PROCESS TO PREPARE AN ALPHA-1-ANTITRYPSINE SOLUTION
DESCRIPTION OF THE INVENTION The invention relates to a process for preparing a solution containing alpha-1-antitrypsin (A1AT) and an A1AT. A1AT is a glycoprotein that has. a molecular weight of approximately 55,000 Daltons and which belongs to the family of serine protease inhibitors. A1AT can inhibit the activity of a variety of proteases, for example trypsin, on which the name of the inhibitor is based for historical reasons. Physiologically, elastase is an objective protease which is so involved especially in processes of tissue and matrix reconstruction and degradation and is released by cells such as granulocytes and this one involved in inflammatory processes. The duration of elastase activity is limited in time and space and is essentially regulated by the A1AT inhibitor. The deregulation of this activity leads to rapid degradation of tissue and may have pathophysiological consequences. In addition, inflammatory processes are initiated and / or promoted. A known example of a reduced or lacking control of elastase is the progress of degradation of local tissue in the lung with associated inflammatory phenomena, which leads to
REF: 169148 progressively to emphysema and is accompanied in this way by, in part significantly, a limited function of the lungs. In the final stage, this can lead to the death of the patient, which can ultimately be prevented only by a lung transplant. These patients suffer from A1AT or A1AT deficiency limited in their functions. The inhibitor is normally produced and secreted in relatively large amounts by the liver and circulates in the blood plasma at relatively high concentrations (a typical concentration is 1.3 mg / ml). In addition, physiologically effective and sufficient concentrations of A1AT are found in organs, especially in lung fluid (epithelial lining fluid), of healthy people. If this A1AT concentration is significantly reduced or if the A1AT present is limited in its functions or is inactive (inactivated), there is an uncontrolled degeneration of the pulmonary tissue with the consequences mentioned above. The reasons for the lack of A1AT or A1AT reduced in its inhibitory function are mainly genetic defects. The mutation commonly called "Z", especially in homozygous individuals (PiZZ), results in the polymerization of A1AT molecules that are already found in the cells that synthesize them. Therefore, the A1AT can no longer reach the circulation, or only in very small amounts. This results, on the one hand, in a lacking inhibitory activity, which manifests itself especially in the lung for prolonged periods of time and, on the other hand, an enrichment of the polymers in the liver cells and thus in functional disorders, corresponding. PiZ heterozygous individuals have a correspondingly reduced inhibitory potential. Additional mutations with similar defects are known. According to current estimates, the prevalence of the PiZZ mutation in the USA is in the order of 1/1600 of the population; therefore, the number of carriers of the mutation is significantly higher, of which only presumably 10% has been identified. Of the patients suffering from functional disorders in the lungs (progressive emphysema) based on a deficiency of A1AT or A1AT reduced in their functions, not all can be treated at present since the A1AT for the treatment and / or prophylaxis is not available sufficiently as an approved medication. An established treatment is based on the intravenous administration of solutions containing A1AT prepared from donor plasmas. The established and currently recommended dosage is 60 mg of A1AT per kg of body weight per week, which corresponds to an average consumption of 16-20 grams of A1AT per patient per month. This in turn corresponds to an amount that is contained on average in 15 liters of plasma. Whereas only part of the inhibitor contained in the plasma of starting material is obtained in pure form as a preparation, a multiple of 15 1 of normal plasma is required as raw material for one patient per month. The total volume of plasma required as raw material to recover A1AT and thus for the permanent treatment of patients is correspondingly large. The established preparation methods for producing A1AT preparations use the paste commonly called Cohn IV1 as a starting material. The latter is prepared by means of the Cohn-Oncley method, which is familiar to an expert, or by means of a modification thereof, based on the fractional separation of plasma proteins with the variation, essentially, of the concentration of ethanol added. , the adjusted pH value and the temperature of the solution. In addition to A1AT, the so-called fraction IV1 usually contains a wide variety of other plasma proteins, partly in amounts already reduced by previous precipitation steps. A variation of this process is the Kistler-Nitschmann method. Accordingly, a fraction that is similar to the Cohn IV1 fraction as well as other fractions containing A1AT, such as the so-called supernatant I + II + III, can also be used as starting materials. Several methods have been described for making a more or less pure A1AT preparation. The use of ion exchange