MXPA99011354A - Alpha 1-antitrypsin preparation and method for the production thereof - Google Patents

Abstract

The invention relates to a native chromatographically purified&agr;1-AT preparation, having a purity of at least 0.7 PE/mg protein and a relative plasma&agr;1-AT activity of at least 120%. The active-inactive&agr;1-AT ratio is greater than in plasma. The invention also relates to a method for producing said preparation and to the use of a carrier material, e.g. an inorganic carrier material such a hydroxylapatite, to separate active&agr;1-AT from inactive&agr;1-AT.

Description

PREPARATION OF ALPHA 1-ANTITRYPSINE AND PROCEDURE FOR THEIR OBTAINION DESCRIPTION OF THE INVENTION: The invention relates to compositions a-1-antitrypsins for their preparation as well as compounds of those compositions. a-1-an i ripsina (aíl-AT) is a protein presented in plasma which, due to its structural and functional properties, is ordered in the Serpina superfamily (serine protease inhibitors), which is why it is also known under the name Protease inhibitor. al-AT is responsible for approximately 90% of the trypsin inhibiting capacity of normal plasma, which is why it is frequently reported as the main plasma serpin (Major plasma serpin). The inhibitory activity of al-AT in reference to elastase has a special physiological significance. al-AT is primarily a protective protein, so that proteolytic enzyme cells left free should be protected. It is synthesized in the liver and secreted as plasma, where it has an average duration of approximately 6 days. The normal plasma concentration of al-AT is 1.3 g / 1. al-AT is a relatively small polar molecule, which quickly reaches the cell fluid and can act there. Human al-AT consists of a single polypeptide chain with 394 amino acid radicals with three positions of REF .: 32185 glycosylation in the asparagine radicals of positions 46, 83 and 247. While in asparagine 46 and 247 there are both two and three side chains of sialisilated carbohydrates antenigos, in asparagine 83 there is only one sialylated side chain (Carrell) and associates Nature 298 (1982), 329-334) The protein chain of al-AT comes in two forms in plasma, where in one form a terminal pentapeptide N is withdrawn because of this carbohydrate side chain, but also because of the possible pentapeptide withdrawal, a complete diverse microheterogeneity is reached, which may vary in one and in the same individual. Thus it is presented that important variations in the proportions of these isoforms of the al-AT molecules during inflammation or decreases in estrogen, which can possibly be attributed, to that because of the stress situation, partially diasialized forms are preferably disintegrated to quickly turn back to normal plasma levels (Patterson Comp Biochem Phisiiol 100 B (3)) 1991) 439 454). The inhibitory effect of the serine protease of al-AT is directed to the formation of stable complexes 1: 1 between al-AT and the protease sought. al-AT therefore acts as a kind of "suicide substrate" with which the following protease reaction of the serine protease is eliminated. The effect of al-AT is primarily inhibited by released radicals, which also occur in the course of inflammation. Physiologically this oxidation ability is likely to be reduced so that in the area immediately following inflammation, the enzyme inhibiting activity of al-AT is decreased, so that proteases can develop in the fight of for example bacteria that cause inflammation. The oxidation lability of al-AT, however, is first and foremost disadvantageous where al-AT or liquid, which contains al-AT, is exposed, as for example in the fluids of the respiratory tract. Thus, the elastic tissue of the lungs before the superficial attack is essentially protected by two inhibitors, the inhibitor of leukocyte protease secreted by humans, which is found mainly in the upper respiratory tract, and al-AT, which predominantly occurs in the lower tract. Although there is usually more than enough inhibitory capacity for the defense of the lower respiratory tract, it can be with too prolonged exposure to free radicals, as for example, with heavy smoking there may be problems (the inhibitory capacity of AT is reduced by half due to heavy smoking). On average, more than 70 qualitative and quantitative variants of human al-AT are known, which is inherited as codominant autosomal allele, from which it is split so that approximately 10% of the European population is a carrier of a pathological variant of al-AT.Al-TA deficiencies are also known and relatively widespread. The most remarkable pathological encounter in dependence on the al-AT gene variant, is a degenerative lung disease that starts early as well as a strong liver disease. Along with this there are also diseases of the kidneys, arthritis and malignant diseases related to a variant of al-AT genes. First of all in lung disease, it is certain that this can be attributed to the corresponding level of al-AT, which due to cumulative proteolytic damage causes a loss of pulmonary elasticity. Thus an intense smoking with a lack of homozygotes