CA1168583A - Blood-coagulation-promoting preparation based on human proteins and a method of producing the same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

In a method of producing a blood-coagulation-promoting preparation based on human proteins and having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), human plasma is treated with sulphated high-polymer carbohydrates and/or with basic ion exchangers, the protein mixture with ge-nerated FEIB-activity is adsorbed on the ion exchanger, and the preparation is gained by elution and concentra-tion.

Description

~16~5~3 The invention relates to a method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity, and a blood-coagulation-promoting preparation produced by the method.
Blood-coagulation-promoting preparations exhibiting factor-VIII-inhibitor-bypassing-activity, in short "FEIBA"
(Factor-Eight-Inhibitor-Bypassing-Activity), are known. In United States patent No. 4,160,025 the production of such a pre-paration is described. It is successfully applied for the treat-ment of patients suffering from haemophilia A and whose blood contains an inhibitor directed against factor VIII. The chemical structure of the FEIBA factor so far has been unknown. It is only known that a protein having a molecular weight of approx-imately lO0,000 ~s involved. The production of the preparation according to the above-mentioned patent specification was carried out by generation from human plasma containing citrate lons in the absence of free calcium ions by treatment with water-insoluble inorganic coagulation-phys~ologically-surface-active substances, such as silicagel or kaolin, and subsequent adsorption and elution, wherein a mixture of factors II, VII, IX and X, of factor FEIBA and of other proteins IS obtained, whose composition has not been described so far.
Although as ment~oned above, the preparation according to United States patent No. 4,160,025 has proved valuable for the treatment of factor V~II inhibitor patients, there is the task of enlarging the sphere of application and further improving the safety of FEIBA preparations, in particular of reducing to a minimum undesired side react~ons, such as thrombogenic and vasoactive effects.

