Jonathan De La Vega | Universidad de Sonora (original) (raw)

Papers by Jonathan De La Vega

Microscopy and Microanalysis, 2018

Journal of Pharmaceutical Sciences, 2013

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine Rbased liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (−46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine R , this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Biotechnology Progress, 2007

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretic... more Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies ... more Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5 mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3 mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285 g pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold—a maximum productivity of 100 g pDNA/(h mL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments—72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74–113% of the values obtained from the latter.

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretic... more Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

Background: Storage stability of plasmid biopharmaceuticals is a critical issue that needs to be ... more Background: Storage stability of plasmid biopharmaceuticals is a critical issue that needs to be addressed during clinical and process development. Objectives: The goal of this work was to evaluate the ability of stabilizers to prolong the stability of plasmid DNA solutions and extend the duration of transgene expression of transfected cells. Methods: A plasmid harboring the GFP gene and Chinese Hamster Ovary (CHO) cells were used as models. Plasmid solutions were formulated with the stabilizer DNAstablePlus TM , 300 mM trehalose and 300 mM cellobiose. The biological activity was monitored by transfecting CHO cells with the preparations using Lipofectamine. Results: Protection against denaturation conferred by DNAstablePlus TM at 60 °C was outstanding, with 94% of the activity preserved after 7 days compared to 76% with trehalose, 70% with cellobiose and <10% without stabilizers. While plasmid DNA stored at room temperature lost 95% of its ability to express GFP in the first month, trehalose, cellobiose and DNAstablePlus TM were able to preserve it for 6, 8 and at least 12 months, respectively. The incorporation of trehalose, cellobiose and DNAstablePlus TM in lipoplexes also contributed to extend the expression of GFP in transfected cells. While a significant loss of GFP-expressing cells (~10%) was observed after 7days with no stabilizers, formulation with DNAsta-blePlus TM , cellobiose and trehalose increased the number of cells GFP-expressing cells to more than 50%. Conclusions: The biological activity of plasmid DNA solutions stored at room temperature was extended several fold by incorporating cellobiose, trehalose and DNAstablePlus TM in the formulations.

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine R-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (−46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine R , this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000–200,000 copy number/cell), transfection efficiency (50% vs. 20%–40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Current Bionanotechnology, 2015

Journal of Chromatography A, 2014

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies ... more Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285μg pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold-a maximum productivity of 100μg pDNA/(hmL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments-72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74-113% of the values obtained from the latter.

Journal of Pharmaceutical Sciences, 2013

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Microscopy and Microanalysis, 2018

Journal of Pharmaceutical Sciences, 2013

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine Rbased liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (−46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine R , this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Biotechnology Progress, 2007

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretic... more Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 40 the column axial dispersion can be neglected should be taken with precaution.

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies ... more Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5 mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3 mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285 g pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold—a maximum productivity of 100 g pDNA/(h mL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments—72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74–113% of the values obtained from the latter.

Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretic... more Breakthrough performance of plasmid DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves (BTC) were significantly affected by inlet flow rate and solute concentration. In the theoretical analysis, a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine, coupled to the solution of the partial differential model equations, was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant, and the forward interaction rate constant, which are the parameters of the membrane transport model. The analysis shows that as inlet concentration or flow rate increases, the deviation of the model from the experimental behavior decreases. The BTCs displacement as inlet concentration increases was explained in terms of a greater degree of column saturation reached and more efficient operation accomplished. The degree of column saturation was not influenced by inlet flow rate. It was necessary to consider in the column model the slight variation in the BTC produced by the axial dispersion, in order to accomplish the experimental curve dispersion. Consequently, the design criteria that for Pe > 40 the column axial dispersion can be neglected should be taken with precaution.

Background: Storage stability of plasmid biopharmaceuticals is a critical issue that needs to be ... more Background: Storage stability of plasmid biopharmaceuticals is a critical issue that needs to be addressed during clinical and process development. Objectives: The goal of this work was to evaluate the ability of stabilizers to prolong the stability of plasmid DNA solutions and extend the duration of transgene expression of transfected cells. Methods: A plasmid harboring the GFP gene and Chinese Hamster Ovary (CHO) cells were used as models. Plasmid solutions were formulated with the stabilizer DNAstablePlus TM , 300 mM trehalose and 300 mM cellobiose. The biological activity was monitored by transfecting CHO cells with the preparations using Lipofectamine. Results: Protection against denaturation conferred by DNAstablePlus TM at 60 °C was outstanding, with 94% of the activity preserved after 7 days compared to 76% with trehalose, 70% with cellobiose and <10% without stabilizers. While plasmid DNA stored at room temperature lost 95% of its ability to express GFP in the first month, trehalose, cellobiose and DNAstablePlus TM were able to preserve it for 6, 8 and at least 12 months, respectively. The incorporation of trehalose, cellobiose and DNAstablePlus TM in lipoplexes also contributed to extend the expression of GFP in transfected cells. While a significant loss of GFP-expressing cells (~10%) was observed after 7days with no stabilizers, formulation with DNAsta-blePlus TM , cellobiose and trehalose increased the number of cells GFP-expressing cells to more than 50%. Conclusions: The biological activity of plasmid DNA solutions stored at room temperature was extended several fold by incorporating cellobiose, trehalose and DNAstablePlus TM in the formulations.

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine R-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (−46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine R , this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000–200,000 copy number/cell), transfection efficiency (50% vs. 20%–40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.

Current Bionanotechnology, 2015

Journal of Chromatography A, 2014

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies ... more Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285μg pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold-a maximum productivity of 100μg pDNA/(hmL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments-72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74-113% of the values obtained from the latter.

Journal of Pharmaceutical Sciences, 2013

This work investigates the impact of quality attributes (impurity content, plasmid charge, and co... more This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models. The plasmid was purified by hydrophobic interaction chromatography (HIC)/gel filtration, and with three commercial kits, which combine the use of chaotropic salts with silica membranes/glass fiber fleeces. The HIC-based protocol delivered a plasmid with the smallest hydrodynamic diameter (144 nm) and zeta potential (-46.5 mV), which is virtually free from impurities. When formulated with Lipofectamine®, this plasmid originated the smallest (146 nm), most charged (+13 mV), and most stable lipoplexes. In vitro transfection experiments further showed that these lipoplexes performed better in terms of plasmid uptake (∼500,000 vs. ∼100,000-200,000 copy number/cell), transfection efficiency (50% vs. 20%-40%), and GFP expression levels (twofold higher) when compared with lipoplexes prepared with plasmids isolated using commercial kits. Overall our observations highlight the potential impact that plasmid purification methodologies can have on the outcome of gene transfer experiments and trials.