A combined liquid chromatography separation method for large-scale plasmid purification

Abstract

The invention provides a plasmid purification method capable of being used for medicine stage nucleic acid pDNA (Plasmid Deoxyribonucleic Acid) vaccine scaled preparation. According to the method, a granular medium column liquid chromatography separation system and an Monoliths column liquid chromatography separation system developed in recent years are used in a combined way; the advantages of the granular medium column liquid chromatography separation system and the Monoliths column liquid chromatography separation system are sufficiently achieved in a mutual complementation way; the excellent effect is achieved; and high practical values are realized.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a purification and separation process for plasmids used in gene vaccines and gene therapy, and more specifically, to a combined liquid chromatography separation method for large-scale plasmid purification. [0002] technical background [0003] Plasmid (pDNA) nucleic acid vaccine is a new type of vaccine under development. It has the advantage of inducing the body to produce a cellular immune response. It is good for the treatment of chronic hepatitis B, AIDS, tuberculosis, cancer and other diseases that require the induction of cellular immunity for which there is no effective vaccine at present. Application prospects, plasmid purification technology has also developed from the laboratory stage to the practical industrialization stage. [0004] Plasmids are circular DNA that can replicate and express independently of chromosomes in Escherichia coli cells. The engineered plasmid is a g...

AI Technical Summary

Problems solved by technology

[0010] In dozens of experiments of the inventors using the official method recommended by BIA to purify the plasmid, the concentration of calcium chloride used in the precipitation of the lysate, the washing of the two monolithic columns, the concentration of the eluent, etc. have a significant impact on the quality and quantity of the purified plasmid. The impact has been carefully studied and found that: 1) adding CaCl to the lysate 2 The precipitate particles combined with RNA can be large or small, the large ones can be removed by centrifugation, and the small ones can only be removed by filtering through a 0.45 / 0.22μm capsule filter, so the quality of the filter used is high, otherwise these small particles may clog the column Pores in the body reduce its service life

2) CaCl 2 Precipitation removes incomplete RNA and removes a considerable portion of the plasmid at the same time, severely reducing its recovery

3) The BIA method relies on the high throughput and high flow rate of its monolithic column to not concentrate the lysate clarified liquid, and because it contains 0.5-1.0M CaCl 2 However, it is necessary to add several times the volume of TE solution to dilute the conductivity to reduce the conductivity, so that the loading volume of the CIM DEAE column is greatly increased and the processing time cannot be shortened; The water flow (for example, the maximum flow rate allowed by the 8ml monolithic column is 100ml / min) is not strong enough and may be partially lost

4) The nucleic acid load of the CIM DEAE weak anion exchange column is 6-8mg / ml (8ml column 48-64mg), due to CaCl 2 The treated lysate still contains a lot of RNA, which compete to occupy the binding sites in the column and reduce the binding and recovery of pDNA, that is, the column cannot fully recover pDNA

The present inventor has carried out many experiments with 8ml CIM DEAE column, and its pDNA recovery mostly is only 1 / 3 of its load of 16-18mg, fails to give full play to the load advantage of CIM DEAE column

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