Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1996-09-30
1998-05-05
Lilling, Herbert J.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 6, 435259, 435267, 435268, 435269, 435270, 435820, C12P 1934, C12N 106
Patent
active
057476632
DESCRIPTION:
BRIEF SUMMARY
The present invention pertains to a process for the depletion or removal of endotoxins from preparations containing active ingredients designated for therapeutical use which are obtained from natural sources by genetic engineering and/or biotechnology by treatment with chromatographic material, as well as the use thereof, an aqueous solution for performing said process, and a kit containing members for performing said process.
Molecular biological processes are increasingly gaining importance in the preparation of medicaments. They include, on one hand, the classical genetic engineering methods for the preparation of medicaments, and also, to a growing extent, the so-called gene therapy in which nucleic acids are introduced in the genome of species to be treated. The removal of endotoxins is becoming of crucial importance therein.
For example, to prepare plasmid DNA on a preparative scale, it is necessary to proliferate the plasmids by means of so-called host cells. These are generally gram-negative enterobacteria, such as mutants of E. coli K-12. Gram-negative bacteria have cell walls which are surrounded by an outer membrane. Located on this membrane are so-called lipopolysaccharids (LPS), also known under the designation endotoxins. Endotoxins are responsible, for instance, for the typical phenomena which accompany a bacterial intoxication, such as inflammatory reactions and fever as well as endotoxic shock.
As noted in German Patents P 44 03 692 and P 44 22 291, in vivo and ex vivo gene therapy involves the use of plasmid DNA for the treatment of genetically caused diseases, such as cystic fibrosis, but also for the treatment of cancer or hemophilia, or for the immunisation against infectious diseases (TIBTECH, Special Issue: Gene Therapy Therapeutic Strategy and Commercial Prospects, May 1993, Vol. 11, No. 5 (112)). It is of crucial importance therein that the administered DNA cause no side-reactions, such as inflammatory or necrotic reactions. It must be ensured, therefore, that the DNA used for such kind of treatment is not contaminated with endotoxins.
Preparations made by genetic engineering and/or biotechnology may also contain endotoxins. Therefore, it is important to remove or deplete the endotoxins to below physiologically safe amounts.
The purification methods presently known, for example, for plasmid DNA from gram-negative bacteria, are not able to completely remove the endotoxins from plasmid DNA. These methods include, for instance, cesium chloride gradient centrifugation or anion exchange chromatography.
Cesium chloride gradient centrifugation is based on the fact that differently sized DNA molecules have different migration velocities in a salt gradient. However, lipopolysaccharides show the same migration bevahior as DNA in a density gradient and thus cannot be effectively separated from DNA.
As compared to anion exchange chromatography, cesium chloride gradient centrifugation is rather time-consuming and uses a number of toxic substances, such as ethidium bromide. Also, cesium chloride has to be removed by an additional dialysis.
Aida and Pabst, in "Removal of endotoxin from protein solutions by phase separation using Triton X 114", J. Immunol. Methods 132, 191-195 (1990), suggest a method for the removal of endotoxins from protein solutions by means of Triton.RTM. X 114 extraction. The solution to be treated is spiked with the detergent Triton.RTM. X 114. The protein-containing aqueous phase is removed following incubation and centrifugation, and the endotoxin-removed or endotoxin-depleted protein is precipitated. As has already been suggested in WO 95/21177, this method may also be used for the extraction of endotoxins from DNA solutions. However, this method is characterized by by a rather high expenditure of work and is applied only after the isolation of the DNA. This method is less suited for the purification of DNA quantities on a preparative, especially industrial, scale.
The object of the invention is to provide a process which is successful in preparing endotoxin-free or endo
REFERENCES:
patent: 4997932 (1991-03-01), Reardon et al.
Colpan Metin
Moritz Peter
Schorr Joachim
Lilling Herbert J.
Qiagen GmbH
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