of a networked multi-unit computer integrated control system. The use of data acquisition hardware and graphical programming tools alleviates tedious programming and maintains potency and flexibility. One application of the control system, the control of a mammalian cell perfusion culture based on a key nutrient glucose concentration, was demonstrated. The control system offers customized user interface for all process control parameters and allows the flexibility for continued improvement and implementation of new tailored functions. The temperature, pH, dissolved oxygen and glucose level were accurately controlled. This revised version was published online in June 2006 with corrections to the Cover Date.     

A systematic concept in bioprocess analysis and design

A systematic concept in bioprocess analysis and design is presented according to an integrating strategy as basis for a biotechnological methodology. A comparison illustrates analogies and significant differences between chemical and biological processes. The study of the interactions between environments and organisms includes influences of physical transport phenomena and also enzyme and metabolic regulations. In both cases the macroscopic principle with a formalkinetic approach is recommended for quantification purposes. The determination of the characteristic-time-regime with characteristic rate constants makesit possible to simplify mathematical modeling using the concepts of the rate-determining-step and quasistationarity. Finally, guidelines are summarized for the use of unstructured and structured kinetic models.     

From a co-production design to an integrated single-cell biorefinery

Engineering microorganisms capable of accumulating multiple products are sometimes attractive because they yield several advantages in balancing the in vivo metabolic flux and restoring the optimal cell physiology. With the development of metabolic engineering and synthetic biology, numerous strategies for minimizing the substrate waste, optimizing the product portfolios, and maximizing the product yield in co-production systems have been designed and applied. This paper reviewed the recent developments in this field and discussed the challenges that may be encountered during the scaling up of the co-production systems. Finally, the importance of product portfolios and biorefinery strategy of single-cell in co-production processes was proposed.     

Visualizing integrated bioprocess designs through "windows of operation".

This paper demonstrates a simple graphical approach for the design and analysis of a bioprocess flowsheet in which process interactions are significant. Results are presented showing how the feasible space for operation can be simulated and used both to address key design and operating decisions and to identify suitable trade-offs between operating variables, such as fermentation growth rate and disruption conditions, in order to achieve prespecified levels of process performance. Using verified models to describe the production and isolation of an intracellular protein alcohol dehydrogenase (ADH) in yeast as a test bed, a series of so-called "windows of operation" are developed at growth rates in the range of 0.06-0.28 h(-1) and for a range of overall process specifications. The effects of altering the process design performance specification as defined by the level of cell debris removal and the overall process productivity on the size and position of the feasible space were investigated to demonstrate the sensitivity of the flowsheet to changes in process objectives. Using the approach it has been possible to visualise the processing trade-offs required to increase performance in terms of the level of cell debris removal by 50% and the overall process productivity by 400% from a defined base level. The approach provides a convenient tool when designing integrated bioprocesses by enabling process options to be compared visually and can help in achieving better process designs and accelerating process development for the biological process industry.     

Searching for the Pareto frontier in multi-objective protein design

The goal of protein engineering and design is to identify sequences that adopt three-dimensional structures of desired function. Often, this is treated as a single-objective optimization problem, identifying the sequence–structure solution with the lowest computed free energy of folding. However, many design problems are multi-state, multi-specificity, or otherwise require concurrent optimization of multiple objectives. There may be tradeoffs among objectives, where improving one feature requires compromising another. The challenge lies in determining solutions that are part of the Pareto optimal set—designs where no further improvement can be achieved in any of the objectives without degrading one of the others. Pareto optimality problems are found in all areas of study, from economics to engineering to biology, and computational methods have been developed specifically to identify the Pareto frontier. We review progress in multi-objective protein design, the development of Pareto optimization methods, and present a specific case study using multi-objective optimization methods to model the tradeoff between three parameters, stability, specificity, and complexity, of a set of interacting synthetic collagen peptides.     

The design and development of an integrated natural products screening database

We designed and developed NEXUS--a new natural products screening database and related suite of software applications--to utilize the spectacular increases in assay capacity of the modern high throughput screening (HTS) environment. NEXUS not only supports seamless integration with separate HTS systems, but supports user-customized integration with external laboratory automation, particularly sample preparation systems. Designed and developed based on a detailed process model for natural products drug discovery, NEXUS comprises two integrated parts: (1) a single schema of Oracle tables and callable procedures and functions, and (2) software "front-ends" to the database developed using Microsoft Excel and Oracle Discovery/2000. Many of the back-end processing functions were written in Programming Language/Structured Query Language (PL/SQL) to provide an Application Programmer's Interface, which allows end users to create custom applications with little input from information technology professionals.     

