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Translating Bioscience into Bioprocessing

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					                     ESACT-UK 17th Annual Meeting

                                    Programme


Thursday 4th January 2007

11.00        Registration desk opens


12.00-12.50 Lunch, 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.

     1.30-2.00      Human Cancer Cell Lines. Quality Control of Cross-
                    Contamination
                    Professor John Masters, University College London, U.K.

     2.00-2.30      Investigating the Cellular Responses to Clinically-Useful DNA
                    Damaging Agents
                    Dr Daniel Lloyd, Department of Biosciences, University of Kent,
                    Canterbury.


2.30-3.00    Keynote Presentation
                    Translating Bioscience into Bioprocessing
                    Professor David James, Dept of Chemical and Process Engineering,
                    University of Sheffield, U.K.


3.00-3.30    Coffee, 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.

     4.00-4.30     The use of Wave Bioreactors in the Pharmaceutical Industry
                   Dr Girish Shah, GlaxoSmithKline R&D, Stevenage, U.K.

     4.30-5.00     The use of FACS for the Selection of Cell Lines with Superior
                   Productivity Characteristics
                   Dr Jon Welsh, Lonza Biologics plc, Slough, Berkshire, U.K.

     5.00-5.15     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.

     5.15-5.30     TruLink: 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

7.30-9.00am Breakfast


9.00-10.40   Session 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.

    9.30-10.00     Molecular Analysis of Stability of Recombinant GS-NS0 Cell Lines
                   Professor Alan Dickson, University of Manchester, U.K.

    10.00-10.20    Initial 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.

    10.20-10.40    Gene Expression Regulation of Cold Inducible RNA Binding Protein
                   (CIRBP)
                   Mr Mohamed Al-Fageeh, University of Kent, Canterbury, Kent, U.K.


10.40-11.45 Coffee, 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.
    12.15-12.45   Microscale and Automation Approaches for Rapid Cell Culture
                  Process Development
                  Professor Gary Lye, Department of Biochemical Engineering, University
                  College London, U.K.

    12.45-1.05    Measurements for Biotechnology – Improving Measurements in the
                  Biopharmaceutical Industry
                  Dr Anna Hills, Biotechnology Group, National Physical Laboratory,
                  Teddington, U.K.


1.05pm            Award of prizes.

1.10pm            Close of Meeting (Jon Green)


1.15-2.15pm       Lunch
  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 (32C) 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 32C for 2, 6, 12 and 24
hours.
        The Influence of Isotype, Glycoform and Epitope
        Specificity on the Functional Profile of Antibody
                          Therapeutics
                     Professor Roy Jefferis, University of Birmingham, U.K.


Multiple parameters have been defined that impact on the functional activity of therapeutic
antibodies. Protein and/or glycoform engineering can generate antibodies with maximal or
minimal potential to activate effector functions. The antibody format may thus be optimised
for particular disease indications, e.g. cell killing or cytokine neutralisation. Early choice of
antibody format for new therapeutics allows integration of key parameters into clone selection
and “Process Analytical Technology” (PAT).
  Microscale and Automation Approaches for Rapid Cell
              Culture Process Development
  Timothy Barrett, Rosario Scott, Andrew Wu, Hu Zhang, Susana Levy, Chris Mason,
                              Farlan Veraitch, Gary Lye


The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering,
University College London, Torrington Place, London, WC1E 7JE, UK.



Experimentation in microplate formats offers a potential platform technology for the
evaluation and optimisation of cell culture conditions. Provided that the results obtained are
quantitative and reproducible, it should be possible to obtain process design data early and
cost effectively in an automated manner. This presentation describes the engineering
characterisation of liquid mixing and gas-liquid mass transfer in microwell systems and their
impact on suspension and adherent cell cultures.

In the case of suspension cultures studies have focussed on therapeutic antibody production.
Here it is important that microwell conditions accurately simulate the ultimate large-scale
performance. Working with both murine hybridoma and CHO-S cells we have characterised
microwells in terms of energy dissipation (via CFD), fluid flow patterns and oxygen transfer
rate as a function of shaking frequency. At low shaking frequencies there is visible cell
settling while at high frequencies there is evidence of decreased cell growth and reduced cell
viability. Using the average energy dissipation rate as a basis for scale translation we have
been able to relate optimum microwell performance to results obtained in shake-flask and
stirred-bioreactors.

