Production of recombinant antibody fragment in Pichia pastoris

Foram estudados a composição e o pH do meio de cultivo para a produção do fragmento de anticorpo (scFv) anti-LDL(-), expresso em Pichia pastoris recombinante. Os experimentos que definiram a composição e pH do meio assim como a concentração inicial de células na fase de indução foram realizados em agitador orbital a 250 rpm, com temperatura de 30 ºC na fase de crescimento e 20 ºC na fase de indução, durante 72 horas, com adição diária de 1% (v/v) de metanol. Para modificação do meio foi realizado um planejamento experimental empregando como variáveis independentes: extrato de soja, casaminoácidos e ureia, os quais substituíram o YNB e a biotina presentes no meio padrão (BMMY). Apesar de haver maior produção no meio com extrato de soja, o meio contendo 10 g.L-1 de casaminoácidos foi selecionado, uma vez que este favoreceu a etapa de purificação. A partir da faixa de pH estudada entre 3,0 e 8,0, determinou-se que o pH 8,0 no início da fase de indução favorece a maior produção. Finalmente, o meio BMMY-CA (pH 8,0) foi utilizado para cultivo em biorreator com volume de trabalho de 10L e a partir deste cultivo foram calculados os parâmetro cinéticos (velocidades de crescimento, de consumo de substrato e de produção, bem como produtividade). Diante do conjunto de experimentos realizados, foi possível otimizar a composição do meio de cultivo e as condições operacionais, que possibilitaram um aumento do rendimento bem como aumento do volume de produção do scFv anti-LDL(-) em biorreator.Composition and pH culture medium for the production of anti- LDL(-) antibody fragment (scFv) were studied in recombinant yeast Pichia pastoris. The experiments that defined medium composition, pH and the initial cell concentration in the induction phase were carried out in baffled shaker flasks at 250 rpm, with temperature at 30°C for growth phase and 20°C for induction phase, during 72 hours with daily addition of 1% (v/v) methanol. A design of experiments employing for medium modification with independent variables: soy extract, casamino acids and urea, which replaced the YNB and biotin present in the standard medium (BMMY) was conducted for medium formulation. Even though, there was an increase in medium production with soy extract, the medium containing 10 g.L-1 casamino acids was selected since it favors the purification step. Through a pH range study (3.0 to 8.0), it was determined that pH 8.0 during induction phase offered higher levels. Then, the best results from shaker flask cultivation (BMMY-CA with casamino acids and pH 8.0) were carried out in 1L bioreactor, showing a biomass and a scFv production increase compared to the standard, BMMY (pH 6.0). Finally, the medium BMMY-CA (pH 8,0) was submitted to a volume scale-up into a 10L bioreactor, which was also used to evaluate kinetic parameters (rates of growth, substrate consumption and production as well as productivity). In conclusion, by optimizing culture medium and operating conditions it was possible to increase yield and scale-up the production of scFv anti-LDL(-) in bioreactor

Desenvolvimento de nanoestruturas poliméricas para encapsulação do antitumoral miltefosina

