WO2004050700A1

WO2004050700A1 - Process for preparating concentrated thrombin solutions and use in fibrin glue

Info

Publication number: WO2004050700A1
Application number: PCT/SE2003/001548
Authority: WO
Inventors: Stefan Winge; Peter Aizawa; Göran Karlsson
Original assignee: Biovitrum AB
Current assignee: Swedish Orphan Biovitrum AB
Priority date: 2002-12-02
Filing date: 2003-10-06
Publication date: 2004-06-17
Anticipated expiration: 2005-06-02
Also published as: AU2003267906A1; SE0203552D0

Abstract

One aspect of the invention relates to a process for the preparation of a concentrated thrombin solution from an initial thrombin solution obtainable by activation of a prothrombin solution, which process comprises subjecting such an initial thrombin solution to the following consecutive steps: (a) heat treatment and (b) hydrophobic interaction chromatography on an aliphatic adsorbent. Another aspect of the invention relates to a thrombin solution having a thrombin concentration higher than about 100,000 IU/ml. A further aspect of the invention relates to the use of a thrombin solution in a component for a fibrin glue.

Description

Process to-? preparing concentrated thro-i-bin solutions and use in fibrin glue.

Technical field of the Invention

The present invention relates to a process for the preparation of a concentrated thrombin solution from an initial thrombin solution obtainable by activation of a prothrombin solution, to a thrombin solution and to the use of such a thrombin 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 (factor II_a, FII_a) • Prothrombin is present in native plasma, and the activation is catalysed by factor X_a (FX_a, activated Stuart factor) . Thrombin, a proteolytic enzyme, catalyses the actual clotting reaction (conversion of fibrinogen into fibrin) . Thrombin cleaves four peptide bonds in the central region of fibrinogen to release four fibrinopeptides . A fibrin molecule devoid of these fibrinopeptides is called fibrin monomer. Fibrin monomers spontaneously assemble into the ordered fibrous arrays called 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 provide necessary cofactors at optimal concentrations and in an optimal environment. Further, in commercial plasma fractionation some cofactors are purified separately and are thus 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.

US 2001/0033837 Al describes the production of a thrombin preparation which is obtained from prothrombin which is, after activation to thrombin, purified by hydrophobic interaction chromatography (HIC) . The adsorbent employed in the HIC is a gel with coupled phenyl radicals or other ligands with a similar hydrophobicity . Before or after the HIC it is also possible in addition to carry out cation exchange chromatography. The chromatographies in this case may be carried out as "positive" (binding of thrombin) or as "negative" (binding of the impurities) chromatography. According to this document, the thrombin solution obtained after chromatographic purification can then be subjected to virus inactivation or virus reduction such as filtration. Virus inactivation or reduction can also take place before the chromatographic purification. A thrombin preparation which contains as stabiliser a noncovalently binding inhibitor, and to which further stabilisers can be added for stabilisation, is also described.

The abovementioned known production process is very synoptic and thus has a great potential for improvements, It is partly focused on the HIC and its combination with cation exchange chromatography. This combination is laborious and does not result in a satisfactory thrombin concentration. Additionally, the hydrophobicity of the phenyl radicals used as adsorbent during the HIC is too high to give an optimal result. The abovementioned process is further focused on stabilisation of the final thrombin preparation. It does not, however, provide for the stabilisation necessary in connection with heat treatment methods for virus inactivation. Such methods are essential when preparing a high quality thrombin solutio .

Hydrophobic affinity chromatography on p-chloro- benzylamido-agarose has been used to charachterise various modified forms of human thrombin (Lundblad, R.L. (1993) Arch . Biochem . Biophys . 302, 109-112) . Elution of native thrombin was performed with either acetonitrile or 1,4-dioxane. Such eluents are harmful to humans and should be avoided in preparative chromatography for the production of thrombin for e.g. medical use.

Summary of the invention

An object of the present invention is to provide an improved process for the preparation of a concentrated thrombin solution. A second object of the invention is to provide a process for the preparation of a concentrated thrombin solution which utilises synergy effects between its steps.

Another object of the present invention is to provide a concentrated thrombin solution. Another object of the invention is to provide a thrombin solution which is essentially free from denatured and degraded thrombin and from other contaminating proteins. Still another object of the invention is to provide a thrombin solution which is substantially pathogen free.

A further object of the present invention is to enable the production of an improved fibrin glue.

