CA1293214C - Process for the preparation of a factor v concentrate - Google Patents
Process for the preparation of a factor v concentrateInfo
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- CA1293214C CA1293214C CA000536687A CA536687A CA1293214C CA 1293214 C CA1293214 C CA 1293214C CA 000536687 A CA000536687 A CA 000536687A CA 536687 A CA536687 A CA 536687A CA 1293214 C CA1293214 C CA 1293214C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
Abstract of the disclosure:
A process for the preparation of a coagulation factor V
concentrate is described, in which a concentrate of this type is prepared from blood plasma or from a fraction of blood plasma, which contain an anticoagulant, with addi-tion of a salt of a bivalent metal in an amount such that the concentration of doubly charged cations is less than the concentration equivalent to the concentration of anti-coagulant.
A process for the preparation of a coagulation factor V
concentrate is described, in which a concentrate of this type is prepared from blood plasma or from a fraction of blood plasma, which contain an anticoagulant, with addi-tion of a salt of a bivalent metal in an amount such that the concentration of doubly charged cations is less than the concentration equivalent to the concentration of anti-coagulant.
Description
`^ 3L~2~3~
, 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 p~asma, which contain an anticoagulant.
There are contributions to the biological phenomenon of hemostasis by the vessels, the blood platelets and plasma ~actors. The primary wound closure is brought about by the synergistic cooperation of vessels and blood plate-lets, which result in what is called primary wound clo-sure, ~hich is then reinforced by plasma coagulation which results in the formation of fibrin. The blood platelets form the reaction surface where plasma coagulation takes place, in which 13 plasma factors are involved, not in-cluding the inhibitors. These plasma factors are pro-teins, mainly glycoproteins, and their action is predom-inantly 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 by an injury. The activation is based on limited proteo-lysis with each of the foregoing factors in the coagula-tion cascade activating the one ~hich follows on the basis of an enzyme/substrate reaction. Essential for the acti-vation are phospholipids, as are found in the form of amixture, which is called platelet factor 3 (PF 3), in the platelets, and calcium ions. Also important are so-c~lled accelerators, proteins which are able to increase the rate of the coagulat;on processO The accelerators which act in conjunct;on ~ith the phospholipids and calc;um9 by forming what are called activation complexes~ include factors V and VIII tF V and F VlII).
F V i~ a ~-globulin ~hich has a molecular ~e;ght of 330,00~ dalton, ;s activated by limited proteolysi~ of thrombin and is inact;vated by protein Ca~ At the sams time, specific activating peptides are produced.
.~
:~29~
The basis of the action of F V as accelerator ;s that it intensifies the interaction between F X a and prothrombin in the presence of calcium and platelet phospholip;ds to such an extent that the rate of thrombin formation is in-creased about 1,000 times. The complex composed of fac-tors 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 f3ctor results in a slowing down of thrombin formation, and thus of coagulation, and, as the biologi-cal consequence, in an increased risk of hemorrhage.
All coagulation factors, irrespective of their enzymatic characteristics, are, comparatively speaking, very un-stable and sensitive to denaturation, at any rate in theform in ~hich they are normally obtained fro~ plasma which has been treated to prevent coagulatiQn. This particu-larly applies to F VIII and F V, the t~o accelerator pro-teins.
Use is made of the instability of factor V in the prepara-tion of F V-deficient plasma: human citrated plasma incu-bated in a water bath at 37C loses its F Y activity within 24 hours, whereas all the other factors remain stable.
Inborn and acquired F V-deficiency states are relatively rare. Nevertheless, there is a number of indications for ~hich 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 ~ith cther 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 reflested in the designation "labile factor" for factor V.
For these rçasons, there ~a~ a need for a process for ob-taining F V from ~hole blood, which guarantees the ~2~
stability of this factor during removal, storage and pro-cessing of the blood in the presence of anticoagulants.
Such a stabilization of F V would make it possible to pre-pare a stable F V plasma for coagulat;on tests and to pre-pare 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 concen-trate.
