IE70455B1

IE70455B1 - Pasteurized glue for joining human or animal tissues

Info

Publication number: IE70455B1
Application number: IE276690A
Authority: IE
Inventors: Marion Steinbach; Jacques Chabbat
Original assignee: Fond Nat Transfusion Sanguine
Priority date: 1989-08-01
Filing date: 1990-07-31
Publication date: 1997-02-26
Also published as: IE902766A1; PT94859A; EP0436712A1; FR2650508A1; DE69023470T2; ATE129903T1; DE69023470D1; PT94859B; WO1991001762A1; DK0436712T3; ES2080155T3; FR2650508B1; EP0436712B1

Abstract

The present invention relates to a process for pasteurising a mixture of proteins mainly containing fibrinogen by heating said mixture in conditions ensuring pasteurisation, in which said mixture is pasteurised in the presence of a monosaccharide and an alcoholic sugar.

Description

The present invention relates to a pasteurized glue for joining human or animal tissues.

It has been known for a long time to use substances promoting blood coagulation in order to stop haemorrhages or to heal wounds.

In the first proposals which were made in this field, tampons or pads of fibrin were first used, then sticking of tissues was subsequently carried out. using blood plasma.

This type of glue is, generally speaking, a concentrate of haemostasis factors which is coagulable by thrombin rich in fibrinogen.

It consists of two essential components which are mixed as required at the time of use. The first component consists of a mixture of proteins extracted from human plasma: fibrinogen, fibronectin and factors XIII (or fibrin-stabilizing factors). The second component consists of thrombin/calcium which can be of animal, for example bovine, origin.

In order to avoid all risk of viral contamination during the application of this glue, it is necessary to perform, during its preparation, a step of viral inactivation of the first component based on proteins of plasma origin. Pasteurization, which is a suitable method for this type of operation, causes, in fact, problems on account of the thermal instability of some proteins present in this first component.

EP 103196 describes a process for the pasteurization of solutions of fibrinogen in the presence of high calcium concentrations, which causes the formation of an insoluble precipitate of fibrin.

EP 37078 describes the pasteurization of aqueous solutions of factor XIII not containing fibrinogen in the presence of at least 10% of a salt of a C3-C10 carboxylic acid.

EP 35204 describes a process for the pasteurization of a mixture of plasma proteins in which the fibrinogen content is less than 60% of the total proteins, in the presence of a polyol. - 2 EP 11739 relates to a process for the purification of factor XIII from placenta, followed by pasteurization of the product obtained. * The object of the present invention is to propose a new pasteurized glue, essentially characterized by the fe process for obtaining the first component extracted from human plasma, this process including a viral inactivation step.

More especially, the present invention relates to a process for the pasteurization of a mixture of proteins principally containing fibrinogen, by heating the said mixture under conditions effecting pasteurization, characterized in that: a) a cryoprecipitate of human plasma is dissolved and then precipitated in the presence of heparin, b) the precipitate obtained is redissolved and constitutes the mixture to be pasteurized, c) the said mixture is pasteurized in the presence of a monosaccharide and a sugar alcohol at 60 °C for 10 hours, or under equivalent pasteurization conditions, and in that the said mixture is free from calcium ion, on condition that the fibrinogen content in the mixture is greater than 60%.

Preferably, and in distinction to many processes of the prior art, in particular the process described in EP-B-0,103,196, the pasteurized mixture of proteins contains no calcium ion, and is preferably free from thrombin inhibitor.

In the preferred application of this process, the mixture of proteins also contains plasma factor XIII, and may be obtained from a cryoprecipitate of human plasma dissolved and precipitated in the presence of heparin; * the precipitate obtained, when redissolved, constitutes the mixture to be pasteurized.

The inventors have, in effect, demonstrated that the presence of a monosaccharide and a sugar alcohol during the pasteurization of the protein solution enables the said proteins to be stabilized, and consequently their biological activities to be preserved.

The concentrations of monosaccharide and sugar alcohol used for stabilizing the proteins naturally depend on the nature and concentration of the proteins in the protein solution and on the actual nature of the monosaccharide and of the sugar alcohol used, and are generally between 1,000 and 1,400 g for the monosaccharide and between 200 and 400 g for the sugar alcohol, added per litre of protein solution.