These and other objects are obtained by the invention as claimed. Thus, in one of its aspects the invention provides a process for the preparation of a concentrated thrombin solution from an initial thrombin solution obtainable by activation of a prothrombin solution, which process comprises subjecting such an initial thrombin solution to the following consecutive steps:

(a) heat treatment and (b) hydrophobic interaction chromatography on an aliphatic adsorbent.

In other aspects, the invention provides a thrombin solution having a thrombin concentration higher than about 100,000 IU/ l and the use of a thrombin solution in a component for a fibrin glue.

A pathogen inactivating method is included in the process to ensure a high virus safety of the biological material. Heat treatment is a well known and efficient such method. However, stabilisers must normally be added to a protein solution to protect the proteins from heat denaturation during heat treatment. Although stabilised, the thrombin solution may contain some heat denatured proteins after being subjected to the heat treatment. Possibly contaminating proteins and inactive thrombin molecules are present in the solution as well. Said stabilisers and protein impurities are removed during the hydrophobic interaction chromatography step.

The use of an aliphatic adsorbent facilitates suitably weak bindings to the target protein to allow desorption of said protein in its native state. It is highly desirable to keep the thrombin in its native state throughout the process.

The combination of the heat treatment step and the subsequent hydrophobic interaction chromatography step offers additional advantages related to synergy effects: The heat treatment step involves the use of salts as stabilisers. Thus, the invention provides subsequent purifying by hydrophobic interaction chromatography, which is a process able to bind thrombin also at high salt levels. A corresponding binding of thrombin at high salt levels can not be achieved by e.g. ion exchange chromatography on a gel with immobilised diethylaminoethyl (DEAE) groups. A thrombin solution having a thrombin concentration higher than about 100,000 IU/ml allows the use of the solution in different applications without causing dilution of other components. This feature is especially advantageous when the thrombin solution is used as a component in a fibrin glue . In addition to the mentioned heat treatment and hydrophobic interaction chromatography steps of the process of the present invention, optional steps can be present. Such optional steps improve the performance of the process by their specific actions as well as by introducing interaction advantages among steps. The following description serves to further clarify the functions and features of each step, whether essential or optional, 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 initial thrombin solution by activation of prothrombin to thrombin. The thrombin solution according to the invention and its use are finally described.

Activation of prothrombin to thrombin

The activation process of prothrombin to thrombin is, in vivo, a very complex system including several cofactors and inhibitors. It is essential to provide necessary cofactors as not all factors involved in the enzymatic cascade are present in plasma. Examples of such factors important for in vivo activation, not present in plasma, are calcium, phospholipids and tissue factor. Further, in commercial plasma fractionation some cofactors are purified separately and not available in the fraction used for preparation of thrombin. One way of obtaining the initial thrombin solution involves an activation process, which is optimised for in vi tro use. Thus, inhibitors are removed and the only cofactor that is needed to convert prothrombin to thrombin is factor X. The initial thrombin solution of the present invention is obtainable by activation of a prothrombin solution. In particular, it is obtainable by activation of a prothrombin solution that is essentially free from FVII, FIX, FVIII, calcium and/or phospholipids.

The initial thrombin solution is obtainable by activation of a prothrombin 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 around 28 %wt, to the prothrombin solution.

The initial thrombin solution is obtainable by activation of a prothrombin 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 around 44 °C.

The initial thrombin solution is obtainable by activation of a prothrombin solution at a pH in the range of from 7 to 9, preferably around 8.

Heat treatment

Pathogen inactivating methods are included among the steps of the process of the present invention to ensure a high virus safety of the biological material. Pasteurisation is a well-known virus inactivation method useful for protein solutions. It is performed at a temperature higher than 50 °C for a period of more than 1 hour, preferably at 60 °C for 10 h. The heat treatment is performed at a pH in the range of from 6.5 to 7.5, preferably about 7.0. However, stabilisers must normally be added to the protein solution to protect the proteins from heat denaturation during heat treatment. The choice of suitable stabilisers for a thrombin solution is discussed in Example 2 below. In the heat treatment step of the present process a preferred stabiliser is sucrose, preferably at a concentration of from 25 to 75 %wt, more preferably around 50 %wt . Other preferred stabilisers are the amino acids alanine (preferably at around 0.25 %wt) , arginine (preferably at around 1.5 %wt) , glycine (preferably at around 0.5 %wt) and lysine (preferably at around 1.3 %wt) . A further preferred stabiliser is NaCl, preferably at around 0.4 %wt . The said stabilisers may be present solely or in any combination, also in combination with other stabilising substances. Most preferred is a combination of all said stabilisers at their respective preferred concentrations.

