Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Patent
1998-12-22
2000-11-07
Therkorn, Ernest G.
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
210656, 2101982, 2105021, 530383, 530413, B01D 1508
Patent
active
061431796
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
The present invention concerns a process for the affinity chromatographic purification of factor VIII by adsorption to immobilized von Willebrand factor.
The purification of human factor VIII (FVIII) from biological fluids such as plasma, plasma fractions or plasma derivatives using affinity chromatographic methods has been known for a long time.
Thus Tuddenham et al. (J. Lab. Clin. Med. 93 (1979), 40-53) describe such a purification procedure using immobilized polyclonal antibodies which were isolated from the plasma of inhibitor patients.
Veerman E. C. I. et al. (Thromb. Res. 33 (1983), 89-93) describe a purification process using monoclonal antibodies to human FVIII.
Furthermore processes for the purification of FVIII are known using immobilized monoclonal murine antibodies to human von Willebrand factor (vWF) in which vWF/FVIII complexes are adsorbed and subsequently vWF-free FVIII is isolated by known methods (cf. e.g. EP-A-0083 438 and U.S. Pat. No. 4,831,118).
However, a disadvantage of these processes is that separation of FVIII from its natural stabilizer, the vWF, has an adverse effect on the stability of FVIII. Moreover it is unfavourable for certain forms of therapy to obtain a completely vWF-free FVIII preparation (Hornsey et al, Thromb. Haemost. 57 (1987), 102-105).
Adsorption of FVIII to immobilized plasmatic vWF is already known (cf. Leyte et al., Biochem. J. 257 (1989), 679-683). However, it was found that only about 1-2% of the plasmatic FVIII activity is bound to immobilized plasmatic vWF. An economic process is inconceivable with such a result.
Hence an object of the present invention was at least to partially eliminate the disadvantages of the state of the art. In particular a process should be provided which enables a high purification efficiency to be obtained in a simple and cost-effective manner.
This object is achieved by a process for the purification of factor VIII by affinity chromatography characterized in that a biological fluid containing factor VIII is contacted with immobilized cellular von Willebrand factor or a derivative thereof under such conditions that factor VIII is adsorbed to the immobilized von Willebrand factor or derivative thereof, impurities are separated and adsorbed factor VIII is eluted. It was surprisingly found that cellular vWF e.g. vWF formed by endothelial cells is, in contrast to plasmatic vWF, excellently suitable after immobilization for purifying FVIII. A vWF produced by endothelial cells (EC-vWF) has namely a substantially higher affinity for FVIII than plasmatic vWF so that it is possible to isolate the FVIII activity from biological fluids in high purity and yield.
Cellular vWF is preferably isolated from cultured human cells, preferably from cultured human endothelial cells e.g. from human endothelial cells of the umbilical vein (HUVEC), bound to the solid phase and incubated with a biological fluid. After separation of impurities (non-binding or unspecifically-binding impurities), the adsorbed FVIII is eluted preferably by applying a salt gradient. The advantages of the process according to the invention are in particular that human material is used exclusively, the purification is less time consuming and the purified FVIII has a low proteolysis rate. This leads to an unusually high specificity of the purified material.
Human plasma, plasma fractions, plasma derivatives e.g. cryoprecipitates or culture supernatants of cells producing FVIII are preferably used as biological fluids. The biological fluid can optionally be subjected to a prior chromatographic purification e.g. an aluminium hydroxide adsorption.
If plasma is used as the biological fluid, a predilution of ca. 1:2 and the addition of proteinase inhibitors is preferred.
It is additionally preferred to keep the biological fluid containing FVIII in contact with the immobilized vWF for a period of at least 30 min. A contact period of at least 1 h is particularly preferred. The incubation temperature is preferably 10 to 37.degree. C.
Monomeric vWF or derivatives thereof e.g.
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Knutson, et al., "Porcine Factor VII:C Prepared By Affinity Interaction With Von Willebrand Factor and Heterologous Antibodies Sodium Dodecyl Sulfate Poly Acrylomide Gel Analysis," Blood 59(3):615-624 (1982) (computer generated abstract).
Muller-Berghaus Gert
Potzsch Bernd
Schwinn Horst
Kerckhoff-Klinik GmbH
Therkorn Ernest G.
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