WO2004050904A1

WO2004050904A1 - Prothrombin purification

Info

Publication number: WO2004050904A1
Application number: PCT/SE2003/001549
Authority: WO
Inventors: Stefan Winge; Peter Aizawa
Original assignee: Biovitrum Ab
Priority date: 2002-12-02
Filing date: 2003-10-06
Publication date: 2004-06-17
Also published as: AU2003267907A1; SE0203551D0

Abstract

One aspect of the invention relates to a process for the preparation of a purified solution of prothrombin and factor X from a fraction of blood plasma obtainable by cryoprecipitation, which process comprises subjecting such a fraction to prepurification steps and to the following steps: (a) metal chelate gel chromatography and (b) affinity gel chromatography. Another aspect of the invention relates to a solution of prothrombin and factor X, wherein the prothrombin has the ability of being activated to thrombin in the substantial absence of other enzymes or cofactors. A further aspect of the invention relates to the use of a solution of prothrombin and factor X for the preparation of a thrombin solution.

Description

PROTHROMBIN PURIFICATION

Technical field of the invention

The present invention relates to a process for the preparation of a purified solution of prothrombin and factor X from a fraction of blood plasma obtainable by cryoprecipitation, to a solution of prothrombin and factor X and to the use of such a solution.

Background art

Blood clots are formed in vivo by a series of zymogen activations. In this enzymatic cascade, the activated form of one factor catalyses the activation of the next factor. One of the last steps of this cascade comprises activation of prothrombin (factor II, FII) to thrombin. Prothrombin is present in native plasma and the activation is catalysed by factor X (FX, Stuart factor) . Thrombin catalyses the actual clotting reaction (conversion of fibrinogen to fibrin). (Stryer, L. (1987) Biochemistry 3rd ed.)

If preparation of a thrombin solution from a plasma source is to be performed in vitro, clottable proteins

(mainly fibrinogen) must be removed from the plasma so as to avoid formation of an insoluble clot when the prothrombin is activated. Additionally, it is essential to prepare selected cofactors in a specific way, as not all factors involved in the enzymatic cascade, in vivo, are present in plasma. Further, in commercial plasma fractionation some cofactors are purified separately and not available in the fraction used for preparation of thrombin. Any substances inhibiting the activation as well as pathogens and other contaminants should be absent .

A process for the preparation of a prothrombin solution, intended for the further activation of prothrombin to thrombin, should thus comprise measures for removal of clottable proteins and maintaining necessary cofactors.

The European patent application 617 049 Al describes a process for the purification of factor IX, factor X and factor II from human plasma or fractions thereof, wherein a solution containing prothrombin complex factors is purified by repeated ion exchange chromatographic separations followed by adsorption chromatography on metal ions. This process is circumstantial as it involves concentration and diafiltering steps between the ion exchange separations and the adsorption separation. It is focused on the purification of factor IX, whereas fractions of prothrombin and factor X are obtained as by- products. The process involves binding of factor IX as well as prothrombin and factor X to the metal ion column, which is not optimal when aiming for a product comprising prothrombin and factor X only.

The European patent application 317 376 Al describes a process for preparing a high purity factor IX concentrate by ion exchange chromatography and subsequent affinity chromatography. Although it is mentioned that it is possible to obtain fractions enriched with prothrombin and factor X, those two components are actually separated from each other during the purification. The process involves binding of factor X to the affinity adsorbent, whereas prothrombin and factor IX are not bound. This procedure is not optimal when aiming for a product comprising both prothrombin and factor X, but not factor IX.

WO 89/05650 and Huiping Wu et al . (1998), "Protein C separation from human blood plasma Cohn fraction IV-1 using immobilized metal affinity chromatography", in: Hudetz and Bruley (eds.) Oxygen Transport to Tissue XX, describes separation methods based on adsorption to immobilized metal ions. However, these texts do not describe complete purification processes for the preparation of a purified solution of prothrombin and factor X.

Summary of the invention

An object of the present invention is to provide an improved process for the preparation of a purified prothrombin solution.

A second object of the present invention is to provide a purified solution of prothrombin and factor X. Another object of the present invention is to provide a prothrombin solution suitable for the subsequent preparation of a thrombin solution.

These and other objects are obtained by the process as claimed. Thus, in one of its aspects, the invention provides a process for the preparation of a purified solution of prothrombin and factor X from a fraction of blood plasma obtainable by cryoprecipitation, which process comprises subjecting such a fraction to prepurification steps and to the following steps:

(a) metal chelate gel chromatography and

(b) affinity gel chromatography.

In other aspects, the invention provides a solution of prothrombin and factor X, wherein the prothrombin has the ability of being activated to thrombin in the substantial absence of other enzymes or cofactors, and the use of such a solution for the preparation of a thrombin solution.