Miltefosine (hexadecylphosphocholine, HePC), a synthetic antitumor designed from natural phospholipids, is clinically approved for cutaneous metastases of breast cancer and cutaneous lymphoma. This drug acts mainly at cellular membrane level, where it accumulates and interferes with lipid metabolism and lipid-dependent signaling pathways leading the cells to apoptosis. However, HePC systemic and peroral administration induces hemolysis and mucosal toxicity, respectively. To overcome these limitations, we investigated the protective properties of colloidal polymeric micelles (PM) composed by Pluronics, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide). We found that both Pluronic composition and concentration modulate the hemolytic profile of incorporated drug (HePC-PM) by increasing the drug amount to cause in vitro hemolysis. Moreover, small-angle X-ray scattering (SAXS) was used to assess structural information of interactions between HePC and PM. Additionally, we showed that HePC-PM prevented mucosal irritation, decreasing bleeding and lysis of blood vessels in a chicken chorioallantoic membrane model. Interestingly, HePC-PM increased the in vitro selective cytotoxicity against cervix tumor cells rather healthy fibroblasts, suggesting a differential uptake of these nanostructures by tumor cells. Furthermore, we also found that HePC induces cytotoxicity and decrease cell survival, migration and proliferation in osteosarcoma cells in vitro. We showed that cytotoxicity by HePC is associated with caspase-3 activation, DNA fragmentation, apoptotic-like bodys formation and inhibition of both constitutive and cytokine-stimulated Akt/PKB phosphorylation. HePC-PM clearly reduces the drug cytotoxic effects. Finally, we demonstrated that Pluronic F127 polymeric micelles are efficient for cargo delivering the encapsulated drug preferentially into tumor cells rather than healthy cells. These findings together suggest that Pluronic F127 PM reduce drug side effects and provide a potential alternative for systemic delivery of HePC, as well as other amphiphilic drugs.Miltefosina (hexadecilfosfocolina, HePC), um fármaco antitumoral sintético desenvolvido a partir de fosfolipídios naturais, é clinicamente aprovada para o tratamento tópico de metástases de câncer de mama e linfomas cutâneos. Atua principalmente nas membranas celulares, onde se acumula e interfere no metabolismo lipídico e nas vias de sinalização dependentes de lipídios levando as células à apoptose. No entanto, quando administrada sistemicamente ou oralmente a HePC induz hemólise e toxicidade de mucosas, respectivamente. Para superar estas reações adversas investigamos os efeitos protetores conferidos por micelas poliméricas coloidais (PM) compostas por Pluronics, copolímeros tribloco de poli(óxido de etileno) e poli(óxido de propileno). Inicialmente, encontramos que a composição e concentração do Pluronic modulam o perfil hemolítico do fármaco encapsulado (HePC-PM), aumentando a quantidade necessária de HePC para causar hemólise in vitro. Além disso, utilizamos o espalhamento de raios-X a baixo ângulo (SAXS) para obter informações estruturais das interações entre HePC e PM. Em seguida, mostramos que HePC-PM preveniu a irritação da mucosa, diminuindo a hemorragia e a vasoconstricção em membrana corioalantóica de ovos embrionados. Estudos in vitro demonstraram que a HePC-PM aumentou seletivamente a citotoxicidade contra células de carcinoma HeLa em relação a fibroblastos saudáveis, sugerindo captação diferencial dessas nanoestruturas pelas células tumorais. Além disso, relatamos que, in vitro, a HePC induz citotoxicidade, diminui a sobrevivência, migração e proliferação osteossarcomas. Esta citotoxicidade está associada à ativação da caspase-3, fragmentação do DNA, formação de corpos apoptóticos e inibição da fosforilação de Akt/PKB. Adicionalmente, HePC-PM reduz os efeitos citotóxicos nestas linhagens. Finalmente, demonstramos que as micelas poliméricas de Pluronic F127 são eficientes para a entrega intracelular fármacos preferencialmente em células tumorais, e em menor grau em células saudáveis. Em conjunto, os dados sugerem que este sistema nanoestruturado reduz a toxicidade da HePC e representa uma alternativa potencial para a administração sistêmica deste e de outros fármacos anfifílicos

Biomoléculas extraídas por SBAs: ejemplos prácticos

The actual biotechnology industry demands fast and economic upstream and downstream processes to purify biomolecules. In this context, different purification techniques, that offer both high recovery and purity to the final product, have been assayed by different research groups. Liquid-liquid extraction with aqueous two-phase systems is one of the most studied methodologies for bio-separation. This technique presents several advantages such as mild conditions of working, cost-effectiveness, short-time consumption and high recovery percentage of the final product. With the aim to present a comparison of liquid-liquid extractions with other techniques, several aqueous two-phase extraction processes of biomolecules are presented in this review. We presented the advantages and disadvantages of them as of the compared systems. In general, the highest final product purities are achieved when different methodologies are combine, being the chromatographic ones the most applied in the last stages for the high purification factor obtained after them. Alternative methodologies, such as aqueous two-phase systems (ATPS), i.e., PEG/salts or ionic liquids; aqueous two-phase micellar systems, using solvents and surfactants; and extractive fermentation with ATPS, are relevant for both cost-effectiveness and time-saving of the purification process.La industria biotecnologica actual exige procesos rápidos y económicos para la producción y la purificación de biomoléculas. En este contexto, diferentes técnicas separativas que ofrezcan un alto rendimiento y una alta pureza del producto final han sido evaluadas por diferentes grupos de investigación. La extracción líquido-líquido con sistemas bifasicos acuosos (SBAs) es una de las metodologías más estudiadas para bio-separación. Esta técnica presenta varias ventajas, tales como condiciones suaves de trabajo, alta relación costo-beneficio, cortos tiempos de consumo y alto porcentaje de recuperación del producto final. Con el objetivo de comparar extracciones líquido-líquido con otras técnicas de separación, diferentes procesos de extracción de biomoléculas seran presentados en este trabajo de revisión. Adicionalmente, se presentaran las ventajas y desventajas de cada uno de ellos. En general, los más altos grados de pureza del producto final fueron obtenidos al combinar diferentes metodologías, siendo las cromatográficas las más aplicados en las últimas etapas debido a los elevados factores de purificación obtenidos después de ellas. Metodologías alternativas como por ejemplo, SBAs basados en mezclas de PEG/sal o liquídos iónicos/sal, sistemas micelares de dos fases acuosas (SMDFAs) formados por solventes o surfactantes; fermentación extractiva utilizando SBA, también resultan ser relevantes debido a sus bajos costos y cortos tiempo en sus procesos de purificación

