Nigel Titchener-Hooker

• University College London

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form o...

Bioprocess development studies often involve the investigation of numerical and categorical inputs via the adoption of Design of Experiments (DoE) techniques. An attractive alternative is the deployment of a grid compatible Simplex variant which has been shown to yield optima rapidly and consistently. In this work, the method is combined with dummy...

Biological medicines are produced by a bioprocess: a series of steps, from cell culture through purification to final formulation and possibly freeze-drying. Such a bioprocess must be demonstrated to be in control over the lifetime of the medicine. It is now acknowledged that optimizing each individual unit operation may not suffice for overall con...

Depth filtration is employed in the production of many biological products. Although high throughput process development is available for most purification operations, options for high throughput depth filtration are limited. In this report, we describe the development and qualification of an ultra scale-down (USD) device for high throughput depth...

BACKGROUND This study ascertains the degree to which bioseparation techniques, considered as alternatives to packed bed chromatography, have been adopted for large-scale biomanufacturing. Such information will be useful to process engineers interested in investigating the potential of these technologies. RESULTSThe results are based on three intern...

The past decade has seen an increasing focus on the issues surrounding climate change and this has triggered international governments to develop environmental legislation and policies for the energy-intensive industries (EIIs) that can help reduce their anthropogenic greenhouse gases (GHG) emissions. The biopharmaceutical industry is a relatively...

The rapid development of purification processes for polysaccharide vaccines is constrained by a lack of analytical tools current technologies for the measurement of polysaccharide recovery and process-related impurity clearance are complex, time-consuming, and generally not amenable to high throughput process development (HTPD). HTPD is envisioned...

The increasing requirement for multivalent vaccines containing diverse capsular polysaccharides has created an unmet need for a fast and straightforward assay for polysaccharide titer. We describe a novel and robust assay for the quantitation of anionic capsular polysaccharides. The binding of hexadecyltrimethyammonium bromide (Hb) to anionic capsu...

In recent years, high throughput screening (HTS) studies have been increasingly employed as an integral element of bioprocess development activities. These studies are often limited by an analytical bottleneck; they generate multiple samples for analysis and the available analytical methods cannot always cope with the added analytical burden. A pot...

During bioprocess development a potentially large number of analytes require measurement. Selection of the best set of analytical methods to deploy can reduce the analytical requirements for process investigation but currently relies on application of heuristics. This paper introduces a generic methodology, Strategic Assay Selection, for screening...

The large-scale manufacture of biological products results in the generation of significant quantities of process information that can be used to inform future design decisions. Currently this information is not exploited to its full potential. The challenge is thus to identify and/or develop tools that allow the utilisation of this valuable resour...

The capacity to locate efficiently a subset of experimental conditions necessary for the identification of an operating envelope is a key objective in many studies. We have shown previously how this can be performed by using the simplex algorithm and this paper now extends the approach by augmenting the established simplex method to form a novel hy...

High throughput approaches to facilitate the development of chromatographic separations have now been adopted widely in the biopharmaceutical industry, but issues of how to reduce the associated analytical burden remain. For example, acquiring experimental data by high level factorial designs in 96 well plates can place a considerable strain upon a...

Flat-sheet membrane discs represent the current standard format used for experimental prediction of the scale-up of normal flow filtration processes. Use of this format is problematic, however, since the scale-down results typically show a 40-55% difference in performance compared to large-scale cartridges depending upon the feedstock used. In this...

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BACKGROUND: Recently, there has been increased interest in employing process analytical technologies (PAT) that combine an understanding of how operating parameters affect product quality together with real-time analysis to achieve efficient process manipulation during operation and thereby ensure that product outputs meet specifications. Primarily...

Pleating of membranes allows a larger surface area to be packed into small volume modules and is an integral feature of cartridges used in a range of biotechnology applications. This work investigates the effect that pleating has upon cartridge performance. Using specially fabricated 2.54cm (1″) sections of a large scale pleated membrane cartridge,...

An agent-based system framework is developed to provide a flexible environment for analysing bioprocesses based on a whole process understanding. In this system, agent components cooperate with each other in performing their tasks. These include the description of the whole process behaviour, evaluating process operating conditions, monitoring of t...