al-AT is doubly dangerous and has a high risk of developing, a serious emphysema already in the age less than 40 years. Against homozygotes of non-smokers it has a shortening of life of approximately 20 years.Al-AT is present both active and inactive in plasma (see for example Pajdak Wy asoc Folia Histochemica et Cytobiologica vol 24 (1986 ) page 169 172) A series of manufacturing processes are known for al-AT, which covered the fractional precipitation of plasma with polyethylene glycol 4000, but also the preparation of different plasma fractions (precipitate IV-1 Cohn fraction or residue A or A + l from Kistler and Nitschmann) (Feldman and Winkelman Blood Separation and Plasma Fractionation (1991) Wiley-Liss Inc., pp. 341-383) In other purifications the fractions of blood found are cleaned for example with DEAE cellulose (Basis et al. (Vopr. Med. Khim. 33 910 (1987) 54-59), being treated with affinity chromatography materials, Basis and Asoc describe a procedure for the purification of al-AT by precipitation of plasma ammonium sulfate and then SEAE-cellulose chromatography and hydroxylapatite chromatography. This procedure must be done constantly in the presence of mercaptoethanol, which protects the protein against the oxidation of groups containing S. Al-AT is obtained after hydroxylapatite chromatography in two fractions, however the two proteins, the al AT and albumin, do not separate completely. In the course of the pre-processing for the present invention it was determined, that with all the procedures hitherto used, but also in a procedure according to Basis et al. and according to the patent EP-0 698 615, it is not possible to completely separate the constituent in plasma with the active al-AT ^ the inactive al-AT completely, or to produce a preparation obtaining active isomers. It is the task of the present invention therefore to establish a procedure with which al-AT preparations can be obtained in which the ratio of active al-AT to inactive al-TA is improved in favor of active al-AT, that is, with which inactive al-AT can be selectively separated from active al-AT. Yet another task of the present invention is to present an improved al-AT preparation compared to existing preparations. The object of the present invention is therefore a preparation based on chromatographically purified native AT-AT, which has a purity of at least 0.7 PE / mg protein and a relative plasma AT-activity of at least 120%. Plasma al-AT activity is defined as the ratio of active to inactive al-At, where this ratio in plasma is taken as 100% of the relative activity of plasma ai-AT. For the preparations prepared according to the invention, this activity relative to AT remains preferably above 130%, still more preferably above 140%, especially above 150%. In special cases, the relative plasma activity of al-AT may also be above 160%. In the products according to the invention, because of the purity of the obtained protein, it approaches the established AT, whereby the relative plasma activity is easy to calculate. The al-AT remains in the blood or in serum in a certain fraction always in an inactive form, that is, it does not have practically inhibitory activity of elastase, but always reacts immunely. The total amount in al-AT (active and inactive) can be determined with all methods in use, for example quantitative immunological methods of antigens, such as ELISA. The causes for the presentation of al-AT can be multiple, for example the al-AT can be due to a dissociation of the molecule or due to disorders in the conformation to be inactive. Since there can normally be up to 20% of al-AT inactive in the blood or in the serum, and this can not be separated by the usual methods, the preparation according to the invention represents an essential advantage with respect to the preparations in question. -AT usual, in which until now always the inactive fraction has been "dragged". As mentioned, the concentration of al-AT in normal plasma remains at 1.3 g / 1, producing a specific activity of 0.77PE / mg (PE plasma units) determined in the elastase inhibition test see example 3 ). The α-AT preparation according to the invention preferably contains at least one isomer with a pT value between 4.3 and 4.4. It has been shown in a surprising manner that exactly because of the presence of this isomer the activity of al-AT is very high. Since the isomer with a pl value between 4.3 and 4.4 due to its acid nature in the process described in the state of the art to obtain al-AT always together with albumin, it must be separated from the fraction gained from al-AT, not being found in any preparation anything worth mentioning fractions of that isomer. In the course of the present invention, however, the relevance of this isomer was recognized in reference to the activity yield and in accordance with the invention were prepared in the manufacturing process preferably only or steps, which did not allow or only did so in a very limited a separation of that isomer from the usual al-AT, so that also most of that acid isomer existed in the final preparation. The distribution