~ccording to the inventIon, there is provided a method of ~ 168583 producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X
and having factor-VIII-inhibitor-bypassing-activity, which preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per ky rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate contain~ng a protein with FEIB-~activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obta~n a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.
The present invention also provides a blood-coagulation-promoting pxeparation comprising human proteins including coagulat~on factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), when prepared by the above method.
The above-defined character~stic features of the prepar-ation, i.e. the absence of thrombogenic activity and of kallikrein activity or prekallikrein activator activity - the latter being responsible for vasoactive effects -, suggest that the preparation has an excellent safety. The thrombosis inducing activity test and the test on kallikrein activity and 1 168~83 prekallikrein activator activity are known to one skilled in the art. They will be described in more detail after the Examples.
The third characteristIc feature of the preparation according to the invention, i.e. the separability by way of affinity-chromatography on dextran sulphate agarose by means of an NaCl gradient is illustrated in Figure 1 of the drawings by way of an example. The method of affinity-chromatography on dextran sulphate agarose ~s known to one skilled in the art (D.S.
Pepper and C. Prowse, Thrombosis Research 11 (1977), 687-692).
Dextran sulphate (,molecular weight 500,000) ~s coupled to CNBr-activated sepharose* 4 B (Pharmacia F~ne Chemicals ~B~ Uppsala !
Sweden). The dextran sulphate sepharose thus o~tained is equi-librated in a 0.4~ w/v tr~sodiumcitrate.2H20 solut~on (pH = 7,4) and filled into a column. The sample to be exami,ned is applied onto the column and then fract~onally eluted with an NaCl solution in 0.4% w~v trisod~umcitrate.2H20 (pH = 7,4) with an increasing NaCl concentration.
I~n F~gure l the fraction numbers of the eluates are plotted on the abscissa. On the left ord~nate the activities of the coagulation factors are plotted in units per ml and on the right ordinate the concentrat~on of the sodium chloride gradient 's plotted in mol~l. The l~near course of the gradient is entered as line G. As can ~e seen from Figure l, the protein w~th factor IX act~v~ty elutes in the regi,Gn of 0.1 to 0.5 molar NaCl, w~th a maximum at 0.3 molar, the protein with FEIB-activity elutes in the reg~on of 0~3 to 0.5 molar NaCl, with a maximum of 0.4 molar. The increas~ng NaCl concen-tration is revealed by the NaCl gradient G reaching from O molar to 0,65 molar NaCl.
Ftnally, as the fourth characterist~c feature of the * Trade Mark preparation according to the invention also the content of globulins when electrophoretically separated is typical, which is explained by way of Figure 2 of the drawings. The upper section of Figure 2 illustrates, by way of an example, the electrophore-tic separation trace of the eluates of the chroma- -tographic separation on dextran sulphate sepharose according to the invention containing proteins with factor IX activity and with FEIB-activity, whereas the lower section of Figure 2 reflects the electrophoretic separation trace of a native human plasma. As will be seen from the traces in F-~gure 2, in this example, for a preparation in accordance with the invention, the main peak in the ~-globulin region amounts to 70% of the total protein. Downstream of the main peak there follows a shoulder ~n the a-globulin reg~on, amounting to 14% of the total protein content, and thereafter one observes a small peak in the ~-globul~n region, which amounts to 16% of the total protein, Advantageously a further characteristic feature of the preparat~on according to the invention resides in the fact that the FEIB-acti~lty, after a one~h~ur incubation ;n factor VIII inhibitor plas~a, IS` preserved by at least 50%. This property ind~cates a longer lasting efficacy when applied to factor YIII inh~bitor pat~ent$.
Advantageously a further characteristic of the preparation according to the invention resides in the fact that the factor IX activity, after a one-hour incubation in factor IX deficient plasma, is preserved by at least 50%. This means that the preparation contains little or only a very slight port~on of activated factor IX. It is known that activated factor IX is inactivated in human plasma. ~ctivated factor IX would cause detrimental thrombogenic effects. It is known from the literature (Proc. Natl. Acad. Sci. USA, Vol. 74, No. 7, 3028-3032, July 1977, "In vitro and in vivo correlation of clotting protease activity: Effect of heparin" by S.N. Gitel, R C. Stephenson and S. Wessler) that it is exactly factor IXa that possesses the highest thrombogenic efficacy as opposed to other activated coagulation factors, such as Xa and IIA
(thrombin)~
In a preferred embodiment of the preparation according to the invention, the preparation further comprises inter-alpha-trypsin-~nhibitor (ITI) in an amount of 0.05 to 5 mg per FEIBA un;t.
The content of IT~ causes the thrombogenicity of the preparation of the invention to be low in the ~essler test.
The method of producing the new blood-coagulation-promoting preparat~on compr~sing human proteins, including c~agulation factors II, VII~, IX and X and having a factor-VIII-inhibitor=bypassing-actlvity, is characterized in that human plasma is treated with sulphated , high molecular weight polymer carbohydrates and/or w~th basic ion exchangers, and the resultant protein mixture having generated FEIB-activity is adsorbed, and the preparation is recovered by elution and concentration.
When carrying out the method it is to be taken care that the plasma and the reactants be kept free from substances that are capable of increas~ng the antithrombin III act~vity, such as heparin or heparinoids.
According to one embodi~ent of the method of the present invention, the plasma at first is briefly treated w~th sulphated, h~gh ~olecular weight polymer carbohydrates, the ~; ;
~ "~

