Small-scale comparison of pseudoperfusion feeding strategies using basal and concentrated feed media

Perfusion has long been the industrial choice for the production of unstable proteins, and nowadays is being intensively studied also for stable proteins due to its ability to keep high viable cell densities and high volumetric productivities over long operation times. However, conventional perfusion is fed with basal medium at high dilution (or perfusion) rates, causing a dilution of the protein of interest (POI) and generating large volumes of harvest to be processed in the purification steps. In this work, small-scale pseudoperfusion experiments were performed to compare the conventional perfusion strategy using basal culture medium (TC-LECC, Xell AG) with a feeding strategy employing a concentrated feed medium (TCX2D, Xell AG) that was originally developed for fed batch processes. The aim was to evaluate if the concentrated feeding strategy could equally enable high cell concentrations and high viabilities, but at higher dilution rates and potentially yielding higher product titers. The cell line CHO DP-12 (ATCC CRL-12445), producing a recombinant monoclonal antibody, was cultivated in spin tubes, and a daily medium exchange to mimick perfusion was initiated in the mid-exponential of growth phase. TC-LECC basal medium and TCX2D concentrated medium were fed at perfusion rates chosen according to their initial glucose concentration, in the range of 0.15 – 1 vvd and 0.05 – 0.35 vvd, respectively. Both processes kept the cell viability higher than 60% for approximately 15 days. Maximum viable cell density of 20.106 mL-1 was reached on day 6 for both processes. During the exponential phase, both feeding strategies provided a sustainable cell growth, though the post-feeding glucose measurements were not equivalent. MAb volumetric productivities were the same for both processes, and these results will guide the implementation of a controlled-fed process in a perfusion bioreactor

Evaluation of pseudo-perfusion feeding strategies for mAb production using a CHO cell line adapted to concentrated feed media

Perfusion has been intensively studied for biopharmaceuticals production due to the high volumetric productivities over long periods in operation. However, high dilution rates (D) used to feed basal media for therapeutic protein production imply in the generation of large harvest volumes, causing a dilution of the product of interest (POI) and increasing purification costs. Previous pseudo-perfusion studies of our group have indicated the possibility of getting a more concentrated POI (a monoclonal antibody produced by a CHO cell line) in the harvest stream using as perfusion feed a concentrated medium (as opposed to basal medium) at lower dilution rates. However, a decrease in cell viability and volumetric productivity (Pv) due to the higher osmolality related to the concentrated media were observed over cultivation time. In the present work, the same CHO cell line was first adapted to higher osmolality levels by using a concentrated feed that was originally designed for fed-batch processes (TCX2D, Xell AG, Germany), instead of using NaCl or another osmolyte to enhance osmolality. Blends at pre-defined ratios of basal medium (TC-LECC, Xell AG, Germany) and concentrated feed were prepared in order to establish an osmolality range between 280 and 380 mOsm kg-1.Cells were gradually passaged in high osmolality media for approximately one month and considered adapted when the viability recovery was higher than 90%. After adaptation of cells, small-scale pseudo-perfusion runs were carried out, starting the change of medium at the mid-exponencial cell growth phase. The glucose ratio (Glcrel), used as a factor of glucose increment, was calculated assuming the basal media as the control condition, and the range of D for each condition was determined as the inverse proportion related to Glcrel. Hence, basal medium (Glcrel =1.0, 280 mOsm kg-1) was fed in the range of 0.15 – 1 vvd, while media mixtures with Glcrel ~ 2.0 (\u3e 350 mOsm kg-1) were fed at 0.07 – 0.50 vvd. Cell viability was kept higher than 90% during at least 17 days of experiment, and viable cell densities (VCD) as high as 50E6 cells/mL were reached for all experimental conditions, including for the higher osmolality level (380 mOsm kg-1). Previous studies using cells not adapted to grow in concentrated media indicated a rapid decrease of viability as the Glcrel increased, probably due to high osmolality. A pseudo steady-state at 40-50E6 cells/mL was established for all conditions through bleeding at rates between 0.02-0.32 vvd, to avoid oxygen depletion. For the conditions of higher osmolality (350 and 380 mOsm kg-1), perfusion rate was increased at 1 vvd at late stage of run in order to avoid glucose starvation. The increase of perfusion rate has promoted a residual glucose concentration higher than the control condition (280 mOsm kg-1, fed only with the basal medium) and hence, a cultivation time longer than 20 days was achieved for both conditions. The profile of accumulated product suggested that a slightly higher osmolality level could enhance product titers over cultivation time. Although high cell density and high viabilities were achieved for all experimental condtions, volumetric productivity (Pv) values were, in average, 50% lower in comparison to the control conditio

