In Situ Near-Infrared (NIR) Versus High-Throughput Mid- Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coil cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R-2) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

Monitoring the ex-vivo expansion of human mesenchymal stem/stromal cells in xeno-free microcarrier-based reactor systems by MIR spectroscopy

Human mesenchymal stem/stromal cells (MSCs) have received considerable attention in the field of cell-based therapies due to their high differentiation potential and ability to modulate immune responses. However, since these cells can only be isolated in very low quantities, successful realization of these therapies requires MSCs ex-vivo expansion to achieve relevant cell doses. The metabolic activity is one of the parameters often monitored during MSCs cultivation by using expensive multi-analytical methods, some of them time-consuming. The present work evaluates the use of mid-infrared (MIR) spectroscopy, through rapid and economic high-throughput analyses associated to multivariate data analysis, to monitor three different MSCs cultivation runs conducted in spinner flasks, under xeno-free culture conditions, which differ in the type of microcarriers used and the culture feeding strategy applied. After evaluating diverse spectral preprocessing techniques, the optimized partial least square (PLS) regression models based on the MIR spectra to estimate the glucose, lactate and ammonia concentrations yielded high coefficients of determination (R2 ≥ 0.98, ≥0.98, and ≥0.94, respectively) and low prediction errors (RMSECV ≤ 4.7%, ≤4.4% and ≤5.7%, respectively). Besides PLS models valid for specific expansion protocols, a robust model simultaneously valid for the three processes was also built for predicting glucose, lactate and ammonia, yielding a R2 of 0.95, 0.97 and 0.86, and a RMSECV of 0.33, 0.57, and 0.09 mM, respectively. Therefore, MIR spectroscopy combined with multivariate data analysis represents a promising tool for both optimization and control of MSCs expansion processes.

Adolescentes e comportamentos de saúde

Muitos jovens hoje, e segundo as tendências apontadas em diversos estudos, muitos mais no futuro, estão em risco de vida, de adquirir doenças, deficiências e incapacidades, entre outras consequências negativas para a saúde. Uma questão que se destaca neste cenário é a origem deste risco. Enquanto que anteriormente as causas da mortalidade e morbilidade nos jovens estavam associadas a factores de ordem biomédica, hoje essas causas estão essencialmente associadas a factores de origem social, ambiental e comportamental. Comportamento e estilo de vida são, então, determinantes cruciais para a saúde, doença, deficiência/incapacidade e mortalidade prematura. Dentro dos estilos de vida que colocam em risco a saúde, o bem-estar e muitas vezes a própria vida dos jovens, encontra-se um largo conjunto de comportamentos, nomeadamente o consumo de substâncias (álcool, tabaco, drogas, medicamentos), a violência, o suicídio, os acidentes, as desordens alimentares, a gravidez na adolescência e as doenças sexualmente transmitidas. Como já foi referido, é evidente que estes comportamentos têm consequências negativas a nível pessoal. Para além deste tipo de consequências encontram-se também as consequências a nível social que se poderão traduzir em diversas dimensões de desvantagem social, nomeadamente na integração social e na independência económica. Outros tipos de “custos” são os encargos económicos que a sociedade tem que suportar para cuidados de saúde, reabilitação e institucionalização dos jovens. A perspectiva de que a construção da saúde e do bem-estar desde o início pode prevenir sérios e dispendiosos problemas para o indivíduo e para a sociedade tem vindo a aumentar nas últimas décadas. Organismos como o Conselho da Europa ou a Organização Mundial de Saúde salientam nas suas directrizes a prevenção primária como uma parte importante dos programas nacionais de saúde e educação. Neste âmbito torna-se importante realçar três aspectos que se destacam nestas directrizes. Em primeiro, a necessidade de uma intervenção preventiva precoce, dado que a flexibilidade da criança e do jovem fazem deles alvos ideais para os programas de prevenção, a serem implementados em contextos vocacionados para a promoção do desenvolvimento do indivíduo, nomeadamente o contexto escolar. Em segundo lugar, a noção de que qualquer intervenção integrada num âmbito preventivo não se deve limitar aos momentos de crise ou a prevenir crises. Deverá para além disso promover e optimizar a capacidade de tomar decisões e, consequentemente, a autonomia do jovem. Em terceiro lugar, a importância de incluir os principais contextos de vida e os seus intervenientes nestes processos, dado estes constituírem uma das principais influências na vida dos adolescentes.

