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This thesis discusses recent advances towards the grand unification of process design, con- trol and operational optimization via multi-parametric programming techniques. First the PARametric Optimization and Control (PAROC) framework is presented together with a prototype implementation for the development of advanced receding horizon policies via multi-parametric programming and their closed-loop implementation. It is shown how state- of-the-art multi-parametric programming algorithms are utilized for the development of explicit optimization policies for a variety of problems (including control, estimation and scheduling) based on high-fidelity models and model approximation steps. Two developments are presented towards the integration of design, control and opera- tional optimization within PAROC; (i) the integration of design and control and (ii) the integration of scheduling and control. For (i) we develop receding horizon optimization for- mulations where the design variables are simultaneously considered, resulting in explicit, design–dependent policies which are then included within a Mixed-Integer Dynamic Opti- mization (MIDO) algorithm minimizing operational and investment costs. For (ii), we de- velop scheduling strategies where the process dynamics and corresponding controller designs are simultaneously considered, resulting in explicit control–dependent scheduling schemes. Surrogate/approximate models are proposed to address the time–scale mismatch between the mid–term schedule and the short–term control optimization problem. Finally, the inte- gration of (i) and (ii) is shown within an overall dynamic optimization problem. The developments are presented via a domestic cogeneration heat and power (CHP) system example and case studies of a tank, a continuously stirred tank reactor and a binary distillation column.

This thesis discusses recent advances in a variety of areas in multi-parametric programming and explicit model predictive control (MPC). First, novel theoretical and algorithmic results for multi-parametric quadratic and mixed-integer quadratic programming (mp-QP/mp- MIQP) problems extend the current state-of-the-art: for mp-QP problems, it is shown that its solution is given by a connected graph, based on which a novel solution procedure is developed. Furthermore, several computational studies investigate the performance of different mp-QP algorithms, and a new parallelization strategy is presented, together with an application of mp-QP algorithms to multi-objective optimization. For mp-MIQP problems, it is shown that it is possible to obtain the exact solution of a mp-MIQP problem without resorting to the use of envelopes of solutions, whose computational performance is compared in a computational study with different mp-MIQP algorithms. Then, the concept of robust counterparts in robust explicit MPC for discrete-time linear systems is revisited and an elegant reformulation enables the solution of closed-loop robust explicit MPC problems with a series of projection operations. This approach is extended to hybrid systems, where the same properties are proven to hold. Finally, a new approach towards unbounded and binary parameters in multi-parametric programming is introduced, and several examples highlight its potential.

Bioprocesses performance is monitored using microbial growth kinetics models. However most of them are empirical and unstructured ignoring molecular and transcriptional interactions thus failing in accurate prediction. Pseudomonas putida mt-2, which harbours the TOL plasmid, is a strain of great biotechnological potential. M-xylene and toluene are commonly utilised by TOL pathway while toluene enables chromosomal ortho-cleavage pathway activation. Herein, the transcriptional kinetics of TOL and key ortho-cleavage promoters which control substrate bioconversion resulting in biomass formation was consistently studied. Thus, revealing the interconnection of the two pathways and promoters’ specific expression patterns. The experimental observations lead to a dynamic model coupling transcriptional regulation to microbial growth kinetics by providing upstream quantitative information. This model enables adequate predicition capability of substrate utilisation and biomass growth under a wide range of initial conditions. However in nature it is uncommon for bacteria to degrade a sole substrate. Therefore P. putida mt-2 cells induction with succinate-toluene, m-xylene-toluene mixtures is studied. The transcriptional kinetics revealed promoters’ bi-modal expression pattern and carbon catabolite repression regardless of the growth conditions. Transcriptional regulation upon entry of m-xylene-toluene mixture was modelled resulting in a mechanistic microbial growth kinetic model development which accurately predicts substrate(s) utilisation and biomass growth patterns. The current double substrate microbial growth kinetic model can more accurately predict the macroscopic phenomena compared to the Monod, Monod-type and competitive enzymatic interaction models.