The number of viable viruses is preferably reduced at least 10⁴ times by the described heat treatment.

Hydrophobic interaction chromatography

Although stabilised, the thrombin solution may contain some heat denatured proteins after being subjected to the heat treatment. Possibly, contaminating proteins and inactive thrombin molecules are present in the solution as well. The amount of said stabilisers and protein impurities is reduced during the hydrophobic interaction chromatography step.

In general, to a hydrophobic interaction chromatography (HIC) medium, proteins are adsorbed in a buffer of high ionic strength and desorbed in a buffer of low ionic strength. The performance of a HIC step is, however, due to a combination of the biochemical characteristics of the protein sample to be processed and different parameters, e.g. hydrophobicity (carbon chain length) of the immobilised ligand, type of chromatography matrix, temperature, pH, concentration of salt, type of salt according to the Hoffmeister series etc.

The use of a short chain aliphatic adsorbent is desirable. Preferably, the adsorbent is a C₄-C₈ alkyl gel. The use of a hydrophobic ligand with a short carbon chain is advantageous. A gel having phenyl or long carbon chain (more than about 8 carbon atoms) ligands may cause too strong bindings to the target enzyme (thrombin) and thus make it difficult to desorb in a native state. More preferably, the adsorbent is a butyl gel. The hydrophobic interaction chromatography is preferably performed so as to bind thrombin to the gel. Impurities are bound to the gel together with thrombin and are preferably washed away with a first sodium citrate solution before elution of thrombin. The first sodium citrate solution preferably has a concentration in the interval of from 0.2 to 1.0 M, more preferably from 0.3 to 0.6 M. Optionally more than one washing may take place. It is then desirable to perform the first washing at a concentration in the upper part of said range and further washings at lower concentrations.

After washing of the gel, thrombin is eluted from the gel by means of lowering the ionic strength. The eluent is water or a second sodium citrate solution of a lower concentration than the first sodium citrate solution, preferably lower than 0.2 M, more preferably about 0.17 M.

Temperature and pH are important process parameters during the hydrophobic interaction chromatography. It is suitably performed at a pH in the interval of from 6.5 to 7.5, preferably about 7.0. The hydrophobic interaction chromatography is suitably performed at a temperature in the interval of from 20 to 30 °C, preferably about 25 °C.

Virus filtration In addition to the abovementioned heat treatment step for virus inactivation, there are legal requirements for further virus reduction of biological material administered to humans. The combination of different virus inactivation and reduction steps improves the biological safety of the product obtainable by the process of the present invention. A suitable method for further virus reduction of the thrombin solution is virus filtration. The thrombin solution is preferably filtered through a filter having a cut-off of about 15-20 nm, corresponding to a molecular weight of about 70,000. Two or more filters in series may optionally be used.

The performance of the virus filtration step (d) is strongly improved in the event that it is performed after the hydrophobic interaction chromatography step (c) . The purity of the thrombin solution resulting from the hydrophobic interaction chromatography allows a faster and more efficient filtering process. Additionally, the virus filtration step is preferably performed before the stabilisation step (see below) , if such a step is present. The virus filter may be clogged at the high protein concentrations reached during stabilisation.

The number of viable viruses is preferably reduced at least 10⁴ times by the described virus filtration.

Formulation The thrombin solution is optionally subjected to a further process step for formulation. The formulation is preferably performed by concentration and desalting of the solution. Concentration and desalting can be performed simultaneously by ultrafiltration of the solution against a buffer solution. Stabilisers are then transferred from the buffer solution to the thrombin solution. The choice of suitable stabilisers for long term storage is discussed in Preparatory example 4 below. Preferably the buffer solution comprises sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt . The buffer solution preferably has a pH of about 7.0. The formulation is performed so as to achieve a thrombin solution of a concentration higher than about 100,000 IU/ml. It is also performed so as to achieve a thrombin solution comprising sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt .

The obtained thrombin solution can be further stabilised by freezing, preferably at a temperature lower than -70 °C. It will then be stable for at least one year. Further stabilisation can also be achieved by freeze-drying.

The purity of the thrombin solution achieved after the disclosed purification method is sufficiently high for in vivo purposes. However, for e.g. standard or stability purposes it may be advantageous to reach an even higher purity. This can be achieved by adding an anion-exchange gel to the thrombin solution, stirring for a suitable period of time (about 1 hour) , and removing the gel by filtration or centrifugation. It is also possible to process the thrombin solution through a column with such a gel or through a positively charged filter. This additional procedure is preferably performed at a stage where the pH and ionic strength of the thrombin solution are at their physiological values, suitably after formulation of the thrombin solution but before final concentration of the same. The purity will typically be > 95 % after such a procedure.