In the case of adherent cells we have focussed on the expansion and differentiation of both
adult and embryonic stem cells for use in drug screening and cell-based therapies. Here the
emphasis is on the maintenance of reproducible and defined culture conditions in order to
minimise phenotypic variability. Bioprocess conditions for automated stem cell culture have
been studied as a function of inoculation cell density, dissolved oxygen levels and the
dynamic variation of parameters such as pH and temperature. The impact of shear and
centrifugal forces have also been evaluated. The results indicate the sensitivity of stem cells
to bioprocess conditions and their impact on the yield and viability of the desired cell
phenotype.
        Measurements for Biotechnology – Improving
       Measurements in the Biopharmaceutical Industry
    Dr Anna Hills, Biotechnology Group, National Physical Laboratory, Teddington, U.K.


The Biotechnology Group at the National Physical Laboratory is concerned with the
metrology of established and novel analytical techniques to support the biopharmaceutical
industry, from analysing protein mixtures or characterising protein interactions to benefit drug
discovery or disease diagnosis, to manufacturing and product characterisation. Sound
measurement practise means efficient discovery and development of drugs and diagnostics,
more certain control of biopharmaceutical production and better communication with
regulators.

Example projects include: detection and quantification of process-related impurities;
strengthening QC of biopharms through establishment of structure function relationships;
improving inter-comparability of in vitro cell based measurements; and development of
methods to image cell/protein attachment on biocompatible surfaces. Work is largely
supported by the Measurements for Biotechnology programme which is centred around four
themes; cell-based technology, gene measurement, product characterisation, and protein
measurement. It was commissioned by the DTI‟s National Measurement System and is
coordinated by the National Physical Laboratory and LGC Ltd.

Note: for more information please contact the programme coordinators or go to
www.mfbprog.org.uk
 POSTER

ABSTRACTS
     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 (32C) 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 32C for 2, 6, 12 and 24
hours.
       The use of Imaging Technologies in the Study of
           Biological Vaccines and Cell Screening
   Lorraine Berry, Rachel Prenata-Blanc and Roland Fleck, NIBSC, Blanche Lane, South
                                 Mimms, EN6 3QG, U.K.

The development of new and novel vaccines, and developments in regenerative medicine,
provides a continual need for new effective testing procedures to ensure their safety,
effectiveness and efficacy. The new imaging facilities at NIBSC provide a range of
microscopy techniques to support the vaccine studies and other work of the institute.

Confocal and epifluorescent microscopy are routinely used for testing and evaluating the
effect of new vaccines. To study the effects of anthrax lethal toxin (LT), the anthrax toxin
receptor (TEM8) was identified on neutrophils derived from NB4 cells (a human
promyelocytic leukaemia cell line) using confocal imaging. The project aim is to develop an
assay measuring toxicity of any remaining LT in anthrax vaccines. Initial experiments indicate
temperature dependent binding, that energy may be required to keep the protective antigen
bound to TEM8, and exposure to LT may impair actin assembly in neutrophils. It is hoped
that these studies will form the basis of cell-based cytotoxicity assays, reducing the reliance
on animal models. Cellular assays are also being developed to investigate vaccine
administration, again utilising confocal microscopy. FITC labelled proteins are being used to
look at binding, uptake and internalisation of antigens, and inhibition of neurotransmitters in
selected cell types to model toxin uptake, and to characterise the induction mechanisms
involved with trans-cutaneous tetanus immunisation.

Electron microscopy has also been used for the detailed study of viruses in attenuated or
inactivated viral vaccines. Current TEM studies include morphological differences between
stable and non-stable forms of the poliovirus, and the evaluation of vaccine constancy in new
HPV vaccines. TEM is routinely employed in cell screening for adventitious agents and viral
contaminants. Regenerative medicine such as gene therapy and stem cell therapy will also
require effective procedures for validation. This includes screening of stem cell lines and the
ultrastructural evaluation of replication of gene therapy vectors. TEM was utilised to
compliment the characterisation of defined cell lines within the UK Stem Cell Initiative; an
initiative dedicated to the use of consistent, quality assured and defined cell lines for research
into regenerative medicines. Morphological differences between cell lines provided a valuable
insight into reproducibility of stem cell differentiation between labs.

