LYTAG-driven purification strategies as a key to integrate and intensify the downstream processing of monoclonal antibodies

Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeutics in the biotechnological and biopharmaceutical industry with more than 250 mAbs currently undergoing clinical trials. High titer producing cultures and complex mixtures containing high cell densities, together with an increasing growing demand for highly pure mAbs is making recovery and purification processes hot targets for improvement and opens important technological challenges in mAbs manufacturing platforms. This work explores the use of an affinity dual ligand based on a choline binding polypeptide tag (LYTAG) fused with the synthetic antibody Z domain (LYTAG-Z) as a tool to integrate and optimized the downstream processing of mAbs. Upon addition of this ligand to an animal cell culture broth, antibody-LYTAG-Z complexes are formed which can be easily captured and separated from host cell impurities by affinity partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol –PEG, as PEG molecules have the ability to binding to the choline binding sites of LYTAG. Integration of clarification and primary mAbs recovery was successfully accomplished using a system composed of 6% PEG 3350 Da and 7% dextran 500,000 Da in which an extraction yield of 89% and a clarification higher than 95% were achieved. IgG-rich phases were further processed by chromatography, using three different strong anion exchange matrices charged with quaternary methyl amines (a choline analogue) – CIMmultus QA, HiTrap Q FF and gPore NW Q. A two-elution method was developed for the separation of the antibody-LYTAG-Z complexe, allowing simultaneous purification of the antibody and recovery of the ligand. The process was successfully scale-up 10000 times allowing a global antibody recovery of 70% with a purity of 89% and enabling 100% cell removal

Promoção da saúde no ambiente escolar no Brasil

OBJETIVO Avaliar os ambientes escolares aos quais estão expostos estudantes do nono ano no Brasil e nas cinco regiões do país segundo diretrizes de promoção da saúde. MÉTODOS Estudo transversal, de 2012, com amostra representativa do Brasil e suas macrorregiões. Escolares do nono ano e gestores de escolas públicas e privadas foram entrevistados. Foi proposto o Escore de Promoção de Saúde no Ambiente Escolar (EPSAE) e foi estimada a distribuição dos escolares segundo esse escore e segundo odds ratio (OR) brutas e ajustadas, por regressão ordinal, para exposição dos escolares a escolas com escores mais elevados, segundo as variáveis independentes. RESULTADOS Um escolar tem mais probabilidade de frequentar escola com EPSAE elevado na região Sul (OR = 2,80; IC95% 2,67–2,93) se a escola for privada privada (OR = 4,52; IC95% 4,25–4,81) e estiver localizada em capital de estado e se o escolar tiver 15 anos de idade ou mais, exercer atividade remunerada ou tiver mãe ou pai com escolaridade superior. CONCLUSÕES São importantes as desigualdades entre regiões do país e entre redes de ensino, tornando clara a necessidade de alocação de recursos e ações que promovam maior equidade.OBJECTIVE Evaluate the school environments to which ninth-year students are exposed in Brazil and in the five regions of the country according to health promotion guidelines. METHODS Cross-sectional study from 2012, with a representative sample of Brazil and its macroregions. We interviewed ninth-year schoolchildren and managers of public and private schools. We proposed a score of health promotion in the school environment (EPSAE) and estimated the distribution of school members according to this score. Crude and adjusted odds ratios (OR) were used, by ordinal regression, to determine the schoolchildren and schools with higher scores, according to the independent variables. RESULTS A student is more likely to attend a school with a higher EPSAE in the South (OR = 2.80; 95%CI 2.67–2.93) if the school is private (OR = 4.52; 95%CI 4.25–4.81) and located in a state capital, as well as if the student is 15 years of age or older, has a paid job, or has parents with higher education. CONCLUSIONS The inequalities among the country’s regions and schools are significant, demonstrating the need for resources and actions that promote greater equity

Synthesis of esters derived from [4-(2-hydroxyethyl)-[1,2,3]triazol-1-yl]-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose

