Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-09-05
2003-09-09
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100
Reexamination Certificate
active
06617443
ABSTRACT:
The invention relates to a method for reducing endotoxin levels or removing endotoxins from biological material. The method according to the invention enables, for example, high-purity plasmid DNA to be obtained from natural sources.
The demand for rapid and efficient methods for obtaining high-purity plasmid DNA from biological sources is constantly increasing owing to the increasing importance of recombinant DNA for exogenous expression or therapeutic applications. In particular, the demand for purification methods which can also be carried out on a larger scale is also increasing.
Virtually all known methods for the purification of, in particular, relatively large amounts of plasmid DNA include a chromatographic purification step. The efficiency of this step generally also determines the efficiency and effectiveness of the purification.
Plasmids are epigenomic circular DNA molecules having a length of between 4 and 20 kB, which corresponds to a molecular weight of between 2.6×10
6
and 13.2×10
6
daltons. Even in their compact form (super coil), plasmid DNA molecules normally have a size of several hundred nm. Owing to these dimensions, they are larger than the pores of most chromatography materials. This in turn causes, inter alia, the poor binding capacities of the separating materials generally used for plasmid DNA.
A further problem in the purification of plasmid DNA is caused by the impurities from which the plasmid DNA is to be separated. These are firstly genomic DNA and RNA. Exactly like plasmid DNA, these molecules have a strongly anionic character and thus a very similar binding behaviour to separating materials.
The removal of endotoxins is at least as complex. Endotoxins are lipopoly-saccharides (LPSs) which are located on the outer membrane of Gram-negative host cells, such as, for example,
Escherichia coli.
During lysis of the cells, LPSs and other membrane constituents are dissolved out in addition to the plasmid DNA. Endotoxins are present in cells in a number of approximately 3.5×10
6
copies per cell (
Escherichia Coli
and Salmonella Typhimurium Cell. and Mol. Biology, J. L. Ingraham et al. Eds., 1987, ASM) and thus exceed the number of plasmid DNA molecules by a factor of more than 10
4
. For this reason, plasmid DNA obtained from Gram-negative host cells often contains large amounts of endotoxins. However, these substances result in a number of undesired side reactions (Morrison and Ryan, 1987, Ann. Rev. Med. 38, 417-432; Boyle et al. 1998, DNA and Cell Biology, 17, 343-348). If it is intended to employ the plasmid DNA for, for example, gene therapy, it is of extreme importance that, for example, inflammatory or necrotic side reactions due to the impurities do not occur. There is therefore a great demand for effective methods for reducing endotoxin concentrations to the lowest possible levels.
Known methods for reducing endotoxin levels are based on a plurality of purification steps, frequently using silica supports, glass powder or hydroxy-lapatite, and on reverse-phase, affinity, size-exclusion and/or anion-exchange chromatography.
Firstly, the host cells are digested by known methods, such as, for example, alkaline lysis. However, other lysis methods, such as, for example, the use of high pressure, boiling lysis, the use of detergents or digestion by lysozyme, are also known.
The plasmid DNA in the medium obtained in this way, a “cleared lysate”, is principally contaminated by relatively small cell constituents, chemicals from the preceding treatment steps, RNA, proteins and endotoxins. The removal of these impurities usually requires a plurality of subsequent purification steps. Purification by means of anion-exchange chromatography has proven particularly advantageous.
However, the dynamic binding capacity of most anion exchangers for plasmid DNA is only about 0.4 mg/ml of separating material. The reason for this low value is that the functional groups are bonded to the support directly or via short spacers and consequently are only available to a limited extent for interactions with the large plasmid DNA molecules.
A further disadvantage of conventional anion-exchange chromatography is that a considerable amount of endotoxins is bound together with the plasmid DNA and cannot be separated off in this way. Plasmid DNA with endotoxin proportions of greater than 500 EU/mg of plasmid DNA is obtained. In order to reduce the endotoxin levels, further purification steps, such as, for example, chromatographic steps (gel filtration) or precipitation with isopropanol, ammonium acetate or polyethylene glycol, are therefore necessary. Purification methods which combine chromatographic methods, such as, for example, anion-exchange chromatography, and additional endotoxin removal steps enable plasmid DNA having an endotoxin content of less than 50 EU/mg of plasmid DNA to be obtained. However, methods of this type are usually complex, time-consuming and of only limited suitability for the purification of relatively large amounts of DNA.
WO 95/21179 describes a method for the reduction of endotoxin levels in which a cleared lysate is firstly pre-incubated with an aqueous salt solution and detergents. This is followed by purification by ion-exchange chromatography, in which the ion-exchange material is washed with a further salt solution, and the plasmid DNA is eluted and subsequently purified further, for example by isopropanol precipitation. This method likewise has the above-mentioned disadvantages.
Instead of a pure anion-exchange chromatography step, WO 99/63076 uses a mixed-mode principle in which from 25 to 90% of an alcohol is added to the washing buffer. This method likewise typically requires a plurality of steps for achieving effective purification.
The object of the present invention was therefore to provide a method for the chromatographic purification of plasmid DNA which firstly gives plasmid DNA having an endotoxin content of less than 50 EU/mg of plasmid DNA without further purification steps and which secondly is also suitable for the purification of large amounts of DNA.
It has been found that plasmid DNA is obtained in very good purity without further precipitation steps if a cleared lysate is pre-incubated with salt-free detergent solution and subsequently purified by means of anion-exchange chromatography on a tentacle support. The method according to the invention is suitable for the purification of small, but in particular also large amounts of plasmid DNA.
In contrast to the disclosure of WO 95/21179, in which the pre-incubation is carried out with a salt-containing detergent solution, it has now been found that the use of a salt-free detergent solution, in particular in combination with subsequent anion-exchange chromatography on tentacle supports, effects very efficient reduction of endotoxin levels.
The present invention therefore relates to a method for the reduction in endotoxin levels in nucleic acids originating from natural, genetic engineering or biotechnological sources, characterised by 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, i.e. under anion-exchange chromatography conditions;
e) elution of the sample by a further increase in the ion strength and/or by a pH change.
In a preferred embodiment, the biological source to be purified contains plasmid DNA.
In a preferred embodiment of the method, a DEAE or TMAE anion exchanger is used.
In a preferred embodiment, the washing buffer used for step d) comprises from 0 to 20% (v/v) of an organic solvent or solvent mixture, in particular one or more alcohols, preferably C1 to C5 alcohols, particularly preferably ethanol and/or isopropanol.
In a furthe
Hendriks Robertus
Lantos Andrea
Wehsling Maria
Ketter James
Merck Patent GmbH
Millen White Zelano & Branigan P.C.
LandOfFree
Method for removing endotoxins does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for removing endotoxins, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for removing endotoxins will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3099942