A method for producing a preparation based on fibrinogen and fibronectin as well as protein compositions obtainable according to this method

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DESCRIPTION

Method for producing a preparation based on fibrinogen and fibronectin, as well as protein compositions that can be obtained according to this method.

The invention relates to a method for producing protein compositions containing fibronectin and fibrinogen, and optionally other ingredients, it also relates to the protein compositions that can be obtained according to this method.

Fibrinogen-based tissue adhesives ("fibrin adhesives") have been known for a long time. They are used to join human or animal tissues or parts of organs, to close wounds, in hemostasis and to aid wound healing, either without seams or reinforced with sutures.

Its mode of action is based on an imitation of the final phase of blood clotting.

Due to the action of thrombin, the (soluble) fibrinogen is initially converted into fibrin monomers that spontaneously aggregate and form a sticky mass, the so-called fibrin clot. Simultaneously, factor XIII (F XIII) present is activated by thrombin in the presence of calcium ions to become factor XIIIa. Thanks to the latter, the added fibrin monomers and also the possibly present fibronectin cross-link to form a heavy polymer due to the formation of new peptide bonds. Due to this crosslinking reaction, the strength of the clot formed increases substantially. The clot generally adheres well to the wound and tissue surfaces, which implies an adhesive and hemostatic effect.

Therefore, fibrin adhesives are often used as two-component adhesives that comprise the fibrinogen component together with a thrombin solution, which additionally contains calcium ions.

A special advantage of a fibrin adhesive is that it does not remain in its application site as a foreign body, but is completely resorbed in the same way as during natural wound healing and is replaced by newly formed tissue. Various cells, for example macrophages and later fibroblasts, migrate into the clot, lyse and reabsorb the clot material and form new tissue.

Although the complicated wound healing procedures so far are by no means completely clear, there is no doubt that the presence of fibronectin in the clot is of crucial importance for cell growth and therefore healing.

A fibrin clot of optimal composition should therefore also contain fibronectin in addition to its main component, fibrinogen.

Although the mode of action of fibrin adhesives is basically equivalent to the natural blood coagulation process, for it to be sufficiently effective (adhesive force, hemostatic effect) a substantially higher concentration of active components (in particular fibrinogen) of the present in the blood, the fibrinogen concentration in human blood reaches about 2.5-3 mg / ml.

It is known that by precipitation of PEG a fibrinogen solution can be obtained with which a satisfactory adhesive force could be achieved already for fibrinogen concentrations below 30 mg / ml in an artificial test system (WO 92/13495); even so, a high density in the fibrin network (which is basically achieved by high concentrations of fibrinogen in the fibrin adhesive) cannot be guaranteed for many of the purposes of tissue adhesion.

Therefore, to ensure optimal efficacy, the fibrinogen content in the fibrin adhesive should be at least 70 mg / ml. However, the production of such a ready-to-use concentrated fibrinogen or fibrin adhesive solutions involves some difficulties.

Because ready-to-use solutions do not remain stable in storage for long periods of time, they must be prepared on demand by reconstitution from lyophilized preparations or by thawing frozen liquid solutions.

Due to the relatively low solubility of fibrinogen and the concurrent high concentration of fibrinogen required in an effective fibrin adhesive, in general this remains more cumbersome and heavy than desired by users despite various proposals for improvement. It is understandable that, in particular, in the field of emergency surgery, a particularly quick and easy availability of a fibrin adhesive is required.

Furthermore, concentrated fibrin adhesive solutions are generally very viscous due to high concentrations of fibrinogen. However, it is advisable that the viscosity is relatively low, not only to improve its handling, but also for certain modes of application of the fibrin adhesive, for example when applied by spray devices (such as Duploject^® with associated spray equipment) or via a catheter.

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Both requirements, that is, the rapid availability and low viscosity of concentrated fibrin adhesive solutions, are more difficult to meet if their preparation (dissolution or thawing, respectively) has to be carried out without further auxiliary means, such as heating or stirring equipment, at room temperature, and whether the fibrin adhesive preparations also contain substances of high molecular weight, in particular fibronectin. For fibronectin too, especially in combination with fibrinogen, is relatively difficult to dissolve and generally leads to decreased solubility and increased viscosity of fibrin adhesives.

Methods for producing fibrinogen-containing preparations that can be used as tissue adhesives include their production from cryoprecipitates, optionally with a subsequent washing step, and precipitation steps with ethanol, ammonium sulfate, polyethylene glycol, glycine o / l-alanine and , their production from plasma within the scope of known plasma fractionation methods, respectively (fc, for example: "Methods of plasma protein fractionation", 1980 ed .: Curling Academic Press, pages 3-15, 33-36, and 57-74, or Blomback B and M., "Purification of human and bovine fibrinogen", Arkiv Kemi 10, 1959, page 415 ff.).

Some suggestions for reducing the viscosity of highly concentrated fibrinogen solutions are already found in the prior art. Thus, for example, both the addition of solubilizers such as substances containing urea or guanidine residues, for example: arginine (fc DE 3203775-A1) or the addition of high concentrations of non-physiological salts are known. However, such tissue adhesives have been shown to have cytotoxic and proliferation inhibitory properties, respectively (Redl et al., Med. Welt 36, 1985, pages 769-776)

In EP 0 804 933 the addition of substances that improve the solubility of fibrinogen is suggested. Said substances are, for example, vitamins, aromatic compounds such as those derived from benzene or phenol, or those derived from heterocyclic compounds such as piperidine, pyridine or pyrimidine. Document WO 95/23167 A describes two components (salt and 6-aminohexanoic acid) that are added to a protein concentrate obtained from frozen total plasma where 6-aminohexanoic acid is used to cancel the affinity of plasminogen towards fibrinogen and not as a precipitating agent.