Leukaemia is defined as a group of haematological diseases (related to blood and blood-forming tissue) characterized by malignant proliferation of myeloblasts or lymphoblasts that replace normal bone marrow elements and infiltrate normal tissues. The study of leukaemia has been hindered by the lack of appropriate in vitro models, which can mimic this microenvironment. It is hypothesized that the fabrication of porous 3-D scaffolds for the biomimetic growth of leukaemic cells in vitro could facilitate the study of the disease in its simulated native 3-D niche. In this study, polyhydroxyalkanoate (PHA), in particular poly(hydroxybutyrate) (PHB) and poly(hydroxybutyrate-co-valerate) (PHBV) porous 3-D scaffolds with an improved thickness (in relative to the conventionally made PHA matrices) are utilized and investigated to model the abnormal 3-D leukaemic cellular growth system in the absence of exogenous cytokines. The polymeric porous 3-D scaffolds were fabricated using an ideal polymer concentration of 4% (w/v). The salt-leaching efficacy and the effect of salt residual on the cell growth media were carried out to validate the significant amount of salt remnant inside the porous materials. The physico-chemical characteristics of the porous 3-D scaffolds such as surface wetting, porosity, BET surface area and pore size distribution were studied by means of drop sessile analyzer (DSA), helium gas pycnometry, mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM). To increase probability of cellular attachment and proliferation, the polymeric scaffold surfaces were treated with O2-rf-plasma (100 W at 10 min) and NaOH (0.6M). Next, in order to improve the in vitro 3-D leukaemic cell culture, two main bone marrow extracellular matrix (ECM) proteins which are collagen type I or fibronectin were immobilized via physical adsorption on the treated surfaces of the polymeric porous 3-D scaffolds. Meanwhile, the in vitro degradation studies were conducted on both polymeric scaffolds with the hydrolytic degradation media of phosphate buffered saline (PBS) and cell growth media. The scaffolds were analyzed and compared for mass loss, morphology and pH changed of the PBS and cell growth media throughout 45 weeks and 9 weeks of the study respectively. Overall, PHB and PHBV displayed a good seeding efficiency (24 h) and excellent leukaemic cellular growth for up to 6 weeks (protein-coated scaffolds), assessed by MTS assay and SEM. Once the abnormal hematopoietic 3-D model (cell lines) was established, a new model to culture human primary acute myeloid leukaemia mononuclear cells (AML MNCs) was studied, compared and validated. All leukaemic cells grew better in PHBV scaffolds coated with 62.5 μg/ml collagen type I and sustained cell growth in the absence of exogenous cytokines. As a result, it was concluded that PHBV-collagen scaffolds may provide and could be used, as a practical model with which to study the biology and treatment of primary AML in an in vitro mimicry without the use of 2-D culture system and animal models.

This thesis describes the fabrication of novel green materials using nanocellulose as the building block. Bacterial cellulose (BC) was used as the nanocellulose predominantly in this work. BC is highly crystalline pure cellulose with an inherent fibre diameter in the nano-scale. A single BC nanofibre was found to possess a Young’s modulus of 114 GPa. All these properties are highly favourable for using BC as a nanofiller/reinforcement in green nanocomposite materials. In this work, the surface of BC was rendered hydrophobic by grafting organic acids with various aliphatic chain lengths. These surface-modified BC was used as nanofiller for poly(L-lactide) (PLLA). Direct wetting measurements showed that the BC nanofibre-PLLA interface was improved due to the hydrophobisation of BC with organic acids. This led to the production of BC reinforced PLLA nanocomposites with improved tensile properties. Nanocellulose can also be obtained by grinding of wood pulp, producing nanofibrillated cellulose (NFC). The surface and bulk properties of one type of NFC and BC were compared in this work. Furthermore, the reinforcing ability of NFC and BC was also studied and it was observed that there is no significant difference in the mechanical performance of NFC or BC reinforced nanocomposites. A novel method based on slurry dipping to coat sisal fibres with BC was developed to modify the surface of natural fibres. This method can produce either (i) a densely BC coating layer or (ii) “hairy” BC coated sisal fibres. Randomly oriented short BC coated sisal fibre reinforced hierarchical composites were manufactured. It was found that hierarchical (nano)composites containing BC coated sisal fibres and BC dispersed in the matrix were required to produce composites with improved mechanical properties. This slurry dipping method was also extended to produce robust short sisal fibre preforms. By infusing this preform with a bio-based thermosetting resin followed by curing, green composites with significantly improved mechanical properties were produced. BC was also used as stabiliser and nano-filler for the production of macroporous polymers made by frothing of acrylated epoxidised soybean oil followed by microwave curing.

