MXPA06000500A - Device for forming joints in concrete works - Google Patents

Abstract

The invention relates to a device for forming contraction joints in concrete works. The invention comprises a plurality of assemblies which are made from a concrete-separating material and which are mounted to rigid linear elements in an alternate manner on one side and then the other of the surface crack line on the upper face of the concrete work surface, leaving spaces therebetween for cracking. The inventive device enables the adjacent slabs formed by the contraction joint to be fixed, thereby improving the resistance performance thereof. The invention is suitable for in situ concrete works, such as roads, channels, esplanades, sewers, tunnels, railways, dykes, etc.

Description

FORMULATION FOR A PHARMACEUTICAL SUBSTANCE OF PROTEIN WITHOUT ADDED HUMAN SERUM ALBUMIN DESCRIPTION OF THE INVENTION The present invention relates to a composition consisting of non-peptidic substances of low molecular weight, which stabilizes protein agents formulated in pharmaceutical substances and thus dispenses with the use of human serum albumin (HSA, for its acronym in English) . The present invention further relates to a pharmaceutical composition, which in addition to the protein agent also contains the composition consisting of non-peptidic substances of low molecular weight. The development of genetic engineering techniques provides a variety of novel pharmaceutical substances whose agents represent proteins. Compared to conventional pharmaceutical substances, whose agents consist of low molecular weight substances, high molecular weight proteins exhibit high efficiency at low amounts of substance and therefore are applied at very low concentrations and doses, respectively. At these concentrations and low doses, respectively, manufacturers of pharmaceutical substances face a problem. Namely, the proteins have the Ref. 169084 property of adhering to solid surfaces. Due to this absorption, a large portion of the applied protein agent can be lost. Of course, this effect is therefore more serious the lower the concentration of the protein that is to be applied. Without an appropriate formulation, the protein agent can even be completely lost. An additional problem of these pharmaceutical substances and protein agents, - respectively, lies in the high instability of the proteins. For example, they can easily be oxidized (cysteine residues, methionine residues), and deaminate (asparagine), respectively, or they can be divided into fragments and aggregated into higher order complexes, respectively. An efficient formulation should avoid these losses of protein agent and should guarantee a stable product. Since the binding of proteins to the surfaces is non-specific, the loss of agent can be avoided by adding an additional protein (non-specific) in high excess. Since this additional protein should preferably not have pharmacological activity at all and should not stimulate the production of antibodies, human serum albumin (HSA) is currently used for these purposes, which can also be obtained from low prices, as it is applied in high quantities as a substitute for plasma. Therefore, currently several pharmaceutical substances (various interferons, growth factors, coagulation factors, toxins and jbotulinum vaccines) are on the market, which contain HSA as a stabilizer. HSA is a product derived from human blood, which therefore can be, despite a mandatory examination, contaminated (such as by virus) and which can provide a spread of disease to the recipient of the pharmaceutical substance containing HSA (especially because new pathogens appear (or may appear) from time to time, which can not be recorded by tests over time). Therefore, the authorities responsible for the approval of pharmaceutical substances are pushing to replace HSA in newly approved pharmaceutical substances. For this reason, HSA should not be used in pharmaceutical substance formulations - as long as they can be substituted by other substances. Human serum albumin (HSA) is due to several particularly useful reasons for the formulation of a protein agent. It is a protein and therefore can inhibit and neutralize, respectively, all non-specific reactions in the protein agent. This applies particularly for reactions at interfaces (liquid-solid, liquid-gas), which can lead to the denaturing of the agent (Henson et al., 1970), Colloid Interface Sci 32, 162-165). The presence of HSA protects against denaturation. In addition, the proteins have an affinity to the surfaces to which they are bound non-specifically by hydrophobic interactions (Norde. (1995) Cali ß _ Mater 5, - -.9-7 = 112). -os-sites-deion-on-surfaces can be saturated by an excess of HSA, so that the protein agent remains in solution, which is in particular mandatory, if the dose of the protein agent is too much. low . In addition, the presence of HSA protects against denaturation processes during filling and optional lyophilization as well as during the storage of the pharmaceutical substance (for example, against oxidative degradation processes or against asparagine deamination). A protein that protects the agent in that way naturally must not present any pharmacological activity on itself, a prerequisite that is satisfied by HSA. The HSA, which is a human protein, should not serve as an antigen, that is, should not stimulate the production of antibodies. However, since HSA is isolated from blood and purified by chemical methods, it can not be strictly excluded, that is, during the purification process neoepitopes are produced, which means new antigenic structures, for which the receptor of the mixture of HSA and protein agent develops antibodies. This could lead to unwanted side reactions. Due to possible collateral reactions, the use of different proteins and a mixture of oligopeptides, respectively, is not desirable. In principle, gelatin can also be considered a stabilizer. It is a protein of