In EP 0373044 A a plasma concentrate is described which is obtained by precipitating plasma with ethanol.

EP 0059265 describes a composition for closing and healing wounds comprising a collagen vehicle, fibrinogen, factor XIII and thrombin. The preparation of this composition includes the formation of an emulsion by mixing the components with ethanol and subsequent evaporation of the latter.

Therefore, an object of the present invention is to eliminate the drawbacks of the known preparations and further improve them, as well as to provide protein compositions having a high fibrinogen content and an easily adjustable fibrinogen to fibronectin ratio; and optionally other ingredients, for example suitable for obtaining an improved ready-to-use tissue adhesive, while maintaining in particular properties such as good cell compatibility or the formation of a physiological fibrin structure after mixing with a thrombin solution . At the same time, the viscosity properties of such protein compositions or pharmaceutical preparations must also be improved.

Another object of the present invention is to provide a simplified and faster method of producing said protein compositions. In particular so that it is also easily possible to carry out on an industrial scale.

According to the invention, these objectives are achieved by a method for the production of protein compositions comprising fibrinogen and fibronectin, which is characterized in that an initial solution containing fibrinogen and fibronectin is treated with a precipitation composition comprising a polyol, in particular polyalkylene glycol or an alcohol of up to four carbon atoms, especially ethanol, or an inorganic salt, especially ammonium sulfate or alkali metal sulfate, and an amino acid, in particular glycine or alanine, so that in a single precipitation step a precipitate is formed containing fibrinogen and fibronectin. This method is characterized in that with the precipitation of the composition through a single precipitation step, an efficient preparation is obtained that preserves the proteins and with a high yield.

After the single precipitation step, the obtained precipitate can be further processed by methods known per se, preferably into a pharmaceutical preparation, in particular a tissue adhesive (fibrin adhesive).

By a solubility modifying component, it is meant a substance that has a precipitating effect or an effect that improves the solubility of at least one of the proteins, fibrinogen or fibronectin, under other given conditions such as temperature, pH, ionic strength, etc. .

Surprisingly, according to the invention it has been found that using two solubility modifying components that are different (one from the other) in cooperation allows a recovery of fibrinogen and fibronectin in the precipitate in large quantities, the precipitation step being the only one of the invention surprisingly less damaging to proteins than individual precipitation of the respective substances.

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Furthermore, it has also been shown that a precipitated composition according to the invention and comprising fibrinogen and fibronectin has many greatly improved properties with a view to its practical application, in particular as regards its viscosity and reconstitution, thus making it possible an easier application of these preparations.

As raw materials to prepare the initial solution, in principle all those that have been used up to now or those that are possible, respectively, of the prior art can be used to prepare said protein compositions.

As raw material for preparing the starting solution or for using directly as starting solution, preferably plasma, in particular human plasma, or a plasma fraction containing fibrinogen and fibronectin, preferably a fraction derived from a cryoprecipitate, respectively, is used.

However, according to the invention it is also possible to use other starting materials or starting solutions containing fibrinogen and fibronectin, for example cell culture supernatants.

The solubility modifying components are chosen such that in the single precipitation step a precipitate is formed which contains fibrinogen and fibronectin in a predetermined and desired ratio.

According to the invention, the components contained in the precipitating composition are chosen such that, under certain conditions, they differ from each other in terms of their solubility-modifying effect; that is, improvement of solubility or precipitation, over fibrinogen and fibronectin, respectively. For example, a component may have a precipitating effect on basically only one or two of the proteins; that is, on fibrinogen or on fibronectin.

However, it is also possible that one of the components has a precipitating effect on both proteins, while the other component has a precipitating effect on only one of the two proteins or that it even increases the solubility of the other protein, that is, that it does not act as a precipitating agent but as a solubilizer.

In particular, the component that modifies the solubility is a substance selected from the group of alcohols, in particular an alcohol with up to four carbon atoms, an inorganic salt, an organic salt, a polyol, in particular polyalkylene glycol, a polyester and an amino acid. . Esters, ketones, and cyclic, heterocyclic, or polycyclic organic compounds can also be used.

As solubility modifying components, in particular ethanol, polyethylene glycol, ammonium or alkaline salts such as ammonium sulfate or alkali sulfate, or preferably the amino acids glycine and (d-) amine are used.

Among the organic polymers, linear polymers, in particular those having an average molecular weight of approximately 200 to 20,000, for example polyalkylene glycols, stand out in particular. Polyethylene glycol has been found to be especially suitable. These water-soluble, non-toxic synthetic polymers, particularly those having a molecular weight between 400 to 10,000, preferably about 4,000, are most preferably used in precipitation due to their preservative effect on fibrinogen and fibronectin. In addition to the particularly preserving and mild effect of the components to be used in the precipitation composition, their safety as regards their use in humans must be taken into account when selecting them.

As a pair of components that can be used according to the invention are, for example, polyethylene glycol (PEG), in particular PEG 4000, which has a precipitating action on both fibrinogen and fibronectin, and as a second component glycine, which in the presence of another component, for example polyethylene glycol, surprisingly not only precipitates fibrinogen preferentially compared to fibronectin, but even acts as a solubilizer for fibronectin. Glycine in a concentration range greater than 0.6 M, a concentration at which only glycine is currently known as a protein precipitating agent, has a particularly good effect on increasing the solubility of fibronectin.

