Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
1999-12-23
2002-08-06
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S659000, C436S161000, C536S063000
Reexamination Certificate
active
06428703
ABSTRACT:
The invention concerns a method for purifying biological macromolecules, e.g. nucleic acids or proteins, from starting materials, in which the macromolecules, contained in a biological solution, are purified on a chromatographic material, and in which endotoxins are partially or completely removed with the aid of a nonionic surfactant. The invention is furthermore based on a chromatographic column for performing this method which contains a chromatographic material enclosed by closure filters.
Gene therapy is increasingly being used for the treatment of certain diseases. For this purpose, preparations which contain macromolecules such as nucleic acids or proteins as their active ingredients are obtained from biological starting material, e.g. from bacteria modified by genetic engineering. The preparations are then introduced into the human body.
It is essential for the success of gene therapy that the preparation be free of toxic substances. Since the preparations in question are usually isolated from bacteria such as the Gram-negative enterobacterium
E. coli
, and these bacteria contain as cell-wall constituents lipopolysaccharides (LPSs) that are also referred to as endotoxins, the removal of such endotoxins is very important. The reason is that intravenously administered preparations can cause fever, inflammatory reactions, or irreversible shock if they contain even small concentrations of endotoxins.
Various methods are known for removing the endotoxins. In a number of methods, this is done by using nonionic surfactants, for example the Triton X-114® surfactant of the Rohm & Haas company, specifically in conjunction with the method utilized for purifying and isolating the macromolecules. With these methods, the biological starting material containing the macromolecules is digested using a common method. Cell digestion can be accomplished by so-called alkaline lysis, by the action of high temperatures (boiling lysis), by the action of mechanical forces (compression/expansion, French press), or by the action of enzymes or detergents that destroy the cell membrane. Cell debris is then removed from the resulting cell lysate by mechanical methods, for example filtration or centrifuging.
In a subsequent step, the resulting lysate (also called “cleared lysate”) is purified chromatographically. This is done by binding the macromolecules contained in the lysate to a chromatographic material having an anion exchange function. What is used in the existing art is a cartridge into which the chromatographic material is inserted. This can preferably be a porous or nonporous, inorganic or organic sorbent modified with an anion exchange function. A washing buffer is added in order to remove undesired substances. This is followed by elution of the macromolecules by adding an elution buffer. The eluted macromolecules are precipitated and are then available for further applications.
To remove the endotoxins, Aida et al. (Journal of Immunological Methods, 132 (1990), 191-195) and Manthorpe et al. (Human Gene Therapy 4 (1993), 419-431) recommend adding Triton X-114® surfactant to the solution with the macromolecules (once they have been chromatographically isolated and purified), and incubating the solution. The endotoxins bound to the surfactant are then removed by phase separation via centrifuging. A phase separation of this kind can be achieved, however, only if temperature conditions are strictly observed, temperature control of the centrifuge that is used being particularly complex. Repeated incubation at temperatures of 50° C. or 40° C. can cause irreversible damage to sensitive molecules.
EP-A-0 308 239 proposes removing endotoxin from an aqueous, purified, biologically usable, macromolecule-containing solution by adding a dialysable surfactant to the solution, and then bringing the solution into contact with a water-insoluble endotoxin sorbent in order to bind the endotoxins to the sorbent. The remaining liquid phase is then separated from the sorbent, and the surfactant is removed by dialysis. This yields a surfactant-free aqueous solution of the biological macromolecule, with a reduced concentration of endotoxins, which is usable for in vivo applications.
A similar method is evident from WO 97/29113. With this method the endotoxins are removed, after purification of the nucleic acids, in a further step using hydroxylapatite chromatography. In both cases, there is an increased outlay in terms of equipment, and the time required is considerable.
WO 95/21177 and WO 95/21179 propose, in addition to other methods for endotoxin removal, adding to the filtered or centrifuged lysate (cleared lysate) a buffer that contains, inter alia, 10% Triton X-100® or Triton X-114®. The solution is preincubated for a long period at low temperatures before being passed on for chromatographic purification. This method has the same disadvantages as the method described above. In addition, with all the methods the removal of endotoxins requires an additional process step, which considerably lengthens and complicates the purification and isolation procedure. In the case of sensitive nucleic acid molecules, this can result in decreased yield.
It is the object of the invention to make available a method for producing biological macromolecules containing little or no endotoxin that can be performed easily, quickly, and thus economically, and is also reliably reproducible. A further object is to make available a chromatographic column for carrying out this method.
According to the present invention, this object is achieved in that the surfactant and the biological solution are brought into contact immediately before or during the purification of the macromolecules on the chromatographic material. The basic concept of the invention is thus to remove endotoxins from the macromolecule-containing biological solution by the fact that the endotoxins enter into interaction with surfactants simultaneously with and in the same place as the chromatographic purification of the biological macromolecule, and thus do not bind to the chromatographic material and consequently can be washed out of the chromatographic material; the washing-out operation can be assisted by the use of detergent-containing solutions. It has been found, surprisingly, that very effective depletion of endotoxins can hereby be achieved with no need for time-consuming incubation and extraction steps. Since endotoxin removal takes place simultaneously with the purification and isolation of the macromolecules on the chromatographic material, a definite economy in terms of time and effort is attained.
The term “surfactant” is also to be understood as a mixture of different surfactants. In this context, the surfactant should belong to the group of the polyethylene glycol ethers, and preferably should be Triton X-114®.
It has proven to be particularly advantageous to supply the nonionic surfactant as a constituent of washing buffers that are used in any case on the chromatographic material. Alternatively or (better yet) in combination therewith, the chromatographic material, and/or a closure filter in a chromatographic column containing said material, should be impregnated with the surfactant. The surfactant should be present in the washing buffer and/or in the chromatographic material and/or in the closure filter at a concentration of 0.1to 10%.
With regard to the second portion of the object, provision is made according to the present invention for the closure filter and/or the chromatographic material itself to be equipped with a nonionic surfactant, for example Triton X-114®.
A porous or nonporous, inorganic or organic sorbent, modified with anion exchange functions, can be used as the chromatographic material. Suitable closure filters are macroporous, sintered glass or a similar plastic, as well as compressed or interwoven synthetic mats made of polyamide, polyester, polypropylene, cellulose, or combinations of said materials. Preferably the closure filter and/or the chromatographic material is impregnated with the surfactant. The surfactant should be pr
Sieber Manfred
Zeidler Robert
Zinn Thomas
Liniak, Berenato, Longacre & White LLC
Macherey Nagel GmbH & Co.
Therkorn Ernest G.
LandOfFree
Method for purifying biological marcromolecules, and... 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 purifying biological marcromolecules, and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for purifying biological marcromolecules, and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2924957