During the metal chelate chromatography step of the process according to the invention, inhibiting proteins of high molecular weight are removed from the solution of prothrombin and factor X. The speed by which subsequent activation of prothrombin to thrombin occurs is strongly increased by the presence of such a step. The increase of activation speed contributes to an effective industrial use of the purified solution of prothrombin and factor X. The increase of activation speed also indirectly contributes to an increased recovery by minimising the degeneration of thrombin, which occurs during the activation period. The affinity gel chromatography step is essential to the process, as it has the function of withholding contaminating proteins (i.a. C4 binding proteins, protein S, factor IX) from the solution of prothrombin and factor X. It also gives the opportunity to make more products (e.g. a factor IX concentrate) from the same plasma source .

The solution of prothrombin and factor X according to the invention is native and of high purity. Thus, the only cofactor that is needed for conversion of prothrombin to thrombin is factor X, as no inhibitors are present. The process according to the invention provides this advantage by utilising a unique combination of chromatographic purification steps. The prothrombin in such a solution can easily be activated to thrombin, without the addition of other enzymes or cofactors, relatively fast and with a satisfactory recovery.

In addition to the metal gel chromatography and affinity gel chromatography steps of the process of the present invention, prepurification steps are present. These steps improve the performance of the process by their specific actions as well as by introducing advantages resulting from interactions among steps. The process utilises a unique combination of chromatography steps together with protective conditions (pH and salts). In addition to the abovementioned steps of the process of the present invention, optional steps can be present. The following description serves to further clarify the functions of each step, whether essential or optional, and its features as well as interactions among steps. However, in a process according to the invention, certain optional steps can be omitted and/or present in a different order. Also described is a possible way of obtaining the starting material of the present process. The solution of prothrombin and factor X according to the invention, and use of said solution, are finally described.

Cryoprecipitation

The starting material for the process according to the invention is a fraction of blood plasma obtainable by cryoprecipitation. Such a fraction is widely available and of a standardised character. Frozen blood plasma is thawed at about 0 °C. The cryoprecipitate is removed by centrifugation and the supernatant is used in the further process . Fractions of plasma obtained by other methods are equally useful as long as their properties are substantially similar to those of fractions obtained by cryoprecipitation .

Prepurification : Anion exchange chromatography for the removal of i . a . clottable proteins

This initial prepurification step is a major waypoint in the commercial processing of plasma products. Clottable proteins as well as other useful proteins, such as antithrombin III, immunoglubulin G and albumin, are separated from the solution of prothrombin and factor X and refined in processes for each component. Removal of clottable proteins is a prerequisite for successful later use of the prothrombin solution for activation to thrombin.

This step is preferably performed under such conditions that prothrombin and factor X are bound to the gel together with fibrinogen, antithrombin III, immunoglobulin G and albumin. Then, undesired substances are washed away, after which prothrombin and factor X are eluted by means of increased salt concentration. In a preferred embodiment of this step, the adsorbent is diethylaminoethyl (DEAE) groups immobilised on a solid phase .

It is preferred to equilibrate the gel with a buffered equilibration solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, before applying the protein solution. Preferably, the total concentration of salt in the equilibration solution is in the range of from about 1 to about 200 mM. The pH of the equilibration solution is preferably in the range of from about 6.5 to about 7.5.

In a preferred embodiment of this step, the gel is washed with a buffered washing solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates. Preferably, the total concentration of salt in the washing solution is in the range of from about 0.01 to about 0.1 M. The pH of the washing solution is preferably in the range of from about 5.5 to about 6.5.

Elution of prothrombin and factor X follows by means of an increased salt concentration. Preferably the elution is performed with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates. Preferably, the total concentration of salt in the elution solution is in the range of from about 0.1 to about 0.5 M. The pH of the elution solution is preferably in the range of from about 5.5 to about 6.5.

Virus inactivation

A pathogen inactivation method may be included among the steps of the process of the present invention to ensure a high virus safety of the biological material. Virus inactivation is preferably performed by the solvent/detergent method. Said method involves contacting the solution of prothrombin and factor X with a solvent (e.g. 0.3 % tri(n-butyl) phosphate (TNBP) ) and a detergent (e.g. 1 % Triton® X-100) . After the solvent/detergent treatment the amount of additives is reduced by addition of soy bean oil and subsequent phase separation.

Affinity gel chromatography

The affinity gel chromatography step is essential to the process as it has the function of withholding contaminating proteins (i.a. C4 binding proteins, protein S, factor IX) from the solution of prothrombin and factor X.

This step is preferably performed under such conditions that prothrombin and factor X pass through the gel, whereas a variety of contaminating proteins are withheld on the gel. It is desirable that prothrombin and factor X both pass the gel, as the process of the present invention aims at a purified solution of said proteins. This is achieved by a suitable adaptation of the process parameters (see below) . In a preferred embodiment of this step, the adsorbent is heparin. The use of heparin as a ligand for the affinity chromatography is advantageous owing to its double functions of both cation effects (due to the negatively charged groups on the heparin molecule) and affinity for specific proteins (such as factor IX) .