Naturally, the present invention is not limited to the combinations PEG and glycine, since the average skilled person will be able to easily find also other components or pairs of components suitable according to the invention by means of simple systematic tests.

To carry out the method of the invention, the skilled person can find suitable components or pairs of components according to the following test scheme.

Under specific conditions such as temperature, pH, ionic strength, etc., and under stirring, aliquots of the initial solution, prepared by the usual methods, are added to precipitation compositions containing a first solubility modifying component, for example a amino acid, and a second different component of them, for example PEG 4000, ethanol or ammonium sulfate. A series of precipitations is carried out in which the concentration of one of the components always remains constant and that of the other varies. The precipitates formed are centrifuged and the total protein content as well as the relative fibrinogen content (Fbg) is determined.

ES 2 263 451 T3 fibronectin (Fn) and albumin (Alb); for example by SDS PAGE (SDS polyacrylamide gel electrophoresis) of the reduced and non-reduced samples, staining with Coomassie blue and performing a densiometric evaluation.

Those components that lead to a protein composition containing the desired fibrinogen and fibronectin are then chosen for the precipitation composition according to the invention. The optimization of the respective component system will be carried out by means of easy to check parameters such as yield, relative content of fibrinogen and fibronectin and viscosity of the preparation obtained at a predetermined fibrinogen content (for example: 70 or 100 mg of fibrinogen per ml).

In a preferred embodiment, the precipitation composition comprises polyalkylene glycol, which is added to the initial solution, in particular at a final concentration of 1 to 12% w / v, preferably 4 to 8% w / v.

In another preferred embodiment, the precipitation composition comprises one or more amino acids that are added to the initial solution, in particular at a final concentration of 0.5 to 3 M, preferably 0.75 to 2 M, in particular 0, 75 to 1.5 M.

An organic or inorganic salt, for example ammonium sulfate, can be included in a concentration of up to 20% w / v, preferably 2 to 20% w / v, in particular between 5 and 15% w / v.

The precipitation composition is preferably added in liquid form.

Other raw material ingredients can also be precipitated by the precipitation composition, for example and in particular factor XIII. The ability of the solubility modifying composition to coprecipitate such substances can also influence the final choice of the individual components of the precipitation composition.

According to a preferred embodiment, an attempt is made to keep plasminogen either in the precipitated protein composition or in the pharmaceutical preparation, quite low. Therefore, a precipitation composition is preferably used in which plasminogen remains for the most part in the supernatant, which means that in the precipitate obtained the plasminogen is reduced with respect to fibrinogen and / or fibronectin compared to the material. cousin.

According to another preferred embodiment, other ingredients can be added to the protein composition, in particular fibrinolysis inhibitors, factor XIII, antibiotics, growth factors, analgesics, anti-inflammatory agents or mixtures thereof. Said additions can be made before, during and after the precipitation step and are preferably made after said step.

During the subsequent processes, in particular in the final formulation, the protein composition according to the invention or the pharmaceutical preparation according to the invention is preferably completely frozen or lyophilized, especially if greater stability is to be achieved during storage. The ready-to-use pharmaceutical preparation according to the present invention is preferably provided in liquid form.

In particular, the method according to the invention has the advantage that well-defined protein compositions can be obtained directly in a simple way, whereas the methods used to date for protein fractionation, especially plasma fractionation, have always tried to obtain proteins. individual as pure as possible, and optionally mixing them again afterwards, to obtain the desired mixture.

Due to the simplicity and brevity of the method according to the invention, the risks of microbial contamination and pyrogen formation during production are also minimized. Furthermore, the sensitive proteins in plasma, for example fibrinogen and fibronectin, are preserved and to a large extent protected from possible denaturation, and this is the reason why the preparations according to the invention have better properties.

Surprisingly, it has been shown that protein compositions or pharmaceutical preparations that can be obtained by a single precipitation step of the combination of the invention, at equally high concentrations of fibrinogen, have approximately a 25% lower viscosity, preferably 30%, especially 40% lower than comparable preparations, for example fibrin adhesives obtained by traditional production methods, in particular by precipitation with a single agent.

By comparable preparations are meant preparations having similar contents of fibrinogen and fibronectin, and optionally more ingredients, and a comparable degree of purity, which show similar good cellular compatibility and which form clots of physiological fibrin structure after mixing with a solution of thrombin.

In an especially preferred embodiment, the protein composition and the ready-to-use pharmaceutical preparation do not contain substantially any other substances that improve the solubility of fibrinogen and neither are said substances added to the preparation.

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Surprisingly, it has been shown that the liquid compositions according to the invention have a markedly lower viscosity compared to known comparable preparations, as a result of which their handling and the use of fibrin adhesives are particularly facilitated.

In particular at room temperature, the lyophilized compositions according to the invention can be reconstituted more easily and quickly than other comparable preparations.

Particularly, when the method of the invention is carried out on a large scale, which is yet another advantage of the present invention, it can be applied on a large scale efficiently and cost-effectively, it is preferable to add the precipitation composition in liquid form. This can be a solution or a suspension.

