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Abstract(s)
O vírus influenza é a causa de uma doença respiratória grave, comummente chamada de gripe, de elevada transmissibilidade e distribuição global e que provoca elevados níveis de mortalidade por todo o mundo. A melhor opção para travar esta doença viral e contagiosa é a vacinação. Apesar das vacinas da gripe convencionais, baseadas na tecnologia de ovos de galinha embrionados, serem normalmente eficazes, apenas conferem proteção contra as estirpes dominantes de cada ano e têm um risco zoonótico associado. Deste modo, são produzidas anualmente novas vacinas para antecipar as estirpes circulantes do vírus, processo que leva meses até estar concluído. As vacinas de DNA, baseadas na tecnologia do DNA recombinante, são uma alternativa promissora relativamente à vacinação convencional uma vez que são efetivas, e têm um processo de produção genérico e fácil de transpor para larga escala, em oposição ao complexo processo de produção associado às vacinas convencionais. Esta terapêutica consiste na expressão de um plasmídeo com os genes que codificam os antigénios de interesse num hospedeiro apropriado, permitindo a sua produção in vivo. No entanto, as vacinas de DNA requerem uma grande quantidade e com elevado grau de pureza, da isoforma superenrolada do DNA plasmídico biologicamente ativa. A cromatografia tem sido a técnica de escolha na purificação do pDNA por ser reprodutível, fiável e fácil de transpor para larga escala. No entanto, as matrizes convencionais usadas para este fim apresentam algumas limitações como as suas baixas propriedades de transferência de massa e baixa capacidade de ligação para moléculas maiores com o pDNA. Neste sentido, os criogéis podem ser utilizados no processo downstream por serem uma boa alternativa às matrizes cromatográficas convencionais, devido aos seus poros grandes e interconectados, boa difusibilidade e capacidade de ligação entre a molécula alvo e o suporte. O ácido barbitúrico, bem como os seus análogos tio- e/ou N,N-di-substituídos podem ser facilmente ligados aos criogéis ativados com grupos epóxido fazendo uso da bem descrita reatividade nucleofílica do C-5 na forma de enol ou enolato.
Deste modo, foram preparados suportes cromatográficos baseados em criogéis derivatizados em condições acídicas e utilizando ligandos barbitúricos representativos como o ácido barbitúrico, tiobarbitúrico e 1,3-difenil-2-tiobarbitúrico, com diferentes concentrações de ligando. Assim, no presente trabalho de investigação, foi testada a purificação do pDNA NTC 7482-41H-VA2 HA com 6.471 kbp que codifica a glicoproteína antigénica de superfície do vírus influenza, hemaglutinina, utilizando suportes de criogéis derivatizados com barbituratos.
Para isto, foram quimicamente sintetizadas colunas de criogel com diferentes densidades de ligando. Inicialmente, foi estudado o perfil de retenção e eluição bem como a separação da isoforma superenrolada do pDNA da isoforma circular aberta, com base nas diferentes matrizes sintetizadas, sendo a coluna de criogel com ácido 1,3-difenil-2-tiobarbitúrico com 5,0 mg/mL, aquela que demonstrou os melhores resultados, sendo por isso a escolhida para os estudos posteriores de separação de isoformas. Este suporte foi explorado através da manipulação de diferentes concentrações de sal e valores de pH, nos equipamentos cromatográficos Flash Protein Liquid Chromatographic e ÄKTA Purifier, numa tentativa de separar a isoforma superenrolada do pDNA como exigido pelas agências reguladoras. A melhor estratégia de separação das isoformas consistiu na aplicação de um gradiente crescente de cloreto de sódio usando 10,0 mM de tampão acetato de sódio com pH 5,0 como passo de ligação, e uma estratégia de eluição por dois passos usando o mesmo tampão com 0,550 M, seguido de 1,0 M de cloreto de sódio, que resultou numa separação parcial das isoformas sc e oc do pDNA do amostra pré-purificada. Na amostra de lisado, a utilização de 10,0 mM de tampão acetato de sódio com pH 5,0 como passo de ligação, e uma estratégia de eluição por dois passos usando o mesmo tampão com 0,263 M, seguido de 2,500 M de cloreto de sódio promoveu a separação da isoforma superenrolada do DNA do RNA, proteínas e outras impurezas com 65,60 % de pureza e um grau de recuperação de 88,0 %. Os resultados obtidos demonstram a aplicabilidade dos criogéis de ácido 1,3-difenil-2-tiobarbitúrico com 5,0 mg/mL na separação e purificação das vacinas de pDNA contra o vírus influenza por cromatografia de afinidade.
