The potential of small molecules to modulate glycosylation by media design

Background and novelty A large number of recent publications demonstrate the effect of cell culture media on post-translational modifications of recombinant proteins1. This study aims to extend the toolbox of media design beyond the commonly known media components. We identified and tested a large variety of cell culture compatible chemical components such as pigments and sugar derivatives in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies. Experimental approach The cells were cultivated in fed-batch mode cultures using shaking 96-deepwell plates, and process performance such as viable cell density, viability and product titer were monitored. Supernatants of each culture were purified and N-glycan analysis was performed by CGE-LIF. The findings were confirmed in 30 mL fed-batch shake tube cultures and/or 15 mL micro-scale bioreactors (ambr15TM system) operated at controlled pH and pCO2. Results and discussion The addition of the components at the beginning of the culture exhibited significant changes of the glycosylation profile of the expressed protein. Furthermore, strategies such as the adjustment of the levels and the supplement addition in the feed instead of the media and maintaining constant media osmolality while increasing the supplement concentration allowed to fine-tune the effect of the components on glycosylation profile and to improve the culture performance. Finally, the use of some of the tested supplements increased peak cell density to levels above 20 mio viable cells/mL and product titer up to 1.5-fold, while maintaining high viability throughout the culture. These results show that media design alone is sufficient to specifically modulate some of the essential protein quality attributes and to increase productivity, which circumvents the need of modifying the gene expression of the cell lin

An exploration of strategic planning and stakeholder engagement for the development of heritage sites in Plovdiv, Bulgaria

This study determined how stakeholders of heritage attractions apply strategic management for their business planning and development. A conceptual framework for strategic heritage planning was created and applied within the case of Plovdiv, Bulgaria. The framework provided a new way of interpreting whether effective strategies were used within the heritage management sector. In addition to offering a lens to view policy planning, the framework led to a stakeholder analysis determining who was governing the heritage sites within the city.The literature review revealed that studies about planning for heritage are neither prescriptive nor descriptive. Instead, they commonly reviewed challenges in planning with valuation, policy learning, implementation and maintenance for safeguarding sites. The new conceptual framework was created based on the gaps, challenges, issues and recommendations presented in the literature for heritage preservation. Each stage is operational and can be used as a guide for good practice or as an audit instrument.Critical realism was the most appropriate research approach because the study was practical and investigated how stakeholders process policy planning in the heritage sector. This study used purely qualitative methods and considered the stakeholders' experiences to give meaning to the situation. Purposive sampling was used and the questions created for the semi-structured interviews focused on stakeholder involvement throughout the phases of the framework.Accordingly, the Interview questions focused on assessment, creation and implementation of policy. Nine stakeholders were interviewed who were directly involved in the policy planning for heritage in Plovdiv. Document analysis was also used assessing the planning strategies highlighted in the Municipal Policy Document for Plovdiv 2014-2020.In terms of the strategic planning and development process of the heritage sites, the findings revealed that managers pay more attention to the assessment and 3 creation phases rather than the implementation phase. With regards to stakeholder involvement, the research showed that few of them were involved at certain stages of the process due to the hierarchy of governance. Academic and managerial recommendations are further discussed in the study

Generating glycan variants for biological activity testing by means of parallel experimental design and multivariate analysis

For more than 20 years, the industry has mainly invested in productivity enhancements. Recently, the focus of cell-culture process development began to shift. The modulation of quality attributes of recombinant therapeutic protein has gained substantial interest as demonstrated by the plethora of recent publications describing the effect of cell culture media on post-translational modifications of recombinant proteins1. Focusing on glycosylation, our team has developed a toolbox of media design beyond the commonly known media components and a rational high-throughput experimental design method. We identified and tested a large variety of novel cell culture compatible chemical components in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies and antibody fusion molecules. The compounds were evaluated in five different parallel 96-DWP fed-batch experiments, considering their mode of biological action. Viable cell density, viability and product titer were monitored and purified supernatants underwent N-glycan analysis by 2AB-UPLC and site-specific glycan-peptide analysis. Multivariate analysis identified the best performing glycosylation modulators, which were confirmed in spin tubes. Intracellular nucleotide and nucleotide sugar levels were analyzed by capillary electrophoresis, the gene expression by next-generation sequencing technologies, and the impact of the generated glycan variants on the biological activity was assessed. Non-targeted metabolite profiling was carried out to build a multivariate model linking metabolites with the glycan fingerprint. The screening experiments in 96-DWP produced a large glycosylation distribution diversity2,3. Subsequent D-optimal quadratic design in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the glycosylation specifications was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications than the best 25% in 96-deepwell plates. Further enhancement enabled us to generate extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. The glycan variants induced significant responses in the respective in vitro biological activity assays. Moreover, metabolites correlating with time-dependent glycan profiling data were pinpointed and the glycan distribution of an external data set predicted. Our data highlight the great potential of cell culture medium optimization to modulate product quality and show the feasibility of the generation of a wide range of glycan variants suitable for biological activity testing. [1] Brühlmann D, Jordan M, Hemberger J, Sauer M, Stettler M and Broly H, Tailoring recombinant protein quality by rational media design, Biotechnology Progress 2015, 31:615–629. [2] Brühlmann D, Muhr A, Parker R, Vuillemin T, Bucsella B, Torre S, La Neve F, Lembo A, Haas T, Sauer M, Souquet J, Broly H, Hemberger J, Jordan M, Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation, Journal of Biotechnology 2017, 252:32-42. [3] Brühlmann D, Sokolov M, Butté A, Sauer M, Hemberger J, Souquet J, Broly H, Jordan M, Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve Biosimilar product quality, Biotechnology and Bioengineering 2017, 114(7):1363-1631

