Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

Ebrahimpour, Maryam; Jahanshahi, Mohsen; Hosenian, Amir H.

doi:10.1365/s10337-010-1676-9

Cited by 8 publications

(5 citation statements)

References 28 publications

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“…Frontiers in Bioengineering and Biotechnology frontiersin.org in other works (Sousa et al, 2008a;Ebrahimpour et al, 2010). Therefore, the equilibration and binding condition were maintained with 150 mM NaCl.…”

Section: Screening Of Desorption Conditionsmentioning

confidence: 99%

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing

Ferreira,

Marie,

Berger

et al. 2024

Front. Bioeng. Biotechnol.

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Since the world first approved gene therapeutics, nucleic acid-based therapies have gained prominence. Several strategies for DNA-based therapy have been approved, and numerous clinical trials for plasmid DNA (pDNA)-based vaccines are currently in development. Due to the rising interest in pDNA for vaccination and gene therapy, plasmid manufacturing must become more effective. One of the most critical steps is downstream processing, involving isolation and purification procedures. To comply with the regulatory guidelines, pDNA must be available as a highly purified, homogeneous preparation of supercoiled pDNA (sc pDNA). This process undertakes several challenges, primarily due to the diversity of molecules derived from the producer organism. In this study, different resins were tested for the adsorption and selective polishing of sc pDNA. To identify optimal pDNA adsorption conditions, batch and column assays were performed with different resins while promoting electrostatic and hydrophobic interactions. The effect of ionic strength, pH, and contact time were evaluated and optimized. Additionally, static and dynamic binding capacities were determined for the selected resins. Analytical chromatography and agarose gel electrophoresis were used to assess the selectivity of the most promising resins toward sc pDNA isoform. Also, genomic DNA, endotoxins, and proteins were quantified to characterize the final sc pDNA quality. At the same time, the recovery and purity yields were evaluated by quantification of sc pDNA after the purification procedure. Overall, the results of the chromatographic assays using agmatine- and arginine-based resins have shown promising potential for sc pDNA polishing. Both resins demonstrated excellent binding capacity for pDNA, with agmatine outperforming arginine-based resin in terms of capacity. However, arginine-based resin exhibited a superior pDNA recovery yield, reaching a notable 52.2% recovery compared to 10.09% from agmatine. Furthermore, both resins exhibited high relative purity levels above 90% for the sc pDNA. The comprehensive characterization of the recovered sc pDNA also revealed a significant reduction in gDNA levels, reinforcing the potential of these prototypes for obtaining high-quality and pure sc pDNA. These findings highlight the promising applications of both resins in scalable pDNA purification processes for gene therapy and biopharmaceutical applications.

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Section: Screening Of Desorption Conditionsmentioning

confidence: 99%

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing

Ferreira,

Marie,

Berger

et al. 2024

Front. Bioeng. Biotechnol.

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“…Therefore, such hydrodynamic studies should be performed in the columns with new design, although most of them were performed in commercial columns. Recently, a simple customized NBG expanded bed column was constructed by the Nanobiotechnology group (Babol University of Technology) (Shahavi et al, 2008;Ebrahimpour et al, 2010). Hydrodynamic parameter determination at various position of the customized NBG column is crucial.…”

Section: Introductionmentioning

confidence: 99%

Investigation of hydrodynamic parameters in a novel expanded bed configuration: local axial dispersion characterization and an empirical correlation study

Taheri

Jahanshahi

Mosavian

et al. 2012

Braz. J. Chem. Eng.

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-Study of liquid behavior in an expanded bed adsorption (EBA) system is important for understanding, modeling and predicting nanobioproduct/biomolecule adsorption performance in such processes. In this work, in order to analyze the local axial dispersion parameters, simple custom NBG (Nano Biotechnology Group) expanded bed columns with 10 and 26 mm inner diameter were modified by insertion of sampling holes. Based on this configuration, the particles and liquid can be withdrawn directly from various axial positions of the columns. Streamline DEAE particles were used as solid phase in this work. The effects of factors such as liquid velocity, viscosity, settled bed height and column diameter on the hydrodynamic parameters were investigated. Local bed voidages in different axial bed positions were measured by a direct procedure within the column with 26 mm diameter. Increasing trend of voidage with velocity at a certain position of the bed and with bed height at a certain degree of expansion was observed. Residence time distribution (RTD) analysis at various bed points showed approximately uniform hydrodynamic behavior in the column with 10 mm diameter while a decreasing trend of mixing/dispersion along the bed height at a certain degree of expansion was seen in the column with 26 mm diameter. Also lower mixing/dispersion occured in the smaller diameter column. Finally, a combination of two empirical correlations proposed by Richardson-Zaki and Tong-Sun was successfully employed for identification of the bed voidage at various bed heights (RSSE=99.9%). Among the empirical correlations presented in the literatures for variation of the axial dispersion coefficient, the Yun correlation gave good agreement with our experimental data (RSSE=87%) in this column.