Growing commercial pressures in the pharmaceutical industry are establishing a need for robust computer simulations of whole bioprocesses to allow rapid prediction of the effects of changes made to manufacturing operations. This paper presents an integrated process simulation that models the cGMP manufacture of the FDA-approved biotherapeutic CroFa...

Biopharmaceutical manufacture is subject to numerous risk factors that may affect operational costs and throughput. This paper discusses the need for incorporating such uncertainties in decision-making tools in order to reflect the inherent variability of process parameters during the operation of a biopharmaceutical plant. The functionalities of a...

The article examines how a small set of easily implemented micro biochemical engineering procedures combined with regime analysis and bioprocess models can be used to predict industrial scale performance of biopharmaceutical protein downstream processing. This approach has been worked on in many of our studies of individual operations over the last...

Single-column chromatography is widely used in the biopharmaceutical industries, although multi-column alternatives in the form of simulated moving bed (SMB) processes are now emerging. It may be difficult, however, to determine which column alternative will be best suited for a given application, and this work sets out to address this issue. A sys...

In this article, a systematic approach is presented which allows the estimation of model parameters including isotherm parameters for equilibrium-dispersive and general rate models including a procedure for how to subsequently select the most appropriate isotherm and model for a given application. The approach consists of three parts: determination...

BACKGROUND: The financial and technical challenges currently being faced by the pharmaceutical industry have prompted the development of computer models that rapidly evaluate manufacturing strategies for commercial‐scale drug production. The utility of a model is enhanced by the application of sensitivity analysis techniques (SA), which seek to qua...

The present paper describes the application of GSA (Global Sensitivity Analysis) techniques to mathematical models of bioprocesses in order to rank inputs such as feed titres, flow rates and matrix capacities for the relative influence that each exerts upon outputs such as yield or throughput. GSA enables quantification of both the impact of indivi...

This paper describes the use of Principal Component Analysis (PCA) as a tool for modeling chromatographic separations. PCA is an analytical technique developed to extract key information out of large data sets and to develop relationships and correlations. The basis of the proposed model is the use of PCA to correlate experimental chromatographic d...

Supercoiled plasmid DNA is susceptible to fluid stress in large-scale manufacturing processes. A capillary device was used to generate controlled shear conditions and the effects of different stresses on plasmid DNA structure were investigated. Computational fluid dynamics (CFD) analysis was employed to characterize the flow environment in the capi...

The selection of appropriate operating conditions for bioprocessing is complex due to the large number of interacting stages and variables. Bioprocesses also operate under tight regulation and therefore tools to assist bioprocess design are of significant utility. Conventional approaches for the analysis of variable sensitivities are inadequate. We...

This work presents a systematic evaluation of the influence of lipids and casein on the performance of a chromatographic capture step for the recovery of a target protein from transgenic milk. Lactoperoxidase (LPO) was spiked at concentrations typical of those to be expected for transgenic proteins in commercial bovine milk and the dynamic adsorpti...

A simulation is described that evaluates the impacts of altering bio-manufacturing processes. Modifications designed to improve production levels, times and costs were assessed, including increasing feed volumes/titres, replacing initial downstream stages with packed or expanded bed affinity steps and removing ion exchange steps. Options were evalu...

A systematic framework is developed in this work to analyse bioprocesses based on a whole process understanding considering the interactions between process operations. This yields a capacity to predict process performance in the case of process variations, and to improve process efficiency within the process design space. An agent-based approach i...

Membrane filtration through 0.2 μm pores is typically the last operation in the production of pharmaceutical grade plasmid DNA. The membrane sterilisation of purified DNA solutions containing plasmids and bacterial artificial chromosomes (BAC) is investigated in this paper. A linear relationship between total DNA transmission and vector size was ob...

Automated experimentation in microwell plate formats is widely used in high throughput drug discovery. Such approaches are now being considered for the study of bioprocess unit operations in order to speed the delivery of new medicines to market. The generation of useful design data from microwell formats requires an understanding of the engineerin...

