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