animal origin, which causes immunological reactions and which could also be a vehicle for pathogens. The use of HSA and another suitable stabilizer for the protein agent, respectively, is also particularly important for pharmaceutical substances, which contain protein agents, which are administered at very low doses, since the proteins are, in particular at low concentrations , extremely unstable and, moreover, join at once to specific binding sites available. As a result, they are lost for therapeutic use. As examples for a protein agent, which is applied at very low doses, the neurotoxins of Clostridium botulinum can be mentioned. These highly active proteins are active at lower amounts (they are, and Clostridium botulinum neurotoxiria type A is, respectively, of all the pharmaceutical substances hitherto developed the only ones, which are administered at the lowest dose (500 pg / This very low amount of protein is lost unless a protective agent is used.It can be taken from the prior art, which essentially describes HSA as a protective agent, that there is a demand to provide alternatives to HSA such as e.stabiliz. In this context, the inventors faced the problem of developing a composition that protects / stabilizes the protein agents in pharmaceutical substances, which is at least as good. as HSA The postulated problem has been solved by the inventor by developing a composition consisting of non-peptidic substances of low molecular weight, which stabilize protein agents formulated in pharmaceutical substances and whose composition therefore dispenses with the use of HSA. This composition is called composition for stabilization, below. According to a first aspect, the present invention relates to an unrelated protein-free composition, the composition can be formulated as pharmaceutical substances with protein agents. This stabilization composition is preferably based on low molecular weight substances, have been produced in accordance with European Pharmacopoe (Ph. Eur.) And are approved as pharmaceutical adjuvants. In particular, the. The composition also dispenses with HSA and ensures not only stable storage of the protein agents and the pharmaceutical substance without loss of agent, respectively, but also eliminates the risk that the respective drug substance administered is contaminated with infectious agents. It is surprising that those "simple" and low molecular weight substances, as used in accordance with the invention for the stabilization composition, show desired performance and can replace HSA. The stabilization composition according to the invention replaces HSA by a combination of different low molecular weight substances that lack collateral reactions, these substances protect against loss of protein agent by adsorption to surfaces as well as by denaturation processes and chemical degradation of dissolved or lyophilized protein agents. In addition, the stabilization composition prevents degradation of the agent while it is stored for a period of >; 6 months at elevated temperature. The composition for stabilization according to the invention has the constituents denoted in claim 1, these constituents are: a) a surface active substance, in particular a non-ionic detergent (surfactant), 5 and b) a mixture of minus two amino acids, wherein at least the two amino acids are either Glu and Gln or Asp and Asn. The stabilization composition according to the invention with a further preferred mode, one or more of the following additional constituents: c) a disaccharide, preferably sucrose (cane sugar), trehalose or lactose, d) ethylenediaminetetraacetic acid (EDTA) ), preferably in the form of one of its salts such as Na4-EDTA. Preferred stabilization compositions according to the invention contain any of constituents a), b) and e), or constituents a), b) and d), or constituents a), b), c) and d). All of these preferred compositions are either soluble in aqueous media or are aqueous solutions. It is advantageous that all the substances used in the stabilization composition are approved as adjuvants for pharmaceutical preparations and therefore they were examined toxicologically in detail, which means that they can be mixed with the stabilization composition according to the invention without further examination. It is surprising that an exactly defined compilation of these simple substances showed the desired performance, namely, to provide a serum-free albumin-free formulation for protein agents. According to a second aspect, the present invention ... relates to. __ a .._ pharmaceutical composition containing a protein agent and the aforementioned stabilization composition, comprising constituents a) • and b), or one of the aforementioned preferred stabilization compositions. The agent and the protein agent, respectively, are preferably prepared in an aqueous solution (dissolved) containing constituents a) and b) and optionally also c) and / or d). This solution can be lyophilized subsequently. If the solution, in fact, is to be lyophilized, the previous addition of constituent c) is particularly advantageous. After lyophilization, the pharmaceutical composition is present as a powder, which can be reconstituted (preferably with water for injection purposes (WFI).) Accordingly, the pharmaceutical composition is preferably present in the form of a dried powder. By freezing or drying under vacuum soluble in aqueous media Before the therapeutic application, the lyophilized composition and the powder, respectively, is preferably reconstituted with water for injection purposes (WFI) The pharmaceutical composition however may also be present in liquid form, preferably as an aqueous solution The preferred pharmaceutical compositions of the present invention contain prjximximum, a,,,,,,. ) mentioned above as a protein a factor VIII similar to the coagulation factor (antihemophilic globulin), a cytokine such as an interferon, in particular such as alpha, beta or gamma, an enzyme such as