Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles.

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.

Application of ER Stress Biomarkers to Predict Formulated Monoclonal Antibody Stability.

For a therapeutic monoclonal antibody (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 subvisible particles (SVPs) can impact 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. The levels of intracellular endoplasmic reticulum (ER) stress perceived by Chinese hamster ovary (CHO) cell lines, the day of mAb harvest, and the relationship with subsequent product stability of two mAbs (denoted A and B), as determined by the SVP content after accelerated stability studies, are reported here. Here, it is shown that 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 can reflect the stability of a mAb, and that biomarkers of such stress could help define culture harvest time as a tool to control SVP formation in formulated mAbs.

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.

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.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) 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, eIFs (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 utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present 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.

Control and regulation of mRNA translation.

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.

Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

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.

Inotropic action of the puberty hormone kisspeptin in rat, mouse and human: cardiovascular distribution and characteristics of the kisspeptin receptor.

Kisspeptins, the ligands of the kisspeptin receptor known for its roles in reproduction and cancer, are also vasoconstrictor peptides in atherosclerosis-prone human aorta and coronary artery. The aim of this study was to further investigate the cardiovascular localisation and function of the kisspeptins and their receptor in human compared to rat and mouse heart. Immunohistochemistry and radioligand binding techniques were employed to investigate kisspeptin receptor localisation, density and pharmacological characteristics in cardiac tissues from all three species. Radioimmunoassay was used to detect kisspeptin peptide levels in human normal heart and to identify any pathological changes in myocardium from patients transplanted for cardiomyopathy or ischaemic heart disease. The cardiac function of kisspeptin receptor was studied in isolated human, rat and mouse paced atria, with a role for the receptor confirmed using mice with targeted disruption of Kiss1r. The data demonstrated that kisspeptin receptor-like immunoreactivity localised to endothelial and smooth muscle cells of intramyocardial blood vessels and to myocytes in human and rodent tissue. [(125)I]KP-14 bound saturably, with subnanomolar affinity to human and rodent myocardium (K(D) = 0.12 nM, human; K(D) = 0.44 nM, rat). Positive inotropic effects of kisspeptin were observed in rat, human and mouse. No response was observed in mice with targeted disruption of Kiss1r. In human heart a decrease in cardiac kisspeptin level was detected in ischaemic heart disease. Kisspeptin and its receptor are expressed in the human, rat and mouse heart and kisspeptins possess potent positive inotropic activity. The cardiovascular actions of the kisspeptins may contribute to the role of these peptides in pregnancy but the consequences of receptor activation must be considered if kisspeptin receptor agonists are developed for use in the treatment of reproductive disorders or cancer.

Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates.

Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed.

Kisspeptins are novel potent vasoconstrictors in humans, with a discrete localization of their receptor, G protein-coupled receptor 54, to atherosclerosis-prone vessels.

The G protein-coupled receptor GPR54 (also designated KISS1) is activated by cleavage products of the KiSS1 protein, the kisspeptins (KP), to act as a molecular switch for puberty. Additionally, KP are potent inhibitors of tumor metastasis and play a role in placentation, both processes involving angiogenesis. Our aim was to investigate whether GPR54 and KP are expressed within normal and diseased human vasculature and what their functional role may be. RT-PCR screening of human blood vessels revealed a discrete localization of GPR54 mRNA in smooth muscle of vessels with the same developmental origins, aorta, coronary artery, and umbilical vein, a pattern confirmed by immunocytochemistry and radioligand binding. Novel ligand [(125)I]KP-13 exhibited saturable and high-affinity binding in aorta smooth muscle sections (dissociation constant K(D) = 0.2 +/- 0.03 nM), and using confocal microscopy, we found colocalization of receptor and peptide to vascular endothelial cells and to the atherosclerotic plaque of coronary artery. RIA detected 13.04 +/- 2.94 and 20.50 +/- 5.00 fmol/g KP in human coronary artery and aorta, respectively. KP-10, KP-13, and KP-54 acted as vasoconstrictors with comparable potency and efficacy in isolated rings of coronary artery (negative logarithm of the EC(50) and maximal response, respectively, as follows: KP-10, 7.89 +/- 0.24 and 33.7 +/- 17.0; KP-13, 8.66 +/- 0.88 and 35.1 +/- 7.9; KP-54, 8.86 +/- 1.11 and 25.7 +/- 5.5) and umbilical vein (negative logarithm of the EC(50) and maximal response, respectively, as follows: KP-10, 8.44 +/- 022 and 24.3 +/- 3.7; KP-13, 8.43 +/- 0.88 and 28.4 +/- 8.6; KP-54, 8.93 +/- 0.39 and 36.9 +/- 5.2). In conclusion, we have detected expression of both peptide and receptor in aorta, coronary artery, and umbilical vein and have shown for the first time that the KP are vasoconstrictors in humans, suggesting a previously undescribed role for GPR54 and KP in the cardiovascular system.