Nerves and blood vessels in degenerated intervertebral discs are confined to physically disrupted tissue

Nerves and blood vessels are found in the peripheral annulus and endplates of healthy adult intervertebral discs. Degenerative changes can allow these vessels to grow inwards and become associated with discogenic pain, but it is not yet clear how far, and why, they grow in. Previously we have shown that physical disruption of the disc matrix, which is a defining feature of disc degeneration, creates free surfaces which lose proteoglycans and water, and so become physically and chemically conducive to cell migration. We now hypothesise that blood vessels and nerves in degenerated discs are confined to such disrupted tissue. Whole lumbar discs were obtained from 40 patients (aged 37–75 years) undergoing surgery for disc herniation, disc degeneration with spondylolisthesis or adolescent scoliosis (‘non‐degenerated’ controls). Thin (5‐μm) sections were stained with H&E and toluidine blue for semi‐quantitative assessment of blood vessels, fissures and proteoglycan loss. Ten thick (30‐μm) frozen sections from each disc were immunostained for CD31 (an endothelial cell marker), PGP 9.5 and Substance P (general and nociceptive nerve markers, respectively) and examined by confocal microscopy. Volocity image analysis software was used to calculate the cross‐sectional area of each labelled structure, and its distance from the nearest free surface (disc periphery or internal fissure). Results showed that nerves and blood vessels were confined to proteoglycan‐depleted regions of disrupted annulus. The maximum distance of any blood vessel or nerve from the nearest free surface was 888 and 247 μm, respectively. Blood vessels were greater in number, grew deeper, and occupied more area than nerves. The density of labelled blood vessels and nerves increased significantly with Pfirrmann grade of disc degeneration and with local proteoglycan loss. Analysing multiple thick sections with fluorescent markers on a confocal microscope allows reliable detection of thin filamentous structures, even within a dense matrix. We conclude that, in degenerated and herniated discs, blood vessels and nerves are confined to proteoglycan‐depleted regions of disrupted tissue, especially within annulus fissures

Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed

The in situ mechanics of cells in the annulus fibrosus of the intervertebral disc

Bibliography: p. 134-160Cells of the intervertebral disc are sparse, yet they are responsible for the secretion and organization of the extracellular matrix. Mechanical factors in vivo and in vitro influence the metabolic activity of the cells, altering the expression of key extracellular matrix molecules. Understanding the mechanobiological response is crucial to our elucidating the underlying mechanisms involved in disc degeneration and disease. However, the basic mechanisms that govern mechanical and biological interactions in situ are largely unknown. Using novel techniques of confocal microscopy, this dissertation has established that the in situ mechanical environment of cells from the annulus fibrosus cannot be directly inferred from tissue loads due to the morphological and micromechanical complexities of the cell and tissue matrix. With a systematic histological investigation, an extensive cellular matrix was described; with, variations in cell shape, arrangement of cellular processes and cytoskeletal architecture found both within and between the defined zones of the outer and inner annulus. Notably, the distinct morphology of cells from the interlamellar septae was identified and described. The in situ mechanical environment of these cells contrasted that of the cells within the lamellar layers. The interlamellar cells experienced a shear environment from the relative motion of the adjacent lamellar layers, while cells within the lamellar layer appeared protected via sliding of the collagen fibrils within the surrounding matrix. The in situ mechanical environment of cells was, therefore, found to be governed by micro and macro structural complexities of the collagen matrix. The detailed understanding of the in situ mechanical and morphological environment of the cells is essential to establishing the role of mechanical loads in low back pain and disc degeneration, and in determining the mechanotransduction mechanisms whereby these loads can alter the expression of matrix molecules. This dissertation provides the first direct knowledge of how mechanical loads applied to the whole disc are transferred to the cells in situ