of the isomer of the preferred preparation thus corresponds to that of the native protein, especially the protein, which is obtained from plasma extractions. In particular, the isomer distribution of the al-AT preparation according to the invention corresponds to the visual evaluation by isoelectric focusing (IEF) on a sample of quadruple bands. The IEF is made especially with the IEF board (registered trademark) PEF IEF ™ amfoline plate obtainable from Pharmacia (pH 4.0 -6.5 or pH 4.0 - 5.0) corresponding to the prescription given by Pharmacia using IEF markers of the Signature Sigma (IEF Mi 3.6 - 6.6). The isomer with a pl value between 4.3 and 4.4 corresponds to the most acid band. This isomer, however, falls exactly in high purity al-AT preparations or totally pure or only to a negligible extent within the limits of the test. The al-AT preparation according to the invention has in particular a high purity, for example a purity of more than 0.8PE / mg, preferably a purity of more than 0.9 PE / mg and more preferably more than 1 PE / mg. mg. It has been shown that purities according to the invention of more than 1.2 PE / mg are achievable according to the invention. The preparation according to the invention usually contains less than 10%, preferably less than 5%, especially less than 2% of inactive al-AT. In accordance with the present invention it is also possible to make available an al-AT preparation, which is essentially free of inactive al-AT. The ratio of active al-AT to inactive AT is insed in the preparation according to the invention, especially higher than that of normal human plasma. It is also in accordance with the present invention possible to obtain an overactive AT-AT indicated as nascent. It is understood in the present invention under the expression called normal, a plasma standardized with respect to the content of al-AT. Standardization as well as the determination of active al-AT can be performed with the methods in use, for example by means of inhibition of elastase or trypsin activity. The preparation according to the invention has essentially the advantage with respect to the preparations according to the state of the art, that especially to pharmaceutical preparations the preparation provided has due to the low content of inactive al-TA, a much more defined effect than the known preparations, which especially is of enormous advantage in medicinal uses. Preferably, a preservative al-AT is available, in which the use of oxidation stabilizers, such as β-mercaptoethanol, can be avoided. "Conservative means in the context of the present invention that al-AT can be sufficiently stable also without the presence of oxidation stabilizers such as β-mercaptoethanol, which, especially considering the large The oxidation ability of al-AT is especially surprising. The preparation according to the invention can be made from blood, plasma, serum, or fractions thereof, but can also be manufactured from cell cultures, especially recombinant cell cultures, or cell-overhanging cultures. Preferably, the monomer content of the preparation according to the invention is at least 95%, preferably at least 98%. A preferred embodiment is also a pharmaceutical preparation, containing the al-AT preparation of the invention, optionally with pharmaceutically tolerable auxiliary substances, as a buffer, stabilizers, additives, anti-oxidants, salts or excipients. Since the present invention is mainly to be applied in the pharmaceutical field, it consists of a preferred embodiment, in the preparation for the inactivation of existing pathogens. In a favorable manner, the preparation is established in a stable form, preferably as freeze-dried or co-solution (deep-cooled), especially as a solution. , which is suitable to be applied as an aerosol or as a spray. The preparation can also be presented in association with liposomes or phospholipids or other forms of micro or nano particles, which is advantageous in certain applications. The subject of the present invention is also a process for the preparation of the preparations al-AT according to the invention by the purification of a fraction containing al-AT, which is preferably obtainable from a human plasma deposit by means of adsorption chromatography, in such a way that the AT is absorbed, if necessary the inactive al-AT is separated, and the active al-AT is gained in a fraction by elution. Under adsorption chromatography according to the present invention is meant such chromatography, in which al-AT is adsorbed on the chromatography material. Finally, the desired fraction can be won by circumvention. For adsorption chromatography, for example, a non-organic chromatography material such as hydroxylapatite, preferably ceramic hydroxylapatite, can be used, and adsorption chromatography can also be carried out as an anion exchange, preferably in the presence of a detergent. As the anion exchanger, preference is given to using materials based on carbohydrates or vinyl polymers, which have been evaluated in laboratory or industrial use, especially commercially available products such as