Computer-based simulation of the recovery of intracellular enzymes and its pilot-scale verification

Accurate and reliable models are required for a range of unit operations if simulations are to be used for accelerating the design and optimisation of bioprocesses. This paper presents results of pilot-plant studies that have been used to verify process simulations for a sequence of operations comprising of cell disruption, fractional protein precipitation and centrifugal separation. These have been tested using the purification of the enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae as being representative of the recovery of a labile intracellular enzyme. Comparison of pilot-plant against simulated data highlights where improvements to the models are required and has resulted in increased confidence in the simulations for a wide range of conditions including the operational scale and the nature of the starting material. Results demonstrate the effectiveness of the verification approach for the development of reliable predictive models to assess the feasibility of process designs and performance of a train of bioprocess operations. UCL is the Biotechnology and Biological Sciences Research Council's sponsored Advanced Centre for Biochemical Engineering and the Council's support is gratefully acknowledged. The authors would like to thank I Ms. N. Abidi for technical assistance.     

On the controllability of continuous fermentation processes

Simulation may be used as a powerful tool for accelerating bioprocess design. This paper demonstrates the use of simulations in exploring the nature and impact of the interactions that exist in a typical bioprocess for the recovery of an intracellular protein. The study shows that an integrated approach to design must be adopted in order to achieve acceptable process designs. Data from a fed-batch fermentation, with verified models for cell harvesting, cell disruption and cell debris removal have been integrated to demonstrate the consequence of process design and operating decisions on the resulting process performance. The trade-offs between product recovery and the extent of cell debris removal for a range of operating conditions have been represented through a series of windows of operation which show how process conditions must be altered in order for given process performance levels to be realised. The capacity to account for process performance including the impact of interactions is seen as a pre-requisite for rigorous bioprocess sequence design and optimisation.     

The application of a Pareto optimisation method in the design of an integrated bioprocess

A rapid method for designing integrated bioprocesses, using a combination of a windows of operation and a Pareto optimisation approach, is described in this paper. Within bioprocesses, multiple objectives are common, and achieving a satisfactory trade-off amongst the design objectives is crucial. Conventional optimisation results in the identification of the best operating policy for a given desired performance but gives little insight into how the process performance changes in the vicinity of the solution. In this paper, we explore the use of a Pareto optimisation technique to locate the optimal conditions for an integrated bioprocessing sequence and the benefits of first reducing the feasible space by the development of a series of windows of operation to provide a smaller search area for the optimisation. The final results are then presented in performance trade-off graphs and look-up tables, which give the design engineer an easily manageable solution set to work with. In this way, the decision-making procedure for design is made faster and more transparent. Two case studies illustrate the results from this integrated design methodology, some of which are counter-intuitive compared with the general design experience.     

A new miniature hydrostatic pressure chamber for microscopy. Strain- free optical glass windows facilitate phase-contrast and polarized- light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature

This paper describes the development of a miniature, temperature- controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase- contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardation (BR) og 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20 times UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40 times phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chambers, as well as methods for shifting and controlling the temperature within the chamber, are included.     

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.     

Non-linear control of continuous bioreactors

Control of bioreactors has achieved importance in the recent years. This may be due to the fact that they are difficult to control which may be attributed to its nonlinear dynamic behavior. The model parameters of the bioreactor also vary in an unpredictable manner. The complexity of the biochemical processes inhibits the accurate modeling and also the lack of suitable sensors make the process state difficult to characterize. Considerable emphasis has been placed on the control of fed-batch fermentors because of their prevalence in industries. However, when production of biomass is to be optimized, continuous operation is desirable. Several procedures are available for the nonlinear control of processes, viz., differential geometric approach, internal model control approach, reference synthesis technique, predictive control design, etc., but the major disadvantage of these approaches is the computational time required to perform the prediction optimization. In this study, a nonlinear controller based on a polynomial discrete time model (NARMAX) is evaluated for its performance on a fermentor. It can be shown that a nonlinear self-tuning controller based on NARMAX model can be extended to the control of fermentors. The response is smooth for both load and setpoint changes even when process parameters are assumed to be zero and uncertainty in parameters are present and in the presence of controller constraints. The control action can be made more or less robust by changing the design parameters appropriately. Therefore, nonlinear self-tuning controller is suitable for control of industrial processes.     