It is preferred to equilibrate the gel with NaCl and/or trisodium citrate at a slightly acidic pH. The NaCl concentration is preferably in the range of from 20 to 100 mM, more preferably from 50 to 100 mM and most preferably around 80 mM. The trisodium citrate concentration is preferably in the range of from 25 to 75 mM, more preferably around 50 mM. It is suitable to perform the equilibration at a pH in the range of from 5.5 to 6.5, more preferably around 6.0. Anion exchange chromatography for the removal of impurities and solvent/detergent chemicals

This step has a twofold aim: further removal of contaminating proteins and removal of solvent/detergent chemicals from the virus inactivation step, in the case where such a step is present. The present step is thus preferably performed after the virus inactivation step, if present.

This anion exchange chromatography step for the removal of impurities and solvent/detergent chemicals is preferably performed under such conditions that prothrombin and factor X are bound to the gel together with impurities and remaining solvent/detergent chemicals . Then, undesired substances are washed away, after which prothrombin and factor X are eluted. In a preferred embodiment of this step, the adsorbent is dietylaminoethyl (DEAE) groups immobilised on a solid phase .

It is preferred to equilibrate the gel with a buffered equilibration solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, before applying the protein solution. Preferably, the total concentration of salt in the equilibration solution is in the range of from about 10 to about 100 mM. The pH of the equilibration solution is preferably in the range of from about 5.5 to about 6.5.

In a preferred embodiment of this step, the gel is washed with a buffered washing solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates. Preferably, the total concentration of salt in the washing solution is in the range of from about 10 to about 100 mM. The pH of the washing solution is preferably in the range of from about 5.5 to about 7.5. Elution of prothrombin and factor X follows by means of an increased salt concentration. Preferably the elution is performed with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates. Preferably, the total concentration of salt in the elution solution is in the range of from about 100 to about 1000 mM. The pH of the elution solution is preferably in the range of from about 6.5 to about 7.5.

Metal chelate gel chromatography

During the metal chelate chromatography step, inhibiting proteins of high molecular weight are removed from the solution of prothrombin and factor X. The speed by which subsequent activation of prothrombin to thrombin occurs is strongly increased by the presence of such a step.

This step is preferably performed under such conditions that prothrombin and factor X pass through the gel, whereas i.a. inhibiting proteins of high molecular weight are withheld on the gel. It is desirable that prothrombin and factor X pass the gel, as this streamlines the purification process by reducing the number of treatments of prothrombin and factor X. This is achieved by careful adaptation of the process parameters (see below) . In a preferred embodiment of this step, the gel is loaded with copper ions. The use of copper ions is advantageous as it allows both prothrombin and factor X to be collected in the fall-through fraction, whereas the impurities stay bound to the chromatography medium. Other metal ions may attract the affinity regions in different ways and thus change the elution order of the proteins. It is preferred to equilibrate the gel with a NaCl solution at about neutral pH. The NaCl concentration is preferably in the range of from 0.1 to 1 M, more preferably around 0.5 M. It is suitable to perform the equilibration at a pH in the range of from 6.5 to 7.5 and most preferably around 7.0. As an alternative to, preferably in addition to, said NaCl equilibration, it is preferred to equilibrate the gel with i idazole. The imidazole concentration is preferably in the range of from 2 to 30 mM, more preferably about 10 mM.

Concentration and desalting

The purified solution of prothrombin and factor X may be concentrated and optionally desalted. The concentration is preferably performed by ultrafiltration. Preferably, concentration and desalting are performed simultaneously. The methods for concentration and desalting are well known in the art.

Prothrombin solution

The prothrombin in the inventive solution of prothrombin and factor X has the ability of being activated to thrombin in the substantial absence of other enzymes or cofactors. Preferably, it is essentially free from factor VII, factor IX, factor VIII, calcium and/or phospholipids . These properties of the purified solution of prothrombin and factor X are advantageous for the intended use thereof (see below) . The solution of prothrombin and factor X according to the present invention is characterised in that it has a prothrombin concentration higher than about 600 IU/ l. The inventive solution of prothrombin and factor X preferably has a ratio of 1 ID^* of prothrombin to 0,6 + 0,1 IU of factor X. This ratio is a prerequisite for successful utilisation of the inventive solution for the intended use (see below) .

Use of the prothrombin solution The activation process of prothrombin to thrombin is, in vivo, a very complex system including several cofactors, inhibitors and charged surfaces (phospo- lipids) . It is essential to provide necessary cofactors, as not all factors involved in the enzymatic cascade are present in plasma. Further, in commercial plasma fractionation some cofactors are purified separately and not available in the fraction used for preparation of thrombin.

The solution of prothrombin and factor X according to the present invention is used for the preparation of a thrombin solution. Such preparation involves an activation process, which is optimised for in vitro use. Thus, inhibitors have been removed from the solution and the only cofactor that is needed for conversion of prothrombin to thrombin is factor X. The preparation is performed by activation of said solution of prothrombin and factor X, said activation comprising adding a salt, preferably sodium citrate, in the concentration range of from 5 to 50 %wt, preferably from 20 to 40 %wt, most preferably about 28 %wt, to the solution of prothrombin and factor X.

The preparation is performed by activation of said solution of prothrombin and factor X at a temperature in the range of from 5 to 50 °C, preferably in the range of from 25 to 50 °C and more preferably about 44 °C. The preparation is performed by activation of said solution of prothrombin and factor X at a pH in the range of from 7 to 9, preferably about 8.

