Download ESACT-UK – 13th Annual Meeting 6th

ESACT-UK – 13th Annual Meeting
6th-7th January 2003, Chancellors Conference Centre, Manchester.
A very big thank you goes to
Ros Gusterson
(GlaxoSmithKline, Beckenham)
who kindly volunteered to produce the meeting report of the 13th ESACT UK
meeting.
If you are interested in producing the meeting report for the 14th meeting in Leicester
please let the committee know by email [email protected]
The following is a summary of the oral presentations:
‘Tissue engineering: Cartilaginous tissues and gene enhancement
chrondrogenic cells’. Professor Tim Hardingham, University of Manchester.
of
The application of tissue engineering as a new strategy for developing treatments for the repair and
regeneration of damaged and diseased tissues was discussed. Particular reference to arthritic disease,
and moreover cartilage repair, as a target for tissue engineering was demonstrated. Chondrocytes play
an important role in cartilage formation by promoting new matrix assembly. Previous work has shown
that chondrocytes isolated from cartilage proliferate in culture, but become fibroblastic and lose the
expression of major cartilage matrix genes. Experiments were performed using retroviral vectors to
transfect cells with the transcription factor SOX9, thereby promoting expression of type II collagen.
Results demonstrated increased expression of type II collagen in chondrocytes and furthermore,
transfer of the cells to alginate or pellet culture produced further enhancement of type II collagen
expression. It was concluded that gene transfer provides great potential in tissue engineering for the
enhancement of cell function.
‘Cell surface proteoglycans are differentially regulated by chondrocytes during adaptation to cell
culture’. Alan Murdoch, University of Manchester.
The importance in changes of chondrocyte cell surface proteoglycans during
enzymatic release from tissue, and on subsequent monolayer cultures was discussed.
The expression levels of eleven cell-surface proteoglycans were measured in human
articular cartilage and cultured chondrocytes. Results showed that the proteoglycan,
glypican-6, was a major transcript of chondrocytes, as measured by RNA extracted
directly from the cartilage. However, although low levels of the proteoglycan CD44
were found in the same tissue, there was a large increase in expression during
overnight enzyme digestion. It was proposed that this may reflect a response of the
chondrocytes to the removal of their immediate peri-cellular matrix. It was therefore
concluded that although it has previously been assumed that freshly isolated cells
have a similar expression to cells in tissue, this may not be the case. In conclusion, the
results showed sensitivity of chondrocyte cell surface proteoglycan expression to cellmatrix interactions. These results may help modulate the response of cultured
chondrocytes to growth factors and chemokines.
‘Regulation of adult stem cell fate by notch signalling’. Phil Jones, University of
Cambridge.
The pathways controlling stem cell division were discussed with particular reference
to the Notch pathway. Previous work has shown that the Notch signalling system
regulates proliferation and differentiation in many tissues. Work here looked at its
role in cell differentiation in C2C12 myoblasts. Results showed that a novel
transcription factor, Hes6, is expressed after activation of the Notch pathway.
Evidence was shown to demonstrate that over-expression of Hes6 inhibits
differentiation, with a substantial reduction in the proportion of cells undergoing cell
cycle withdrawal. Work was also performed in Xenopus embryos, where Hes6 can be
found in myogenic precursors. These results showed an expansion of the myotome,
but suppression of terminal muscle differentiation and disruption of the somite
formation at the tail bud stage. Furthermore, analysis of the Hes6 mutants indicated
that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but
that protein-protein interactions are, suggesting that Hes6 mediates its effects by
protein-protein interactions rather than by acting as a transcription factor. Thus, it was
concluded that Hes6 plays a novel role in multiple stages of muscle formation.
‘Health and safety legislation in practice’. Liz Pollit, HSE.
This talk provided an overview of the health and safety legislation involved in work with cells, with
particular reference to the regulations for genetically modified organisms. Areas discussed included the
dangers and risks involved in working with biological agents, the classification and containment
measures required, inactivation of waste, and the standards expected by inspectors.
‘GMO biosafety and conforming with the GMO2000 regulations in a
manufacturing environment’. Martin Beadmore, Lonza group.
The issues involved in conforming to the regulatory requirements of the GMO Regulations 2000 were
discussed. These included the prevention of cross-contamination, problems encountered with
adventitious agents, validated methods of waste inactivation (both chemical and physical), the training
of staff for operator safety, and general risk assessment and emergency plans.
‘Regulatory issues surrounding cell therapy and tissue engineering products’.
Daniel Galbraith, Q-One Biotech Ltd.