Although F ~ can easily be denatured, it has already been isolated in active form from plasma. This has been pos-sible by adding to fresh citrated plasma, from which the prothrombin factors had been removed, synthetic protease inhibitors to protect against enzymatic breakdo~n of F V.
Addit;ons of this type are not suitable for the prepara-tion 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 o~ factor F V from plasma, from which the cryoprecipitate has been removed, by chromatography on an anion exchanger has also already been d;sclosed. The lo~
yield is disadvantageous in this process.
lt has been found, surprisingly, that the loss of F V ac-tivity when it is obtained from plasma is a function of the citrate concentration when citrate is used an antico-agulant. Whereas F V is relatively stable at 20C and 5 mmol/l citrate, denaturation occurs as the sitrate con-centration is ;ncreased.
It has not been possible to use a citrate concentration below 5 mmol/l if coagula~ion ~as to be avoided~
It has also been found, surprisingly~ that a loss of acti-vity can be reduced by addition of doubly charged cations, preferably caLcium ions. ~hereas the F V activity in cit-rated plasma, which normally contains tri-Na citrate in a concentration of 11 ~mol/l, falls to values near 0% of 3~
normal after 21 hours at 37C, the retent;on of activity is 35% if a soLution of this type contains 2.5 mmol/l cal-cium ions, and is 70% if it contains S mmol/l calcium ions.
The invention relates to a process for the preparation of a concentrate of coagulation factor V from an anticoagu-lant-containing human blood plasma or from an anticoagu-lant-containing fraction of human blood plasma, which com-prises addition of a salt of a bivalent metal, in an amount such that the concentration of doubly charged cat-ions ~hich is reached is less than the concentration equi-valent to the concentration of anticoagulant, to a plasma of ~his 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 calcium, in particular calcium chloride or acetate. The concentration of doubly charg~d cations is preferably at least 1 mmol/l, particularly preferably more than 2 mmoL/l.
The action does not necessarily increase continuousLy w;th the concentration, it may pass through a maximum.
Of course, the concentration must not be so high that the mixture coagulates.
Anticoagulants are, in particular~ salts o~ citric acid or heparin.
It ;s not necessary to add the calcium ;mmed;ately 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 plas~a has been stored at 3-5C for this time. Addition of calcium to frozen and rethawed plasma or to th supernatant after re-moval of cryoprecipitate like~ise markedLy stabilizes F V.
Fig. 1 sho~s the dependence of the F V activi~y on the time and the calcium concentration. The pH ~3S as in ~3Z~
citrated plasma 7.5-7.8, and the temperature was 4C.
This entailed ~rozen human citrated plasma obta;ned from 9 parts by vo~ume of whole blood and one part by volume of sodium citrate, and having a final citrate concentrat;on of 10 mmol/l, being thawed and dialyzed against-a phys;ological buffer having a cit-rate concentration of 10 mmol/l and a calcium concentra-tion 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 O.S, d of 1 and e of 2 mmol/l. Whereas the F V act;vity remained virtually constant for 16 hours at a concentration of 2 mmol/l calcium in the citra~e-1S contain;ng F V solution, the activity fell as a functionof 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 contain-;ng 10 mmol/l sod;um citrate is sufficient to s~abilizethe 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 obt~ined by removing blood from a brachial vein into a tri-Na citrate solut;on~
A citrated plasma of this type whish has been partially recalcified is suitable as starting mater;al for obtain-ing an F V concentrate.
When 1 USP U/ml heparin ~as added to fresh human citrated plas~a ~tinal tri-Na citr2te concentration: 11 m~ol/l) and this solution was maintained at 20C~ the F V activity decreased to a considerably gre~ter extent than in the control ~ithout ~ddition of heparin. However, ;t is pos-sible by addition of 5 mmol/L caLciu~ ch~oride to reduce drastically this loss of activity even under these ~2~32~9~
conditions.