Brief description of the drawings Figure 1 shows SDS-PAGE analysis of the purification on the chelating Sepharose FF gel according to Example 2 below. Figure 1A shows the analysis under non-reducing conditions and Figure IB under reducing conditions .

Figures 2-4 show the effects of pH and temperature on the activation of prothrombin according to results from Preparatory example 1. Figure 5 shows the effect of temperature on the activation of prothrombin according to results from Preparatory example 1.

Figure 6 shows the effect of salt concentration on the activation of prothrombin according to results from Preparatory example 1.

Examples

Some illustrative examples are provided for a better understanding of the process of the present invention and its embodiments. The prepara tory example explains the basis for the use of the inventive solution of prothrombin and factor X by illustrating experimentation aimed at revealing suitable parameter settings. The compara tive examples illustrate procedures being less advantageous alternatives to parts of the process of the invention, which alternatives do not form part of the invention as claimed. Finally, the analysis methods used are explained in detail.

Example 1

Part 1 : Cryoprecipitation and prepurification

1200 kg of frozen blood plasma were thawed to 0 °C and the resulting cryoprecipitate was removed by centrifugation .

The resulting cryosupernatant was processed through a 70 liter column (bed height 11 cm) packed with DEAE Sepharose FF (Pharmacia Biotech) gel. The gel was equilibrated with a solution of 150 mM sodium chloride and 1 mM sodium phosphate at pH 7.0 before applying the cryosupernatant. In the column, coagulation factors, including prothrombin, factor IX and factor X, were separated from the cryosupernatant. The gel was then washed with 0.07 M sodium citrate at pH 6.0. The remaining proteins (including prothrombin, factor IX and factor X) were eluted with 0.2 M sodium citrate at pH 6.0. The elute was concentrated to a defined protein concentration using an ultrafilter system (Biomax-8, Millipore) . The properties of the eluate, compared to the cryosupernatant, are shown in Table 1 below.

Part 2 : Virus inactivation

The concentrated eluate was solvent/detergent (Triton X-100/tri-n-butylphosphate) treated for 6 h at 22 °C to inactivate all present enveloped viruses. Thereafter, 5 % soybean oil was added to reduce the amount of virus inactivating chemicals. After phase- separation of the oil, the remaining protein solution was diluted (1+3) with 0.08 M sodium chloride.

Part 3 : Affinity gel chromatography

The diluted protein solution was applied to a 70 liter column packed with Heparin Sepharose FF gel (Amersham Pharmacia Biotech) . The gel was equilibrated with a solution of 80 mM sodium chloride and 50 mM trisodium citrate at pH 6.0 before applying the protein solution. The non-binding protein fraction (including prothrombin/FX) was collected. The properties of the passing fraction compared to prothrombin/FX-containing fractions from earlier stages of the process are shown in Table 1.

Table 1

FII FX/FII FVII/FII FIX/FII FVIII/F! recovery activity activity activity activit; (%) ratio ratio ratio ratio

Cryo 100 1.0 1.0 1.0 < 0.05 centrifugate (part 1)

DEAE-eluate 87 0.9 < 0.05 0. < 0.05 (part 1)

Non-binding 87 0.8 < 0.05 < 0.05 < 0.05 fraction (part 3)

Part 4 : Further prepurification

The prothrombin/FX fraction from part 3 was further processed through a 14 litre column (bed height 20 cm) packed with DEAE Sepharose FF (Pharmacia Biotech) gel. The gel was equilibrated with a solution of 80 mM sodium chloride and 50 mM sodium citrate at pH 6.0 before applying the protein solution. On the gel, the proteins were bound and separated from remaining residues of virus inactivating chemicals. The gel was then washed with 10 column volumes of equilibration buffer . Thereafter, the proteins (including prothrombin/FX) were eluted with 0.2 M sodium citrate at pH 6.0.

Part 5 : Metal chelate gel chromatography

Chelating Sepharose FF gel in a 14 litre column (bed height 20 cm) was charged with copper ions to its maximum capacity. The column was preequilibrated with 5 bed volumes of elution buffer (30 mM imidazole, 20 mM phosphate and 0.5 M sodium chloride at pH 7) to avoid bleeding of copper ions during the run. The equilibration was performed with 3 bed volumes of equilibration buffer (2 mM imidazole, 20 mM phosphate and 0.5 M sodium chloride at pH 7.0). The prothrombin rich fraction from part 4 was prepared (NaCl was added to 0.5 M and pH was adjusted to 7.0) and applied to the column. A linear gradient from 2 to 30 mM imidazole was run. Three different peaks could be observed. Peak number 1 contained prothrombin and FX. Peaks number 2 and 3 mainly contained proteins of high molecular weight. Results from the metal chelate gel chromatography are shown in Table 2.

Table 2

Sample FII FX FX/FII FII recove :ry recovery activity purity

(%) (%) ratio (Iϋ/mg)

Before 100 100 0.69 3.3 chelate gel

Peak 1 86 87 0.67 6.4

Peak 2 0 0 - 0

Peak 3 0 0 - 0

Example 2

Parts 1-4

Performed as in Example 1.

