ESACT-UK 17th Annual Meeting
Programme
Thursday 4th January 2007
11.00 Registration desk opens
12.00-12.50Lunch, registration, and trade stand set-up.
1.00-2.30 Session 1 (Chair Jon Green)
Maintenance of Cell Lines & Mammalian Cell Systems for Oncology
1.00-1.30 Cell Line Issues and Good Cell Culture Practice
Professor Glyn Stacey, Director of the UK Stem Cell Bank,
National Institute for Biological Standards and Control.
1.30-2.00Human Cancer Cell Lines. Quality Control of Cross-Contamination
Professor John Masters, University College London, U.K.
2.00-2.30Investigating the Cellular Responses to Clinically-Useful DNA
Damaging Agents
Dr Daniel Lloyd, Department of Biosciences, University of Kent, Canterbury.
2.30-3.00Keynote Presentation
Translating Bioscience into Bioprocessing
Professor David James, Dept of Chemical and Process Engineering, University of Sheffield, U.K.
3.00-3.30Coffee, networking, trade stands and chance to view posters
3.30-5.30 Session 2 (Chair Julian Hanak)
Application of Technologies to Mammalian Cell Bioprocessing
Sponsored by Wave Biotech
3.30-4.00 Scale-up of Stem Cell Culture for Drug Discovery
Ms Julie Kerby, Stem Cell Sciences, U.K.
4.00-4.30The use of Wave Bioreactors in the Pharmaceutical Industry
Dr Girish Shah, GlaxoSmithKline R&D, Stevenage, U.K.
4.30-5.00The use of FACS for the Selection of Cell Lines with Superior Productivity Characteristics
Dr Jon Welsh, Lonza Biologics plc, Slough, Berkshire, U.K.
5.00-5.15Measurement and Control of Viable Cell Density in a cGMP Mammalian Cell Bioprocessing Facility
J. P. Carvell, Aber Instruments Ltd, Science Park, Aberystwyth, U.K.
5.15-5.30TruLink: a Technology for Improving On-Line Process Analysis and Enabling Easier PAT Implementation
John Bonham-Carter, Finesse LLC, Mälarhöjdsvägen 37, 12940, Hägersten, Stockholm, Sweden.
5.30-5.45 ESACT-UK AGM
6.00-7.30 Wine reception, Poster Session, trade stands and networking.
7.30-l2.00 Dinner followed by bar open. Note that posters will be on site to view.
Friday 5th January 2007
7.30-9.00amBreakfast
9.00-10.40Session 3 (Chair Mark Smales)
Systems Biology for Improved Bioprocessing
Sponsored by Broadly Technologies
9.00-9.30 Exploring the CHO Proteome to Improve Bioprocess Productivity
Dr Paula Meleady, National Institute for Cellular Biotechnology, Dublin City University, Ireland.
9.30-10.00Molecular Analysis of Stability of Recombinant GS-NS0 Cell Lines
Professor Alan Dickson, University of Manchester, U.K.
10.00-10.20Initial Identification of Low Temperature and Culture Stage Induction of miRNA Expression in Suspension CHO-K1 Cells
Dr Patrick Gammell, National Institute for Cellular Biotechnology, Dublin City University, Ireland.
10.20-10.40Gene Expression Regulation of Cold Inducible RNA Binding Protein (CIRBP)
Mr Mohamed Al-Fageeh, University of Kent, Canterbury, Kent, U.K.
10.40-11.45Coffee, networking, trade stands and chance to view posters
11.45-Session 4 (Chair Tracey Sambrook / Jon Green)
Product Enhancement and Quality Control
Sponsored by Sigma-Aldrich Fine Chemicals (SAFC)
11.45-12.15 The Influence of Isotype, Glycoform and Epitope Specificity on the Functional Profile of Antibody Therapeutics
Professor Roy Jefferis, University of Birmingham, U.K.
12.15-12.45Microscale and Automation Approaches for Rapid Cell Culture Process Development
Professor Gary Lye, Department of Biochemical Engineering, University College London, U.K.
12.45-1.05Measurements for Biotechnology – Improving Measurements in the Biopharmaceutical Industry
Dr Anna Hills, Biotechnology Group, National Physical Laboratory, Teddington, U.K.
1.05pmAward of prizes.
1.10pmClose of Meeting (Jon Green)
1.15-2.15pmLunch
TALK
ABSTRACTS
Cell Line Issues and Good Cell Culture Practice
Professor Glyn Stacey, Director of the UK Stem Cell Bank, National Institute for Biological Standards and Control.
Human Cancer Cell Lines. Quality Control of Cross-Contamination
Professor John Masters, University College London, U.K.
