Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Albumin
Patent
1994-10-24
1997-10-14
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Albumin
530362, 530363, 530412, 530416, 530417, 530418, 530427, 210645, 210651, 210656, 210660, C07K 100, A23J 100, B01D 1508
Patent
active
056774249
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method for purifying an aqueous solution of raw albumin.
The solution may be any aqueous solution of raw albumin. It is more particularly an albumin solution obtained from the blood plasma of a mammal such as a bovine, horse, pig, sheep or rabbit and preferably a solution obtained from human plasma.
2. Description of the Related Art
It is well known to separate out from blood, by centrifugation, on the one hand, the globules and, on the other hand, the blood plasma. It has for many years been known that the plasma contains, besides albumin which constitutes the majority of the dissolved material, a whole series of other molecules, in particular proteins, the extraction of which is particularly valuable bearing in mind their therapeutic uses. Methods have thus been proposed for fractionated precipitation by a precipitation agent at variable pH, ionic strengths and temperature. The process which is currently the most widely used is that of Cohn (see COHN J. et al. J. Am. Chem. Soc. 72, 465-474/1950) or variants of this process in which the precipitation agent used is ethanol. According to the said Cohn process, the plasma is cooled to -30.degree. C. and then warmed to +2.degree. C., which thereby results in a cryoprecipitate containing anti-hemophilia factor VIII, fibrinogen and fibronectin. The supernatant, referred to as "supernatant I", is separated from the abovementioned precipitate; its pH is lowered to 5.85.+-.0.05 and ethanol is added until the ethanol concentration is 19 % by volume, the temperature being lowered, progressively as the ethanol is added, to -5.degree. C. A precipitate is thus obtained, referred to as "precipitate (II+III)", containing in particular the gamma-globulins and a supernatant, referred to as "supernatant (II+III)" containing the albumin and impurities. The supernatant thus obtained is taken up and the alcohol content is increased until an ethanol concentration of 40% by volume is obtained, the temperature being lowered to approximately -8.degree. C. A precipitate is thus obtained, referred to as "precipitate IV", and a supernatant, referred to as "supernatant IV", which contains the albumin with a degree of purity of approximately 94 to 97% by weight of proteins. Supernatant IV serves as starting material for the desired albumin solutions, but it contains a large amount of ethanol; according to a first technique, supernatant IV may be dialysed directly against physiological serum, but it is then necessary to use a very large amount of water and the method is thus slow and expensive; according to another technique, the pH of supernatant IV is lowered to 4.80.+-.0.05, which causes the albumin to precipitate: this precipitate, referred to as "precipitate V", is separated out and is redissolved in physiological serum, the remainder of the ethanol being extracted by dialysis.
To complete the purification of albumin, it has already been proposed to use chromatographic techniques, in particular by ion exchange (see in particular Curling J. M., Methods of Plasma Protein Fractionation, Ed. J. Curling, Acad. Press, 77-91 (1980); Tayot J. L. et al., Methods of Plasma Protein Fractionation, Ed. J. Curling, Acad. Press, 149-160 (1980); ion exchangers!, Ann. Pharm. Fr. 39, 403-409 (1981)). The principle behind these methods is to bind the albumin to a specific support, as a function of the pH and of the ionic strength, and thereby to eliminate in the effluent some of the impurities which are found in the albumin solution treated by chromatography. Unfortunately, although these techniques allow the quality of the albumin solutions obtained to be enhanced substantially, they are difficult and expensive to implement. In actual fact, the treated albumin solutions contain approximately 4% of diverse impurities to be extracted. Given that the albumin is bound to the packing material of the chromatography column, it is necessary to bind 96% of the material treated and, since the binding of albumin per gram of packi
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Graafland Hubert
Rucheton Marcel
Stefas Elie
Mohamed Abdel A.
Tsang Cecilia J.
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