144 research outputs found
Implementierung einer kontinuierlichen ProzessfĂŒhrung zur ProduktivitĂ€tssteigerung bei der Kultivierung tierischer Zellen
Durch technologische Innovationen in der ProzessĂŒberwachung und die Entwicklung von Single-Use AusrĂŒstung rĂŒcken kontinuierliche Kultivierungsprozesse zur Herstellung monoklonaler Antikörper (mAK) zunehmend in den Fokus der biopharmazeutischen Industrie. Daher existieren neben zahlreichen Design of Experiment (DoE) AnsĂ€tzen zur ProduktivitĂ€tssteigerung im etablierten Fed Batch Betrieb vermehrt auch Transferprotokolle in Perfusionsprozesse, bei denen vielfach höhere UmsĂ€tze durch Hochzelldichte-Kultivierungen erreicht werden. Hierbei werden derzeit die KapazitĂ€tsgrenzen der verwendeten membranbasierten Zellretentionssysteme im Hinblick auf die Trenneffizienz und die Produktausbeute erreicht, sodass der Bedarf nach neuen Methoden zur Zellabtrennung steigt.
Im Rahmen dieser Arbeit wurden mehrere AnsĂ€tze zur ProduktivitĂ€tssteigerung einer CHO-Zelllinie untersucht. Eine Steigerung der zellspezifischen ProduktivitĂ€t um 8 % in einem etablierten Fed Batch Prozess wurde durch eine pH-Wert Erhöhung innerhalb der exponentiellen Wachstumsphase erreicht. ZusĂ€tzlich besteht ein nicht-linearer Zusammenhang zwischen der induzierten ProduktivitĂ€t und einer Zunahme des Zelldurchmessers. Die maximale Lebendzellzahl konnte auĂerdem durch eine semi-kontinuierliche FĂŒtterungsstrategie um 3,1 Mio. Zellen/mL gesteigert werden.
Im zweiten Ansatz wurde ein kontinuierlicher quasi-Steady State im Chemostaten bei 8 Mio. Zellen/mL mit einer mAK-Ausbeute von 0,2 g/L*Tag erreicht. ZusĂ€tzlich konnte eine reversible glucoseinduzierte Wachstumsinhibierung der Zellen bei > 8 g/L (+1 Tag) mit erhöhter spezifischer ProduktivitĂ€t detektiert werden. FĂŒr eine Steigerung der kontinuierlichen mAK-Ausbeute durch erhöhte Zelldichten wurde ein Perfusionsprozess mittels einer 3D-gedruckten Zellseparationsspirale im Hinblick auf die Trenneffizienz und die StabilitĂ€t der Zellretention evaluiert. Neben einer Designoptimierung wurde die ProzessstabilitĂ€t durch die Implementierung einer automatischen SpĂŒlung und einer FlussratenĂŒberwachung mit webbasierter Steuerung gesteigert. Die LangzeitstabilitĂ€t des verwendeten Druckmaterials sowie die Scherstressbildung wurden in einem Perfusionsprozess > 500 h evaluiert
Establishment of a Perfusion Process with Antibody-Producing CHO Cells Using a 3D-Printed Microfluidic Spiral Separator with Web-Based Flow Control
Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methodsâsuch as perfusion processesâare being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devicesâsuch as tangential flow filtrationârely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10â17 Ă 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor
Implementation of QbD strategies in the inoculum expansion of a mAb production process
The quality by design approach was introduced to the biopharmaceutical industry over 15 years ago. This principle is widely implemented in the characterization of monoclonal antibody production processes. Anyway, the early process phase, namely the inoculum expansion, was not yet investigated and characterized for most processes. In order to increase the understanding of early process parameter interactions and their influence on the later production process, a risk assessment followed by a design of experiments approach was conducted. The DoE included the critical parameters methotrexate (MTX) concentration, initial passage viable cell density and passage duration. Multivariate data analysis led to mathematical regression models and the establishment of a designated design space for the studied parameters. It was found that the passage duration as well as the initial viable cell density for each passage during the inoculum expansion have severe effects on the growth rate and viability of the early process phase. Furthermore, the variations during the inoculum expansion directly influenced the production process responses. This carry-over of factor effects highlights the crucial impact of early process failures and the importance of process analysis and control during the first part of mAb production processes. © 2020 The Authors. Engineering in Life Sciences published by Wiley-VCH Gmb
Stressâinduced increase of monoclonal antibody production in CHO cells
Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit humanâlike glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stressâinduced cell size expansion and increased specific productivity was investigated. CO(2) and macronutrient supply of the cells during a 12âday fedâbatch cultivation process were tested as stress factors. Shake flasks (500Â mL) and a smallâscale bioreactor system (15Â mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cellâspecific productivity. The achieved stressâinduced increase in cellâspecific productivity of up to 94.94.9%â134.4% correlates to a cell diameter shift of up to 7.34Â ÎŒm. The highest final product titer of 4Â g/L was reached by glucose oversupply during the batch phase of the process
Stress-induced increase of monoclonal antibody production in CHO cells
Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human-like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress-induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12-day fed-batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small-scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell-specific productivity. The achieved stress-induced increase in cell-specific productivity of up to 94.94.9%â134.4% correlates to a cell diameter shift of up to 7.34 ÎŒm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process
Optimization of a mAb production process with regard to robustness and product quality using quality by design principles
Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambrÂź15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the N-1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2 Ă 106 cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing. © 2022 The Authors. Engineering in Life Sciences published by Wiley-VCH GmbH
Monitoring cell productivity for the production of recombinant proteins by flow cytometry : An effective application using the cold capture assay
Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically timeâconsuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively timeâeffective, and potentially cheaper alternative for monitoring the productivity of a cell culture
Characterising innovations in maternal and newborn health based on a common theory of change: lessons from developing and applying a characterisation framework in Nigeria, Ethiopia and India.
Government leadership is key to enhancing maternal and newborn survival. In low/middle-income countries, donor support is extensive and multiple actors add complexity. For policymakers and others interested in harmonising diverse maternal and newborn health efforts, a coherent description of project components and their intended outcomes, based on a common theory of change, can be a valuable tool. We outline an approach to developing such a tool to describe the work and the intended effect of a portfolio of nine large-scale maternal and newborn health projects in north-east Nigeria, Ethiopia and Uttar Pradesh in India. Teams from these projects developed a framework, the 'characterisation framework', based on a common theory of change. They used this framework to describe their innovations and their intended outcomes. Individual project characterisations were then collated in each geography, to identify what innovations were implemented where, when and at what scale, as well as the expected health benefit of the joint efforts of all projects. Our study had some limitations. It would have been enhanced by a more detailed description and analysis of context and, by framing our work in terms of discrete innovations, we may have missed some synergistic aspects of the combination of those innovations. Our approach can be valuable for building a programme according to a commonly agreed theory of change, as well as for researchers examining the effectiveness of the combined work of a range of actors. The exercise enables policymakers and funders, both within and between countries, to enhance coordination of efforts and to inform decision-making about what to fund, when and where
Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy
CurePSP Foundation, the Peebler PSP Research Foundation, and National Institutes on Health (NIH) grants R37 AG 11762, R01 PAS-03-092, P50 NS72187, P01 AG17216 [National Institute on Aging(NIA)/NIH], MH057881 and MH077930 [National Institute of Mental Health (NIMH)]. Work was also supported in part by the NIA Intramural Research Program, the German National Genome Research Network (01GS08136-4) and the Deutsche Forschungsgemeinschaft (HO 2402/6-1), Prinses Beatrix Fonds (JCvS, 01â0128), the Reta Lila Weston Trust and the UK Medical Research Council (RdS: G0501560). The Newcastle Brain Tissue Resource provided tissue and is funded in part by a grant from the UK Medical Research Council (G0400074), by the Newcastle NIHR Biomedical Research Centre in Ageing and Age Related Diseases to the Newcastle upon Tyne Hospitals NHS Foundation Trust, and by a grant from the Alzheimerâs Society and Alzheimerâs Research Trust as part of the Brains for