Human cancer cell lines are widely used research tools. But for almost all the human cancer cell lines used, there is no proof that they were derived from either the individual or the tissue claimed. Many human cancer cell lines are derived from different tissues, individuals or even species to that claimed.
The first continuous cell line derived from a human cancer, HeLa, was developed in 1952 from a glandular cancer of the cervix. In 1967, using isozyme analysis, it was shown that many of the human continuous cell lines available were contaminated with HeLa cells. These false cell lines include KB, HEp-2, Int407, Chang liver and WISH cells. Surprisingly, 40 years later these cells continue to be used respectively as models of skin and head and neck cancer, fetal intestine, hepatocytes and amniotic cells, despite being unequivocally HeLa cells derived from a cervical cancer.
Cell lines can easily be authenticated. DNA profiling uses highly polymorphic short tandem repeats (STRs) for forensic purposes, including paternity testing and to identify crime suspects. DNA profiling provides an unambiguous method for identifying cell lines indefinitely, from whatever laboratory or other source the cells are obtained. DNA profiling is reproducible between laboratories and is inexpensive.
Investigating the Cellular Responses to Clinically-Useful DNA Damaging Agents
Sara Bhana, Amanda J. Weeks, Catherine E. M. Hogwood, C. Mark Smales and Daniel R. Lloyd, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, U.K.
Humans are constantly exposed to a wide variety of DNA damaging agents through environmental exposure to UV rays in sunlight, carcinogens in cigarette smoke and industrial wastes, and dietary mutagens. Human cells have evolved to respond to such DNA damage with mechanisms such as cell cycle arrest, apoptosis and DNA repair, so that the cell does not replicate a damaged DNA template. These mechanisms have important consequences for the clinical efficacy of cancer chemotherapies whose mode of action is to induce cytotoxic DNA damage in target tumour cells. The work in our laboratory currently focuses on such DNA damaging agents and how the cellular environment can influences therapeutic outcome. Using monolayer cultures of human cells, we have investigated the cellular response to cisplatin, a chemotherapeutic drug used in the treatment of testicular cancer and other solid tumours. In particular, we have focussed on the role of the p53 tumour suppressor protein, whose gene is mutated in around 50% of human cancers. We have found that, in the presence of p53, efficient removal of cisplatin-induced DNA damage from the genome takes place, while in the absence of p53 this damage persists. The results indicate that p53 is required for efficient repair of cisplatin-induced DNA damage in human cells. We are currently investigating how this p53-dependent DNA repair interacts with other p53-regulated cellular responses to DNA damage, and its relative influence on the ultimate fate of the cell. The results suggest that the p53 status of tumours may influence therapeutic outcome of cancer therapies based on damaging DNA. We are also developing this work to evaluate the cellular responses to DNA-damaging radionuclide therapy, with particular emphasis on the correlation of DNA damage and toxicity with subcellular location of the active radioisotopes. Our current focus is to use the results from these studies to inform the development and subsequent evaluation of novel DNA-damaging radiopharmaceuticals.
Translating Bioscience into Bioprocessing
Professor David James, Professor of Bioprocess Engineering, Dept of Chemical and Process Engineering, University of Sheffield, U.K.
Mammalian cell based recombinant protein production systems can now achieve volumetric productivities of over 5g/L, and the upwards trend continues. However, at a fundamental level, what do we really know about an engineered organisms’ ability to successfully function in vitro? Rapid cell proliferation, high specific productivity, appropriate product processing and acquired adaptation to environment are all crucial success factors that are obtained by high-throughput screening of clonal derivatives rather than directed manipulation. Genetic heterogeneity within the cell population is utilised effectively, but blindly.
Industrial mammalian cell technology is still largely locked into a high-throughput selection modus operandi based on simple paradigms for gene expression and metabolic flux. The future is a systems-based biotechnology where predictability will arise from a mathematical understanding of complexity. How do we begin the transition?
Scale-up of Stem Cell Culture for Drug Discovery
Julie Kerby and Hazel Thomson, Stem Cell Sciences plc, U.K.
Embryonic Stem (ES) cells are extraordinary cells, capable of proliferating in a pluripotent state indefinitely and of differentiating spontaneously into all cell types in vivo and many in vitro. The manipulation and modification of ES cells by processes such as directed differentiation and genetic modification have lead to their potential application in biopharmaceutical areas such as cellular therapy and drug discovery.