Colour filtering in a-SiC : H based p-i-n-p-i-n cells: A trade-off between bias polarity and absorption regions

A large area colour imager optically addressed is presented. The colour imager consists of a thin wide band gap p-i-n a-SiC:H filtering element deposited on the top of a thick large area a-SiC:H(-p)/a-Si:H(-i)/a-SiC:H(-n) image sensor, which reveals itself an intrinsic colour filter. In order to tune the external applied voltage for full colour discrimination the photocurrent generated by a modulated red light is measured under different optical and electrical bias. Results reveal that the integrated device behaves itself as an imager and a filter giving information not only on the position where the optical image is absorbed but also on it wavelength and intensity. The amplitude and sign of the image signals are electrically tuneable. In a wide range of incident fluxes and under reverse bias, the red and blue image signals are opposite in sign and the green signal is suppressed allowing blue and red colour recognition. The green information is obtained under forward bias, where the blue signal goes down to zero and the red and green remain constant. Combining the information obtained at this two applied voltages a RGB colour image picture can be acquired without the need of the usual colour filters or pixel architecture. A numerical simulation supports the colour filter analysis.

Optical confinement and colour separation in a double colour laser scanned photodiode (D/CLSP)

Large area n-i-p-n-i-p a-SiC:H heterostructures are used as sensing element in a double colour laser scanned photodiode image sensor (D/CLSP). This work aims to clarify possible improvements, physical limits and performance of CLSP image sensor when used as non-pixel image reader. Here, the image capture device and the scanning reader are optimized and the effects of the sensor structure on the output characteristics discussed. The role of the design of the sensing element, the doped layer composition and thickness, the read-out parameters (applied voltage and scanner frequency) on the image acquisition and the colour detection process are analysed. A physical model is presented and supported by a numerical simulation of the output characteristics of the sensor.

Estudo do uso de hidrotalcites de Mg-Al como catalisadores heterogéneos para produção de biodiesel

A transesterificação de óleos vegetais ou gorduras animais com um álcool de baixo peso molecular é o principal processo utilizado na produção de biodiesel. Actualmente os processos industriais utilizam catalisadores homogéneos para acelerar a reacção. No entanto a utilização de catalisadores heterogéneos, no processo de transesterificação, tem sido sugerido por vários investigadores pois, são amigos do ambiente e podem ser regenerados e reutilizados portanto possibilitam a utilização de processos contínuos. Neste contexto, a utilização de hidrotalcites Mg-Al, como catalisadores heterogéneos para produção de biodiesel foi investigada neste trabalho experimental. As hidrotalcites com diferentes razões molares Mg/Al (Mg/Al=1, 2, 3 e 4) foram preparadas pelo método de co-precipitação. As diversas matrizes catalíticas obtidas, calcinadas a diferentes temperaturas, foram caracterizadas por difracção de raios X (DRX), análise térmica (TG-DSC), espectroscopia de infravermelhos (MIR), microscopia electrónica de varrimento (SEM) e isotérmicas de adsorção com azoto (BET). Estes catalisadores foram testados na metanólise de óleos vegetais para produzir biodiesel. As hidrotalcites Mg/Al=2, HT2A e HT2B (preparada com metade da quantidade de NaOH) calcinadas a 507 ºC e 700 ºC, respectivamente, foram as que apresentaram melhores resultados ao catalisar a reacção com um rendimento em éster superior a 97%, utilizando 2.5% da massa de catalisador, em relação à massa do óleo, razão molar metanol/óleo igual a 12, temperatura reaccional de 65 ºC durante 4h. Foi também investigada a reutilização do catalisador e o efeito da temperatura de calcinação. Constatou-se que o catalisador hidrotalcite HT2B apresentou melhor comportamento catalítico pois permitiu catalisar a reacção de transesterificação até três ciclos reaccionais, convertendo em ésteres 97%, 92% e 34% no primeiro, segundo e terceiro ciclos reaccionais, respectivamente. A análise de, algumas propriedades do biodiesel obtido como, o índice de acidez, a viscosidade e o índice de iodo mostraram que os resultados obtidos estão dentro dos valores limite recomendados pela norma EN 14214. Em anexo apresenta-se uma comunicação à First International Conference on Materials for Energy, Karlsruhe, 2010.