It has also been shown that it is convenient for the precipitate obtained after the single precipitation step to be washed at least once with a wash buffer. In addition to a suitable buffer system, the wash buffer preferably contains tranexamic acid, lysine, ε-aminocaproic acid, detergents or mixtures of these substances. It may also contain fibrinolysis inhibitors or protease inhibitors, of plasma, animal or plant origin, or genetically or synthetically engineered inhibitors, respectively. These substances can be added separately or can already be in the raw material.

A preferred protease inhibitor is, for example, aprotinin, in particular genetically engineered aprotinin. Another preferred fibrinolysis inhibitor is tranexamic acid (trans-4- (aminomethyl) cyclohexanoic acid, t-AMCHA).

The protein precipitate obtained can be used as such or it can be further processed into a protein composition or a pharmaceutical preparation by methods known per se. Such processing methods include, for example, various purification steps such as treatment (washing) with a cold wash buffer, or formulation as a pharmaceutical preparation, respectively.

These compositions or final preparations of the invention are especially suitable for tissue adhesion, hemostasis and to aid or accelerate wound healing.

The plasma which is preferably used as raw material in the present invention is preferably preselected, for example according to the method of WO 96/35437, whereby microbial contamination is almost completely excluded. Preferably only raw material is used in which the absence of pathogens, in particular viruses and / or prions, is found.

In another preferred embodiment, at least one step is provided to inactivate or weaken possible pathogens present.

To inactivate the pathogens preferably a surfactant and / or thermal treatment is carried out (dry or steam treatment); for example a solid state heat treatment, in particular a steam treatment according to EP-0 159 311 or EP 519 901 or EP 674 531.

Other treatments for inactivating pathogens also comprise treatment with chemical or chemical / physical methods, for example with chaotropic substances according to WO 94/13329, DE 4434538 or EP0131740 (solvent), or by photoinactivation.

Weakening by filtration, in particular ultrafiltration, preferably in the presence of virus binding agents, for example Aerosil, is also a preferred method of weakening viruses within the scope of the present invention.

According to the invention, inactivation by heat treatment, solvent treatment, detergent treatment or combinations (simultaneous or consecutive) of these treatments, as well as optionally filtration, are particularly preferred.

The inactivation can be carried out before or after filtration. Preferably, two independent inactivations are carried out, for example one treatment before the single precipitation step and a second treatment after it.

In another preferred embodiment, the protein composition is characterized in that it contains factor XIII. Factor XIII is preferably added and its content is, for example, at least 80 units per gram of fibrinogen, preferably at least 100 U / g of fibrinogen. If there are also substances that inhibit the fibrin crosslinking reaction, for example possible antibiotics present in the preparation, the factor XIII content is preferably increased to at least 500 U / g of fibrinogen. Preferably, factor XIII is added as a purified, inactivated virus product and separately (a factor XIII preparation produced according to EP 0 637 451 is especially preferred).

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In another preferred embodiment, the protein composition is characterized by a low plasminogen content. The plasminogen content is preferably at most 1.6 mg / g of fibrinogen, especially less than 0.8 mg / g of fibrinogen and in particular less than 0.3 mg / g of fibrinogen.

Advantageously, the protein composition according to the invention comprises fibronectin and fibrinogen in a proportion of between 0.02 to 0.5, preferably from 0.02 to 0.25, also preferably from 0.02 to 0.2, especially 0 .04 to 0.16, in particular about 0.1 (0.05 to 0.15); that is, unlike the very pure fibrinogen preparations (WO 94/20524), it still contains considerable amounts of fibrinogen.

The protein composition may further comprise active ingredients such as antibiotics, growth factors, analgesics, or combinations thereof.

In a preferred embodiment, the protein composition comprising fibrinogen and fibronectin in a fibronectin / fibrinogen ratio of 0.02 to 0.5 according to the invention is characterized in that

a) as a solution it contains at least 70 mg of fibrinogen / ml or it can be reconstituted or liquefied in said solution,

b) at 20 ° C it has a viscosity of at most 350 cSt, preferably at an osmolarity of less than 500 mOsm, especially less than 400 mOsm,

c) after mixing with a thrombin-CaCl solution₂ forms an opaque clot with a physiological fibrin structure and

d) does not contain an addition of a solubility enhancing substance exhibiting a group containing benzene, pyridine, piperidine, pyrimidine, morpholine, pyrrole, imidazole, pyrazole, furan, thiazole or purine.

In another preferred embodiment, the protein composition according to the invention comprises fibrinogen and fibronectin in a fibronectin / fibrinogen ratio of 0.02 to 0.5, characterized in that

a) as a solution it contains at least 70 mg of fibrinogen / ml or it can be reconstituted or liquefied in said solution,

b) at 20 ° C it has a viscosity of at most 150 cSt, preferably at an osmolarity of less than 500 mOsm, especially less than 400 mOsm,

c) after mixing with a thrombin-CaCl2 solution, it forms an opaque clot with a physiological fibrin structure and

d) contains one or more solubilizing substances with a group containing benzene, pyridine, piperidine, pyrimidine, morpholine, pyrrole, imidazole, pyrazole, furan, thiazole or purine in a maximum total concentration of 150 mM.

The protein compositions or pharmaceutical preparations of the invention are versatile in terms of their use. In particular, the preparations according to the invention are used for tissue adhesion, hemostasis and / or wound healing.