Thrombin solution

The thrombin solution of the present invention is characterised in that it has a thrombin content higher than about 100,000 IU/ml. Such a high thrombin concentration is desirable in several applications as it decreases the dilution of other components being present in the application. Such a high concentration may also facilitate stable long term storage. The thrombin solution of the present invention preferably has a purity higher than about 1000 IU/mg. High purity is a prerequisite for safe medical and surgical applications as well as for stable storage conditions . The thrombin solution of the invention is stable for more than 12 months frozen.

Use of the thrombin solution

Fibrin glues are applied to e.g. tissue wounds and consist substantially of a thrombin component and a fibrinogen component. The components are mixed upon use and an adhesive, fibrin, is formed. In several applications a highly viscous glue is desired to prevent the glue from draining off its point of application. The thrombin solution according to the present invention is thus particularly useful for use in a component for a fibrin glue, as its high concentration prevents unnecessary dilution of the glue. The thrombin component is preferably a composition further comprising hyaluronic acid. Hyaluronic acid is a non-toxic substance enhancing the viscosity of the glue. Hyaluronic acid is known to have positive effects on wound healing. Such a composition may comprise from 50 to 5000, preferably around 500, IU thrombin/ml of hyaluronic acid. The composition of the inventive thrombin solution and hyaluronic acid is stable for at least 2 months at 3 °C. The thrombin solution according to the invention can also be used as a glue component or to prevent blood loss on its own, using fibrinogen from a sieving wound. One such application is to apply the thrombin solution to a plaster.

Brief description of the drawings

Figures 1-3 show the effects of pH and temperature on the activation of prothrombin according to results from Preparatory example 1. Figure 4 shows the effect of temperature on the activation of prothrombin according to results from Preparatory example 1.

Figure 5 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 preparatory examples explain the basis for the process of the invention. Thus, a procedure for obtaining the initial thrombin solution is illustrated as well as experimentation aimed at revealing suitable parameter settings for the procedures of the invention. The comparative examples illustrate known procedures being less advantageous alternatives to the present invention. Finally, the analysis methods used are explained in detail.

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) 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 1-3. 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 4. 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) 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 5. The yield of thrombin is increased by increasing concentration of trisodium citrate.

Preparatory example 2: Sodium citrate activation of prothrombin

The prothrombin in 1561.7 g of a solution of prothrombin and factor X 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 1: 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 1. Sodium citrate activation is preferable as it is faster and gives a higher recovery of thrombin.

Table 1

Thrombin Activation recovery (%) time (h)

Sodium citrate activation 44 18

(Preparatory example 2)

Self activation 24 91

(Comparative example 1) Preparatory example 3: Stabilisation during heat treatment

To a thrombin solution containing 711 IU thrombin/ml having a purity of 1582 IU/mg, different stabilisers according to Table 2 were added. The solution was then heat treated at 60 °C for 10 h. Subsequently the samples were analysed regarding biological activity of thrombin and the recovery was calculated. As can be seen in Table 2, thrombin can be successfully stabilised with different substances during heat treatment.

Table 2

Stabiliser Thrombin recovery* (%)

1.5 M ammonium sulphate, pH 6.5 67

2 M glycine, 50 %wt sucrose, pH 6.8 112

2 M glycine, 50 %wt sucrose, 0.1 M EDTA, pH 6.8 100 0.5 M sodium citrate, 50 %wt sucrose, pH 6.5 112 Potassium acetate, sucrose, pH 6.5 108

1 M sodium citrate, 20 %wt sucrose, pH 7.5 110

* Thrombin recovery > 100 % may be due to activation of prothrombin residues present in the thrombin solution or to measurement inaccuracy.

Example 1 : Heat treatment and hydrophobic interaction chromatography

Heat treatment

A sample of the initial thrombin solution (16.8 kg) was diluted to 2.5 times its original volume with a 10 mM disodium phosphate solution of pH = 6.8. Stabilisers were added in the following amounts: 0.24 %wt alanin, 1.52 %wt arginine, 0.54 %wt glycine, 0.42 %wt sodium chloride,

1.31 %wt lysine and 50.4 %wt sucrose. The solution was heat treated for 10 h at 60 °C. The thrombin recovery after heat treatment was 90 %.

