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Minerva Biotecnologica 2007 December;19(4):133-8

Copyright © 2007 EDIZIONI MINERVA MEDICA

lingua: Inglese

Optimization of alkaline lysis protocol and salt precipitation steps in the production of plasmid DNA

Bo H., Li H, Shao H., Huang S.

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Large-scale processes used to manufacture grams of plasmid DNA should be cGMP compliant, economically feasible, and environmentally friendly. Alkaline lysis and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as extraction and prepurification steps, respectively. This work describes a study of alkaline lysis protocol and calcium chloride precipitation. The results showed that, when the ratio of resuspension buffer and bacteria weight reached to 11:1 (v/g), the yield of pDNA would reach to the highest and would not increase with adding the resuspension buffer dosage. By compared the different ratio of resuspension buffer, lysis solution and neutralizing salt solution, we found that the different ratio did not affect the yield of the total pDNA (supercoiled pDNA and open circular pDNA) when the lysis solution dosage was the same amount. But, when the lysis solution dosage was the same, the supercoiled pDNA yield as the ratio was 1:1:1 would be higher than the yield as the ratio was 1:2:1. An experimental design indicated that calcium chloride concentrations could be reduced down to 0.13 M, resulting in an acceptable supercoiled plasmid DNA recovery (>90%) and a removal of >89% of RNA. The dosage of calcium chloride is inferior to that of other precipitation agents, such as ammonium sulfate. In conclusion, it is possible to reduce substantially the mass of lysis agents and precipitation agents used without affecting supercoiled plasmid DNA yield and recovery. This directly translates into an improvement of the process economics and in a reduction of the environment impact of the process.