Fed-batch and perfusion culture dominate mammalian cell culture production processes. In this paper, a decision-support tool was employed to evaluate the economic feasibility of both culture modes via a case study based upon the large-scale production of monoclonal antibodies. The trade-offs between the relative simplicity but higher start-up costs...

A key issue within the biopharmaceutical industry is the potential feasibility of retrofits as a means of reducing operating costs and improving process efficiency. This paper presents a framework, designed to capture both process and business aspects of such a decision. This allows analysis of the key economic and technical elements and the overal...

Growing pressures in the bioprocess industries are driving the need for simulations that rapidly evaluate strategies for achieving improvements in large-scale production. This paper presents a prototype simulation that evaluates the financial and technical impacts of developing and implementing a range of manufacturing changes to a pre-existing ind...

The selection of appropriate operating conditions for bioprocessing is complex due to the large number of interacting stages and variables. Models for each processing stage require significant numbers of variables and thus the whole process model will consist of a large number of variables overall. Interactions typically exist within a bioprocess m...

This study presents an experimental approach to measure the density distribution of expanded bed adsorption (EBA) matrices. We report on the use of a series of solutions of caesium trifluoroacetate (CsTFA) of varying density spun in a laboratory centrifuge so as to separate representative matrix samples on the basis of bead density. Mass data was u...

The risk of a pandemic with a virulent form of influenza is acknowledged by the World Health Organization (WHO) and other agencies. Current vaccine production facilities would be unable to meet the global requirement for vaccine. As a possible supplement a DNA vaccine may be appropriate, and bioprocess engineering factors bearing on the use of exis...

Fermentations carried out at 450-L and 20-L scale to produce Fab' antibody fragments indicated a serious problem to control levels of dissolved oxygen in the broth due to the large oxygen demand at high cell densities. Dissolved oxygen tension (DOT) dropped to zero during the induction phase and it was hypothesised that this could limit product for...

Chromatographic separation is a pre-eminent downstream processing operation for the isolation of biological products, and has excellent resolution so is almost indispensable in the manufacturing of biopharmaceuticals for human diagnostic and therapeutic purposes. The complexity of chromatographic process dynamics, the large number of parameters inv...

A comparison between two feeding strategies in a gas blending fermentation process to produce Fab’ by Escherichia coli was carried out at 20 L scale. A pH-stat feeding strategy was proposed to address the issue of glycerol oscillations that occur in repeated batch fermentations. This feeding strategy was based on direct coupling of growth and conco...

Given the time, cost and risk associated with drug development, biopharmaceutical companies typically need to have a portfolio of drugs in development to be successful. Current pressures of cost and speed to market are driving the need for more effective means of assessing the value and risks of such drug portfolios. This paper presents research to...

Scale-down is a methodology that combines the use of very small volumes of process fluid in dedicated devices to predict accurately the behaviour of process-scale biotechnological unit operations and for the production of comparable material for use in further devices which, taken together, facilitate the mimic of a complete full-scale process. Thi...

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a...

This work demonstrates an experimental method for studying breakthrough behaviour in expanded beds. The behaviour of beds made with differently sized particles were studied at varying flowrates. The use of a dimensionless residence time measurement allowed a more valid comparison of breakthrough characteristics in expanded bed operation by compensa...

This paper discusses the need for decisional-support tools in the biotech industry and presents the configuration of a prototype tool for modelling bioprocesses and regulatory compliance activities such as quality control (QC), quality assurance (QA) and batch documentation in a biopharmaceutical plant. The impact of employing a range of manufactur...

Quality control (QC)/quality assurance (QA) and batch documentation form key parts of the in-process validation procedure for any biopharmaceutical. The activities of QC/QA and batch documentation rather than the manufacturing steps themselves are usually rate limiting in a plant (Ramsay, 2001). Thus, managing QC/QA and batch documentation becomes...

In this paper, optimal operating policies for HPLC separation processes based on a closed-loop recycling strategy are presented. Successful application of closed-loop recycling HPLC processes had been reported, mainly in the pharmaceutical industry, but there is still limited knowledge about the dynamic behaviour of this process and little work on...

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 identificat...