Hydrophobic interaction chromatography The heat treated thrombin solution was purified using a 14 litre butyl Sepharose FF (Amersham Biosciences) hydrophobic interaction chromatography column, under the following conditions:

The heat treated thrombin solution was diluted (1 part of protein solution and 3 parts of aqueous buffer) to reach a salt content of 155 mg-of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid) . The gel (equilibrated with the same salt solution) was loaded with 12 mg protein/ml gel at a flow of 2 column volumes/h. The column was thereafter washed with 2 column volumes of equilibration buffer and 5 column volumes of an aqueous wash buffer which included 108 mg of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid).

The fraction which did not bind to the column and the wash fractions were discarded. The bound thrombin fraction was eluted with an aqueous buffer containing 50 mg of trisodium citrate and 3.6 mg of disodium phosphate per gram of solution (pH was adjusted to 7.0 using citric acid) . The fractions were analysed according to thrombin recovery, thrombin activity and protein recovery. As can be seen in Table 3, a specific activity of more than 1500 IU/mg and a thrombin recovery of about 50 % is obtained with this purification method. Table 3

Sample Thrombin Thrombin activity Protein recovery (%) (IU/mg) • recovery (%)

Before 100 662 100 Wash 30 338 58 Eluate 47 1624 19

Comparative example 2: Alternatives to hydrophobic interaction chromatography

Two alternative purification methods, polyethylene glycol (PEG) precipitation and anion exchange chromatography on a gel with immobilised diethylaminoethyl (DEAE) groups, were compared to hydrophobic interaction chromatography.

Polyethylene glycol precipitation

A thrombin solution containing 7785 IU thrombin/ml and 18,1 mg protein/ml, and having a purity of 430 IU/mg was used as initial material. Portions of the initial material were adjusted to different pH (6.0, 6.5, 7.0, 7.5 and 8.0) and polyethylene glycol (PEG-4000) was added to different concentrations (10, 15 and 20 %wt) . The resulting solutions were stirred for 1 h at 25 °C and thereafter filtered through a 0.8 μm filter. The clear filtrates were analysed regarding protein content and thrombin activity. As can be seen in Table 4, the maximum activity achieved was slightly above 600 IU/mg, with no decrease in thrombin recovery. Table 4

PEG concentration pH Thrombin Thrombin

(%wt) recovery (%) activity (IU/mg)

Initial material 7.0 100 430

10 6.0 96 606

15 6.0 54 482

20 6.0 20 273

10 6.5 107 643

15 6.5 71 511

20 6.5 32 360

10 7.0 111 612

15 7.0 79 541

20 7.0 44 508

10 7.5 100 508

15 7.5 90 581

20 7.5 49 430

10 8.0 100 545 15 8.0 84 576 20 8.0 47 432

Anion exchange chromatography on a DEAE gel 9658 g of a solution comprising 2749 IU thrombin/ml and 6,4 mg protein/ml, and having a purity of 429 IU/ml, in a buffer solution of 0.02 M sodium phosphate at pH 7.5, was purified using a 32 litre DEAE Sepharose FF (Amersham Biotech) chromatography column, under the following conditions: The thrombin solution was diluted (1 part protein solution and 9 parts of 0.02 M sodium phosphate, pH 7.5) and applied to the gel (equilibrated with 0.02 M sodium phosphate, pH 7.5), 2 mg protein/ml gel was loaded to the column with a flow of 5 column volumes/h. The column was thereafter washed with 2 column volumes of equilibration buffer and the thrombin fraction was eluted with 5 column volumes of a solution of 0.02 M sodium phosphate and 0.05 M sodium citrate, pH 7.5. The fraction was analysed according to thrombin recovery, thrombin activity and protein recovery. As can be seen in Table 5, a thrombin activity of more than 1100 IU/mg and a recovery of 100 %, is obtained with this purification method.

Table 5

Sample Thrombin Thrombin activity Protein recovery (%) (IU/mg) recovery (%)

Before 100 429 100

Eluate 100 1125 38

As can be seen from Tables 3, 4 and 5, the hydrophobic interaction chromatography gives the best purification, compared to PEG precipitation and anion exchange chromatography on a DEAE gel .