According to a preferred embodiment of the invention, glucose is preferably used as the monosaccharide and sorbitol as the sugar alcohol, at concentrations of 1,200 g for the glucose and of 300 g for the sorbitol, added per litre of protein solution.

According to the invention, the protein solution is pasteurized at a temperature of 60°C for 10 hours, or under equivalent pasteurization conditions, that is to say treatment temperatures and times sufficient to enable all trace of virus present in the product, especially hepatitis virus, to be neutralized. This pasteurization will be performed under conditions akin to physiological conditions in order not to disrupt the proteins. Thus, the pH used will preferably be equal to 8.

The invention also relates to a glue for joining human or animal tissues, of a type containing two components : a) a mixture of proteins extracted from human plasma, principally containing fibrinogen and factor XIII, and b) thrombin/calcium, mixed as required at the time of sticking, characterized in that the mixture of proteins principally containing fibrinogen and factor XIII is pasteurized by the process described above.

The detailed description of an embodiment of the invention, given below without implied limitation, will enable other features of the invention to be demonstrated.

The cryoprecipitate is obtained by known techniques from human plasma thawed at a temperature of 0 to 4°C and then centrifuged. The cryoprecipitate is then redissolved in a pH 7 Tris buffer. The protein concentration is then 15 to 35 g/1.

The mixture of proteins is precipitated from the solution obtained with heparin. 10 to 40 U/ml of heparin j and a temperature of 10 to 35°C will preferably be employed.

The precipitate obtained is then recovered by centrifugation and the supernatant removed.

The precipitate obtained is then solubilized in a pH 8 buffer containing stabilizing agents, especially amino acids such as glycine and arginine, as well as fibrinolysis inhibitors such as e-aminocaproic acid and aprotinin (plasmin inhibitor). The protein concentration of the solution is 40 to 70 g/1 in this case. At this stage, it is possible to envisage a clarifying filtration step in order to remove insoluble matter.

It is hence only at the point of the pasteurization step that the actual pasteurization stabilizers, that is to say the monosaccharide and the sugar alcohol, are introduced. The protein solution containing these stabilizers is then heated to a temperature of 60°C for 10 hours.

In order to remove the stabilizers after the viral inactivation treatment and to concentrate the proteins, common methods of the precipitation, dialysis or filtration type, for example, may be employed.

Thus, the pasteurized solution may be diluted in ( a buffer at a pH lying between 6 and 7 containing 0.015 M NaCl/5 mM trisodium citrate/25 mM e-aminocaproic acid/17 mM arginine. The solution, diluted in a 1:5 to 1:10 (V/V) ratio, is cooled to a temperature of between -1° and -5°. The proteins are precipitated by the addition of alcohol to 10% final, a tenperature below -1°C and above -5°C being maintained, the precipitate is recovered after centrifugation and the supernatant is removed.

The centrifugation pellet is then redissolved with a pH 7.5 buffer containing 0.06 M NaCl/1 mM trisodium citrate/60 mM glycine/20 mM e-aminocaproic acid/20 mM arginine. The solution obtained is dialysed against the same buffer and then concentrated to attain a protein concentration of 30 to 40 g/1.

At this stage, agents promoting solubility of the product after lyophilization, such as polysorbate and/or sodium caprylate, may be added. The solution is then filtered under sterile conditions using filters having a porosity of 0.2 μπι, distributed in bottles and then lyophilized.

The lyophilisate can be taken up with a solution of water for injections which can contain a plasmid inhibitor (for example aprotinin). The solution obtained should contain a minimum concentration of fibrinogen of 70 g/1.

The reconstituted product contains fibrinogen and fibronectin in the following proportions: 65 to 85% and 7 to 14%, respectively, relative to the total protein. It also contains FXIII as well as heparin (50 to 200 U/ml) .

The example given below without implied limitation will enable other features of the present invention to be demonstrated.

