Professor Mark Smales

Professor of Industrial Biotechnology
Director: Industrial Biotechnology Centre


Professor Mark Smales is 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 (a) biotherapeutic recombinant protein yields and quality, (b) manufacture of gene therapies, (c) re-tuning of cell metabolism via synthetic biology approaches. His group in particular focusses upon the investigation of cultured mammalian cells for the purposes of producing biotherapeutic proteins for the treatment of disease, for the generation of diagnostics and for manufacturing of gene therapies. 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. A further aspect of his work is around mRNA translation and its control. He has a particular interest in how both initiation and elongation message specific control is achieved by the cell when under specific stresses, particularly in response to cold-shock.
Mark is Director of the Industrial Biotechnology Centre and a member of the Industrial Biotechnology and Synthetic Biology Research Group.
ORCID ID: 0000-0002-2762-4724

Research interests

Cell and metabolic engineering, therapeutic recombinant protein biotechnology, gene therapy, vaccine technology, control of mRNA translation 

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, control of mRNA translation, and ore recently its application to manufacturing of gene therapies and vaccines. The laboratory has extensive experience in gene expression analysis 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 research grants and studentships. 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.


MSc-R project available for 2020/21

Generation of Coronavirus protein antigens for development of diagnostics and novel vaccine approaches (Joint supervision with Prof Martin Warren)
This project will build upon the on-going work in the Smales and Warren lab’s at Kent, and the Warren lab at the Quadram Institute in Norwich, to recombinantly produce key protein antigens from the coronavirus and then utilise these in diagnostics and the development of new vaccine candidates based on incorporation into bacteria microcompartments. The project will be undertaken in collaboration with Mologic, commissioned by the Government to develop diagnostics for those who have, or have had, COVID-19, based upon antigen-antibody based tests. The Warren lab is the world-leading lab on the generation, manipulation and biochemical characterisation of pdu based microcompartments whilst the Smales lab is a world-leading laboratory in the development of systems for the generation of complex glycoproteins in mammalian cells and their application to diagnostics development. Candidates will be involved in the cloning, expression and characterisation of the major protein components of the virus and various truncations and modifications thereof, and subsequent attachment of these to bacterial microcompartments such that these can be used to generate proof-of-concept data on the utility of this approach to elicit an appropriate immune response indiciative of good vaccine candidates. Additional Research costs: £1500

Engineering of Mammalian Cells for Enhanced Biotherapeutic Protein and Gene Therapy Production
Mammalian cells are a major expression system for the production of biotherapeutic recombinant proteins used to treat a range of diseases and for the generation of gene therapies. The Chinbese hamster ovary (CHO) cell is the current industrial system of choice for the expression of complex, post-translationally modified recombinant biopharmaceutical proteins for use in humans in the clinic to treat a variety of diseases. Current CHO host cells have been manipulated and ‘optimised’ for the commercial manufacture of monoclonal antibodies (mAbs) in particular, however many new protein biologics in development are difficult to express (DTE) in these systems. As such the product yields and quality are not sufficient to be used in the clinic and potentially life changing and saving drugs thus never reach the market. The programme of work described here will take a novel approach to solving this challenege and provide a fundamental understanding of the impact of metabolic engineering on the CHO cell factory and its ability to produce biotherapeutic proteins at high yield and quality, particularly those that are DTE in CHO cell or any other system.
Additional Research costs: £1500


Showing 50 of 97 total publications in the Kent Academic Repository. View all publications.