In summary, the imaging facilities at NIBSC provide valuable support for its researchers.
Capabilities range from fluorescence and confocal microscopy on live cells through to cryo-
preparation of specimens for electron microscopy. These techniques, coupled with X-ray
microanalysis, cryo-tomography, 3D visualization and immuno-specific labelling, will allow
greater detailed studies to be achieved, and to meet the ever-emerging challenges of
biological standardization.
   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.
    EFFECTS OF SERUM AND GROWTH FACTORS ON HEK
        293 PROLIFERATION AND APOPTOSIS AND
              ADENOVIRUS PRODUCTIVITY
                          Angela Buckler1 and Mohamed Al-Rubeai1,2
1
 Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham
B15 2 TT, U.K.
2
 School of Chemical & Bioprocess Engineering, University College Dublin
Belfield, Dublin 4, Ireland.


The effect of serum on cell growth and virus production in both basal and serum free media
was examined in Human Embryonic Kidney (HEK) 293. A 40 fold decrease in virus titre was
observed in Dulbecco‟s Modified Eagles Medium (DMEM) cultures supplemented with 10%
FCS when compared to 1% FCS. However, a 90 fold increase in virus titre was observed in
DMEM supplemented with 1% FCS as compared with EX-CELLTM 293 serum free culture.
Similar effect was also seen for the efficiency of gene transfer as indicated by the expression
level of GFP. The results demonstrated that although higher serum concentration has a
negative and dose-dependent effect on titre its supplementation in small quantity is essential
for virus productivity. The effect of a variety of Insulin-like growth factors on proliferation and
apoptosis of HEK 293 cells as well as on infection and specific productivity was also studied
in serum free media. It was found that LONGTM R3 IGF-1, an analogue of IGF-1 promotes cell
proliferation and inhibits apoptosis more effectively than Insulin and IGF-1. Nutrient
deprivation and viral infection were found to be the main factors inducing apoptotic cell death
in HEK cells. Similarly, cultures supplemented with LONGTM R3 IGF-1 showed relatively
higher virus productivity in comparison to other cultures but virus productivity was found to be
significantly (p<0.05) higher when there were no growth factors. The implication of these
findings for the development of two separate processes for cell expansion and virus
production will be discussed in respect to serum, growth factors and basal formulations.
    Stem-Like Cells are Stably Entrapped within both
 Established Cell Lines and Short-Term Cultures Derived
          from Glioblastoma Multiforme (GBM)
                               Sarah Brown and John L Darling

Neuro-oncology Research Group, Research Institute in Healthcare Science, School of
Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB,
United Kingdom


Glioblastoma multiforme, the commonest malignant brain tumour in adults, represents a
formidable clinical challenge. Even with optimal therapy survival rarely exceeds a year from
diagnosis and there is a pressing need to develop new therapies based on an understanding
of the biology of these tumours. It has been recognised for many years that most surgical
specimens of GBM give rise to short-term cultures that appear to be composed of replicating
glial-like cells and that about half of these short-term lines will establish in culture. Limited
studies have suggested that surgical specimens of GBM (and perhaps some other types of
brain tumour) have subpopulations of CD133 positive stem-like cells and it has been
hypothesised that the persistence of these cells in vitro might contribute to the success in
generating established cell lines from these tumours.

In serum-free stem cell medium (Chemicon), using CD133 antibody conjugated to a magnetic
bead separation system (Miltenyi Biotech), subpopulations of cells have been isolated from
four short-term cell lines, IN46, IN859, IN1056 and IN1528 (passage level 7-24) and a well
characterised established cell line, U251MG (passage level 628-634). The proportion of
CD133 positive cells isolated from U251MG ranged between 0.23 and 10.5% and between
1.7 and 6.7% in short-term cell lines. CD133 positive cells grown in stem cell medium
appeared to be rounded and loosely adherent to the substratum. They grew slowly and
formed “neurosphere‟-like structures after about three weeks in culture following regular
refeeding. CD133 negative cells were, in contrast, more substrate adherent and did not form
neurospheres in vitro. Returning either population of cells to normal growth medium (Ham‟s
F-10 supplemented with 10% foetal calf serum) resulted in a rapid change in phenotype
where the cultures reverted to a form resembling the parental cell lines. Immunophenotyping
of the cells indicated that both subpopulations of U251MG cells paradoxically expressed the
mature astrocyte-specific intermediate filament marker glial fibrillary acidic protein (GFAP)
and nestin, an intermediate filament usually associated with neural stem cells. The SRY-
related HMG box transcription factor SOX1 was also expressed in both populations of cell but
not in the nucleus. Bmi1, a Polycomb group repressor essential for driving neural stem cell
proliferation probably by repressing genes involved in senescence, was expressed at high
levels of the nuclei in CD133 positive cells but not or at much lower levels in CD133 negative
cells.

