Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Patent
1999-01-07
2000-05-02
Minnifield, Nita
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
436514, 436516, 430381, 430383, 430384, 430413, 430416, 430417, 430420, 430427, G01N 33558, G01N 33561, A61K 3514, A23J 100
Patent
active
060571640
DESCRIPTION:
BRIEF SUMMARY
The present invention pertains to a method for the aptitude testing of protein fractions containing factor VIII the further processing of which comprises a pasteurizing step, as well as the utilization of batches found to be unsuitable.
Factor VIII preparations are predominantly prepared from cryoprecipitate. Applicant's preparations are prepared by purification of the redissolved cryoprecipitate with aluminum hydroxide, virus inactivation with solvents/detergents and subsequent anion exchange chromatography. Due to sharpened safety directions for virus inactivation, a pasteurizing step has been additionally performed in which the preparations are heated at 63.degree. C..+-.1.degree. C. in the presence of a stabilizer for 10 hours following the first virus inactivation.
Whereas the preparations virus-inactivated according to the solvent/detergent method alone have been applied without difficulties for years, the preparations additionally subjected to the pasteurizing step exhibited unexpected and unforeseeable inhibitor formations in patients pretreated with factor VIII. Such inhibitor formations occurred after about six weeks or later. Intensive studies had the result that such inhibitor formations had occurred in an accumulated fashion in patients treated with a particular batch or with batches of a particular origin of the cryoprecipitate.
Further intensive studies had the result that those were the only batches which contained small quantities, but altogether detectable and identifiable quantities, of factor VIII fragments within a range of from 20 to 50 kD. These fragments could not be found in any other batch or in products from competitors.
When these relationships were found, it was further revealed that the early occurrence of inhibitor formation after about six weeks had only been observed in those patients which were exclusively treated with the preparations from particular batches whereas patients in which such inhibitor formation occurred later had been treated with preparations from different batches. Thus, the conclusion could be drawn with very high certainty that those contaminants in the factor VIII containing protein fractions employed, namely cryoprecipitate of a particular origin, cause the inhibitor formations. In addition, it was established that such inhibitor formation did not occur in those patients which had already been treated for years with preparations of the same origin, but in which the pasteurizing step had not yet been performed. Thus, it has to be considered now that these fragments of factor VIII are subjected to changes in the pasteurizing step which then cause the inhibitor formation.
Thus, it has been the object of the present invention to develop an aptitude test for protein fractions containing factor VIII the further processing of which comprises a pasteurizing step. This object is achieved in a surprisingly simple manner by examining the entire starting material for fragments within a range of from 20 to 50 kD. If such fragments are not present, the starting material can be employed for the preparation of a twofold virus-inactivated product. If, in contrast, such fragments are present, then additional measures are necessary for processing such starting material into useful preparations free of side effects.
BRIEF DESCRIPTION OF THE DRAWINGS
Each of FIGS. 1 and 2 is a chromatogram of a separation performed by SDS polyacrylamide gel electrophoresis.
FIG. 3 is a chromatogram of size exclusion (chromatography) performed on Fractogel BioSEC.
FIG. 4 is a chromatogram of size exclusion (chromatography) performed on Superose 6.
FIG. 5 is a chromatogram of size exclusion (chromatography) performed in tandem, first, on Fractogel BioSEC and, second, on Superose 6.
FIG. 6 represents comparative results of size exclusion chromatography performed in tandem (as described above) on Fractogel BioSEC and Superose 6.
FIG. 7 is a chromatogram of size exclusion (chromatography) affected between proteins and detergents.
One possibility of further processing is the use of such start
REFERENCES:
patent: 5259951 (1993-11-01), Arright et al.
Josic et al, Purification of factor VIII and von Willbrans factor from human plasma by anion -exchange chromatography. J.chromatography, vol. 662, pp. 181-190, 1994.
Arrighi et al, Factor VIII: c Concentrate virus inactivated: progress in purification by using classic chromatographic methods. Vox Sang, von. 64, pp. 13-18, 1993.
Bihoreau et al, Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor, European Journal of Biochemistry, 185, 111-118, 1989.
Buchacher Andrea
Josic Djuro
Stadler Monika
Baskar Padma
Minnifield Nita
Octapharma AG
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