Example 2 : Virus filtration

To the thrombin solution of Example 1 (eluate, 21 kg, 1.5 mg/ml) sodium chloride was added to a concentration of 1 M. The solution was then nano-filtered through one DV20 (1 ², Pall) filter using dead-end filtration technique (2 bar pressure) . Thereafter the filter was washed with 5 kg of buffer (0.02 mol/1 sodium phosphate, 1 mol/1 sodium citrate pH 7.0). The filtrate (27 kg) was analysed regarding thrombin recovery, thrombin activity and protein recovery, see Table 6. Table 6

Sample Thrombin Thrombin activity Protein recovery (%) (IU/mg) recovery (%)

Before 100 1100 100 filtration Filtrate 95 1100 95

Preparatory example 4 : Stability study of purified thrombin

To ensure a stable purified thrombin drug, suitable stabilisers must be added to the final product. To samples of a protein solution comprising 252 IU thrombin/ml and 1.36 mg protein/ml, having a purity of 540 IU thrombin/mg protein, in a buffer solution of 0.05 M trisodium citrate at pH 7.0, different candidates of stabilisers, according to Tables 8 and 9, were added. The samples were then incubated at 37 °C, to accelerate the denaturation of the protein, (Table 7) or 2-8 °C (Table 8) and subsequently analysed for biological activity of thrombin after different periods of time. Finally the thrombin recovery was calculated. As can be seen in Tables 7-8, some of the stabilisers have better effects than others.

In Tables 7 and 8 : Lys = lysine Arg = arginine Gly = glycine Asp = aspartate Trp = tryptophan Hya = hyaluronic acid EACA = ε-amino caproic acid I = 0.15 M Lys, Arg, Gly, pH = 7.0 K = I + 5 % saccharose, pH = 7.0 Table 7 ( incubation at 37 °C )

Stabiliser pH Thro b.in recovery (%)

1 2 4 8 12 week weeks weeks weeks weeks

0.15 M Lys 7.0 75 55 59 24 22

0.15 M Lys 6.5 74 52 55 26 25

0.30 M Lys 7.0 84 64 69 33 35

0.60 M Lys 7.0 84 65 71 34 36

0.15 M Arg 7.0 85 62 67 32 37

0.15 M Arg 7.5 71 49 42 18 16

0.15 M Arg 6.5 85 66 72 33 40

0.30 M Arg 7.0 80 53 44 28 22

0.15 M Lys, Arg, 7.0 88 70 60 42 35

Gly

0.15 M Lys, Arg + 7.0 86 63 56 36 31

0.5 M ^"( 3iy

I + ! 5 ^■ ' . saccharose 7.0 84 72 72 45 38

I + ( D.I D7 % Hya 74 63 52 41 28

I + ( D.: L4 % Hya 79 69 52 33 29

0.15 M Arg(L) + 7 73 65 64 36 43

0.15 M Arg(D)

K + ( D.l D7 % Hya 84 68 52 40 43

0.15 M Arg + Gly 7 70 57 44 27 27

0.15 M EACA 7 70 56 40 24 22

0.15 M Arg + 7 68 57 39 26 28

0.30 M Gly

0.15 M Asp 7 38 18 7 2 25

0.15 M Trp 7 2 1 0 0 0 Table 8 (incubation at 2-8 °C)

Stabiliser pH Thrombin recovery* (%)

2 weeks 4 weeks

0.15 M Lys 7.0 91 130

0.15 M Lys 6.5 81 119

0.30 M Lys 7.0 88 116

0.60 M Lys 7.0 83 115

0.15 M Arg 7.0 94 136

0.15 M Arg 7.5 85 122

0.15 M Arg 6.5 95 125

0.30 M Arg 7.0 85 93

0.15 M Lys, Arg, Gly 7.0 90 99

0.15 M Lys, Arg + 0.5 M Gly 7.0 80 81

I + 5 ^■ ' . saccharose 7.0 97 102

I + 0.1 D7 % Hya 85 93

0.15 M Arg(L) + 0.15 M Arg(D) 7 87 86

K + 0.1 D7 % Hya 94 82

0.15 M Arg, Gly 7 82 70

0.15 M EACA 7 87 75

0.15 M Arg + 0.30 M Gly 7 80 72

0.15 M Asp 7 87 75

0.15 M Trp 7 76 65

Example 3: Formulation

27.4 kg of the protein solution from Example 2, containing 2057 IU thrombin/ml and 1.2 mg protein/ml, having a purity of 1714 IU thrombin/mg protein, arid being buffered with 1 M sodium chloride and 0.01 M sodium phosphate at pH 7.0, was concentrated and diafiltered (ultrafiltration system: 5 x 0.5 m² Biomax-5, Millipore) against 10 volumes of diafiltration buffer (5 %wt sucrose, 0.8 %wt sodium chloride, 2.9 %wt arginine, 2.5 %wt lysine and 1.0 %wt glycine, pH 7.0). The solution was further concentrated to the minimum process volume of the system (5 kg) . The thrombin concentrate, which contained about 3000 IU/ml thrombin, was further concentrated to a thrombin concentration higher than 150,000 IU/ml, about 150 g, and then further concentrated on a smaller ultrafiltration system (3 x 0.1 m² Biomax-5, Millipore) . The properties of the resulting concentrate are shown in Table 9. A ratio of thrombin "clot" concentration to thrombin concentration (substrate) of about 1 indicates that the thrombin molecules are present in their active form.

