An investigation into the role of chromatin modifying elements on the production of recombinant antibodies from CHO Cells

Stable cell line generation requires the gene of interest to become stably integrated into the host cells genome. The generation of stable cell lines by random integration produces large variation in clonal expression and stability that is due, in part, to the nature of the chromatin it has integrated into. Elements have been identified that can modify the chromatin structure and have been shown to protect transgcne expression from the effects of the neighbouring chromatin environment. The aim of the study was to investigate whether the identified chromatin structure modifying elements (UCOE, MAR, STAR and HS4) could increase the level and stability of antibody expression in stable cell lines in the mammalian expression systems used at UCB. A series of expression constructs were generated to assess the effect of the chromatin structure modifying elements on antibody expression in stable cell lines. Initially seven different vectors for each clement that possessed different combinations of the individual elements were assessed as pooled and clonal stable cell populations which indicated that the elements increased antibody expression by varying amounts and optimal vector configurations were different for the elements. The optimised vectors were subsequently used in a comparative side-by-side study with expression levels from pooled and stable cell lines being analysed. A subset of clones harbouring each of the five test constructs (Ab535 control, 1.5kb A2UCOE, MAR X_S29, STAR 7 and cHS4 tandem) derived from the pooled stable cell lines were genetically characterised to determine copy number and clonal diversity. Clones were cultured long-term in the absence of selection pressure which resulted in a decrease in expression being observed in all of the clones. The decline in antibody expression was accompanied by a decrease in mRNA levels which was not caused by a loss of either HC or LC gene copies from the genome. Analysis of DNA methylation patterns across the hCMV-MIE promoter regions demonstrated that this epigenetic regulatory mechanism was involved in the silencing of a number of clones which exhibited a decreasein productivity

Platform based screening strategies that deliver reliable and high quality continous biomanufacturing processes

The challenge during mammalian cell line and upstream process development is to identify and isolate stable, high expressing cell lines producing product with the appropriate critical product quality attributes rapidly, reproducibly and with relative ease. Current platform processes are based on a defined set of hierarchical screening strategies utilised to identify key cellular performance criteria required for fed-batch culture (Porter et al 2010a,b). The application of continuous biomanufacturing principles has introduced a paradigm shift, due to their inherent advantage of higher productivity which can facilitate the implementation of smaller process equipment and result in cost-effective, lean and agile manufacturing facilities. However, as we move from fed-batch to continuous manufacturing we must re-evaluate and leverage the correct platform technologies (host cell line, expression vector, cell line development process, cell culture media/feed, process control) to rapidly identify the correct cellular performance criteria that are important for continuous biomanufacturing processes. Furthermore, whereas the adoption of robust and reproducible platform processes have been widely adopted for fed-batch processes, optimal upstream continuous processes performance still largely relies on the optimisation of key bioprocess parameters which are optimised in an ad-hoc manner during process development. To increase speed-to-clinic we show the application of both a new cell line development and continuous upstream production platform methodology which has been successfully utilised to establish reliable and high quality continuous upstream biomanufacturing processes for multiple CHO-DG44 derived cell lines and recombinant monoclonal antibody products. References Porter AJ, Dickson AJ, Racher AJ. (2010a) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Realising the potential in bioreactors. Biotechnol Prog 26(5):1446-54 Porter AJ, Racher AJ, Preziosi R, Dickson AJ (2010b) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation. Biotechnol Prog 26(5): 1455-64

Cases of advanced visual field loss at referral to glaucoma clinics - more men than women?

Purpose In many medical conditions ‘late presentation’ of disease is more of a problem for men than women. Risk of sight loss from glaucoma is certainly greater in those detected with advanced disease. We performed a retrospective study to test the hypothesis that men are more likely than women to have advanced visual field loss at referral to glaucoma clinics. Methods We used 152 918 Humphrey visual fields from 32 147 patients from three regionally different hospitals in England; no other clinical data were made available apart from patient's age, sex and examination dates. The study population was defined as patients with measureable visual field loss in at least one eye at referral to glaucoma clinics. Cases of advanced visual field loss as defined by the Enhanced Glaucoma Severity Staging method at the first visit to secondary care were used as a proxy measure for late presentation of glaucoma. Age-adjusted relative risk (RR) was calculated as the ratio of the proportion of men to women with this proxy measure. Results Median (interquartile range) age and MD (worse eye) for 3733 men and 4264 women was 72 (63, 79) and 74 (64, 81) years and −6.4 (−11.7, −3.8) and −6.3 (−11.0, −3.8) dB respectively. Overall proportion of patients with advanced visual field loss at referral to glaucoma clinics was slightly higher in men (25.0%) than in women (22.3%); this difference was statistically significant (p < 0.01). Overall age-standardised RR was statistically significant (1.16; p < 0.001); a person with late presentation of glaucoma is 16% (95% confidence interval: 7–25%) more likely to be a man than a woman. Conclusions A large number of patients with glaucomatous visual field defects are estimated to have advanced loss in at least one eye on referral to secondary care in England; risk for men more likely presenting with late disease is slightly greater than for women