Part 5 : Metal chelate gel chromatography Chelating Sepharose FF gel in a 14 litre column (bed height 20 cm) was charged with copper ions to its maximum capacity. The column was preequilibrated with 5 bed volumes of elution buffer (30 mM imidazole, 20 mM phosphate and 0.5 M sodium chloride at pH 7) to avoid bleeding of copper ions during the run. The equilibration was performed with 3 bed volumes of equilibration buffer (2 mM imidazole, 20 mM phosphate and 0.5 M sodium chloride at pH 7.0) . The prothrombin rich fraction from part 4 was applied to the column which had been equilibrated with 10.4 mM imidazole, 0.5 M NaCl and 20 M phosphate. The proteins of high molecular weight were bound to the gel and the non-binding fraction, comprising prothrombin and FX, was collected (Table 3) .

Table 3

Sample FII FX FX/FII FX FII recovery recovery activity purity purity { % ) { % ) ratio (IU/mg) (IU/mg)

Before 100 100 0.68 2.57 3.79 chelate gel

Non- 100 100 0.69 4.66 6.71 binding raction

Figure 1 shows SDS-PAGE analysis of the purification on the chelating Sepharose FF gel. Figure 1A shows the analysis under non-reducing conditions and Figure IB under reducing conditions . The patterns are explained in Table 4.

Table 4

Pattern Fraction

1 Thrombin marker

2 Prothrombin marker

3 Before chelating Sepharose FF gel

4 After chelating Sepharose FF gel

5 After chelating Sepharose FF gel (concentrated)

6 High molecular weight fraction separated with chelating Sepharose FF gel Example 3

Parts 1-3

Performed as in Example 1.

Part 4 : Further prepurification

The prothrombin fraction from part 3, was further processed through a 14 litre column (bed height 20 cm) packed with DEAE Sepharose FF (Amersham Pharmacia Biotech) gel. The gel was equilibrated with 67 mM (10 mS/cm, 25 °C) sodium citrate at pH 5.8 before applying the protein solution, and then washed with 10 column volumes equilibration buffer and 5 column volumes of a buffer comprising 50 mM sodium chloride and 20 mM sodium phosphate at pH 6.7. The proteins were bound and separated from remaining residues of virus inactivating chemicals. The remaining proteins (including prothrombin/FX) were eluted with a buffer comprising 700 mM sodium chloride and 20 mM sodium phosphate at pH 6.7. During the washing procedure, the purity of the prothrombin/FX-fraction was increased.

Part 5 : Metal chelate gel chromatography

The eluate obtained from part 4 was further processed through a 10 litre column (bed height 14 cm) packed with chelating Sepharose FF (Amersham Pharmacia Biotech) gel which had been charged with copper ions. The gel was equilibrated with a soiution of 0.5 M sodium chloride, 10.4 mM imidazole and 20 mM sodium phosphate at pH 7.0 before applying the protein solution. The proteins that did not bind to the gel (prothrombin/FX) were collected. The purity of the protrombin/FX-fraction was further increased in this part.

Part 6: Concentration and desalting The protein fraction was concentrated and diafiltered against 10 volumes of a solution of 50 mM sodium chloride and 20 mM sodium phosphate at pH 6.7, using an ultrafilter system (2.5 m²Biomax-8, Millipore) The properties of the resulting solution are shown in Table 5.

Table 5

Sample FII FII FX FX/FII recovery purity purity activity

(%) (IU/mg) (IU/mg) ratio

Example 3 100 7.29 4.57 0.63 (part 6)

Comparative example 1: Purification without metal chelate gel chromatography

Parts 1-4 were performed as in Example 1. The elute was concentrated and diafiltered against 10 volumes of 50 mM sodium citrate at pH 6.0, using a ultrafilter system (2.5 m² Biomax-8, Millipore) . The properties of the resulting solution are shown in Table 6.

Table 6

Sample FII FII FX FX/FII recovery purity purity activity

(%) (IU/mg) (IU/mg) ratio

Comparative 100 3.58 2.29 0.64 example 1 Preparatory example 1: Activation parameters for the activation of prothrombin to thrombin

Experiments performed during early development of the method involve addition of thrombin to the solution to be activated. However, such an addition causes traceability problems in the products to be manufactured. Thus, the addition was excluded in later development and does not form part of the invention as claimed, although it is present in some parts of this preparatory example.

Influence of pH and temperature

A solution of prothrombin (7.29 IU/ml) and FX (4.57 IU/ml), which was obtained according to the present invention, was subjected to activation of prothrombin to thrombin under different conditions. The effects of pH and temperature on the activation in the presence of 25 %wt of trisodium citrate and an initial addition of thrombin (300 IU/ml) were studied. Activation was performed at pH = 6.5, 7.25 and 8 and at 25, 31 and 37 °C. The results are shown in Figures 2-4. The yield of thrombin is increased by increasing pH and the reaction time is decreased by increasing temperature. Among the studied conditions, the fastest activation with the highest yield is thus obtained at pH = 8 and 37 °C. The effect of temperature on the activation in the presence of 25 %wt of trisodium citrate without addition of thrombin was studied. Activation was performed at pH = 7.8 at 38 and 44 °C. The results are shown in Figure 5. The reaction time is noticeably shorter at the higher temperature.