  • Roobol, A., Roobol, J., Smith, M., Carden, M., Hershey, J., Willis, A. and Smales, C. (2020). Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis. Metabolic Engineering [Online] 59:98-105. Available at:
    There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.
  • Vito, D., Eriksen, J., Skjødt, C., Weilguny, D., Rasmussen, S. and Smales, C. (2020). Defining lncRNAs Correlated with CHO Cell Growth and IgG Productivity by RNA-Seq. iScience [Online] 23. Available at:
    How the long non-coding RNA (lncRNA) genome in recombinant protein producing Chinese hamster ovary (CHO) cell lines relates to phenotype is not well described. We therefore defined the CHO cell lncRNA transcriptome from cells grown in controlled miniature bioreactors under fed-batch conditions using RNA-Seq to identify lncRNAs and how the expression of these changes throughout growth and between IgG producers. We identify lncRNAs including Adapt15, linked to ER stress, GAS5, linked to mTOR signaling/growth arrest, and PVT1, linked to Myc expression, which are differentially regulated during fed-batch culture and whose expression correlates to productivity and growth. Changes in (non)-coding RNA expression between the seed train and the equivalent day of fed-batch culture are also reported and compared with existing datasets. Collectively, we present a comprehensive lncRNA CHO cell profiling and identify targets for engineering growth and productivity characteristics of CHO cells.
  • Budge, J., Knight, T., Povey, J., Roobol, J., Brown, I., Singh, G., Dean, A., Turner, S., Jaques, C., Young, R., Racher, A. and Smales, C. (2020). Data for engineering lipid metabolism of Chinese hamster ovary (CHO) cells for enhanced recombinant protein production. Data in Brief [Online] 29:105217. Available at:
    The data presented in this article relates to the manuscript entitled ‘Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production’, published in the Journal Metabolic Engineering [1]. In the article here, we present data examining the overexpression of the lipid metabolism modifying genes SCD1 and SREBF1 in CHO cells by densitometry of western blots and by using mass spectrometry to investigate the impact on specific lipid species. We also present immunofluorescence data at the protein level upon SCD1 and SREBF1 overexpression. The growth profile data during batch culture of control CHO cells and CHO cells engineered to overexpress SCD1 and SREBF1 during batch culture are also reported. Finally, we report data on the yields of model secretory recombinant proteins produced from control, SCD1 or SREBF1 engineered cells using a transient expression systems.
  • Budge, J., Knight, T., Povey, J., Roobol, J., Brown, I., Singh, G., Dean, A., Turner, S., Jaques, C., Young, R., Racher, A. and Smales, C. (2019). Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production. Metabolic Engineering [Online] 57:203-216. Available at:
    Chinese hamster ovary (CHO) cell expression systems have been exquisitely developed for the production of recombinant biotherapeutics (e.g. standard monoclonal antibodies, mAbs) and are able to generate efficacious, multi-domain proteins with human-like post translational modifications at high concentration with appropriate product quality attributes. However, there remains a need for development of new CHO cell expression systems able to produce more challenging secretory recombinant biotherapeutics at higher yield with improved product quality attributes. Amazingly, the engineering of lipid metabolism to enhance such properties has not been investigated even though the biosynthesis of recombinant proteins is at least partially controlled by cellular processes that are highly dependent on lipid metabolism. Here we show that the global transcriptional activator of genes involved in lipid biosynthesis, sterol regulatory element binding factor 1 (SREBF1), and stearoyl CoA desaturase 1 (SCD1), an enzyme which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, can be overexpressed in CHO cells to different degrees. The amount of overexpression obtained of each of these lipid metabolism modifying (LMM) genes was related to the subsequent phenotypes observed. Expression of a number of model secretory biopharmaceuticals was enhanced between 1.5-9 fold in either SREBF1 or SCD1 engineered CHO host cells as assessed under batch and fed-batch culture. The SCD1 overexpressing polyclonal pool consistently showed increased concentration of a range of products. For the SREBF1 engineered cells, the level of SREBF1 expression that gave the greatest enhancement in yield was dependent upon the model protein tested. Overexpression of both SCD1 and SREBF1 modified the lipid profile of CHO cells and the cellular structure. Mechanistically, overexpression of SCD1 and SREBF1 resulted in an expanded endoplasmic reticulum (ER) that was dependent upon the level of LMM overexpression. We conclude that manipulation of lipid metabolism in CHO cells via genetic engineering is an exciting new approach to enhance the ability of CHO cells to produce a range of different types of secretory recombinant protein products via modulation of the cellular lipid profile and expansion of the ER.
  • Povey, J., Saintas, E., Aderemi, A., Rothweiler, F., Zehner, R., Dirks, W., Cinatl, J., Racher, A., Wass, M., Smales, C. and Michaelis, M. (2019). Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines. Cells [Online] 8:1194. Available at:
    The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.
  • Talbot, N., Mead, E., Davies, S., Uddin, S. and Smales, C. (2019). Application of ER Stress Biomarkers to Predict Formulated Monoclonal Antibody Stability. Biotechnology Journal [Online] 14:1900024. Available at:
    For a therapeutic mAb to reach the clinic, the molecule must be produced at an appropriate yield and quality, then formulated to maintain efficacy and stability. The formation of sub‐visible particles (SVPs) can impact on product stability and is monitored during formulation development however, the potential of a mAb to form such species can be influenced throughout the whole bioprocess. We investigate levels of intracellular ER stress perceived by cells, day of mAb harvest and the relationship to subsequent product stability of two mAbs (denoted A and B), produced in CHO cell lines, as determined by SVP content after accelerated stability studies. We show the propensity of mAb A to form SVPs can be predicted by transcript expression of biomarkers of cellular ER stress, heavy/light chain transcript and polypeptide amounts, and harvest day. Further, mAb A material harvested on day 9 of culture was more stable, in terms of SVP formation, than material harvested on day 13. These data suggest that ER stress perceived by CHO cells during culture can reflect the stability of a mAb, and that biomarkers of such stress could help define culture harvest time as a tool to control or reduce SVP formation in formulated mAbs.
  • Pekle, E., Smith, A., Rosignoli, G., Sellick, C., Smales, C. and Pearce, C. (2019). Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single Cell Level. Biotechnology Journal [Online]:1800675. Available at:
    Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the
    generation of high-producing clonal cell lines. During cell line development, cell cloning using
    fluorescence activated cell sorting (FACS) has the potential to combine isolation of single cells
    with sorting based on specific cellular attributes that correlate with productivity and/or growth,
    identifying cell lines with desirable phenotypes for manufacturing. This study describes the
    application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single
    cell level to identify cell attributes predictive of productivity. IFC assays to quantify organelle
    content, and recombinant heavy (HC) and light (LC) chain polypeptide and mRNA amounts in
    single cells were developed. The assays were then validated against orthogonal standard flow
    cytometry, western blot and qRT-PCR methods. We describe how these IFC assays may be
    used in cell line development and show how cellular properties can be correlated with
    productivity at the single cell level, allowing the isolation of such cells during the cloning
    process. Our analysis found HC polypeptide and mRNA to be predictive of productivity early
    in the culture, however specific organelle content did not show any correlation with