Embryonic stem cells (ESCs) are promising as therapeutic material since they are pluripotent (potentially differentiate into any mature cell) and have “limitless” self-renewal capacity. To achieve widespread clinical utility, ESC cultures have to be designed to meet specific process requirements (e.g. quantity, quality etc.). Currently, most pluripotent stem cell (PSC) cultures are fragmented protocols relying on operator–intensive processing, as 2D monolayers on tissue culture plastic, at ambient O2 conditions. Incidentally, such culture conditions are sub-optimal, often leading to unscheduled stem cell behaviour. This thesis examines how ESC bioprocesses can be improved. Culture environment effects on ESCs are investigated, as well as computational tools for in silico design. I demonstrate how critical culture parameters and mathematical modelling can be exploited to improve the undifferentiated expansion of ESCs. Beginning with 3D murine ESCs (mESCs) cultures, 1) dynamic rotary cultures were demonstrated to improve self-renewal signalling activity, yielding improved proliferation of mESCs with higher “stemness” levels. 2) Culture metabolism was another critical factor. During batch feeding, metabolites accumulate within the culture environment especially at later stages in culture, causing stresses that impair ESC proliferation and “stemness”, independent of growth factor levels. In contrast, perfusion feeding maintained well-regulated culture environments that promoted the expansion of highly “naïve” mESCs. 3) Computational approaches can complement bioprocess design. Mathematical models identified novel multi-scale interactions within the bioprocess and effectively simulated bioreactor fluid dynamics. 4) As a means to further optimize the bioprocess, alternative signalling factors were combined with dynamic perfusion cultures in reduced (5%) O2 conditions, which generated increased cell yields having high “stemness” levels at half the costs. In conclusion, numerous ‘standard’ culture conditions were found to be sub-optimal for mESC culture, emphasizing the need for improved bioprocesses using rational design based on stem cell bioscience. It is anticipated that these integrated stem cell bioprocesses, can improve product yield and quality at reduced costs. Such bioprocess strategies will facilitate the usage of PSCs as therapeutics.

Even relatively simple microorganisms, which have been extensively studied, are hosts to a complex network of interconnected processes occurring on diverse time scales. The multilevel nature of the regulatory network of cells and the interactions occurring at the intra cellular level further augment this complexity (Yokobayashi et al., 2003). Attempts to wholly model the function of even a single cell are currently non trivial, if not impossible, as the amount of delicate intracellular measurements required to validate such a model is exhaustive both in terms of labour and cost. Uncertainties introduced both on the parameter identifiability and on the mechanistic level further complicate this task. The large number of biological data generated with the advancement of a variety of high-throughput experimental technologies demand for the development of comprehensive mathematical model building methods able to capture the complex phenomena occurring within a cell (Covert et al., 2001). Borrowing the fundamental research principles from the Process Systems Engineering paradigm, mathematical modelling of biological systems can provide a systematic means to quantitatively study the characteristics of the multilevel interactions that occur in cell culture. In the present thesis, an integrated modelling framework is established that can ensure the seamless interaction of experimental biology with the development of quantitative mathematical descriptions of biological systems. The use of model-based techniques can facilitate the reduction of unnecessary experimentation and reduce labour and operating costs by identifying the most informative experiments and providing strategies to optimise and automate the bio-process at hand. Paving the way towards a ‘closed-loop’ approach for bio-process automation (Kiparissides et al., 2011), the work herein presents a biological model development framework following a step by step approach, highlighting challenges and “real life” problems associated with each stage of model development. By organising available information in a systematic way, unnecessary experimentation is avoided and models with a priori objectives can be established to guide the in vivo process through the in silico representation. The proposed methodology combines macroscopic and subcellular model development, parameter estimation, global sensitivity analysis, model based design of experiments and selection of optimal feeding policies via dynamic optimisation methods in a fromalised structure. The combined mathematical and experimental framework for the control and optimisation of mammalian cell culture systems, presented herein, is experimentally validated via the succesfull model based optimisation of antibody secreting GS-NS0 cell cultures.