In another preferred embodiment, the protein compositions or pharmaceutical preparations of the invention can also be used to produce a fibrin-based biomatrix. For this, the protein composition or the pharmaceutical preparation of the invention is added with a suitable enzyme to convert fibrinogen into fibrin, preferably thrombin, and the fibrin formed is used either in its fresh state or after lyophilization and rewetting as a material. carrier for growing cells or as a so-called biomatrix (see for example WO 99/15209).

The biomatrix of the invention can be present in various forms, for example in the form of a sponge, in sheets, microbeads or as flakes.

The fibrin-based biomatrix of the invention is especially suitable for cell growth, in particular of human cells. As examples of cells capable of being cultured or growing by means of this fibrin matrix, keratinocytes, fibroblasts, chondrocytes are mentioned. Said biomatrix is ​​also suitable for healing wounds or for replacing tissue, in particular as a substitute for the skin. Another embodiment of the present invention is a pharmaceutical preparation comprising a protein composition according to the present invention.

The invention is explained in more detail below by means of the following examples and figures, to which, however, it is not restricted.

Fig. 1: shows the dependence of the fibronectin / fibrinogen ratio (Fn / Fbg) in the precipitate on the PEG concentration in the precipitation mixture (glycine concentration: 1 M).

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Fig. 2: shows the dependence of the fibronectin / fibrinogen ratio (Fn / Fbg) in the precipitate on the glycine concentration in the precipitation mixture (PEG concentration: 6.5% w / v).

Fig. 3: shows the dependence of the fibronectin / fibrinogen ratio (Fn / Fbg) in the precipitate on the concentration of //-alanine in the precipitation mixture (ethanol concentration: 2% w / v).

Fig. 4: shows the dependence of the fibronectin / fibrinogen ratio (Fn / Fbg) in the precipitate on the ammonium sulfate concentration in the precipitation mixture (^ -alanine concentration: 1 M).

Fig. 5: shows the dependence of the fibronectin / fibrinogen ratio (Fn / Fbg) in the precipitate on the jd-alamna concentration in the precipitation mixture (ammonium sulfate concentration: 5% w / v).

Examples

Example 1

Cryoprecipitated human plasma prepared according to methods known per se was dissolved with a 4-fold quantity of a buffer solution containing 20 mM sodium citrate, 120 mM sodium chloride, 5 mM tranexamic acid, as well as 1,200 IU of heparin / l. The pH was adjusted to 7.3 and filtered until clarification.

Aliquots of this solution were added to the solution containing glycine and polyethylene glycol 4000 (PEG 4000), stirring at room temperature, so that in each case a final concentration of 1 M of glycine and final concentrations of PEG 4000 ranging between 1 and 10% (w / v).

The precipitates formed were centrifuged and the relative content of fibrinogen (Fbg), fibronectin (Fn) and albumin (Alb) was determined by SDS PAGE (SDS polyacrylamide gel electrophoresis) of the reduced and non-reduced samples, staining with Coomassie blue and performing a densiometric evaluation (see also EP 0 345 246, Examples 1-37).

The results are summarized in Table 1; the fibronectin / fibrinogen ratio versus PEG concentration is also plotted in Fig. 1.

TABLE 1

Dependence of the protein composition of the precipitate on the content of PEG in the precipitation mixture (glycine concentration 1 M)

<td>% (w / v) PEG</td><td>% Fibrinogen Protein</td><td>% Protein Fibronectin</td><td>% Albumin Protein</td><td>Fibronectin / Fibrinogen</td>

<td> 1</td><td> 84</td><td> 3</td><td> 5</td><td> 0,04</td>

<td> 2</td><td> 83</td><td> 4</td><td> 5</td><td> 0,05</td>

<td> 3</td><td> 82</td><td> 5</td><td> 4</td><td> 0,06</td>

<td> 4</td><td> 81</td><td> 7</td><td> 4</td><td> 0,09</td>

<td> 5</td><td> 80</td><td> 8</td><td> 4</td><td> 0,10</td>

<td> 6</td><td> 78</td><td> 9</td><td> 3</td><td> 0,12</td>

<td> 7</td><td> 77</td><td> 10</td><td> 3</td><td> 0,13</td>

<td> 8</td><td> 76</td><td> 11</td><td> 3</td><td> 0,15</td>

<td> 9</td><td> 75</td><td> 13</td><td> 2</td><td> 0,17</td>

<td> 10</td><td> 74</td><td> 14</td><td> 2</td><td> 0,19</td>

The example shows how the protein precipitates according to the invention can be obtained from a mixture of plasma proteins by a single precipitation with a mixture of two components (here, glycine and PEG), the protein precipitates of which contain variable amounts of fibrinogen in addition to the main component,

ES 2 263 451 T3 fibrinogen, where the fibronectin / fibrinogen ratio can be adjusted as desired within certain limits (for example by choosing the agents according to Example 1 in a range of 0.02-0.2), choosing appropriately the concentrations of agents and the ratio of one to the other, respectively. Such protein precipitates are suitable, for example, for the production of fibrinogen-based tissue adhesives (fibrin adhesives).

Example 2

Cryoprecipitated human plasma was dissolved in a manner similar to Example 1, the pH was adjusted to 7.3 and filtered until clarification. Aliquots of this solution were added in a similar way to Example 1 with solutions containing glycine and PEG 4000, so that in each case a final concentration of 6.5% (w / v) of PEG was obtained, and final concentrations of glycine ranging from 0.2 to 1 M. The precipitates formed were centrifuged analogously to Example 1 and analyzed.