a urokinase or a streptokinase, an activator of plasminogen or ultrapure neurotoxin (for a definition of "ultrapure neurotoxin" see below) and a neurotoxin complex, respectively, of Clostridium botulinum, especially types A, B, C, D, E, F or G of Clostridium botulinum. Since clostridial toxins are formulated in lower amounts as pharmaceutical substances, particularly their ultrapure form, they are the preferred protein. In particular, ultrapure neurotoxins of type A and B are preferred. Further preferred pharmaceutical compositions of the present invention contain, in addition to the aforementioned constituents a) and b) and the agent, in addition to constituent c) defined above, constituent d) defined above , or these two constituents together. In both compositions according to the invention are at least the two amino acids (i) aspartic acid and asparagine, or (ii) glutamic acid and glutamine. However, preferably the compositions contain at least three (aspartic acid, asparagine, glutamic acid; acid-aspartic acid, asparagine, glutamine; aspartic acid, glutamic acid, glutamine; asparagine, ___? _lutámico acid. glutamine) of these four amino acids, or even all four (aspartic acid, asparagine, glutamic acid and glutamine). Preferably the individual amino acids are used in concentrations of 20 to 200 mM, preferably 20 to 100 mM, in particular 50 mM. This corresponds in the case of filling 0.5 ml of starting solution in the amount of 1.3 mg to 14.7 and 1.3 to 7.4 mg, respectively, preferably about 3.7 mg per amino acid in the powder after drying. In another preferred embodiment of both compositions of the present invention, the surface active substance (the surfactant) is a non-ionic detergent, preferably a polysorbate (such as Polysorbate 20 or Polysorbate 80) or poloxamer (such as Poloxamer 184 or Poloxamer 188). If the pharmaceutical composition is present in liquid form, the polysorbate portion is preferably 0.01 to 0.5% by weight, preferably a 0.2% by weight portion. This corresponds to the amount of 0.05 to 2.5 mg, preferably 1 mg of polysorbate, after, for example, a freeze-drying process of 0.5 ml of starting solution. In a further preferred embodiment of both compositions of the present invention, the disaccharide is sucrose, trehalose or lactose. Sucrose is especially preferred. It is __] ^ a_ ^ omp ^ sJ._cj._ón_farnacéutica de__conf.oxmi.dad with the invention provided as a solution, the solution preferably contains 2 to 10% by weight, more preferred 5% by weight of the disaccharide, in particular of sucrose . In addition, as already described, the use of a constituent d), a complex former (chelator) having an additional stabilizing effect. The concentration of ethylenediaminetetraacetic acid in the starting solution of the pharmaceutical composition is preferably 0.1 to 1.0 mM, in particular 5 mM. Both types of compositions according to the present invention preferably have a pH value of 5.0 to 8.5, more preferred a pH of 6.0 to 8.0, in particular 6.0 to 7.0 and 6.5, respectively. The pH value is optionally, if necessary and desired, respectively, adjusted with NaOH. The initially mentioned neurotoxins of Clostridium botulinum, in particular Clostridium botulinum neurotoxins of type A and B, have to be formulated for pharmaceutical purposes in very low doses. For the Thus, the quality of the stabilization composition according to the invention can be verified particularly well by means of this agent, which is extremely complicated to handle. Other protein agents are formulated in considerably greater amounts; -HSA-by-so-can-be-easily-implemented by the stabilization compositions according to the invention. Clostridium botulinum toxin type A (trade name Botox ™, Allergan, Dysport ™, Ipsen) has been applied for many years to therapy of different forms of dystonias (eg, blepharospasm, torticollis), of spasticitis, for the treatment of hyperhidrosis, but also in the Cosmetic area to remove wrinkles from the face. This agent is a protein complex, which is synthesized by bacteria that grow anaerobically . { Clostridium Jbotulinum). The active agent in this protein complex is a protein with a molecular weight of 150 kD, the botulinum neurotoxin (BoNT). This toxin and neurotoxin, respectively, acts on the motor endplate and inhibits the transduction of the nerve impulse to the muscle and therefore leads to a paralysis of this muscle. This mechanism of action allows to apply the neurotoxin in diseases, in which the stimulus transduction is pathologically changed, that is, an increased acetylcholine release occurs. The botulinum toxins and neurotoxins, respectively, currently on the market are all based on the toxin complex of Clostridium botulinum, ie the neurotoxin. Essentially, the active molecule is embedded in an assembly of proteins with different molecular weights: these are vari_as__hemagglutinins ..- (15 kD, ._ 19-kD, _3 _- ^ kD - ^ - 52 kD) as well as a non-hemagglutinic non-toxic protein (NTNH, 120 kD). Without these so-called protective proteins, the isolated neurotoxin is very unstable and is easily degraded by proteases. Protective proteins and complex-forming proteins, respectively, are therefore for the actual function of the available nerve cell, but they play a role in the stabilization of the susceptible neurotoxin. On the other hand, there are indications that these complex-forming proteins can exert an immunostimulatory function, which could be responsible for the production of antibodies in 5-10% of patients, which inevitably leads to the end of therapy with this type of neurotoxin (" without secondary responder "). In addition, it has been taken into account that patients are forced with foreign protein (complex-forming proteins), which pharmacologically is not absolutely necessary. Therefore, it makes sense to apply the pure neurotoxin free of complex forming protein as an agent, but it needs, because of its low dose and lability, a particular efficient formulation.

