Lipidomics for robust high performance process development

As the biopharmaceutical industry reduces the risk of potential contaminations by adventitious agents and increases process yields, high performance cell culture processes have been developed that rely on animal-free peptide-free, protein-free chemically defined and lipid-free media. These processes rely on cell lines that have been adapted to these lipid-free production conditions and have developed very effective lipid production capacities. These lipid-production capacities result in new challenges in the harvest and purification steps such as filterability, ability to clean resins and resin reuse. This oral presentation will show case how lipidomics can provide insights and opportunities to control the interactions between high performance bioreactor production processes, harvest conditions and purification. Results obtained across large scale production processes of three different monoclonal antibodies will be displayed. The importance of controlling lipid biosynthesis and the presence of lipids in the cell culture fluid prior to affinity capture chromatography will be discussed. Three different control strategies will be showcased and their pros and cons in terms of process yields, robustness of the harvest and impact on the purification process post-harvest explaine

Curation of a CHO DG44 genome scale model and application to support cell culture development process

Genome scale models (GSM) have become a useful tool to connect different omics dataset into a single computational framework, thus giving a good overview of the flux distribution and metabolites interconnections in a specific environmental condition. A community genome-scale metabolic network reconstruction of Cricetulus griseus and cell line specific models have been recently developed 1. The main objectives with the use of the published CHO DG44 model were to enhance industrial bioprocess performance by suggesting genetic or metabolic targets, as well as strategies for medium optimization, and by bringing more fundamental knowledge about CHO cell metabolism. In a first step, some corrections were required in order to improve the biological relevancy of the predicted intracellular fluxes. The optimization method chosen was Parsimonious Flux Balance Analysis, based on the assumption that the cell is using a minimum amount of enzymes to reach a maximized objective value, under steady state. As the predictions were generating a lot of infeasible cycles, silencing of amino acid transporters that do not involve protons or sodium in the model allowed to reduce the incoming flow of amino acids and led to disappearance of infeasible cycles in the flux distribution solution. Four reactions involved in central carbon metabolism were manually added in the model, and some reactions were removed from the model to improve predictions such as the cytosolic enzyme fumarase, mainly localized in mitochondria, or L-asparaginase which is not reported to be present in CHO cells. As initial predictions for lactate production rate were overestimated compared to experimental measurements, the assumption of lipid accumulation during cell culture was added in the form of a constraint for a minimal level of triglyceride synthesis in the model (Figure 1). In a second step, the accuracy of the prediction from the curated model was tested with three independent data set obtained from a fed-batch experiment with a CHO DG44 cell line producing a monoclonal antibody in 2L stirred tank glass bioreactors. For modelling with GSM, pre-calculated input values are required in order to constraint the model with the environmental conditions, and thus to give a prediction that is representative of the experimental condition. Uptake rates of essential nutrients initially present in extracellular medium and consumed as the cells grow were used as the limit for a maximum uptake rate in the model. The objective function chosen was maximization of growth rate or maximization of specific productivity. As a result, correlation coefficients between experimental value and prediction indicate a good fit for growth rate and specific productivity (Qp) (Figure 2). Predicted amino acid consumption rates were comparable to experimental values, indicating the accuracy of the predicted stoichiometric requirements for cell growth and energy, except for 19% of the fluxes studied (Figure 3). As the extracellular flux values seem to correlate with experimental data, prediction of intracellular flux rates were analyzed at different timepoints of the culture, showing the activation of multiple metabolic pathways. Based on the results obtained, optimization of cell culture medium was performed to identify the limiting metabolites during the process that could lead to an increased growth rate and Qp. Please click Additional Files below to see the full abstract

Efeito da terapia de espelho no tratamento da dor fantasma em pacientes amputados: revisão bibliográfica

