Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

University to business technology transfer — UK and USA comparisons

University to business technology transfer offers specific challenges, beyond those encountered in industry more widely. This paper examines the issues in university to business technology transfer in the UK and USA and presents the results of a survey of UK and US university technology transfer officers. Findings indicate significant differences in the motivations of universities in each country to transfer technology, the consistency of university technology transfer policies and the accessibility of university technologies to business. The study also looks at perceived barriers to university to business technology transfer and offers suggestions for possible improvements to the process. © 2006 Elsevier Ltd. All rights reserved.

Simulation modelling for business

Simulation modelling has been used for many years in the manufacturing sector but has now become a mainstream tool in business situations. This is partly because of the popularity of business process re-engineering (BPR) and other process based improvement methods that use simulation to help analyse changes in process design. This textbook includes case studies in both manufacturing and service situations to demonstrate the usefulness of the approach. A further reason for the increasing popularity of the technique is the development of business orientated and user-friendly Windows-based software. This text provides a guide to the use of ARENA, SIMUL8 and WITNESS simulation software systems that are widely used in industry and available to students. Overall this text provides a practical guide to building and implementing the results from a simulation model. All the steps in a typical simulation study are covered including data collection, input data modelling and experimentation.

Improving returnable transport equipment management with an autonomous approach

Challenges of returnable transport equipment (RTE) management continue to heighten as the popularity of their usage magnifies. Logistics companies are investigating the implementation of radio-frequency identification (RFID) technology to alleviate problems such as loss prevention and stock reduction. However, the research within this field is limited and fails to fully explore with depth, the wider network improvements that can be made to optimize the supply chain through efficient RTE management. This paper, investigates the nature of RTE network management building on current research and practices, filling a gap in the literature, through the investigation of a product-centric approach where the paradigms of “intelligent products” and “autonomous objects” are explored. A network optimizing approach with RTE management is explored, encouraging advanced research development of the RTE paradigm to align academic research with problematic areas in industry. Further research continues with the development of an agent-based software system, ready for application to a real-case study distribution network, producing quantitative results for further analysis. This is pivotal on the endeavor to developing agile support systems, fully utilizing an information-centric environment and encouraging RTE to be viewed as critical network optimizing tools rather than costly waste.

Development of real-time process control systems using formal techniques

A major application of computers has been to control physical processes in which the computer is embedded within some large physical process and is required to control concurrent physical processes. The main difficulty with these systems is their event-driven characteristics, which complicate their modelling and analysis. Although a number of researchers in the process system community have approached the problems of modelling and analysis of such systems, there is still a lack of standardised software development formalisms for the system (controller) development, particular at early stage of the system design cycle. This research forms part of a larger research programme which is concerned with the development of real-time process-control systems in which software is used to control concurrent physical processes. The general objective of the research in this thesis is to investigate the use of formal techniques in the analysis of such systems at their early stages of development, with a particular bias towards an application to high speed machinery. Specifically, the research aims to generate a standardised software development formalism for real-time process-control systems, particularly for software controller synthesis. In this research, a graphical modelling formalism called Sequential Function Chart (SFC), a variant of Grafcet, is examined. SFC, which is defined in the international standard IEC1131 as a graphical description language, has been used widely in industry and has achieved an acceptable level of maturity and acceptance. A comparative study between SFC and Petri nets is presented in this thesis. To overcome identified inaccuracies in the SFC, a formal definition of the firing rules for SFC is given. To provide a framework in which SFC models can be analysed formally, an extended time-related Petri net model for SFC is proposed and the transformation method is defined. The SFC notation lacks a systematic way of synthesising system models from the real world systems. Thus a standardised approach to the development of real-time process control systems is required such that the system (software) functional requirements can be identified, captured, analysed. A rule-based approach and a method called system behaviour driven method (SBDM) are proposed as a development formalism for real-time process-control systems.

Pharmaceutical compounding and dispensing

This is a detailed and practical guide to the theory and practice of extemporaneous compounding and dispensing, and a source of reference to extemporaneous formulae. Pharmacists have been responsible for compounding medicines for centuries and there is currently a dearth of current information on the topic, yet it is still taught in schools of pharmacy and required in community and hospital departments and by "specials" manufacturers and in development of new products in industry. This is a modern, detailed and practical guide to the theory and practice of extemporaneous compounding and dispensing, which will equip readers with the knowledge required for producing extemporaneous formulations safely and effectively.

