GB2079292A - Blood Clotting Factor Production - Google Patents

Abstract

The 3-4% PEG precipitate of cryoprecipitate which is normally discarded during the process of obtaining high purity Factor VIII concentrate from human blood has been found to be usable as a starting material for the production of a Von Willebrand clotting factor concentrate which when intravenously administered to a Von Willebrand haemophiliac either as it is (when in liquid form) or else together with a pharmaceutically acceptable carrier in as an effective clotting amount is capable of bringing about blood clotting at the locus of bleeding in a Von Willebrands disease sufferer. A liquid concentrate of this invention may be prepared by solubilising a 3-4% PEG precipitate with a solubiliser which can take up most of the Von Willebrand factor present in the precipitate, preferentially precipitating fibrinogen from the thus obtained solution, treating the fibrinogen free supernatant with an adsorbent which will adsorb clotting factors II, VII, IX and X, subsequently treating the supernatant obtained with a precipitant which is capable of precipitating all of the protein present and then solubilising the thus obtained precipitate with a solubilizer capable of taking up most of the Von Willebrand factor present. The liquid concentrate of Von Willebrand factor obtained may, if desired, be lyophilised.

Description

SPECIFICATION Blood Clotting Factor Production The present invention relates to the production of a blood clotting factor and more particularly relates to a process for the production of a Von Willerbrand factor concentrate, to pharmaceutical formulations containing the concentrate, and to its use in combatting Von Willerbrand's disease.

Generally, high purity Factor VIII concentrate is obtained from human blood in the following manner. Blood is taken from a donor and is spun to remove red blood cells. Thereafter, the plasma obtained is spun to remove platelets and the supernatant is frozen to a low temperature. After a suitable period of time, which is normally at least two days, the frozen material is thawed. This thawing results in a precipitate, called a cryoprecipitate, which is then solubilised after centrifugation in a Tris type buffer. After solubilisation, the solution obtained is subjected to a double precipitation process using polyethylene glycol (PEG) as the precipitant. The first precipitation involves treatment of the solution with 34% PEG with the purpose of precipitation of fibrinogen.This precipitate, known as the 34% PEG precipitate of cryoprecipitate is normally discarded. The next stage, after removal of the aforementioned precipitate, is to subject the resultant supernatant to a second precipitation using 10% PEG whereby a high purity Factor VIII concentrate is obtained.

Typical methods for obtaining such concentrates are described in the following articles:-- Johnson A. J., Newman J., Howell M. B. et al, "Purification of Antihemophilic factor (AHF) for clinical and experimental use", Thromb, Diath, Haemorrh, Suppl 26: 377, 1967 and Newman J, Johnson A.

J., Karpatkin M. H. et al. "Methods for the production of clinically effective intermediate- and high purity Factor VIII concentrates" Br. J.

Haematol.21:1, 1971.

It has now been surprisingly found that a concentrate of Von Willerbrand factor can be obtained from the aforementioned 34% PEG precipitate of cryoprecipitate by-product formed in the production of high purity Factor VIII concentrate.

Accordingly, the present invention provides a process for the production of a concentrate of Von Willerbrand Factor which comprises the steps of: (i) solubilising a 34% PEG precipitate of cryoprecipitate with a solubiliser capable of taking up substantially all of the Von Willebrand factor present in said precipitate; (ii) adding to the solution thus obtained a precipitant which is capable of preferentially precipitating fibrinogen with regard to the other components of the solution; (iii) treating the supernatant obtained with an adsorbent capable of adsorbing clotting factors 11, VII, IX and X; (iv) treating the supernatant obtained with a precipitant capable of precipitating substantially all of the protein present; and (v) solubilising the precipitate thus obtained with a solubiliser capable of taking up substantially all of the Von Willebrand factor in said precipitate.

Preferably the process of the present invention further comprises the step of: (vi) freeze drying the solution obtained in step (v).

The solubiliser used in step (i) of the process of the present invention is advantageously a buffer, preferably a Tris type buffer. It is further preferred for such a buffer to contain one or more inorganic salts, and a buffer comprising Tris and NaCI has been found to be particularly suitable for this step.

Optionally the buffer may contain a sugar component, glucose being a particularly suitable sugar, and the most preferred buffer for the purposes of step (i) of the process of the present invention is NaCI 0.4M, Tris 0.02M optionally together with 3% glucose. Step (i) is generally carried out at a temperature below 370C, preferably at 200C and generally at a pH of from 5 to 9, preferably from 6 to 8, more preferably from 6.5 to 7.5 and most preferably at 6.8.

