Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Patent
1992-07-19
1994-04-19
Drodge, Joseph W.
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
424550, 514 21, 530344, 530855, B01D 1102, A61K 3562
Patent
active
053043103
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
Efficient and economic purification methods play a central part in the preparation of chemical and biological products. During the course of the development of biotechnology and genetic engineering in recent years increasing importance has become attached, in particular, to the development of methods for purifying proteins, peptides and vitamins on the industrial scale.
An important step in the isolation of most cellular constituents is the fractionation of the starting material or of an intermediate during the course of the purification. Used for this purpose are, as a rule, (fractional) precipitations or partition chromatographies. To carry out precipitations, the properties of the solvent are altered in such a way, by adding appropriate reagents, that the solubility of the protein greatly decreases and it precipitates. The solubility can be altered by adding salts such as ammonium sulfate or organic solvents such as ethanol or by changing the pH or the temperature (Green, A. A. & Hughes W. L. in "Methods in Enzymology", volume I, pp. 67-90 (1955); Belter et al, 1988, pages 100 ff. in "Bioseparations", eds.: Belter, P. A., Cussler, E. L. & Hu, W. -S.).
Liquid-liquid two-phase partition chromatographies have also been employed for a long time for purifying biomolecules. In the 1940s and 1950s, exclusively organic/aqueous two-phase systems containing water-immiscible organic solvents were used. Although these are very suitable for concentrating and purifying low molecular weight biomolecules such as antibiotics and vitamins (Schlugerl, K., 1987, in "Separations for Biotechnology", eds.: Verral, M. S. & Hudson, M. J. pp. 260-269), there are strict limits on their use for isolating proteins in particular. In particular, the denaturation and precipitation of the proteins in the organic/aqueous system preparer (sic) great problems (Johansson G., 1985, in "Partitioning in aqueous two-phase systems", Acad. Press, pp. 161-225).
Another method for liquid-liquid two-phase extraction was developed by Albertson in the late 1950s. He used 2-phase systems with two aqueous phases and was able to show that these are very suitable for, in particular, the concentration of proteins (Nature 182, 709 (1958)). Since then, almost exclusively aqueous 2-phase systems have been used and further developed for the extraction of proteins with 2-phase systems. However, because of the high costs of starting materials for phase-induced polymers such as polyethylene glycol or dextran, aqueous 2-phase extractions are carried out only rarely on the industrial scale.
The invention relates to a method for concentrating and purifying biomolecules, which comprises dissolving the biomolecule in a mixture of water and/or of a buffer and of a water-miscible organic solvent, and concentrating it in one of the phases which separate in the subsequent phase separation brought about by the addition of salts, where the pH at which the concentration takes place, the solvents suitable for the extraction of the biomolecule, and the nature of the salt are determined in a series of tests.
Suitable biomolecules are, in particular, vitamins and, preferably, proteins and peptides. To carry out the method, the biomolecule is first introduced into water or a buffer and/or directly into an aqueous/organic solvent system.
Care must be taken when the organic solvent is added to the aqueous phase that the protein to be purified remains in solution. If the protein does not remain in solution, it is advisable to alter the pH, the temperature, the proportion and nature of the organic solvent, the nature of the buffer and its concentration, the protein concentration and the nature and concentration of the salt. If another substance precipitates on addition of the organic solvent, it is removed by centrifugation or filtration. Particularly suitable solvents are 1- and 2-propanol, acetone, THF and acetonitrile. Most suitable for purifying hirudin are n-butanol and 2-propanol.
The salt is added to the solution obtained in this way for the phase separation. Exa
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Kreimeyer Andreas
Lang Kurt
BASF - Aktiengesellschaft
Drodge Joseph W.
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