Purification of nucleic acid from a sample containing nucleic acid and endotoxin

• US 10,457,931 B2
• Filed: 04/03/2017
• Issued: 10/29/2019
• Est. Priority Date: 04/06/2016
• Status: Active Grant

• Current Assignee: Axagarius GmbH & Co. KG
• Original Assignee: Axagarius GmbH & Co. KG
• Inventors: Heymans, Stefan, Rosenbaum, Thorsten
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US15/477,671
• Publication Number:
  US 20170292122A1
• Time in Patent Office: 939 Days
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

Disclosed is a process for purifying nucleic acids, especially plasmid DNA, from a nucleic acid-containing, biologic sample, consisting of (a) preparation of a fluid sample containing nucleic acid and endotoxin each in dissolved form; (b) precipitation at least of nucleic acid and endotoxin from the fluid sample; (c) washing of the components of the fluid sample precipitated out in step b) in order to at least partially remove the endotoxin with at least one washing solution, which contains at least one amine compound with at least two carbon atoms and with a molar mass of ≤500 g/mol and; at least one organic solvent different from the aforementioned amine compound and has a pH-value (20° C.) in the range from pH 3.0 to pH 8.5, and; (d) dissolution of the remaining nucleic acid from the washed, precipitated constituents from step c) using a dissolving buffer and collection of the dissolved nucleic acids in a separate receptacle. The process is suitable for the effective and economical removal of endotoxins.

Claims

1. A process for purifying nucleic acids from a biologic sample containing nucleic acid, comprising the following steps:

a) preparing a fluid sample containing nucleic acid and endotoxin each in dissolved form;

b) precipitating at least the nucleic acid and endotoxin from the fluid sample onto the surface of a solid carrier material selected from the group consisting of mineral carrier materials, quartz fibers, silica, glass, aluminum oxide, zeolite, titanium dioxide, zirconium dioxide, and combinations thereof;

c) washing the precipitated constituents of the liquid sample from step b) to at least partially remove the endotoxin with a washing solution which contains

(i) an amine compound of formula (I),

2. The process according to claim 1, wherein in step b) at least nucleic acid and endotoxin are precipitated by adsorption or precipitation.

3. The process according to claim 1, wherein in step b) at least nucleic acid and endotoxin are separated as solids by precipitation, comprising

i) collecting the precipitated nucleic acid and entotoxin on the surface of a solid carrier material, or

ii) compacting the precipitated nucleic acid and entotoxin by centrifugation.

4. The process according to claim 1, wherein the amine compound is selected from the group consisting of triethylamine, triethanolamine, 2-amino-2-(hydroxymethyl)-propane-1,3-diol, 2,2-bis(hydroxy-methyl)-2,2′

,2″

-nitrilotriethanol, 1,3-bis[tris(hydroxymethyl)methylamino]propane, and mixtures thereof.

5. The process according to claim 1, wherein the washing solution of step c) contains the amine compound in a concentration of 200 mmol/L to 800 mmol/L.

6. The process according to claim 1, wherein the nitrogen atom of the amino group in the amine compound is at least 50% protonated.

7. The process according to claim 1, wherein the washing solution contains the C₁-C₆-monoalcohol in a total concentration of 20 to 80% by weight relative to the total weight of the washing solution.

8. The process according to claim 1, wherein the washing solution of step c) has a pH-value (20°

C.) in the range from pH 3.0 to 7.5.

9. The process according to claim 1 wherein at least 50% by weight of the endotoxin of the fluid sample are removed in the process removed.

10. A method of claim 1 comprising utilizing a washing solution comprising

(i) an amine compound of formula (I),

11. The method according to claim 10 wherein the nucleic acid and the endotoxin are bound to the surface of a solid carrier material.

12. A kit for purifying nucleic acids comprising a washing solution, which contains

(i) an amine compound of formula (I),