Desirably, but not essentially, after step (i) and before step (ii), the temperature of the solution is lowered to, for example 100C and the pH is adjusted to 6 by the addition of e.g. citric acid if the pH is originally above 6 and by the addition of e.g. sodium hydroxide if the pH is originally below 6. The solution is then kept at that temperature for a time such as half an hour before proceeding to step (ii).

A typical precipitant for use in step (ii) of the process of the present invention is glycine which may be added to the solution obtained from step (i) in dry form or as a solution although it is generally more convenient to add glycine.

Preferably the precipitant, such as glycine, is added to a final concentration of approximately 1 .8M, preferably over a period of time such as 10 minutes rather than all in one addition.

Preferential precipitation of fibrinogen by use of a suitable precipitant such as glycine may be aided by lowering the pH and/or temperature of the solution during precipitant addition.

After step (ii) the precipitate obtained is generally separated out by gross filtration, preferably on a sterile gauze, and the supernatant retained. In step (iii) the supernatant is treated with an adsorbent capable of removing clotting factors II, VII, IX and X, and it has been found that aluminium hydroxide is particularly suitable for that purpse. Advantageously, sufficient Al (OH)3 is added to bring the final concentration thereof to up to 10%, preferably from 4 to 6% although more may be added if desired. The Al (OH)3 may be added in any convenient form, although the addition of 20% Al (OH)3 is preferred.The adsorbent used in this step should be allowed to have sufficient contact with the components of the solution which it is to adsorb, and in this connection it is advantageous to subject the mixture containing the adsorbent to continuous stirring during this stage. Generally, if stirring is carried out, a contact time of about 5 to 10 minutes is sufficient.

After step (iii) the adsorbent and the components adsorbed thereon are generally separated from the mixture by filtration, or more preferably by centrifugation, preferably at room temperature, for a period such as 1 5 minutes and at a force of generally at least 6,000 g, and the supernatant is retained.

In step (iv) the supernatant thus obtained is treated with a precipitant which is capable of precipitating substantially all of the protein present in the supernatant and it has been found that PEG is particularly suitable for this step, optionally together with a second suitable precipitant such as albumin. If PEG is used, it may be added in solid form or as a solution, although addition of a PEG solution is preferred, advantageously in an amount such that the final PEG concentration is up to 25%, most preferably 10%. A suitable PEG solution for addition has a concentration of about 30% and with advantage such PEG solutions are added at a low temperature, for example 40C. If albumin is used in addition to a first precipitant it is conveniently added in an amount of 0.5% (final concentration).

Precipitation of protein in this step may be aided by lowering the temperature of the mixture to, for example 1 OOC by means of, for instance, a refrigerated alcohol bath. Constant stirring also aids precipitation and with great advantage, the pH of the supernatant to which the precipitant is to be added is slightly on the acid side of neutral, e.g. pH6, and this slight acidity may be achieved, if the supernatant does not already have such a pH by the addition of a suitable amount of an acidic or basic reagent as the circumstances dictate.

After step (iv) the precipitate required for the next stage is generally separated from the mixture by filtration, or more preferably, by centrifugation in a centrifuge conveniently at a rotational speed of at least 32,000 r.p.m.

The fine precipitate thus obtained from step (iv) is subjected to a solubilising step, that is, step (v) of the process of the present invention. The solubiliser used in step (v) is advantageously a buffer, preferably a buffer containing Tris and/or citrate. It is further preferred for such a buffer to contain one or more inorganic salts, and buffers comprising Tris and/or citrate together with NaCI have been found to be particularly suitable for this step. Optionally the buffer used contains a sugar component such as glucose and/or a buffering agent such as glycine. The most preferred buffers for the purposes of step (v) of the process of the present invention are 0.1 5M NaCI, 0.02M citrate and 0.1 M glycine and also 0.1 so NaCI, 0.02M citrate, 0.03M Tris and 1% glucose.Step (v) is generally carried out at a temperature below 37"C, preferably at 200C and generally at a pH of from 5 to 9, preferably from 6 to 8, more preferably from 6.5 to 7.5 and most preferably at 6.8.

