Professor Mark Smales
Professor of Industrial Biotechnology
School of Biosciences
- 01227 (82)3746
Professor Mark Smales is currently Professor of Industrial Biotechnology in the School of Biosciences at the University of Kent. The group headed by Mark has a number of on-going projects whose objectives are to further advance our understanding of biotechnological products and processes at the fundamental biological or chemical level to enable their manipulation and control for improved biotherapeutic recombinant protein yields and quality. His group in particular focusses upon the investigation of cultured mammalian cells for the purposes of producing biotherapeutic proteins for the treatment of disease and for the generation of diagnostics. This includes upstream and downstream bioprocessing and embracing and utilising novel technologies such as genome editing to engineering cell systems and tune them for the desired use.
Mark is Director of the Centre for Molecular Processing and a member of the Industrial Biotechnology and Synthetic Biology Research Group.back to top
Also view these in the Kent Academic Repository
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Adherent CHO Cell Stained for Actin. CHO cells are the mammalian cell industrial workhorse.
Cell engineering and therapeutic recombinant protein biotechnology
The focus of the research in the laboratory is to work on aspects relating to improving our understanding of the biology that underpins bioprocessing and recombinant protein production from cell expression systems, particularly in vitro cultured mammalian expression systems. The laboratory is recognised internationally for its work using cultured mammalian cells for in vitro research purposes, particularly in relation to investigating the cellular constraints on recombinant protein productivity. The laboratory has extensive experience in proteome analysis and gene expression in mammalian cell systems, specifically recombinant gene expression, and is ideally placed with strong biotechnological and industrial links to exploit this technology. Currently the laboratory is funded via a number of BBSRC, EPSRC and industrial (particularly with Lonza Biologics plc) research grants and studentships, with research focussing upon investigating the molecular responses in mammalian cells upon cold-shock, the post-transcription limitations upon recombinant protein production in mammalian cells, the role of mRNA translation and its control in determining recombinant protein yields at physiological and sub-physiological temperatures, investigation of the unfolded protein response to identify novel targets for manipulation of mammalian cells in order to enhance recombinant protein production, and the investigation of protein stability with regard to formulation and delivery of biotherapeutic proteins. The work is primarily focussed upon determining the biological mechanisms that underpin each of these areas, but we are also actively involved in applying this knowledge to develop new and improved systems and processes for the biotechnology industry. Below is a sample of some of the areas we are currently interested in investigating but this is by no means inclusive. We are always interested in discussing potential projects and collaborations with academic and industrial colleagues. If you are interested, or wish to discuss graduate opportunities (Masters and PhD) or vacancies within the laboratory, please contact us.
Chemical and structural integrity of proteins in the glassy state – Protein formulation and stability
A model peptide that shows how chemical modification (lower image) can result in conformation change
This work is undertaken in collaboration with Dr Mark Howard from the School of Biosciences and Dr Roger Parker from the Institute of Food Research
There is an industrial requirement to preserve proteins prior to their use in order to maintain biological authenticity. In vitro, further degradations compromise solution state stability including hydrolysis which is generally observed at sequences containing aspartic acid (Asp-Pro is particularly susceptible), diketopiperazine formation when glycine is in the third position from the N-terminus and disulphide cross-linking reactions. As a result of the susceptibility of proteins and peptides to chemical modification, various strategies have been developed for their in vitro preservation in order to prevent such modifications. One strategy proposes that preservation will be achieved by putting the protein in the glassy state. The aim of this programme of work is to determine the physical and chemical basis and mechanisms of globular protein stability in the glassy state. There are two industrially relevant strategies for achieving stability using glass states, first; a general minimisation of molecular mobility and, second; by additionally using specific interactions in polyelectrolyte complexes. Here we are applying the following key methodologies:
- The determination of chemical and structural modifications to peptides during glassy state stabilisation and storage.
- Characterisation of glass transition temperatures and structural relaxation rates in proteins, peptides and their mixtures with stabilising additives.
- Measurement of local and global mobility in proteins and protein matrix glasses using EPR spin labels and probes.
- NMR structural analysis of the modifications to model peptides and proteins.
- Determine the conditions under which macromolecular complexation proceeds and characterise its consequent effect on probe mobility and chemical stability.
These methodologies will allow us to acquire: (1) a comprehensive view of the chemistry and structural changes occurring in proteins during glassy state stabilisation, and (2) a physico-chemical understanding of those factors controlling stabilisation. It is anticipated that information arising from this project will enable: (a) the development of improved methodologies for the preservation of foodstuffs and therapeutic proteins, (b) establish a database of potential modifications to proteins under industrially relevant processing conditions, and (c) develop monitoring systems to detect trace amounts of modified proteins.
Individual objectives suitable for assessing the outcome of the work are:
- Identification of stabilising treatments, additives and product formulations that result in protein stabilisation/destabilisation and modification.
