Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE
SPECIFICATION
(ORIGINAL)
Application Number: S Lodged: let 7Z2-1F L7 Class Int. Class 61 0823 Complete Specification Lodged: Accepted: Published: Priority: Related Art:
I
Name of Applicant: Address of Applicant: Actual Inventor: Address for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany NORBERT HEIMBURGER, GERHARDT KUMPE and WILFRIED WORMSBACHER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the invention entitled: A PROCESS FOR THE PREPARATION OF A FACTOR V CONCENTRATE The following statement is a full description of this invention, including the best method of performing it known to us TIIE COMMISSIONER OF PATENTS.
Edwd. Waters Sons, Melbourne.
-la- BEHRINGWERKE AKTIENGESELLSCHAFT 86/B 010 Ma 556 Dr. Ha/Bn A process for the preparation of a factor V concentrate The invention relates to a process for the preparation of a factor V concentrate from human blood plasma or from a fraction of blood plasma, which contain an anticoagulant.
There are contributions to the biological phenomenon of hemostasis by the vessels, the blood platelets and plasma factors. The primary wound closure is brought about by the synergistic cooperation of vessels and blood platelets, which result in what is called primary wound closure, which is then reinforced by plasma coagulation which 0o"o results in the formation of fibrin. The blood platelets 0 00 ooo form the reaction surface where plasma coagulation takes place, in which 13 plasma factors are involved, not inc luding the inhibitors. These plasma factors are pro- S teins, mainly glycoproteins, and their action is predominantly that of serine proteases. The latter circulate in the blood in the form of inactive precursors and are activated in response to stimuli such as are represented o by an injury. The activation is based on limited proteo- 0 0 o lysis with each of the foregoing factors in the coagulation cascade activating the one which follows on the basis o o of an enzyme/substrate reaction. Essential for the activation are phospholipids, as are found in the form of a mixture, which is called platelet factor 3 (PF in the Splatelets, and calcium ions. Also important are so-called accelerators, proteins which are able to increase the rate of the coagulation process. The accelerators which act in conjunction with the phospholipids and calcium, by forming what are called activation complexes, include factors V and VIII (F V and F VIII).
F V is a B-globulin which has a molecular weight of 330,000 dalton, is activated by limited proteolysis of thrombin and is inactivated by protein Ca. At the same time, specific activating peptides are produced.
L_ _I 2 The basis of the action of F V as accelerator is that it intensifies the interaction between F X a and prothrombin in the presence of calcium and platelet phospholipids to such an extent that the rate of thrombin formation is increased about 1,000 times. The complex composed of factors X a, phospholipids, calcium and FVa is designated prothrombin activator or prothrombinase. As appropriate for the accelerator action of F V, a reduction in this coagulation factor results in a slowing down of thrombin formation, and thus of coagulation, and, as the biological consequence, in an increased risk of hemorrhage.
All coagua ion factors, irrespective of their enzymatic characteristics, are, comparatively speaking, very un- S 15 stable and sensitive to denaturation, at any rate in the form in which they are normally obtained from plasma which has been treated to prevent coagulation. This particularly applies to F VIII and F V, the two accelerator proteins.
20 C0 0t Use is made of the instability of factor V in the preparation of F V-deficient plasma: human citrated plasma incu- .o bated in a water bath at 370C loses its F V activity within 24 hours, whereas all the other factors remain stable.
Inborn and acquired F V-deficiency states are relatively o rare. Nevertheless, there is a number of indications for Swhich a F V concentrate appears indicated. However, owing to the instability and susceptibility to denaturation, no such concentrate is yet available. This is very much in contrast with other coagulation factors, all of which are available for therapy in the form of concentrates. This illustrates the particular instability of this coagulation factor, which is reflected in the designation "labile factor" for factor V.
For these reasons, there was a need for a process for obtaining F V from whole blood, which guarantees the 3 stability of this factor during removal, storage and processing of the blood in the presence of anticoaguLants.
Such a stabilization of F V would make it possible to prepare a stable F V plasma for coagulation tests and to prepare F V-containing plasma for replacement or as starting material for the preparation of an F V concentrate.
The object of the invention is to prepare an F V concentrate.
Although F V can easily be denatured, it has already been isolated in active form from plasma. This has been possible by adding to fresh citrated plasma, from which the prothrombin factors had been removed, synthetic protease inhibitors to protect against enzymatic breakdown of F V.