A Simple Contrast Matching Rule for OSEM Reconstructed PET Images with Different Time of Flight Resolution

Background: Time-of-Flight (TOF) is a leading technological development of Positron Emission Tomography (PET) scanners. It reduces noise at the Maximum-Likelihood solution, depending on the coincidence–timing–resolution (CTR). However, in clinical applications, it is still not clear how to best exploit TOF information, as early stopped reconstructions are generally used. Methods: A contrast-recovery (CR) matching rule for systems with different CTRs and non-TOF systems is theoretically derived and validated using (1) digital simulations of objects with different contrasts and background diameters, (2) realistic phantoms of different sizes acquired on two scanners with different CTRs. Results: With TOF, the CR matching rule prescribes modifying the iterations number by the CTRs ratio. Without TOF, the number of iterations depends on the background dimension. CR matching was confirmed by simulated and experimental data. With TOF, image noise followed the square root of the CTR when the rule was applied on simulated data, while a significant reduction was obtained on phantom data. Without TOF, preserving the CR on larger objects significantly increased the noise. Conclusions: TOF makes PET reconstructions less dependent on background dimensions, thus, improving the quantification robustness. Better CTRs allows performing fewer updates, thus, maintaining accuracy while minimizing noise

Transportation Analysis Informing Transportation Planning and Policy at ODOT

The Transportation Planning Analysis Unit (TPAU) at ODOT helps to provide information to a large variety of transportation plans, projects, and policy questions. This allows customers to make better informed decisions and to maximize limited resources. In order to fill this role TPAU and the Oregon modeling community have a fairly large toolbox of models and analysis tools and procedures. This transportation seminar will give an overview of the role and services that TPAU provides and the different tools and processes used to fulfill that role.https://pdxscholar.library.pdx.edu/trec_seminar/1047/thumbnail.jp

A UTILIZAÇÃO DO GPS NA AQUISIÇÃO DE DADOS HIDROLÓGICOS

Este artigo apresenta a aplicação do Sistema de Posicionamento Global - GPS na locação de marcos referenciais para o nivelamento de réguas linimétricas e seções batimétricas instaladas ao longo dos rios Paraná, Ivinheima e Baía no trecho entre Porto São José e o Porto 18, Brasil. O método de posicionamento utilizando o Sistema de Posicionamento Global apresentou maior eficiência do que os obtidos por métodos tradicionais

Measurement of change in health status with Rasch models

Background: The traditional approach to the measurement of change presents important drawbacks (no information at individual level, ordinal scores, variance of the measurement instrument across time points), which Rasch models overcome. The article aims to illustrate the features of the measurement of change with Rasch models. Methods: To illustrate the measurement of change using Rasch models, the quantitative data of a longitudinal study of heart-surgery patients (N=98) were used. The scale "Perception of Positive Change" was used as an example of measurement instrument. All patients underwent cardiac rehabilitation, individual psychological intervention, and educational intervention. Nineteen patients also attended progressive muscle relaxation group trainings. The scale was administered before and after the interventions. Three Rasch approaches were used. Two separate analyses were run on the data from the two time points to test the invariance of the instrument. An analysis was run on the stacked data from both time points to measure change in a common frame of reference. Results of the latter analysis were compared with those of an analysis that removed the influence of local dependency on patient measures. Statistics t, \u3a72 and F were used for comparing the patient and item measures estimated in the Rasch analyses (a-priori \u3b1=.05). Infit, Outfit, R and item Strata were used for investigating Rasch model fit, reliability, and validity of the instrument. Results: Data of all 98 patients were included in the analyses. The instrument was reliable, valid, and substantively unidimensional (Infit, Outfit<2 for all items, R=.84, item Strata range=3.93-6.07). Changes in the functioning of the instrument occurred across the two time, which prevented the use of the two separate analyses to unambiguously measure change. Local dependency had a negligible effect on patient measures (p 65.8674). Thirteen patients improved, whereas 3 worsened. The patients who attended the relaxation group trainings did not report greater improvement than those who did not (p=.1007). Conclusions: Rasch models represent a valid framework for the measurement of change and a useful complement to traditional approaches. \ua9 Anselmi et al