Status Review for Anadromous Atlantic Salmon (Salmo salar) in the United States

https://digitalmaine.com/commerce_feddocs/1003/thumbnail.jp

Time to get real: the case for critical action research in purchasing and supply management

In fragile and often complex supply chains, PSM failures continue to be reported in the media, often with severe economic, social and environmental consequences. To encourage organisations to engage in responsible PSM, we need engaged research. In this paper we argue that Action Research (AR) is an influential, participative method to challenge the more dominant versions of PSM impacts, which tend to focus only on the positive, and often only monetised elements of what is valued. AR places change at the core of the research process, requiring critical reflexive practice of the impact of assumptions, values and actions on others. We argue that PSM research has more potential for influence if it starts from a ‘real’ problem anchored in practice, and that crucially, the problem itself should be challenged dialogically by scholars, practitioners and diverse stakeholders. Critical AR can reframe performance from a technical, company-centric notion to explore broader relationships between inputs and outputs over a longer time frame. We explore the risks and rewards of Critical AR for PSM scholars and draw conclusions on our role as engaged advocates of change

Axial Globe Position Measurement: A Prospective Multicenter Study by the International Thyroid Eye Disease Society

Identify a reproducible measure of axial globe position (AGP) for multicenter studies on patients with thyroid eye disease (TED)

Predicting the Tolerated Sequences for Proteins and Protein Interfaces Using RosettaBackrub Flexible Backbone Design

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others

Self-Potential as a Predictor of Seawater Intrusion in Coastal Groundwater Boreholes

This work was supported by the Natural Environment Research Council in the UK, as part of the Science and Solutions for a Changing Planet Doctor Training Partnership, run by the Grantham Institute for Climate Change at Imperial College London. We thank Southern Water for access to the boreholes at Saltdean and Balsdean. We thank Southern Water and Atkins Global for funding the installation of the equipment. We also thank Dr Amadi Ijioma for providing a prototype of the electrodynamic modelling code in MATLAB, which has since been adapted for use in a coastal chalk aquifer. Three anonymous reviewers are thanked for their comments, which greatly helped to improve the manuscript. The data used in this paper are in the tables, figures and cited information. The authors have no conflicts of interest to declare.Peer reviewedPublisher PDFPublisher PD

Observation of two new Ξb−\Xi_b^- baryon resonances

Two structures are observed close to the kinematic threshold in the $\Xi_b^0 \pi^-$ mass spectrum in a sample of proton-proton collision data, corresponding to an integrated luminosity of 3.0 fb$^{-1}$ recorded by the LHCb experiment. In the quark model, two baryonic resonances with quark content $bds$ are expected in this mass region: the spin-parity $J^P = \frac{1}{2}^+$ and $J^P=\frac{3}{2}^+$ states, denoted $\Xi_b^{\prime -}$ and $\Xi_b^{*-}$. Interpreting the structures as these resonances, we measure the mass differences and the width of the heavier state to be $m(\Xi_b^{\prime -}) - m(\Xi_b^0) - m(\pi^{-}) = 3.653 \pm 0.018 \pm 0.006$ MeV$/c^2$, $m(\Xi_b^{*-}) - m(\Xi_b^0) - m(\pi^{-}) = 23.96 \pm 0.12 \pm 0.06$ MeV$/c^2$, $\Gamma(\Xi_b^{*-}) = 1.65 \pm 0.31 \pm 0.10$ MeV, where the first and second uncertainties are statistical and systematic, respectively. The width of the lighter state is consistent with zero, and we place an upper limit of $\Gamma(\Xi_b^{\prime -}) < 0.08$ MeV at 95% confidence level. Relative production rates of these states are also reported.Comment: 17 pages, 2 figure