The presence of stem-like cells entrapped within cultures derived from surgical biopsy
material from patients with GBM may account for the relative ease with which these cultures
establish in vitro and that studies aimed at specifically targeting these cells might result in
more effective therapies.
    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.
 Optimising Transient Transfection in Mammalian (Serum
    Free) cells to Generate Early-Phase Recombinant
           Antibodies for in-vitro/vivo Studies
                                 H.Hailu, D.James, S.Colley

At UCB Celltech there is a need for high throughput transient antibody generation, at around
100Abs/year and between 2 to100mg quantities for in-vitro and in-vivo experiments. We are
therefore, interested in rapid efficient large scale transient systems.

Our best yields are from using an „in-house designed‟ electroporator, which regularly
achieves 5 fold higher levels of expression than our PEI (Polyethyleneimine) method, and on
occasions, are above 100mg/L for CHOs cells. However, the approach requires time-
consuming centrifugation steps, and so we are still utilizing PEI as an alternative transfection
method in both 293F and CHOs(Invitrogen) cells.

Using PEI, we regularly achieve expression levels between 5 to 40mg/L in 293F cells, but
have not achieved this in CHOs cells. In an attempt to overcome this we have looked at
different CHO media from various commercial sources (Sigma, Cambrex, Ajinomoto, an in-
house formulations), in addition we have examined the effect of different supplements
derived from plant hydrolysates to increase yields.
We have also attempted to increase antibody expression by altering the ratio of single
antibody chain plasmid DNAs encoding the separate heavy and light chain antibody genes.

The results from these various experiments will be presented to illustrate our approach to
rapid medium through put antibody generation.
 Global Cellular Responses of in vitro Cultured Human Cells to
    the Chemotherapeutic DNA-Damaging Agent cisplatin
                 Catherine E Hogwood, C Mark Smales, and Daniel R Lloyd

                 Dept of Biosciences, University of Kent, Canterbury, Kent.

Cells are exposed to a variety of DNA damaging agents on a daily basis, hence mechanisms
have evolved to maintain the integrity of the genome. To date three major responses to DNA
damage have been described; cell cycle arrest, DNA repair and apoptosis. The initial aims of
this investigation were to optimise the growth of the selected model cellular system, HT1080
fibrosarcoma clones that exhibit varying p53 expression due to the presence of a stably
transfected RNA interference construct expression short hairpin RNA directed against p53
mRNA. At various time points after exposure to 50M cisplatin, HT1080 cells were lysed in
order to obtain total protein extracts. Western blot analysis on the resulting protein extracts
confirmed differential p53 expression levels in the clones investigated. This analysis showed
that two of the clones, clone 2 and 24, express dramatically decreased p53 levels,
presumably due to the presence of a detectable knockdown via RNAi, whereas clones 14
and 26 express relatively „normal‟ levels of p53. Further, these studies clearly demonstrated
cisplatin-induced upregulation, along with the anticipated transactivation of p21 in the p53-
proficient clones. As p53 is known to play a key role in the cellular response mechanisms to
DNA damage, the HT1080 clones expressing differing levels of p53 will subsequently be
used to identify candidate p53-regulated proteins for RNAi to probe their role in the DNA
damage response. In order to identify these RNAi targets, a proteomic analysis (i.e. 2D-
PAGE) approach will be used to identify those proteins that are up-regulated in a p53-
dependent manner in the presence cisplatin. The expression of these target proteins will then
be silenced/downregulated by RNAi. The effect of silencing specific proteins on cell survival,
DNA repair and the proteome will then be investigated. This project will ultimately result in a
greater understanding of how cells respond to DNA damage and help in the identification of
major control points within the network of the DNA damage responses.
      Evaluation of the Effect of Culture Media on Dome
                  Formation in Caco-2 cells
1
Sally Lees, Damian Marshall*, Julie Davies*, 1Ross Hawkins.
1
 NIBSC, Blanch Lane, South Mimms, EN6 3QG; * LGC, Queens Road, Teddington,
TW110LY.