Development of perfusion processes for mAb production aiming at high cell densities sustained by low cell-specific perfusion rates

Perfusion processes have been employed for approximately 25 years in the biopharmaceutical industry for the production of labile biotherapeutics, at high dilution rates and low residence times. In the last 5 years, due to the higher volumetric productivities, more compact bioreactors and possibility of integration to continuous downstream processing, perfusion has become increasingly popular also for the production of stable biopharmaceuticals, such as monoclonal antibodies (mAbs), thus allowing for process strategies involving higher residence times in the bioreactors. Viable cell densities (VCD) higher than 100 million cells per mL have been reached in the last years in perfusion processes using tangential filtration systems, such as TFF and ATF, as cell retention devices. However, these very high cell density cultures have usually been achieved through the application of high dilution rates, requiring large medium preparation facilities, large footprint for medium and harvest storage, and large capture chromatography systems to process the harvested supernatant. The aim of the present study was to develop perfusion processes for stable biotherapeutics operating at low cell-specific perfusion rates, as a way to minimize liquid handling in perfusion-based plants. The CHO-DP12 cell line (ATCC, USA) secreting a humanized mAb was used as model system. Cells were cultured in stirred-tank bioreactors, and processes based on two cell retention devices were evaluated: a CS10 inclined lamella settler (Biotechnology Solutions, USA) and an ATF2 (Repligen, USA). A chemically defined, animal-derived component free medium (TC-LECC, Xell AG, Germany) was used as basal medium. In some runs, a concentrated nutrient solution commercialized to be used as feed for fed-batch processes (TCX2D, Xell AG, Germany) was used to fortify the basal medium, aiming at a reduction of the cell-specific perfusion rate (CSPR). In a one-month perfusion process using the inclined settler and fed with basal medium, steady states lasting one week each were established at CSPRs progressively decreasing from 46 to 18 pL/cell/day. This allowed to sustain a VCD of approximately 50 million cells per mL at viabilities \u3e90% at a dilution rate (harvest + bleed) of approximately 0.9 vvd. Under these conditions, a metabolic shift towards lactate consumption was observed, and an increase in mAb titer was achieved for the lower CSPRs. Based on these results, the same feeding strategy (basal medium alone, using a CSPR of approximately 18 pL/cell/d) was employed in a one-month ATF-based perfusion process with the aim of establishing very high cell density processes. A peak VCD of 136 million cells per mL was obtained, and a smooth perfusion operation at an average of 120 million cells per mL was successfully established, with viabilities higher than 87% over the entire run (Fig. 1). In comparison with the pioneering work by Clincke et al. (2013, doi: 10.1002/btpr.1704), the CSPR used in the present work is approximately 3-fold lower than theirs, and no issues regarding pumping the cell suspension in and out of the ATF cartridge were experienced, in spite of the similar high cell densities achieved. In a further attempt to decrease even more the CSPR, we investigated a gradual fortification of the basal medium by adding a concentrated feed solution in increasing proportions (up to 40% by volume), which enabled a reduction of CSPR down to 13 pL/cell/d in an ATF-based perfusion run kept at 40 million cells per mL for over 30 days. However, a progressive decrease in cell viability, especially at the highest nutrient concentrations tested, was observed, being possibly associated to the osmolality increase caused by the gradual medium fortification. Overall, these results have shown the feasibility of achieving very high cell densities at CSPRs below 20 pL/cell/d using basal medium only, and further studies are currently ongoing to further improve the process. Please click Additional Files below to see the full abstract

Impactos da previsão sazonal de chuva sobre as previsões de vazões no sistema interligado sul/sudeste