Constraining local structure can speed up folding by promoting structural polarization of the folding pathway

The pathway which proteins take to fold can be influenced from the earliest events of structure formation. In this light, it was both predicted and confirmed that increasing the stiffness of a beta hairpin turn decreased the size of the transition state ensemble (TSE), while increasing the folding rate. Thus, there appears to be a relationship between conformationally restricting the TSE and increasing the folding rate, at least for beta hairpin turns. In this study, we hypothesize that the enormous sampling necessary to fold even two-state folding proteins in silico could be reduced if local structure constraints were used to restrict structural heterogeneity by polarizing folding pathways or forcing folding into preferred routes. Using a Gō model, we fold Chymotrypsin Inhibitor 2 (CI-2) and the src SH3 domain after constraining local sequence windows to their native structure by rigid body dynamics (RBD). Trajectories were monitored for any changes to the folding pathway and differences in the kinetics compared with unconstrained simulations. Constraining local structure decreases folding time two-fold for 41% of src SH3 windows and 45% of CI-2 windows. For both proteins, folding times are never significantly increased after constraining any window. Structural polarization of the folding pathway appears to explain these rate increases. Folding rate enhancements are consistent with the goal to reduce sampling time necessary to reach native structures during folding simulations. As anticipated, not all constrained windows showed an equal decrease in folding time. We conclude by analyzing these differences and explain why RBD may be the preferred way to constrain structure.     

Optimization of glucose isomerase reactor: optimum operating temperature mode

The optimum temperature operation mode required to achieve high fructose productivity is studied for immobilized glucose isomerase (GI) packed bed reactor. In this study, the reactor design equation based on reversible Michaelis-Menten kinetics assumes both thermal enzyme deactivation and substrate protection. The optimization problem is formulated as a discretized constrained nonlinear programming problem (NLP). The formulation is expressed in terms of maximization of fructose productivity as the objective function subject to reactor design equation, kinetic parameter equations, substrate protection factor equation and feasibility constraints. The constraints are discretized along the reactor operating period by employing piecewise polynomial approximations. Approximately 7% improvement in terms of fructose productivity is achieved when running the reactor at the optimum decreasing temperature operation mode as compared to the constant optimum isothermal operation.     

Avian cooperative breeding: Old hypotheses and new directions

In cooperatively breeding birds, individuals that appear capable of reproducing on their own may instead assist others with their breeding efforts. Research into avian cooperative breeding has attempted to reconcile the apparent altruism of this behaviour with maximization of inclusive fitness. Most explanations of cooperative breeding have suggested that philopatry is enforced by ecological constraints, such as a shortage of resources critical to breeding. Non-dispersers may then benefit both directly and indirectly from contributing at the nest. Recent research has shown that such benefits may be sufficient to promote philopatry, without the need for ecological constraints, and emphasizes that consideration of both costs and benefits of philopatry is essential for a comprehensive approach to the problem. The growing body of data from long-term studies of different species should combine with an improved phylogenetic perspective on cooperative breeding, to provide a useful base for future comparative analyses and experimentation.     

A structured approach to design and operation of biotransformation processes

A structured approach to design and operation of biotransformation (bioconversion) processes, based on previous case studies, has been developed. This requires knowledge of the key characteristics of a biotransformation which determine the constraints on process selection. The approach is illustrated for five biotransformations, two enzymic and three microbial. Some generic problems such as low water solubility and volatility of reactants, reactant and product toxicity have been identified. The microblal oxidations of toluene and fluorobenzene to toluenecis-glycol and fluorocatechol respectively byPseudomonas putida have been used to illustrate how these constraints may be overcome by addition of tetradecane as a second liquid phase, use of a membrane oxygenator and introduction ofin situ product removal.     