Influence of salt concentration

A solution of prothrombin and FX (total protein concentration = 30 mg/ml) , which was obtained according to the present invention, was subjected to activation of prothrombin to thrombin at different salt conditions. The effect of the trisodium citrate concentration on the activation in the presence of an initial addition thrombin (300 IU/ml) was studied. Activation was performed with 13, 20 and 26 %wt trisodium citrate at pH = 8 at 25 °C. The results are shown in Figure 6. The yield of thrombin is increased by increasing concentration of trisodium citrate.

Example 4 : Sodium citrate activation of prothrombin The prothrombin in 1561.7 g of a solution of prothrombin and factor X that was obtained according to the present invention was activated to thrombin by the following procedure. 624.7 g of trisodium citrate was added to the solution and pH was adjusted to 8.0 by addition of 22.7 ml of 0.5 M sodium hydroxide. The solution was incubated under continuous stirring for 18 hours at 44 °C.

Comparative example 2: Self activation of prothrombin Starting material for this example was a solution of prothrombin and FX with the following properties : concentrated prothrombin/FX fraction with an absorbance at 280 nm of 200, pH 6.0, buffer 0.05 M sodium citrate, prothrombin concentration 600 IU/ml and factor X concentration 300 IU/ml. The protein solution was tempered in a water bath to 22-23 °C and 60 μl of a solution containing 20 mg/ml sodium hydroxide was added to 1 g of protein solution (pH = 8.0) under continuous stirring. Further 40 μl of a solution containing 2 M calcium chloride was added to 1 ml of protein solution (80 mM CaCl₂) . The solution was stirred at 22-23 °C for 91 h, whereafter precipitated (milky looking solution) calcium citrate was removed using centrifugation (2 h, 3000 r/min) and filtration (pore sizes 5 μm, 1.2 μm and 0.8 μm in series) . The obtained clear solution contained 34,390 IU/ml thrombin (thrombin recovery 24 %) and had a thrombin activity of 734 IU/mg.

Self activation and sodium citrate activation of prothrombin are compared in Table 7. Sodium citrate activation is preferable as it is faster and gives a higher recovery of thrombin.

Table 7

Thrombin Activation recovery (%) time (h)

Sodium citrate activation 44 If (Example 4)

Self activation 24 91

(Comparative example 2)

Analysis methods

Prothrombin antigen analysis (FII:Ag)

Prothrombin presence was detected using an assay based on the antibody "sandwich" principle according to the following.

Rabbit anti-human prothrombin antibodies (A0325, obtained from DAKO) are coated in the wells of a microplate. In a first immunologic reaction, these antibodies bind the prothrombin in the sample. A second antibody, peroxidase conjugated rabbit anti-human prothrombin (P0446, obtained from DAKO), is added, which results in a "sandwich". Subsequent washing steps remove unbound conjugate between prothrombin and the second antibody. Peroxidase activity, which is proportional to the prothrombin content in the sample, is determined by addition of TMB (3, 3' , 5, 5' -tetramethylbenzidine) . The reaction is stopped with 25^"% (4.5 mol/1) sulfuric acid and the absorbance is read at 450 nm against a reagent blank. The prothrombin content of each lot of Standard Human Plasma from Dade Behring, Cat. No. ORKL21, is presented by the manufacturer in the data sheet as percent of normal plasma value.

Factor X antigen analysis (FX:Ag)

Presence of factor X was detected using an assay based on the antibody "sandwich" principle according to the following.

Rabbit anti-human factor X antibodies (A0373, obtained from DAKO) are coated in the wells of a microplate. In a first immunologic reaction, these antibodies bind the factor X in the sample. A second antibody, peroxidase conjugated rabbit anti-human factor X antibody (P0379, obtained from DAKO) , is added, which results in an antibody "sandwich". Subsequent washing steps remove unbound conjugate. Peroxidase activity, which is proportional to the factor X content in the sample, is determined through addition of TMB (3, 3', 5,5'- tetramethylbenzidine) . The reaction is stopped with 25 % (4.5 mol/1) sulfuric acid and the absorbance is read at 450 nm against a reagent blank. The factor X content of each lot of Standard Human Plasma from Dade Behring, Cat. No. 0RKL21, is presented by the manufacturer in the data sheet as percent of normal plasma value.

SDS-PAGE

Proteins are denatured by the anionic detergent sodium dodecyl sulfate (SDS) to yield negatively charged complexes of protein-SDS. The charge density will be about the same for all proteins and separation is obtained mainly according to molecular mass by electrophoresis in polyacrylamide gels (PAGE) in the presence of SDS. The migration distance will be inversely proportional to the logarithm of the molecular mass. SDS- PAGE of proteins by this method is run under either reducing or non-reducing conditions . Reducing conditions (addition of reducing sample buffer followed by heating of the samples) will cleave disulfide bonds of proteins and the subunits can be separated if they differ in size. In non-reducing conditions, the denatured proteins are separated by molecular size..The protein bands separated by electrophoresis are visualized by silver staining. Evaluation is done visually by judging the appearances of standards and samples.

