ESACT-UK – 12th Annual Meeting
3rd-4th January 2002, Churchill College, Cambridge

This two-day symposium consisted of 18 primary oral presentations given by delegates originating from industry and academia. A selection of poster presentations was also available.
Ideas and techniques concerned with improving industrial cell processes were presented. An interesting review ‘Evolution of Animal Cell Technology’ was presented by Professor Bryan Griffiths. The difficulty of scale-up bottle necks was highlighted that related well with the implementation of virus barriers.
The baculovirus was also an area of interest with improvements in quantification moving away from the standard plaque assay.
The last session focused on proteomics. Three speakers touched on the use of 2D gel as a use for identifying mechanisms of action of drugs and toxins, quantifying the protein content of cells and assessing changes in protein expression during culture. New techniques such as difference gel electrophoresis (DiGE) were brought to our attention.
The following is a summary of the primary oral presentations:
‘Cell-mediated cancer gene therapy’. Dr Stuart Naylor, Oxford Biomedica
The combination of two novel targeting strategies for gene-based cancer therapy was discussed: cell-based gene delivery to the tumour site using macrophages combined with transcriptional targeting of therapeutic gene expression.
Macrophage Activated Killer Cells ‘armed’ with the therapeutic P450 gene, ex vivo, inherently target tumour cells and deliver the therapeutic to the tumour. The optimised promoter (hypoxia response element-HRE) also targets the gene expression to hypoxia (predominant feature in solid tumours)
Pre-clinical data was presented to show that macrophage infiltration of the tumour and expression of P450 was successful. The delivery of P450 was subsequently shown to convert the pro-drug cyclophosphamide (CPA) to cytotoxic anti-tumour metabolites.
Future work was discussed using stem cell derived monocytes (StemGen) due to the inappropriate use of macrophages for intravenous administration.
‘Evaluation of retrovirus production systems’, James N. Warnock, University of Birmingham
The large-scale production of retrovirus vectors as tools for gene therapy was discussed.
Data from a two-stage process was shown, in which the temperature was dropped from 37°C to 32°C following inoculation. Comparisons of microcarrier types and production systems were made and a recommendation that Immobasil FS (Ashby Scientific) or Fibracel™ (Bibby Sterilin) microcarriers and spinner baskets have the highest optimum potential for scale up.
‘Peptides as transfection agents’, M. Rouquette, Cobra Therapeutics
The use of cationic peptides as non-viral gene delivery systems was discussed. The idea of using cationic peptides as natural DNA condensing agents, which form condensed entities with plasmid DNA, for transfection in vitro, was explored. Two such peptides were reported on; CL22 was shown to transfect primary dendritic and endothelial cells, in the presence of chloroquine. CP36 being endosomolytic and therefore not requiring chloroquine for release from the endosome was shown to transfect endothelial cells at a comparably improved rate to DOPEC and Polyethylenimine (PEI).
Serum was also shown to play an important role, and cell-line dependency of both the peptides and serum was shown as significant.
‘New developments in baculovirus expression vector systems’. Robert Possee, NERC Institute of Virology and Environmental Microbiology
A novel approach to produce recombinant baculovirus was described. Firstly, the removal of the chitinase gene from the baculovirus genome. This was to prevent the insect cells from liquefying themselves and therefore improve viability of the cells. This also improved secretion of the recombinant protein from the nucleus of the virus-infected cells.
Secondly, the baculovirus genome, lacking part of ORF1629 gene, was inserted with a Bac replicon and maintained in E. coli.. Virus DNA was recovered and mixed with a baculovirus transfer vector containing a foreign gene and used to co-transfect insect cells. A genetically homogenous virus stock was generated and could be amplified to provide working stocks.
This method was described to be an improved process for (a) infected cell viability and (b) production of recombinant proteins in baculovirus infected cells.
‘Improved and rapid (18h) assays for baculoviruses’, Chris Mannix: GlaxoSmithKline
Three methods for quantification of baculovirus were compared; the traditional plaque assay, a modified plaque assay and a growth inhibition assay. The modified plaque assay was described as a simplified version of the traditional assay where the infection is carried out in suspension and the growth inhibition assay is based on the inhibition of insect cells caused by the virus infection. Both the modified plaque assay and the growth inhibition assay were shown to have results of between 1.5 and 2 times higher than the traditional assay.