The regulatory needs for tissue engineered products were discussed in this talk. The
potential risks were outlined, including the safety issues involved in process design,
product characterisation, and most importantly, the possibility of viral infection of
stem cell products. The safety testing of these products is problematic due to the
limited shelf life of many of the materials and it was therefore concluded that novel
strategies are required for many of these treatments.
‘Select T robotics system’. Phil Offin, Automation Partnership.
An overview of automated cell culture was given, with an example of the development of a SelecT
machine created by The Automation Partnership (TAP) and a consortium of six cell biologists from
major biopharmaceutical companies. The idea was to develop a flexible, automated system for the cell
culture of different cell lines. The advantages of the SelecT were discussed, including its ability to
produce consistent results, multiple batch processing, and its ability to function unattended during ‘out
of hours’ work.
Recombinant baculovirus as mammalian cell gene-delivery vectors. Tom Kost,
GlaxoSmithKline, North Carolina.
This talk focused on the advantages of recombinant baculovirus, for the delivery of foreign genes into
mammalian cells. Some history of the baculovirus system was provided. This included details of the
insect as a member of the arthropod family to the vector construction (comprising the polyhedrin gene)
and use of the insect host cell line Sf9. The talk then proceeded to cover properties of the system,
methods of gene transfer into mammalian cells, with particular reference to the BacMam virus used at
GSK and possibilities to increase gene expression using the histone deacetylase inhibitors trichostain
and sodium butyrate. Advantages of the BacMam system were outlined including its lack of
cytotoxicity, its ability to transduce a host of cell lines, the possibility to perform transient or stable
transfections and its large capacity for gene copy insertion. Three applications of the baculovirus for
viral vector production were given, including hepatitis B, expression of the estrogen receptor and
KATP channel expression. The talk was concluded with the biosafety considerations when using the
baculovirus expression system.
‘Apoptosis and its suppression in hepatocytes’. Nyaradzo Mukewena, University
of Birmingham.
This talk discussed apoptosis in the immortalised rat hepatocyte cell line HepZ. Results showed that
hepatocytes die by apoptosis induced by nutrient deprivation, and moreover that apoptosis induced by
staurosporine (STS), could be prevented by caspase-8 inhibitor. Furthermore, the inhibitors of the
mitochondrial membrane potential were shown not to have any protective effect over STS-induced
apoptosis. Conversely over-expression of the transcription factor bcl-2 in HepZ cells was shown to
reduce cell death induced by nutrient depletion and STS.
‘Definition of stress indicators in NS0 myeloma cells’. Renata Cudna, University
of Manchester.
This presentation outlined the cell regulatory processes that lead to apoptosis, with particular attention
to the role of the endoplasmic reticulum (ER) in the stress response. Two transcription factors of the
stress signalling pathway were investigated, growth arrest and DNA damage gene 153 (GADD153),
and X-box binding protein-1 XBP-1. Previous work has shown that GADD153 expression is increased
in response to stress conditions, and that XBP-1 is induced as part of the ER stress response with
tunicamycin, and activates the ER chaperones. More specifically, results here showed that GADD153
expression is induced prior to the onset of apoptosis in response to stress conditions. Furthermore, data
showed that stress enhanced XBP-1 mRNA expression was processed unconventionally allowing for
the expression of functional protein. These findings therefore suggest a potential use of GADD153 and
XBP-1 as stress indicators of apoptosis in NSO myeloma cells.
‘Expression profiles of GS-NS0 system under different osmotic pressures’. MonHan Wu. Imperial College, London.
This talk outlined the importance of understanding the basic cellular processes of the GS-NS0
mammalian cell culture system to enable improved strategies for process optimisation by highlighting
the importance of varying osmotic pressures on cells. To do this the biology, analysis and application
of the cell process needs to be understood. The biology involves gene and protein expression,
metabolism and population dynamics; the analysis involves understanding the sequence, function and
linkage database; and the application covers areas of metabolic engineering, plasmid design and
process control. In order to achieve this, the use of DNA microarrays has been used to investigate the
expression and metabolic profiles of the GS-NSO system under different osmotic pressures.
Preliminary results identified up to 300 genes which are up or down regulated, involved in ion
transport, accumulation of osmolytes, cell cycle distribution and proliferation, cytoskeletal organisation
and cell metabolism.
‘Enhancement of productivity by over-expression of p21CIP1 in proliferationcontrolled CHO-GS system’. Jing-Xui-Bi, University of Birmingham.