This shows that it is possible to replace citrate more or less by heparin and to stabilize the F V by calcium. Thus, i~ is also possible in this way to achieve an anticoagu-lant effect without removing the calcium which is neces-sary for the stabilization of F V.
However, ~ith calcium it is possib~e to stabilize F Y 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~
I~ is possible to use as F V-containing fraction~ which can be employed as starting material for the prepara-tion of a concentrate, the plasma supernatant after re-moval of cryoprecipitate. F V can be stabilized by cal-cium even in cryoprecipitate-free plasma. This also app-lies to the steps in the operation which can be used for obtaining an F V concentrate, for example chromatography o~ cryoprecipitate-free plasma on an an;on exchanger.
F V determination: F V can be determined by the method of Koller, Loeliger and Duckert ~Perlick, Gerinnungslabora-torium in Klinik and Praxis ~The coagulation laboratoryin hospitaLs and general pract;ce)~ publ;sher: VE~ Georg Thieme, Leipzig, 1960).
This entails 0.1 ml of F V-deficient plasma being mixed ~ith 0.1 ml of a defined dilution of norma~ plasma. This mixture is incubated at 37C for 30 sec~ Then 0.2 ml of a ca~cium-cont~ining thromboplastin is ~dded, 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 con-centrate which is ~o be examined, d;lutions thereo~ in barbital buffer are used in place of normal plasma in the test ~hich is described above, and the c~ott;ng t;me is :~2~3321~
determined. The F V concentration can be fsund by refer-ence to a calibration graph obtained with 3 normal plasma dilution series.
One unit of F V is equivalent to the F V activity of 1 ml of normal plasma.
The examples wh;ch 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 -25C and, after 24 hours, thawed again, partially re-calcified and incubated at 37C. 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 mmol or 5 mmol~l Ca2~.
Incu- Residual F V activity in %
bation Frozen plasma Frozen plasma Frozen plasma time~ithout addi- + 2.5 mmoltl ~ 5 mmol/l CaZ+
tions Ca2~ _ _ 4 h 59 81 81 6 h 46 77 81 21 h 10 38 74 3Zl~
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 2-4C, and the residue which remained ;nsoluble, the cryo-precipitate, ~as removed by centrifugation. To remove the PPSB factors~ 237 ml of an Al(OH)3 suspension contain-ing 1 9/100 mL were added to the supernatant at 15C, the mixture was stirred for 15 min, the precipitate was re-moved by centrifugation, and 1 ~SP U/ml heparin was added to the solutionn The solut;on was divided into 2 portions each of Z350 ml.
CaCl2 was added to portion 1 un~il the concentration was 5 mmol/l, and portion 2 was left as it ~as. Both were processed to give an F V concentrate under conditions which differed only in that all the solvents, na~ely the equi-libration, washing and dialysis media, ~or portion 1 con-tained S mmol/l CaCl2.
To obtain F V, 32 g of moist packed DEAE-Sephadex~R) A 50 ~h;ch had been equilibrated at pH 7~5 with a soLution con-taining 60 mmol/l NaCl, 20 mmol/l tri-Na citrate, and for portion 1 additionally 5 mmol/l CaCl2, were added to each port;on, and the mixture was st;rred at 15C for 1 h.
The anion exchanger resin was removed from the supernatant9 transferred into a coluMn and ~ashed free of plasma res;-dues ~ith 1.5 l of phys;ological NaCl solution.
Elution was carried out ~ith a solutisn containing 1 moltlNaCl~ 1 USP U/ml heparin, 0.2 U/ml AT IlI, and for por-~ion 1 additionally 5 mmol/l CaCl2. T~e ~luate ~as col-lected in 3 ~ractions of 30 ml, 70 ml and 180 ml. A~mon-ium sulphate was added to fractions 2 and 3 un~il 60%
saturation was reached, and the prec;pitate ~as dissolved in 5 ml o~ phys;ological saline, dialyzed and freeze-dried.
3'~
_ 9 _ Whereas in the processing with ~he addition of calcium chloride the ent;re F Y activ;ty present after the alu-m;num hydroxide adsorption was recovered ;n the lyoPh;l;-sate, ;t ~as only 20X of th;s without caLcium.