In all variants, protein production was 95%. The fibronectin / fibrinogen ratio with respect to the glycine concentration is summarized in Table 2 and further graphically represented in Fig. 2.

TABLE 2

Dependence of the protein composition of the precipitate on the glycine content in the precipitation mixture (PEG concentration 6.5% w / v)

<td>Glycine mol / l</td><td>Fibronectin / Fibrinogen</td>

<td> 0,2</td><td> 0,16</td>

<td> 0,4</td><td> 0,16</td>

<td> 0,6</td><td> 0,16</td>

<td> 0,8</td><td> 0,14</td>

<td> 1,0</td><td> 0,11</td>

Complementing Example 1, this example shows that per se known protein precipitating agents, such as glycine, in no way act solely as precipitating agents when used in combination with PEG, but rather, under certain conditions, glycine, it can act for example as a fibronectin solubilizer, This circumstance occurs at a concentration in which up to now only glycine was known as a protein precipitation agent.

The following Examples 3-5 present comparable preparation compositions obtained according to the prior art.

Example 3

A plasma cryoprecipitate was dissolved as in Example 1. While stirring at room temperature, glycine was added to a final concentration of 2 mol / L. The protein precipitate formed was centrifuged and analyzed as in Example 1. The fibrinogen had almost completely precipitated. The relative content of fibrinogen was 86% of the total amount of proteins, the relative content of fibronectin was 1.5%. The fibronectin / fibrinogen ratio was therefore 0.017.

Example 4

A plasma cryoprecipitate was dissolved as in Example 1. While stirring at room temperature, PEG 4000 was added to a final concentration of 10% (w / v). The protein precipitate formed was centrifuged and analyzed as in Example 1. The fibrinogen had almost completely precipitated. The relative content of fibrinogen was 72% of the total amount of proteins, the relative content of fibronectin was 16%. The fibronectin / fibrinogen ratio was therefore 0.22.

Example 5

A plasma cryoprecipitate was dissolved as in Example 1. While stirring at room temperature, PEG 4000 was added to a final concentration of 10% (w / v). The protein precipitate formed was centrifuged, redissolved and glycine was added to the solution in a final concentration of 2 M, while stirring at temperature.

ES 2 263 451 T3 environment. The protein precipitate formed was centrifuged again and analyzed as in Example 1. The relative content of fibrinogen was 93% of the total amount of proteins, the relative content of fibronectin was 1.5%. The fibronectin / fibrinogen ratio was therefore 0.016.

Examples 3-5 show that with the known methods of the prior art it is not possible to arrive, in one step or even in several consecutive steps, at protein mixtures having a determined fibronectin / fibrinogen ratio, as when, for For example, a combination of glycine and PEG is used in the concentrations used in Example 1 or 2, to arrive at a ratio ranging from 0.02 to 0.2.

Example 6

Production of a lyophilized preparation according to the invention, with two independent virus inactivation steps

Cryoprecipitated human plasma prepared according to methods known per se was dissolved with a 4-fold amount of a buffer solution (LP1) containing 20 mM sodium citrate, 120 mM sodium chloride, 5 mM tranexamic acid (t-AMCHA), as well as 1,200 Heparin IU / l and filtered until clarification. Subsequently, the so-called solvent / detergent treatment (SD) was carried out to inactivate the possible enveloped viruses present. To do this, a mixture of Triton X-100, Tween 80 (Polysorbate-80K) and tri-n-butylphosphate (TNBP) was added to give final concentrations of 1%, 0.3% and 0.3% (v / v ) respectively. After stirring for 1 hour at room temperature, it was filtered again until clarity. The solution with the same volume of a solution containing 2M glycine and 13% (w / v) PEG 4000 (precipitation composition) was added with stirring at room temperature for 30 minutes and centrifuged. The sediment was ground and redissolved in LP1 containing 1% Tween 80, and the precipitation was repeated in the same way. To remove SD and precipitation reagents, the pellet was ground and cold-treated (0-2 ° C) 2 times with a 10-fold amount of a buffer solution containing Na₃10 mM citrate and 5 mM t-AMCHA in the presence of small amounts of Tween 80. The washed pellet was then dissolved in a buffer solution containing Na₃-citrate 10 mM and t-AMCHA 5 mM. After adjusting the protein concentration to 40 g / l, the whole solution was lyophilized. To effect a further virus inactivation, the lyophilized material was adjusted to a residual humidity of 7-8% and heated for 10 hours at 60 ° C plus 1 hour at 80 ° C under oxygen exclusion. The lyophilisate thus treated was dissolved in a 20 mM niacinamide solution with a protein concentration of 38 g / l, mixed with 6 g of pasteurized human albumin / l, and the pH was adjusted to 7.3.

The solution was sterilized by filtration, placed in final 5.0 ml containers (small glass bottles) under sterile conditions, and lyophilized.

The final product thus obtained constituted a ready-to-use solution when dissolved with 2.0 ml of water for injection (WFI) or with 2.0 ml of a 50 mM t-AMCHA solution, with a fibrinogen content of more than 70 mg / ml. This solution, for example, can be used as an adhesive for fabrics.

In principle, instead of using pure water, the freeze-dried end product can also be dissolved in aqueous solutions containing additional active substances, for example fibrinolysis inhibitors, coagulation factor XIII, antibiotics, growth factors, analgesics, etc.