- Structural characterisation of protein modifications.
- Identification of matrices for minimising general and localised probe mobility.
- Formulation of an overall physico-chemical model relating chemical and physical stability of proteins in the glassy state.
We are also interested in developing high concentration monoclonal antibody liquid form
Cold stress response in in vitro cultured mammalian cells and its application for the enhancement of recombinant protein yields
Many living organisms have adapted sophisticated strategies to allow their survival over a dynamic range of temperatures. The response to elevated temperatures has been extensively studied in both prokaryotic and eukaryotic systems and generally involves the induction of heat-shock proteins (HSPs), a family of proteins that are highly conserved between all organisms from bacteria to mammals. In contrast to the HSP response, the mechanisms involved in the response to sub-physiological temperatures are poorly understood and have been studied in few organisms. A number of plant genes are induced by low temperature stress, and in prokaryotes cold stress induces several well-characterised cold-shock proteins (CSPs).
By contrast, the response of eukaryotic cells to cold-shock and the biological mechanisms that govern cellular response to sub-physiological temperatures are not well understood. Cold-stress exposures cells to two major stresses; those relating to changes in temperature and those related to changes in oxygen concentration due to higher dissolved oxygen concentrations at reduced temperatures. Although our understanding of the cold-shock response in eukaryotes is limited, several studies have demonstrated that induced CSPs are key determinants in the adaptation to growth and survival at lower temperatures although little is known about what effect changes in dissolved oxygen concentrations may play in these responses. What is becoming clear is that exposing eukaryotic cells to sub-optimal temperatures invokes a coordinated response involving modulation of the cell cycle, metabolism, transcription, translation, and the cell cytoskeleton. Moreover, the response of eukaryotes to cold stress has been implicated in adaptive thermogenesis, cold tolerance, storage of tissue, organs and cells, therapeutic treatment of brain damage, and as a method to improve recombinant protein production in mammalian cells.
We have a number of projects whose focus is to identify proteins and mechanisms involved in the molecular response(s) governing cellular adaptation to sub-physiological temperatures (37ºC, cold stress) and is pertinent to understanding how these responses are coordinated. We are also interested in the response of mammalian cells upon recovery from cold-shock. These projects investigate a number of areas of the cold stress response from the global responses as determined by global proteome approaches, and more specific investigations studying how cold stress effects the cell cytoskeleton, both in collaboration with Dr Martin Carden, to structural dynamic studies of the cold shock inducible protein CIRP and its binding to RNA ligands with Dr Richard Williamson. We are also interested in how recombinant protein production can be enhanced at lower culture temperatures and the mechanisms responsible for this and the relationship to mRNA stability and mRNA translation.
Our proposed mechanism outlining the major responses initiated upon cold stress perception in mammalian cells
Determination of the post-transcriptional constraints that limit recombinant protein yield during bioprocessing from in vitro cultured mammalian cells for enhanced production
The ability of industrially relevant expression systems to produce recombinant protein (rP) has advanced considerably in recent years. However, despite such advances our understanding of the cellular processes that determine/limit rP yield from in vitro cultured mammalian cells remains poor. Thus, our understandings of those cellular processes that constrain or limit rP production (rPP) during bioprocessing are poorly understood and open to conjecture. What is clear is that the constraints are ultimately determined by many different parameters and that cell growth and cell specific productivity alone cannot determine recombinant protein yields. The laboratory therefore has a large number of projects on-going within this area to identify the key mechanisms and genes/proteins that determine or limit recombinant protein yield from in vitro cultured mammalian cells. Some of these projects are in collaboration with Dr Martin Carden, Dr Peter Klappa, Professor Mick Tuite and Dr Tobias von der Haar within the school via funding from the Bioprocessing Research Industry Club (BRIC) whilst others are funded from Research Councils or Industry. We are particularly interested in the modulation of translation and metabolic pathways, the makeup of mRNAs, the role of chaperones and foldases, and the role of the unfolded protein response in recombinant protein production. We are using a combination of genomic/proteomic, molecular biology and protein biochemistry approaches to investigate this large area. We envisage determination of the key processes and mechanisms that determine rPP will allow the modelling of cell performance, allowing the prediction and selection of high producing clones and the development of new approaches for cell engineering to improve recombinant protein yields.