, 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 p~asma, which contain an anticoagulant.
There are contributions to the biological phenomenon of hemostasis by the vessels, the blood platelets and plasma ~actors. The primary wound closure is brought about by the synergistic cooperation of vessels and blood plate-lets, which result in what is called primary wound clo-sure, ~hich is then reinforced by plasma coagulation which results in the formation of fibrin. The blood platelets form the reaction surface where plasma coagulation takes place, in which 13 plasma factors are involved, not in-cluding the inhibitors. These plasma factors are pro-teins, mainly glycoproteins, and their action is predom-inantly 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 by an injury. The activation is based on limited proteo-lysis with each of the foregoing factors in the coagula-tion cascade activating the one ~hich follows on the basis of an enzyme/substrate reaction. Essential for the acti-vation are phospholipids, as are found in the form of amixture, which is called platelet factor 3 (PF 3), in the platelets, and calcium ions. Also important are so-c~lled accelerators, proteins which are able to increase the rate of the coagulat;on processO The accelerators which act in conjunct;on ~ith the phospholipids and calc;um9 by forming what are called activation complexes~ include factors V and VIII tF V and F VlII).
F V i~ a ~-globulin ~hich has a molecular ~e;ght of 330,00~ dalton, ;s activated by limited proteolysi~ of thrombin and is inact;vated by protein Ca~ At the sams time, specific activating peptides are produced.
.~
:~29~
The basis of the action of F V as accelerator ;s that it intensifies the interaction between F X a and prothrombin in the presence of calcium and platelet phospholip;ds to such an extent that the rate of thrombin formation is in-creased about 1,000 times. The complex composed of fac-tors 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 f3ctor results in a slowing down of thrombin formation, and thus of coagulation, and, as the biologi-cal consequence, in an increased risk of hemorrhage.
All coagulation factors, irrespective of their enzymatic characteristics, are, comparatively speaking, very un-stable and sensitive to denaturation, at any rate in theform in ~hich they are normally obtained fro~ plasma which has been treated to prevent coagulatiQn. This particu-larly applies to F VIII and F V, the t~o accelerator pro-teins.
Use is made of the instability of factor V in the prepara-tion of F V-deficient plasma: human citrated plasma incu-bated in a water bath at 37C loses its F Y activity within 24 hours, whereas all the other factors remain stable.
Inborn and acquired F V-deficiency states are relatively rare. Nevertheless, there is a number of indications for ~hich 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 ~ith cther 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 reflested in the designation "labile factor" for factor V.
For these rçasons, there ~a~ a need for a process for ob-taining F V from ~hole blood, which guarantees the ~2~
stability of this factor during removal, storage and pro-cessing of the blood in the presence of anticoagulants.
Such a stabilization of F V would make it possible to pre-pare a stable F V plasma for coagulat;on tests and to pre-pare 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 concen-trate.
Although F ~ can easily be denatured, it has already been isolated in active form from plasma. This has been pos-sible by adding to fresh citrated plasma, from which the prothrombin factors had been removed, synthetic protease inhibitors to protect against enzymatic breakdo~n of F V.
Addit;ons of this type are not suitable for the prepara-tion 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 o~ factor F V from plasma, from which the cryoprecipitate has been removed, by chromatography on an anion exchanger has also already been d;sclosed. The lo~
yield is disadvantageous in this process.
lt has been found, surprisingly, that the loss of F V ac-tivity when it is obtained from plasma is a function of the citrate concentration when citrate is used an antico-agulant. Whereas F V is relatively stable at 20C and 5 mmol/l citrate, denaturation occurs as the sitrate con-centration is ;ncreased.