Resumo: A otimização da geração de energia nos aproveitamentos hidrelétricos do sistema elétrico Brasileiro requer a previsão de vazões afluentes com resolução mensal. Atualmente, aplicamse procedimentos estocásticos para estimar os cenários de vazões futuras, admitindo-se que todas as seqüências de vazões observadas no passado têm a mesma probabilidade de ocorrer no futuro. O objetivo desse trabalho é avançar sobre o tema da previsão sazonal de vazões, de forma que as vazões futuras passem a ter probabilidades de ocorrência condicionadas pelo estado e processos do sistema climático. A integração das previsões sazonais de chuva produzidas por um conjunto de modelos climáticos dinâmicos e estatísticos é o elemento novo que muda o paradigma atual de isoprobabilidade dos eventos climáticos futuros. Este estudo investiga os índices de acerto das previsões de vazões mensais, com horizonte de três meses, para os principais aproveitamentos hidrelétricos do sistema Sul-Sudeste, com a aplicação de um modelo chuva-vazão que usa a previsão climática de chuva como dado de entrada. As previsões climáticas sazonais de chuva foram fornecidas pela integração das previsões produzidas por dois modelos climáticos de circulação global e por um modelo estatístico produzidas no projeto EUROBRISA. A questão científica que este estudo investiga é se o estado atual da ciência em previsão climática sazonal é adequado para produzir previsões de vazões mensais que sejam úteis para a otimização da geração hidrelétrica. A investigação do índice de acerto das previsões de vazão mensal foi realizada em três aproveitamentos hidrelétricos selecionados para amostrar as principais bacias do sistema elétrico Sul-Sudeste. A análise dos índices de acerto das previsões de vazão foi desenvolvida para o período 1987- 2001, em que a versão 2007 do projeto EUROBRISA produziu rodadas longas de previsão climática sazonal de chuva

Hydrocyclones for single-use perfusion application

Hydrocyclones (HC) are very compact devices that promote solid-liquid separation under the action of a centrifugal field. Despite the small size, HCs have a large processing capacity and do not suffer from clogging. Therefore, several publications explored HCs as a potential cell retention device in perfusion applications in the last 20 years, but limited to non-disposable lab-scale bioreactors and to relatively low cell densities (up to ~10 million cells/mL). Even though the absence of moving parts may streamline the HC manufacturing, the performance of solid-liquid separation is highly dependent on the HC internal geometry. Said that, hydrocyclones can be produced by 3D printing, making them a promising alternative for the integration of cell retention devices in single-use bioreactor bags. The performance of hydrocyclones also depends on the attachment configuration to the bioreactor and cell concentration of the feed suspension. In this work, at first rapid batch tests were carried out to evaluate the impact of: (i) cell concentration; (ii) diameter of connector installed in the recirculation loop; and (iii) controlled harvest flow rate enabled by a peristaltic pump (520U model, Watson Marlow). The main response considered was their effect on HC separation efficiency. The stainless-steel HC2015 designed for mammalian cell separation (Pinto et al., 2008) was selected for the preliminary batch tests, and also used as a benchmark for plastic prototypes produced by 3D-printing techniques. Afterwards, the same HC2015 was installed in a 50-L single-use bag (XDR50 Xcellerex, GE Healthcare) specially customized for a perfusion cultivation with a mAb producer CHO cell line. The stainless-steel HC2015 when operating at 2.3 bar provided a total separation efficiency (Et) up to 96%, and a centrifugal separation efficiency (E´) of 82% for a CHO cell suspension at 24E6 viable cells per mL Concentrated cells recovered by the underflow port did not show decrease in viability compared to the feed suspension. The reduction of a TC connector size from 19.7 to 12.7 mm resulted in the total filling of the recirculation loop with liquid, disrupting the formation of the desirable umbrella-pattern discharge of the underflow and reducing cell retention. The use of a peristaltic pump to control the overflow flow rate equivalent to perfusion rates of 1 and 2 RV (reactor volume) per day in 40-L bioreactor working volume resulted in a reduction of the E´ values and a consequent increase of cell concentration in the harvest stream. The reduction in the separation efficiency was probably due to a disturbance of the liquid flow pattern inside the HC, since it was observed that the typical gas core coming out from the overflow was absent. These features were taken into account in the HC operation in the single-use 50-L perfusion bioreactor, and a cell concentration of 50E6 cells per mL was successfully achieved with a cell-specific perfusion rate (CSPR) as low as 20 pL per cell per day. The harvest stream consisted of a natural cell bleeding leaving the overflow outlet. Moreover, the lower cell viability and average diameter in the overflow evidenced the preferential retention of viable cells returning into the bioreactor, thus providing a healthier culture environment. A 3D-printed hydrocyclone with equivalent geometry to the stainless-steel HC2015 was made and presented slightly lower separation efficiencies. Further studies proposing materials with a smoother surface and investigating further 3D-printing techniques are currently ongoing. Pinto, R. C.V., Medronho, R. A., Castilho, L. R. (2008). Separation of CHO cells using hydrocyclones. Cytotechnology, 56(1), 57–67. doi:10.1007/s10616-007-9108-