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Current members of the Smales group are:
Tanya Knight Laboratory Manager
Dr Jane Povey Postdoctoral Research Associate
Development of predictive tools for the isolation of highly productive cell lines
I joined the Department of Biosciences in 2005 following a Daphne Jackson Fellowship based at Pfizer in Sandwich. My current Post Doc project is involved in improving and developing new methods for screening and selecting recombinant mammalian cell lines. Previously at Kent I worked on a project investigating protein stability in the glassy state. I have a background in protein biochemistry and have previously worked in industry for Welcome (now Murex) Diagnostics, Evans Medical and Cortecs Diagnostics
Dr Catherine Hogwood Postdoctoral Research Associate
Cell line development based on process understanding of host cell protein interactions
My PhD at Kent focussed on investigating the global cellular responses to the chemotherapeutic agent cisplatin with respect to differential p53 expression levels. The three main responses to DNA damage; cell cycle arrest, apoptosis and DNA damage are largely studied independently, however it is hypothesised that these processes come together to form an complex integrated network to ensure the integrity of the genome is protected. An in vitro model cell system and a proteomic approach were applied in which to investigate the hypothesis that considerable crosstalk occurs between the main responses to DNA damage. Through the coupling of 2D-proteomic analysis with mass spectrometry a number of proteins were identified that were up-/down-regulated in response to cisplatin exposure in p53 expressing and p53 knockdown cells in order to further define the role(s) of p53 in the integrated network of responses to DNA damage. This work was undertaken collaboratively between the Lloyd and Smales groups at Kent.
Currently I am working on a project in the Smales group funded through the BRIC initiative (BBSRC, EPSRC and Industrial members) focussed upon the identification/characterization of the major host cell protein (HCPs) contaminants in the cell culture supernatant at harvest of cultured Chinese hamster ovary cells engineered to express therapeutic recombinant proteins. This knowledge should facilitate the development of knowledge based approaches to design more efficient or alternative purification strategies and the rational selection and/or engineering of host cell lines to limit the levels of such problematic HCPs. The project utilises a combination of interrelated approaches to investigate the HCP profile from monoclonal antibody producing cell lines during culture and subsequent downstream processing to determine if these processes and the HCP complement itself can be manipulated to improve, or offer alternatives for, downstream bioprocessing. The direct outcomes of this research and benefits to the bioprocessing industry will be (i) a defined CHO HCP profile for a model system and an understanding of how this changes/accumulates during fermentation and at harvest, (ii) knowledge as to whether the target protein changes the HCP profile and the ease with which these are removed, (iii) an understanding of the HCPs removed by particular chromatographic steps and techniques, (iv) alternative methods of monitoring and measuring HCPs present during bioprocessing, (v) determination of the effects of eliminating specific HCPs on cell phenotype and subsequent downstream bioprocessing, and (vi) the design of novel or alternative processes to remove HCPs via either up- or down-stream approaches.
Dr Angelica Ozanne Postdoctoral Research Associate
Unravelling and engineering the role of trace metals and recombinant therapeutic protein synthesis and heterogeneity from Chinese hamster ovary cells
Dr Barrie Rooney Postdoctoral Research Associate
‘Dipstick’ test tackles fatal sleeping sickness
Barrie has been working on a project to develop a quick and simple diagnosis method, similar to a dipstick pregnancy test, to fight a deadly sleeping sickness. The test to diagnose Human African Trypanosomiasis (HAT) just requires a pin-prick blood sample and will remove the need to take complex equipment into remote areas of sub-Saharan Africa. Existing tests rely on extracts directly from the dangerous parasite, but now the scientists at the School of Biosciences have designed a way to test for the disease more easily and safely, and therefore more cheaply.
The next generation test was developed by Dr Barrie Rooney and Professor Mark Smales, together with School colleagues, working with international medical charity Medecins Sans Frontieres (MSF) in research funded by the Biotechnology and Biological Sciences Research Council (BBSRC) Flexible Interchange Programme (FLIP).
Millions of people are at risk of HAT, which is usually fatal if untreated, with patients falling into a coma before death. Around 5,000 cases are reported each year, with severe social and economic costs, and some areas at risk remain uncovered by surveillance and control efforts. The disease is caused by the parasite Trypanosoma brucei gambiense (T.b. gambiense) and spread by the bite of infected tsetse flies.
Dr Rooney has been involved with MSF mobile HAT screening teams in central African countries for over 10 years. Traditional testing involves a large team in remote areas doing time consuming microscopic work, and painful lumbar punctures, which requires electricity and refrigeration. By combining the latest genome databases and old fashioned fermentation techniques the researchers have come up with a fast, simple way of making robust and reliable tests. The new tests are designed to be heat stable and user-friendly like a dipstick pregnancy test.
The paper “Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs”) is published in PLOS Neglected Tropical Diseases.
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Year 2 BI503 - Cell Biology
- BI518 - Molecular Biology and Genetics
Final Year BI601 - Skills for Biochemists
- BI602 - Cellular Communication I
- BI637 - Forensic DNA Analysis
- BI631 - Skills for Biomedical Scientists II
- BI626 - Integrated Endocrinology and Metabolism
- Member of BBSRC Bioprocessing Research Industry Club (BRIC) steering panel
- Executive Editor, Biotechnology Letters
- Member of Editorial Board, Biotechnology and Applied Biochemistry
- Member of BioprocessUK strategic steering group
- Member of ESACT-UK committee
- Teach on UCL Biochemical Engineering MBI programme