Therefore, the prerequisite for the use of complex protein-free neurotoxin (the complex protein-free neurotoxin is occasionally also referred to as an ultrapure neurotoxin) as an agent in a pharmaceutical substance ... was: jarsarrollar = - ^ - -one = _, pharmaceutical composition that guarantees the stability of the biological function of the ultrapure neurotoxin for a longer period. This prerequisite has been met by the inventor in providing the stabilization composition in accordance with the invention. The two pharmaceutical substances based on the type A neurotoxin concurrently on the market contain as an essential stabilizer HSA (Botox ™ contains for 100 units of agenté 0.5 mg of HSA and additionally 0.9 mg of sodium chloride, while Dysport ™ contains 500 units of agent 0.125 mg of HSA and also 2.5 mg of lactose). Neurobloc ™, based on the B-type toxin complex, consists of the case of 2000 units of a solution with 500 μg / ml HSA, 0.01 M sodium succinate and 0.1 M sodium chloride. As described above, the HSA serves in particular the purpose of preventing the adsorption of the toxin to the walls of bottles (glass jars, syringes, cannulas) and to protect against denaturation. Without the albumin in the serum (or substances that substitute this effect), the toxin is inevitably lost. This is mainly due to the fact that the amount of neurotoxin in these pharmaceutical substances is very low (Botox ™ contains 5 ng, Dysport ™ 20 ng of toxin complex per package unit ("bottle")). As long as no other protein is present, the sufficiently present non-specific phoxotne_ binding sites are occupied by the toxin. In the presence of a high excess (> 50,000 times), as in the aforementioned pharmaceutical substances, the binding sites are occupied by HSA, so the neurotoxin complex remains in solution. The probability that the ultrapure complex free neurotoxin adsorbs to the solid surface of the container is considerably higher, because the amount of pure neurotoxin protein for a dose of 100 units is only 500 pg. The patent application WO 01/58472 describes a formulation for a Clostridium botolinum type A complex, consisting essentially of hydroxyethyl starch. Examples are given in which the formulation is stable for one year. With neurotoxin-free protein-forming ultrapure complex, therefore exams are not described. However, it is established that "the toxin protein has a marked instability on the removal of the hemagglutinin protein". In addition, in the case of ultrapure botulinum toxin it is noted that the "pure botulinum toxin is labile but has limited practical utility for preparing a pharmaceutical composition. "However, it is not mentioned that the formulation described on the basis of hydroxyethyl starch is also efficient to stabilize the ultrapure neurotoxin. - - - - Un-or-rmu-1 The ultra-pure neurotoxin-containing HSA-deficiency is described in US Patents 5,512,547 and 5,756,468. In the first patent, a formulation containing HSA as well as trehalose and maltotriose or related sucrose is described. patent specifies a formulation, which contains in addition to these saccharides methionine or cysteine.