Issues in the development of an industrial bioprocess advisory system

The background and motivation for the construction of a fault detection and advisory system for an industrial fermentation process plant are described. Here, the knowledge extracted from the operators (implemented in the form of production rules) is integrated with multivariate data-based methods for fault detection. The industrial benefits arising from this integrated system include: (1) reduced variability, (2) increased mean performance levels, (3) reduced operator-training time and (4) knowledge management in the broader organization.     

Use of operating windows in the assessment of integrated robotic systems for the measurement of bioprocess kinetics

This study examines the utility of an automated liquid handling robot integrated with a microwell plate reader to enable the rapid acquisition of bioprocess kinetic data. The relationship between the key parameters for liquid handling accuracy and precision and the sample detection period has been characterized for typical low-viscosity (<2.0 mPa x s) aqueous and organic phases and for a high-viscosity aqueous phase (60 mPa x s), all exhibiting Newtonian rheology. The use of a simple graphical method enables the suitability of a given automation platform to be assessed once the user has determined the minimum sample detection period and the minimum accurate and precise dispense volume. This provides for a reduction in the duration of any experiment by maximizing well usage within each microwell plate. The suitability of employing an integrated automation platform to gather kinetic data for systems typical of those encountered in bioprocessing is analyzed via a series of case studies. Application to alkaline cell lysis, where disruption is complete within 120 s, showed that the range of available dispense volumes and the number of wells that can be utilized is limited. In contrast, analysis of a system exhibiting slow process kinetics, the fermentation of Escherichia coli TOP10 pQR239 in microwell plates, demonstrated that, for a typical sample detection period of 30 min, the only restrictions on the degree of well utilization are the liquid handling accuracy and precision and the volume capacity of the liquid handling robot. Finally, liquid-liquid extraction, an example of a kinetically independent operation, was also examined. In this case, only a single equilibrium measurement is required, which means that the only restrictions to the utilization of the integrated devices are the liquid handling accuracy and precision. Integrated automation platforms represent a powerful process development tool over traditional experimental methods used for bioprocess development. Smaller volumes of reagent and sample can be used to achieve greater throughput, while high levels of reproducibility and sensitivity are maintained.     

Applications of modelling for bioprocess design and control in industrial production

The on-line measurement of the relevant parameters and the control conception for three production processes for fine chemicals by fermentation and biotransformation at the 15 m³ scale were developed. The models describe the bioprocesses which successfully result in fully automated manufacturing steps. Modelling also proved to be a valuable tool for a better insight into biochemical fundamentals of the processes. Moreover, proper use of data logging, modelling and process control was important for quality, since two processes were controlled on-line and quality relevant deviations were registered early. Finally, combining modelling with simulation, we could drastically reduce both development time and cost.List of Symbols F l/h flux - V l volume - U₀ g/l nicotinonitrile concentration influx - U g/l actual nicotinonitrile concentration - q_ug/gh specific educt (=nicotinonitrile) transformation rate - x g/l biocatalyst concentration - p₀ g/l nicotinamide concentration influx - p g/l actual nicotinamide concentration - q_pg/gh specific product (=nicotinamide) formation rate - k parameter loss of activity - q_{u, max}g/gh max. specific educt transformation rate - K_ug/l saturation constant for nicotinonitrile - K_ig/l inhibition constant for nicotinonitrile - K_iig/l inhibition constant for nicotinamide - MW_Ag/mol molecular weight for nicotinonitrile - MW_Bg/mol molecular weight for nicotinamide - NS Nicotinic acid - 6-HNS 6-Hydroxynicotinic acid - r_{NS, 6HNS} g/lh 6-HNS production rate - r_{6HNS, X} g/lh biomass production rate - r_{NS, 6HNS, max} g/lh max. 6-HNS production rate - S_NS g/l actual NS concentration - K_{S, NS} g/l saturation constant for NS - K_{i, 6HNS} g/l inhibition constant for 6-HNS - K_o2 g/l saturation constant for oxygen - r_{6HNS, X, max} g/lh max. biomass production rate - S_6HNS g/l actual 6-HNS concentration - K_{ii, NS} g/l inhibition constant for NS - RQ mol/mol respiration quotient - S_xylg/l actual xylene concentration - K_{i, xyl}g/ inhibition constant for xylene - K_{i, DMPY}g/ inhibition constant for 2,5-dimethylpyrazine - r_Xg/lh biomass production rate - r_{X, max}g/lh max. biomass production rate - K_{s, xyl}g/l saturation constant for xylene - S_DMPYg/l actual concentration of DMPY - K_{i, MPCA}g/ inhibition constant for MPCA - K_O2g/ saturation constant for oxygen - S_MPCAg/l actual MPCA concentration - S_O2g/l actual oxygen concentration - r_MPCAg/lh MPCA production rate - r_{MPCA, max}g/lh max. MPCA production rate - k_lgl inhibition constant for the intermediates - k_{s, DMPY}gl saturation constant for DMPY     