~8~83 protein mixture having generated FEIB-activity is then adsorbed on an ion exchanger having a dextran structure and eluted and concentrated immediately thereafter.
According to another embodiment of the method of the present invention, the plasma is treated with an ion exchanger having a dextran structure; after at least two hours of exposure the protein mixture with generated FEIB-activity, adsorbed on the ;on exchanger, is eluted and then concentrated. With this embodiment, the generation of the FEIB-activity depends on the period of exposure. This may last up to 48 hours, The ind~vidual s-teps of the production process are not critIcal and may vary within a large range; thus the pH may be from 6 to 9, the temperature may range from 0 to 40C, the amounts of dextran sulphate used may be from 0.1 to 500 mg~l plasma, and DEAE-Sephadex* may be used ~n an amount of from 0.01 to 10 g/l plasma. As a starting material for the method of the invent~on, not only nati~e human plasma, but also plasma fractions, e.g., cryosupernatant and Cohn-I-supernatant (8% v/v alcohol) may be used.
The preparation according to the ~nvention and -the method for its production wIll be explained in more detail by the follow-ing examples, the methods of determ~nation applied ~eing explained and the res~ults ~e~ng shown in the * Trade Mark ~16~583 Tables following the Examples.
Example 1:
1,000 l of fresh frozen human citrated plasma are thawed at 0 to +4C and the resultiny cryoprecipitate is separated by centrifugation at +2C. To the resulting "cryosupernatant" 10 g of dextran sulphate (molecular weight 500,000) are added at a native pH of 7.7 and stirred for 15 minutes at + 4 C, with the substance FEIBA being generated.
Thereafter 500 g of the anion exchanger DEAE-Sephadex A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) are added and stirred at +4 C for half an hour, the generated FEI~A-substance together with the factors of the prothrom-bin complex (II, VII, IX, X) and inert proteins being ad-sorbed on the insoluble DEAE-Sephadex.
The DEAE-Sephadex is separated immediately after the adsorption procedure by centrifugation or filtration; the supernatant plasma may be used for recovering gamma-glo-bulin and albumin.
The DEAE-Sephadex is subjected to a double washing process; at first the DEAE-Sephadex is stirred with 50 l of a solution consisting of 4 g/l trisodiumcitrate.2H20, 7 g/l sodium chloride and 18 g/l disodium hydrogen phos-phate.12H20 in distilled water, pH 7.5,for 15 minutes at +4 C. After separation by filtration the DEAE-Sephadex is stirred with 50 l of a solution consisting of 4 g/l tri-sodiumcitrate.2H20 and 7 g/l sodium chloride in distilled water, pH 7.5, for 15 minutes at + 4 C and then again is separated by filtration.
For elu-tion the DEAE-Sephadex is stirred with 25 l of 1 ~8583 a solution consisting of 30 g/l sodium chloride and 1 g/l trisodiumcitrate.2H20 in distilled water, pH 7.0, for 20 minutes at +4 C. The eluate con-taining the generated FEIBA substance, the factors of the prothrombin complex (II, VII, IX, X) as well as inert protein, is gained by filtration, the DEAE-Sephadex is discarded. The eluate is dialyzed over night against 1,000 l of distilled water at +4 C, then frozen and subjected to a first lyophili-zation process. In the resulting bulk-material the FEIB-activity is determined according to the method describedin U.S. patent No. 4,160,025.
.For the production of the pharmaceutically applicable preparation with FEIB-activit~ the bulk-material is dis-solved in so much distilled pyrogen-free water that the FEIB-activity amounts to between 10 and 50 FEIBA units per ml (in the present case 25 FEIBA units per ml). After the addition of the salts required for establishing isoton-icity and adjusting the pH to between 7.0 and 7.5 the so-lution is cleared through membrane filters and at last is sterile-filtered through a 0.2 ~um membrane filter.The so-lution is filled into the final containers under sterile conditions in 20 ml portions, deepfrozen and lyophilized.
Example 2:
1,000 l fresh frozen human citrated plasma are thawed at 0 to +4 C and the resulting cryoprecipitate is sepa-rated by centrifugation at +2 C. To the resultin~ "cryo-supernatant" 500 g of the anion exchanger DEAE-Sephadex A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) are added at a native pH of 7.7 and stirred for half an hour at +4 C, the factors of the prothrombin complex (II, VII, ~1~85~3 IX, X) and inert proteins being adsorbed on the DEAE-Sephadex.
Thereafter the mixture is allowed to stand for 12 hours at +4 C; during this "contact time" the FEIBA sub-stance is generated.
The DEAE-Sephadex, after a 12-hour "contact time", is separated by centrifugation or filtration; the supernatant plasma may be used for recovering gamma-globulin and al-bumin.
The further processing of the DEAE-Sephadex (double washing, elution etc.) is effected in the same manner as described in E~ample 1.
The thrombosis induci:ngactivity test according to Wessler, which is described in the literature, i.e. in J. Appl. Physiol 14 (1959), 943-946, "Biologic Assay of a Thrombosis-Inducing Activity in Human Serum" by Stanford Wessler, Stanley M. Reimer and Mindel C. Sheps, is per-formed in the following manner:
3 rabbits are used per test. The animals are narcotized with Nembutal; after an additional local anesthesia the heart-side vena jugularis is laid open, two ligatures being prepared at a distance of 1 to 2 cm.
The preparation to be tested is now injected within 15 seconds in the desired dosis into the ear vein opposite the vena jugularis laid open. Within 10 to 25 seconds after the injection of the preparation the prepared ligatures are contracted. The isolated vein segment now remains in situ in the rabbit for 10 minutes. Then the vein section is removed from the animal and dissected in a Petri dish in vfv~
~,~ 30 a 5 ~ sodium citrate solution, the contents being evaluated _ g _ according to the following scheme.
0 = no clot 1 = few macroscopically visible fibrin particles