EXAMPLE 1: Starting with 280 1 of plasma, cryoprecipitation enabled 2 kg of precipitate (cryoprecipitate) to be recovered. The proteins participating in the composition of the glue are obtained after redissolving this precipitate (1 kg/4 litres) in a 20 mM Tris buffer, pH 7, followed by precipitation at a temperature of 25°C after the addition of 32 U/ml of heparin. The suspension obtained is centrifuged, and the pellet (approximately 1 kg) is recovered and then redissolved in 70 mM trisodium citrate/0.1 M glycine/0.03 M NaCl/50 mM e-aminocaproic acid/50 mM arginine/100 U/ml aprotinin, pH 8 (1 kg/1 litre) . Heating of the liguid for 10 hours at 60°C is then performed after protein-stabilizers, namely 2,400 g of glucose and 600 g of sorbitol, have been added to 2 litres of solution for a final volume of approximately 4 litres (taking into account the dilution due to the addition of the sugars) . After pasteurization, the solution is diluted 10-fold in 5 mM citrate/0.051 M NaCl/25 nN e-aminocaproic acid/17 mM arginine, pH 7. The solution obtained is then precipitated with alcohol at % final, at a temperature of between -2 and -3°C. The > precipitated proteins are recovered in the centrifugation pellet. The latter is then redissolved in 1 mM trisodium citrate/60 mM NaCl/20 mM e-aminocaproic acid/ 60 mM glycine, pH 7.5.

The proteins are then concentrated to a content of 27-30 g/1 for a final volume of 1.5 1. The product is then distributed in bottles after sterilizing filtration, and thereafter lyophilized. The concentration ofcoagulable proteins may be adjusted according to the volume of reconstitution of the lyophilisate. For a protein content of 110 g/1, the product obtained has 77 g/1 of coagulable fibrinogen, 12 g/1 of fibronectin and an aprotinin content of 2,000 KlU/ml (solvent used for reconstitution of the lyophilisate).

Claims

1. Process for the pasteurization of a mixture of proteins principally containing fibrinogen, by heating the said mixture under conditions effecting pasteurization, characterized in that a) a cryoprecipitate of human plasma is dissolved and then precipitated in the presence of heparin, b) the precipitate obtained is redissolved and constitutes the mixture to be pasteurized, c) the said mixture is pasteurized in the presence of a monosaccharide and a sugar alcohol at 60°C for 10 hours, or under equivalent pasteurization conditions, and in that the said mixture is free from calcium ion, on condition that the fibrinogen content in the mixture is greater than 60%.

2. Process according to Claim 1, characterized in that the mixture is free from thrombin inhibitor.

3. Process according to Claim 1, characterized in that the mixture contains plasma factor XIII.

4. Process according to one of Claims 1 to 3, characterized in that the pasteurization is performed in the presence of glucose and sorbitol.

5. Process according to one of Claims 1 to 4, characterized in that the quantity of monosaccharide used is between 1,000 and 1,400 g to be added per litre of solution, and the quantity of sugar alcohol between 200 and 400 g to be added per litre of solution.

6. Process according to Claim 5, characterized in that the quantities of glucose and sorbitol to be added per litre of solution are 1,200 g/1 and 300 g/1, respectively.

7. Process according to one of Claims 1 to 6, characterized in that the precipitation in the presence of heparin is performed at a temperature of 10 to 35°C.

8. Process according to one of Claims 1 to 7, characterized in that the pasteurization step is performed at protein concentrations of 40 to 70 g/1.

9. Process according to one of . Claims 1 to 8, characterized in that the precipitate before pasteurization contains other stabilizing agents and fibrinolysis inhibitors.

10. Glue for joining human or animal tissues, of the type containing two components: 5 a) a mixture of proteins extracted from human plasma, principally containing fibrinogen and factor XIII, and b) thrombin/calcium, mixed as required at the time of sticking, characterized 10 in that the mixture of proteins principally containing fibrinogen and factor XIII is pasteurized by the process according to one of Claims 1 to 9.

11. A process according to Claim 1 for the pasteurization of a mixture of proteins, substantially as herein15 before described and exemplified.

12. A pasteurized mixture of proteins whenever obtained by a process claimed in a preceding claim.

13. A glue, according.to Claim 10, substantially as hereinbefore described.