Work here summarised the regulation of the arrest of cellular growth and multiplication (cytostasis) on
monoclonal antibody production using an inducible p21 CIP1 cyclin dependent kinase (CDK) inhibitor
expression system. Results showed that induction of cell cycle arrest in G1 correlates with monoclonal
antibody production rate. Arresting cells at different phases of the batch growth curve without medium
change did not affect either the degree of cell cycle arrest or the productivity level, although cells at the
end of exponential/stationary phase lost their viability at a faster rate due to nutrient depletion. When
nutrients were available, cells arrested at the end of the exponential phase outperformed cells from
other batch culture phase in their productivity. Furthermore, cell size increased upon cell cycle arrest
compared to the average proliferating cells. This was accompanied by an increase in mitochondrial
activity, and moreover an increase in antibody productivity. These results therefore demonstrated the
possibility of uncoupling cell cycle and productivity to achieve higher production rates without
increasing cell number. It also provided further evidence to suggest that cell size, not cell cycle, is the
major cellular determinant of productivity.
‘Development of improved chemically defined mammalian cell fermentation process’. Allan
Maxwell, Lonza group.
This talk outlined the need for process optimisation for large-scale production of
biopharmaceuticals using protein-free chemically defined processes. Attention was
also given to maximising facility productivity for process optimisation as well as the
need for efficient platform processes. The strategy employed by Lonza using a fedbatch process was discussed whereby manipulation of the nutritional and
physicochemical environment led to over a 3-fold increase in recombinant antibody
production using GS-NS0 cells, in an animal component free, chemically defined
process.
‘Influence of cell culture media on the adherence of cells on microcarriers’.
Gerald Blueml, Amersham Biosciences.
Research here focussed on the influence of different media components, such as bivalent cations, iron
concentrations, hydro-colloids and other biopolymers of non-animal origin, and on the attachment and
detachment process in microcarriers to improve colonisation of cells, to allow cell harvesting and
promote scale-up. Results showed that carboxymethyl dextran and low ferric citrate concentrations
enhanced the adherence of CHO cells, and that this increased their shear resilience resulting in higher
cell densities. In addition, iron-dextran was shown to enhance adherence of CHO cells whilst dextran
sulphate decreased the adherence of the cells.
‘Recombinant arylsulfatase B production with the fluidised bed: Process stability
and scale up’. Markus Durrschmid, Institute of Applied Microbiology, Vienna.
The fluidised bed process was discussed here as a method for the large-scale
production of arylsulfatase B (ASB). The enzyme ASB is absent in patients suffering
with the lysosomal storage disorder Maroteaux-Lamy VI, and is a protein with
mannose rich glycosylation sites, which has to be produced and harvested in its
precursor state for its successful application in enzyme replacement therapy. The
fluidised bed reactor proved to be a robust and reliable system with stable production
of ASB and an economically viable process. Indeed results comparing the fluidised
bed reactor with the stirred tank reactor showed that the percentage of ASB-precursor
protein was significantly higher in the fluidised bed system than in the stirred tank
reactor.
‘Optimising bioprocess manufacturing’ Richard Spurling, BioPharm Services
Ltd.
This presentation outlined how an engineering perspective can help the companies in the
pharmaceutical industry. The goals of BioPharm Services are to produce an efficient and cost effective
system coupled with production optimisation. The importance of tracking time was discussed with
reference to Gantt charts showing example processes and their elapsed times. Case studies were also
given to demonstrate the above considerations, one such example was the rationale behind disposable
technology. It was concluded that the analysis of a manufacturing strategy should be an integral part of
clinical development.
‘Fed-batch cultivation of cells: model based, adaptive control in comparison to
alternative control strategies’. Ralf Pörtner, University of Hamburg.
The difficulties encountered with the fed-batch system were reviewed here, and the
need for new strategies discussed, with particular reference to the Open-LoopFeedback-Optimal (OLFO) controller system. The problems with fed-batch include
differences in metabolism between different cell lines, non-physiological culture
conditions, the low number of online process parameters, and the risk of
contamination during sampling. The combination of a fed-batch process and OLFO
control was discussed providing a new approach for cell culture process control,
coupling an efficient cultivation concept to a capable process control strategy. The
significant advantage of the system is its universal character, since it is not bound to a
certain cell line or type of reactor. It therefore represents a solution with good
transferability to diverse cell cultivation. It was concluded that the adaptive, modelbased OLFO controller is a valuable tool for the fed-batch control of hybridoma cell
cultures.
‘A miniature bioreactor designed for high throughput automated cell culture’.
Sally Lamping, University College London.
Due to the need for increased efficiency of high throughput screening in industry, the potential use of a
miniature bioreactor design as a replacement for the multi-well plates was discussed. The miniature
bioreactor can hold a working volume of 6ml, with its major dimensions the same as those of a 24microwell plate. It is made from stainless steel and mimics the stirred tank reactor with a set of three
micro-fabricated flat open turbine impellers in the centre of the vessel. In addition, the bioreactor has
probes to read oxygen transfer rate, pH and cell growth.