A prototype Streamline-Phenyl matrix was evaluated in a hydrophobic interaction mode for the direct recovery of alcohol dehydrogenase (ADH) from yeast cell homogenate. At 5% breakthrough of ADH, a yield of 100% was obtained for a dynamic expanded bed capacity of 240 U(ADH)/ml matrix with a purification factor of 9.2. This compared with a dynamic ca...

Traditionally most downstream bioprocesses have been operated without real-time knowledge of product and key contaminants, yielding little confidence in their operation and the impact on subsequent operations. A rapid UV-vis spectral prediction technique has been successfully demonstrated for the at-line characterization of a large scale continuous...

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was p...

A simple engineering framework that enables the rapid representation of the performance of liquid chromatographic separations is provided in this paper. The fractionation diagram and its associated maximum purification factor versus product yield, and contamination index versus product yield diagrams, may be derived directly from chromatographic da...

This chapter discusses a systematic procedure for isotherm determination. The isotherm parameters for the candidate isotherms by estimation methods based on a minimal set of experimentally obtained elution profiles is determined. The chapter describes the isotherm model that can accommodate the displacement and competitive effects that characterize...

Time to market, cost effectiveness, and flexibility are key issues in today's biopharmaceutical market. Bioprocessing plants based on fully disposable, presterilized, and prevalidated components appear as an attractive alternative to conventional stainless steel plants, potentially allowing for shorter implementation times, smaller initial investme...

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimat...

A decision-support tool is presented for modelling both the technical and business aspects of biopharmaceutical manufacture. The use of the tool for risk analysis is demonstrated through a case study that uses Monte Carlo simulation to imitate the randomness inherent in manufacturing subject to technical and market uncertainties. The analysis demon...

Detailed mathematical models of chromatographic separation processes is of crucial importance due to the increased application of this separation technique in the downstream processes of the biotechnological, pharmaceutical and fine chemical industries. In this paper, a detailed dynamic model of a high performance liquid chromatographic unit was ex...

In a previous paper, the stages of biopharmaceutical process development were defined and it was shown that a body of experience is accumulating, which can be used in the selection of the best process. In this second article, new methods are described by which faster development of such processes can be achieved.

This paper describes the development of a hierarchical framework for modelling biopharmaceutical manufacture. Emphasis is placed on how a closer integration of bioprocess and business process modelling can be achieved by capturing common information in an object-oriented enviroment. The steps to use the developed decision-support software tool for...

Conventional control of expanded-bed adsorption (EBA), like that of packed-bed chromatography, is based upon off-line measurements of the column eluant. The relatively high-void volumes in EBA systems means that this approach can lead to significant performance losses caused by the inability to achieve tight control of breakthrough. This problem is...

The height of a stable expanded bed depends upon the physical properties of the inlet liquid of which viscosity, density and flow rate have the most significant influence. A change in any of these parameters will subject the top of the bed to fluctuations and effective control would require monitoring the position of the top of the bed. In this wor...

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for th...

This paper presents a case study for use in the teaching of bioprocess design. Taking the production and isolation of an intracellular protein from S. cerevisae , it demonstrates how undergraduates can use a range of data to construct and then to investigate the range of process flowsheet options available for such a process duty. The paper conside...

The purification of an intracellular product from a complex mixture of contaminants after cell disruption is a common problem in processes downstream of fermentation systems. This is particularly challenging for the recovery of particulate (80 nm in diameter) multimeric protein products, named virus-like particles (VLPs), from cell debris and other...

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...

The recovery of plasmid DNA from cells is achieved by a two-step chemical reaction which usually employs sodium hydroxide and sodium dodecyl sulphate followed by neutralisation with a chilled solution of potassium acetate. It is important that the gelatinous floc of chromosomal DNA with proteins debris is not broken down since fragments of chromoso...

The disruption of recombinant E. coli cells containing a 76.8kb plasmid DNA was achieved by the chemical lysis method in a coaxial cylinder rheometer which allowed in situ measurements of rheological changes to be carried out as the lysis reaction proceeded. For the cases studied the cell lysis time was found to be approximately 30s. Moreover, the...

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that...