Practical application of ligand efficiency metrics in lead optimisation

The use of composite metrics that normalise biological potency values in relation to markers of physicochemical properties, such as size or lipophilicity, has gained a significant amount of traction with many medicinal chemists in recent years. However, there is no consensus on best practice in the area and their application has attracted some criticism. Here we present our approach to their application in lead optimisation projects, provide an objective discussion of the principles we consider important and illustrate how our use of lipophilic ligand efficiency enabled the progression of a number of our successful drug discovery projects. We derive, from this and some recent literature highlights, a set of heuristic guidelines for lipophilicity based optimisation that we believe are generally applicable across chemical series and protein targets.     

Control of substrate concentration in a continuous bioprocess

This paper is the first one presenting an application of the control law based on the ratio of measured and optimal values for a real fermentation pilot plant operated in continuous mode. A controller of this type takes into account the process variables and may be designed by using classical linear methods. A simple algorithm is applied for a real CSTR to control the substrate concentration. The results obtained confirm that this approach offers the possibility to combine simplicity and effectiveness in bioprocess control.     

HIVToolbox, an integrated web application for investigating HIV

Many bioinformatic databases and applications focus on a limited domain of knowledge federating links to information in other databases. This segregated data structure likely limits our ability to investigate and understand complex biological systems. To facilitate research, therefore, we have built HIVToolbox, which integrates much of the knowledge about HIV proteins and allows virologists and structural biologists to access sequence, structure, and functional relationships in an intuitive web application. HIV-1 integrase protein was used as a case study to show the utility of this application. We show how data integration facilitates identification of new questions and hypotheses much more rapid and convenient than current approaches using isolated repositories. Several new hypotheses for integrase were created as an example, and we experimentally confirmed a predicted CK2 phosphorylation site. Weblink: [http://hivtoolbox.bio-toolkit.com].     

Multi-fold enhancement in vitamin E (alpha-tocopherol) production via integration of bioprocess optimisation and metabolic engineering in cell suspension of sunflower

Journal of Plant Biochemistry and Biotechnology - Alpha-tocopherol, a highly active form of the antioxidant vitamin E in humans, effectively scavenges free radicals and protects the cell membranes....     

Plasmid stability in a recombinant mammalian cell bioprocess

Summary A simple experimental method is devised to determine the fraction of plasmid-harboring cells in a bioprocess employing recombinant mammalian cells. The fraction of plasmid-harboring cells decreased as serum content in the growth medium decreased. The relatively higher increase in the generation time of the plasmid-harboring cell was primarily responsible for this decrease. The mathematical expression obtained for this fraction in terms of the two parameters, i.e. the generation time ratio and the plasmid-loss probability, could represent the experimental data extremely well. The numerical values of these parameters could show the inherent insight of the system. It was found that the data plot against time can draw us to a misleading conclusion of the absence of the effect of serum concentration.     

Overcoming substrate inhibition during biological treatment of monoaromatics: recent advances in bioprocess design

The biological removal of monoaromatic compounds from contaminated environments, usually arising from industrial activity, is challenging because of the inherent toxicity of these compounds to microorganisms, particularly at the concentrations that can be encountered in industrial waste streams. A wide range of bioprocess designs have been proposed and tested with the aim of achieving high removal efficiencies, with varying degrees of technical success, and potential for practical implementation. This review reports on the progress on variations of well-known themes made in the last 3–4 years, as well as new bioprocess technologies that address the cytotoxicity of monoaromatics directly. Areas for further research are also proposed.     

Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high‐throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high‐throughput disruption methods exist. The development of an automated, miniaturized, high‐throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high‐pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA‐based methods to mimic large‐scale homogenization processes. These results demonstrate that AFA‐mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130–140, 2018