2 = some small thrombi

3 = two or more large thrombi

4 = one single thrombus filling up the entire isolated vein segment.
The test is valued positive in the case of a ~-re-action~ It is essential for the preparation according to the invention that upon injection of the preparation which contains at least 2 units of FEIBA per kg of experimental animal, no 4-reaction takes place.
The determination of the kallikrein activity and the prekallikrein activator activity is carried out in the following way.
KALLIKREIN:
1. Method:
Kallikrein amidolytically splits para-nitroanilin (pNA) from a specific chromogenic substrate. The concentration of pNA is photometrically measured at a wave length of 405 nm.
2. Reagents:
Buffer:
Solution A: 3.03 g "TRIS" and 1.7 g imidazole are dis-solved in 500 ml 0.1n hydrochloric acid and water is added up to 1,000 ml.
Solution B: 4.04 g "TRIS" and 2.27 g imidazole are dis-solved in 500 ml 0.1n hydrochloric acid and water is added up to 1,000 ml.
Solution C: 11.69 g sodium chloride are dissolved with ~1~85~3 water to 1,000 ml.
Solutions A and B are mixed until a pH of 7.9 is reached.
To this mixture the same volume of solution C is added.

Chromogenic substrate S-2302 (Kabi/ Stockholm): H-D-d~
prolyl-L-phenylalanyl-L-arginine-p-~aR~d-dihydro-chloride 1 millimolar solution of S-2302: 25 mg in 41 ml of water.
Sample:
The sample is dissolved in the original volume and used in the test undiluted.
3. Test:
In a water bath at a temperature of 37 C
1.0 ml buffer preheated to 37 C
0.1 ml sample 0.2 ml chromogenic substrate S-2302 are pipetted into a plastic tube. This mixture is charged into a photometer heated to 37 C and the increase of the optical density per minute (~OD/min) at a wave length of 405 nm with a path length of 10 mm is measured. The ac-0 tivity of a sample is expressed in ~OD/min.PREKALLIKREIN ACTIVATOR:
1. Method:
From a purified prekallikrein preparation (PKK) kallikrein (KK) is generated by means of a prekallikrein activator (PKKA). The kallikrein amidolytically splits para-nitro-aniline (pNA) from a specific chromogenic substrate. The concentration of pNA is photometrically measured at a wave length of 405 nm.
2. Reagents:
Buffer and chromogenic substrate correspond to the re-~168583 agents described in connection with the kallikrein deter-mination.
Prekallikrein preparation:
The production of the preparation is effected according to a prescription of Harpel, modified by M. S. Horowitz (New York Blood Center). Therein human citrated plasma is treated with a DEAE-cellulose. The fraction that is not bound to the DEAE-cellulose contains the pre-kallikrein.
Positive control (standard):
As standard (= reference value) an albumin preparation of the Burau of Biologics (soB) of the Food and Drug Ad-ministration, Bethesda, Maryland 20205, U.S., is used. This preparation contains a prekallikrein activator. The kalli-krein generation with this BoB-standard represents the re-ference value 1 and is equated with 100 %.
Sample:
The sample is dissolved in the original volume and used in the test undiluted.
3. Test:
In a water bath at a temperature of 37 C
0.05 ml prekallikrein preparation 0.05 ml sample a) BoB-standard for the reference value b) test sample (in a second test mixture) are pipetted into a plastic tube. After an incubation of 10 minutes at 37 C
0.7 ml buffer solution 0.1 ml chromogenic substrate S-2302 are pipetted. This mixture is charged into a photometer heated to 37C and the increase in the optical density

