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  • Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2010-09-03)
Clarence M Ongkudon, Michael K Danquah
ABSTRACT

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 (0.1-1.8mL/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 (0.3-0.7M) 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 400nm pore size of monolith in 0.7M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0M at 3%B/min.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Diethylamine hydrobromide, 98%
Sigma-Aldrich
Diethylamine hydrochloride, ReagentPlus®, 99%
Sigma-Aldrich
Diethylamine, purified by redistillation, 99.5%
Sigma-Aldrich
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Sigma-Aldrich
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