Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/3340
Título: Novel affinity chromatography processes for the purification of plasmid DNA using small aromatic molecules
Autor: Nunes, Catarina Caramelo
Orientador: Tomaz, Cândida Ascensão Teixeira
Almeida, Paulo Jorge da Silva
Marcos, João Carlos Nunes
Palavras-chave: Cromatografia de afinidade
Purificação de plasmídeos
Purificação de DNA
Berenit-Sepharose
DAPP-Sepharose
Data de Defesa: Out-2013
Resumo: Molecular therapies are gaining importance as an effective therapeutic approach for various types of diseases. The most efficient vectors used to introduce the therapeutic genes are of viral origin however, non-viral vectors based on pDNA are gaining popularity due to their superior safety and easy of production. These factors have increased the demand for high quantities of pharmaceutical grade plasmid DNA (pDNA). Therefore, the research for more efficient pDNA purification protocols has also increased. Moreover, the final pDNA product must meet stringent quality criteria established by the regulatory agencies. Liquid chromatography is the method of choice for the purification of pDNA, since it is simple, robust, versatile and high reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes towards designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. With these facts in mind, the aim of this work was to develop new chromatographic methods for the purification of pharmaceutical grade pDNA, with the purpose of improving the overall procedures to more effective, simple, economic and environmental-friendly ones. The minor groove binder berenil and the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) where chosen to be used as ligands in pDNA chromatographic purification studies. They were immobilized to an epoxy-activated Sepharose matrix using a relatively mild curing method, without a catalyst and with quite small ligand:Sepharose weight ratios. Berenil binds pDNA preferentially through hydrophobic interactions but other types of interaction contributions cannot be neglected. It was shown to be quite effective at separating and purifying pDNA from clarified and non-clarified cell lysates, using three different approaches, although isoform resolution was not obtained in either case. Using mild amounts of ammonium sulphate in the eluent, berenil-Sepharose support was able to purify distinct pDNA of two different sizes from clarified cell lysates. Moreover, the ability was continual when the clarification process was replaced by a second chromatographic run. In all cases plasmid solutions were in accordance to the specifications of a pharmaceutical product, however the yields were quite different: two consecutive chromatographic runs lead to lower recoveries (33%) and smaller pDNA molecules have higher recoveries using one run through the column (85% vs 45%). A negative chromatography approach was also performed with berenil-Sepharose, showing some advantages in terms of salt usage as well as procedure time. In this case the recovery yield was quite good (87%) and although pDNA solutions had a comparable purity to that obtained with the other approaches, the gDNA reduction was not so effective. DAPP is slightly A-T specific and binds DNA through non-covalent, reversible stacking interactions of the condensed aromatic moiety into two successive base pairs, while the phenyl residue gets inserted into the minor groove. In addition, protonated DAPP molecules bind to DNA much strongly due to the generation of strong electrostatic interactions. Plasmid DNA binding to DAPP-Sepharose varies with pH and is affected by the presence of salt in the eluent. In fact, total retention of clarified lysate components was only possible with a pH below DAPP's free state pKa (5.8) and the presence of salt destabilizes that same retention. So, the elution of bound species was simply performed by adding small amounts of sodium chloride to the buffers. These features were successfully applied for purification of sc pDNA with two distinct sizes that were obtained according to the regulatory agencies specifications. Once more, the recovery yield of the smaller pDNA molecule was higher than the one obtained for the largest one (94% vs 65%). In conclusion, DAPP-Sepharose showed exceptional characteristics to be used as an affinity support for the purification of pharmaceutical grade sc pDNA. In comparison with berenil- Sepharose, it uses much smaller amounts of salt, with less economic and environmental impact, while improving the quality and yield of the obtained plasmid fractions. Moreover, it is able to separate sc pDNA from linear and oc isoforms even in complex lysates. Moreover, combining DAPP-Sepharose chromatography with other optimized production, extraction and clarification procedures, can offer a number of advantages for pharmaceutical pDNA purification. Also, since the most significant disadvantage of this DAPP-Sepharose support is the relatively low capacity for pDNA, which in turn is strongly related to the solid matrix used, other more stable stationary phases with low pressure drops and interconnected macropores, that allow a high mass transfer of solutes, are quite fascinating alternatives.
URI: http://hdl.handle.net/10400.6/3340
Designação: Doutoramento em Bioquímica
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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