5 ~ 3 per minute (aOD/min) is measured at a wave length of 405 nm with a path length of 10 mm. The activity of a sample OD/min) is expressed in % of the BoB-standard.
The characterization of the preparation according to the invention by an affinity-chromatographic separation of thepreparation on dextran sulphate sepharose has already been described in connection with Fig. 1.
The electrophoretic separation of the proteins with factor IX activity and with FEIB-activity, which is re-ferred to in connection with Fig. 2, can be carried outin the following manner.
As the carrier for the electrophoretic separation of the various proteins a cellulose-acetate membrane serves, which is wetted with the electrophoresis buffer (p~ 8.6, ionic strength 0.075). On this membrane the samples to be analyzed - together with a normal human plasma as standard - are applied and then separated in the electric field; the separation is effected in that differently charged proteins migrate at different speeds in the electric field. To this end the membrane provided with the samples is treated in a special cell filled with the electropharesis buffer for 16 to 18 minutes at a voltage of 250 volts and an initial current intensity of 4 to 6 milliamperes.
After termination of the separation procedure the various proteins are rendered visible by putting the mem-brane into a fixing and dyeing solution. After some rinsing baths the membrane is made transparent in a iurther bath, applied onto a glass plate and dried in a drying chamber at 100 C.

~16~583 The dried membrane is then analyzed in an automati-cally registering and integrating densitometer, which produces separation curves as illustrated in Fig. 2.
The various proteins appear as different peaks whose areas are proportional to the relative percentage values of the corresponding proteins, these areas being determined by the automatic integration of the densito-meter.
The allocation of the various peaks or proteins of the test sample to defined proteins or protein groups is effected by comparing them with the peaks of the normal human plasma simultaneously analy~ed as standard. During the electrophoretic analysis the latter is separated ir,to5protein groups which by de-finition are designated as follows - in the order of decreasing mobility in the electric field: albumin, ~-globulins, ~-globulin, fibrinogen, and ~-globulin.
The definition of the FEIBA unit as well as its determination (potency test) is described in U.S. patent No. 4,160,025. The determination of the activity of the coagulation factors II, VII, IX
and X is also described in the above mentioned U.S. patent No. 4,160,025.
The determination of the residual activity of FEIBA after incubation in factor VIII inhibitor plasma is carried out in den following manner.
1. Reagents:
The reagents to be used are described in U.S. patent No. 4,160,025 in connection with the determination llfi8583 of the FEIBA units (potency test).
2. Test:
From a preparation adjusted to 50 FEIBA units/ml t'ne following dilutions are prepared with a solution of 7 g/l sodium chloride and 7 g/l sodium citrate.2H20 as diluent:
1 : 2, 1 : 4, 1 : 8, 1 : 16, 1 : 32 and 1 : 64. From these

