A process for supercoiled plasmid DNA purification based on multimodal chromatography

Silva-Santos, A. Rita; Alves, Cláudia P.A.; Prazeres, Duarte Miguel F.; Azevedo, Ana M.

doi:10.1016/j.seppur.2017.03.042

Cited by 20 publications

(10 citation statements)

References 28 publications

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“…The mixture then proceeded to buffer exchange and a concentration to 0.5 L using TFF prior to anion exchange chromatography. This first purification step was a capture step where all anion components were attached onto the column and salt ionic strength was increased in proportion to its concentration; the product was then eluted ( Stadler et al, 2004 ; Sun et al, 2013 ; Silva-Santos et al, 2017 ). In our experiment, our pDNA was eluted with Tris-EDTA buffer containing 1 M NaCl while impurities such as remaining RNA came out with lower salt concentrations at 0.6 M, as displayed in Figure 1A .…”

Section: Resultsmentioning

confidence: 99%

Toward QbD Process Understanding on DNA Vaccine Purification Using Design of Experiment

Hocharoen

Noppiboon

Kitsubun

2021

Front. Bioeng. Biotechnol.

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DNA vaccines, the third generation of vaccines, are a promising therapeutic option for many diseases as they offer the customization of their ability on protection and treatment with high stability. The production of DNA vaccines is considered rapid and less complicated compared to others such as mRNA vaccines, viral vaccines, or subunit protein vaccines. However, the main issue for DNA vaccines is how to produce the active DNA, a supercoiled isoform, to comply with the regulations. Our work therefore focuses on gaining a process understanding of the purification step which processes parameters that have impacts on the critical quality attribute (CQA), supercoiled DNA and performance attribute (PA), and step yield. Herein, pVax1/lacZ was used as a model. The process parameters of interest were sample application flow rates and salt concentration at washing step and at elution step in the hydrophobic interaction chromatography (HIC). Using a Design of Experiment (DoE) with central composite face centered (CCF) approach, 14 experiments plus four additional runs at the center points were created. The response data was used to establish regression predictive models and simulation was conducted in 10,000 runs to provide tolerance intervals of these CQA and PA. The approach of this process understanding can be applied for Quality by Design (QbD) on other DNA vaccines and on a larger production scale as well.

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Section: Resultsmentioning

confidence: 99%

Toward QbD Process Understanding on DNA Vaccine Purification Using Design of Experiment

Hocharoen

Noppiboon

Kitsubun

2021

Front. Bioeng. Biotechnol.

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“…Fractogel TMAE [22] sc pDNA Capto TM adhere resin [23] Lactoferrin Sulfanilic acid-modified chitosan mini-spheres [24] Nucleic Acids Sepharose CL-6B treated with 1,4-butanediol diglycidyl ether [25] mcDNA Sephacryl S-1000 SF matrix [26] mcDNA Cadaverine modified monolith [27] pre-miRNA-29 L-arginine-Sepharose 4B gel [28] sc pDNA…”

Section: Adenoviral Vectormentioning

confidence: 99%

“…The resin Fractogel TMAE showed a great binding performance and real potential to purify adenovectors comparatively to the other three tested resins that presented lower dynamic binding capacity. The purification of supercoiled plasmid DNA (sc pDNA) was described by Silvia-Santos et al [ 23 ], who tested a cationic multi-modal support, the Capto TM adhere. The authors isolated the sc pDNA isoform from other impurities, such as RNA and open circular pDNA isoform, with a recovery yield of over 90%.…”

Section: Biomolecules Purification By Preparative Chromatographymentioning

confidence: 99%

Supported Ionic Liquids Used as Chromatographic Matrices in Bioseparation—An Overview

et al. 2022

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Liquid chromatography plays a central role in biomanufacturing, and, apart from its use as a preparative purification strategy, either in biopharmaceuticals or in fine chemicals industries, it is also very useful as an analytical tool for monitoring, assessing, and characterizing diverse samples. The present review gives an overview of the progress of the chromatographic supports that have been used in the purification of high-value products (e.g., small molecules, organic compounds, proteins, and nucleic acids). Despite the diversity of currently available chromatographic matrices, the interest in innovative biomolecules emphasizes the need for novel, robust, and more efficient supports and ligands with improved selectivity. Accordingly, ionic liquids (ILs) have been investigated as novel ligands in chromatographic matrices. Given herein is an extensive review regarding the different immobilization strategies of ILs in several types of supports, namely in silica, Sepharose, and polymers. In addition to depicting their synthesis, the main application examples of these supports are also presented. The multiple interactions promoted by ILs are critically discussed concerning the improved selectivity towards target molecules. Overall, the versatility of supported ILs is here considered a critical point to their exploitation as alternatives to the more conventional liquid chromatographic matrices used in bioseparation processes.

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“…Several RNase‐free bioprocesses based on anion‐exchange chromatography have consequently been developed, however, they are rather complex and time‐consuming, and need an additional operational unit to accommodate the resulting burden of RNA impurity . Alternatives to packed‐bed chromatography have been developed in the last 15 years, and include now a variety of approaches that include, but is not limited to functionalized ‘monolithic’ rods, (composite) synthetic membranes and (magnetic) micro‐to‐nano particles . All the mentioned systems present comparative advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

pDNA capture using grafted adsorbents

Singh

Dsouza

Yelemane

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND: 'Expanded' composite materials are of interest as an alternative, or as a supplement, to packed-bed chromatography during bioproduct recovery and purification. Functionalized non-woven fabrics and mega-porous bodies are examples of systems that showed promise. However, there is scarce information on their suitability to capture and release plasmid DNA (pDNA), an important type of product employed in gene therapy. RESULTS: Composite adsorbents were prepared using either chemical (CG-DEAE-NW) or gamma-irradiated graft-polymerization (GIR-DEAE-MP), and subsequently modified to have diethylamino ethanol (DEAE) functionality. Capture experiments showed that pDNA can actually reversibly bind to the two mentioned adsorbents, with capacity values of 2.4 and 1.3 mg per mL, respectively. These values are in the range of what can be expected from commercial beaded adsorbents but lower that the values expected from monoliths. CONCLUSIONS: Expanded materials, due to their high voidage, may present limited capacity for pDNA. However, such materials are able to bind proteins and other contaminants from bacterial lysate, opening the way for their utilization in the 'negative' mode.

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A process for supercoiled plasmid DNA purification based on multimodal chromatography

Cited by 20 publications

References 28 publications

Toward QbD Process Understanding on DNA Vaccine Purification Using Design of Experiment

Toward QbD Process Understanding on DNA Vaccine Purification Using Design of Experiment

Supported Ionic Liquids Used as Chromatographic Matrices in Bioseparation—An Overview

pDNA capture using grafted adsorbents

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