Biomolecules extracted by ATPs: practical examples

The actual biotechnology industry demands fast and economic upstream and downstream processes to purify biomolecules. In this context, different purification techniques, that offer both high recovery and purity to the final product, have been assayed by different research groups. Liquid-liquid extraction with aqueous two-phase systems is one of the most studied methodologies for bio-separation. This technique presents several advantages such as mild conditions of working, cost-effectiveness, short-time consumption and high recovery percentage of the final product. With the aim to present a comparison of liquid-liquid extractions with other techniques, several aqueous two-phase extraction processes of biomolecules are presented in this review. We presented the advantages and disadvantages of them as of the compared systems. In general, the highest final product purities are achieved when different methodologies are combine, being the chromatographic ones the most applied in the last stages for the high purification factor obtained after them. Alternative methodologies, such as aqueous two-phase systems (ATPS), i.e., PEG/salts or ionic liquids; aqueous two-phase micellar systems, using solvents and surfactants; and extractive fermentation with ATPS, are relevant for both cost-effectiveness and time-saving of the purification process.La industria biotecnologica actual exige procesos rápidos y económicos para la producción y la purificación de biomoléculas. En este contexto, diferentes técnicas separativas que ofrezcan un alto rendimiento y una alta pureza del producto final han sido evaluadas por diferentes grupos de investigación. La extracción líquido-líquido con sistemas bifasicos acuosos (SBAs) es una de las metodologías más estudiadas para bio-separación. Esta técnica presenta varias ventajas, tales como condiciones suaves de trabajo, alta relación costo-beneficio, cortos tiempos de consumo y alto porcentaje de recuperación del producto final. Con el objetivo de comparar extracciones líquido-líquido con otras técnicas de separación, diferentes procesos de extracción de biomoléculas seran presentados en este trabajo de revisión. Adicionalmente, se presentaran las ventajas y desventajas de cada uno de ellos. En general, los más altos grados de pureza del producto final fueron obtenidos al combinar diferentes metodologías, siendo las cromatográficas las más aplicados en las últimas etapas debido a los elevados factores de purificación obtenidos después de ellas. Metodologías alternativas como por ejemplo, SBAs basados en mezclas de PEG/sal o liquídos iónicos/sal, sistemas micelares de dos fases acuosas (SMDFAs) formados por solventes o surfactantes; fermentación extractiva utilizando SBA, también resultan ser relevantes debido a sus bajos costos y cortos tiempo en sus procesos de purificación.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Marine-derived fungi: diversity of enzymes and biotechnological applications

The ocean is considered to be a great reservoir of biodiversity. Microbial communities in marine environments are ecologically relevant as intermediaries of energy, and play an important role in nutrient regeneration cycles as decomposers of dead and decaying organic matter. In this sense, marine-derived fungi can be considered as a source of enzymes of industrial and/or environmental interest. Fungal strains isolated from different substrates, such as invertebrates, decaying wood, seawater, sediments and mangrove detritus, have been reported to be producers of hydrolytic and/or oxidative enzymes, with alginate lyase, amylase, cellulase, chitinase, glucosidase, inulinase, keratinase, ligninase, lipase, nuclease, phytase, protease and xylanase being among the enzymes produced by fungi of marine origin. These enzymes present temperature and pH optima ranging from 35 to 70 ºC, and 3.0 to 11.0, respectively. High-level production in bioreactors is mainly performed using submerged-state fermentation. Certain marine-derived fungal strains present enzymes with alkaline and cold-activity characteristics, and salinity is considered an important condition in screening and production processes. The adaptability of marine-derived fungi to oceanic conditions can be considered an attractive point in the field of fungal marine biotechnology. In this review, we focus on the advances in discovering enzymes from marine-derived fungi and their biotechnological relevance