6 dilutions a 1 : 10 dilution in factor VIII inhibitor plasma (0.05 ml pre-diluted sample+-0.45 ml factor-VIII-inhibitor plasma) is each prepared.
These 1 : 10 dilutions in factor VIII inhibitor plasma ("incubation mixtures") are then analyzed immediately and after one hour of incubation at 37 C according to the following test scheme:
0.1 ml "incubation mixture"
0.1 ml phospholipid-kaolin suspension incubation at 37 C for 1 minute 0.1 ml m/40 calcium chloride.
The time from the addition of calcium chloride to the clot formation is taken with a timer like in the potency test.
3. Calculation of the residual activity:
Analogously to the description in the potency test, a cali-bration curve is established with the coagulation times of the immediately determined dilutions (undiluted s~nple =
50 FEIBA units/ml). The activities (FEIBA units/ml) of the various dilutions incubated for one hour are then calculated by using the calibration curve and are expressed in percent of the individual activities of the non-incubated dilutions.
The mean values of the activities thus calculated produce the average residual activity of the sample after an hour of incubation, expressed in percent of the initial activity 1~8583 prior to incubation.
The determination of the residual activity of factor IX after incubation in factor IX deficient plasma is carried out in the following manner:
1. Reagents:
Factor IX deficient plasma: citrated plasma of a patient suffering from severe haemophilia B (factor IX below 1 %).
Phospholipid/kaolin suspension: PTT reagent of Immuno Dia-gnostica Ges.m.b.H. For the test the required amount of factor IX deficient plasma is mixed with an equal volume of the phospholipid/kaolin suspension, incubated for 5 minutes at 37 C and then stored in an ice bath during the test period.
Citrate/saline solution as diluent for samples: 7 g/l trisodium citrate.2H20, 7 g/l sodium chloride.
Calcium chloride m/20 (0.05 molar): storing at 37 C during the test period.
2. Test:
From a preparation adjusted to 50 factor IX units/ml 7 geometric dilutions (1 : 2, 1 : 4 etc. until 1 : 128) are prepared with the citrate/saline solution. From the undi-luted sample as well as from the 7 geometric dilutions a 1 : 10 dilution in factor IX deficient plasma is each prepared (0.05 ml prediluted sample + 0.45 ml factor IX
deficient plasma).
These 1 : 10 dilutions in factor IX deficient plasma ("incubation mixtures") are then analy~ed immediately and after an hour of incubation at 37 C according to the following test scheme, each incubation mixture being di-luted 1 : 10 with the citrate/saline solution prior to 1 ~6~583 determination:
0.2 ml mixture of factor IX deficient plasma and phospho-lipid/kaolin 0.1 ml "incubation mixture", 1 : 10 diluted with citrate/
saline solution incubation at 37 C for 1 minute 0.1 ml m/20 calcium chloride The time from the addition of calcium chloride to the clot formation is taken by a timer.
3. Calculation of the residual activity:
A calibration curve is established with the coagula~ion times of the immediately determined dilutions (undiluted sample = 50 factor IX units/ml) by plotting the coagulation times against the corresponding dilutions on double logarith-mic graph paper. The activities (factor IX units/ml) of the various dilutions incubated for one hour are then calculated by using the calibration curve and are expressed in percent of the respective activities of the non-incubated dilutions.
The mean values of the activities thus calculated produce the average residual activity of the sample after one hour of incubation, expressed in percent of the initial activity prior to incubation.
Immunologic determination of the inter-alpha-trypsin-inhibitor (ITI):
1. Method:
The determination is effected according to the Ouchterlony- -technique, wherein a specific antibody diffuses in an agar medium against an antigen-containing sample. The antigen specifically reacts with the antibody, forming an immune 0 precipitation p-e-ak that is valued as positive reaction.