The product obtained according to the method described above corresponded in its protein content and composition basically to the preparation of adhesive for fabrics described in EP 804933 A2 (Example 2). The fibronectin / fibrinogen ratio was about 0.09, the plasminogen content about 0.15 mg / g of fibrinogen. After mixing the ready-to-use tissue adhesive solution with an equal volume of a thrombin-CaCl solution₂, viscoelastic opaque physiological clots were formed.

Despite the broad agreement with the product described in document EP 804933, the tissue adhesive according to the invention is characterized by having a marked lower viscosity, with an identical content of a substance that improves the solubility of fibrinogen (50 mM niacinamide).

This is at least surprising, since the preparation according to the invention had not been subjected to one, but two independent virus inactivation steps; and furthermore, the steam treatment had been carried out under worse conditions (10 hours, 60 ° C plus 1 hour at 80 ° C). From experience, such heat treatment leads to poorer solubility and higher viscosity.

Thus, surprisingly, it has been found that t-AMCHA, in addition to its known antifibrinolytic effect, reduces the viscosity of fibrinogen-containing solutions. In particular, t-AMCHA further reduces the viscosity of the fabric adhesive solutions obtained according to the invention (see Table 3).

ES 2 263 451 T3

TABLE 3

Viscosity (cSt) in the presence of 50 mM niacinamide

<td>Temperature (° C)</td><td>Tissue adhesive according to the invention</td><td>Tissue adhesive according to the invention + t-AMCHA 50 mM</td><td>Tissue adhesive according to EP 804933A2 (Example 2)</td>

<td> 20</td><td> 99</td><td> 78</td><td> 277</td>

<td> 25</td><td> 76</td><td> 63</td><td> 132</td>

<td> 30</td><td> 56</td><td> 52</td><td> 81</td>

<td> 37</td><td> 39</td><td> 34</td><td> 53</td>

Example 7

Production of a fully frozen tissue adhesive according to the invention with two independent virus inactivation steps

Until filter sterilization, the production was carried out in a similar manner to Example 6. Subsequently, the filter sterilized solution was once more lyophilized under sterile conditions, dissolved in a concentrated tissue adhesive solution, filled into containers. end (disposable syringes) and stored fully frozen. Before use, it is only necessary to defrost it.

The ready-to-use fabric adhesive solution had the same properties as the dissolved preparation of Example 6.

Before the filter sterilized diluted tissue adhesive solution is again lyophilized and dissolved in a concentrated state, it can also be directly evaporated under mild vacuum into a concentrated tissue adhesive solution.

Method for measuring viscosity

To standardize the measurement method, initially a sample of the tissue adhesive solution is frozen at &lt;-20 ° C. To determine its viscosity, the sample is thawed in a water bath at the desired measurement temperature, incubated for approximately 30 minutes at that temperature, and then its viscosity is determined in a temperature-controlled capillary viscometer.

Subsequently, the sample can be incubated at a higher temperature in the viscometer, and the measurement can be repeated at that temperature. The individual measurements are carried out in series at increasing temperatures. If they are measured in the inverted sequence, false values ​​(too low) could be obtained, since equilibrium is only achieved slowly at lower and lower temperatures.

Example 8

A plasma cryoprecipitate was dissolved as in Example 1, the pH was adjusted to 7.3 and filtered until clarification. Similar to Example 1, aliquots of this solution were added to solutions containing ^ -alanine and ethanol, although not at room temperature, but at 0 ° C, so that in each case an ethanol concentration of 2% was obtained. (v / v), and different concentrations of ^ -alanine in a range of 0-1 M. The precipitates formed in a manner similar to Example 1 were centrifuged and analyzed. Fig. 3 graphically represents the Fn / Fbg ratio dependent on the concentration of ^ -alanine.

TABLE 4

Dependence of the protein composition of the precipitate on the content of β-alanine in the precipitation mixture (ethanol concentration: 2% o)

<td>^ -alanine (M)</td><td>Fn / Fbg</td>

<td> 0,0</td><td> 0,43</td>

<td> 0,2</td><td> 0,34</td>

<td> 0,4</td><td> 0,30</td>

ES 2 263 451 T3 (Continued)

<td>β-alanine (M)</td><td>Fn / Fbg</td>

<td> 0,6</td><td> 0,26</td>

<td> 0,8</td><td> 0,25</td>

<td> 1,0</td><td> 0,23</td>

Similar to Example 2, this example shows that per se known protein precipitating agents, such as β-alanine, when used in combination with ethanol, by no means only act as precipitating agents, but can also act , under certain conditions of β-alanine, as solubilizers of fibrinogen.

The following Examples 9-11 show compositions of comparable preparations obtained according to the prior art.

Example 9

A plasma cryoprecipitate was dissolved as in Example 1. β-alanine was added, stirring at 0 ° C, until obtaining a final concentration of 2 M. The protein precipitate formed was centrifuged and analyzed as in Example 1. The content The relative fibrinogen content was 74% of the total proteins, the relative fibronectin content was 16%. The Fn / Fbg ratio was therefore 0.22.

Example 10

A plasma cryoprecipitate was dissolved as in Example 1. Ethanol was added, stirring at 0 ° C, until a final concentration of 2% (v / v) was obtained. The protein precipitate formed was centrifuged and analyzed as in Example 1. The relative content of fibrinogen was 67% of the total protein, the relative content of fibronectin was 29%. The Fn / Fbg ratio was therefore 0.43.