The need to use HSA in a formulation for the ultrapure neurotoxin is demonstrated in a publication as well (Goodenough et al. (1992) Appl Environm Microbiol 58 3426-3428.). The pharmaceutical composition according to the invention can be produced for example as follows: A solution of the agent (e.g., of a Clostridium botulinum neurotoxin) is diluted with the composition for stabilization (in the form of an aqueous solution) at a concentration of 1.0-1.2 ng / ml (= 200 units / ml) and subsequently sterile filtered. With 0.5 ml of this dilution, jars are filled, it is lyophilized or dried under vacuum and stored until the therapeutic application. A bottle therefore contains a lyophilized composition or a vacuum dried powder with approximately 100 units of the neurotoxin. To be administered to the patient, the lyophilized composition or powder is reconstituted with 2-8 ml of WFI, as indicated. The stabilization composition described in accordance with the present invention guarantees a complete recovery of the protein agent (the neurotoxin) after dilution, filtration in sterile form, filling and lyophilization. The lyophilized composition is at 37 ° C stable for more than 6 months.

EXAMPLE 1 It was verified whether the use of the stabilization composition according to the invention provides a higher recovery compared to a phosphate pH regulator and a composition of a phosphate and polysorbate pH regulator, respectively. All excipients used have been obtained from manufacturers at a pharmaceutical grade. Clostridium botulinum neurotoxin type A can be obtained from List Biological Laboratories, Inc. Campell, California, E.U.A. and occurred, respectively, according to DasGupta, B.R. (1984) Toxicon 3, 415-424. A solution of Clostridium botulinum neurotoxin type A (168 μg / ml) was diluted with a stabilization composition according to the invention at a concentration of 0.5 μg / ml. The stabilization composition was 50 mM with respect to aspartic acid, asparagine, glutamic acid and glutamine, contained 0.05% by weight of Polysorbate 20 and had a pH of 7.5. _5_ A-1.2 ng / ml dictional dilution - (= - 200-LD50 / ml) was carried out with several solutions (see table 1). After filtration on a 0.22 μ filter, a glass bottle was filled with 0.5 ml of these additional diluted solutions (6 R, Münnerstádt) and stored at 37 ° C. The bottles were sealed with rubber stoppers. After being stored for 15 hr, the concentration of the neurotoxin in the individual solutions was determined by a conventional enzyme immunoassay (EIA).

TABLE 1 twenty The selected formulation resulted in complete recovery after incubation at 37 ° C.

EXAMPLE 2 It was examined whether a pharmaceutical composition according to the invention with 200 units of neurotoxin type A / ml (1.2 ng / ml) is stable over a long period. From a supply solution with 0.5 μg / ml of Clostridium botulinum neurotoxin type A, a dilution with a concentration of 1.2 ng / ml was produced when using the compositions listed in table 2 according to example 1. After. filtration in sterile form, jars were filled with 0.5 ml of these compositions, which were then sealed with rubber stoppers. After storage for 15 hr at 4 ° C and 37 ° C, the amount of type A neurotoxin was determined in an ELISA.

After 8 months of storage at 4 ° C, the biological activity in the flasks was determined by means of an ex vivo assay. Therefore, the activity of the compositions was determined by the mouse diaphragm assay (Wohlfahrt_K et al (1997) Naunyn-Schmiedebergs Arch.

Phar col. 355, 225-340) TABLE 2 A solution consisting of the amino acids Asn, Asp, Gln and Glu (each 50 mM) and Polysorbate 80 was stable at 4 ° C for at least 8 months.

- - EXAMPLE 3 The stabilizing effect of different mixtures of amino acids was examined. The pharmaceutical compositions were again adjusted to a concentration of 1.2 ng of Clostridium botulinum neurotoxin type A / ml (200 units / ml) (the dilutions were carried out according to example 1 and with the solutions listed in table 3 , respectively) and stored at 4 ° C after filtering in sterile form on a 0.22μ filter. The results of a determination of neurotoxin in the enzyme immunoassay are shown in Table 3.

TABLE 3 A mixture of all four amino acids resulted in a complete recovery of the agent.

