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Authors
Abstract(s)
A cromatografia é uma poderosa ferramenta preparativa e analítica e um dos métodos de
purificação mais importantes aplicados em processos “downstream” no ramo da
Biotecnologia. A cromatografia de afinidade é uma das técnicas de eleição na purificação de
DNA plasmídico (pDNA), sendo o ligando usado um dos fatores principais para o sucesso do
processo. A purificação cromatográfica de pDNA requer suportes altamente porosos devido ao
seu alto peso molecular e à elevada viscosidade das suas soluções. Os criogéis são materiais
poliméricos macroporosos com canais interconectados, sendo uma alternativa aos suportes
particulados convencionais na purificação de pDNA. O 3,8-diamino-6-fenilfenantridina (DAPP)
e o tosilato de 5-pent-4-en-1-il-fenantridínio são ligandos de origem sintética e apresentam
na sua estrutura um braço espaçador e carga positiva, o que os torna potencialmente
adequados para a imobilização num criogel usando e o metacrilado de 2-hidroxietilo (HEMA)
como co-monómero, para posteriores estudos cromatográficos. Neste projeto, foram testados
dois métodos de imobilização do ligando à matriz e dois suportes de controlo. Foram
efetuados vários ensaios cromatográficos, de retenção e de separação das isoformas
superenrolada (sc) e circular aberta (oc) de pDNA. Adicionalmente, testou-se a separação da
isoforma sc a partir de uma amostra de lisado clarificado. Os resultados mostram que o DAPP
(5 mg/mL) e o HEMA são os ligandos com melhores perfis de retenção sendo por isso
escolhidos para os estudos de separação de isoformas. Tanto o DAPP como o HEMA mostraram
seletividade para a isoforma sc usando como passo de ligação uma solução tampão de acetato
de sódio 10 mM pH 5,0 e como passo de eluição, tampão acetato de sódio 10 mM pH 5,0 na
presença de NaCl. Quanto à amostra de lisado clarificado, apenas o HEMA revelou capacidade
de separação da isoforma sc dos contaminantes RNA e proteínas. Estes estudos iniciais
permitiram demonstrar o potencial da aplicabilidade de criogéis com ligandos sintéticos para
a separação e purificação de pDNA por cromatografia de afinidade.
Chromatography is a powerful analytical and preparative tool and one of the major methods applied in downstream purification processes in the biotechnology field. Affinity chromatography is one of the techniques of choice in purification processes of plasmid DNA (pDNA) and the ligands are the main factor for a successful process. Purification of pDNA requires highly porous matrices due to their high molecular weight and high viscosity of the solutions. The cryogels are macroporous polymeric materials with interconnected channels becoming an alternative to the conventional particulate supports in the purification of pDNA. The 3,8-Diamino-6-phenylphenanthridine (DAPP), tosylate 5-(pent-4 -en-yl)phenanthridinium and 2-hydroxyethyl methacrylate (HEMA) are synthetic ligands with an easy synthesis that present in their structure one spacer arm and positive charge which made them potentially suitable ligands for immobilization onto to a cryogel and subsequent chromatographic studies. In this study two immobilization methods of ligand to the matrix and two control supports were studied. Several chromatographic experiments were carried out: initially, the retention, and separation of supercoiled (sc) isoform from open circular (oc) isoform in a sample of concentrated pDNA and then separation of sc isoforms from impurities in a sample of clarified lysate. The results showed that DAPP 5 (mg/mL) and HEMA have the best retention profiles being chosen for the isoforms separation studies. Both DAPP and HEMA showed selectivity for the sc isoform using 10 mM sodium acetate buffer pH 5, as binding step, and two step elution with a 10 mM sodium acetate buffer pH 5 in the presence NaCl. Only HEMA showed ability to separate sc isoform from impurities such as RNA and proteins in the clarified lysate sample. These initial studies have demonstrated the potential applicability of cryogels with synthetic ligands to pDNA separation and purification by affinity chromatography.
Chromatography is a powerful analytical and preparative tool and one of the major methods applied in downstream purification processes in the biotechnology field. Affinity chromatography is one of the techniques of choice in purification processes of plasmid DNA (pDNA) and the ligands are the main factor for a successful process. Purification of pDNA requires highly porous matrices due to their high molecular weight and high viscosity of the solutions. The cryogels are macroporous polymeric materials with interconnected channels becoming an alternative to the conventional particulate supports in the purification of pDNA. The 3,8-Diamino-6-phenylphenanthridine (DAPP), tosylate 5-(pent-4 -en-yl)phenanthridinium and 2-hydroxyethyl methacrylate (HEMA) are synthetic ligands with an easy synthesis that present in their structure one spacer arm and positive charge which made them potentially suitable ligands for immobilization onto to a cryogel and subsequent chromatographic studies. In this study two immobilization methods of ligand to the matrix and two control supports were studied. Several chromatographic experiments were carried out: initially, the retention, and separation of supercoiled (sc) isoform from open circular (oc) isoform in a sample of concentrated pDNA and then separation of sc isoforms from impurities in a sample of clarified lysate. The results showed that DAPP 5 (mg/mL) and HEMA have the best retention profiles being chosen for the isoforms separation studies. Both DAPP and HEMA showed selectivity for the sc isoform using 10 mM sodium acetate buffer pH 5, as binding step, and two step elution with a 10 mM sodium acetate buffer pH 5 in the presence NaCl. Only HEMA showed ability to separate sc isoform from impurities such as RNA and proteins in the clarified lysate sample. These initial studies have demonstrated the potential applicability of cryogels with synthetic ligands to pDNA separation and purification by affinity chromatography.
Description
Keywords
Criogéis Cromatografia de Afinidade Dapp Dna Plasmídico Fenantridinas Hema Purificação