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em FisioterapiaIntrodução: A terapia de espelho tem sido utilizado como um dos métodos de tratamento para a dor fantasma no amputados que trabalha com estratégia de feedback visual. Objetivo: verificar o efeito da Terapia de Espelho no alívio da dor fantasma em amputados. Metodologia : Pesquisa computorizada nos motores de busca b-on, na base de dados Pubmed e na Science direct com o intuito de identificar estudos experimentais e quasi-experimentais, publicados nos últimos 10 anos em inglês, que abordassem a aplicação da terapia de espelho no alívio da dor fantasma em pessoas amputadas dos membros. Resultados : Foram incluídos 6 estudos experimentais, 4 deles randomizados controlados, com um total de 167 participantes. 2 estudos avaliaram a terapia de espelho nos MI e 2 nos MS, 3 compararam a terapia de espelho e prática mental e 1 a terapia de espelho e o TENS. Conclusão : A terapia de espelho parece mostrar benéficos no alívio da dor fantasma e na consciencialização do membro fantasma, contudo são necessários mais estudos para demonstrar a sua eficácia.Introduction: Mirror therapy has been referred to treatment method for phantom limb pain of the amputees which work with visual feedback strategy. Objective: Analyze several protocols about the mirror therapy (MT) effects on the management of phantom limb pain (PLP). Methodology: Were conducted computerized search in web of knowledge, b-on, Pubmed and ScienceDirect data bases, to identify last 10 years studies that evalued the effects of MT in populations of patients experiencing PLP after limb amputation. Results: Were inclued 6 experimental studies including 4 randomized controlled trials, with a total of 167 participants. 2 studies evalued the effect of MT on lower limb amputees e 2 on upper limb amputees, 3 compared MT and mental visualization and 1 compared MT and TENS. Conclusion: MT appear to be an effective therapy for reducing PLP and increase awareness of phantom limb. However, further studies are required to demonstrate efficiency of MT.N/

Efeito da terapia de espelho no tratamento da dor fantasma em pacientes amputados: revisão bibliográfica

Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciada em FisioterapiaIntrodução: A terapia de espelho tem sido utilizado como um dos métodos de tratamento para a dor fantasma no amputados que trabalha com estratégia de feedback visual. Objetivo: verificar o efeito da Terapia de Espelho no alívio da dor fantasma em amputados. Metodologia : Pesquisa computorizada nos motores de busca b-on, na base de dados Pubmed e na Science direct com o intuito de identificar estudos experimentais e quasi-experimentais, publicados nos últimos 10 anos em inglês, que abordassem a aplicação da terapia de espelho no alívio da dor fantasma em pessoas amputadas dos membros. Resultados : Foram incluídos 6 estudos experimentais, 4 deles randomizados controlados, com um total de 167 participantes. 2 estudos avaliaram a terapia de espelho nos MI e 2 nos MS, 3 compararam a terapia de espelho e prática mental e 1 a terapia de espelho e o TENS. Conclusão : A terapia de espelho parece mostrar benéficos no alívio da dor fantasma e na consciencialização do membro fantasma, contudo são necessários mais estudos para demonstrar a sua eficácia.Introduction: Mirror therapy has been referred to treatment method for phantom limb pain of the amputees which work with visual feedback strategy. Objective: Analyze several protocols about the mirror therapy (MT) effects on the management of phantom limb pain (PLP). Methodology: Were conducted computerized search in web of knowledge, b-on, Pubmed and ScienceDirect data bases, to identify last 10 years studies that evalued the effects of MT in populations of patients experiencing PLP after limb amputation. Results: Were inclued 6 experimental studies including 4 randomized controlled trials, with a total of 167 participants. 2 studies evalued the effect of MT on lower limb amputees e 2 on upper limb amputees, 3 compared MT and mental visualization and 1 compared MT and TENS. Conclusion: MT appear to be an effective therapy for reducing PLP and increase awareness of phantom limb. However, further studies are required to demonstrate efficiency of MT.N/

Novel modeling methodology to predict product quality and cell culture performance in fed-batch and perfusion cultures

The acceleration of biopharmaceutical process development is difficult when traditional experience-based sequential approaches are used. As a result, fully optimized and well understood cell culture processes prior to scale-up are rare. Here we show that an accurate, scalable and simple model able to predict cell growth, cell metabolism, titer and some product quality attributes will significantly accelerate process development, improve process development outcomes and reduce development and production costs. Please click Additional Files below to see the full abstract

Predictive macroscopic models of cell growth, metabolism and monoclonal antibody production of fed-batch processes at various scales

Recently, the pharmaceutical industry is increasingly focusing on early drug development which comes with increasing constraints to accelerate process development, reduce costs and demonstrate a deep understanding cell culture processes. However, cellular metabolism is very complex and by far not fully understood. Cells can be cultivated in various types of bioreactors applying sophisticated feeding strategies mostly based on experience and series of experiments. Modern systems biology promises modeling of such processes on the basis of a system-wide understanding of cellular processes but is still unable to deliver predictive models in due time at reasonable cost. Practically applicable, predictive models are highly demanded in industry for the purpose of process optimization and control. To this end, we developed a systematic methodology for metabolic and cell growth modeling that is directly applicable in an industrial environment. We demonstrate that the models developed are able to predict a wide range of new experimental cell culture conditions. Please click Additional Files below to see the full abstract