Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review)

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review)

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

Design of experiments to study the impact of process parameters on droplet size and development of non-invasive imaging techniques in tablet coating

Atomisation of an aqueous solution for tablet film coating is a complex process with multiple factors determining droplet formation and properties. The importance of droplet size for an efficient process and a high quality final product has been noted in the literature, with smaller droplets reported to produce smoother, more homogenous coatings whilst simultaneously avoiding the risk of damage through over-wetting of the tablet core. In this work the effect of droplet size on tablet film coat characteristics was investigated using X-ray microcomputed tomography (XμCT) and confocal laser scanning microscopy (CLSM). A quality by design approach utilising design of experiments (DOE) was used to optimise the conditions necessary for production of droplets at a small (20 μm) and large (70 μm) droplet size. Droplet size distribution was measured using real-time laser diffraction and the volume median diameter taken as a response. DOE yielded information on the relationship three critical process parameters: pump rate, atomisation pressure and coating-polymer concentration, had upon droplet size. The model generated was robust, scoring highly for model fit (R2 = 0.977), predictability (Q2 = 0.837), validity and reproducibility. Modelling confirmed that all parameters had either a linear or quadratic effect on droplet size and revealed an interaction between pump rate and atomisation pressure. Fluidised bed coating of tablet cores was performed with either small or large droplets followed by CLSM and XμCT imaging. Addition of commonly used contrast materials to the coating solution improved visualisation of the coating by XμCT, showing the coat as a discrete section of the overall tablet. Imaging provided qualitative and quantitative evidence revealing that smaller droplets formed thinner, more uniform and less porous film coats.

Design of improved membrane protein production experiments:quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright © 2005 The Protein Society.

Some studies relating to the design of shallow, fluidized bed boilers

The concept of shallow fluidized bed boilers is defined and a preliminary working design for a gas-fired package boiler has been produced. Those areas of the design requiring further study have been specified. Experimental investigations concerning these areas have been carried out. A two-dimensional, conducting paper analog has been developed for the specific purpose of evaluating sheet fins. The analog has been generalised and is presented as a simple means of simulating the general, two-dimensional Helmholtz equation. By recording the transient response of spherical, calorimetric probes when plunged into heated air-fluidized beds, heat transfer coefficients have been measured at bed temperatures up to 1 100°C. A correlation fitting all the data to within ±10% has been obtained. A model of heat transfer to surfaces immersed in high temperature beds has been proposed. The model solutions are, however, only in qualitative agreement with the experimental data. A simple experimental investigation has revealed that the effective, radial, thermal conductivities of shallow fluidized beds are an order of magnitude lower than the axial conductivities. These must, consequently, be taken into account when considering heat transfer to surfaces immersed within fluidized beds. Preliminary work on pre-mixed gas combustion and some further qualitative experiments have been used as the basis for discussing the feasibility of combusting heavy fuel oils within shallow beds. The use of binary beds, within which the fuel could be both gasified and subsequently burnt, is proposed. Finally, the consequences of the experimental studies on the initial design are considered, and suggestions for further work are made.

Structural behaviour and design of load-bearing falsework

Tne object of this research was to investigate the behaviour of birdcage scaffolding as used in falsework structures, assess the suitability of existing design methods and make recommendations for a set of design rules. Since excessive deflection is as undesirable in a structure as total collapse, the project was divided into two sections. These were to determine the ultimate vertical and horizontal load-carrying capacity and also the deflection characteristics of any falsework. So theoretical analyses were developed to ascertain the ability of both the individual standards to resist vertical load, and of the bracing to resist horizontal load.Furthermore a model was evolved which would predict the horizontal deflection of a scaffold under load using strain energy methods. These models were checked by three series of experiments. The first was on individual standards under vertical load only. The second series was carried out on full scale falsework structures loading vertically and horizontally to failure. Finally experiments were conducted on scaffold couplers to provide additional verification of the method of predicting deflections. This thesis gives the history of the project and an introduction into the field of scaffolding. It details both the experiments conducted and the theories developed and the correlation between theory and experiment. Finally it makes recommendations for a design method to be employed by scaffolding designers.

Design of a genetic algorithm for bi-objective flow shop scheduling problems with re-entrant jobs

This paper presents a simulated genetic algorithm (GA) model of scheduling the flow shop problem with re-entrant jobs. The objective of this research is to minimize the weighted tardiness and makespan. The proposed model considers that the jobs with non-identical due dates are processed on the machines in the same order. Furthermore, the re-entrant jobs are stochastic as only some jobs are required to reenter to the flow shop. The tardiness weight is adjusted once the jobs reenter to the shop. The performance of the proposed GA model is verified by a number of numerical experiments where the data come from the case company. The results show the proposed method has a higher order satisfaction rate than the current industrial practices.

Structural design of contact lens-based drug delivery systems; in vitro and in vivo studies of ocular triggering mechanisms

This study identifies and investigates the potential use of in-eye trigger mechanisms to supplement the widely available information on release of ophthalmic drugs from contact lenses under passive release conditions. Ophthalmic dyes and surrogates have been successfully employed to investigate how these factors can be drawn together to make a successful system. The storage of a drug-containing lens in a pH lower than that of the ocular environment can be used to establish an equilibrium that favours retention of the drug in the lens prior to ocular insertion. Although release under passive conditions does not result in complete dye elution, the use of mechanical agitation techniques which mimic the eyelid blink action in conjunction with ocular tear chemistry promotes further release. In this way differentiation between passive and triggered in vitro release characteristics can be established. Investigation of the role of individual tear proteins revealed significant differences in their ability to alter the equilibrium between matrix-held and eluate-held dye or drug. These individual experiments were then investigated in vivo using ophthalmic dyes. Complete elution was found to be achievable in-eye; this demonstrated the importance of that fraction of the drug retained under passive conditions and the triggering effect of in-eye conditions on the release process. Understanding both the structure-property relationship between drug and material and in-eye trigger mechanisms, using ophthalmic dyes as a surrogate, provides the basis of knowledge necessary to design ocular drug delivery vehicles for in-eye release in a controllable manner.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.