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The growing demand of plasmid DNA for gene therapy and third generation vaccines has encouraged the development of simple, robust and scalable production bioprocesses. Bearing in mind the purification step, the membrane separation processes may be a viable and cost-effective option that may be used without compromising the plasmid integrity. In this work, an operation of tangential flow ultrafiltration in a hollow-fiber module was used for the pre-purification of plasmid pVAX1-NH36 from clarified lysates of Escherichia coli DH5α culture. The ultrafiltration system was characterized using model solutions to determine the membrane resistance, plasmid concentration on the membrane wall and the system mass-transfer coefficient. In the analysis, a model based on Darcy's law and the stagnant film layer was used. These results allowed to estimate an optimal flux of 0.0016 cm/s and an optimal bulk concentration for the diafiltration process of 5.50 µg/mL. The concentration of lysates containing the plasmid of int...

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA for third-generation vaccines. The process development requires conduct basic studies in batch format to determine the characteristic parameters of the biomolecule-adsorbent system. In this work, the equilibrium and kinetic performance of the plasmid pCI adsorption onto ion-exchange perfusive particles POROS 50 HQ was studied. The isotherm obtained was appropriately described by the Langmuir model, with the parameters q m = 29.15 mg PCI/mL of adsorbent and K d = 0.0145 mg de pCI/mL. These values show a greater capacity and more specific adsorption of pCI onto the anion-exchange (AE) perfusive particles then onto AE adsorptive membranes previously studied. The kinetics of adsorption was described adequately by a lumped resistance model using a kinetic constant of k 1 = 0.0067 mL/mg s.

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

The interest in gene therapy and production of vaccines based on plasmid DNA (pDNA) has increased in recent years because they are novel techniques for the treatment or prevention of genetic diseases or infections. A typical bioprocess for the production of pDNA includes four stages: fermentation, primary recovery, intermediate recovery, and final purification. The crucial stage in the process is the capture of pDNA, as it is essential to provide good quality material for the purification stage. In this work, the behavior of tangential flow ultrafiltration (TFUF) in the intermediate recovery of plasmid pVAX1-LipL32 from lysates of E. coli ferments was investigated as an alternative to recovery operations that use chemical agents harmful to the environment. The concentration profile of the nucleic acids in both filtrate and retentate were determined by global analysis. The plasmid integrity during the recovery stages was verified at several intervals by means of electrophoresis analysis. A simple mathematical model was obtained to describe the process behavior that can be used as a straightforward method for the scale-up of the recovery process.