Mathematical modelling and control of agitated extraction columns

Liquid-liquid extraction has long been known as a unit operation that plays an important role in industry. This process is well known for its complexity and sensitivity to operation conditions. This thesis presents an attempt to explore the dynamics and control of this process using a systematic approach and state of the art control system design techniques. The process was studied first experimentally under carefully selected. operation conditions, which resembles the ranges employed practically under stable and efficient conditions. Data were collected at steady state conditions using adequate sampling techniques for the dispersed and continuous phases as well as during the transients of the column with the aid of a computer-based online data logging system and online concentration analysis. A stagewise single stage backflow model was improved to mimic the dynamic operation of the column. The developed model accounts for the variation in hydrodynamics, mass transfer, and physical properties throughout the length of the column. End effects were treated by addition of stages at the column entrances. Two parameters were incorporated in the model namely; mass transfer weight factor to correct for the assumption of no mass transfer in the. settling zones at each stage and the backmixing coefficients to handle the axial dispersion phenomena encountered in the course of column operation. The parameters were estimated by minimizing the differences between the experimental and the model predicted concentration profiles at steady state conditions using non-linear optimisation technique. The estimated values were then correlated as functions of operating parameters and were incorporated in·the model equations. The model equations comprise a stiff differential~algebraic system. This system was solved using the GEAR ODE solver. The calculated concentration profiles were compared to those experimentally measured. A very good agreement of the two profiles was achieved within a percent relative error of ±2.S%. The developed rigorous dynamic model of the extraction column was used to derive linear time-invariant reduced-order models that relate the input variables (agitator speed, solvent feed flowrate and concentration, feed concentration and flowrate) to the output variables (raffinate concentration and extract concentration) using the asymptotic method of system identification. The reduced-order models were shown to be accurate in capturing the dynamic behaviour of the process with a maximum modelling prediction error of I %. The simplicity and accuracy of the derived reduced-order models allow for control system design and analysis of such complicated processes. The extraction column is a typical multivariable process with agitator speed and solvent feed flowrate considered as manipulative variables; raffinate concentration and extract concentration as controlled variables and the feeds concentration and feed flowrate as disturbance variables. The control system design of the extraction process was tackled as multi-loop decentralised SISO (Single Input Single Output) as well as centralised MIMO (Multi-Input Multi-Output) system using both conventional and model-based control techniques such as IMC (Internal Model Control) and MPC (Model Predictive Control). Control performance of each control scheme was. studied in terms of stability, speed of response, sensitivity to modelling errors (robustness), setpoint tracking capabilities and load rejection. For decentralised control, multiple loops were assigned to pair.each manipulated variable with each controlled variable according to the interaction analysis and other pairing criteria such as relative gain array (RGA), singular value analysis (SVD). Loops namely Rotor speed-Raffinate concentration and Solvent flowrate Extract concentration showed weak interaction. Multivariable MPC has shown more effective performance compared to other conventional techniques since it accounts for loops interaction, time delays, and input-output variables constraints.

The PCF and the question of intellectual workers : the crisis of social ideology

The aim of this thesis is to examine the approach of the Parti Communiste Francais (from 1956 to 1982) to the emergence of new strata of salaried `intellectual workers' (technicians, engineers, low to middle managers in industry and commerce, scientific researchers, teachers etc) parallelled by the gradual diminution of the traditional industrial working class which forms the core of the Party's support base. This examination is carried out in the context of the debate in France (initiated in the 1950s by social theorists of the Left) on the class membership and role of these strata. The reason for the emergence of such a debate is that in a society given to both a rapid evolution of its social structure and an increased polarisation between Left and Right, a precise knowledge of the objective and subjective determinations of new strata would enable parties to the Left to make proper distinctions between potential allies and adversaries. The thesis posits the view that the PCF has failed to make correct distinctions between its potential allies and adversaries and has thus pursued unsuccessful alliance strategies. The thesis contributes towards a scientifically-based understanding of one of the reasons governing the PCF's steady decline since the 1950s.

The influence of composition on the optical properties of electrodeposited gold

In industry the colour of a gold alloy electrodeposit is checked by visual comparison with standard panels. The aims of the present work have been to access the application of spectrophotmetric techniques to the measurement of the colour of gold alloy electrodeposits and to examine the factors that influence the colour of thin deposits. The minimum thickness of deposit required to produce its final colour and completely hide the underlying substrate was measured and found to depend on the nature of the substrate, the plating solution and the operating conditions. Bright and matt electrodeposits were studied. The influence of alloying gold by adding copper, silver and indium to the plating solution were investigated. CIE chromaticity coordinates were calculated from spectrophotometric data using a computer programme written for the purpose. The addition of silver to a simple gold bath caused the colour of the deposit to change from yellow through green to near white in a smooth progression as the amount of silver in solid solution steadily increased. The colour of deposits formed when additions of copper were made was complicated by the formation of intermediate phases. À colour in the blue region of the spectrum was obtained in a few experiments investigating the influence of indium additions to the gold bath.