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“…Generally, the design of the column configuration is another critical effective factor in the formation of excellent hydrodynamic properties. Recently, a simple customized Nano Biotechnology Group (NBG) EBA column was constructed by our group (Babol University of Technology) and was employed successfully for recovery and purification of nano/bioproducts [1,2,20,21]. In our previous work, an ordinary NBG column was modified to examine axial changes of clean feedstock (buffer) hydrodynamic parameters within this new design contactor (unpublished data).…”

Section: Introductionmentioning

confidence: 99%

Hydrodynamic Characteristics and Adsorption Particularity of Nanobiological Feedstock Along the Bed Height in a Novel Chromatography Column

2012

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Expanded bed adsorption (EBA) is a practical method for the separation of nanoparticulates. In order to analysis the local hydrodynamic and adsorption behavior of nanoparticle (NP)-based biological feedstock, a modified Nano Biotechnology Group EBA column with a 26-mm inner diameter was used to withdraw liquid from different axial positions of the column. Fabricated egg albumin (EA) NPs with an average size of 70 nm were employed as a model system and viral size/charge mimic to assess the relationship between hydrodynamic and adsorption performance of NPs at the different column regions. The effects of influential factors, including flow velocity and initial concentration of NPs, on NP hydrodynamic behavior and adsorption kinetics along the bed height were investigated. NP hydrodynamic studies confirmed that non-uniform behavior dominated the system and a decreasing trend of liquid mixing/dispersion with increase of bed height was observed in this column. The results demonstrated an increase in the mixing/dispersion at certain bed heights with the increase in both the velocity and feed initial concentration. Breakthrough curves were measured at various column points to determine the adsorption performance [dynamic binding capacity (DBC) and yield] in different bed positions/zones. Yield and DBC of NPs were improved along the bed height, whereas liquid velocity had the opposite effect. Increasing the initial concentration of NPs enhanced only the DBC. Separation of EA NPs under optimal conditions was 87 %, which is an excellent result for a one-pass frontal chromatography method. KeywordsEBA chromatography Á Modified NBG column Á EA NPs Á Axial hydrodynamic behavior Á Axial adsorption performance List of symbols B o Bodenstein number C Exit concentration of NPs in bed effluent (kg m -3 ) C o Initial concentration of NPs (kg m -3 ) C 9Exit concentration that has left the 9-cm sample port and is measured at this port (kg m -3 ) C 12Exit concentration that has left the 12-cm sample port and is measured at this port (kg m -3 )Volume of feed loaded onto the column until C 9 reaches a certain degree of NP breakthrough (m 3 ) V f,12Volume of feed loaded onto the column until C 12 reaches a certain degree of NP breakthrough (m 3 ) V l Liquid volume above the particles in the sampling syringe (m 3 ) V L,0-9 Liquid volume present in the bed voidage for the 0-to 9-cm column zone (m 3 ) V L,0-12 Liquid volume present in the bed voidage for the 0-to 12-cm column zone (m 3 ) V s,0-9 Settled volume of adsorbent in the 0-to 9-cm column zone (m 3 ) V s,9-12 Settled volume of adsorbent in the 9-to 12-cm column zone (m 3 ) V o Volume of the particles in the sampling syringe (m 3 )Greek letters e Bed voidage e i Local bed voidage at ith layer of bed d Variance (s)

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Adsorption Strategy of Plasmid DNA Nanoparticulate: Preparative Purification by a Simple Custom Expanded Bed Column

Cited by 8 publications

References 28 publications

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing

Investigation of hydrodynamic parameters in a novel expanded bed configuration: local axial dispersion characterization and an empirical correlation study

Hydrodynamic Characteristics and Adsorption Particularity of Nanobiological Feedstock Along the Bed Height in a Novel Chromatography Column

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