However, in order to become a viable screening alternative, it is imperative that the culture processes for growth and differentiation are robust and reproducible, and the cells are generated in a sufficient quantity. Current approaches used for culture of ES cells operate at a manual bench scale. Scale-up by the generation of multiple parallel manual processes is unattractive because of potential variability of output; ES cells will spontaneously differentiate and are prone to genetic changes if not grown under strictly controlled conditions. The use of serum free media and automation or bioreactors introduces the consistency that is highly desirable to generate cells on a scale suitable for high throughput screening and has the added advantage of using technology that is already in place in many companies.
Stem Cell Sciences (SCS) has investigated means for large-scale production of murine ES cells, and their subsequent differentiation into neural subtypes. In the automated system, CompacT SelecT TM, cells were propagated in T-flasks as an adherent monolayer, passaged a number of times and transferred into 96 well assay plates for subsequent analysis. In the bioreactor, cells were propagated in suspension on microcarriers over 8 days then harvested manually. Cells harvested from the cultures were analysed by trypan blue exclusion, clonal assay, expression of Oct-4 (stem cell marker) and monolayer neural differentiation.
The use of human stem cells and their differentiated progeny in drug discovery is highly desirable as they offer the chance to screen on more physiologically relevant cells. The transfer of scale up technologies from mouse to human ES is becoming more realistic but remains a significant challenge.
The use of Wave Bioreactors in the Pharmaceutical Industry
Dr Girish Shah, GlaxoSmithKline R&D, Stevenage, U.K.
The use of disposable equipment for biotechnology applications offers a wealth of advantages including reduction of preparation time, elimination of cleaning and sterilization steps and cross-contamination, and a greater ease of use. These benefits are likely to contribute to significant cost savings in time and capital. The upward trend toward cultivating animal cells for the production of recombinant proteins (including monoclonal antibodies) is poised to continue for the foreseeable future, often requiring manufacture of hundreds of milligrams to gram quantities of recombinant proteins to support early evaluations. Wave Bioreactors are increasingly being used in the Pharmaceutical Industry for these and many other purposes. Apart from cultivating animal cells, Wave Bioreactors are also being used in vaccine production and cultivating plant cells for the production of secondary metabolites.
The use of FACS for the Selection of Cell Lines with Superior Productivity Characteristics
Dr Jon Welsh, Lonza Biologics plc, Slough, Berkshire, U.K.
Measurement and Control of Viable Cell Density in a cGMP Mammalian Cell Bioprocessing Facility
J. P. Carvell, Aber Instruments Ltd, Science Park, Aberystwyth, U.K.
Of the available on-line biomass assays, the radio-frequency (RF) impedance method has a clear advantage for cGMP because it is an unambiguous reflection of viable cell biovolume rather than the total number of cells. Although other more approximate methods are available for cells in suspension, RF impedance is practically the only on-line method available for cells in suspension, attached to micro-carriers and immobilized cells at often high densities. Data are presented to show how live cell concentrations and conductivity derived from a RF Impedance derived instrument, the Aber™ Biomass Monitor, have been used in a cGMP environment. The recent trend has been to use the for process control and an example will be shown of using the instrument to maintain a constant level of live HeLa cells grown in suspension under perfused cytostat conditions.
TruLink: a Technology for Improving On-Line Process Analysis and Enabling Easier PAT Implementation
Warren, Randy*; West, Larry*; Bonham-Carter, John§;
*Finesse LLC, 9351 Irvine Blvd, Irvine, 92618, CA, USA;
§Finesse LLC, Mälarhöjdsvägen 37, 12940, Hägersten, Stockholm, Sweden.
There are many tools and instruments for analysing samples throughout the bioprocess chain, of which the majority are currently off-line, labour intensive and subject to variation. Ideally, as many measurements as possible should be taken on-line, automatically, and subject to risk assessment or quality parameters, such that there is confidence for the result to be used in a closed-loop control setting. This should lead to less variation in process outcomes, and allow the process to be moved closer to an optimum operating strategy.
There are several difficulties that must be solved to realise this result, which include for example, avoiding “islands of automation”; connecting currently off-line instruments to become on-line; allowing the control system to “own” and control the analysers; ensuring the control system assesses the risks and accuracy from each measurement etc. In theory these difficulties can be solved today, but in practice, very few try and almost no-one achieves the goal.
Finesse has set up an organisation called TruLink, which is open to any organisation, individual or company, which has similar goals to the OPC foundation, the IEEE standards or the fieldbus technology standards. TruLink is a technology that can be implemented by vendors of analysis equipment to ensure that biomanufacturing companies can properly implement the vendors’ equipment in a PAT environment. TruLink will be described in detail, giving examples of how it can be used in an operational environment and the subsequent benefits.