  • Jossé, L., Zhang, L. and Smales, C. (2018). Application of microRNA Targeted 3?UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools. Biotechnology Journal [Online] 13:1800129. Available at:
    The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3?UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near?complementary binding to the 3?UTR region of transcripts are harnessed. From the literature, the authors identified let?7f as a highly abundant, invariant miR in CHO cells. Three 3?UTR targets of the let?7f miR are then cloned in the DHFR host 3?UTR to determine the impact on gene expression (HMGA2 3?UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down?regulation of luciferase activity is mediated by the nature of the 3?UTR and its ability to bind let?7f. The same 3?UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44?derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody?producing cells with low MTX concentrations.
  • Tamošaitis, L. and Smales, M. (2018). Meta-analysis of publicly available Chinese hamster ovary (CHO) cell transcriptomic datasets for identifying engineering targets to enhance recombinant protein yields. Biotechnology Journal [Online] 13:1800066. Available at:
    Transcriptomics has been extensively applied to the investigation of the CHO cell platform for
    the production of recombinant biotherapeutic proteins to identify transcripts whose expression
    is regulated and correlated to (non)desirable CHO cell attributes. However, there have been
    few attempts to analyse the findings across these studies to identify conserved changes and
    generic targets for CHO cell platform engineering. Here we have undertaken a meta-analysis
    of CHO cell transcriptomic data and report on those genes most frequently identified as
    differentially expressed with regard to cell growth (?) and productivity (Qp). By aggregating
    differentially expressed genes from publicly available transcriptomic datasets associated with
    ? and Qp, using a pathway enrichment analysis and combining it with the concordance of
    gene expression values, we have identified a refined target gene and pathway list whilst
    determining the overlap across CHO transcriptomic studies. We find that only the cell cycle
    and lysosome pathways show good concordance. By mapping out the contributing genes we
    have constructed a transcriptomic ‘fingerprint’ of a high-performing cell line. This study
    provides a starting resource for researchers who want to navigate the complex landscape of
    CHO transcriptomics and identify targets to undertake cell engineering for improved
    recombinant protein output.
  • Vito, D. and Smales, C. (2018). The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions. Biotechnology Journal [Online] 13:1800122. Available at:
    The role of non?coding RNAs in determining growth, productivity and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non?coding RNAs have received less attention. One such class are the non?coding RNAs known collectively as long non?coding RNAs (lncRNAs). We have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. We report on those lncRNAs present in a model host CHO cell line under batch and fed?batch conditions on two different days and relate the expression of different lncRNAs to each other. We demonstrate that the mouse microarray was suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT?PCR. We then further analysed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed?batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. We discuss the implications for the publication of this rich dataset and how this may be used by the community.
  • Gourbatsi, E., Povey, J. and Smales, C. (2018). The effect of formulation variables on protein stability and integrity of a model IgG4 monoclonal antibody and translation to formulation of a model ScFv. Biotechnology Letters [Online] 40:33-46. Available at:
    Objectives: There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in ‘platform’ formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations.
    Results: We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett–Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules.
    Conclusions: The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules.
  • Godfrey, C., Mead, E., Daramola, O., Dunn, S., Hatton, D., Field, R., Pettman, G. and Smales, C. (2017). Polysome Profiling of mAb Producing CHO Cell Lines Links Translational Control of Cell Proliferation and Recombinant mRNA Loading onto Ribosomes with Global and Recombinant Protein Synthesis. Biotechnology Journal [Online] 12:1700177. Available at:
    mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5? untranslated regions that might be utilised to enhance recombinant transcript translation.
  • Almeida, A., Pinto, R., Smales, C. and Castilho, L. (2017). Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells. Biotechnology Letters [Online] 39:1109-1120. Available at:
    Objectives: To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4–8 °C.
    Results: NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance.
    Conclusions: Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, l-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.
  • Migani, D., Smales, C. and Bracewell, D. (2017). Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein (HCP) release in Chinese hamster ovary cells. Biotechnology Progress [Online] 33:666-676. Available at:
    Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145–166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase.
  • Bastide, A., Peretti, D., Knight, J., Grosso, S., Spriggs, R., Pichon, X., Sbarrato, T., Roobol, A., Roobol, J., Vito, D., Bushell, M., von der Haar, T., Smales, C., Mallucci, G. and Willis, A. (2017). RTN3 Is a Novel Cold-Induced Protein and Mediates Neuroprotective Effects of RBM3. Current Biology [Online] 27:638-650. Available at:
    Cooling and hypothermia are profoundly neuroprotective, mediated, at least in part, by the cold shock protein, RBM3. However, the neuroprotective effector proteins induced by RBM3 and the mechanisms by which mRNAs encoding cold shock proteins escape cooling-induced translational repression are unknown. Here, we show that cooling induces reprogramming of the translatome, including the upregulation of a new cold shock protein, RTN3, a reticulon protein implicated in synapse formation. We report that this has two mechanistic components. Thus, RTN3 both evades cooling-induced translational elongation repression and is also bound by RBM3, which drives the increased expression of RTN3. In mice, knockdown of RTN3 expression eliminated cooling-induced neuroprotection. However, lentivirally mediated RTN3 overexpression prevented synaptic loss and cognitive deficits in a mouse model of neurodegeneration, downstream and independently of RBM3. We conclude that RTN3 expression is a mediator of RBM3-induced neuroprotection, controlled by novel mechanisms of escape from translational inhibition on cooling.
  • Saintas, E., Abrahams, L., Ahmed, G., Ajakaiye, A., AlHumaidi, A., Ashmore-Harris, C., Clark, I., Dura, U., Fixmer, C., Ike-Morris, C., Mato Prado, M., Mccullough, D., Mishra, S., Schoeler, K., Timur, H., Williamson, M., Alatsatianos, M., Bahsoun, B., Blackburn, E., Hogwood, C., Lithgow, P., Rowe, M., Yiangou, L., Rothweiler, F., Cinatl jr, J., Zehner, R., Baines, A., Garrett, M., Gourlay, C., Griffin, D., Gullick, W., Hargreaves, E., Howard, M., Lloyd, D., Rossman, J., Smales, C., Tsaousis, A., von der Haar, T., Wass, M. and Michaelis, M. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at:
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Feary, M., Racher, A., Young, R. and Smales, C. (2016). Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis. Biotechnology Progress [Online] 33:17-25. Available at:
    In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines.
  • Jossé, L., Xie, J., Proud, C. and Smales, C. (2016). mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells. Biochemical Journal [Online] 473:4651-4664. Available at:
    Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.
  • Williams, M., Sánchez, E., Aluri, E., Douglas, F., MacLaren, D., Collins, O., Cussen, E., Budge, J., Sanders, L., Michaelis, M., Smales, C., Cinatl, J., Lorrio, S., Krueger, D., de Rosales, R. and Corr, S. (2016). Microwave-assisted synthesis of highly crystalline, multifunctional iron oxide nanocomposites for imaging applications. RSC Advances [Online] 6:83520-83528. Available at:
    We report a reproducible single-step, microwave-assisted approach for the preparation of multifunctional magnetic nanocomposites comprising superparamagnetic iron oxide (Fe3O4) cores, a polyelectrolyte stabilizer and an organic dye with no requirement for post-processing. The stabilisers poly(sodium 4-styrenesulfonate) (PSSS) and sodium polyphosphate (SPP) have been thoroughly investigated and from analysis using electron microscopy, dynamic light scattering measurements, magnetic hysteresis and magnetic resonance (MR) imaging, we show that the higher degree of Fe3O4 nanoparticle crystallinity achieved with the PSSS stabiliser leads to enhanced magnetic behaviour and thus better contrast agent relaxivity compared to the less crystalline, poorly defined particles obtained when SPP is employed as a stabiliser. We also demonstrate the potential for obtaining a multifunctional magnetic-fluorescent nanocomposite using our microwave-assisted synthesis. In this manner, we demonstrate the intimate link between synthetic methodology (microwave heating with a polyelectrolyte stabilizer) and the resulting properties (particle size, shape, and magnetism) and how this underpins the functionality of the resulting nanocomposites as agents for biomedical imaging.
  • Lintern, K., Pathak, M., Smales, C., Howland, K., Rathore, A. and Bracewell, D. (2016). Residual on column host cell protein analysis during lifetime studies of protein A chromatography. Journal of Chromatography A [Online] 1461:70-77. Available at:
    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC–MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC–MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.
  • Chiverton, L., Evans, C., Pandhal, J., Landels, A., Rees, B., Levison, P., Wright, P. and Smales, C. (2016). Quantitative definition and monitoring of the host cell protein proteome using iTRAQ: a study of an industrial mAb producing CHO-S cell line. Biotechnology Journal [Online] 11:1014-1024. Available at:
    There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.
  • Vasilev, N., Smales, C., Schillberg, S., Fischer, R. and Schiermeyer, A. (2016). Developments in the production of mucosal antibodies in plants. Biotechnology Advances [Online] 34:77-87. Available at:
    Recombinant mucosal antibodies represent attractive target molecules for the development of next generation biopharmaceuticals for passive immunization against various infectious diseases and treatment of patients suffering from mucosal antibody deficiencies. As these polymeric antibodies require complex post-translational modifications and correct subunit assembly, they are considered as difficult-to-produce recombinant proteins. Beside the traditional, mammalian-based production platforms, plants are emerging as alternative expression hosts for this type of complex macromolecule. Plant cells are able to produce high-quality mucosal antibodies as shownby the successful expression of the secretory immunoglobulins A (IgA) andM(IgM) in various antibody formats in different plant species including tobacco and its close relative Nicotiana benthamiana, maize, tomato
    and Arabidopsis thaliana. Importantly for biotherapeutic application, transgenic plants are capable of synthesizing functional IgA and IgM molecules with biological activity and safety profiles comparable with their native mammalian counterparts. This article reviews the structure and function of mucosal IgA and IgM antibodies and summarizes
    the current knowledge of their production and processing in plant host systems. Specific emphasis is given to consideration of intracellular transport processes as these affect assembly of the mature immunoglobulins, their secretion rates, proteolysis/degradation and glycosylation patterns. Furthermore, this review provides an outline of glycoengineering efforts that have been undertaken so far to produce antibodies with homogenous human-like glycan decoration.Webelieve that the continued development of our understanding of the plant cellular machinery related to the heterologous expression of immunoglobulins will further improve the production levels, quality and control of post-translational modifications that are ‘human-like’ from plant systems and enhance the prospects for the regulatory approval of such molecules leading to the commercial exploitation of plant-derived mucosal antibodies.
  • Knight, J., Bastide, A., Peretti, D., Roobol, A., Roobol, J., Mallucci, G., Smales, C. and Willis, A. (2016). Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis. RNA [Online] 22:623-635. Available at:
    The RNA exosome is essential for 3? processing of functional RNA species and degradation of aberrant RNAs in eukaryotic cells. Recent reports have defined the substrates of the exosome catalytic domains and solved the multimeric structure of the exosome complex. However, regulation of exosome activity remains poorly characterized, especially in response to physiological stress. Following the observation that cooling of mammalian cells results in a reduction in 40S:60S ribosomal subunit ratio, we uncover regulation of the nuclear exosome as a result of reduced temperature. Using human cells and an in vivo model system allowing whole-body cooling, we observe reduced EXOSC10 (hRrp6, Pm/Scl-100) expression in the cold. In parallel, both models of cooling increase global SUMOylation, leading to the identification of specific conjugation of SUMO1 to EXOSC10, a process that is increased by cooling. Furthermore, we define the major SUMOylation sites in EXOSC10 by mutagenesis and show that overexpression of SUMO1 alone is sufficient to suppress EXOSC10 abundance. Reducing EXOSC10 expression by RNAi in human cells correlates with the 3? preribosomal RNA processing defects seen in the cold as well as reducing the 40S:60S ratio, a previously uncharacterized consequence of EXOSC10 suppression. Together, this work illustrates that EXOSC10 can be modified by SUMOylation and identifies a physiological stress where this regulation is prevalent both in vitro and in vivo.
  • Gourbatsi, E., Povey, J., Uddin, S. and Smales, C. (2016). Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system. Biotechnology Letters [Online] 38:589-596. Available at:
    The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic.
    Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme.
    Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.
  • Mead, E., Masterton, R., Feary, M., Obrezanova, O., Zhang, L., Young, R. and Smales, C. (2015). Biological Insights into the Expression of Translation Initiation Factors from Recombinant CHOK1SV Cell Lines and their Relationship to Enhanced Productivity. Biochemical Journal [Online] 472:261-273. Available at:
    Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAb) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop; eukaryotic initiation factors (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A) binding protein (PABP) 1 and PABP interacting protein 1 (PAIP1) across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilise knowledge of the amounts of these factors to build predictive models for, and use cluster analysis to identify, high-producing cell lines. This study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity.
  • Bracewell, D., Francis, R. and Smales, C. (2015). The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control. Biotechnology and Bioengineering [Online] 112:1727-1737. Available at:
    The use of biological systems to synthesize complex therapeutic products has been a remarkable success. However, during product development, great attention must be devoted to defining acceptable levels of impurities that derive from that biological system, heading this list are host cell proteins (HCPs). Recent advances in proteomic analytics have shown how diverse this class of impurities is; as such knowledge and capability grows inevitable questions have arisen about how thorough current approaches to measuring HCPs are. The fundamental issue is how to adequately measure (and in turn monitor and control) such a large number of protein species (potentially thousands of components) to ensure safe and efficacious products. A rather elegant solution is to use an immunoassay (enzyme-linked immunosorbent assay [ELISA]) based on polyclonal antibodies raised to the host cell (biological system) used to synthesize a particular therapeutic product. However, the measurement is entirely dependent on the antibody serum used, which dictates the sensitivity of the assay and the degree of coverage of the HCP spectrum. It provides one summed analog value for HCP amount; a positive if all HCP components can be considered equal, a negative in the more likely event one associates greater risk with certain components of the HCP proteome. In a thorough risk-based approach, one would wish to be able to account for this. These issues have led to the investigation of orthogonal analytical methods; most prominently mass spectrometry. These techniques can potentially both identify and quantify HCPs. The ability to measure and monitor thousands of proteins proportionally increases the amount of data acquired. Significant benefits exist if the information can be used to determine critical HCPs and thereby create an improved basis for risk management. We describe a nascent approach to risk assessment of HCPs based upon such data, drawing attention to timeliness in relation to biosimilar initiatives. The development of such an approach requires databases based on cumulative knowledge of multiple risk factors that would require national and international regulators, standards authorities (e.g., NIST and NIBSC), industry and academia to all be involved in shaping what is the best approach to the adoption of the latest bioanalytical technology to this area, which is vital to delivering safe efficacious biological medicines of all types.
  • Knight, J., Bastide, A., Roobol, A., Roobol, J., Jackson, T., Utami, W., Barrett, D., Smales, C. and Willis, A. (2015). Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation. Biochemical Journal [Online] 465:227-238. Available at:
  • Roobol, A., Roobol, J., Bastide, A., Knight, J., Willis, A. and Smales, C. (2015). p58(IPK) is an Inhibitor of the eIF2? Kinase GCN2 and its Localisation and Expression Underpin Protein Synthesis and ER Processing Capacity. Biochemical Journal [Online] 465:213-225. Available at:
  • Tunjung, W., Cinatl, J., Michaelis, M. and Smales, C. (2015). Anti-Cancer Effect of Kaffir Lime (Citrus Hystrix DC) Leaf Extract in Cervical Cancer and Neuroblastoma Cell Lines. Procedia Chemistry [Online] 14:465-468. Available at:
    Previous study showed that kaffir lime leaf contains alkaloid, flavonoid, terpenoid, tannin and saponin. The objective of this study was to examine the cytotoxic effect of kaffir lime leaf extract on cervical cancer and neuroblastoma cell lines. The method used for this research to determine cell viability was an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed that an ethyl acetate extract had an IC50 for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental cells of 40.7 ?g · mL–1, 28.4 ?g · mL–1, 14.1 ?g · mL–1, and 25.2 ?g · mL–1 respectively. Furthermore, the IC50 of chloroform extracts for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental were 17.6 ?g · mL–1, 18.9 ?g · mL–1, 6.4 ?g · mL–1, and 9.4 ?g · mL–1 respectively. These data showed that kaffir lime extract reduces the viability of cervical and neuroblastoma cell lines and may have potential as anti-cancer compounds.
  • Rooney, B., Piening, T., Büscher, P., Rogé, S. and Smales, C. (2015). Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs). PLOS Neglected Tropical Diseases [Online] 9:e0004271. Available at:
    The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials.
  • An ultra scale-down approach identifies host cell protein differences across a panel of mAb producing CHO cell line variants (2015). Biotechnology Journal [Online] 11:415-424. Available at:
    During the manufacture of biopharmaceutical products, the final product must lie within strict pre-set specifications, for example the host cell protein (HCP) content. A number of specific HCPs have been identified in particular products and the interactions between product/HCPs have also been recently investigated; however, a comparison of the HCP dynamics between related cell lines and their response to early downstream processing to aid process development and cell line selection has not been published. We have utilised a proteomic approach coupled with an ultra scale-down study to determine the HCP profile dynamics, at harvest and during early downstream processing, across a panel of recombinant GS-CHOK1SV antibody producing cell lines. The results reveal that cell culture viability upon harvest has the greatest impact upon shear sensitivity and HCP concentration. Whilst the general HCP population/profile was broadly similar across the cell lines, the actual amounts of some specific HCPs in the supernatant differed and a number of cell line specific differences in the response to early downstream processing were observed. We anticipate that such knowledge can now be applied to cell line selection and downstream processing development to target reduction/removal of general and specific problematic HCPs before and during downstream processing.
  • Povey, J., O’Malley, C., Root, T., Martin, E., Montague, G., Feary, M., Trim, C., Lang, D., Alldread, R., Racher, A. and Smales, C. (2014). Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling. Journal of Biotechnology [Online] 184:84-93. Available at:
    Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10 L bioreactor model of Lonza's large-scale (up to 20,000 L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10 L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10 L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines’ performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.
  • Mead, E., Masterton, R., von der Haar, T., Tuite, M. and Smales, C. (2014). Control and regulation of mRNA translation. Biochemical Society transactions [Online] 42:151-154. Available at:
    Translational control is central to the gene expression pathway and was the focus of the 2013 annual Translation UK meeting held at the University of Kent. The meeting brought together scientists at all career stages to present and discuss research in the mRNA translation field, with an emphasis on the presentations on the research of early career scientists. The diverse nature of this field was represented by the broad range of papers presented at the meeting. The complexity of mRNA translation and its control is emphasized by the interdisciplinary research approaches required to address this area with speakers highlighting emerging systems biology techniques and their application to understanding mRNA translation and the network of pathways controlling it.
  • Roobol, A., Roobol, J., Carden, M., Smith, M., Hershey, J., Bastide, A., Knight, J., Willis, A. and Smales, C. (2014). The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h. Biochemical Journal [Online] 458:213-224. Available at:
    eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.
  • Hogwood, C., Bracewell, D. and Smales, C. (2014). Measurement and control of host cell proteins (HCPs) in CHO cell bioprocesses. Current Opinion in Biotechnology [Online] 30:153-160. Available at:
    Chinese hamster ovary (CHO) cells are widely used for the production of biotherapeutic recombinant proteins for a range of molecules including monoclonal antibodies and Fc-fusion proteins. Regulatory requirements for the final product include the removal of host cell proteins (HCPs) to acceptable amounts (<100 ppm). Recent research has begun to unravel the extent to which upstream process conditions and subsequent product recovery and purification processes impact upon the HCP profile. A number of upstream parameters, including the selection of the cell line, the culturing process (e.g. feeding regime, culture temperature), cell viability at time of harvest/culture duration and cell shear sensitivity can all influence the resulting HCP profile. Further, the molecule itself plays an important role in determining those HCPs that are retained throughout a bioprocess and HCPs can co-elute with the target product during purification. Measurement and monitoring of HCPs is usually undertaken using ELISA technology, however alternative approaches are also now emerging that complement ELISA and allow the detection, identification and monitoring of specific HCPs. Here we discuss our understanding of how the process itself influences those HCPs present throughout the production process and the challenges in their monitoring, measurement and removal.
  • Masterton, R. and Smales, C. (2014). The impact of process temperature on mammalian cell lines and the implications for the production of recombinant proteins in CHO cells. Pharmaceutical Bioprocessing [Online] 2:49-61. Available at:
