Method for removing endotoxins

• US 6,617,443 B2
• Filed: 09/05/2002
• Issued: 09/09/2003
• Est. Priority Date: 03/06/2000
• Status: Expired due to Fees

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Hendriks, Robertus, Wehsling, Maria, Lantos, Andrea
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US10/220,763
• Publication Number:
  US 20030068810A1
• Time in Patent Office: 369 Days
• Field of Search: 536/25.41, 435/320.1
• US Class Current: 536/25.41
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Abstract

The invention relates to a method for removing endotoxins from nucleic acids. The endotoxins are removed by pre-incubating the nucleic acids in a salt-free detergent solution and subsequent anion exchange chromatography on a tentacle anion exchanger.

Claims

1. Method for the reduction in endotoxin levels in nucleic acids originating from natural, genetic engineering or biotechnological sources, comprising the following steps:

a) preparation of a medium which contains the nucleic acids to be purified;

b) pre-incubation of the medium from step a) with a salt-free detergent solution;

c) application of the incubation solution from step b) to anion-exchanger material whose functional groups are bonded to tentacles on the surface of the support;

d) washing of the anion exchanger, in which the impurities are washed out by increasing the ion strength and/or by pH changes;

e) elution of the sample by a further increase in the ion strength and/or by a pH change.

2. Method for reducing endotoxin levels according to claim 1, wherein the biological source to be purified contains plasmid DNA.

3. Method according to claim 1, wherein the anion exchanger contains DEAE or TMAE as functional groups.

4. Method according to claim 1, wherein the medium prepared in step a) has been obtained from a natural source by a digestion method.

5. A method according to claim 4, wherein the digestion method is alkaline lysis, centrifugation, filtration or precipitation.

6. A method according to claim 1, wherein the medium which contains the nucleic acids to be purified contains single-stranded or double-stranded RNA or DNA, RNA/DNA hybrids, DNA fragments, oligonucleotides, amplified DNA or RNA, BACs, or plasmid DNA.

7. A method according to claim 1, wherein the anion exchanger is washed by a buffer that contains an organic solvent or solvent mixture up to 20% by volume.

8. A method according to claim 7, wherein the solvent is or the solvent mixture comprises ethanol or methanol or both ethanol and methanol.

9. A method according so claim 1, wherein the medium is a cleared lysate.

10. A method according to claim 1, wherein the functional groups of the anion-exchanger material are bonded to monomer units of polymer chains which are polymerised onto a base support.

11. A method according to claim 1, wherein the tentacles are copolymers that comprise methacrylate.

12. A method according to claim 1, wherein the anion-exchanger material are prepared by reacting a tentacle support containing reactive groups of formula I with a compound of formula II,

wherein

R¹,R² and R³, independently of one another, are H or CH₃,

R⁴ is H, alkyl having 1-5 carbon atoms or aryl having 6-12 carbon atoms,

U is — O—

or —

NH—

, and

n is an integer of 1 to 5,

13. A method according to claim 12, wherein

R¹ and R² are H,

R³ is CH₃,

R₄ is H,

U is —

O, and

n is 1.

14. A method according to claim 12, wherein

R⁵ is H, methyl or ethyl, and

m is an integer of 2 to 3.

15. A method according to claim 12, wherein the compound of formula II is ethanol amine.

16. A method according to claim 1, wherein the support contains a base polymer which comprises polyamide, polyvinyl alcohol or copolymers of (meth)acrylate derivatives or comonomers containing aliphatic hydroxyl groups, and optionally contains groups of formula III,

wherein

R¹,R² and R³, independently of one another, are H or CH₃,

R⁴ is H, alkyl having 1-5 carbon atoms or aryl having 6-12 carbon atoms,

U is —

O—

or —

NH—

,

n is an integer of 1 to 5, and

one radical X is OH, and the other X is —

NR⁷R⁸ or —

N⁺R⁷R⁸R⁹, in which

R⁷ and R⁸, independently of one another, are H or alkyl having 1-5 carbon atoms, and

R⁹ is alkyl having 1-5 carbon atoms,

with the proviso that, if X is —

N⁺R⁷R⁸R⁹, then R⁷ and R⁸ are not H.

17. A method according to claim 1, wherein the anion exchanger contains DMAE as functional groups.

18. A method according to claim 1. wherein the salt-free detergent solution contains less than 1 mM salt and is present in the medium in 0.01 to 30% by volume.

19. A method according to claim 1, wherein the medium which contains the nucleic acids to be purified contains plasmid DNA.

20. A method according to claim 7, wherein the buffer contains 10 to 15% of ethanol and/or isopropanol.