a-SiCH Based Devices as Optical Demultiplexers

In this paper we present results on the use of a multilayered a-SiC:H heterostructure as a wavelength-division demultiplexing device (WDM) for the visible light spectrum. The WDM device is a glass/ITO/a-SiC:H (p-i-n)/ a-SiC:H(-p) /Si:H(-i)/SiC:H (-n)/ITO heterostructure in which the generated photocurrent at different values of the applied bias can be assigned to the different optical signals. The device was characterized through spectral response measurements, under different electrical bias. Demonstration of the device functionality for WDM applications was done with three different input channels covering wavelengths within the visible range. The recovery of the input channels is explained using the photocurrent spectral dependence on the applied voltage. The influence of the optical power density was also analysed. An electrical model, supported by a numerical simulation explains the device operation. Short range optical communications constitute the major application field, however other applications are also foreseen.

A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R 2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R 2 = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

Comparative analysis of near and mid-infrared spectroscopy to monitor recombinant cyprosin production

Infrared spectroscopy, either in the near and mid (NIR/MIR) region of the spectra, has gained great acceptance in the industry for bioprocess monitoring according to Process Analytical Technology, due to its rapid, economic, high sensitivity mode of application and versatility. Due to the relevance of cyprosin (mostly for dairy industry), and as NIR and MIR spectroscopy presents specific characteristics that ultimately may complement each other, in the present work these techniques were compared to monitor and characterize by in situ and by at-line high-throughput analysis, respectively, recombinant cyprosin production by Saccharomyces cerevisiae. Partial least-square regression models, relating NIR and MIR-spectral features with biomass, cyprosin activity, specific activity, glucose, galactose, ethanol and acetate concentration were developed, all presenting, in general, high regression coefficients and low prediction errors. In the case of biomass and glucose slight better models were achieved by in situ NIR spectroscopic analysis, while for cyprosin activity and specific activity slight better models were achieved by at-line MIR spectroscopic analysis. Therefore both techniques enabled to monitor the highly dynamic cyprosin production bioprocess, promoting by this way more efficient platforms for the bioprocess optimization and control.

Comparing wind generation profiles: A circular data approach

The importance of wind power energy for energy and environmental policies has been growing in past recent years. However, because of its random nature over time, the wind generation cannot be reliable dispatched and perfectly forecasted, becoming a challenge when integrating this production in power systems. In addition the wind energy has to cope with the diversity of production resulting from alternative wind power profiles located in different regions. In 2012, Portugal presented a cumulative installed capacity distributed over 223 wind farms [1]. In this work the circular data statistical methods are used to analyze and compare alternative spatial wind generation profiles. Variables indicating extreme situations are analyzed. The hour (s) of the day where the farm production attains its maximum daily production is considered. This variable was converted into circular variable, and the use of circular statistics enables to identify the daily hour distribution for different wind production profiles. This methodology was applied to a real case, considering data from the Portuguese power system regarding the year 2012 with a 15-minutes interval. Six geographical locations were considered, representing different wind generation profiles in the Portuguese system.In this work the circular data statistical methods are used to analyze and compare alternative spatial wind generation profiles. Variables indicating extreme situations are analyzed. The hour (s) of the day where the farm production attains its maximum daily production is considered. This variable was converted into circular variable, and the use of circular statistics enables to identify the daily hour distribution for different wind production profiles. This methodology was applied to a real case, considering data from the Portuguese power system regarding the year 2012 with a 15-minutes interval. Six geographical locations were considered, representing different wind generation profiles in the Portuguese system.