chromatography for the enrichment of A1AT, especially by means of anion exchangers, has been reported repeatedly (Gray et al., 1960; Crawford et al., 1973; Chan and collaborators, 1973; etc). However, this preparation step alone does not produce an A1AT preparation having a purity corresponding to the state of the art. Therefore, other preparation steps are used, partly in combination with ion exchangers. For example, adsorption or precipitation methods, such as incubation with polyethylene glycol (US-A-4,379,087), with adsorbents of zinc chelates or heparin (US-A-4, 629, 567) or others are used. These methods are used for purification(additional) of the A1AT, but in each of them a more or less large loss of product yield must be tolerated. In principle, the product loss increases as the number of preparation steps increases. In addition, this is often accompanied by an extension of the preparation time, which can decrease both the integrity and activity of the A1AT and increase the cost of production. In addition to protein preparation steps, so-called virus inactivation steps or virus abatement steps are an essential part of the preparation processes for protein products prepared from plasma. In addition to the commonly called SD method (solvent / detergent), which inactivates the corresponding viruses by damaging its protective lipid envelope, thermal inactivation methods, for example pasteurization (heat treatment for 10 hours at 60 ° C), are Apply to increase security against viruses. Filtration through "nanofilters" retains viruses, whether they are enveloped by lipids or free of lipids, usually depending on the size of the viruses. The state of the art is to integrate two process steps, each of which is effective by itself and is based on different principles together in a production process for maximum security against viruses. Depending on the protein, stabilizers, for example amino acids or sugars, are added during these process steps to stabilize the protein. Therefore, these must be subsequently removed from the solution containing A1AT. In the SD method, detergents are added as active agents for the inactivation of viruses, which must be removed in the further course of the preparation process by means of suitable methods. For this purpose, adsorption to hydrophobic matrices has been established, such as chromatography with immobilized CÍ8 chains. This chromatography is again accompanied by yield losses of the product and includes the disadvantages mentioned above of each (additional) chromatographic step in a method. In addition, these matrices are repeatedly used as a rule, that is, expensive and time-consuming matrix regeneration steps are required. Accordingly, an alternative, effective and rapid method for the qualitative removal of detergents that dispenses with a chromatographic step has been described (WO 94/26287, US-A-5, 817, 765). In this way, the protein solution containing detergent is brought to superphysiological concentrations (> 0.5 M) of a salt, for example Na citrate, to form. detergent-containing particles, which can be separated simply by means of filtration, for example. In the following, this method refers to a "detergent / salt displacement" method. In the examples of WO 94/26287, the "detergent / saline displacement" method is applied to three proteins isolated in solution, which are transferrin, antithrombin III and albumin. In the examples, the process leads to a recovery of protein activity of 95%, respectively, and to the reduction of the detergent concentration. If the method is applied under conditions, such that the yield of the target protein is not affected too much, often the concentration of Triton "11 in the product is still high." In example 4 of WO 94/26287, the inventors are capable to recover 95% of albumin activity, but obtain a product comprising Triton X-100"250 ppm and TNBP 35 ppm. Especially when producing medical preparations, concentrations of Triton X-100"11 greater than 50 ppm, preferably greater than 10 ppm should be avoided and it is generally desirable to reduce the detergent contents as much as possible. process to develop an A1AT preparation that results in a product of high purity and safety as effective as quickly as possible., during the process, the activity and / or the quality of the A1AT should not be adversely affected and the detergents should be removed at levels that are acceptable for medical preparations. Another objective of the invention was to provide a method for the purification of solutions comprising A1AT, during which other protein components are removed. Preferably, the A1AT solution should also be reduced for other components such as lipids or viruses. The goal is achieved by means of a process to prepare an A1AT from solutions containing A1AT, which comprises the steps consisting of: (a) attaching a solution containing A1AT to ion exchange chromatography; (b) adding detergents and optionally a solvent to inactivate the viruses enveloped with lipids; (c) followed by increasing the salt concentration for the salt displacement of the detergents. In addition, the problem of the invention is solved by the modalities defined