It has not been possible to use a citrate concentration below 5 mmol/l if coagula~ion ~as to be avoided~
It has also been found, surprisingly~ that a loss of acti-vity can be reduced by addition of doubly charged cations, preferably caLcium ions. ~hereas the F V activity in cit-rated plasma, which normally contains tri-Na citrate in a concentration of 11 ~mol/l, falls to values near 0% of 3~
normal after 21 hours at 37C, the retent;on of activity is 35% if a soLution of this type contains 2.5 mmol/l cal-cium ions, and is 70% if it contains S mmol/l calcium ions.
The invention relates to a process for the preparation of a concentrate of coagulation factor V from an anticoagu-lant-containing human blood plasma or from an anticoagu-lant-containing fraction of human blood plasma, which com-prises addition of a salt of a bivalent metal, in an amount such that the concentration of doubly charged cat-ions ~hich is reached is less than the concentration equi-valent to the concentration of anticoagulant, to a plasma of ~his 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 calcium, in particular calcium chloride or acetate. The concentration of doubly charg~d cations is preferably at least 1 mmol/l, particularly preferably more than 2 mmoL/l.
The action does not necessarily increase continuousLy w;th the concentration, it may pass through a maximum.
Of course, the concentration must not be so high that the mixture coagulates.
Anticoagulants are, in particular~ salts o~ citric acid or heparin.
It ;s not necessary to add the calcium ;mmed;ately 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 plas~a has been stored at 3-5C for this time. Addition of calcium to frozen and rethawed plasma or to th supernatant after re-moval of cryoprecipitate like~ise markedLy stabilizes F V.
Fig. 1 sho~s the dependence of the F V activi~y on the time and the calcium concentration. The pH ~3S as in ~3Z~
citrated plasma 7.5-7.8, and the temperature was 4C.
This entailed ~rozen human citrated plasma obta;ned from 9 parts by vo~ume of whole blood and one part by volume of sodium citrate, and having a final citrate concentrat;on of 10 mmol/l, being thawed and dialyzed against-a phys;ological buffer having a cit-rate concentration of 10 mmol/l and a calcium concentra-tion 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 O.S, d of 1 and e of 2 mmol/l. Whereas the F V act;vity remained virtually constant for 16 hours at a concentration of 2 mmol/l calcium in the citra~e-1S contain;ng F V solution, the activity fell as a functionof 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 contain-;ng 10 mmol/l sod;um citrate is sufficient to s~abilizethe 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 obt~ined by removing blood from a brachial vein into a tri-Na citrate solut;on~
A citrated plasma of this type whish has been partially recalcified is suitable as starting mater;al for obtain-ing an F V concentrate.
When 1 USP U/ml heparin ~as added to fresh human citrated plas~a ~tinal tri-Na citr2te concentration: 11 m~ol/l) and this solution was maintained at 20C~ the F V activity decreased to a considerably gre~ter extent than in the control ~ithout ~ddition of heparin. However, ;t is pos-sible by addition of 5 mmol/L caLciu~ ch~oride to reduce drastically this loss of activity even under these ~2~32~9~
conditions.
This shows that it is possible to replace citrate more or less by heparin and to stabilize the F V by calcium. Thus, i~ is also possible in this way to achieve an anticoagu-lant effect without removing the calcium which is neces-sary for the stabilization of F V.
However, ~ith calcium it is possib~e to stabilize F Y 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~
I~ is possible to use as F V-containing fraction~ which can be employed as starting material for the prepara-tion of a concentrate, the plasma supernatant after re-moval of cryoprecipitate. F V can be stabilized by cal-cium even in cryoprecipitate-free plasma. This also app-lies to the steps in the operation which can be used for obtaining an F V concentrate, for example chromatography o~ cryoprecipitate-free plasma on an an;on exchanger.
F V determination: F V can be determined by the method of Koller, Loeliger and Duckert ~Perlick, Gerinnungslabora-torium in Klinik and Praxis ~The coagulation laboratoryin hospitaLs and general pract;ce)~ publ;sher: VE~ Georg Thieme, Leipzig, 1960).