  • Ashton, L., Hogwood, C., Tait, A., Kuligowski, J., Smales, C., Bracewell, D., Dickson, A. and Goodacre, R. (2014). UV resonance Raman spectroscopy: a process analytical tool for host cell DNA and RNA dynamics in mammalian cell lines. Journal of Chemical Technology and Biotechnology [Online] 90:237-243. Available at:
  • Al-Fageeh, M. and Smales, C. (2013). Alternative Promoters Regulate Cold Inducible RNA-Binding (CIRP) Gene Expression and Enhance Transgene Expression in Mammalian Cells. Molecular Biotechnology [Online] 54:238-249. Available at:
    The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding (CIRP) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.
  • Hogwood, C., Bracewell, D. and Smales, C. (2013). Host cell protein dynamics in recombinant CHO cells. Bioengineered [Online] 4:288-291. Available at:
    During the production of recombinant protein products, such as monoclonal antibodies, manufacturers must demonstrate clearance of host cell impurities and contaminants to appropriate levels prior to use in the clinic. These include host cell DNA and RNA, product related contaminants such as aggregates, and importantly host cell proteins (HCPs). Despite the importance of HCP removal, the identity and dynamics of these proteins during cell culture and downstream processing (DSP) are largely unknown. Improvements in technologies such as SELDI-TOF mass spectrometry alongside the gold standard technique of ELISA has allowed semi-quantification of the total HCPs present. However, only recently have techniques been utilized in order to identify those HCPs present and align this with the development of approaches to monitor the dynamics of HCPs during both fermentation and downstream processing. In order to enable knowledge based decisions with regards to improving HCP clearance it is vital to identify potential problematic HCPs on a cell line and product specific basis. Understanding the HCP dynamics will in the future help provide a platform to rationally manipulate and engineer and/or select suitable recombinant CHO cell lines and downstream processing steps to limit problematic HCPs.
  • Hogwood, C., Tait, A., Koloteva-Levine, N., Bracewell, D. and Smales, C. (2013). The dynamics of the CHO host cell protein profile during clarification and protein A capture in a platform antibody purification process. Biotechnology and Bioengineering [Online] 110:240-251. Available at:
    Recombinant protein products such as monoclonal antibodies (mAbs) for use in the clinic must be clear of host cell impurities such as host cell protein (HCP), DNA/RNA, and high molecular weight immunogenic aggregates. Despite the need to remove and monitor HCPs, the nature, and fate of these during downstream processing (DSP) remains poorly characterized. We have applied a proteomic approach to investigate the dynamics and fate of HCPs in the supernatant of a mAb producing cell line during early DSP including centrifugation, depth filtration, and protein A capture chromatography. The primary clarification technique selected was shown to influence the HCP profile that entered subsequent downstream steps. MabSelect protein A chromatography removed the majority of contaminating proteins, however using 2D-PAGE we could visualize not only the antibody species in the eluate (heavy and light chain) but also contaminant HCPs. These data showed that the choice of secondary clarification impacts upon the HCP profile post-protein A chromatography as differences arose in both the presence and abundance of specific HCPs when depth filters were compared. A number of intracellularly located HCPs were identified in protein A elution fractions from a Null cell line culture supernatant including the chaperone Bip/GRP78, heat shock proteins, and the enzyme enolase. We demonstrate that the selection of early DSP steps influences the resulting HCP profile and that 2D-PAGE can be used for monitoring and identification of HCPs post-protein A chromatography. This approach could be used to screen cell lines or hosts to select those with reduced HCP profiles, or to identify HCPs that are problematic and difficult to remove so that cell-engineering approaches can be applied to reduced, or eliminate, such HCPs. Biotechnol. Bioeng. 2013; 110: 240–251.
  • Bracewell, D. and Smales, C. (2013). The challenges of product- and process-related impurities to an evolving biopharmaceutical industry. Bioanalysis [Online] 5:123-126. Available at:
  • Wagstaff, J., Masterton, R., Povey, J., Smales, C. and Howard, M. (2013). 1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture. PLoS ONE 8:e77195-e77195.
    We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon
    a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of
    CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic
    culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems
    including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero.
    We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and
    alanine with both cell lines responding in a similar manner and comparable to previously reported data. However,
    analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in
    response to temperature was cell line specific. The significance of our results is presented using principal component
    analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our
    approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism
    upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of
    metabolites in culture supernatant to improve growth or productivity.
  • Peters, S., Smales, C., Henry, A., Stephens, P., West, S. and Humphreys, D. (2012). Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability. Journal of Biological Chemistry [Online] 287:24525-24533. Available at:
    The integrity of antibody structure, stability, and biophysical characterization are becoming increasingly important as antibodies receive increasing scrutiny from regulatory authorities. We altered the disulfide bond arrangement of an IgG4 molecule by mutation of the Cys at the N terminus of the heavy chain constant domain 1 (CH1) (Kabat position 127) to a Ser and introduction of a Cys at a variety of positions (positions 227–230) at the C terminus of CH1. An inter-LC-CH1 disulfide bond is thus formed, which mimics the disulfide bond arrangement found in an IgG1 molecule. The antibody species present in the supernatant following transient expression in Chinese hamster ovary cells were analyzed by immunoblot to investigate product homogeneity, and purified product was analyzed by a thermofluor assay to determine thermal stability. We show that the light chain can form an inter-LC-CH1 disulfide bond with a Cys when present at several positions on the upper hinge (positions 227–230) and that such engineered disulfide bonds can consequently increase the Fab domain thermal stability between 3 and 6.8 °C. The IgG4 disulfide mutants displaying the greatest increase in Fab thermal stability were also the most homogeneous in terms of disulfide bond arrangement and antibody species present. Importantly, mutations did not affect the affinity for antigen of the resultant molecules. In combination with the previously described S241P mutation, we present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules.
  • Mead, E., Chiverton, L., Spurgeon, S., Martin, E., Montague, G., Smales, C. and von der Haar, T. (2012). Experimental and In Silico Modelling Analyses of the Gene Expression Pathway for Recombinant Antibody and By-Product Production in NS0 Cell Lines. PLoS ONE [Online] 7:e47422. Available at:
    Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.
  • Tait, A., Hogwood, C., Smales, C. and Bracewell, D. (2011). Host cell protein dynamics in the supernatant of a mAb producing CHO cell line. Biotechnology and Bioengineering [Online] 109:971-982. Available at:
    The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.
  • Kotov, N., Bates, D., Gizatullina, A., Gilaziev, B., Khairullin, R., Chen, M., Drozdov, I., Umezawa, Y., Hundhausen, C., Aleksandrov, A., Yan, X., Spurgeon, S., Smales, C. and Valeyev, N. (2011). Computational modelling elucidates the mechanism of ciliary regulation in health and disease. BMC Systems Biology [Online] 5:143. Available at:
    Ciliary dysfunction leads to a number of human pathologies, including primary ciliary dyskinesia, nephronophthisis, situs inversus pathology or infertility. The mechanism of cilia beating regulation is complex and despite extensive experimental characterization remains poorly understood. We develop a detailed systems model for calcium, membrane potential and cyclic nucleotide-dependent ciliary motility regulation.

