Patricia Guerrero-Germán

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.

Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography

A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow‐fiber tangential filtration and frontal anion‐exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30‐fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane‐based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.

Segregated growth kinetics of Escherichia coli DH5α‐NH36 in exponential‐fed perfusion culture for pDNA vaccine production

The clinical demand of plasmid DNA (pDNA) has been increasing constantly. An exponential‐fed perfusion (EFP) culture is a new mode for plasmid production for clinical trials and commercialization. However, the culture conditions may lead to cell filamentation and growth cessation. In this study, the variation of the physiological state and the plasmid contents of Escherichia coli DH5α hosting pVAX1‐NH36 in an EFP culture for application as a Leishmaniasis vaccine was investigated. The culture performance was monitored using flow cytometry (FC) and real‐time quantitative PCR. The FC studies showed a high viability of cell population and a constant distribution of complexity and size. A high homogeneity of pDNA (>95 % of supercoiled) was obtained, which might be attributed to a better culture environment. The obtained plasmid specific and volumetric yields of 1.8 mg/g dcw and 36.5 mg/L represent typical values for laboratory‐scale plasmid production in a defined medium. A segregated kinetic model of the perfusion system was developed and fitted to the experimental data (R2 > 0.96). A practical conclusion of this work is that a space–time yield analysis of a bioprocess requires a viability evaluation. This new strategy of culture operation might help in the efficient production of pDNA for therapeutic use.

Plasmid DNA pre-purification by tangential flow filtration

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 Darcys 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 interest was performed by constant flow in batch mode achieving a 41% of impurities removal. These results suggest the use of a combination of concentration--diafiltration steps for the pre-purification of the plasmid lysates processed. The electrophoretic and high-performance liquid chromatography by hydrophobic interaction analysis showed that the plasmid supercoiled isoform maintained its integrity and no appreciable plasmid loss was present.

Methylene Blue Loaded PLGA Nanoparticles: Combined Emulsion, Drug Release Analysis and Photodynamic Activity

The use of polymeric nanoparticles for the control release of photosensitizer compounds such as methylene blue represents a promising option for cancer treatment. Methylene blue (MB) has been of great interest in many areas of clinical medicine, from neurological disorders to cancer chemotherapy [12]. It can be used in photodynamic therapy, which consists on the application of MB in the area of interest, and then activated by light at 665 nm producing reactive oxygen species that leads to the death of the target cell via oxidative damage. Polymeric nanoparticles loaded with methylene blue (MB-PNP) were prepared by using a combined emulsification technique [4-5]. Briefly, MB is dissolved in deionized water and into a DCM solution containing MB and PLGA. The mixture is emulsified at 22% of amplitude (26.5 μm) by sonication. Next, an aqueous solution of 5% w/v PVA is added into the mixture and a second emulsification is carried during at 75% amplitude (90 μm). The solvent is evaporated under magnetic stirring, at room temperature. MB-PNPs are washed by three centrifugation cycles and freeze-dried for further characterization. All experiments were performed by triplicate.

Batch Equilibrium and Kinetic Studies of Plasmid pCI Adsorption onto Perfusion Particles

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.

Simulation of Frontal Protein Affinity Chromatography Using MATLAB

In this study a transport model that includes axial dispersion in the bulk liquid, pore diffusion, external film resistance and finite kinetic rate, was used to mathematically describe a frontal affinity chromatography system. The corresponding differential equations system was solved in a simple and accurate form by using the numerical method of lines implemented in MATLAB. The solution was compared with experimental data from literature and the analytic Thomas solution. The frontal affinity chromatography of lysozyme to Cibacron Blue Sepharose was used as a model system. A good fit to the experimental data was obtained with the simulated runs of the transport model using this methodology. This approach was used to perform a parametric analysis of the experimental frontal affinity system. The solution of the transport model results in simple way to predict frontal affinity performance as well a better understanding of the fundamental mechanisms responsible for the separation.

Low intensity sonosynthesis of iron carbide@iron oxide core-shell nanoparticles

Here we demonstrate a simple method for the organic sonosynthesis of stable Iron Carbide@Iron Oxide core-shell nanoparticles (ICIONPs) stabilized by oleic acid surface modification. This robust synthesis route is based on the sonochemistry reaction of organometallic precursor like Fe(CO)5 in octanol using low intensity ultrasonic bath. As obtained, nanoparticles diameter sizes were measured around 6.38 nm ± 1.34 with a hydrodynamic diameter around 25 nm and an estimated polydispersity of 0.27. Core-Shell structure of nanoparticles was confirmed using HR-TEM and XPS characterization tools in which a core made up of iron carbide (Fe3C) and a shell of magnetite (γ-Fe2O3) was found. The overall nanoparticle presented ferromagnetic behavior at 4 K by SQUID. With these characteristics, the ICIONPs can be potentially used in various applications such as theranostic agent due to their properties obtained from the iron oxides and iron carbide phases.

Evaluation of a combined emulsion process to encapsulate methylene blue into PLGA nanoparticles

The delivery of photosensitizer compounds using biodegradable nanoparticles could improve dosage, controlled release and its bioavailability. In this study, methylene blue (MB) loaded PLGA nanoparticles (MB-PNP) are prepared by a new approach combining single and double emulsification techniques. Comparisons of MB-PNP obtained with the combined and the individual techniques are presented. Nanoparticles are characterized by dynamic light scattering, laser Doppler electrophoresis and scanning electron microscopy. Particles prepared by the combined technique presented hydrodynamic diameters of 186 nm. The sizes of MB-PNP obtained from the single emulsion technique are similar to the combined technique, while the diameter of particles prepared by double emulsion increased from 201 nm to 287 nm as the TDL increased. MB-PNP displayed an average zeta potential between −21 mV and −28 mV for all formulations. MB loading ranges between 0.3–1.4%, while the encapsulation efficiency ranges from 8–14%, both depending on the TDL and the preparation technique. In vitro release studies show a monophasic release profile that was analyzed by considering the mechanisms of initial burst, drug diffusion and a combination of them. Experimental results could be better described using a mathematical model of release that simultaneously combines the mechanisms of initial burst and drug diffusion. The approach presented to encapsulate MB and also to analyze the drug release could be extended to other drugs with partial solubility.

A STRAIGHTFORWARD METHOD TO SCALE-UP PLASMID DNA INTERMEDIATE RECOVERY BY TANGENTIAL FLOW ULTRAFILTRATION

ABSTRACT 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.