Desirably, but not essentially, after step (v) and before any subsequent processing, the temperature of the solution is lowered, for example, to 1 O0C and the pH is adjusted to 6 by the addition of e.g. citric acid if the pH is originally above 6 and by the addition of e.g. sodium hydroxide if the pH is originally below 6. The solution is then kept at that temperature for a time, such as half an hour before proceeding with any further processing.

The solubilised precipitate (which is, of course, a liquid Von Willebrand factor concentrate of the present invention) is then preferably filtered to remove any insoluble products, and this filtered material may then be stored as such or else, more preferably, if freeze-dried by any of the methods well known in the art for freeze-drying. The freeze-dried material is a solid Von Willebrand factor concentrate of the present invention.

The concentrates of the present invention, when administered intravenously as a suitable formulation and in an effective amount, are capable of bringing about blood clotting in humans suffering from Von Willebrands disease.

Preferably the active ingredient i.e. the Von Willebrand clotting factor is administered in an amount corresponding to about 40 units per kilogramme body weight of the patient. The above mentioned filtered liquid Von Willebrand Factor concentrate may be administered as it is or else together with a pharmaceuticaily acceptable carrier or diluent as an intravenous formulation.

However, it is preferred to administer the freezedried solid Von Willebrand Factor concentrate dispersed in a pharmaceutically acceptable carrier or diluent. This is because for a given dose of Von Willerbrand factor, a solid concentrate containing that dose can be formulated as an injectable solution, for example in sterile water, having one quarter the volume of a liquid concentrate containing that same dose.

According to another aspect of the present invention therefore there is provided an intravenous pharmaceutical formulation which comprises a Von Willebrand concentrate of the present invention together with a pharmaceutically acceptable carrier or diluent.

The present invention also provides a method of clotting blood at the locus of bleeding of a Von Willebrand haemophiliac which comprises administering an effective clotting amount of a Von Willebrand factor concentrate of the present invention.

The present invention will be further illustrated with reference to the following Examples in which Example 1 describes a preferred embodiment of the process of the present invention and Example 2 describes a clinical test showing the effectiveness of the concentrates obtained by the process of Example 1 in a patient suffering from Von Willebrand's disease.

Example 1 34% PEG precipitate in the preparation of FactorVlll:C concentrate from cryoprecipitate (1 kg) was solubilised in 8 iitres of a buffer containing 0.4M NaCI, 0.02M Tris and 0.1 M EACA at pH 6.8. Dry glycine was then progressively added over a period of 10 minutes to a final concentration of 1.8M. The heavy precipitate which formed was removed by gross filtration on sterile gauze. The pH of the supernatant was then adjusted to 6.8 and 20% Al (OH)3 was added with constant stirring to obtain a final concentration of 4% aluminium hydroxide.

The average normal contact time of the Al (OH)3 step was 5-10 minutes. The mixture obtained was then centrifuged in 1 litre plastic cups for 1 5 minutes at 6,000 g at room temperature. The supernatants were collected and pooled and the pH thereof adjusted to 6. A 30% PEG 4000 solution at 40C was added to the pooled material to a final PEG concentration of 10%, and the mixture obtained was subsequextly put into a refrigerated alcohol bath and the temperature was lowered to 1 00C under constant stirring conditions. After 5 minutes at 1 OOC, the precipitate was collected by centrifugation in a Sharples centrifuge at 32,000 r.p.m. using an input flow rate of 400 ml/min.The fine precipitate was then dissolved with constant stirring in a buffer at pH 6.8 containing 0.15M NaCI, 0.02M citrate and 0.1 M glycine. Approximately 800 ml of this buffer was used for each kilogramme of precipitate. The solubilised precipitate was then filtered through a sterile gauze at room temperature to remove any insoluble particles and was subsequently filtered through a Millipore (Registered Trade Mark) sandwich filter composed of 5, 1.2, 0.8 0.65, 0.45, 0.30 and 0.20 micron filters. The material thus filtered was then freeze-dried in 100 ml aliquots.

The above procedure was carried out twelve times and the final products analysed. The mean results of the final product was: Proteins 19.6+4.1 mg/ml Fibrinogen 9.0+4.5 mg/ml F Vlil :C 3.3+0.9 u/ml F Vlil: AG 6.5+2.2 u/ml and FVIll :VWF 10.1+2.9 u/ml The specific activity of F VEIL: VWF was found to be 0.52+0.2 u/mg and the average yield from starting material in terms of F VEIL: AG was 28%.