The effect of cycling dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

The antibiotic, difficidin, and its hydroxylated derivative, oxydifficidin, were synthesised by cultures of Bacillus subtilis grown on a complex medium in batch culture at dissolved oxygen tensions (DOT) of 15, 20 and 40% air saturation. During part of the growth phase the DOT was cycled about the control value and the effect on growth and antibiotic production observed. In fermentations with cycling at 15 and 20% DOT the growth yields were lower than for the fermentations done at constant DOT throughout. There appears to be a complex interaction between growth rate and difficidin production rate which led to a reduced specific production rate at 15% DOT as a result of cycling.UCL is the Biotechnology and Biological Sciences Research Council Interdisciplinary Research Centre for Biochemical Engineering and the Council's support is gratefully acknowledged. The authors wish to thank Merck & Co. for provision of the difficidin and oxydifficidin used to calibrate the HPLC assay.     

Subjective interpretation,laboratory error and the value of forensic DNA evidence: Three case studies

This article discusses two factors that may profoundly affect the value of DNA evidence for proving that two samples have a common source: uncertainty about the interpretation of test results and the possibility of laboratory error. Three case studies are presented to illustrate the importance of the analyst's subjective judgments in interpreting some RFLP-based forensic DNA tests. In each case, the likelihood ratio describing the value of DNA evidence is shown to be dramatically reduced by uncertainty about the scoring of bands and the possibility of laboratory error. The article concludes that statistical estimates of the frequency of matching genotypes can be a misleading index of the value of DNA evidence, and that more adequate indices are needed. It also argues that forensic laboratoires should comply with the National Research Council's recommendation that forensic test results be scored in a blind or objective manner.Editor's commentsThe author treats the timely and important issue of laboratory error. Readers will need to read the paper by Lempert in this volume for an alternative interpretation of the 1989 proficiency testing of Cellmark diagnostics.     

Workshop--Predicting the structure of biological molecules

This April, in Cambridge (UK), principal investigators from the Mathematical Biology Group of the Medical Research Council's National Institute of Medical Research organized a workshop in structural bioinformatics at the Centre for Mathematical Sciences. Bioinformatics researchers of several nationalities from labs around the country presented and discussed their computational work in biomolecular structure prediction and analysis, and in protein evolution. The meeting was intensive and lively and gave attendees an overview of the healthy state of protein bioinformatics in the UK.     

Use of Monte Carlo simulations for improved facility fit planning in downstream biomanufacturing and technology transfer

Biologics manufacturing is capital and consumable intensive with need for advanced inventory planning to account for supply chain constraints. Early-stage process design and technology transfer are often challenging due to limited information on process variability regarding bioreactor titer, process yield, and product quality. Monte Carlo (MC) methods offer a stochastic modeling approach for process optimization where probabilities of occurrence for process inputs are incorporated into a deterministic model to simulate more likely scenarios for process outputs. In this study, we explore MC simulation-based design of a monoclonal antibody downstream manufacturing process. We demonstrate that this probabilistic approach offers more representative outcomes over the conventional worst-case approach where the theoretical minimum and maximum values of each process parameter are used without consideration for their probability of occurrence. Our work demonstrates case studies on more practically sizing unit operations to improve consumable utilization, thereby reducing manufacturing costs. We also used MC simulations to minimize process cadence by constraining the number of cycles per unit operation to fit facility preferences. By factoring in process uncertainty, we have implemented MC simulation-based facility fit analyses to efficiently plan for inventory when accounting for process constraints during technology transfer from lab-scale to clinical or commercial manufacturing.     

Human genetic banking: altruism, benefit and consent

This article considers how we should frame the ethical issues raised by current proposals for large-scale genebanks with on-going links to medical and lifestyle data, such as the Wellcome Trust and Medical Research Council's 'UK Biobank'. As recent scandals such as Alder Hey have emphasised, there are complex issues concerning the informed consent of donors that need to be carefully considered. However, we believe that a preoccupation with informed consent obscures important questions about the purposes to which such collections are put, not least that they may be only haphazardly used for research (especially that of commercial interest)--an end that would not fairly reflect the original altruistic motivation of donors, and the trust they must invest. We therefore argue that custodians of such databases take on a weighty pro-active duty, to encourage public debate about the ends of such collections and to sponsor research that reflects publicly agreed priorities and provides public benefits.     