EXAMPLE 4 It was examined at what pH value the formulations developed gave the highest recovery. The formulation was prepared at pH values from 6.0 to 8.0 at a concentration of 1.2 mg / ml Clostridium botulinum neurotoxin type A and stored at 37 ° C after filtration on a 0.22 μ filter. The results of a determination of neurotoxin in the enzyme immunoassay are shown in Table 4.

TABLE 4 The best recoveries were achieved at pH values of 6.0 and 6.5.

EXAMPLE 5 It was examined at what concentration of the four amino acids aspartic acid, asparagine, glutamic acid and glutamine achieve the highest recovery. When starting from a dilution of Clostridium botulinum type A neurotoxin with 0.5 μg / ml, an additional dilution at 1.2 μg / ml was produced and filled with 6R bottles at a dose of 0.5 ml per bottle after filtration on a filter of 0.22 μ. After sealing with a rubber stopper they were stored at 4 ° C for 15 hr and subsequently the amount of neurotoxin / vial was determined.

TABLE 5 a TABLE 5 b In the case of the liquid formulation, a concentration of 50 mM amino acids proved efficient for the recovery of the agent.

EXAMPLE 6 It was examined which amino acid composition resulted in the highest recovery after drying by _5 freezing, where EDTA was not used in this approach. The solution with which it was filled (0.5 ml) was dried by freezing and stored overnight at 4 ° C. The lyophilized compositions were reconstituted in 0.5 ml of water for injection purposes. The concentration of neurotoxin was determined by enzyme immunoassay.

TABLE 6 fifteen twenty The highest recovery of the agent in lyophilized compositions was achieved when all four amino acids were present in the composition and at a concentration of 50 mM.

EXAMPLE 7 .. At the e_mp_ez_a_r .___ a_ depart, -of a- predilution. from . a Clostridium botulinum type neurotoxin in a solution (which was 50 mM each for aspartic acid, asparagine, glutamic acid and glutamine, and 0.5 mM for EDTA, and the solution also contained 0.2% by weight of Polysorbate 80 and 5% in sucrose weight and having a pH of 6.5), a final dilution of 1.26 ng of neurotoxin type A / ml (200 U / ml) in a solution of identical composition was produced and filtered on a 0.22 μ membrane filter . 0.5 ml of it were applied with a pipette in 6 R glass bottles and subsequently lyophilized. The lyophilized compositions were dissolved in WFI. Content. agent (neurotoxin type A) was determined in the enzyme immunoassay. 96% by weight of the agent was detected. To verify the biological activity of the recovered agent, the lyophilized compositions were dissolved and tested on the diaphragm. The lyophilized composition of a bottle contained 110 units (corresponding to 110% recovery).

EXAMPLE 8 In analogy to Example 7, lyophilized compositions were produced and subsequently stored at ... 3.7-_C -.___ D After three months, the agent content was determined in the immunoassay. 94% by weight of the. Input agent were detected. The verification of the activity / vial in the biological test (diaphragm test) resulted in a content of 102 units / vial.

EXAMPLE 9 When starting from a predilution of interferon beta in one solution (which was for one approach without, for the other with, 0.5 mM EDTA), it was 50 mM each for aspartic acid, asparagine, glutamic acid and glutamine, and the The solution also contained 0.2% by weight of Polysorbate 80 and 5% by weight of sucrose and had a pH of 7.0, final dilutions with 20 μg / ml (4 international units Mi / ml) in solutions with the corresponding composition were produced and filtered over a 0.22 μ membrane filter. 1 ml of each filtrate was pipetted into 6 R glass bottles and subsequently lyophilized. The lyophilized compositions were dissolved in WFI. The content of agent (interferon beta) was determined in the enzyme immunoassay. 18.8 (without EDTA) and 19.6 μg (0.5 M EDTA), respectively, of the agent were detected. To check the biological activity of the recovered agent, the lyophilized compositions were dissolved and the activity was determined in the conventional bioassay (inhibition - of the cytopathic effect on VERO cells compared to the reference standard). 94 and 95%, respectively, of the input biological activity were recovered.