An evaluation of certain aspects of an occupational health service

This thesis has been concerned with obtaining evidence to explore the proposition that the provision of occupational health services as arranged at the present time represents a misallocation of resources. The research has been undertaken within the occupational health service of a large Midlands food factory. As the research progressed it became evident that questions were being raised about the nature and scope of occupational health as well as the contribution, in combating danger at work, that occupational health services can make to the health and safety team. These questions have been scrutinized in depth, as they are clearly important, and a resolution of the problem of the definition of occupational health has been proposed. I have taken the approach of attempting to identify specific objectives or benefits of occupational health activities so that it is possible to assess how far these objectives are being achieved. I have looked at three aspects of occupational health; audiometry, physiotherapy and pre-employment medical examinations as these activities embody crucial concepts which are common to all activities in an occupational health programme. A three category classification of occupational health activities is proposed such that the three activities provide examples within each category. These are called personnel therapy, personnel input screening and personnel throughput screening. I conclude that I have not shown audiometry to be cost-effective. My observations of the physiotherapy service lead me to support the suggestion that there is a decline in sickness absence rates due to physiotherapy in industry. With pre-employment medical examinations I have shown that the service is product safety oriented and that benefits are extremely difficult to identify. In regard to the three services studied, in the one factory investigated, and because of the immeasurability of certain activities, I find support for the proposition that the mix of occupational health services as provided at the present time represents a misallocation of resources.

Rationalisation and improvement of a company's engineering design procedures

There is a great deal of literature about the initial stages of innovative design. This is the process whereby a completely new product is conceived, invented and developed. In industry, however, the continuing success of a company is more often achieved by improving or developing existing designs to maintain their marketability. Unfortunately, this process of design by evolution is less well documented. This thesis reports the way in which this process was improved for the sponsoring company. The improvements were achieved by implementing a new form of computer aided design (C.A.D.) system. The advent of this system enabled the company to both shorten the design and development time and also to review the principles underlying the existing design procedures. C.A.D. was a new venture for the company and care had to be taken to ensure that the new procedures were compatible with the existing design office environment. In particular, they had to be acceptable to the design office staff. The C.A.D. system produced guides the designer from the draft specification to the first prototype layout. The computer presents the consequences of the designer's decisions clearly and fully, often by producing charts and sketches. The C.A.D. system and the necessary peripheral facilities were implemented, monitored and maintained. The system structure was left sufficiently flexible for maintenance to be undertaken quickly and effectively. The problems encountered during implementation are well documented in this thesis.

Aircraft rotable inventory optimisation: development of a new solution

Analysis of the use of ICT in the aerospace industry has prompted the detailed investigation of an inventory-planning problem. There is a special class of inventory, consisting of expensive repairable spares for use in support of aircraft operations. These items, called rotables, are not well served by conventional theory and systems for inventory management. The context of the problem, the aircraft maintenance industry sector, is described in order to convey some of its special characteristics in the context of operations management. A literature review is carried out to seek existing theory that can be applied to rotable inventory and to identify a potential gap into which newly developed theory could contribute. Current techniques for rotable planning are identified in industry and the literature: these methods are modelled and tested using inventory and operational data obtained in the field. In the expectation that current practice leaves much scope for improvement, several new models are proposed. These are developed and tested on the field data for comparison with current practice. The new models are revised following testing to give improved versions. The best model developed and tested here comprises a linear programming optimisation, which finds an optimal level of inventory for multiple test cases, reflecting changing operating conditions. The new model offers an inventory plan that is up to 40% less expensive than that determined by current practice, while maintaining required performance.

Isolation, characterisation and application of novel microbial protein cross-linking enzymes

Microbial transglutaminase is favoured for use in industry over the mammalian isoform, and hence has been utilized, to great effect, as an applied biocatalyst in many industrial areas including the food and textiles industries. There are currently only a limited number of microbial TGase sources known. A number of organisms have been screened for transglutaminase activity using biochemical assays directed towards TGase catalyzed reactions (amine incorporation and peptide cross-linking assay). Of those organisms screened, TGase was identified in a number of isolates including members of the Bacillus and Streptomyces families. In addition, a protein capable of performing a TGase-like reaction was identified in the organism Pseudomonas putida that was deemed immunologically distinct from previously described TGase isoforms, though further work would be required to purify the protein responsible. The genuses Streptoverticillium and Streptomyces are known to be closely related. A number of micro-organisms relating to Streptomyces mobaraensis (formerly Streptoverticillium mobaraensis) have been identified as harboring a TGase enzyme. The exact biological role of Streptomyces TGase is not well understood, though from work undertaken here it would appear to be involved in cell wall growth. Comparison of the purified Streptomyces TGase proteins showed them to exhibit marginally different characteristics in relation to enzymatic activity and pH dependency upon comparison with Streptomyces mobaraensis TGase. In addition, TGase was identified in the organism Saccharomonospora viridis that was found to be genetically identical to that from S. mobaraensis raising questions about the enzymes dissemination in nature. TGase from S. baldaccii was found to be most diverse with respect to enzymatic characteristics whilst still retaining comparable E(y-glutamyl) lysine bond formation to S. mobaraensis TGase. As such S. baldaccii TGase was cloned into an expression vector enabling mass production of the enzyme thereby providing a viable alternative to S. mobaraensis TGase for many industrial processes.