Blood shortage is one of the biggest concerns of the World Health Organization. The reason: while around 30% of the industrialized world population will require a life-saving transfusion sometime during their lives, only 6% of the total population actually donates blood that can only be stored for up to 42 days. Despite the huge efforts that have been made to awaken the population to proactively donate blood (according to the World Health Organization 2009a, apporximately 85 million units of blood are collected annually worldwide), there is still a large shortage around the world. Hence, other alternatives have been suggested to address this. Since the first attempts, in the late 1940s, to produce an alternative to blood donation in the laboratory, several breakthroughs were made, most of them focusing on the synthetic route: the search for a chemical molecule that could replace the main vital function of red blood cells (RBCs) - oxygenation of the body cells (Kimball 1994). Nevertheless, several issues, mainly regarding the stability and controlled oxygen release by these molecules, still pose serious barriers for the success of this option (Kimball 1994). Nevertheless, several issues, mainly regarding the stability and controlled oxygen release by these molecules, still pose serious barriers for the success of this option (Kimball 1994). Hence, the focus on blood substitutes is now being directed to blood itself. My PhD project in the Department of Chemical Engineering of Imperial College London aimed at mimicking nature's bioreactor to produce human blood: the Bone Marrow (BM), a three-dimensional (3D) structure comprised of a vascularized support matrix, cellular constituents, and humoral factors. The BM 3D spatial configuration generates micro-concentration gradients that modulate cellular self-renewal, differentiation and apoptosis - mimicked by a 3D scaffold that can be rendered bioactive by coating with extracellular proteins. On the other hand, the BM vascular system ensures the supply of nutrients and removal of harmful metabolites, as well as the collection of maturing cells formed in the marrow cavity - mimicked by an intricate selectively-permeable membrane system that both renews the microenvironment and harvests mature RBCs. I have combined these two bio-inspired characteristics of the marrow into the first ex vivo 3D dual hollow fibre bioreactor (DHFB) that allows addressing mass transfer challenges faced by state-of-the-art technology and the continuous production and release of RBCs. This system was shown to be biocompatible, and allowed the differentiation of cord blood stem cells into mature enucleated RBCs under a cocktail composed of 100ng.mL-1 stem cell factor and 2,000U.ml-1 erythropoietin over 31 days. Mature enucleated RBCs could be prpduced exclusively ex vivo in a 3-dimension feeder-free culture. This technology has the potential to allow cost-effective production of clinically-relevant numbers of red blood cells with selective cell harvesting in a closed hematopoietic system.

Embryonic stem cells (ESCs) are known for their ability to be maintained almost indefinitely in an undifferentiated, proliferating state with the potential to give rise to all the cell types. Current strategies for the differentiation of ESCs are limited by their inability to control differentiation resulting in a heterogeneous cell population. Addressing this limitation, it has been previously reported that treatment with HepG2-conditioned medium (HepG2-CM) enhances the formation of multipotent mesodermal progenitors from ESCs. This promotes greater control of ESC differentiation in a lineage-specific fashion possibly resulting in efficient skeletal differentiation, which is an observation demonstrated by our group. In this study, by regulating culture time, preferential differentiation to either the osteogenic or cardiomyogenic lineage from murine ESCs was achieved using HepG2-CM in a three-dimensional integrated bioprocess. In addition, an automatable and scalable bioprocess was developed through the design, fabrication, and testing of a novel perfusion bioreactor system that has improved mineralised cellular construct generation. Finally, an animal pilot study was conducted to evaluate the efficacy and toxicity of our mineralised cellular constructs

