A scalable high-cell-density Escherichia coli culture method was established to obtain pharmaceutical grade plasmid DNA (pDNA), together with an optimized purification process. The effects of several components of the medium, such as carbon and nitrogen sources that ensure bacterial nutritional needs, were studied. The operation parameters, such as temperature, shaking and aeration, were set and the optimum values of cell growth and specific pDNA productivity in culture were determined. The subsequent purification process for pharmaceutical grade pDNA was implemented, by combining RNA precipitation with ammonium sulfate and two successive chromatographic steps consisting of size exclusion chromatography and reverse phase-high performance liquid chromatography. This work comprised the first report on the use of reverse phase to purify DNA for application in humans.

A scalable high-cell-density Escherichia coli culture method was established to obtain pharmaceutical grade plasmid DNA (pDNA), together with an optimized purifi cation process. The effects of several components of the medium, such as carbon and nitrogen sources that ensure bacterial nutritional needs, were studied. The operation parameters, such as temperature, shaking and aeration, were set and the optimum values of cell growth and specifi c pDNA productivity in culture were determined. The subsequent purifi cation process for pharmaceutical grade pDNA was implemented, by combining RNA precipitation with ammonium sulfate and two successive chromatographic steps consisting of size exclusion chromatography and reverse phase-high performance liquid chromatography. This work comprised the fi rst report on the use of reverse phase to purify DNA for application in humans.

FDA added "Therapeutic plasmid DNA vector" to the list of well-characterized biotechnology product and gene therapy has been moved rapidly from laboratory scale to clinical trials.As a matter of urgency, it is essential to develop new protocols to obtain highquality plasmids with high yields.The understanding, optimization and validation of steps, from pDNA design and host strain selection to mass-cultivation and purification, are crucial if this novel vaccine technology will become commercially successful (Prather et al., 2003).Innovative methodologies and new engineering tools are needed to expand the window of opportunity for process design.The advantages of the procedure described over existing technology to produce pharmaceutical grade pDNA for gene therapy include a high cell density culture, improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal derived components or raw materials.By employing this simple, scalable and applicable approach we concluded successfully clinical trial (phase I); and currently is in Phase II using the pIDKE2 plasmid; which is the main component CIGB's candidate vaccine against Hepatitis C virus.The principal topics of this chapter are: Fermentation process including design of high-cell-density culture, Downstream process using tangential flow filtration and chromatography, which are widely accepted methods for pDNA purification and act as orthogonal techniques platform, Scale up of bioprocess, Quality control of pDNA manufacturing and finally regulatory aspects.

The importance of somatotropin as a growth promoting agent and immune-stimulator has long been recognized and its potential application in the fish farming industry has been an active research area. In the work reported here, we sought to improve the stability of a previously obtained truncated somatotropin by applying a 60 °C heat shock to the culture supernatant containing this molecule, and then compared its effects with and without heat shock on larval growth and immune functions. We observed that the treatment with heat shock at 60 °C enhanced protein stability, growth and innate immune functions in tilapia larvae.

Membrane chromatography offers a good solution to the challenge of developing an efficient chromatographic procedure for plasmid DNA purification. The large convective pores of anion exchange membranes allow plasmid DNA to access all the anionic binding sites of the membrane at high flow rates. Here we demonstrate that the pIDKE2 plasmid can be purified from a recombinant Escherichia coli lysate using a Sartobind D membrane combined with size exclusion chromatography to render material with 95% purity and an overage yield of 50%. This process yields therapeutically suitable plasmid DNA that meets all regulatory requirements.

Growth hormone (GH) is a single chain polypeptide of approximately 22 kDa, produced by the pituitary gland and with pleiotropic functions among vertebrates. It mainly regulates body growth, being also involved in reproduction, immunity and osmoregulation in teleost fish. In order to obtain the tilapia (Oreochromis hornorum) growth hormone (tiGH) in Pichia pastoris cells, its gene was cloned into expression vectors in both with and without a heterologous secretion signal. The tiGH, obtained either intracellularly or extracellularly in P. pastoris cells, was characterized showing its production as associated to the cellular rupture precipitate with an approximate molecular weight of 22 kDa; or being secreted with an approximate molecular weight of 18 kDa, respectively. The mass spectrometry analysis of the recombinant protein obtained in the culture supernatant corroborated the identity of the protein as tiGH but lacking 46 aminoacids of its carboxyl terminal sequence. The tiGH biological activity of P. pastoris intact cells producing this protein was carried out in tilapia larvae (Oreochromis sp.), showing, for the first time, that it is possible to stimulate fish growth by immersion baths with recombinant tiGH-producing yeast. On the contrary what was previously postulated for mammals, the evaluation of P. pastoris cells expressing the truncated variant of tiGH demonstrated that this protein is also able to stimulate growth and immune system in fish. This is the first report of a biologically active, truncated GH variant in fish.

A novel purification method was developed for recovering the plDKE2 plasmid, which encodes a polyprotein encompassing amino acids 1-650 of the hepatitis C virus (HCV) polyprotein, from recombinant Escherichia coli. Bacterial cells were harvested and subjected to alkaline lysis. After centrifugation, the host contaminant RNA was removed from the clarified alkaline lysate using a highly loaded size-exclusion chromatography and the eluted fraction was applied to reverse-phase media: POROS R1 50. Finally, a second size-exclusion chromatography step was carried out to purify the plasmid DNA from other small molecular-weight contaminants. Analytical methods proved that the purified plasmid DNA had a purity of 95% after Sephocryl S 1000. Plasmid identity was confirmed by restriction enzyme digestion. Biological activity of the purified plasmid was confirmed in vivo, immunized mice developed a positive antibody response against all HCV structural antigens. This procedure offers an alternative to traditional methods that use organic reagents, mutogenic and toxic compounds, and animal-derived enzymes. Although the yields are lower when using this method, it is scalable and free of animal-derived substances and organic solvents.