This entails 0.1 ml of F V-deficient plasma being mixed ~ith 0.1 ml of a defined dilution of norma~ plasma. This mixture is incubated at 37C for 30 sec~ Then 0.2 ml of a ca~cium-cont~ining thromboplastin is ~dded, 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 con-centrate which is ~o be examined, d;lutions thereo~ in barbital buffer are used in place of normal plasma in the test ~hich is described above, and the c~ott;ng t;me is :~2~3321~
determined. The F V concentration can be fsund by refer-ence to a calibration graph obtained with 3 normal plasma dilution series.
One unit of F V is equivalent to the F V activity of 1 ml of normal plasma.
The examples wh;ch 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 -25C and, after 24 hours, thawed again, partially re-calcified and incubated at 37C. 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 mmol or 5 mmol~l Ca2~.
Incu- Residual F V activity in %
bation Frozen plasma Frozen plasma Frozen plasma time~ithout addi- + 2.5 mmoltl ~ 5 mmol/l CaZ+
tions Ca2~ _ _ 4 h 59 81 81 6 h 46 77 81 21 h 10 38 74 3Zl~
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 2-4C, and the residue which remained ;nsoluble, the cryo-precipitate, ~as removed by centrifugation. To remove the PPSB factors~ 237 ml of an Al(OH)3 suspension contain-ing 1 9/100 mL were added to the supernatant at 15C, the mixture was stirred for 15 min, the precipitate was re-moved by centrifugation, and 1 ~SP U/ml heparin was added to the solutionn The solut;on was divided into 2 portions each of Z350 ml.
CaCl2 was added to portion 1 un~il the concentration was 5 mmol/l, and portion 2 was left as it ~as. Both were processed to give an F V concentrate under conditions which differed only in that all the solvents, na~ely the equi-libration, washing and dialysis media, ~or portion 1 con-tained S mmol/l CaCl2.
To obtain F V, 32 g of moist packed DEAE-Sephadex~R) A 50 ~h;ch had been equilibrated at pH 7~5 with a soLution con-taining 60 mmol/l NaCl, 20 mmol/l tri-Na citrate, and for portion 1 additionally 5 mmol/l CaCl2, were added to each port;on, and the mixture was st;rred at 15C for 1 h.
The anion exchanger resin was removed from the supernatant9 transferred into a coluMn and ~ashed free of plasma res;-dues ~ith 1.5 l of phys;ological NaCl solution.
Elution was carried out ~ith a solutisn containing 1 moltlNaCl~ 1 USP U/ml heparin, 0.2 U/ml AT IlI, and for por-~ion 1 additionally 5 mmol/l CaCl2. T~e ~luate ~as col-lected in 3 ~ractions of 30 ml, 70 ml and 180 ml. A~mon-ium sulphate was added to fractions 2 and 3 un~il 60%
saturation was reached, and the prec;pitate ~as dissolved in 5 ml o~ phys;ological saline, dialyzed and freeze-dried.
3'~
_ 9 _ Whereas in the processing with ~he addition of calcium chloride the ent;re F Y activ;ty present after the alu-m;num hydroxide adsorption was recovered ;n the lyoPh;l;-sate, ;t ~as only 20X of th;s without caLcium.
Claims (9)
1. A process for the stabilization and preparation of a stable concentrate of coagulation factor V from an anticoagulant-containing human blood plasma or from an anticoagulant-containing fraction of human blood plasma, which comprises adding to human blood plasma containing an anticoagulant or a fraction of human blood plasma containing an anticoagulant, a salt of a bivalent metal to stabilize coagulation factor V in said plasma or fraction of said plasma, said salt of a bivalent metal being added 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; and, obtaining a stable Factor V concentrate.
2. The process as claimed in claim 1, wherein the metal salt is a soluble salt of calcium.
3. The process as claimed in claim 1, wherein the metal salt is calcium chloride or calcium acetate.
4. The process as claimed in claim 1, wherein the concentration of doubly charged cations is at least 2 mmol/l.
5. The process as claimed in claim 1, wherein the anticoagulant is citrate, heparin, phosphate-dextrose or citrate-dextrose or a mixture of such anticoagulants.