Additions of this type are not suitable for the preparation of a standard plasma or for the preparation of pure F V since they are toxic and block the interaction of F V with other coagulation factors.
The obtaining of factor F V from plasma, from which the cryoprecipitate has been removed, by chromatography on an anion exchanger has also already been disclosed. The low yield is disadvantageous in this process.
It has been found, surprisingly, that the loss of F V activity when it is obtained from plasma is a function of S the citrate concentration when citrate is used an anticoagulant. Whereas F V is relatively stable at 20°C and 5 mmol/l citrate, denaturation occurs as the citrate concentration is increased.
It has not been possible to use a citrate concentration below 5 mmol/l if coagulation was to be avoided.
It has also been found, surprisingly, that a loss of activity can be reduced by addition of doubly charged cations, preferably calcium ions. Whereas the F V activity in citrated plasma, which normally contains tri-Na citrate in a concentration of 11 minol/l, falls to values near 0% of 4 normal after 21 hours at 37 0 C, the retention of activity is 35% if a solution of this type contains 2.5 mmol/l calcium ions, and is 70% if it contains 5 mmol/l calcium ions.
The invention relates to a process for the preparation of a concentrate of coagulation factor V from an anticoagulant-containing human blood plasma or from an anticoagulant-containing fraction of human blood plasma, which comprises addition of a salt of a bivalent metal, in an amount such that the concentration of doubly charged cations which is reached is less than the concentration equivalent to the concentration of anticoagulant, to a plasma of this type or to a fraction of this type, and an F V concentrate being obtained in a known manner.
Suitable bivalent metals are Ca, Mg or Mn.
A metal salt of this type is preferably a soluble salt of Scalcium, in particular calcium chloride or acetate. The concentration of doubly charged cations is prefrrably at least 1 mmol/l, particularly preferably more than 2 mmol/l.
The action does not necessarily increase continuously with the concentration, it may pass through a maximum.
Of course, the concentration must not be so high that the mixture coagulates.
Anticoagf lants are, in particular, salts of citric acid) prospi-'e dCes+ros-e C-ore ce/Ctfoe .or heparin.
It is not necessary to add the calcium immediately after removal of the bLood. An advantageous effect is still achieved even if calcium is added up to 12 hours after the removal of blood, in particular if the plasma has been stored at 3-5 0 C for this time. Addition of calcium to frozen and rethawed plasma or to the supernatant after removal of cryoprecipitate likewise markedly stabilizes F V.
Fig. 1 shows the dependence of the F V activity on the time and the calcium concentration. The pH was as in ~rX_ 5 citrated plasma 7.5-7.8, and the temperature was 4 0
C.
This entailed frozen human citrated plasma obtained from 9 parts by volume of whole blood and one part by volume of sodium citrate, and having a final citrate concentration of 10 mmol/l, being thawed and dialyzed against a physiological buffer having a citrate concentration of 10 mmol/l and a calcium concentration of 0.25, 0.5, 1 or 2 mmol/l.
In Fig. 1, a corresponds to a calcium concentration of 0, b of 0.25, c of 0.5, d of 1 and e of 2 mmol/L. Whereas the F V activity remained virtually constant for 16 hours at a concentration of 2 mmol/l calcium in the citratecontaining F V solution, the activity fell as a function of time at lower calcium concentrations.
It is evident from the foregoing that a concentration of calcium ions of at least 2 mmol/l Ca2+ in a medium containing 10 mmol/l sodium citrate is sufficient to stabilize the F V in a freshly obtained citrated plasma, a frozen citrated plasma or a cryoprecipitate-depleted citrated plasma.
The citrated plasma mainly used for routine coagulation analyses is obtained by removing blood from a brachial vein into a tri-Na citrate solution.
A citrated plasma of this type which has been partially recalcified is suitable as starting material for obtaining an F V concentrate.
When 1 USP U/ml heparin was added to fresh human citrated plasma (final tri-Na citrate concentration: 11 mmol/l) and this solution was maintained at 20 0 C, the F V activity decreased to a considerably greater extent than in the control without addition of heparin. However, it is possible by addition of 5 mmol/l calcium chloride to reduce drastically this Loss of activity even under these 6 conditions.
This shows that it is possible to replace citrate more or Less by heparin and to stabilize the F V by calcium. Thus, it is also possible in this way to achieve an anticoagulant effect without removing the calcium which is necessary for the stabilization of F V.