Equipment design considerations for large scale cell culture

Equipment design is frequently recognized as a key component in the success of GMP biologics manufacturing, but is not always implemented with full appreciation of the processing implications. In the case of mammalian cell culture, there are some recognized issues and risks that develop when transitioning to a large scale of operation. The developing demand for cell culture production capacity in the biopharmaceutical industry has led to a progressive increase in the scale of operation in the last decade. This review will provide a high level summary of the documented process difficulties unique to serum-free large scale (LS) cell culture, analyze the engineering constraints typical of these processes, and suggest some practical equipment design considerations to enhance the productivity, reliability and operability of such systems under GMP manufacturing conditions. A systems approach will be used to establish a good LS bioreactor design practice, providing a discussion on gas distribution, agitation, vessel design, SIP/CIP and control issues. This revised version was published online in July 2006 with corrections to the Cover Date.     

Visualizing feasible operating ranges within tissue engineering systems using a “windows of operation” approach: A perfusion‐scaffold bioreactor case study

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio‐regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade‐offs between different performance criteria is a challenge. A “windows of operation” tool that graphically represents feasible operating spaces to achieve user‐defined levels of performance has previously been described by researchers in the bio‐processing industry. This paper demonstrates the value of “windows of operation” to the tissue engineering field using a perfusion‐scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell‐seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow‐rate. Using cyclooxygenase‐2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the “windows of operation” methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow‐rate that achieved user‐defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Biotechnol. Bioeng. 2012; 109: 3161–3171. © 2012 Wiley Periodicals, Inc.     

Demonstration of the use of windows of operation to visualize the effects of fouling on the performance of a chromatographic step

The high resolution afforded by packed bed chromatography makes it an indispensable operation in the downstream processing of therapeutic molecules. Packed bed performance is however inherently susceptible to changes in feed stream characteristics and fouling processes. The impact of fouling is seldom considered during the early stages of bioprocess development which is concerned with the selection of purification conditions. Instead these are performed with rigorously clarified feeds. Under such conditions, chromatography is effectively treated as an isolated step, independent from its preceding unit operations. In this study, we demonstrate how windows of operation could be used to visualize the impact of changes in the preceding clarification step on the fouling response of a subsequent cation exchange capture step. Laboratory columns (2,5 and 12 cm height) were subjected to varying fouling challenges of Escherichia coli lysate containing different amounts of solids carried over from the previous step. Changes in trans‐column pressure drop and breakthrough of the target protein (Fab′) were monitored. The limits of operability of the resin were determined with respect to the process material's properties. This information was used to extract the parameters for the adsorption kinetics used in the general rate (GR) model to create windows of operation for manufacturing scale operation. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Unequal protection for patient rights: The divide between university and health ethics committees

Despite recommendations from the Cartwright Report ethical review by health ethics committees has continued in New Zealand without health practitioners ever having to acknowledge their dual roles as health practitioners researching their own patients. On the other hand, universities explicitly identify doctor/research-patient relations as potentially raising conflict of role issues. This stems from the acknowledgement within the university sector itself that lecturer/research-student relations are fraught with such conflicts. Although similar unequal relationships are seen to exist between health resarchers and their patients, the patient/subjects are not afforded the levels of protection that are afforded student/subjects. In this paper we argue that the difference between universities and health research is a result of the failure of the Operational Standard Code for Ethics Committees to explicitly acknowledge the vulnerability of the patient and conflict of interests in the dual roles of health practitioner/researcher. We end the paper recommending the Ministry of Health consider the rewriting of the Operational Standard Code for Ethics Committees, in particular in the rewriting of section 26 of the Operational Standard Code for Ethics Committees. We also identify the value of comparative ethical review and suggest the New Zealand's Health Research Council's trilateral relationship with Australia's NHMRC (National Health and Medical Research Council) and Canada's CIHR (Canadian Institute of Health Research) as a useful starting point for such a process.