by claims 1 to 19. The process for preparing the A1AT from solutions containing A1AT, for example from the reconstructed plasma fraction IV1 (Cohn), in principle consists of of only two very efficient process steps in terms of its A1AT enrichment: specifically the chromatography by means of an anion exchanger and the treatment of inactivation of SD virus with Triton X-lOO1111 and TnBP, followed by the saline displacement of agents for the inactivation of viruses. It has been discovered that the last mentioned step can also be used very efficiently in solutions containing A1AT. The process of the invention is especially useful when the solution containing A1AT comprises significant amounts of proteins other than A1AT. Surprisingly, under the conditions of the process, this process step can not only be used to remove the detergents for virus inactivation, but also has a significant purification effect with respect to the separation of any impurities of proteins, lipoproteins and lipids. is present without adversely affecting the performance of the A1AT. In combination with the above-mentioned anion exchange chromatography, essentially an A1AT product having a purity of > 90%, preferably > 95% and contains the A1AT in its active form. The process step of the solvent-detergent treatment and the salt displacement recover the active A1AT in a yield of > 80% If for example the reconstituted IV1 paste is used as the starting material, the process of the invention allows the purification of A1AT due to the removal of a-2-macroglobulin, haptoglobin, a-1-acid glycoprotein, IgG, IgA and IgM of solutions comprising the A1AT. In the specific embodiments of the invention, the solution comprising A1AT after the salt displacement step recovers < 10% a-2-macroglobulin, < 40% haptoglobin, < 10% a-1-acid glycoprotein, < 10% IgG, < 10% IgA and / or < 10% IgM, with reference to the A1AT solution before the solvent / detergent treatment, which is for example the eluate of a previous anion exchange chromatography. In the preferred embodiments of the invention, the initial solution comprising A1AT consists of up to 50, up to 20 or up to 10% (w / w) of other proteins. The initial solution preferably comprises at least 1, 2 or 5% (w / w) of other proteins. Still further, it was also observed that the process of the invention is also surprisingly useful as a step of inactivating and reducing virus. Although A1AT is recovered in high yields, the product is not only reduced with respect to other protein components but also with respect to viruses. This is especially useful for viruses not coated with lipids, because viruses coated with lipids will already be inactivated by the detergent treatment. In this way, the process of the invention is also a process for the inactivation of viruses, especially viruses not coated with lipids, in solutions comprising the A1AT. The discovery that a "detergent / saline displacement" method described in WO 94/26287 is applicable in the present case, leads to a removal of detergent at acceptable levels in medical preparations and also results in a highly specific enrichment is surprising. of the A1AT. One would assume that the method of WO 94/26287 would result in products having reduced detergent concentrations, which however could still be too high for pharmaceutical products., and in which all proteins and other components except detergents are recovered in similar proportions. Therefore, one would not assume that the process of WO 94/26287 would be useful in the purification of a protein from other proteins. The use of hydrophobic (interaction) chromatography (HIC), for example in Phenyl-Sepharose ™, is desirable if an A1AT product having an even higher degree of purity is to be obtained. The performance of this step comes to mind, in particular, subsequent to the detergent / saline displacement treatment, since the binding of proteins to a hydrophobic matrix or ligands is usually mediated in the presence of a superphysiological salt concentration. The elution of the bound proteins is then effected, for example, by reducing the salt concentrations. Correspondingly, after the removal of the detergent "displaced by salts" directly. after a rnable decr in salt concentration (by dilution or ultrafiltration / diafiltration; UF / DF), the solution containing A1AT can be contacted with the hydrophobic matrix and the chromatography can be performed in a manner familiar to the skilled person. In addition to the SD treatment of the A1AT-containing solution, at least one additional step for virus inactivation, virus removal and / or prion removal can be integrated into the process, for example a thermal inactivation of the solution viruses which contains A1AT. These steps can be carried out, in particular, in solution directly after the salt displacement process step since the amounts of salts contained in the solution, especially sodium citrate, serve as stabilizers during the heat treatment. As another possibility, the thermal treatment of the lyophilized product comes to mind. Alternatively or in addition, any suitable filtration method for the removal of viruses or prions may be included. Especially, the