    The model describes the intimate relationship between calcium and potassium ionic concentrations inside and outside of cilia with membrane voltage and, for the first time, describes a novel type of ciliary excitability which plays the major role in ciliary movement regulation. Our model describes a mechanism that allows ciliary excitation to be robust over a wide physiological range of extracellular ionic concentrations. The model predicts the existence of several dynamic modes of ciliary regulation, such as the generation of intraciliary Ca2+ spike with amplitude proportional to the degree of membrane depolarization, the ability to maintain stable oscillations, monostable multivibrator regimes, all of which are initiated by variability in ionic concentrations that translate into altered membrane voltage.

    Computational investigation of the model offers several new insights into the underlying molecular mechanisms of ciliary pathologies. According to our analysis, the reported dynamic regulatory modes can be a physiological reaction to alterations in the extracellular environment. However, modification of the dynamic modes, as a result of genetic mutations or environmental conditions, can cause a life threatening pathology.
  • Roobol, A., Roobol, J., Carden, M., Bastide, A., Willis, A., Dunn, W., Goodacre, R. and Smales, C. (2011). ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53. Biochemical Journal [Online] 435:499-508. Available at:
    In vitro cultured mammalian cells respond to mild hypothermia (27–33 °C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31–33 °C) and its complete arrest at the lower range (27–28 °C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. In the present paper, we report that the exposure of Chinese-hamster ovary-K1 cells to mildly hypothermic conditions activates the ATR (ataxia telangiectasia mutated- and Rad3-related kinase)–p53–p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38MAPK (p38 mitogen-activated protein kinase) is also involved in activation of p53 upon mild hypothermia, this is probably the result of activation of p38MAPK by ATR. Furthermore, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR–p53–p21 pathway. Therefore we provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation, which is known to lead to cell cycle arrest.