Example 2 A clinical trial was performed on a patient with severe Von Willebrand's disease. The patient received an intravenous dose equivalent to 25 u/kg of F VIII :AG (approx 40 u/kg F VIII :VWF), the formulation administered-being a freeze-dried product produced in accordance with Example 1 in an amount containing the aforementioned dose dispersed in sterile water. The results of the quantitative bleeding times were:- Before transfusion: 900 yl 30 Minutes post transfusion 880,ul 2 hours post transfusion 88 yl 6 hours post transfusion 49 yl 1 2 hours post transfusion 840 ssl The value in normal subjects, ie: those not suffering from any form of Haemophilia is less than 40 yI.

The quantitative bleeding test used was that described in the article: "The standardisation of certain factors in the cutaneous 'venostasis' bleeding time technique", Ivy A. C, Nelson D and B-uches G(1940) J. Lab. Clin. Med.

26,1812.

Claims (25)

Claims

1. A process for the production of a concentrate of Von Willebrand Factor which comprises the steps of: (i) solubilising a 34% PEG precipitate of cryoprecipitate with a solubiliser capable of taking up substantially all of the Von Willebrand factor present in said precipitate; (ii) adding to the solution thus obtained a precipitant which is capable of preferentially precipitating fibrinogen with regard to the other components of the solution; (iii) treating the supernatant obtained with an adsorbent capable of adsorbing clotting factors II, VII, IX and X; (iv) treating the supernatant obtained with a precipitant capable of precipitating substantially all of the protein present; and (v) solubilising the precipitate thus obtained with a solubiliser capable of taking up substantially all of the Von Willebrand factor in said precipitate.

2. A process as claimed in Claim 1 which further comprises the step of: (vi) freeze-drying the solution obtained in step (v).

3. A process as claimed in Claim 1 or Claim 2 wherein the solubiliser used in step (i) is a buffer.

4. A process as claimed in Claim 3 wherein the buffer contains one or more inorganic salts.

5. A process as claimed in any of Claims 1 to 4 wherein step (i) is carried out at a temperature below 370C and at a pH of from 5 to 9.

6. A process as claimed in Claim 5 wherein the temperature is 200C.

7. A process as claimed in Claim 5 or Claim 6 wherein the pH is 6.8.

8. A process as claimed in any of Claims 1 to 7 wherein between steps (i) and (ii), the temperature of the solution is lowered to 10C and the pH is adjusted to 6.

9. A process as claimed in any of Claims 1 to 8 wherein the precipitant used in step (ii) is glycine.

10. A process as claimed in any of Claims 1 to 9 wherein the adsorbent used in step (iii) is Al(OH)3.

11. A process as claimed in Claim 10 wherein the Al (OH)3 is added to a concentration of up to 10%.

12. A process as claimed in any of Claims 1 to 11 wherein the precipitant used in step (iv) comprises PEG.

1 3. A process as claimed in Claim 12 wherein the precipitant further comprises albumin.

14. A process as claimed in Claim 12 or 13 wherein the final concentration of PEG added is up to 25%.

1 5. A process as claimed in any of Claims 1 to 1 4 wherein step (iv) is carried out at a pH of 6.

16. A process as claimed in any of Claims 1 to 1 5 wherein the solubiliser used in step (v) is a buffer.

1 7. A process as claimed in Claim 16 wherein the buffer comprises Tris and/or citrate.

18. A process as claimed in Claim 16 or 17 wherein the buffer contains one or more inorganic salts.

19. A process as claimed in any of Claims 1 to 1 8 wherein step (v) is carried out at a temperature of below 370C and at a pH of from 5 to 9.

20. A process as claimed in Claim 19 wherein the temperature is 200C.

21. A process as claimed in Claim 19 or Claim 20 wherein the pH is 6.8.

22. A process as claimed in any of Claims 1 to 21 wherein the temperature of the solution is lowered to 10C and the pH thereof is adjusted to 6 immediately after step (v) and before any further processing.

23. A process for the preparation of a concentrate of Von Willebrand Factor substantially as herein described with reference to Exampe 1.

24. A concentrate of Von Willebrand Factor whenever prepared by a process as claimed in any of Claims 1 to 23.

25. An intravenous pharmaceutical formulation which comprises a concentrate as claimed in Claim 24 together with a pharmaceutically acceptable carrier or diluent.