nanofiltration is familiar to the skilled person and can be integrated into the process preferably with commercially available filters having pore sizes within a range of 15 to 20 nm or any suitable filtration to remove viruses and / or prions. The virus removal should be improved by the addition of amino acids, preferably a concentration of 0.1 M for each amino acid. In a preferred embodiment, glycine is added at a concentration greater than 0.2 M. A preferred method is described by Yokoyama et al. (2004, Vox Sanguinis 86, 225-229). The preparation containing A1AT according to the invention can be obtained, initially, by means of a process which is characterized by a combination of the following steps: (a) subjecting a solution containing A1AT to anion exchange chromatography; (b) optionally heparin affinity chromatography YJ ° 1 ^ hydrophobic interaction chromatography (HIC); (c) adding detergents and optionally a solvent to inactivate the viruses enveloped by lipids; (d) followed by the saline displacement of these chemicals and protein impurities; (e) subject to at least one more step of inactivation and / or virus removal. Solutions containing A1AT obtained from plasma and its fractions or recombinant or transgenically expressed A1AT are generally suitable. In a preferred embodiment of the process, the doughIV1 (Cohn) is reconstituted with water or a buffered solution, more preferably with a solution buffered with 20 M Tris to achieve a solution ratio with > 3: 1 (preferably> 10: 1), subsequently contacted with an ion exchange gel, preferably an anion exchange gel, in a preferred embodiment a DEAE-Sepharose "11 (Amersham), more preferably DEAE-Sepharose" Fast flow, the gel is washed and the A1AT is eluted by increasing the ionic strength. Inactivation of the virus is effected, for example, by means of the method according to EP-A-0 131 740. The optional addition of stabilizing agents is followed by the addition of virus inactivating agents, preferably Triton X-100, Polysorbat 80 (Tween 80), TnBP and / or caprylic acid / caprylate, preferably at final concentrations of = 0.1% (w / w) of Triton and Tween 80,
> 0.03% (w / w) of TnBP, caprylic acid / caprylate > 0.1 mM. After an appropriate incubation time, preferably
> 0.1 hours, more preferably > 1 hour, a > 4 ° C, more preferably a > 15 ° C, -the concentration of salt increases, especially at a concentration of > 0.5 M, especially with citrate. The particles formed with which they are removed, especially by means of filtration. Washing again the filters or the separated particles can lead to an increase in the A1AT yield. This can be followed by an additional step of virus inactivation, preferably pasteurization in the presence of sodium citrate > 0.5 M, amino acids, sugars or mixtures of these substances. The subsequent decrease in the concentration of the added substances is preferably effected by means of ultra- / diafiltration. More preferably, the subsequent separation of virus particles is effected by means of nanofilters, preferably by means of filters having pore sizes of 15-20 nm. The A1AT obtained in this way can be stored as a liquid or a frozen preparation, or lyophilized by methods familiar to the skilled person. The A1AT of the invention defined by claims 12 to 17 is different from the preparations of the state of the art. The preparations of EP436086 and DE4407837 do not have the high purity of the preparation of the invention and an IgA content of < 1 mg / ml. The A1AT product obtained in this way can be administered as a solution by the subcutaneous, intramuscular, topical route or as an aerosol, preferably by the intravenous route. As a dry material, it can also be used for inhalation in powder form. Application in a mixture with other solutions is possible, for example, for intravenous application. A possible dosage is, for example, 60 mg / kg of body weight per week or 250 mg / kg per month. Another preferred process includes HIC in addition to the aforementioned process steps, preferably HIC by means of a phenyl matrix, preferably after SD treatment and saline displacement of virucidal agents and other impurities, discussed above. Preferably, a negative purification is performed, that is, the valuable substance A1AT passes the unbound chromatographic matrix and the undesirable substances are bound and in this way. removed from the process solution. In another preferred process which may include the aforementioned HIC, chromatography is performed on immobilized heparin, preferably by means of heparin-5-sepharose or heptarin-fractogel. In this way, the solution containing A1AT is contacted with the heparin gel in a column or in a batch process. The enriched A1AT passes the unbound column or is bound to the supernatant after gel separation. This process step is performed
preferably before or after the ion exchange chromatography mentioned above or after the salt displacement of the detergent and the reduction of the ionic strength of the solution, for example, by dialysis. 15. According to the invention, a medicament containing an A1AT according to the invention is also claimed as a single active ingredient or in combination with anti-inflammatory agents, preferably steroids, NSAIDs and the use of the A1AT according to the invention to prepare a