Ultra-long mode-locked Er-droped fibre lasers

The development of ultra-long (UL) cavity (hundreds of meters to several kilometres) mode-locked fibre lasers for the generation of high-energy light pulses with relatively low (sub-megahertz) repetition rates has emerged as a new rapidly advancing area of laser physics. The first demonstration of high pulse energy laser of this type was followed by a number of publications from many research groups on long-cavity Ytterbium and Erbium lasers featuring a variety of configurations with rather different mode-locked operations. The substantial interest to this new approach is stimulated both by non-trivial underlying physics and by the potential of high pulse energy laser sources with unique parameters for a range of applications in industry, bio-medicine, metrology and telecommunications. It is well known, that pulse generation regimes in mode-locked fibre lasers are determined by the intra-cavity balance between the effects of dispersion and non-linearity, and the processes of energy attenuation and amplification. The highest per-pulse energy has been achieved in normal-dispersion UL fibre lasers mode-locked through nonlinear polarization evolution (NPE) for self-modelocking operation. In such lasers are generated the so-called dissipative optical solitons. The uncompensated net normal dispersion in long-cavity resonatorsusually leads to very high chirp and, consequently, to a relatively long duration of generated pulses. This thesis presents the results of research Er-doped ultra-long (more than 1 km cavity length) fibre lasers mode-locked based on NPE. The self-mode-locked erbium-based 3.5-km-long all-fiber laser with the 1.7 µJ pulse energy at a wavelength of 1.55 µm was developed as a part of this research. It has resulted in direct generation of short laser pulses with an ultralow repetition rate of 35.1 kHz. The laser cavity has net normal-dispersion and has been fabricated from commercially-available telecom fibers and optical-fiber elements. Its unconventional linear-ring design with compensation for polarization instability ensures high reliability of the self-mode-locking operation, despite the use of a non polarization-maintaining fibers. The single pulse generation regime in all-fibre erbium mode-locking laser based on NPE with a record cavity length of 25 km was demonstrated. Modelocked lasers with such a long cavity have never been studied before. Our result shows a feasibility of stable mode-locked operation even for an ultra-long cavity length. A new design of fibre laser cavity – “y-configuration”, that offers a range of new functionalities for optimization and stabilization of mode-locked lasing regimes was proposed. This novel cavity configuration has been successfully implemented into a long-cavity normal-dispersion self-mode-locked Er-fibre laser. In particular, it features compensation for polarization instability, suppression of ASE, reduction of pulse duration, prevention of in-cavity wave breaking, and stabilization of the lasing wavelength. This laser along with a specially designed double-pass EDFA have allowed us to demonstrate anenvironmentally stable all-fibre laser system able to deliver sub-nanosecond high-energy pulses with low level of ASE noise.

Polymer degradation and stability

This edition of Polymer Degradation and Stability is dedicated to papers which were presented in the session on ‘Chemical Modification’ at the Third International Conference on Modification Degradation and Stabilisation of Polymers (MoDeSt) held in Lyon in August 2004. This was the third meeting of the MoDeSt Society which was set up in the year 2000 under the chairmanship of Professor Franco LaMantia; the earlier meetings were held in Palermo (2000) and Budapest (2002). The overall goal of the MoDeSt Society is to promote the latest research carried out in University laboratories, in public organisations and in industry through publications, and organisation of biannual scientific conferences and workshops that act as forums for scientific developments and for promoting networking between academics and industrialists from across the field of polymer modification, degradation and stabilisation. In September 2004, Professor Norman Billingham was elected as Chairman of the Society. The conference was organised by Dr Alain Michel and Veronique Bounor-Legare of the University Claude Bernard, Lyon 1, attracting over 250 delegates with the ‘Chemical Modification’ Session alone enjoying 54 oral presentations and many more poster presentations. We are all very grateful to the organisers for running such a successful and enjoyable meeting. I wish to thank my co-editors, Professors Philippe Dubois and Domenico Acierno and Dr Alain Michel, for their cooperation in the editorial task of the papers published in this special issue. On behalf of the editors of this issue, I wish to express our appreciation to the Editor-in-Chief of Polymer Degradation and Stability, Professor Norman Billingham, for the support and help received during the preparation of this special issue.