Endotoxin reduction in nucleic acid purification

Details Endotoxin reduction in nucleic acid purification Endotoxin reduction in nucleic acid purification

1998-04-22

2001-02-27

Whisenant, Ethan (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025410, C536S025420, C435S320100

Reexamination Certificate

active

06194562

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
TECHNICAL FIELD
This invention relates generally to methods for selectively reducing the concentration of endotoxins from a solution containing endotoxins and nucleic acids, such as a solution derived from the disruption of gram-negative bacteria or a solution of nucleic acids contaminated with endotoxins from the environment. This invention also relates to methods for isolating nucleic acids, such as plasmid DNA, chromosomal DNA, total RNA, mRNA, or RNA/DNA hybrids using a silica matrix.
BACKGROUND
Generally speaking, an endotoxin is a lipopolysaccharide material found in the cell wall of most gram-negative bacilli, including
Escherichia coli
. During lysis of bacterial cell, such as is done to release plasmids from
E coli
transformants, endotoxins are released into the lysate produced thereby. Endotoxin contamination in a nucleic acid sample can adversely limit the utility of the sample, particularly in applications which are sensitive to such contamination. For example, the transfection efficiency of several different cultured eukaryotic cell lines, including HeLa, Huh
7
, COS
7
, and LMH, have been shown to be sharply reduced in the presence of endotoxins. Weber, M. et al. 1995,
BioTechniques
19(6):930-939. Endotoxins have also been found to be toxic to primary human cells, such as primary human skin fibroblasts and primary human melanoma cells, in the presence of entry-competent adenovirus particles. Cotten, M. et al. 1994,
Gene Therapy
1:239-246. Endotoxins have also been shown to produce striking pathophysiological reactions when introduced into animals, including high fever, vasodilation, diarrhea and, in extreme cases, fatal shock. Morrison, David C. 1987,
Ann Rev. Med.
38:417-32.
Endotoxins are not readily separated from nucleic acids, particularly from plasmid DNA.
QIAGEN Plasmid Purification Handbook
(January 1997), p. 55 (hereinafter, the “Handbook”). Endotoxins tend to form mycellar structures which have a similar density, size, and charge distribution on the outer surface of the mycells Id.. As a result, endotoxins co-purify with nucleic acids, particularly with plasmid DNA, in most nucleic acid isolation procedures used today. For example, endotoxins appear in the same band as the DNA-ethidium bromide complex in the cesium chloride gradients used to separate plasmid DNA from other materials in a bacteria lysate Id. and Cotten et al., supra. Endotoxins also co-migrate and co-elute with plasmid DNA from size exclusion and from anion exchange resins.
Handbook
, p. 55.
Phase separation with Triton X-114 has been used to reduce endotoxin levels in solutions of plasmid DNA. However removal of endotoxins with Triton X-114 requires multiple, labor-intensive, phase separation steps, with some plasmid DNA lost in each extraction step. Shigui L. et al.
Clinical Biochemistry
1997, 30(6): 455-463. Various groups of researchers have obtained mixed results with Triton X-114 extraction, with some obtaining unsatisfactory endotoxin removal from plasmid DNA solutions. See, e.g. Montbriand, P. M. et al. 1996
J. Biotechnol
44:43-46; Manthorpe, M et al. 1993,
Hum. Gene Ther.
4: 419-431.
Affinity sorbents have also been developed for the removal of endotoxins from protein or DNA solutions. See, e.g. Anspach, F. B. et al. 1995
J. Chromatography
711:81-92 [protein purification]; Montbriand, supra, p. 44 [DNA purification]. For example, polymyxin B, a cationic polypeptide which stoichiometrically binds to a portion of the LPS molecule, has been linked to various resins, such as Sepharose or agarose, and used to remove endotoxins from a solution of DNA. Molvig, J. and Baek, L. (1987)
Immunochemistry
26:611-619 [Sepharose resin]; Montbriand, supra at 44 [agarose resin]. Affinity resins are generally difficult to synthesize and costly to purchase. Affinity resins also present problems not presented by any of the other, traditional, methods of nucleic acid isolation and/or endotoxin removal described briefly above.
One research group, Montbriand et al., conducted a study of the effectiveness of various types of affinity resins and resin formats in the removal of endotoxins from plasmid DNA. Montbriand, supra. That group removed endotoxins from solutions of plasmid DNA with a polymyxin B-sepharose affinity resin, using gravity flow chromatography, spin column chromatography, and bulk chromatography. Montbriand et al. reported that bulk chromatography resulted in the most efficient purification of DNA, but polymyxin B release into the DNA solution purified therewith was problematic in some applications, such as in-vivo administration of purified DNA to humans. Montbriand et al. obtained some plasmid DNA which were relatively free of endotoxins, using polymyxin-agarose resins. But, all three techniques used by that research group to purify DNA with the resins resulted in a significant loss of DNA.
Some molecular biology companies have developed kits for use in isolating plasmid DNA for transformation of eukaryotic cells, under conditions where endotoxins are digested and separated from the DNA. One such kit is the “EndoFree Plasmid Maxi Kit” sold by QIAGEN, Inc. (Santa Clarita, Calif.) Instructions for use of that kit and detailed descriptions of all but one of the solutions provided for use with that kit are provided the
QIAGEN Plasmid Purification Handbook
. The only solution whose composition is not described in the Handbook is “Buffer ER”. The Handbook also fails to provide any information about the composition of the material in the “QIA-tip” used to bind DNA after Buffer ER is used to “remove endotoxins” from the lysate solution filtered according to the isolation procedure described therein. The DNA isolation procedure described in EndoFree Plasmid Maxi Kit portion of the
Handbook
involves multiple steps many of which result in a loss of plasmid DNA.
A group of researchers, Wicks et al., recently compared use of a QIAGEN plasmid DNA kit with a lysozyme digestion step to a similar kit which did not include any enzymes or other materials designed to remove endotoxins. Wicks, I. P. et al. (March 1995)
Human Gene Therapy
6:317-323, at p. 319. They found endotoxins in the range of 50-500 &mgr;g/ml in plasmid DNA isolated using the kit with a lysozyme treatment step, and endotoxin concentrations of over 3 mg/ml using kits with no such digestion step. Id. Wicks et al. also note, however, that use of both plasmid DNA isolation procedures resulted in a significant loss in the amount of DNA isolated, compared to a considerably more complex procedure described therein. Specifically, Wicks et al. described a plasmid DNA and endotoxin removal procedure, wherein plasmid DNA was isolated from spheroplasts, and wherein endotoxins where removed from the DNA isolated from the spheroplasts, using polymyxin B-agarose chromatography. Id. at 318-20.
Note, incidentally, that the QIAGEN EndoFree Plasmid Maxi kit is designed solely for plasmid DNA isolation. The alkaline lysis step in the first part of the procedure inherently degrades any RNA molecules present in the bacteria cells. The neutralization and filtration steps are expressly designed to precipitate and remove chromosomal DNA. in short, the QIAGEN kit is clearly not suited for the isolation of any species of nucleic acid other than plasmid DNA.
The present invention addresses the need for methods and materials for treating solutions of nucleic acids containing endotoxins to reduce the concentration of endotoxins contained therein. The invention described herein below offers a rapid and efficient means for removing endonucleases from such solutions, thereby providing purified nucleic acids which can be used in a variety of biological applications, including transfection of cultured cells and in vivo administration of nucleic acids to organisms which are susceptible to sepsis.
BRIEF SUMMARY OF THE INVENTION
The method of this invention uses a first sili

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