A literature survey for the performance of high through-put drug screens highlighted that
Caco-2 cells are maintained in a wide selection of different culture media. The same literature
survey also showed there are large discrepancies in the data obtained using Caco-2 cells
between laboratories.

In order to investigate the possible role of culture medium in data variability, cells from
various suppliers were grown in a selection of media for 3 passages. The cells were then
grown to 14 days post confluence, the appearance of domes and the cell monolayer was
recorded by photography. Cells maintained for 50 passages in MEM or DMEM were then
differentiated in both MEM and DMEM . The dome formation was quantified by measuring
the surface area and frequency. TEER and Rhodamine 123 transport were also examined.

Cells that were maintained in DMEM were demonstrated to have a reduced number of
domes formed, even when returned to MEM. These cells also appear to form multiple cell
layers and have reduced TEER values compared to those cultured in MEM.

In conclusion, the behaviour of Caco-2 cells is influenced not only by the current culture
media but also the media they have been maintained in historically.
The Effect of Harvest Time and Means on Glycosylation
Pattern of a Monoclonal Antibody
    Cecilia Qvist1, Helen Baldascini1, Mark Smales2, Atul Mohindra3, Andrew Racher3, Michael
                                 Hoare1 and Shaun Bilsborough4
1
  Dept of Biochemical Engineering, University College London, WC1E 7JE, London
2
  Research School of Biosciences, University of Kent, CT2 7NJ, Canterbury
3
  Lonza Biologics plc, 228 Bath Road, SL1 4DX, Slough
4
  Agilent Technologies, Lakeside, Cheadle Royal Business Park, SK8 3GR, Stockport

The method of production of therapeutic proteins affects their molecular structure and
glycosylation pattern, which are crucial to properties such as stability and efficacy. Choice of
cell line together with culture conditions will have an impact on the glycoforms of the product.
Further processing of the protein, such as centrifugation as the first step to remove cells, can
also affect the structural properties. A key challenge in the production of these glycoproteins
is to produce a consistent glycoform profile between batches. Here we describe methods for
the routine screening of intact antibodies to determine glycosylation status using liquid
chromatography and time of flight mass spectrometry.
This study is looking at the effect of harvest time on the molecular structure of recombinant
IgG. We have compared the glycosylation status of whole recombinant IgG at different
stages of fermentation. Furthermore we have applied ultra scale-down tools developed at
UCL to allow better prediction of the effects of early stage cell recovery by centrifugation on
the structural authenticity of the protein. While there are major effects on the contamination
loads affecting subsequent purification, analysis by isoelectric focusing showed little change
in the profile of the antibody structures. Mass spectrometry analysis, as reported in this
paper, allows a more in-depth investigation into the effect of bioprocessing conditions on the
chemical structure of the antibodies and in particular to focus on the interaction between the
cell culture and cell recovery stages.
       Improving Biopharmaceutical Production from
       Mammalian Cells using Hypothermic or Hypoxic
                   Culturing Conditions
                             Sarah J Scott and C Mark Smales

  Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent

The mammalian cold shock protein Rbm3 is a glycine-rich RNA binding protein believed to
be involved in the control of (post)transcriptional events. A number of regulatory elements
have been identified within the 5‟UTR of Rbm3, including a putative 22nt IRES module
thought to be responsible for its cold inducible expression. We have utilised dicistronic
reporter gene plasmids containing either the full length 5‟UTR or the 22nt IRES from Rbm3 to
investigate translational control elements within these sequences. Preliminary results show
increased IRES mediated translation upon cold shock, but also increased expression of cap-
dependant translated reporter genes. It is not currently clear if this increase is due to
enhanced translation, increased transcription or increased mRNA stability. Investigations into
the effect of hypoxic conditions on Rbm3 5‟UTR mediated translation show that hypoxia
induces translation via the 22nt IRES module, suggesting a common link between cold-shock
and hypoxic responses. The results of these ongoing investigations will determine whether
the regulatory elements contained within the Rbm3 5‟UTR can be used to improve
biopharmaceutical production from mammalian cells under conditions of hypothermia and/or
hypoxia
    Prediction of Recombinant Protein Production in Insect
       Cell-Baculovirus System using Flow Cytometric
                         Technique
                   Kalbinder Singh Sandhu1 and Mohamed Al-Rubeai 1,2 *
1
 Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham
B15 2 TT, U.K.
2
 School of Chemical & Bioprocess Engineering, University College Dublin
Belfield, Dublin 4, Ireland.