‘Productivity of a recombinant chemokine binding protein (murine gamma herpesvirus protein) in two expression systems, Baculovirus versus CHO’, Dr Rodney Smith (Xenova Group)
The presentation focussed on the advantages and disadvantages associated with the production of rCBP by Baculovirus and CHO expression systems. The Baculovirus expression system (BES) is much simpler than that used for CHO, as it required the inoculation of a 10L production vessel with a single ampoule. This is incubated for 22 days and yields ~2g of purified product. The use of CHO cells requires a 21-day incubation of 24 ¥ 850cm2 roller bottles, which are then pooled and incubated for a further 12 days in serum-free medium before they are harvested. This method yields only ~96mg of product. The productivity for BES is approximately 2.1¥10-8 mg/cell/day, that of CHO has been found to be 10 times lower than this. Purification of the harvested material is carried out by diafiltration, concentration, and the use of a one-step Nickel chelate column. Recoveries for BES and CHO were found to be 87% and 83% respectively. Improvements to the BES for the future include a change to a serum-free process, the attainment of higher-density cultures (to eventually enable scale-up) and a method of supplementing depleted medium components. Improvements that are required for the CHO method include the development of a serum-free suspension, the development of a high cell density perfusion or fed batch (which would prolong the production phase) and a scale-up of the procedure beyond 10L.
‘Towards scale-up of transient recombinant protein expression in mammalian cell culture’, Andrew Tait (University College London)
This project was devised in order to minimise the time taken to achieve a gene-to-protein conversion of a potentially therapeutic molecule. This would remove the need to develop stably expressing cell lines during drug development work. The model used aimed to increase the transient expression of the luciferase gene by transfecting it into host NS0 cells on a plasmid. The addition of polyethylenimine (PEI) causes DNA to condense into a PEI-DNA complex, allowing it to be transfected into the host cells more easily. The main disadvantage of using PEI is that it is cytotoxic. Also, NS0 cells containing PEI-DNA have a lower growth rate than the control samples. However, it does buffer the endosome and has the capacity to mediate large-scale transient gene expression.
‘Validation of the gamma irradiation process for serum’, Richard Festen (JRH Biosciences)
The validation of this process will ensure that when medium components are treated with gamma radiation, they will exhibit a required reduction in extraneous agents but will remain viable in order to support culture growth. The study was carried out at both pilot and production scales. The validation study carried out by JRH provided data on the optimum temperature, configuration of material within the irradiation chamber and the dose range to which each item within the chamber was exposed. The goal was to ensure that each item placed into the chamber received a guaranteed consistent dose of radiation, but that each item remained viable following treatment. FBS irradiated at 25-35kGy exhibited a 5-6 logs inactivation of Bovine Viral Diarrhoea, Infectious Bovine Rhinotracheitis and Parainfluenza Type 3.
‘The evolution of animal cell technology’, Professor Bryan Griffiths
Professor Griffiths gave an overview of the advancements made in animal cell biotechnology since its inception in the 1950s, from the manufacture of the first viral vaccine (the Salk Polio Vaccine) in 1954. He presented the factors responsible for the introduction of the strict safety regulations now in operation and need for the standardisation of experimental procedures. Animal cell biotechnology has come full-circle since the Salk Polio Vaccine. At first, processes were developed to produce large cell densities capable of in turn producing large quantities of complex active molecules; recently processes have begun to be scaled down, as cells are now seen as products in themselves and not merely as factories for the production of therapeutic agents.
‘The development of new control strategies for fed-batch mammalian cell culture’, Adrian Chowdhary (University College London)
This presentation outlined the stages involved in the preparation of a metabolic model for a selected GS-NS0 cell line. The model is a metabolic flux analysis model, involving 37 reactions that highlight the cellular metabolism by the measurement of nutrient concentrations. In addition to the models itself, it has been necessary to develop and evaluate new technology that will allow the collection of cell free samples on a continuous basis. These must be compatible with large-scale GMP manufacture. It is hoped that this technology will eventually be used in a GMP manufacturing environment as a validated monitoring system.