Table 9

Sample Thrombin "clot' Thrombin Ratio of thrombin concentration activity "clot" cone, to

(IU/ml) (IU/mg) thrombin cone, (substrate)

Concentrate 264,000 1800 1.1

Human blood plasma comprises 1 IU prothrombin/ml. In vivo conversion of 1 IU prothrombin theoretically results in 238 IU thrombin. The purification method used to obtain the concentrate presented in Table 9, has a recovery of 45 IU thrombin from 1 ml of human blood plasma. Analysis methods

Thrombin ^,clot" concentration

The analysis of thrombin "clot" concentration is performed in compliance with the requirements of the

European Pharmacopoeia. Thrombin standard and samples are diluted with buffer and added to a standard amount of fibrinogen. Since the concentration of fibrinogen is high and constant, the rate of clot formation is primarily determined by the thrombin concentration in the sample. The clotting time is inversely proportional to the thrombin concentration. The thrombin "clot" concentration analysis only detects active thrombin. The unit used for thrombin "clot" concentration is IU/ml.

Thrombin concentration (substrate)

Factor Ila 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 factor Ila activity of the sample, is read photometrically at 405 nm against a reagent blank. The thrombin concentration (substrate) analysis detects total thrombin (active and denatured forms) . The unit used for thrombin concentration is IU/ml.

Ratio of thrombin "clot" concentration to thrombin concentration (substrate)

The ratio of thrombin "clot" concentration to thrombin concentration (substrate) reflects the amount of active thrombin compared to the total amount. A ratio < 1 indicates that denatured thrombin molecules are present.

Thrombin recovery "Thrombin recovery" is a comparison of the total amount of proteins after and before an operation performed on a protein solution. Based on the analyses of thrombin concentration (substrate) (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) ] .

Protein concentration

Total protein concentration was measured as absorbance in a 1 cm cuvette at 280 nm against a diluent in a spectrophotometer from Amersham Pharmacia Biotech, model Ultraspec II, alternatively Ultraspec 2000. If necessary, the samples were diluted with saline to obtain an absorbance between 0.1 and 1.0. An absorptivity of 1.0 ml cm^-1 mg^-1 was assumed for all samples. The unit used for protein concentration is thus mg/ml .

Protein recovery

"Protein recovery" is a comparision of the total amount of proteins after and before an operation performed on a protein solution. Based on the analysis of protein concentration (see above), the protein recovery is calculated as

protein recovery = (amount of protein after) / (amount of protein before) = [ (protein concentration after) x

(volume of solution after) ] / [ (protein concentration before) x (volume of solution before) ] .

Thrombin activity

Based on the analyses of thrombin concentration and protein concentration (see above) , the thrombin activity, or thrombin purity, is calculated as thrombin activity = (thrombin concentration) / (protein concentration) .

Virus inactivation Purification processes for pharmaceutical products derived from human or animal sources must contain virus reduction steps to ensure a safe product. This is stated by regulative authorities and specific procedures for the validation of such reduction steps are recommended. Model viruses (e.g. HIV, parvoviruses, hepatitis viruses) are added to the product before the inactivation step and the content of them is analysed after the inactivation. Thus, a reduction factor is determined for each model virus and each inactivation step.

Claims

1. A process for the preparation of a concentrated thrombin solution from an initial thrombin solution obtainable by activation of a prothrombin solution, which process comprises subjecting such an initial thrombin solution to the following consecutive steps:

(a) heat treatment and

(b) hydrophobic interaction chromatography on an aliphatic adsorbent.

2. A process according to claim 1, wherein the adsorbent is a C₄-Ce alkyl gel, preferably a butyl gel.

3. A process according to claim 1 or 2, wherein the hydrophobic interaction chromatography is performed so as to bind thrombin to the gel.

4. A process according to any one of the preceding claims, wherein the adsorbent is washed with a first sodium citrate solution before elution of thrombin.

5. A process according to claim 4, wherein the first sodium citrate solution has a concentration in the interval of from 0.2 to 1.0 M, preferably from 0.3 to 0.6 M.