The baculovirus expression vector system (BEVS) utilising the Autographa californica
mononuclear polyhedrosis virus (AcMNPV) is widely becoming the system of choice for the
production of many recombinant protein products due to the high yields obtained. There is
however a need to develop a simple reliable at-line method to monitor the production of
recombinant proteins that have no intrinsic reporter properties. Here we utilise flow cytometry
to measure cell size, granularity and DNA content in a single step analyses and correlate
these parameters to the production of the recombinant protein β-galactosidase. Clear
correlations between these parameters and productivity are made with forward and side
scatter signals showing the highest correlation coefficients. Measuring these parameters
does not require any processing of the cells from culture to analysis. These parameters can
therefore be used successfully to predict at-line the amount of recombinant protein product in
a BEVS system.


Keywords: Baculovirus, Insect cells, Monitoring, Recombinant protein, Flow cytometry.
     Isolation of Stable Clones Secreting High Titres of
    Therapeutic Protein using CellXpress™ Technology
  Dr Linda Somerville, Mark Gerber, Jennifer Cresswell, Nan Lin and Kevin Kayser, SAFC
             Biosciences, Cell Line Engineering, U.K. and St Louis MO 63103


Selection of stable highly secreting recombinant cells is critical for robust biopharmaceutical
manufacturing processes. If a high quality clone is not established serious issues can arise
such as low or unstable protein yield and ineffective use of costly resources. The LEAP™
(Laser-Enabled Analysis and Processing) platform combines in-situ imaging with laser
manipulation to efficiently identify, purify and monitor expansion of high secreting clones. It
also allows for rapid analysis of cell population heterogeneity. This presentation shows data
using the CellXpress™ software module on the LEAP™ platform to isolate and characterise
several high-secreting single-cell clones. By combining CellXpress™ with media screening in
multi-well plates we evaluate multiple prospective clones for productivity and process
stability.
     The pH Response in Mammalian Cells and the Effect on
        Recombinant Protein Volumetric Productivity in
    Industrially Important Chinese Hamster Ovary Cell Lines
    Zoë L Towler1, Andrew Racher2, Robert Young2, Atul Mohindra2, and C Mark Smales1
1
Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK
2
Lonza Biologics plc, 228 Bath Road, SL1 4DX, Slough

Mammalian cells, particularly Chinese hamster ovary (CHO) cells, are used to produce a
range of therapeutic recombinant proteins (rP). Our current ability to produce the levels of
therapeutic proteins required in the market place is limited and much research has recently
focussed upon increasing the ability of expression systems to produce higher yields of rP in
order for supply to meet the increasing demand for these products. Culture parameters such
as pH have previously been shown to affect the volumetric productivity of mammalian
cultures. However, the cellular and molecular mechanisms by which reduced pH leads to
prolonged viability and increased recombinant protein productivity is currently unknown. A
Lonza Biologics IgG4 producing CHO cell line, LB01 was grown in small scale batch and fed-
batch cultures and in larger scale airlift bioreactors operated in fed-batch mode under
controlled conditions. The pH of bioreactor cultures was shifted from 7.2 to 6.8 either on day
5 during exponential growth phase or day 8 during stationary phase. Control cultures were
maintained at pH 7.2. The phase of growth cultures were in at the time of the pH shift
resulted in different changes in growth, metabolism and volumetric productivity.
 Rapid Selection of Clonal High Producing Cell Lines in a
            Chemically Defined Environment

Steven Watters, Kerensa J. Jones, Irene Bramke, Edward Richer, Julian Burke
Genetix Ltd. Queensway, New Milton, Hampshire. BH25 5NN, U.K.

The Genetix ClonePixFl technology dramatically shortens timelines by selecting high yielding
clonal cells early in the process of antibody development. This can be done in a chemically
defined environment removing the need for later adaptation stages and large-scale liquid
handling of low producing clones. Using thousands of colonies of cells grown clonally in
methyl cellulose based semi-solid media, Genetix ClonePixFL measures antibody secretion
by fluorescent detection and isolates the most productive colonies to a 96-well plate. This is
now a proven technology for working within the animal free process with improvements in cell
line productivity not just of a few percent but multiple times, combined with increased stability
and clonality.

