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1.
Biotechnol Bioeng ; 118(9): 3610-3617, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33713416

RESUMO

Hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSC-HLC) are expected to have important applications in drug screening and regenerative medicine. However, hiPSC-HLC are difficult to produce on a large-scale to obtain relevant numbers for such applications. The aim of this study was to implement a novel integrated strategy for scalable production of hiPSC-HLC and demonstrate the applicability of dielectric spectroscopy to monitor hiPSC expansion/differentiation processes. We cultured hiPSC as three-dimensional (3D) aggregates in stirred-tank bioreactors (STB) operated in perfusion with an in situ capacitance probe. Dissolved oxygen concentration and dilution rate were controlled along the process and after 5 days of cell expansion, the hepatic differentiation was integrated in sequential steps for 28 days. The hiPSC were able to grow as 3D aggregates and the expression of hepatic markers and albumin production after differentiation confirmed that hepatocyte differentiation improved when compared to 2D culture. These hiPSC-HLC exhibited functional characteristics of hepatocytes including glycogen storage and drug metabolization capacity. Our results also show a good correlation between the cell permittivity measured online and the aggregate biovolume measured by standard offline methods, demonstrating for the first time the potential of dielectric spectroscopy to monitor hiPSC expansion and differentiation in STB.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Espectroscopia Dielétrica , Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Hepatócitos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
2.
Biotechnol Bioeng ; 116(11): 2803-2814, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31317525

RESUMO

The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines. Here, we applied fluorescence spectroscopy as a real-time monitoring tool for recombinant adeno-associated virus (rAAV) production in the IC-BEVS. Fluorescence spectroscopy is simple, yet sensitive and informative. To overcome the strong fluorescence background of the culture medium and improve predictive ability, we combined artificial neural network models with a genetic algorithm-based approach to optimize spectra preprocessing. We obtained predictive models for rAAV titer, cell viability and cell concentration with normalized root mean squared errors of 7%, 4%, and 7%, respectively, for leave-one-batch-out cross-validation. Our approach shows fluorescence spectroscopy allows real-time determination of the best time of harvest to maintain rAAV infectivity, an important quality attribute, and detection of deviations from the golden batch profile. This methodology can be applied to other biopharmaceuticals produced in the IC-BEVS, supporting the use of fluorescence spectroscopy as a versatile PAT tool.


Assuntos
Reatores Biológicos , Dependovirus/crescimento & desenvolvimento , Modelos Biológicos , Animais , Dependovirus/genética , Células Sf9 , Espectrometria de Fluorescência , Spodoptera
3.
Biotechnol Bioeng ; 114(1): 195-207, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27477740

RESUMO

Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-13 C]glucose or [U-13 C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary 13 C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.


Assuntos
Adenoviridae/isolamento & purificação , Adenoviridae/metabolismo , Infecções por Adenovirus Humanos , Isótopos de Carbono/metabolismo , Análise do Fluxo Metabólico/métodos , Cultura de Vírus/métodos , Adenoviridae/química , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Isótopos de Carbono/análise , Linhagem Celular , Glutamina/metabolismo , Humanos , Modelos Biológicos
4.
Sci Rep ; 6: 23529, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27004747

RESUMO

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.


Assuntos
Adenovirus Caninos/fisiologia , Glucose/farmacocinética , Glutamina/farmacocinética , Células Madin Darby de Rim Canino/virologia , Análise do Fluxo Metabólico/métodos , Animais , Isótopos de Carbono/farmacocinética , Proliferação de Células , Transformação Celular Viral , Cães , Regulação da Expressão Gênica , Glicólise , Lipogênese , Células Madin Darby de Rim Canino/metabolismo , Via de Pentose Fosfato
5.
Curr Opin Biotechnol ; 30: 161-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25035940

RESUMO

Protein biopharmaceuticals are mainly produced in mammalian cells which can perform human-like post-translational modifications crucial to protein function. Subject to high variability, these critical quality attributes should be monitored and controlled during the manufacturing process. However, the large time requirements for analysis have been a bottleneck. Recent advances towards automated and high-throughput techniques, combined with multivariate data analysis, are increasingly providing relevant process knowledge in near real-time. New or re-designed analytical tools suited for monitoring product quality are starting to fit in this landscape. Moreover, omics technologies are expanding our understanding of how intracellular mechanisms and the extracellular milieu influence protein quality and quantity, reshaping the adoption of Process Analytical Technology (PAT) and Quality by Design (QbD) in the biopharmaceutical industry.


Assuntos
Indústria Farmacêutica/métodos , Controle de Qualidade , Proteínas Recombinantes/análise , Animais , Linhagem Celular , Indústria Farmacêutica/normas , Humanos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico
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