However, with calcium it is possible to stabilize F V not only in plasma but also in fractions prepared therefrom.
It is possible to use as anticoagulants for this all those which are normally used.
It is possible to use as F V-containing fraction, which S 15 can be employed as starting material for the preparation of a concentrate, the plasma supernatant after re- °oo moval of cryoprecipitate. F V can be stabilized by calo cium even in cryoprecipitate-free plasma. This also applies to the steps in the operation which car be used for obtaining an F V concentrate, for example chromatography of cryoprecipitate-free plasma on an anion exchanger.
F V determination: F V can be determined by the method oc of of Koller, Loeliger and Duckert (Perlick, Gerinnungslaboratorium in Klinik and Praxis (The coagulation laboratory in hospitals and general practice), publisher: VEB Georg Thieme, Leipzig, 1960).
This entails 0.1 ml of F V-deficient plasma being mixed with 0.1 ml of a defined dilution of normal plasma. This mixture is incubated at 37 0 C for 30 sec. Then 0.2 ml of a calcium-containing thromboplastin is added, and the time until a clot appears is determined. A reference graph is constructed in this manner.
For the quantitative F V determination of a plasma or concentrate which is to be examined, dilutions thereof in barbital buffer are used in place of normal plasma in the test which is described above, and the clotting time is 7 determined. The F V concentration can be found by reference to a calibration graph obtained with a normal plasma dilution series.
One unit of F V is equivalent to the F V activity of 1 ml of normal plasma.
The examples which follow illustrate the invention.
Example 1 Stabilization of F V in frozen plasma Fresh human citrated plasma was obtained from 1 part of 110 mmol/l tri-Na citrate and 9 parts of whole blood by removal of the blood cells. The plasma was frozen at 2 5 0C and, after 24 hours, thawed again, partially recalcified and incubated at 37 0 C. The F V activity was measured at particular times during the incubation: Table 1 F V activity in thawed plasma incubated without additions and with 2.5 mmo or 5 mmo Ca2+ and with 2.5 mmol or 5 mmol/l Ca 0 0, co
C
Incubation time Frozen plas without add tions Residual F V activity in ma Frozen plasma Frozen plasma i- 2.5 mmol/l 5 mmol/l Ca2+ Ca 2 0 100 100 100 4 h 59 81 81 6 h 46 77 81 21 h 10 38 74 8 Example 2 Stabilization of F V while a plasma fraction is being obtained from citrated plasma 4.74 L of frozen human citrated plasma were thawed at 2- 4°
C
and the residue which remained insoluble, the cryoprecipitate, was removed by centrifugation. To remove the PPSB factors, 237 ml of an AL(OH) 3 suspension containing 1 g/100 ml were added to tie supernatant at 15 0 C, the mixture was stirred for 15 min, the precipitate was removed by centrifugation, and 1 USP U/ml heparin was added to the solution.
The solution was divided into 2 portions each of 2350 mL.
CaCL2 was added to portion 1 until the concentration was 5 mmol/l, and portion 2 was left as it was. Both were processed to give an F V concentrate under conditions which differed only in that all the solvents, namely the equilibration, washing and dialysis media, for portion 1 cono 20 tained 5 mmol/L CaCL 2 To obtain F V, 32 g of moist packed DEAE-Sephadex
R
A which had been equilibrated at pH 7.5 with a solution containing 60 mmol/l NaCL, 20 mmol/l tri-Na citrate, and for portion 1 additionally 5 mmol/l CaCL 2 were added to each portion, and the mixture was stirred at 15 0 C for 1 h.
The anion exchanger resin was removed from the supernatant, transferred into a column and washed free of plasma residues with 1.5 L of physiological NaCI solution.
Elution was carried out with a solution containing 1 mol/l NaCI, 1 USP U/ml heparin, 0.2 U/ml AT III, and for portion 1 additionally 5 mmol/L CaCL 2 The eluate was collected in 3 fractions of 30 ml, 70 ml and 180 ml. Ammonium sulphate was added to fractions 2 and 3 until saturation was reached, and the precipitate was dissolved in 5 ml of physiological saline, dialyzed and freeze-dried.
I~ 9 -9- Whereas in the processing with the addition of calcium chloride the entire F V activity present after the aluminum hydroxide adsorption *jas recovered in the lyophilisate, it was only 20% of this without calcium.
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