Drug – bio-affecting and body treating compositions – Fermentate of unknown chemical structure – Having a known elemental analysis
Patent
1981-02-11
1983-06-07
Rosen, Sam
Drug, bio-affecting and body treating compositions
Fermentate of unknown chemical structure
Having a known elemental analysis
260112R, A61K 3514
Patent
active
043870929
DESCRIPTION:
BRIEF SUMMARY
In U.S. Pat. No. 4,069,216 there is described and claimed a process for preparing a factor VIII concentrate wherein it comprises the stage consisting in maintaining a buffer solution containing factor VIII and about 6% polyol at a temperature of about 0.degree. to 5.degree. C. until precipitation occurs.
The resulting precipitate is made up of concentrated, purified factor VIII or antihemophilic factor, also called AHF.
According to a particular embodiment, also described and claimed in the aforementioned patent, this process is preceded by a stage consisting in using a plasma solution or plasma cryoprecipitate as the starting product and adding about 4% polyol to said solution, eliminating the resulting fibrinogen precipitate and using the supernatant as the starting product in the process mentioned above.
This preliminary stage was performed at a temperature of about 20.degree. to 30.degree. C.
It has now been discovered that this preliminary purification stage can be replaced by another preliminary stage that consists in treating a factor VIII solution to be purified with a slight amount of polyol corresponding to a concentration less than 4%, while cooling at a temperature below 15.degree. C. Thus, the major part of the fibrinogen and other impurities such as, for example, cold insoluble globulins (CIG) are eliminated by precipitation. By operation at low temperature, a purification is obtained at least as good as with the process at ambient temperature of the aforementioned patent, while a smaller amount of polyol is used.
According to preferred embodiments, this preliminary stage of the present invention can further be characterized by the following points, taken individually or in combination.
The starting product, such as those mentioned in the aforementioned patent, is used in the form of a buffer solution containing factor VIII to be purified; the starting product is, for example, a cryoprecipitate buffer solution or a plasma solution containing factor VIII. The impurities present in the starting product are fibrinogen and CIGs.
Said starting solution is buffered at a pH between 6 and 7, and preferably 6.0 to 6.5.
Polyol is added, preferably in a concentration of about 1 to 3%.
In a general way, the indications of concentrations, in the present invention, are by weight/volume.
The polyol can be, for example, polyethyleneglycol, preferably polyethyleneglycol 4000.
As other usable polyols, there will be cited in particular sequenced polymers of the polyoxyethylene-polyoxypropylene-polyoxyethylene type such as those sold under the name of Pluronic F-68.
Cooling is at a temperature between 0.5.degree. and 15.degree. C., generally between about +1.degree. and +5.degree. C. and in particular between about 2.degree. and 4.degree. C.
Stirring is for a time that can vary from 5 minutes to 3 hours, preferably for about an hour, while cooling is maintained to keep the selected temperature, then centrifuging is performed still while cooling at a temperature below 15.degree. C., preferably between +1.degree. and +5.degree. C., as indicated above.
The resulting precipitate is eliminated and the supernatant is used as the starting product of the process described in said patent.
The following example illustrates the invention without, however, limiting it.
EXAMPLE
Cryoprecipitate (487 g) which is dissolved in 1920 ml of a buffer solution consisting of an aqueous solution of 0.02 M trisodium citrate and 0.1 M glycine with a pH 6.9 is used as the starting point.
After complete dissolving, the pH is adjusted to 6.4 with citric acid and PEG 4000 is added in a 2% concentration (38.4 g). Cooling is to +2.degree. C. and stirring is performed for an hour. Centrifuging is done with the temperature kept at +2.degree. C. The precipitate is eliminated and the supernatant collected (1670 ml).
Then the supernatant is treated according to the process of the aforementioned patent. For this, the pH of the supernatant is adjusted to 6.9 by addition of NaOH, then 68 g of PEG (6%) are added still at +2.degree. C. After stirring
REFERENCES:
patent: 3631018 (1971-12-01), Shanbrom et al.
patent: 3770631 (1973-11-01), Fekete et al.
patent: 4069216 (1978-01-01), Shanbrom
patent: 4085095 (1978-04-01), Bick et al.
Liautaud Jacques
Shanbrom Edward
Rosen Sam
Societe Anonyme dite: "L'Oreal"
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