6. The process as claimed in claim 1, wherein the anticoagulant is heparin.
7. The process as claimed in claim 1, wherein the anticoagulant is a mixture of a salt of citric acid and heparin.
8. The process as claimed in claim 6, wherein the heparin is present in concentrations of 1-5 USP U/ml blood or plasma.
9. The process as claimed in claim 1, wherein the salt of a bivalent metal is added immediately after removal of the blood, after storage of the plasma at 3 - 5 °C, or after freezing and thawing of the plasma.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3615558.6 | 1986-05-09 | ||
| DE19863615558 DE3615558A1 (en) | 1986-05-09 | 1986-05-09 | METHOD FOR PRODUCING A FACTOR V CONCENTRATE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1293214C true CA1293214C (en) | 1991-12-17 |
Family
ID=6300403
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000536687A Expired - Lifetime CA1293214C (en) | 1986-05-09 | 1987-05-08 | Process for the preparation of a factor v concentrate |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0244834B1 (en) |
| JP (1) | JP2540541B2 (en) |
| AT (1) | ATE73667T1 (en) |
| AU (1) | AU610823B2 (en) |
| CA (1) | CA1293214C (en) |
| DE (2) | DE3615558A1 (en) |
| ES (1) | ES2031849T3 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012143507A1 (en) | 2011-04-22 | 2012-10-26 | Kedrion S.P.A. | Novel process for the preparation of a virus-inactivated fv concentrate starting from human plasma, scalable to industrial level |
Families Citing this family (29)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4981952A (en) * | 1988-10-04 | 1991-01-01 | Eli Lilly And Company | Method for the purification of vitamin K-dependent proteins |
| DE4418635C2 (en) * | 1994-05-27 | 1997-07-24 | Immuno Ag | Pharmaceutical composition for the prevention and treatment of bleeding disorders |
| ATE214282T1 (en) | 1997-06-05 | 2002-03-15 | Global Hemostasis Inst Mgr Ab | PREVENTING THE COAGULATION OF BLOOD, BLOOD PLASMA OR SYNOVIAL FLUID PRODUCTS |
| SE9804248D0 (en) * | 1998-12-07 | 1998-12-07 | Global Hemostasis Inst Mgr Ab | Anti-coagulation with calcium containing citrate solution |
| US20030205538A1 (en) | 2002-05-03 | 2003-11-06 | Randel Dorian | Methods and apparatus for isolating platelets from blood |
| US7832566B2 (en) | 2002-05-24 | 2010-11-16 | Biomet Biologics, Llc | Method and apparatus for separating and concentrating a component from a multi-component material including macroparticles |
| US7992725B2 (en) | 2002-05-03 | 2011-08-09 | Biomet Biologics, Llc | Buoy suspension fractionation system |
| DE10392686T5 (en) | 2002-05-24 | 2005-07-07 | Biomet Mfg. Corp., Warsaw | Apparatus and method for separating and concentrating liquids containing multiple components |
| US20060278588A1 (en) | 2002-05-24 | 2006-12-14 | Woodell-May Jennifer E | Apparatus and method for separating and concentrating fluids containing multiple components |
| US7845499B2 (en) | 2002-05-24 | 2010-12-07 | Biomet Biologics, Llc | Apparatus and method for separating and concentrating fluids containing multiple components |
| WO2006086200A1 (en) | 2005-02-07 | 2006-08-17 | Hanuman Llc | Plasma concentrator device |
| US7694828B2 (en) | 2005-04-27 | 2010-04-13 | Biomet Manufacturing Corp. | Method and apparatus for producing autologous clotting components |
| US8567609B2 (en) | 2006-05-25 | 2013-10-29 | Biomet Biologics, Llc | Apparatus and method for separating and concentrating fluids containing multiple components |