EXAMPLE 10 When starting from a predilution of the blood coagulation factor VIII in a solution (which was 50 mM each for aspartic acid, asparagine, glutamic acid and glutamine and 0.5 mM for EDTA, and which also contained 0.2% by weight of Polysorbate and 5% by weight of sucrose and having a pH of 7.3), a final dilution with 250 international units / ml in a solution of identical composition was produced and filtered on a 0.22 μ membrane filter. In a focus, Polysorbate 20 was used, in the other, Polysorbate 80. 1 ml of each of the filtrates obtained was applied with a pipette in 6 R glass bottles and subsequently lyophilized. The lyophilized compositions were dissolved in WFI. The content of agent (clotting factor of blood VIII) was determined in the conventional coagulation test. 238 (P 20) and 245 (P 80) international units per bottle, respectively, were detected.

EXAMPLE 11 When starting from a predilution of streptokinase in solution (which was 50 mM each for aspartic acid, asparagine, glutamic acid and glutamine and 0.5 mM for EDTA, and which also contained 0.2% by weight of Polysorbate 80 and 2.5, 5 and 7.5% by weight of sucrose, respectively, and having a pH of 7.0), a final dilution of 250,000 international units / ml in corresponding composition solutions was produced and filtered on a 0.22 μ membrane filter. . 1 ml of each filtrate was applied with a pipette in 6 R glass bottles and subsequently lyophilized. The lyophilized compositions were dissolved in WFI. The content of agent (streptokinase) was determined in the standard fibrinolysis assay. 236,500, 247,000 and 242,500 international units per bottle, respectively, were detected. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (6)

2. Having described the invention as above, the content of the following claims is claimed as property. A composition free of human serum albumin for stabilization of protein agents in pharmaceutical substances, characterized in that it comprises the following constituents: a) a surface active substance, preferably a detergent (surfactant) nonionic, and b) a mixture of at least two amino acids, wherein at least the two amino acids are either Glu and Gln or Asp and Asn. 2. The composition according to claim 1, characterized in that it also comprises at least one of the following constituents: c) a disaccharide, preferably sucrose (sugar cane), trehalose or lactose, d) ethylenediaminetetraacetic acid (EDTA), preferably in the form of one of its salts such as Na4-EDTA.
3. 3. The composition according to claim 1 or 2, characterized in that it comprises constituents a), b) and e), constituents a), b) and d) or constituents a), b), c) and d).
4. 4. The composition according to any of the preceding claims, characterized in that the composition is either soluble in aqueous media or is present as an aqueous solution. 5. A pharmaceutical composition characterized in that it comprises a protein agent and the composition for stabilization according to any of the preceding claims. 6. The pharmaceutical composition according to claim 5, characterized in that the pharmaceutical composition is present as a powder dried by freezing or drying under vacuum, which is soluble in aqueous media. The pharmaceutical composition according to claim 5 or 6, characterized in that the protein agent is a coagulation factor such as factor VIII (antihemolytic globulin), a cytokine such as an interferon, in particular interferon alpha, beta or gamma , an enzyme such as urokinase or streptokinase, a plasminogen activator, or an ultrapure neurotoxin and a neurotoxin complex, respectively, of Clostridium botulinum, in particular of Clostridium botulinum, of type A or B. 8. The composition for stabilization of compliance with any of claims 1 to 4 or the pharmaceutical composition according to any of claims 5 to 7, characterized in that at least the two amino acids are (i) aspartic acid, asparagine, glutamic acid; (ii) aspartic acid, asparagine, glutamine; (iii) aspartic acid, 5-glutamic acid, glutamine; (iv) asparagine, glutamic acid, glutamine; or (v) aspartic acid, asparagine, glutamic acid and glutamine. 9. The composition according to claim 8, characterized in that the concentrations of the amino acids 10 individual are in each case from 20 to 200 pM, more preferred from 20 to 100 rrM, in particular 50 rrM. 10. The composition according to claim 8 or 9, characterized in that the surface active substance is a non-ionic detergent. 11. The composition according to any of claims 8 to 10, characterized in that the non-ionic detergent is a polysorbate such as Polysorbate 20 or Polysorbate 80 or a poloxamer such as Poloxamer 184 or 188. 12. The composition in accordance with any of the 20 claims 8 to 11, characterized in that the disaccharide is sucrose, trehalose or lactose. 13. The composition according to any of claims 8 to 12, characterized in that the pH value of the composition in solution is from
5. 5.0 to 8.5, in particular 6.0 to 8.0, 25 preferred to 6.0 to 7.0 and
6. 6.5, respectively