Book section

  • Jossé, L., Smales, C. and Tuite, M. (2012). Engineering the Chaperone Network of CHO Cells for Optimal Recombinant Protein Production and Authenticity. In: Recombinant Gene Expression. Springer New York, pp. 595-608. Available at:
    All proteins fold into a defined three-dimensional shape that is compatible with the cellular role and/or biological activity of those proteins. Molecular chaperones are a family of proteins whose role is to assist the folding and targeting of proteins in both normal and stressed cells. The rational manipulation of chaperone levels in a cell line engineered to produce a defined recombinant protein (rP) can significantly improve both the achievable steady-state levels and authenticity of a wide range of recombinant proteins. Here, we describe the methodology associated with expressing a variety of molecular chaperones in Chinese hamster ovary (CHO) lines in order to improve their recombinant protein production capacity. These chaperones include both those that facilitate the folding of the polypeptide chain (i.e. Hsp70, Hsp40) and those that can re-fold proteins that have misfolded in the cell (i.e. ClpB/Hsp104). This latter property is particularly important given the propensity for highly expressed recombinant proteins to misfold in the “foreign” cellular environment.


  • Pekle, E. (2019). Chinese Hamster Ovary Cell Line Development via Imaging Flow Cytometry and Directed Evolution.


  • AJAYI, F. (2019). Reprogramming of Sterol Biosynthesis in Chinese Hamster Ovary Cells for Enhanced Recombinant Protein Production.
    The biopharmaceutical industry is a multi-billion dollar global market with projections that within the next 5 to 10 years, up to 50% of all drugs in development will be biopharmaceuticals. The majority of these drugs are recombinantly produced protein based therapeutics with the demand for such new therapeutic products continuing to increase, broadening the range of medical conditions that they are being used to treat. Chinese hamster ovary (CHO) cells are the preferred and most commonly
    used system for large scale production of recombinant glycoproteins for therapeutic use due to their ability to perform complex human-like post-translational modifications required for biological activity. CHO cells also display robust growth, high productivity and stability, attributes that have enabled them to become widely used. Whilst there have been large increases in the yield and quality of recombinant protein that can be obtained from mammalian expression systems over the years, particularly CHO cells, many recombinant proteins remain difficult to express in CHO cells or any other system. Many of these proteins are highly complex, highly glycosylated, large and unstable proteins. As such, there remains a need to develop CHO cell systems that are able to produce such difficult to express proteins rapidly in higher yield and quality. The endoplasmic reticulum (ER) is a key compartment in the secretory pathway of mammalian cells and during the production of recombinant proteins in CHO cells this has reportedly been a potential bottleneck and site of
    perceived cellular stress as a result of the load imposed on the cell by the recombinant protein.
    This study has investigated two sterol reductases; Transmembrane 7 Superfamily member 2 (TM7SF2) and 7-dehydrocholesterol reductase (DHCR7). ER resident proteins involved in the biosynthesis of cholesterol that have been reported to influence endoplasmic reticulum (ER) and
    nuclear envelope (NE) expansion when over-expressed in mammalian cell lines. The work described here therefore set out to determine whether overexpression of these sterol reductases could enhance the secretory capacity of CHO cells by expansion of the ER and hence lead to increased yields of difficult to express recombinant proteins compared to those cells where these were not overexpressed. The TM7SF2 and DHCR7 genes were cloned into a mammalian expression vector with a hygromycin selection marker to allow for the selection of stable cell lines where the genes had been integrated into the genome. Stable CHO cells overexpressing TM7SF2 and DHCR7 were generated to
    assess the impact on cell growth and stable and transient recombinant protein expression, monitored using western blot techniques and microscopy analysis. Control cell lines over-expressing GFP (but not the sterol reductases) and the pcDNA3.1Hygro (empty) vector were also developed. Stable
    expression levels of the sterol reductases were determined in heterogeneous pools and then used for limited dilution cloning to isolate clonal cell lines with differing sterol reductase expression which were further investigated. Growth profile assays were undertaken to monitor any toxicity effect of over-expression of TM7SF2 and DHCR7 on CHO cells. Transient expression studies of the recombinant bio therapeutic protein etanercept, a 150 kDa biomolecule and erythropoietin (EPO), a 36 kDa recombinant protein, were carried out to investigate the production capacity of lipid engineered CHO cells with reference to controls. Immunocytolocalisation studies were also undertaken to investigate pools and clones of lipid targets predicted to be ER-localized or associated. A YFP tag was present on the N-terminal of TM7SF2 and DHCR7 molecules and was used to monitor expression by fluorescence in stable CHO cells.
    The data from these experiments show that both TM7SF2 and DHCR7 engineered CHO cells had improved cell growth and culture viability over the course of 10-day experimental study compared to the control cells. Western blot studies showed that both TM7SF2 and DHCR7 could be stably over-expressed in CHO cells and the amount of expression varied among the generated clones. Further, the high expressers of TM7SF2 and DHCR7 were identified and investigated for EPO and etanercept production and expression levels of these recombinant biomolecules were observed to be proportional to the levels of TM7SF2 and DHCR7 expressions in CHO cells. More importantly, TM7SF2 and DHCR7 engineered cells showed increased expression levels of these recombinant products when compared with pcDNA3.1Hygro controls as determined by western blot analysis of the amount
    of secreted recombinant target protein in the cell culture supernatant. Confocal images also showed clones expressed different levels of YFP signal which related to expression levels observed when western blot techniques were used. In summary, the data presented here shows that the manipulation of cellular circuits such as cholesterol biosynthesis has the potential to enhance cellular growth and recombinant protein yield. By designing new hosts and cellular circuits to reprogramme
    the CHO cell ER there is the potential of expanding the secretory capacity and/or subsequent secretory vesicle system.
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