2006 Annual Meeting Report

2006 Annual Meeting Report

ESACT-UK 16th Annual Meeting

9th-10th January 2006,

Gilbert Murray Conference Centre, University of Leicester

The 16th annual ESACT-UK meeting was held once again at Leicester and delegates generally agreed that the programme was excellent. A wide range of topics and interests were covered, with a larger portion of invited speakers presenting than in previous years. Below, each presentation is summarized.

‘What is the Therapeutic Potential of Human Embryonic Stem Cells?’

Dr Stephen Minger, King’s College London

This presentation began with an introduction to what stem cells are and described their development. Stem cells are pluripotent cells that can differentiate into different cell types. For this reason embryonic stem cells have therapeutic potential for targeting such things as CNS and endocrine disorders, cardiac, retinal and hepatic regeneration and joint and bone destruction. The developments required for therapeutic applications of human embryonic stem cells were discussed. These necessary developments include the use of animal-free, chemically defined media, the control of differentiation for the generation of specific cell populations, and the testing of therapeutically important cell populations in animal models.

‘Regenerative Medicine Bioprocessing – The Process is the Product’. Dr Chris Mason, University College London

Regenerative medicine includes the use of whole cells e.g. stem cells, and laboratory engineered tissues for clinical therapies. The targets of such clinical therapies are primarily those that do not heal easily with conventional medicine such as venous ulcers. Regenerative medicine bioprocessing is currently an expensive manual process. In order for cell and engineered tissue therapies to become routine clinical applications, process automation is necessary. The main issues that must be addressed with regards to automation of whole cell bioprocess engineering were discussed. These include consistent expansion, differentiation under controlled conditions, reliable distribution, and control of quality. These key issues are being addressed to secure a new UK industry in regenerative medicine bioprocessing.

‘The Role of Whole Cell Bioprocessing in Regenerative Medicine’. Dr Paul Kemp, Intercytex

Whole cell bioprocessing is the system of manufacturing living cells for regenerative medical therapies in order to correct a defect or disease. The sources and types of cells that are used were described. Individual steps in the manufacture of whole cells from procurement, isolation, expansion, assembly/packaging and transport through to clinical application were each considered. Some of the possible ways in which whole cell bioprocessing could be developed for the benefit of regenerative medicine were discussed.

’Using Yeast as a Host for Protein Production’. Dr Roslyn Bill, Aston University

This presentation focussed on the use of yeast as a host, primarily for the production of membrane proteins such as G-protein coupled receptors. Membrane proteins are important because they are ubiquitous, play pivotal roles in all cellular processes and are key drug targets. However, the structures of this class of protein are under represented in the Protein Data Bank. The advantages and disadvantages of bacterial, insect, mammalian and yeast cells as hosts for recombinant protein production were compared. Yeast combine the advantages of eukaryotes with the ease of handling of prokaryotes. The use of Saccharomyces cerevisiae and Pichia pastoris as hosts for the production of recombinant proteins were discussed, particularly with regards to their response to different culture conditions.

‘Alternative Antibody Formats Amenable to Microbial Expression’. Dr David Glover, UCB Celltech

Monoclonal antibodies (mAbs) of the IgG format are commonplace therapeutics for the treatment of a wide range of diseases. However, whilst IgG may be the optimal antibody format in some applications, for others, sub-fragments or engineered domains may be preferred. Due to the requirement of complex protein folding and post-translational modifications, mAbs are usually produced using mammalian expression systems. The use of the monovalent Fab' unit as a simpler alternative antibody format which can be produced using bacterial or yeast expression systems was presented. Microbial expression systems have advantages over mammalian expression systems in terms of scale, cost and time. The use of linker technology to confer or modify the mode of action, valency, cell killing and optimised half-life of Fab' antibody fragments was described.

‘Automating Cell Culture for Gene Expression: Facing the Challenge’. Dr Owen Jenkins, GlaxoSmithKline

This presentation discussed the essential application of automated cell culture to solve the challenges of gene expression. The production of recombinant proteins is essential to all stages of drug discovery; however, the redesign and generation of constructs, use of alternative expression systems and purification protocols can be highly resource-intensive and time-consuming. Multiple expression constructs grown under different conditions in different expression systems were run in parallel using automated technologies. The technologies used for automation of the various steps involved and prospects for the future were discussed.

‘The Development and Use of Cell-Based Assays in High Throughput Screens’. Dr Paul Hayter, Pfizer Global Research and Development

This talk presented the principles involved in the development and use of cell-based assays in high-throughput screening (HTS). Live cell assays have the advantage that compounds can be screened for activity against the drug target in a more representative physiological background. The process of generating cell-lines suitable for HTS and the adaptation of cell-based assays to automated systems for cell production and assay execution was described. In the development of a β-lactamase reporter gene assay β-lactamase expressing cells were isolated by flow assisted cell sorting (FACS) and a reporter cell line was generated. Clones with the desired properties were screened and the assay was miniaturised and optimised. Finally scale up of cell supply for HTS was achieved using automated technology.

‘Cell Supply for Drug Discovery: The use of Continuous Culture and Frozen Cells to support SAR screening’. Dr Stephen Rees, GlaxoSmithKline

This talk began with a discussion of the objectives of SAR (Structure Activity Relationship) screening and the principles of cell based assays in drug discovery. Cell based assays are used in high-throughput SAR screening to identify novel molecules with activity at target proteins and then during the optimisation phase to generate potential drugs for therapeutic use. However, multiple cell lines must be maintained in culture for the duration of the optimisation program which may take several years. Cell culture automation was successfully used to maintain more than 30 cell lines in weekly production for SAR screening. The impact of cryopreserved ‘assay ready’ cells to minimise variability in the screening assay was discussed.

