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US 2010/0248336 A1Published application
Methods for purifying nucleic acids
OwnerRobert Bridenbaugh, Warren Dang +1inventors
InventorsRobert Bridenbaugh, Warren Dang, Lee B. Bussey
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Abstract
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.
Claims20
A method for purifying plasmid DNA from bacterial cells, the method comprising the following steps:
- a) contacting the cells with a lysis solution, thereby forming a lysis mixture;
- b) flowing the lysis mixture through a first static mixer to obtain a lysed cell solution;
- c) contacting the lysed cell solution with a precipitation solution;
- d) flowing the lysed cell solution and the precipitation solution through a second static mixer thereby forming a precipitation mixture;
- e) centrifuging the precipitation mixture, thereby forming a pellet and a clarified solution comprising the plasmid DNA;
- f) neutralizing either the precipitate solution or the clarified solution;
- and,
- g) contacting the clarified solution with a positively charged ion exchange chromatography resin, wherein the plasmid DNA is eluted from the ion exchange chromatography resin with a saline step or continuous gradient;
- thereby forming a purified plasmid DNA solution.
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Dependencies are inferred from the claim text.