Methods for purifying nucleic acids
Methods are provided for producing highly purified compositions of nucleic acids by using a highly streamlined and readily automated process. The methods use static mixers for lysing cells and precipitating debris, following by centrifugation and ion exchange chromatography. The process may include a purification step using tangential flow ultrafiltration. A scaleable process for producing pharmaceutical grade plasmid DNA, useful for gene therapy, is provided.
1. A method for purifying plasmid DNA from a solution containing plasmid DNA, comprising the steps:
a) contacting said solution with a precipitation solution;
b) flowing the solution obtained in step a) through a static mixer under conditions of flow that provide Reynolds numbers in the range of about 50 to 500;
c) further purifying said plasmid DNA using an open tangential flow ultrafiltration (TFU) under conditions that promote formation of a gel layer and said plasmid DNA is retained in a retentate solution;
d) contacting said retentate solution obtained in step c) with an ion exchange chromatography resin under conditions in which plasmid DNA is bound to said resin; and
e) eluting said plasmid DNA from said resin using a step gradient or a continuous gradient of a salt solution with increasing conductivity.
2. The method of claim 1 , further comprising exposing said solution obtained by step c) to a ribonuclease (RNAase).
3. The method of claim 1 , said ion exchange chromatography resin being an anion exchange chromatography resin.
4. The method of claim 1 , said ion exchange resin being a strong anion exchange chromatography resin.
5. The method of claim 1 , wherein said TFU is carried out using an ultrafiltration membrane having a molecular weight cutoff (MWCO) of 300 K Daltons, said plasmid DNA having sizes in the range of 2 Kb to 15 Kb being retained in said retentate solution.
6. The method of claim 1 , wherein said TFU is carried out using an ultrafiltration membrane having a MWCO of 500 K Daltons, said plasmid DNA having sizes in the range of 15 Kb to 50 Kb being retained in said retentate solution.
7. The method of claim 1 , wherein said TFU is carried out using an ultrafiltration membrane having a MWCO of 1000 K Daltons, said plasmid DNA having a size greater than 40 Kb being retained in said retentate solution.