Exploring the CHO Proteome to Improve Bioprocess Productivity
Dr Paula Meleady, National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Very little is known about the cellular and molecular mechanisms that govern the production of complex proteins by engineered mammalian cells. The majority of the work to date has largely concentrated on optimisation of cell culture conditions (e.g. media formulation), bioreactor design, and improvements in the design of expression vectors. To improve protein productivity from mammalian cells it is generally recognised that a greater understanding of the cellular machinery at both the DNA and protein level is required. Chinese hamster ovary (CHO) cells are one of the most important cell lines used for production of protein biopharmaceuticals, and display a range of growth, cellular productivity and metabolic phenotypes for large-scale cell culture processes. These ‘industrially-relevant’ cellular phenotypes can serve as useful benchmarks for judging the suitability of recombinant CHO cell performance in high productivity fed-batch cell culture processes. In collaboration with Wyeth Biopharma (Andover, US and Grangecastle, Dublin, Ireland) we are undertaking differential proteomic expression profiling experiments using 2D Difference Gel Electrophoresis (DIGE) and Mass Spectrometry (using both MALDI-ToF MS and LC-MS/MS). Replicate recombinant CHO samples were collected under appropriate cell culture conditions from both shake flask and bioreactor cultures representing a number of phenotypic categories such as high cell growth rate, high cell density, cellular productivity (Qp), etc. and compared. A large number of proteins have been found to be differentially regulated from the various phenotypic categories, and have been identified.
Molecular Analysis of Stability of Recombinant GS-NS0 Cell Lines
Professor Alan Dickson, University of Manchester, U.K.
Initial Identification of Low Temperature and Culture Stage Induction of miRNA Expression in Suspension CHO-K1 Cells
Patrick Gammell,, Niall Barron, Niraj Kumar & Martin Clynes, National Institute for Cellular Biotechnology, Dublin City University, Ireland.
Here we describe the first miRNA analysis carried out on hamster cells. Chinese hamster ovary (CHO) cell lines are the most important cell line for the manufacture of human recombinant biopharmaceutical products. During biphasic culture, an initial phase of rapid cell growth at 370C is followed by a growth arrest phase induced through reduction of the culture temperature. Growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase. Using miRNA bioarrays generated with probes against human, mouse and rat miRNAs, we have identified a number of differentially expressed miRNAs in CHO-K1 when comparing cells undergoing exponential growth at 370C and stationary phase cells at 310C. Five miRNAs were selected for qRT-PCR analysis using specific primer sets to isolate and amplify mature miRNAs. During this analysis, two known growth inhibitory miRNAs, miR-21 and miR-24 were identified as being upregulated during stationary phase growth induced either by temperature shift or during normal batch culture by both bioarray and qRT-PCR. This data offers a novel insight into the potential of miRNA regulation of CHO-K1 growth and may provide new approaches to rational engineering of both cell lines and culture processes to ensure optimal conditions for recombinant protein production.
Key words:
CHO-K1; Batch Suspension Culture; Temperature Shift; miRNA Bioarray; qRT-PCR.
Gene Expression Regulation of Cold Inducible RNA Binding Protein (CIRBP)
Mohamed B. Al-Fageeh and C. Mark Smales, Dept of Biosciences, University of Kent, Canterbury, Kent, U.K.
The mechanisms of cold-shock responses in mammalian cells are not fully understood, however a number of studies have now shown that reduced temperature cultivation of mammalian cells can lead to enhanced cell specific and volumetric recombinant protein productivity although the effect appears to be cell type and product dependent. Several recent studies have reported that mammalian cells actively and rapidly respond to mild hypothermia (32C) via the overexpression of two major cold-shock proteins, namely Cold Inducible RNA Binding protein (Cirbp) and RNA binding Motif Protein (Rbm3). The molecular mechanisms of Cirbp induction during cold-shock is far from clear. Results from the current investigation show that the Cirbp 5`-UTR is unexpectedly very short, only 82bp long. Interestingly, when the cold-shock period is reduced to 6 hours, the Cirbp 5`-UTR is significantly longer (125bp). Furthermore, transient transfection assay results showed that the longer 5`-UTR of Cirbp was able to significantly enhance the translation of the luciferase reporter gene when transfected into CHO-K1 and NIH/3T3 cells. Interestingly, the translation efficiency of the reporter gene was not improved by cold-shock or hypoxic growth conditions. In addition, 10bp 5`3` unidirectional deletion analysis of the longer 5`-UTR revealed that the full length 5`-UTR is required to enhance reporter gene translation. Quantitative PCR analysis showed that the transcription of the shorter Cirbp transcript is predominant and significantly induced within two hours of cold-shock. Surprisingly, the longer Cirbp mRNA was detectable at much lower levels when cells were cold-shocked at 32C for 2, 6, 12 and 24 hours.