The synthesis of six novel esters containing a triazole ring and an acetylated glucose residue are presentedFCT and FEDER, for National NMR Network (Bruker Avance II 400). We are also grateful for research grant VZ MSMT-0021627501, Czech Republi

Synthesis of esters derived from [4-(2-hydroxy-ethyl)-[1,2,3]triazol-1-YL-2,3,4,6-tetra-o-acetylglucopyranose

2,3,4-Tri-O-benzyl-alpha-D-methylglucoside was prepared and reacted with several acids: benzoic, phenylacetic, 2-(3-bromo-propoxy)-benzoic, acetylsalicylic and 4-(toluene-4-sulfonylamino)-benzoic. The products were isolated with low to fair yields and fully characterized by usual analytical techniquesFundação para a Ciência e Tecnologia and FEDER, for National NMR Network (Bruker Avance III 400). We are also grateful for research grant VZ MSMT-0021627501, Czech Republic

Função pulmonar de crianças com leucemia aguda na fase de manutenção da quimioterapia1

ResumoObjetivoO objetivo desse estudo foi avaliar a função pulmonar de crianças com leucemia aguda.MétodosTrata‐se de um estudo observacional do tipo analítico transversal com 34 crianças, divididas nos grupos A (17 crianças com leucemia aguda na fase de manutenção do tratamento quimioterápico) e B (17 crianças saudáveis). Os grupos foram pareados em relação ao sexo, idade e altura. A espirometria foi mensurada utilizando um espirômetro Microloop Viasys®, de acordo com as recomendações da American Thoracic Society e European Respiratory Society. As pressões respiratórias máximas foram mensuradas utilizando um manovacuômetro digital MVD300 (Globalmed®). As pressões inspiratória máxima e expiratória máxima foram mensuradas a partir do volume residual e da capacidade pulmonar total, respectivamente.ResultadosO grupo A apresentou diminuição significativa da pressão inspiratória máxima quando comparado ao grupo B. Não foram observadas diferenças entre os dados espirométricos dos dois grupos avaliados, bem como entre os valores de pressão inspiratória máxima e pressão expiratória máxima do grupo A com os limites inferiores propostos como referência.ConclusãoAs crianças com leucemia aguda, linfoide ou mieloide não apresentam mudança das variáveis espirométricas e da pressão expiratória máxima durante o período de manutenção do tratamento quimioterápico; no entanto, há uma diminuição da pressão inspiratória máxima.AbstractObjectiveThe aim of this study was to assess the pulmonary function of children with acute leukemia.MethodsCross‐sectional observational analytical study that enrolled 34 children divided into groups A (17 with acute leukemia in the maintenance phase of chemotherapy) and B (17 healthy children). The groups were matched for sex, age and height. Spirometry was measured using a spirometer Microloop Viasys® in accordance with American Thoracic Society and European Respiratory Society guidelines. Maximal respiratory pressures were measured with an MVD300 digital manometer (Globalmed®). Maximal inspiratory pressures and maximal expiratory pressures were measured from residual volume and total lung capacity, respectively.ResultsGroup A showed a significant decrease in maximal inspiratory pressures when compared to group B. No significant difference was found between the spirometric values of the two groups, nor was there any difference between maximal inspiratory pressure and maximal expiratory pressure values in group A compared to the lower limit values proposed as reference.ConclusionChildren with acute leukemia, myeloid or lymphoid, during the maintenance phase of chemotherapy exhibited unchanged spirometric variables and maximal expiratory pressure; However, there was a decrease in inspiratory muscle strength

Minimal intervention in dentistry : which is the best approach for silorane composite restoration repairs?