medicine for the treatment of A1AT deficiency, degenerative phenomena in the lungs, such as pulry fibrosis and emphysema. Suitable forms of application of the medicament containing A1AT are known per se for the person
expert. In particular, all forms of application for proteins are suitable, for example, parenteral applications, intravenous or inhalation administrations. The process according to the invention is further illustrated by the following example: Example 1 For reconstitution, the Cohn IV1 frozen paste is dissolved in 10 mM Tris solution at a weight ratio of 1: 9 with stirring for 3 hours at a time. alkaline pH Anion exchange chromatography A preferred chromatographic material for this process step is DEAE-Sepharose FF (Fast Flow), although conditions can also be adapted by the skilled person for each anion exchange material. A chromatographic column packed with DEAE-Sepharose FF is equilibrated with a solution of 20 mM Tris (pH 8.0). Then, the dissolved paste Cohn IV1 is applied at pH 8.0. The washing is carried out with an equilibrium absorber, followed by a washing step with a buffer solution. The A1AT bound to the matrix can then be eluted by washing the column with 20 mM Tris, 0.075 M NaCl, pH 8.0. The specific activity of the A1AT solution obtained in this way is approximately 0.5 PEU / mg of protein (PEU: the equivalent unit of plasma, corresponds to the amount or activity of A1AT that is on average in one milliliter of human plasma) . The column is eluted with a buffer with a high salt content (for example, 2 M NaCl) and the chromatographic gel can subsequently be regenerated by methods known per se. Solvent / detergent treatment The eluate obtained in this way is concentrated by ultrafiltration. Subsequently, a previously mixed solution of Triton X-100, TnBP and water for pharmaceutical use (WFI) is added to reach a final concentration of 1% (w / w) of Triton X-100 and 0.3% (p / p) of TnBP. The SD treatment is carried out for 4 hours at 20 ° C with gentle agitation. Saline displacement For the removal of SD reagents and the precipitation of associated, undesirable proteins, the A1AT-containing solution is diluted with a solution containing 1.5 M sodium citrate and 20 mM Tris at pH 7.0. The addition at a concentration of 1 M citrate is carried out for at least 15 minutes with stirring. Subsequently,
, the process solution is incubated for at least one hour with gentle shaking. The whitish precipitated product that formed is subsequently removed by filtration. This decreases the concentration of reagents, SD to less than 10 ppm and the associated, undesirable proteins and the denatured A1AT are also separated. After this production step, the specific activity of A1AT is at least 0.8 PEU / mg (PEU: the equivalent unit of plasma, corresponds to the amount or activity of the A1AT that is on average in one milliliter of human plasma). Nanofiltration After the removal of low molecular weight substances by means of UF / DF, the solution obtained in this way is filtered through filters with a nominal exclusion size of 15-20 nm, such as DV20 filters of the company Pall, in order to further increase the virus safety of the A1AT product. Results The concentrations of Triton X-100 and TNBP in the product after filtration were lower than 5 ppm. The amount of A1AT and other protein components in the product was determined and compared to the amounts determined before the solvent / detergent treatment. The following recoveries were calculated: A1AT > 80% α-2-macroglobulin < 10% haptoglobin < 40% a-1-acid glycoprotein < 10% IgG <; 10% IgA < 10% IgM < 10% The results show that the A1AT of the product was specifically recovered in high amounts, while the product was significantly reduced in terms of other protein components. This is further illustrated by Figure 1, which shows the result of an SDS-PAGE of the solution before the solvent / detergent treatment (line 2) and after the salt displacement step (line 3). AlAT corresponds to the broad band-slightly higher than 50kD (compared to the molecular weight marker on line 1). The other various protein components clearly detectable in the starting solution are significantly reduced. EXAMPLE 2 By modifying the process described in Example 1, the A1AT-containing eluate of the ion exchange chromatography is contacted with heparin-sepharose "1 *. The A1AT passes the heparin-sepharose column" 11 without being bound. If the batch operation is used, the A1AT remains in the supernatant. The additional processing of the eluate of the column or of the supernatant of the batch variant is carried out as described in example 1. The product obtained in this way is characterized by an increased purity. Figure 1 shows the result of an SDS-PAGE under reducing conditions, gradient 4-20%, Coomassie stain, 5 μg protein / line. Line 1: Molecular weight marker; line 2: probe of a solution according to example 1 before the solvent / detergent treatment; line 3: probe of a solution according to example 1 after the salt displacement step. 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.