‘Profiling Antibody Fragments in Cellular Systems’. Matt Sleeman, Cambridge Antibody Technology

This presentation began by describing single chain variable fragments (scFv). scFv fragments are the variable regions of antibodies which bind to specific antigens. These scFv fragments can be produced using phage display technology to produce libraries of different scFv fragments. Cell assays are widely used to screen panels of antibodies and antibody fragments of potential therapeutic use to be tested against a specific antigen. A range of cell based assays have been used to screen for antibody fragments such as scFv fragments with the appropriate properties in terms of their mode of action, specificity, affinity, potency and cross-reactivity.

‘Success of BEVS in Generating Kinase Proteins for Structural Study’. Joanna Long, Vertex Pharmaceuticals Ltd

This talk discussed the use and methodology of the Baculovirus expression system (BEVS) in producing significant yields of target protein for crystallographic study. A number of case studies detailing the strategy behind construct design, expression, purification and crystallisation of novel kinases including Aurora kinase proteins were described. Aurora kinases are potential drug targets that have frequently been found to be deregulated in human cancers and the biological depletion of Aurora activity has been shown to block proliferation. The use of BEVS in generating kinase proteins was shown to be successful as the crystal structure of Aurora A kinase revealed unique features that could potentially be exploited for drug design.

‘OSCARTM – A New Gene Amplification System for the Production of Therapeutic Proteins in Cultured Mammalian Cells’. Professor David Melton, University of Edinburgh

Gene amplification systems are routinely used to maximise the recombinant protein yield of industrially important mammalian cells. The most commonly used gene amplification systems (dihydrofolate reductase [DHFR] and glutamine synthetase [GS]) require multiple rounds of amplification, specialised media and toxic chemicals. This presentation discussed a new system that has been developed called OSCARTM (One Step Construct Amplification Routine). OSCARTM is a system where selection and amplification occur in a single step, providing rapid selection of clones producing high levels of protein. This amplification system is based on a series of partially disabled minigene vectors for hypoxanthine phosphoribosyltransferase (HPRT) which is essential for purine synthesis. The OSCARTM system was shown to compare very favourably against the DHFR and GS systems in adherent CHO cells particularly in terms of recombinant protein yield and stability, time and cost.

‘Understanding How a Chromatin Insulator Protects Against Chromosomal Silencing’. Dr Adam West, University of Glasgow

This talk began with an introduction to chromatin and histone structure. DNA methylation and histone modification have profound effects on whether a gene is expressed. The chicken β-globulin 5’ boundary element HS4 has been shown to have insulator activity, preventing chromosomal silencing by interfering with communication between the promoter and enhancer and preventing DNA methylation. It was found that insulator elements can manipulate epigenetic processes that affect chromatin structure, including histone modification and DNA methylation patterns. Transgenic constructs were shown to be protected from chromosomal position effect silencing when flanked by HS4. The functions of each of the five ‘footprints’ of HS4 were established.

‘Apoptosis Indicators in CHO Cells: Emergence of TRB3’. Qian Sun, University of Manchester

Apoptosis is the main mode of cell death in industrially important CHO cells, limiting the attainable viable biomass and hence volumetric recombinant protein yield. The identification of apoptosis indicators in CHO cells upon tunicamycin treatment was discussed. The addition of tunicamycin was used to induce apoptosis and the mRNA expression of several regulatory genes involved in cell cycle progression, growth arrest and apoptosis were assessed. mRNA encoding GADD153, p27 and TRB3 were up-regulated shortly after treatment with tunicamycin, with expression peaks at 12h, 20h and 28h, respectively, followed immediately by apoptosis. These results suggest that these genes can be monitored as markers of different stages towards apoptotic cell death. Engineering the expression of these genes could generate cells for enhanced biomass and hence increased recombinant protein production.

‘The Molecular Responses and Functional Competence of In Vitro Cultured Mammalian Cells at Sub-physiological Temperatures’. Rosalyn Marchant, University of Kent

This presentation discussed the effects of sub-physiological (‘cold-shock’) culturing on mammalian cells. CHO cells cultured at sub-physiological temperatures resulted in prolonged generation time, a prolonged period of maintained cell viability and changes in both volumetric and cell specific recombinant protein production rates. Cold-shock was shown to effect translation efficiency; the relative levels of capped dependent mRNA translation decreased whilst Internal Ribosome Entry Site (IRES) mediated translation increased. Immunoblot analysis showed a general increase in the relative levels of various stress related proteins. The importance of these findings in terms of the development of novel gene expression technologies that exploit the molecular responses to cold-shock in order to optimise recombinant protein production in mammalian cells was also discussed.

‘Functional Genome-wide Analysis of Antibody-Producing NSO GS Cell Line Cultivated in Different Temperatures’. Dr Halina Swiderek, University College Dublin

The result of lowering culture temperature on cell cycle, survivability, cell growth and productivity of CHO cells has been widely published. It has been suggested that lowering culture temperature is useful for increasing recombinant protein production in mammalian cell lines. However, results reported so far suggest that this phenomenon is cell line specific. Genome-wide analyses and proteomic examination were performed on antibody producing NS0 6A1 cells cultivated at 20°C, 34°C and 37°C. Several genes were shown to be up or down regulated under different culture temperatures. There were both differences and similarities in the cellular and extracellular proteins detected in samples from NS0 6A1 cultures grown at the different temperatures.

Zoë Towler