Karen Coopman

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor.

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

The translation of cell-based therapies: clinical landscape and manufacturing challenges

Cell-based therapies have the potential to make a large contribution toward currently unmet patient need and thus effective manufacture of these products is essential. Many challenges must be overcome before this can become a reality and a better definition of the manufacturing requirements for cell-based products must be obtained. The aim of this study is to inform industry and academia of current cell-based therapy clinical development and to identify gaps in their manufacturing requirements. A total of 1342 active cell-based therapy clinical trials have been identified and characterized based on cell type, target indication and trial phase. Multiple technologies have been assessed for the manufacture of these cell types in order to facilitate product translation and future process development.

Large-scale expansion and exploitation of pluripotent stem cells for regenerative medicine purposes: beyond the T flask

Human pluripotent stem cells will likely be a significant part of the regenerative medicine-driven healthcare revolution. In order to realize this potential, culture processes must be standardized, scalable and able to produce clinically relevant cell numbers, whilst maintaining critical biological functionality. This review comprises a broad overview of important bioprocess considerations, referencing the development of biopharmaceutical processes in an effort to learn from current best practice in the field. Particular focus is given to the recent efforts to grow human pluripotent stem cells in microcarrier or aggregate suspension culture, which would allow geometric expansion of productive capacity were it to be fully realized. The potential of these approaches is compared with automation of traditional T-flask culture, which may provide a cost-effective platform for low-dose, low-incidence conditions or autologous therapies. This represents the first step in defining the full extent of the challenges facing bioprocess engineers in the exploitation of large-scale human pluripotent stem cell manufacture.

Comparative Effects of the Endogenous Agonist Glucagon-Like Peptide-1 (GLP-1)-(7-36) Amide and the Small-Molecule Ago-Allosteric Agent “Compound 2” at the GLP-1 Receptor

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca2+ signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated Gαs in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca2+] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum‐free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707.

Large-scale compatible methods for the preservation of human embryonic stem cells: Current perspectives

Human embryonic stem cells (hESCs) and hESC‐derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality‐controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time‐consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high‐throughput drug screen and how many cells can be preserved using these techniques. Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here, the potential to lyophilize hESCs for long‐term storage is considered as one such alternative.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors

Abstract Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.

Differential regulation of prostaglandin E biosynthesis by interferon-γ in colonic epithelial cells

Cyclooxygenase (COX)‐2 expression and activity in response to pro‐inflammatory cytokines TNFα and IFNγ was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. TNFα induced concentration‐ and time‐dependent upregulation of COX‐2 mRNA, protein and prostaglandin (PG)E2 synthesis. Co‐stimulation of TNFα with IFNγ resulted in reduced COX‐2 mRNA and protein expression. IFNγ had no effect on the stability of TNFα‐induced COX‐2 mRNA. TNFα‐induced PGE2 biosynthesis was significantly enhanced by the simultaneous addition of IFNγ and was COX‐2 dependent. The combination of IFNγ and TNFα induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE2 synthesis. These results suggest that, in terms of PGE2 biosynthesis, IFNγ plays a negative regulatory role at the level of COX‐2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFNγ in inflammatory diseases.

Low temperature cell pausing: an alternative short-term preservation method for use in cell therapies including stem cell applications

Encouraging advances in cell therapies have produced a requirement for an effective short-term cell preservation method, enabling time for quality assurance testing and transport to their clinical destination. Low temperature pausing of cells offers many advantages over cryopreservation, including the ability to store cells at scale, reduced cost and a simplified procedure with increased reliability. This review will focus on the importance of developing a short-term cell preservation platform as well highlighting the major successes of cell pausing and the key challenges which need addressing, to enable application of the process to therapeutically relevant cells.

The use of bioreactors as in vitro models in pharmaceutical research.

Bringing a new drug to market is costly in terms of capital and time investments, and any development issues encountered during late-stage clinical trials can often be the result of in vitro-in vivo extrapolations (IVIVE) not accurately reflecting clinical outcome. In the discipline of drug metabolism and pharmacokinetics (DMPK), current in vitro cellular methods do not provide the 3D structure and function of organs found in vivo; therefore, new dynamic methods need to be established to aid improvement of IVIVE. In this review, we highlight the importance of model progression into dynamic systems for use within drug development, focusing on devices developed currently in the areas of the liver and blood-brain barrier (BBB), and the potential to develop models for other organ systems, such as the kidney. We discuss the development of dynamic 3D bioreactor-based systems as in vitro models for use in DMPK studies.