Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1995-10-23
1999-01-12
Huff, Sheela
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530362, 530363, 530395, 530380, C07K 1400
Patent
active
058592130
DESCRIPTION:
BRIEF SUMMARY
This application is a 35 U.S.C. 371 national stage filing of international application PCT/FR94/00143, filed Feb. 2, 1994.
The present invention relates to a protein composition obtained as a secondary product in the production of albumin from human blood plasma, to a method for producing it, to the glycoprotein which it contains and to the use of the said glycoprotein in albumin-stabilizing agents and as an agent for allowing antibodies to be detected and/or assayed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph that shows the turbidity of the albumin solutions A and B as a function of time.
FIG. 2 is a graph that shows as the protein concentrations increases, the turbidity of albumin decreases.
In the state of the art, it is known that, in order to obtain solutions of human albumin, blood plasma is fractionated either by the Cohn method (see Cohn J. et al., J. Amer. Chem. Soc, 72, 465-474-(1950)), or by a derived method in which successive thermal and ethanolic treatments are carried out or a similar method in which a precipitation agent other than ethanol (especially ether, ammonium sulfate, polyethylene glycol or caprylic acid) is used. According to the Cohn method, which is, in practice, the one used most commonly, the plasma is cooled to -30.degree. C. and then warmed to -2.degree. C., which 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 ethanolic concentration is 19% by volume, the temperature being lowered 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 ethanolic concentration of 40% by volume is obtained, the temperature being lowered to about -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 in a degree of purity of about 94 to 97% by weight. The supernatant IV serves as a starting material for the desired albumin solutions, but it contains a large amount of ethanol; according to a first technique, the supernatant IV may be dialyzed directly against physiological saline, but a very large amount of water then needs to be used and the method is thus slow and expensive; according to another technique, the pH of the supernatant IV is lowered to 4.80.+-.0.05, thereby causing the albumin to precipitate out: this precipitate, referred to as "precipitate V", is separated out and is redissolved in physiological saline, the rest of the ethanol being extracted by dialysis.
The aqueous solutions obtained after dialysis of the "supernatant IV" or of the "precipitate V" which is redissolved contain albumin and about 4% by weight, relative to the total weight of protein material, of other so-called secondary or contaminating proteins and/or polymers.
According to FR-A-2,690,444, the albumin is separated from the other proteins by a liquid phase chromatographic method in which, after dialysis, the aqueous solution containing the albumin is passed through at least one so-called "hydrophobic" chromatography column filled with a particulate material capable of retaining some of the proteins other than the albumin; in order to complete the separation, it is also proposed to pass the aqueous albumin solution through at least one affinity chromatography column containing a neutral particulate support or a particulate support close to neutrality charged with a polysulfated compound. The effluent obtained by this method consists of a solution of purified albumin, the majority of the secondary proteins being bound either to the hydrophobic chromatography column(s) or to the affinity chromatography column(s).
REFERENCES:
patent: 4289690 (1981-09-01), Peska et al.
M. Burstein et al, "Polysulfates, anionic detergents, sodium phosphotungstate and the electrophoretic mobility of plasma proteins", Chemical Abstracts, vol. 89, No. 19, Nov. 1978, Abstract No. 159658, p. 256.
Lozier et al, "Complete amino acid sequence of human plasma beta 2-glycoprotein I", Proceedings of the National Academy of Sciences of USA, vol. 81, Jun. 1984, pp. 3640-3644.
Li et al BiochemJ vol. 267 261-264, 1990.
Sofer etal BioTechniques vol. 1 No. 4 198-203, 1983.
Bonnerjea et al Bio/Technology vol. 4 955-958, 1986.
Medhi et al Gene vol. 108 No. 2 293-298, 1991.
Graafland Hubert
Rucheton Marcel
Stefas Elie
Huff Sheela
Reeves Julie E.
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