The method has been shown to work with a variety of cells types both adherent and
suspension, serum containing and serum free chemically defined. The process is
demonstrated here using suspension CHO as an example. This is an area where the
technology has made huge improvements to current timelines by research on optimal growth
of colonies and the assessment of long-term stability. The high throughput nature of the
ClonePixFl allows screening of 4000 colonies/ hour which are ranked in terms of relative
target expression. Picking of the top 1-5 % means that downstream processing is limited to
dealing only with the very best clones, and 95%- 99% are discarded. Highly selective
fluorescent imaging ensures only the most desirable clones are picked, and the use of
multiple wavelengths (multiplexing) enables the study of several parameters in a single read -
expression of the target protein and clone viability/stability, for example.
      Cellular Toxicity and DNA Damage Induced by the
           Radiopharmaceutical Indium111 Oxinate
                   Amanda J. Weeks, Philip J. Blower1 and Daniel R. Lloyd

Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK
1
 Division of Imaging Sciences, King‟s College London, Guy‟s Hospital Campus, St. Thomas‟
Street, London SE1 9RT, UK

Indium111 oxinate is a radiopharmaceutical that decays by electron capture with a half life of
67 hours, emitting gamma radiation with energies of 172 KeV and 246 KeV. Indium 111 oxinate
is used in nuclear medicine for the in-vitro labelling of separated blood cells, which are then
administered to patients to investigate inflammation at the sites of infection and abscesses.
Indium111 has also been evaluated recently as a potential tumour therapy. It is therefore of
interest to investigate the DNA-damaging effects and cytotoxicity associated with cellular
exposure to indium111. We have investigated cellular toxicity of indium 111 oxinate in HT1080
fibrosarcoma cells and MCF-7 breast epithelial cells using the colorimetric MTT assay and
the clonogenic survival assay. The results were compared with data obtained from similar
experiments in which cells were treated with equimolar amounts of indium 111 oxinate that had
been subjected to several weeks of decay, in order to determine the degree of cytotoxicity
attributable to radioactive emissions. The single cell gel electrophoresis (Comet) assay was
also used to determine DNA damage associated with exposure to indium 111 oxinate. HT1080
cells that were subjected to 10 MBq/ml indium 111 oxinate exhibited 0.1 % cellular survival in
the clonogenic survival assay compared to untreated control, while treatment with a molar
equivalent of the decayed indium111 oxinate exhibited 25 % survival compared with the same
control. MCF-7 cells exhibited zero % cellular survival when subjected to treatment with 5
MBq/ml compared to untreated control, while treatment with decayed indium 111 oxinate
exhibited 38 % survival compared with the same control. The Comet assay showed DNA
damage only at doses where cell toxicity was observed. The results demonstrate that
radioactive emissions associated with 10 MBq/ml indium 111 oxinate elicit significant DNA
damage and cytotoxicity in HT1080 and MCF-7 cells. Studies are continuing to investigate
whether indium111 is taken up into the nucleus. These data will be compared with ongoing
cellular uptake studies to correlate intracellular dosage and nuclear proximity with cytotoxicity
and DNA damage.
    Proteomic Analysis of Urine to Detect Biomarkers in
           Pancreatic Ductal Adenocarcinoma
       Mark E. Weeks, D. Hariharan, T. Radon, N.R. Lemoine, T. Crnogorac-Jurcevic


Centre for Molecular Oncology, Institute of Cancer and the CR-UK Clinical Centre, Barts and
The London, Queen Mary's School of Medicine and Dentistry, 1st Floor, John Vane Science
Centre, Charterhouse Square, London


Pancreatic cancer is the fourth leading cause of cancer related deaths in the western world
and it remains difficult to detect early and treat successfully. The disease is very hard to
control and can only can be cured if it is found at an early stage. There is therefore a real and
urgent need to find non-invasive disease markers that will enable earlier intervention and
improve patient prognosis. Urine is easily and readily obtainable body fluid that may be a
source of cancer biomarkers. The application of proteomic techniques to screen the urine of
patients for changes occurring at the molecular level could potentially revolutionise the
detection and management of pancreatic cancer, thus saving valuable lives.

				
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Description: Translating Bioscience into Bioprocessing