Bioprocess integration for human mesenchymal stem cells: from up to downstream processing scale-up to cell proteome characterization

Human mesenchymal stem cells (hMSC) are relevant cell-based products for autologous and allogeneic therapies. To deliver the required cell numbers and doses to therapy, scaling up production and purification processes (at least to the liter-scale) while ensuring high purity, viability and maintaining cells’ critical quality attributes (CQA) and functionality is essential [1]. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissues (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2 L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50 x 106 cell.L-1.day-1), expansion factors (14 - 16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30 - 50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (\u3e 95%) hMSC could be recovered without compromising cell’s CQA of viability, identity and differentiation potential. “Omic” tools in combination with standard analytical assays allow for a better cell characterization, increasing product and process understanding [2] and are thus fundamental for process development. Thus, alongside the standard quality assays for evaluating hMSC’s CQA, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC’ CQA. Overall, through sensitivity, robustness and throughput, this type of workflow provided the identification of specific signatures of the final product. Therefore, it proves to be essential to understand the cells’ final quality as well as to evaluate the impact of manufacturing at different stages of processing. References: [1] Pattasseril J et al, BioProcess Int. 2013, 3, 38–46. [2] Campbell A et al, Stem Cells Transl. Med. 2015, 4, 1155–1163. The authors acknowledge UniMS – Mass Spectrometry Unit team (ITQB-NOVA/iBET, Oeiras, Portugal), iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), and Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding the project CARDIOSTEM (MITP-TB/ECE/0013/2013), and the grants SFRH/BD/51940/2012 (MIT-Portugal), SFRH/BD/52302/2013, SFRH/BD/52481/2014, SFRH/BPD/86513/201

Engineering scalable manufacturing of high-quality human MSC for cell therapy: From up to downstream processing integration to cell proteome characterization

Human mesenchymal stem cells (hMSC) are relevant cell therapy products for autologous and allogeneic therapies. To deliver the required cell numbers and doses to therapy, scaling up production and purification processes (at least to the liter-scale) while ensuring high purity, viability and maintaining cells’ critical quality attributes (CQA) and functionality is essential. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting, volume reduction and washing unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissues (AT-MSC). Single-use technologies were adopted at different steps of the manufacturing workflow to support process integration and scale-up. BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2 L single-use stirred tank bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50 x 106 cell.L-1.day-1), expansion factors (14 - 16 fold) and cell recovery yields (\u3e80%). For the volume reduction and washing steps, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30 - 50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC performed better allowing recovering 18% more cells after a volume reduction factor of 50 without compromising cell’s CQA of viability, identity and differentiation potential. “Omic” tools in combination with standard analytical assays allow for a better cell characterization, increasing product and process understanding and are thus fundamental for process development. Thus, alongside the standard quality assays for evaluating hMSC’s CQA, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC’ CQA. Overall, through sensitivity, robustness and throughput, this type of workflow provided the identification of specific signatures of the final product. Therefore, it proves to be essential to understand the cells’ final quality as well as to evaluate the impact of manufacturing at different stages of processing. The authors acknowledge UniMS – Mass Spectrometry Unit team (ITQB-NOVA/iBET, Oeiras, Portugal), iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), and Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding the project CARDIOSTEM (MITP-TB/ECE/0013/2013), and the grants SFRH/BD/51940/2012 (MIT-Portugal), SFRH/BD/52302/2013, SFRH/BD/52481/2014, SFRH/BPD/86513/2012

Measles outbreak after 12 years without endemic transmission, Portugal, February to May 2017

We report a measles outbreak in two Portuguese health regions (Algarve and Lisbon and the Tagus Valley) since February 2017, and which by 31 May resulted in 28 confirmed cases, of which 16 were unvaccinated. Thirteen cases were healthcare workers. One unvaccinated teenager died. Genotype B3 was identified in 14 cases from both regions. This outbreak occurs after 12 years without endemic measles transmission, and in a context of high measles vaccination coverage and immunity.info:eu-repo/semantics/publishedVersio

Patient-physician discordance in assessment of adherence to inhaled controller medication: a cross-sectional analysis of two cohorts

