Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1994-06-02
2001-05-29
Guzo, David (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S380000, C530S381000, C530S382000, C530S383000, C530S418000, C530S416000, C530S419000, C530S420000
Reexamination Certificate
active
06239261
ABSTRACT:
The invention relates to a process for the preparation of a purified and pasteurized von Willebrand factor concentrate, and to such a concentrate which has been prepared by this process and is suitable for the treatment of von Willebrand syndrome.
The latter syndrome is characterized by a congenital deficiency and/or defect of von Willebrand protein.
There is a need for a pure and virus-safe von Willebrand factor concentrate because increasingly better purified factor VIII:C concentrates, which now contain only traces of von Willebrand factor, are being used for the treatment of hemophilia A.
Since von Willebrand patients have to receive life-long therapy, with high doses in some cases, a product of great purity and safety is indicated. Advantageous preparations are low in fibrinogen, immunoglobulins and isoagglutinins.
In the plasma, von Willebrand factor circulates in a concentration of 5-10 mg/l and in the form of a non-covalently bonded complex with factor VIII, the so-called antihemophilic globulin. In cryoprecipitate, von Willebrand factor is greatly enriched as von Willebrand factor/factor VIII complex and can be isolated therefrom or from plasma or plasma fractions using known fractionation methods.
German Offenlegungsschrift 3,504,385 (U.S. Pat. No. 4,578,218) discloses a process for the treatment of factor VIII complex, in which a factor VIII preparation is bound to an insoluble matrix which carries free sulfate groups, for example dextran sulfate, but evidently no separation of factor VIII complex into factor VIII:C and von Willebrand factor is possible in this case.
GB 2,079,292 describes a process for obtaining a von Willebrand factor from cryoprecipitate, but this does not separate factor VIII:C from von Willebrand factor either.
EP 0,022,052 (U.S. Pat. No. 4,210,580) describes a process in which plasma is treated with sodium heparin, whereupon fibronectin precipitates out together with von Willebrand factor. Antihemophilic factor is obtained from the supernatant. The precipitate is chromatographed on DEAE-cellulose, and fibronectin is obtained. It is stated that when agarose gel is used for the chromatography von Willebrand factor is eluted in the void volume. However, the amounts of heparin used are costly and gel filtration is a bottleneck for preparation on the industrial scale. In addition, toxic KSCN is used.
In European Patent 0,083,483 it is stated, on the state of the art, that J.Lab.Clin.Med. 93, 40 (1979) describes a process for separating factor VIII:C and von Willebrand factor, where the separation is brought about by immuno-adsorption. However, only the factor VIII:C is obtained in sufficient purity in this process.
Another process for separating these two factors is described in Brit.J.Haematol. 43, 669 (1979), with aminohexyl-agarose being used. This process is also unsuitable for obtaining von Willebrand factor.
EP 0,083,483 itself describes a process for obtaining factor VIII:C which contains only small amounts of von Willebrand factor. However, no process for obtaining von Willebrand factor is described therein.
It is common to all these processes that they do not lead to complete dissociation of the complex with subsequent quantitative liberation of a native F VIII:C and vWF. None of these processes describes a pasteurized, and thus virus-safe, product. In order to protect the vWP from proteolytic decomposition during the purification, toxic substances such as DFP and soybean trypsin inhibitor or else buffers such as KSCN and NaN
3
are used in these processes. Finally, these processes have disadvantages because they contain steps which represent a bottleneck for large-scale manufacture. These include, for example, gel filtrations, i.e. separation according to molecular weight or chromatographic steps using a salt gradient for the elution.
The present invention describes a process with which it is possible, surprisingly, to dissociate the complex of factor VIII:C and von Willebrand factor and to isolate von Willebrand factor purified and pasteurized in high yield. The object of this invention is to obtain a purified, pasteurized, and thus virus-safe, coagulating therapeutic agent for the treatment of von Willebrand syndrome.
The invention relates to a process for the preparation of a pasteurized von Willebrand factor concentrate, which comprises a solution which contains von Willebrand factor (vWF) as complex with F VIII:C in a buffer of pH 5.5 to 6.5, which contains amino acids and has a carbohydrate concentration of 5-30% w/w, being treated with an anion exchanger to which F VIII:C binds, and the von Willebrand factor concentrate being obtained from the solution.
It is possible to use as starting material for the preparation of a vWF concentrate solutions in which the vWF is present as complex with F VIII:C, for example plasma and fractions obtained therefrom, such as cryoprecipitate, Cohn fraction I or else supernatants and extracts from cell cultures.
The starting material, preferably cryoprecipitate or an intermediate fraction obtained therefrom, can have been pasteurized.
The vWF can be protected from thermal inactivation during the pasteurization by the addition of carbohydrates, preferably sucrose, preferably in concentrations of 10-60% (w/w), and/or amino acids, preferably glycine, preferably in concentrations of 0.5-3.0 mol/l, and/or calcium salts, preferably 1-20 mmol/l. It is also possible by these measures simultaneously to prevent the precipitation of acid-sensitive proteins, for example of fibronectin.
The carbohydrates act not only to protect the proteins from thermal inactivation or denaturation, but also as solubilizers in the acidic pH range from 6.5 to 5.5, particularly for fibrinogen and vWF in this context, in that they surprisingly prevent precipitation.
After an adsorption of the F VIII:C onto the ion exchanger, the vWF can be kept in solution at pH 5.5, and from this the fibrinogen can be removed by addition of glycine in concentrations of 0.5-3 mol/l, preferably 2.7 mol/l, and the vWF can be precipitated from the glycine supernatant with NaCl concentrations corresponding to 2-15% (v/v), preferably 6% (w/v).
The prepurified vWF intermediate product can be pasteurized a second time to increase the virus safety.
The pasteurized and highly purified vWF can be sterilized by filtration and lyophilized, for example with glycine (2% w/v), albumin (0.5%) in citrated (0.02 mol/l) NaCl (0.06 mol/l) as stabilizers.
The conditions for the dissociation and purification can be transferred to large-scale manufacture.
In contrast to antihemophilic cryoprecipitate, crude cryoprecipitate or cryofractions which have hitherto been used for the treatment of vW syndrome, the product according to the invention is virtually free of ballast proteins.
The process according to the invention complies with the stringent requirements relating to purity, yield and virus safety: viruses which are possibly present are eliminated, together with the ballast proteins, by the purification process, and are inactivated by a pasteurization. The specific activity of a product prepared by the described process is above 100 U of F VIIIR:CoF/mg of protein.
The procedure can be as follows:
Dissolved cryoprecipitate which is greatly enriched in von Willebrand factor and factor VIII and from which the factors of the prothrombin complex have been removed by an Al(OH)
3
adsorption is stabilized in a manner known per se by addition of carbohydrates, amino acids and calcium ions to protect against thermal inactivation and is heated in aqueous solution at 60° C., preferably for 10 h.
The pasteurized solution can be diluted with a buffer of physiological conductivity (12-15 mS) and a pH of 5.5 and the composition 0.2 mol/l lysine and 0.2 mol/l sodium acetate to twice the volume, the pH of the solution adjusted to 5.5 and an anion exchanger added at 20° C.
Under these conditions, the factor VIII binds to basic ion exchangers with DRAK and QAR as functional groups bonded to “SEPHADEX”, “SEPHAROSE”, cellulose or “FRACTOGEL” as matrix, whereas vWz remains in solution. Thes
Heimburger Norbert
Kumpe Gerhard
Wellner Klaus
Aventis Behring GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Guzo David
Leffers, Jr. Gerald G.
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