Factor VIII (heavy chain) antigen analysis (FVIII:Ag)

Factor VIII was determined according to the following method. A microtiter plate is coated with a monoclonal mouse antibody specific for antigenic determinants of the heavy chain (90 kDa) of human coagulation factor VIII. After incubation of the plate with samples, a second monoclonal mouse antibody conjugated with alkaline phosphatase is added. This antibody binds to free antigenic determinant of the heavy chain of factor VIII, thus forming a "sandwich". The signal, which is proportional to the amount of factor VIII :Ag in the sample, is generated through an "ELISA amplification system" as follows.

A substrate containing NADPH is added. The alkaline phosphatase of the conjugate antibody dephosphorylates NADPH to NADH. After incubation, an amplifier solution containing diaphorase, alcohol dehydrogenase, ethanol and INT-violet is added. NADH is then oxidized by diaphorase to NAD⁺, while INT-violet is reduced to a coloured dye, formazan. Through alcohol dehydrogenase, NAD⁺ was reduced back to NADH, which then can participate once again in the reaction. Adding 0.3 M H₂S0₄ stops this "signal amplifying" reaction cycle. The amount of coloured end product (formazan) , which amount is proportional to the amount of factor VIII :Ag in the sample, was then deter- mined photometrically at 490 nm against a reagent blank. Factor IX One-Stage Clotting assay (FIX:C)

Factor IX was determined according to a method based on the ability of a factor IX sample to shorten the coagulation time of a factor IX deficient plasma. The factor IX sample is incubated with factor IX deficient plasma and phospholipid/ellagic acid for five minutes at 37 °C. Calcium is then added and the time for the formation of the clot is measured in one step on an Amelung KC4A micro instrument.

Factor VII :C (substrate assay)

The method is based on the two-stage principle, and was performed using microplate technique. In stage one, factor X is activated to factor X_a via the extrinsic pathway. In stage two, the generated factor X_a hydrolyses the chromogenic substrate S 2222, thus liberating the chromophoric group para-nitroaniline . The hydrolysis is stopped with acid and the colour (yellow) is read photometrically at 405 nm against a reagent blank. The generated factor X_a, and thus the intensity of the colour, is proportional to the factor VII activity of the sample. The method measures the functional factor VII activity independently of the degree of activation. The factor VII activity was expressed in international units (IU) as defined by the 1st

International Standard for Factors II, VII, IX and X in Plasma (84/665). Limit of quantification 0.05 IU/ml.

Thrombin concentration Thrombin hydrolyses the chromogenic substrate S 2238 and thereby liberates the chromoforic group para- nitroaniline (pNa) . The hydrolysis is stopped with acid and the yellow color intensity, which is proportional to the thrombin activity of the sample, is read photometrically at 405 n against a reagent blank. The unit used for thrombin concentration is IU/ml. Thrombin recovery

"Thrombin recovery" is a comparison of the amount of thrombin after and before an operation performed on a protein solution. Based on the analyses of thrombin concentration (see above) , the thrombin recovery is calculated as

thrombin recovery = (amount of thrombin after) / (amount of thrombin before) = [ (thrombin concentration after) x (volume of solution after) ] / [ (thrombin concentration before) x (volume of solution before) ] .

Claims

1. A process for the preparation of a purified solution of prothrombin and factor X from a fraction of blood plasma obtainable by cryoprecipitation, which process comprises subjecting such a fraction to prepurification steps and to the following steps:

(a) metal chelate gel chromatography and

(b) affinity gel chromatography.

2. A process according to claim 1, wherein said prepurification comprises the following step:

(c) anion exchange gel chromatography for removal of impurities and/or solvent/detergent chemicals present.

3. A process according to claim 1 or 2, wherein said prepurification comprises the following step:

(d) anion exchange gel chromatography for removal of any clottable proteins, albumin and/or gammaglobulins present .

4. A process according to any one of the preceding claims, wherein step (a) is performed in such a way that prothrombin and factor X pass through the metal chelate gel without binding.

5. A process according to any one of the preceding claims, wherein the metal chelate gel comprises copper ions.

6. A process according to claim 5, wherein the metal chelate gel is equilibrated with NaCl, preferably at a concentration of from 0.1 to 1 M, more preferably about 0.5 M.

7. A process according to any one of claims 5 or 6, wherein the metal chelate gel is equilibrated at a pH of from 6.5 to 7.5, preferably about 7.0.

8. A process according to any one of claims 5-7, wherein the metal chelate gel is equilibrated with imidazole, preferably at a concentration of from 2 to 30 mM, more preferably about 10 mM.

9. A process according any one of the preceding claims, wherein step (b) is performed in such a way that prothrombin and factor X pass through the affinity gel.

10. A process according to any one of the preceding claims, wherein the affinity gel is a heparin gel.

11. A process according to claim 10, wherein the heparin gel is equilibrated with sodium chloride, preferably at a concentration of from 20 to 100 mM, more preferably from 50 to 100 mM, most preferably about 80 mM.