Biotech companies need to reduce time to market and to raise the efficiency of processing in order to lower costs. This paper presents results of the application of computer-based simulations for the rapid formulation of integrated bioprocesses designs and demonstrates the use of simulations to explore the nature and impact of the interactions that...

Models derived from a computational fluid dynamics (CFD) package (CF4X) were used to predict the fate of micro-organisms lost in aerosols as an incidental feature of the normal operation of bioprocess equipment. The model predicts the tracks of particles from their assumed source to their point of capture in an Aerojet cyclone impinger. In a set of...

Principal component analysis (PCA) was used to analyse the behaviour of a chromatographic separation as its scale increased. Three 4.6 mm diameter columns identical in every respect except for column length (25, 15 and 5 cm), were used to generate the data from a test system based on the reversed-phase HPLC separation of crude erythromycin on a pol...

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high m...

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plas...

SDS–alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high m...

Models for high pressure homogenisation developed using natural bakers' yeast were applied to a recombinant yeast strain. The models were found to be generic in nature and generally adequate for initial process design (&#4530%). More accurate descriptions (&#4510%) of the release of protein engineered enzyme product, the release of total soluble pr...

This paper examines the selection of the appropriate representation of chromatogram data prior to using principal component analysis (PCA), a multivariate statistical technique, for the diagnosis of chromatogram data sets. The effects of four process variables were investigated; flow rate, temperature, loading concentration and loading volume, for...

Numerical simulations are presented showing the effects of operating and geometrical parameters on the transition of laminar to Taylor vortex flow for induced rotational-axial flow in the gap of a pair of rotating cylinders. These simulations indicate that annular rotational flow becomes more stable in the presence of a small degree of axial flow a...

A method is described for the scale-down of a disc stack centrifuge which reduces the number of separating discs and also the liquid and solid hold-up of the centrifuge bowl. This is to enable a reduced volume of process material to be used for study of clarification. Scale-down is achieved in stages using a series of interlocking inserts to suit p...

In determining performance during microbial cultivation, two main features are of importance: the nature of the micro-environment at the site of the cultivation and the microbial response towards this specific environment. It must be remembered that organisms perform according to their genetic capability and according to the nature of the environme...

Experiments were carried out aimed at establishing the effects of equipment scale down on the disruption of Baker's yeast cells in high pressure homogenisers. Data are reported on the cell debris particle size distribution (PSD) and on total protein release as a function of the applied pressure for two valve geometries and three scales of operation...

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...

The work reported in this paper examines the use of principal component analysis (PCA), a technique of multivariate statistics to facilitate the extraction of meaningful diagnostic information from a data set of chromatographic traces. Two data sets mimicking archived production records were analysed using PCA. In the first a full-factorial experim...

Experimental data are given for the solid pressure distributions in liquid chromatographic columns for two commercially available agarose-based compressible gel supports. The data show that for a given liquid velocity the pressure drop increases exponentially with height from the top surface. However, beyond a threshold liquid velocity, flow instab...

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 preci...

Saccharomyces cerevisiae was grown in aerobic continuous culture on a defined minimal medium, with glucose (40 g.l−1) as the growth-limiting carbon source, to acquire knowledge useful in process design and for model-based control. Steady-state concentrations of biomass, glucose, ethanol and activities of model products alcohol dehydrogenase, hexoki...

Experimental data relating to the breakage of isoelectric Soya protein precipitates in a mechanically agitated bioreactor are provided and examined in the light of a proposed mechanistic model which relates the size of the maximum attainable aggregate diameter to the energy dissipation rate in the vessel. The analysis suggests that protein precipit...

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 ident...

Measurements of size distributions are provided for the breakage of commercial packed bakers' yeast cells as a function of operating pressure and number of passes through a Manton Gaulin high-pressure homogenizer. A two parameter model was developed, based upon the use of a Boltzmann function, to simulate the changes in size distribution that accom...

The development of a model for simulating the recoveries by pilot-scale disc-stack centrifugation of whole yeast cells, yeast cell debris and protein precipitates prepared by ammonium sulphate salting-out is presented. The model is based on the grade efficiency concept and incorporates the effects of hindered settling at high biomass concentrations...