Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F)
Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clari...
| Main Authors: | , |
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| Format: | Journal Article |
| Published: |
Elsevier BV
2010
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| Online Access: | http://hdl.handle.net/20.500.11937/35861 |
| _version_ | 1848754610996510720 |
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| author | Ongkudon, C. Danquah, Michael |
| author_facet | Ongkudon, C. Danquah, Michael |
| author_sort | Ongkudon, C. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7. M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400. nm pore size of monolith in 0.7. M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0. M at 3%B/min. © 2010 Elsevier B.V. |
| first_indexed | 2025-11-14T08:43:09Z |
| format | Journal Article |
| id | curtin-20.500.11937-35861 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T08:43:09Z |
| publishDate | 2010 |
| publisher | Elsevier BV |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-358612017-09-13T15:30:50Z Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) Ongkudon, C. Danquah, Michael Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7. M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400. nm pore size of monolith in 0.7. M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0. M at 3%B/min. © 2010 Elsevier B.V. 2010 Journal Article http://hdl.handle.net/20.500.11937/35861 10.1016/j.jchromb.2010.08.011 Elsevier BV restricted |
| spellingShingle | Ongkudon, C. Danquah, Michael Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title | Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title_full | Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title_fullStr | Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title_full_unstemmed | Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title_short | Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F) |
| title_sort | process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcdna3f) |
| url | http://hdl.handle.net/20.500.11937/35861 |