This study aimed to evaluate surface treatments, adhesives and composites for repairing silorane based restorations. One hundred and twenty truncated cones (2 mm smaller diameter and 4 mm larger diameter) made of silorane composite were divided in 12 g

MÍDIA E PSICOLOGIA: UMA REVISÃO DE LITERATURA

A presente pesquisa teve como objetivo realizar uma revisão de literatura a fim de identificar a produção sobre mídia desenvolvida pela área da psicologia no Brasil. Para tanto, foram feitos levantamentos em bases de dados nacionais, utilizando duas combinações de descritores: mídia e psicologia; comunicação e psicologia. Com os dados obtidos foi possível verificar a exígua produção sobre o tema. Por meio da análise dos artigos foram identificados autores e instituições que se destacam na produção do conhecimento sobre o assunto. Também, verificou-se que a mídia aparece nos artigos tanto como objeto de estudo, quanto como instrumento para coleta de informações. A análise apontou que o interesse pela psicologia em pesquisar a mídia aumentou na última década. Nos últimos anos, o meio digital vem ganhando destaque entre as pesquisas. Por fim, espera-se que esta pesquisa, além de apresentar um panorama da produção acadêmica da psicologia brasileira sobre mídia, possa apontar as lacunas, indicando novas e urgentes possibilidades de investigação sobre a interface mídia e psicologia

Perfil fermentativo, composição química e estabilidade aeróbia de silagens de brotos de mandioca com palma forrageira

Objetivou-se avaliar o efeito de níveis de palma forrageira (0, 15, 30 e 45%) em silagens de brotos de mandioca sobre o perfil fermentativo, composição química e estabilidade aeróbica. Quatro tratamentos foram testados com 5 repetições, totalizando 20 silos. A inclusão de palma forrageira nas silagens de broto de mandioca promoveu aumento nas perdas por gases (P < 0,001), capacidade tampão (P < 0,001), pH (P = 0,033), matéria mineral (P < 0,001), carboidratos totais (P < 0,001), carboidratos não fibrosos (P < 0,001) e diminuição nos teores de matéria seca (P < 0,001), matéria orgânica (P < 0,001), extrato etéreo (P = 0,002) e proteína bruta (P < 0,001). Efeito quadrático foi observado para perdas por efluentes (P < 0,001), sendo as maiores perdas por efluentes (94.78 kg/t natural matter) obtidas com a inclusão de 30% de palma forrageira nas silagens de brotos de mandioca. A inclusão de palma forrageira na silagem de brotos de mandioca promoveu redução do pH durante a exposição ao oxigênio (P = 0,008). A inclusão de palma forrageira em até 45% na silagem de brotos de mandioca provoca alterações nas características fermentativas e nutricionais, porém, os valores encontrados nas silagens estão de acordo com os padrões de silagens de boa qualidade.The aim was to evaluate the effect of the cactus pear levels (0, 15, 30, and 45%) in cassava shoot silages on the fermentation profile, chemical composition, and aerobic stability. Four treatments were tested with five repetitions, totaling 20 silos. The inclusion of cactus pear in cassava shoots silages promoted an increase in gas losses (P < 0.001), buffering capacity (P < 0.001), pH (P = 0.033), mineral matter (P < 0.001), total carbohydrates (P < 0.001), non-fiber carbohydrates (P < 0.001) and a decreasing in dry matter (P < 0.001), organic matter (P < 0.001), ether extract (P = 0.002), and crude protein (P < 0.001) content. A quadratic effect was observed for effluent losses (P < 0.001), with greater effluent losses (94.78 kg/t natural matter) obtained with the inclusion of 30% cactus pear in cassava shoots silages. Cactus pear inclusion in cassava shoots silage promoted a reduction in the pH during oxygen exposure (P = 0.008). Including cactus pear in up to 45% of cassava silage, shoots cause changes in the fermentation and nutritional characteristics. However, values found in the silages are by good quality standards

Sustainable strategies based on glycine–betaine analogue ionic liquids for the recovery of monoclonal antibodies from cell culture supernatants