The influenza virus is the cause of a severe respiratory disease, commonly called flu, with high transmissivity, global distribution and high levels of mortality all over the world. The best option to prevent this viral and contagious disease is vaccination. Despite the conventional influenza vaccines, based on embryonated chicken eggs technology, are usually effective, they only provide protection against the dominant strains each year and have a zoonotic risk associated. Thus, new vaccines must be produced annually to anticipate circulating virus strains, a process which takes months to be completed. DNA vaccines are based on recombinant DNA technology and are a promising alternative to conventional vaccines, since they are effective, and have a generic production process and easy to upscale contrary to the complex production process associated to conventional vaccines. This therapy consists of an expression plasmid with the gene encoding the antigen of interest in a suitable host, allowing their production in vivo. However, to scale-up this technology, pDNA vaccines require a large quantity of highly pure biologically active supercoiled (sc) plasmid DNA (pDNA) with pharmaceutical grade which is obtained by the purification steps at downstream processing level. Chromatography has been the technique of choice in pDNA purification because is reproducible, reliable and easy to transpose to large scale. However, conventional matrices used for this purpose have some limitations such as their low mass transfer properties and low binding capacity for larger molecules such as pDNA. In this regard, cryogels can be used in downstream process to be a good alternative to conventional chromatography matrices, due to its large pores and interconnected diffusivity and good binding capacity between the target molecule and the support. Barbituric acid, their thio and/or N,N-disubstituted analogs can be easily linked to epoxy based cryogels using the very well-known reactivity of the C-5 as enol or enolate. In the present study, chromatographic supports were prepared using cryogels derivatized with barbiturates such as barbituric acid, thiobarbituric and 1,3-diphenyl-2-thiobarbituric at different concentrations. Thus, the purification of a plasmid NTC 7482-41H-VA2 HA encoding the surface glycoprotein of influenza, hemagglutinin was performed using cryogels derivatized with barbiturates with different ligand densities as supports. Initially, the retention and elution profile was studied as well as the separation of pDNA sc isoform isoform of open circular, in different matrices. The cryogel with 1,3-diphenyl-2-thiobarbituric acid (5,0 mg / ml) has shown the best results and was therefore chosen for further studies separation of isoforms using different salt concentrations and pH values , in the Flash Protein Liquid Chromatographic and ÄKTA Purifier equipments,. The better separation strategy was applied using 10,0 mM sodium acetate buffer at pH 5,0 as binding step and the elution was achieved with two steps using the same buffer containing 0,550 M, followed by 1,0 M chloride sodium. The lysate was purified using 10,0 mM sodium acetate buffer at pH 5,0 as binding step, followed by two steps using the same buffer with 0,263 M and by 2,50 M sodium chloride respectively. The separation of sc isoform from RNA, proteins and other impurities was performed and a 65.60 % purity and degree of recovery of 88,0 % was obtained. The results demonstrate the potential applicability of cryogels derivatized with acid 1,3-diphenyl-2-thiobarbituric in the separation and purification of of influenza pDNA vaccines by affinity chromatography.
The influenza virus is the cause of a severe respiratory disease, commonly called flu, with high transmissivity, global distribution and high levels of mortality all over the world. The best option to prevent this viral and contagious disease is vaccination. Despite the conventional influenza vaccines, based on embryonated chicken eggs technology, are usually effective, they only provide protection against the dominant strains each year and have a zoonotic risk associated. Thus, new vaccines must be produced annually to anticipate circulating virus strains, a process which takes months to be completed. DNA vaccines are based on recombinant DNA technology and are a promising alternative to conventional vaccines, since they are effective, and have a generic production process and easy to upscale contrary to the complex production process associated to conventional vaccines. This therapy consists of an expression plasmid with the gene encoding the antigen of interest in a suitable host, allowing their production in vivo. However, to scale-up this technology, pDNA vaccines require a large quantity of highly pure biologically active supercoiled (sc) plasmid DNA (pDNA) with pharmaceutical grade which is obtained by the purification steps at downstream processing level. Chromatography has been the technique of choice in pDNA purification because is reproducible, reliable and easy to transpose to large scale. However, conventional matrices used for this purpose have some limitations such as their low mass transfer properties and low binding capacity for larger molecules such as pDNA. In this regard, cryogels can be used in downstream process to be a good alternative to conventional chromatography matrices, due to its large pores and interconnected diffusivity and good binding capacity between the target molecule and the support. Barbituric acid, their thio and/or N,N-disubstituted analogs can be easily linked to epoxy based cryogels using the very well-known reactivity of the C-5 as enol or enolate. In the present study, chromatographic supports were prepared using cryogels derivatized with barbiturates such as barbituric acid, thiobarbituric and 1,3-diphenyl-2-thiobarbituric at different concentrations. Thus, the purification of a plasmid NTC 7482-41H-VA2 HA encoding the surface glycoprotein of influenza, hemagglutinin was performed using cryogels derivatized with barbiturates with different ligand densities as supports. Initially, the retention and elution profile was studied as well as the separation of pDNA sc isoform isoform of open circular, in different matrices. The cryogel with 1,3-diphenyl-2-thiobarbituric acid (5,0 mg / ml) has shown the best results and was therefore chosen for further studies separation of isoforms using different salt concentrations and pH values , in the Flash Protein Liquid Chromatographic and ÄKTA Purifier equipments,. The better separation strategy was applied using 10,0 mM sodium acetate buffer at pH 5,0 as binding step and the elution was achieved with two steps using the same buffer containing 0,550 M, followed by 1,0 M chloride sodium. The lysate was purified using 10,0 mM sodium acetate buffer at pH 5,0 as binding step, followed by two steps using the same buffer with 0,263 M and by 2,50 M sodium chloride respectively. The separation of sc isoform from RNA, proteins and other impurities was performed and a 65.60 % purity and degree of recovery of 88,0 % was obtained. The results demonstrate the potential applicability of cryogels derivatized with acid 1,3-diphenyl-2-thiobarbituric in the separation and purification of of influenza pDNA vaccines by affinity chromatography.
Description
Keywords
Barbituratos Criogéis Cromatografia de Afinidade Gripe Influenza Pdna Purificação Vacinas de Dna