Dementia Resarch Project. We acknowledge the contribution of many tissue samples from the Harvard Brain Tissue Resource Center. We also acknowledge the 'Human Genetic Bank of Patients affected by Parkinson Disease and parkinsonism' (http://www.parkinson.it/dnabank.html) of the Telethon Genetic Biobank Network, supported by TELETHON Italy (project n. GTB07001) and by Fondazione Grigioni per il Morbo di Parkinson. The University of Toronto sample collection was supported by grants from Wellcome Trust, Howard Hughes Medical Institute, and the Canadian Institute of Health Research. Brain-Net-Germany is supported by BMBF (01GI0505). RdS, AJL and JAH are funded by the Reta Lila Weston Trust and the PSP (Europe) Association. RdS is funded by the UK Medical Research Council (Grant G0501560) and Cure PSP+. ZKW is partially supported by the NIH/NINDS 1RC2NS070276, NS057567, P50NS072187, Mayo Clinic Florida (MCF)Research Committee CR programs (MCF #90052030 and MCF #90052030), and the gift from Carl Edward Bolch, Jr., and Susan Bass Bolch (MCF #90052031/PAU #90052). The Mayo Clinic College of Medicine would like to acknowledge Matt Baker, Richard Crook, Mariely DeJesus-Hernandez and Nicola Rutherford for their preparation of samples. PP was supported by a grant from the Government of Navarra ("Ayudas para la RealizaciĂłn de Proyectos de InvestigaciĂłn" 2006â2007) and acknowledges the "Iberian Atypical Parkinsonism Study Group Researchers", i.e. Maria A. Pastor, Maria R. Luquin, Mario Riverol, Jose A. Obeso and Maria C Rodriguez-Oroz (Department of Neurology, ClĂnica Universitaria de Navarra, University of Navarra, Pamplona, Spain), Marta Blazquez (Neurology Department, Hospital Universitario Central de Asturias, Oviedo, Spain), Adolfo Lopez de Munain, Begoña Indakoetxea, Javier Olaskoaga, Javier Ruiz, JosĂ© FĂ©lix MartĂ MassĂł (Servicio de NeurologĂa, Hospital Donostia, San SebastiĂĄn, Spain), Victoria Alvarez (Genetics Department, Hospital Universitario Central de Asturias, Oviedo, Spain), Teresa Tuñon (Banco de Tejidos Neurologicos, CIBERNED, Hospital de Navarra, Navarra, Spain), Fermin Moreno (Servicio de NeurologĂa, Hospital Ntra. Sra. de la Antigua, Zumarraga, Gipuzkoa, Spain), Ainhoa Alzualde (NeurogenĂ©tics Department, Hospital Donostia, San SebastiĂĄn, Spain)
Genetic architecture of sporadic frontotemporal dementia and overlap with Alzheimer's and Parkinson's diseases.
BACKGROUND: Clinical, pathological and genetic overlap between sporadic frontotemporal dementia (FTD), Alzheimer's disease (AD) and Parkinson's disease (PD) has been suggested; however, the relationship between these disorders is still not well understood. Here we evaluated genetic overlap between FTD, AD and PD to assess shared pathobiology and identify novel genetic variants associated with increased risk for FTD. METHODS: Summary statistics were obtained from the International FTD Genomics Consortium, International PD Genetics Consortium and International Genomics of AD Project (n>75â
000 cases and controls). We used conjunction false discovery rate (FDR) to evaluate genetic pleiotropy and conditional FDR to identify novel FTD-associated SNPs. Relevant variants were further evaluated for expression quantitative loci. RESULTS: We observed SNPs within the HLA, MAPT and APOE regions jointly contributing to increased risk for FTD and AD or PD. By conditioning on polymorphisms associated with PD and AD, we found 11 loci associated with increased risk for FTD. Meta-analysis across two independent FTD cohorts revealed a genome-wide signal within the APOE region (rs6857, 3'-UTR=PVRL2, p=2.21Ă10-12), and a suggestive signal for rs1358071 within the MAPT region (intronic=CRHR1, p=4.91Ă10-7) with the effect allele tagging the H1 haplotype. Pleiotropic SNPs at the HLA and MAPT loci associated with expression changes in cis-genes supporting involvement of intracellular vesicular trafficking, immune response and endo/lysosomal processes. CONCLUSIONS: Our findings demonstrate genetic pleiotropy in these neurodegenerative diseases and indicate that sporadic FTD is a polygenic disorder where multiple pleiotropic loci with small effects contribute to increased disease risk
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