6. A process according to any one of the preceding claims, wherein thrombin is eluted from the adsorbent with a second sodium citrate solution.

7. A process according to claim 6, wherein the second sodium citrate solution has a lower concentration than the first sodium citrate solution.

8. A process according to claim 6 or 7, wherein the second sodium citrate solution has a concentration lower than 0.2 M, preferably about 0.17 M.

9. A process according to any one of the preceding claims, wherein the hydrophobic interaction chromatography is performed at a pH in the interval of from 6.5 to 7.5, preferably about 7.0.

10. A process according to any one of the preceding claims, wherein the hydrophobic interaction chromatography is performed at a temperature in the interval of from 20 to 30 °C, preferably about 25 °C.

11. A process according to any one of the preceding claims, wherein the heat treatment is performed at a temperature of from 50 to 70 °C for a period of from 5 to 15 hours, preferably at 60 °C for 10 h.

12. A process according to any one of the preceding claims, wherein the heat treatment is performed at a pH in the range of from 6.5 to 7.5, preferably about 7.0.

13. A process according to any one of the preceding claims, wherein the initial thrombin solution during the heat treatment is stabilised by sucrose, preferably at a concentration of from 25 to 75 %wt, more preferably around 50 %wt .

14. A process according to any one of the preceding claims, wherein the initial thrombin solution during the heat treatment is stabilised by alanine, preferably at around 0.25 %wt; arginine, preferably at around 1.5 %wt; glycine, preferably at around 0.5 %wt; and/or lysine, preferably at around 1.3 %w .

15. A process according to any one of the preceding claims, wherein the initial thrombin solution during the heat treatment is stabilised by NaCl, preferably at around 0,4 %wt .

16. A process according to any one of the preceding claims, wherein the heat treatment is performed so as to achieve a virus reduction of at least 10⁴ times.

17. A process according to any one of the preceding claims, which further comprises subjecting such an initial thrombin solution to the following step:

(c) virus filtration.

18. A process according to claim 17, wherein the virus filtration is performed so as to achieve a virus reduction of at least 10⁴ times.

19. A process according to any one of the preceding claims, which further comprises subjecting such an initial thrombin solution to the following step:

(d) formulation.

20. A process according to claim 19, wherein the formulation is performed by ultrafiltration of the solution against a buffer solution.

21. A process according to claim 20, wherein the buffer solution comprises sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt .

22. A process according to claim 20 or 21, wherein the buffer solution has a pH of about 7.0.

23. A process according to any one of claims 19-22, performed so as to achieve a thrombin solution with a concentration higher than about 100,000 IU/ml.

24. A process according to any one of claims 19-23, performed so as to achieve a thrombin solution comprising at least one of arginine, glycine, lysine, sucrose, and sodium chloride.

25. A process according to any one of claims 19-24, performed so as to achieve a thrombin solution comprising sucrose, preferably from 2 to 10 %wt; NaCl, preferably from 0.5 to 1.5 % wt; arginine, preferably from 2 to 4 %wt; lysine, preferably from 1 to 4 %wt; and/or glycine, preferably from 0.5 to 1.5 %wt .

26. A process according to any one of the preceding claims, wherein said initial thrombin solution is obtainable by activation of a prothrombin solution that is essentially free from FVII, FIX, FVIII, calcium and/or phospholipids .

27. A process according to any one of the preceding claims, wherein said initial thrombin solution is obtainable by activation of a prothrombin 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 around 28 %wt, to the prothrombin solution.

28. A process according to any one of the preceding claims, wherein said initial thrombin solution is obtainable by activation of a prothrombin 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 around 44 °C.

29. A process according to any one of the preceding clams, wherein said initial thrombin solution is obtainable by activation of a prothrombin solution at a pH in the range from 7 to 9, preferably around 8.

30. A process according to any one of the preceding claims, wherein step (c) , when included, is performed after step (b) .

31. A process according to any one of the preceding claims, wherein step (c) , when included, is performed before step (d) , when included.

32. A thrombin solution having a thrombin concentration higher than about 100,000 IU/ml.

33. A thrombin solution according to claim 32, having a purity higher than about 1000 IU/mg protein.

34. A thrombin solution, obtainable by the process according to anyone of claims 1-31.

35. Use of a thrombin solution according to anyone of claims 32-34 in a component for a fibrin glue.

36. Use according to claim 35, whereby the component is a composition further comprising hyaluronic acid.

37. Use according to claim 36, whereby the composition comprises from 50 to 5000, preferably around 500, IU thrombin/ml of hyaluronic acid.