Monoclonal antibodies (mAbs) are of crucial interest for therapeutic purposes, particularly in vaccination and immunization, and in the treatment of life-threatening diseases. However, their downstream processing from the complex cell culture media in which they are produced still requires multiple steps, making mAbs extremely high-cost products. Therefore, the development of cost-effective, sustainable and biocompatible purification strategies for mAbs is in high demand to decrease the associated economic, environmental and health burdens. Herein, novel aqueous biphasic systems (ABS) composed of glycine–betaine analogue ionic liquids (AGB-ILs) and K2HPO4/KH2PO4 at pH 7.0, the respective three-phase partitioning (TPP) systems, and hybrid processes combined with ultrafiltration were investigated and compared in terms of performance as alternative strategies for the purification and recovery of anti-human interleukin-8 (anti-IL-8) mAbs, which are specific therapeutics in the treatment of inflammatory diseases, from Chinese Hamster Ovary (CHO) cell culture supernatants. With the studied ABS, mAbs preferentially partition to the IL-rich phase, with recovery yields up to 100% and purification factors up to 1.6. The best systems were optimized in what concerns the IL concentration, allowing to take advantage of IL-based three-phase partitioning approaches where a precipitate enriched in mAbs is obtained at the ABS interface, yielding 41.0% of IgG with a purification factor of 2.7 (purity of 60.9%). Hybrid processes combining the two previous techniques and an ultrafiltration step were finally applied, allowing the recovery of mAbs from the different fractions in an appropriate buffer solution for further biopharmaceutical formulations, while allowing the simultaneous IL removal and reuse. The best results were obtained with the hybrid process combining TPP and ultrafiltration, allowing to obtain mAbs with a purity higher than 60%. The recyclability of the IL was additionally demonstrated, revealing no losses in the purification and recovery performance of these systems for mAbs. The biological activity of anti-IL-8 mAbs is maintained after the several purification and recovery steps, indicating that the novel ABS, three-phase partitioning and hybrid processes comprising AGB-ILs are promising and sustainable strategies in mAbs downstream processing.publishe

INTRAOPERATIVE ANTICOAGULATION MONITORIZATION IN VASCULAR SURGERY – DOES A BLIND DOSIS FITS ALL?

Introduction: Unfractionated heparin (UFH) has been used for decades to prevent thrombotic events during vascular surgery. Although it is known that UFH has a complex and nonlinear pharmacokinetics, with great individual variability, anticoagulation monitorization in vascular surgery is not routine and a standard empirical dose is often used. Activated clotting time (ACT) has been shown to be a simple, reliable and inexpensive way to monitor UFH anticoagulant effect, being routinely used during cardiac surgery. However, heparinisation remains a dilemma in vascular surgery and few studies emphasized the role of anticoagulation monitoring in this setting. Objectives: To investigate whether a fixed heparin dose of 5000 IU in arterial vascular surgery results in adequate and homogeneous heparinisation in all patients. Secondary endpoints: to identify preoperative factors for heparin response, intraoperative events and outcomes. Methods: This observational prospective pilot study included 30 consecutive patients undergoing arterial vascular surgery. ACT monitoring was performed before clamping and at 3, 30 and 60 minutes after 5000 IU UFH bolus. Preoperative and intraoperative data were also accessed. A target ACT of ≥ 200 s was set, taking in account of the lowest ACT value admitted by vascular surgery recommendations. Results: The average ACT value increased to 210.20 ± 28.82 s (1.61 ± 0.25 times vs baseline) 3 minutes after bolus, then declined to 191.60 ± 21.86 s and 173.4 ± 21.37 s after 30 and 60 minutes, respectively. Three minutes after UFH bolus, 53% patients had ACT ≥ 200 s, decreasing to one third and 7% at 30 and 60 minutes, respectively. Even when weight-based, a correlation between heparin dose per kilogram and ACT change was not found (r = 0.187; p = 0.322). There was also no correlation between ACT values and preoperative hemoglobin, platelet count, creatinine clearance or INR. There was a positive correlation between preoperative aPTT and intraoperative ACT measurements (r = 0.432; p = 0.017). There was no difference between ACT values and previous antithrombotic/anticoagulant therapy and between intraoperative ACT and intraoperative blood loss. Conclusions: This study confirms that administrating a fixed or even a weight-based heparinisation is insufficient to provide consistent anticoagulation levels in all patients. Perioperative anticoagulation should be monitored and ACT-based. Larger clinical RCT's are warranted