12. A process according to any one of claims 10 or 11, wherein the heparin gel is equilibrated with trisodium citrate, preferably at a concentration of from 25 to 75 mM, more preferably about 50 mM.

13. A process according to any one of claims 10-12, wherein the heparin gel is equilibrated at a pH of from 5.5 to 6.5, preferably about 6.0.

14. A process according to claim 2, wherein step (c) is performed so as to bind prothrombin and factor X to the anion exchange gel.

15. A process according to claim 2 or 14, wherein step (c) is performed on a diethylaminoethyl gel.

16. A process according to claim 15, wherein the diethylaminoethyl gel is equilibrated with a equilibration solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt of from about 10 to about 100 mM, before applying the protein solution.

17. A process according to claim 16, wherein the diethylaminoethyl gel is equilibrated with said equilibration solution at a pH in the range of from about 5.5 to about 6.5.

18. A process according to any one of claims 15-17, wherein the diethylaminoethyl gel is washed with a washing solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt in the range of from about 10 to about 100 mM, after applying the protein solution.

19. A process according to claim 18, wherein the diethylaminoethyl gel is washed with said washing solution at a pH in the range of from about 5.5 to about 7.0.

20. A process according to any one of claims 18-19, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt higher than in the washing solution.

21. A process according to any one of claims 15-20, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt in the range of from about 100 to about 1000 M.

22. A process according to any one of claims 20 or 21, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with said elution solution at a pH in the range of from about 6.5 to about 7.5.

23. A process according to claim 3, wherein step (d) is performed so as to bind prothrombin and factor X to the anion exchange gel.

24. A process according to claim 3 or 23, wherein step (d) is performed on a diethylaminoethyl gel.

25. A process according to claim 24, wherein the diethylaminoethyl gel is equilibrated with a equilibration solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt in the range of from about 1 to about 200 mM, before applying the protein solution.

26. A process according to claim 25, wherein the diethylaminoethyl gel is equilibrated with said equilibration solution at a pH in the range of from about 6.5 to about 7.5.

27. A process according to any one of claims 24-26, wherein the gel is washed with a washing solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt in the range of from about 50 to about 400 mM, after applying the protein solution.

28. A process according to claim 27, wherein the diethylaminoethyl gel is washed with said washing solution at a pH in the range of from about 5.5 to about 6.5.

29. A process according to any one of claims 27-28, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt higher than in the washing solution.

30. A process according to any one of claims 24-29, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with an elution solution comprising one or more salt(s) chosen from the group consisting of chlorides, citrates and phosphates, at a total concentration of salt in the range of from about 0.1 to about 0.5 M.

31. A process according to any one of claims 29 or 30, wherein prothrombin and factor X are eluted from the diethylaminoethyl gel with said elution solution at a pH in the range of from about 5.5 to about 6.5.

32. A process according to any one of the preceding claims, which further comprises subjecting such a fraction to the following step: (e) virus inactivation, preferably by the solvent/detergent method.

33. A process according to any one of the preceding claims, which further comprises subjecting such a fraction to the following step: (f) concentration, preferably by ultrafiltration.

34. A process according to any one of the preceding claims, which further comprises subjecting such a fraction to the following step: (g) desalting.

35. A process according to any one of the preceding claims, wherein step (b) is performed prior to step (a) .

36. A process according to any one of the preceding claims, wherein steps (c) , (d) , and/or (e) , when included, are performed prior to step (a) .

37. A process according to any one of the preceding claims, wherein step (e) , when included, is performed prior to step (c) , when included.

38. A process according to any one of the preceding claims, wherein steps (f) and (g) , when included, are performed simultaneously.

39. A process according to any one of the preceding claims, wherein the steps, when included, are performed in the following order: (d) , (e) , (b) , (c) , (a), (f) , (g) .

40. A solution of prothrombin and factor X, wherein the prothrombin has the ability of being activated to thrombin in the substantial absence of other enzymes or cofactors.

41. A solution according to claim 40, which is essentially free from FVII, FIX, FVIII, calcium and/or phospholipids .

42. A solution according to claim 40 or 41, which has a prothrombin concentration higher than about 600 IU/ml.

43. A solution according to any one of claims 40-42, having a ratio of 1 IU of prothrombin to about 0,5-0,7 IU of factor X, preferably to 0,6 IU of factor X.

44. A solution obtainable by the process according to any one of claims 1-39.

45. Use of a solution according to any one of claims 40-44 for the preparation of a thrombin solution.

46. Use according to claim 45, wherein the preparation is performed by activation of said solution, said activation comprising adding a salt, preferably sodium citrate, in the concentration range of from 5 to 50 %wt, preferably from 20 to 40 %wt, most preferably about 28 %wt, to the solution.

47. Use according to claim 45 or 46, wherein the preparation is performed by activation of said solution at a temperature in the range of from 5 to 50 °C, preferably in the range of from 25 to 50 °C and more preferably about 44 °C.

48. Use according to any one of claims 45-47, wherein the preparation is performed by activation of said solution at a pH in the range of from 7 to 9, preferably about 8.