‘Process Limitations in Biological Product Formation’, Tim Clayton (GlaxoSmithKline)
The presentation described some of the impacts of increased cell yields and process yields on the operation of commercial scale bioprocesses using antibody production as the example. He described the issues for development is to achieve a product that is safe, pure and active. Bottlenecks in processes are critical for economic and quality reasons. One change in a process can have effects downstream. For example, a scale effect: increased growth at 80 L is not always equivalent at 8000 L. By using fed-batch to increase the product concentration the bottleneck in down stream purification was eliminated. However, new bottlenecks were introduced such as, low O2, feed mixing, and volume changes. These led to cell damage, foaming and a fire risk if pure O2 was to be used. Therefore, Dr Clayton concluded that any biological product formation process must be considered as a whole, with consideration of changes resulting from alterations to specific steps
‘Application of on-line combinatorial calorometry to monitor the intensity of anaerobic pathways in the growth and productivity of genetically engineered mammalian cells’. D. Olomolaiye (Institute of Biological Sciences, University of Wales, Aberystwyth)
Physical parameters like pH are easy to measure with probes. However, metabolic fluxes are much more difficult to measure, and the cost of suitable equipment is usually prohibitive. In the study described, an on-line heat conductive micro-calorimeter was used to measure the instantaneous heat flow of the culture. This can be indicative of metabolic activity. A simultaneous measurement of the change in capacitance of the culture was taken with a dielectric spectrometer. These two data sets were used to estimate the viable biomass of the culture. The use of this method was validated using CHO320 cells producing IFNg.. The heat of the culture decreases before the cell viability starts to decrease, despite the close correlation between the two measurements at the start of the culture
‘Suppression of apoptosis in industrial-scale biological production systems’, Dr. Angelo Perani (Lonza Biologics)
This presentation was an overview of an EU-funded study on the benefits of apoptosis suppression for the manufacture of recombinant antibodies. Apoptosis is a mechanism that eliminates individual cells when they become damaged or are not required. Factors inducing apoptosis in bioreactors include oxidative stress, nutrient deprivation, waste accumulation, hydrodynamic forces, and pH variation. Cell lines can be engineered to exhibit apoptosis resistance. The most common method is by the over-expression of the anti-apoptotic protein bcl-2, which prevents damage to mitochondria. The team from Lonza engineered a GS-NS0 cell line which over-expressed bcl-2 (6A1-bcl2) by transfecting cell line 6A1(100)3 with the bcl-2 gene. The control line exhibited the onset of apoptosis on day 4 of the culture, while 6A1-bcl2 had a delayed onset of apoptosis on day 9 of the culture and therefore had much higher growth. They were able to show that apoptosis was decreased by 40% and growth increased by 40%. However, the productivity of 6A1-bcl2 was disappointing. This may have been because the original cell line was selected on the basis of bcl-2 expression and not high productivity. Currently, bcl-2-expressing cell lines are being evaluated for the manufacture of recombinant antibodies.
‘Proteomic and genomic comparisons of drug-treated cells and tissues’, Dr Mike Furness (Iconix Pharmaceuticals)
Dr Furness gave an in-depth insight into the uses of proteomics in the study of drug action. The data presented included 2D gel electrophoresis of proteins and cDNA microarray analyses of clonal HepG2 cell populations treated with various drugs. The cDNA microarrays allow us to visualise the differences in protein expression between various types of tissues and cell lines when treated with the same drug. It is hoped that in the future, expression array studies, pharmacological bioassay studies, chemical indexing studies, and literature creation studies will be combined to produce compound profiles, or “Drug Matrices”.
‘Quantitative proteomics’, Dr Kathryn Lilley (Cambridge Centre for Proteomics, University of Cambridge)
The main focus of Dr Lilley’s presentation was the introduction of the concept of Difference Gel Electrophoresis (DiGE) as an aid to high-throughput comparative protein analysis. This technique involves the use of distinct fluorescent cyanine dyes in order to allow two different proteins to be run simultaneously on the same 2D gel. It avoids the problems associated with gel to gel variability in comparative analysis of wild-type versus mutant proteins. The wild-type protein is labelled with cy3 (green) and the mutant form is labelled with cy5 (red). The samples are then pooled and subjected to 2D polyacrylamide gel electrophoresis (PAGE). The technique is sensitive and reproducible; quantification is achieved by calculation of cy3/cy5 ratios.
Lilley also described two non-quantitative techniques. (1) MALDI-TOF, which can provide protein mass fingerprints for hydrophobic and low abundance species. (2) MUDPIT or multi-dimensional protein identification technology.
‘Analysis of mammalian NS0 cell proteome’, Elizabeth Sage, (The University of Kent)
Elizabeth used 2D gel electrophoresis to identify specific alterations in functional gene expression that enable murine myeloma NSO cells in culture to maintain high-level recombinant monoclonal antibody production. By harvesting NS0 cells in culture at three time points, she showed that changes in gene expression during batch culture occurred. This was concurrent to changes in the rate of cellular protein synthesis, alterations in metabolic activity and specific production antibody. A stress protein was observed to be over-expressed in response to stimuli.
Many Thanks go to GlaxoSmithKline, for compiling this meeting report.