1~6~8~

2. Reagents:Rabbit antiserum against ITI, Behringwerke AG, Marbury/
Lahn, sRD
Agar: A solution of 1.25 g agar, 0.9 g sodium chloride and 100 mg sodium azide in 100 ml water is briefly boiled, and the hot homogenous solution is poured onto plates yielding a layer of about 2 mm thickness. Into the cooled solidified gel holes having a size of about 2 mm are punched in two rows at a distance of 5 mm.
Standard and sample:
As calibration substance a protein standard serum of Beh-ringwerke with a definea content of ITI serves. From this reference serum a geometric dilution series in a physiologic sodium chloride solution (9 g NaCl/l) is prepared. The sample to be tested is treated like the standard.
3. Test:
Into one hole row of the agar the dilutions of the cali-bration substance or sample to be tested are charged, in the adjacently arranged hole row the undiluted specific antiserum is pipette-d. The agar plate thus charged is in-cubated at 37 C for 15 hours. Afterwards the reading of the immune precipitations is effected.
~. Calculation of the ITI concentration:
As a measure for the ITI concentration of a sample the di-lution step is chosen at which a precipitation is just visible ("titer" of the sample). The ITI concentration of the sample to be tested is calculated as follows:

titer of test sample x ITI concentratlon of standard titer of standard The ITI concentration is indicated in mg ~ (mg/100 ml).

The preparations produced according to Examples 1 and 2 were analyzed according to the preceding assay methods;
the results are summarized in the following Table.
The illustrations in Fig. 1 (affinity-chromatographic separation) and Fig. 2 (electrophoretic separation) of the drawings correspond to the data of Example 2 in the follow-ing Table.

_ __ _ ., C~
Z Z Z Z
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o ~ ~ o U~ ~ ~ ~ o o o o C~ ~ . . . . . I
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~ 1:':1 IL~ O O
O
~ ~l O ,Y
.~ ~ ~ ~ O C.) t_) :z æ ~;
O G~ O ~ ~ ~
a) ~ ~D ~ ~ ~ ~ ~ ~ ~
~ X ~ ~ ~
~ ~1 H O O O O
m ~ ~r ~
~ o o o _ . _ _ E~ . ~ O

o ~ a o a ~ ~ U~ ~ ~ ~
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~ ~ ~ ~ ~ ~ O O ~ ~ ~ .
v o ~ v ~1 Ul ~ ~ Q O ~
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~J ~ H ~ ~ ~ O O S~
,1 H H ~C a) ~ ~ I ~ X ~1 ~
H ~ H X t~ >1 ~I H ~) ~ , O ~J (d ~ ~
S~ Q O ,1 ,~C (j O ~3 o s:~ a) o m ~ o ~ ~ ~ m H V V V V S l O IH V X H X
~1 ~ ~ ~! ~ ~1 ~ (a td ~ Id E~ ~ ~ ~ O ~
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o h _ __ . .. ... . r :--r~l ~
~ )C

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h ~ ~ ~ O O Ln O

H ~:1 1:4-- -- ~ O
_ _ _ __ 4~ ~ ~ ~ ~ C~
o ~ m~ .~ ~
~ 1l:l ~1 H R
~ m ~ H X ~
~d H O ~1 ~ ~1 1--1 ~::
I .Y ~ O O ,~
h ~ h a~ ~1 5-1 ~a~ ~ ~ ~ ~ o ~ o a) ~ ~ ~ ~ ,, ~ ~ ~ ~ ,J
~_ U~ ~ ~ ~ }~ r~ O rl O ~a 1~ ~ O ~ 1 O 0 ~1 0 rl t~ ~1 ~H rJ 4 ~S ~ ~ ~ ~ ~ ~1 ):: ~ H ~ u~ ~ ~ ~:
h h` `
~ O O~ ~ ~ ~ h ~1 ~ ~ ~ ,~:
O ~: ~ rl rl ~1 r~ X 1~ 0 rd O P~
O ~ O ~ ~ ~ rl ~S rl ~ rl O ~ h ~1 ~) O h 4-1 Q Q Q X ~1 ~ S ,1 ~
~ ~ O O O rl (1~ u~ Q u~ Q h Q o ~ ~I X a) ~ a) ~ a~ ~ ~ ~ ~ ~ ~ ~ o E~ ~ ~1 1 1 I aS h ~: ~
3 ~ ~ . o\O .,1 o\O .,1 H
o

Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following charact-eristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) The preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

2. A blood-coagulation-promoting preparation comprising human proteins including coagulation factors II, VII, IX and X
and factor-VIII-inhibitor-bypassing-activity (FEIBA), whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

3. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, (d) the separation trace obtained when the eluate contain-ing the protein with factor IX activity and the eluate containing the protein with FEIB-activity are electrophoretically separated by a method as hereinbefore defined corresponds to an .alpha.- and .beta.-globulin content characterised by: in the .alpha.-globulin region, a main peak corresponding to a content of 60 to 80% of the total protein together with a shoulder of 10 to 20% of the total protein downstream of the main peak, and thereafter in the .beta.-globulin region a small peak corresponding to a content of 10 to 20% of the total protein, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

4. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, (d) the protein with factor IX activity elutes at an NaCl concentration of 0.1 to 0.5 molar and said protein with FEIB-activity elutes at an NaCl concentration of 0.3 to 0.5 molar, the maximum factor IX activity eluting at 0.3 molar and the maximum FEIB-activity eluting at 0.4 molar, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

5. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, (d) the preparation exhibits FEIB-activity which, after one-hour incubation in factor VIII inhibitor plasma, is preserved by at least 50%, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

6. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaC1 concentration than said protein with FEIB-activity, (d) the preparation exhibits factor IX activity which after a one-hour incubation in factor IX deficient plasma, is preserved by at least 50%, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

7. A method of producing a blood-coagulation-promoting preparation comprising human proteins, including coagulation facators II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), the preparation having the following characteristics:
(a) the preparation is free of thrombogenic activity in doses of up to at least 2 units of FEIBA per kg rabbit in the thrombosis inducing activity test according to Wessler, (b) the preparation is free of kallikrein activity and free of prekallikrein activator-activity, measured in an aqueous solution of said preparation with a FEIBA concentration of up to at least 10 units per ml, (c) the preparation is affinity-chromatographically separable on dextran sulphate agarose by means of an NaCl gradient so as to obtain an eluate containing a protein with factor IX activity and an eluate containing a protein with FEIB-activity, said protein with factor IX activity eluting at a lower NaCl concentration than said protein with FEIB-activity, (d) the preparation further including a content of inter-alpha-trypsin-inhibitor (ITI) of 0.05 to 5 mg per FEIBA unit, which method comprises treating human plasma with sulphated high-polymer carbohydrates and/or with basic ion exchangers so as to obtain a protein mixture with generated FEIB-activity, adsorbing said protein mixture, and recovering said preparation by elution and concentration.

8. A method as set forth in claim 1, 3 or 4, wherein said human plasma at first is briefly treated with sulphated high-polymer carbohydrates, said protein mixture with generated FEIB-activity is then adsorbed on an ion exchanger on dextran basis and is then immediately eluted and concentrated.

9. A method as set forth in claim 5, 6 or 7, wherein said human plasma at first is briefly treated with sulphated high-polymer carbohydrates, said protein mixture with generated FEIB-activity is then adsorbed on an ion exchanger on dextran basis and is then immediately eluted and concentrated.

10. A method as set forth in claim 1, 3 or 4, wherein said human plasma is treated with an ion exchanger on dextran basis, and after at least two hours of exposure, said protein mixture with generated FEIB-activity, adsorbed on said ion exchanger, is eluated and concentrated.

11. A method as set forth in claim 5, 6 or 7, wherein said human plasma is treated with an ion exchanger on dextran basis, and after at least two hours of exposure, said protein mixture with generated FEIB-activity, adsorbed on said ion exchanger, is eluted and concentrated.