Example 11

A plasma cryoprecipitate was dissolved as in Example 1. Ethanol was added, stirring at 0 ° C, until a final concentration of 2% (v / v) was obtained. The formed protein precipitate was centrifuged, redissolved and β-alanine was added to the solution with stirring at 0 ° C until obtaining a concentration of 2 M. The newly formed protein precipitate was centrifuged and analyzed as in Example 1. The relative fibrinogen content was 77% of the total proteins, the relative fibronectin content was 16%. The Fn / Fbg ratio was therefore 0.21.

Examples 9-11 show that with the known methods of the prior art it is not possible, in one step and even in several consecutive steps, to arrive at protein mixtures having defined Fn / Fbg ratios as when used, for example , a combination of β-alanine and ethanol in the concentrations used in Example 8, to arrive at a ratio ranging between 0.23-0.34.

Example 12

A plasma cryoprecipitate was dissolved as in Example 1, the pH was adjusted to 7.3 and filtered until clarification. Similar to Example 1, aliquots of this solution were added to solutions containing β-alanine and ammonium sulfate, so that, in each case, a concentration of 1 M β-alanine and different concentrations of ammonium sulfate were obtained. in a range between 5-15% (w / v). The precipitates formed were centrifuged in a manner similar to Example 1 and analyzed. The results are summarized in Table 5; Fig. 4 graphically represents the Fn / Fbg ratio depending on the concentration of ammonium sulfate.

TABLE 5

Dependence of the protein composition of the precipitate on the content of ammonium sulfate in the precipitation mixture (concentration of β-alanine: 1 M)

<td>% (w / v) ammonium sulfate</td><td>(% of Proteins) Fbg</td><td>(% of Proteins) Fn</td><td>(% of Proteins) Alb</td><td>Fn / Fbg</td>

<td> 5</td><td> 69</td><td> 5</td><td> 0</td><td> 0,07</td>

<td> 10</td><td> 78 6 12</td><td> 0</td><td> 0,15</td><td></td>

<td> 15</td><td> 78</td><td> 16</td><td> 0</td><td> 0,21</td>

ES 2 263 451 T3

Similar to Example 1, this example shows how protein precipitates can be obtained from a mixture of plasma proteins by a single precipitation with a two-component precipitation composition (here / l-alanine and ammonium sulfate), whose protein precipitates contain variable amounts of fibronectin, wherein the fibronectin / fibrinogen ratio can be adjusted as desired within certain limits (for example by selecting the components according to Example 12 on a scale ranging from 0.07 to 0.21); by means of the suitable choice of the concentrations of the components and the proportion of some with respect to others, respectively.

Example 13

A plasma cryoprecipitate was dissolved as in Example 1, the pH was adjusted to 7.3 and filtered until clarification. Similar to Example 1, aliquots of this solution were added to solutions containing / l-alanine and ammonium sulfate, so that, in each case, an ammonium sulfate concentration of 5% (w / v) was obtained. , and different concentrations of / l-alanine in a range between 0.6-1.4 M. The precipitates formed were centrifuged in a manner similar to Example 1 and analyzed. In Fig. 5 the Fn / Fbg ratio is graphically represented in relation to the / l-alanine concentration.

TABLE 6

Dependence of the protein composition of the precipitate on the content of β-alanine in the precipitation mixture (ammonium sulfate concentration: 5%)

<td>/ l-alanine (M)</td><td>Fn / Fbg</td>

<td> 0,6</td><td> 0,19</td>

<td> 1,0</td><td> 0,07</td>

<td> 1,4</td><td> 0,04</td>

Similar to Example 2, this example shows that per se known protein precipitating agents, such as / l-alanine, when used in combination with ammonium sulfate, by no means act solely as precipitating agents, but rather they can also act, under certain conditions, with / l-alanine, as fibrinogen solubilizers.

The following Examples 14-16 show compositions of comparable preparations obtained according to the prior art.

Example 14

A plasma cryoprecipitate was dissolved as in Example 1. / l-alanine was added, stirring at room temperature to obtain a final concentration of 2 M. The protein precipitate formed was centrifuged and analyzed as in Example 1. The relative content of fibrinogen amounted to 82% of the total proteins, the relative content of fibronectin was 1%. The Fn / Fbg ratio was therefore 0.01.

Example 15

A plasma cryoprecipitate was dissolved as in Example 1. Ammonium sulfate was added, stirring at room temperature until a final concentration of 15% (w / v) was obtained. The protein precipitate formed was centrifuged and analyzed as in Example 1. The relative content of fibrinogen was 76% of the total protein, the relative content of fibronectin was 16%. The Fn / Fbg ratio was therefore 0.21.

Example 16

A plasma cryoprecipitate was dissolved as in Example 1. Ammonium sulfate was added, stirring at room temperature until a final concentration of 5% (w / v) was obtained. The protein precipitate formed was centrifuged, redissolved and l-alanine was added to the solution with stirring at room temperature to obtain a final concentration of 2 M. The protein precipitate formed was centrifuged again and analyzed as in Example 1 . The relative content of fibrinogen was 84% ​​of the total proteins, the relative content of fibronectin was 1%. The Fn / Fbg ratio was therefore 0.01.

Examples 14-16 show that with the known methods of the prior state of the art it is not possible to arrive, in one stage or even in several consecutive stages, at mixtures of proteins that have a certain ratio of fibronectin / fibrinogen, as when, for For example, a combination of l-alanine and ammonium sulfate is used in the concentrations used in Example 13, to arrive at a ratio ranging from 0.04-0.19.