Acute Myeloid Leukaemia (AML) is a cancer of hematopoietic stem cells that develops in the three-dimensional (3-D) niches provided by the bone marrow microenvironment in vivo. The study of AML has been hampered by the lack of appropriate ex vivo models, which can mimic this microenvironment. It was hypothesised that the fabrication of scaffolds for the biomimetic growth of leukemic cells ex vivo could facilitate the study of the disease in its native 3-D niche. The growth of different leukemic cell lines was first evaluated, namely K- 562, HL-60 and Kasumi-6 on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials: poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate) (PMMA), poly (D, L-lactade) (PDLLA), poly (caprolactone) (PCL), and polystyrene (PS). These results were compared with two commercially available scaffolds from BD™ Biosciences. Overall, out of all the scaffolds, PLGA and PU displayed the best seeding efficiency and leukemic cellular growth, assessed by MTS assay, scanning electron microscopy and immunohistochemistry. In order to improve the ex vivo 3-D leukemic cell culture, PLGA and PU scaffolds were coated with bone marrow extracellular matrix (ECM) proteins, collagen (62.5 or 125 μg/ml) and fibronectin (25 or 50 μg/ml) and a combination of both proteins: collagen + fibronectin (62.5 + 25 μg/ml) respectively. Once the abnormal hematopoietic 3-D model was established, a new model to culture normal hematopoietic cord blood mononuclear cells was studied and compared. All 3 leukemic cell lines and cord blood cells grew better in PU scaffolds coated with collagen type I using the low concentration and sustained growth in the absence of exogenous cytokines. As a result, it was concluded that PU-collagen scaffold could provide a practical model with which to study the biology and treatment of primary AML in an ex vivo mimicry without the use of animal models.

Stem cell therapies and tissue engineering strategies are required for the clinical treatment of respiratory diseases. Previous studies have established protocols for the differentiation of airway epithelium from stem cells but have involved costly and laborious culture methods. The aim of this thesis was to achieve efficient and reproducible maintenance and differentiation of embryonic stem cells to airway epithelium, in 2D and 3D culture, by developing appropriate bioprocessing technology. Firstly, the 2D differentiation process of human and murine ES cells into pulmonary epithelial cells was addressed. The main finding in was that the proportion of type II pneumocytes, the major epithelial component of the gas-exchange area of lung, differentiated with this method was higher than that obtained in previous sudies, 33% of resultant cell expressed the specific marker surfactant protein C (SPC) compared with up to 10%. Secondly, the maintenance and differentiation was carried out in 3D. A protocol was devised that maintained undifferentiated human ES cells in culture for more than 200 days encapsulated in alginate without any feeder layer or growth factors. For ES cell differentiation in 3D, a method was devised to provide a relatively cheap and simple means of culture and use medium conditioned by a human pneumocyte tumour cell line (A549). The differentiation of human and murine ES cells into pulmonary epithelial cells, particularly type II pneumocytes, was found to be upregulated by culture in this conditioned medium, with or without embryoid body formation. The third step was to test whether this differentiation protocol was amenable to scale-up and automation in a bioreactor using cell encapsulation. It was possible to show that encapsulated murine ES cells cultured in static, co-culture or rotating wall bioreactor (HARV) systems, differentiate into endoderm and, predominantly, type I and II pneumocytes. Flow cytometry revealed that the mean yield of differentiated type II pneumocytes was around 50% at day 10 of cultivation. The final stage of the work was to design and produce a perfusion system airlift bioreactor to mimic the pulmonary microenvironment in order to achieve large scale production of biologically functional tissue. The results of these studies thus provide new protocols for the maintenance of ES cells and their differentiation towards pulmonary phenotypes that are relatively simple and cheap and can be applied in bioreactor systems that provide for the kind of scale up of differentiated cell production needed for future clinical applications.