We aimed to compare patient's and physician's ratings of inhaled medication adherence and to identify predictors of patient-physician discordance.(SFRH/BPD/115169/2016) funded by Fundação para a Ciência e Tecnologia (FCT); ERDF (European Regional Development Fund) through the operations: POCI-01-0145-FEDER-029130 ('mINSPIRERS—mHealth to measure and improve adherence to medication in chronic obstructive respiratory diseases—generalisation and evaluation of gamification, peer support and advanced image processing technologies') cofunded by the COMPETE2020 (Programa Operacional Competitividade e Internacionalização), Portugal 2020 and by Portuguese Funds through FCT (Fundação para a Ciência e a Tecnologia).info:eu-repo/semantics/publishedVersio

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

Introduction: The ROS1 gene rearrangement has become an important biomarker in NSCLC. The College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology testing guidelines support the use of ROS1 immunohistochemistry (IHC) as a screening test, followed by confirmation with fluorescence in situ hybridization (FISH) or a molecular test in all positive results. We have evaluated a novel anti-ROS1 IHC antibody (SP384) in a large multicenter series to obtain real-world data. Methods: A total of 43 ROS1 FISH-positive and 193 ROS1 FISH-negative NSCLC samples were studied. All specimens were screened by using two antibodies (clone D4D6 from Cell Signaling Technology and clone SP384 from Ventana Medical Systems), and the different interpretation criteria were compared with break-apart FISH (Vysis). FISH-positive samples were also analyzed with next-generation sequencing (Oncomine Dx Target Test Panel, Thermo Fisher Scientific). Results: An H-score of 150 or higher or the presence of at least 70% of tumor cells with an intensity of staining of 2+ or higher by the SP384 clone was the optimal cutoff value (both with 93% sensitivity and 100% specificity). The D4D6 clone showed similar results, with an H-score of at least 100 (91% sensitivity and 100% specificity). ROS1 expression in normal lung was more frequent with use of the SP384 clone (p < 0.0001). The ezrin gene (EZR)-ROS1 variant was associated with membranous staining and an isolated green signal FISH pattern (p = 0.001 and p = 0.017, respectively). Conclusions: The new SP384 ROS1 IHC clone showed excellent sensitivity without compromising specificity, so it is another excellent analytical option for the proposed testing algorithm

La renovación de la palabra en el bicentenario de la Argentina : los colores de la mirada lingüística

El libro reúne trabajos en los que se exponen resultados de investigaciones presentadas por investigadores de Argentina, Chile, Brasil, España, Italia y Alemania en el XII Congreso de la Sociedad Argentina de Lingüística (SAL), Bicentenario: la renovación de la palabra, realizado en Mendoza, Argentina, entre el 6 y el 9 de abril de 2010. Las temáticas abordadas en los 167 capítulos muestran las grandes líneas de investigación que se desarrollan fundamentalmente en nuestro país, pero también en los otros países mencionados arriba, y señalan además las áreas que recién se inician, con poca tradición en nuestro país y que deberían fomentarse. Los trabajos aquí publicados se enmarcan dentro de las siguientes disciplinas y/o campos de investigación: Fonología, Sintaxis, Semántica y Pragmática, Lingüística Cognitiva, Análisis del Discurso, Psicolingüística, Adquisición de la Lengua, Sociolingüística y Dialectología, Didáctica de la lengua, Lingüística Aplicada, Lingüística Computacional, Historia de la Lengua y la Lingüística, Lenguas Aborígenes, Filosofía del Lenguaje, Lexicología y Terminología

Precoating Effects in Fine Steelmaking Dust Filtration

Particle emissions into the atmosphere can cause extensive damage to the environment and human health. To improve the efficiency in the collection of submicronic particles, new filtration media appeared on the market due to new textile technologies and equipment, such as filter media developed with polytetrafluoroethylene (PTFE) membranes; however, these are more expensive. A filter coating technique called precoating is a more economical alternative that could enhance filtration efficiency. This paper aimed to evaluate the operational parameters of precoating for micro- and nanoparticle filtration and compare the results with those obtained from the PTFE membrane. For this purpose, filtration cycles were performed, using precoated polyester with hydrated lime and dolomitic limestone, polyester with a PTFE membrane, and steel industry ultrafine dust. The results showed that the precoated polyester had a longer cycle duration and lower pressure drop than the polyester with a PTFE membrane. Therefore, precoating was shown to be a great alternative to be used in bag filters in steel mills because it presented high collection efficiency for submicronic particles, in addition to increasing the bag lifespan with less energy expenditure