| US7806276B2 (en) | 2007-04-12 | 2010-10-05 | Hanuman, Llc | Buoy suspension fractionation system |
| US8328024B2 (en) | 2007-04-12 | 2012-12-11 | Hanuman, Llc | Buoy suspension fractionation system |
| EP2620139B1 (en) | 2008-02-27 | 2016-07-20 | Biomet Biologics, LLC | Interleukin-1 receptor antagonist rich solutions |
| WO2009111338A1 (en) | 2008-02-29 | 2009-09-11 | Biomet Manufacturing Corp. | A system and process for separating a material |
| US8187475B2 (en) | 2009-03-06 | 2012-05-29 | Biomet Biologics, Llc | Method and apparatus for producing autologous thrombin |
| US8313954B2 (en) | 2009-04-03 | 2012-11-20 | Biomet Biologics, Llc | All-in-one means of separating blood components |
| US9011800B2 (en) | 2009-07-16 | 2015-04-21 | Biomet Biologics, Llc | Method and apparatus for separating biological materials |
| US8591391B2 (en) | 2010-04-12 | 2013-11-26 | Biomet Biologics, Llc | Method and apparatus for separating a material |
| US9642956B2 (en) | 2012-08-27 | 2017-05-09 | Biomet Biologics, Llc | Apparatus and method for separating and concentrating fluids containing multiple components |
| US9895418B2 (en) | 2013-03-15 | 2018-02-20 | Biomet Biologics, Llc | Treatment of peripheral vascular disease using protein solutions |
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| US20140271589A1 (en) | 2013-03-15 | 2014-09-18 | Biomet Biologics, Llc | Treatment of collagen defects using protein solutions |
| US9950035B2 (en) | 2013-03-15 | 2018-04-24 | Biomet Biologics, Llc | Methods and non-immunogenic compositions for treating inflammatory disorders |
| US9550028B2 (en) | 2014-05-06 | 2017-01-24 | Biomet Biologics, LLC. | Single step desiccating bead-in-syringe concentrating device |
| US11738120B1 (en) | 2022-04-14 | 2023-08-29 | Cormedix Inc. | Synthesis of taurolidine, purity profiles and polymorphs |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3101752A1 (en) * | 1981-01-21 | 1982-08-26 | Behringwerke Ag, 3550 Marburg | "METHOD FOR PURIFYING THE BLOOD COAGINING FACTORS II, VII, IX AND / OR X AND PREPARATIONS PRODUCED THEREFORE" |
-
1986
- 1986-05-09 DE DE19863615558 patent/DE3615558A1/en not_active Withdrawn
-
1987
- 1987-05-06 DE DE8787106523T patent/DE3777440D1/en not_active Expired - Fee Related
- 1987-05-06 AT AT87106523T patent/ATE73667T1/en not_active IP Right Cessation
- 1987-05-06 EP EP87106523A patent/EP0244834B1/en not_active Expired - Lifetime
- 1987-05-06 ES ES198787106523T patent/ES2031849T3/en not_active Expired - Lifetime
- 1987-05-08 JP JP62110951A patent/JP2540541B2/en not_active Expired - Lifetime
- 1987-05-08 CA CA000536687A patent/CA1293214C/en not_active Expired - Lifetime
- 1987-05-08 AU AU72642/87A patent/AU610823B2/en not_active Ceased
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012143507A1 (en) | 2011-04-22 | 2012-10-26 | Kedrion S.P.A. | Novel process for the preparation of a virus-inactivated fv concentrate starting from human plasma, scalable to industrial level |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0244834B1 (en) | 1992-03-18 |
| ATE73667T1 (en) | 1992-04-15 |
| AU610823B2 (en) | 1991-05-30 |
| ES2031849T3 (en) | 1993-01-01 |
| JPS62267235A (en) | 1987-11-19 |
| JP2540541B2 (en) | 1996-10-02 |
| DE3615558A1 (en) | 1987-11-12 |
| DE3777440D1 (en) | 1992-04-23 |
| EP0244834A3 (en) | 1989-01-04 |
| AU7264287A (en) | 1987-11-12 |
| EP0244834A2 (en) | 1987-11-11 |
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