DEAE-Sephacel®, DEAE Sephadex®, DEAE Sepharose® CLSB, DEAE Sepharose® Fast Flow, QAS Sephadex®, Q-Sepharose® Fast Flow, Q-Sepharose Big Beats®, Q-Sepharose® High Performance (Signature Pharmacia); DEAE-trisacryl, DEAE Sphrodex®, Q-hyper D® (Signature Sepracor) DEAE Toyopearl® _QAE Toyopearl®, Toyopearl Super Q® (Pirma Tosohaas, Fractogel® EMDT-TMAE or other farctogel materials, Licrospher 1000TMAE®, Licrospher 1000DEAE® and Licrospher 4000 DMAE® (Fa Merck) Macroprep DEAE®, Macroprep Q® (BioRAd); Protein PAK DEAE® (FA Warters) where the treatment with a strong anion exchanger, for example Q-Sepharose is especially preferred The exact conditions, in which the achievement or gain of active al-AT is carried out, can also vary according to the starting material, but these are known to the person skilled in the art for each material, knowing the present invention and easy to obtain without effort or to an optimum, preferably the starting material is not incubated on or before the adsorption chromatography or only for a time. very short (for example a few minutes) the chromatography of adsorption or obtaining of al-AT, is carried out especially under such conditions that the isomer remains obtained with an isomer with a value pl re 4.30 and 4.4-especially 4.34 and 4.39 and it is especially preferred that it be maintained after purification or treatment. These conditions are easy to carry to an optimal state by the technician testing several elution dampers for the adsorption materials that are used each time, since the isomer with a pl value between 4.3 and 4.4 or fraction is contained in that isomer, being easy deletion by IEF. Here it must be especially noted that this acid isomer does not separate with albumin. This is for the technician without problem, for example with gel electrophoresis or on IEF test of the eluted fractions (and the detection of al-TA acid and albumin). In another preferred embodiment, no chromatography is carried out in a cation exchanger, especially no cation exchange chromatography with a low pH value.

Under detergent (surfactant) is usually understood an active organic substance on the boundary surface, especially synthetic organic products. According to the invention, non-anionic detergents, such as polyether, especially alkyl / phenol, polyglycol ether, products of fatty acid ethoxylation, fatty acid amides, fatty amines, fatty alcohols, amino-oxides, etc. are preferably used. fatty acid, such as polyalcohols, and sugar esters. The detergent acts especially on the protein in a non-denaturing manner. Especially preferred for the purpose of the present invention is a surfactant from the group of polysorbates (for example Tween) and a surfactant from the Triton group. The adsorption chromatography can in the process according to the invention, according to a preferred embodiment, receive another treatment for example a precipitation, filtration, gel filtration, treatment with an inorganic carrier material, or a chromatographic purification before or after after . As the preferred inorganic carrier material, hydroxylapatite has been distinguished, where the ceramic hydroxylapatite is especially preferred. In another preferred embodiment, the adsorption chromatography is combined with an anion exchanger, preferably in the presence of a detergent, with an adsorption of hyoxylapatite. In the course of the elaboration of the present invention it has been presented as surprising, that under special circumstances it is made possible, or also by treatment with inorganic materials such as hydroxylapatite, a separation of the active al-AT from inactive al-AT is performed. . This effect then occurs especially when al-AT and is basically separated from albumin. Therefore, it is also the treatment of an al-AT preparation previously purified with the inorganic carrier material, especially with hydroxylapatite, appropriate alone, to produce a preparation according to the invention. Preferably the albumin is removed in a high amount by percent from the starting material. The separation of active al-AT is preferably carried out by fractional elution. The treatment with hydroxylapatite preferably represents the final purification step. It has been surprisingly shown that although in a known way al-AT is extremely oxidizable, the process according to the invention can also be carried out without the application of ß mercaptoe ano, or another buffer of oxidation, which especially should be avoided in pharmaceutical preparations. The process according to the invention is then preferably carried out in the absence of β-mercaptoethanol or other oxidation stabilizers, ie no buffers are used, which contain a stabilizer. The starting material comes preferably from a plasma extraction, especially from one that is produced from blood donations of individuals with normal AT. For the process of the invention, better plasma or plasma fractions are used, especially a decreased fraction of albumin, preferably a Cohn V precipitate, corresponding essentially to a Cohn IV-A fraction (according to Cohn Review 28 (1941) 395-417 ). In another preferred embodiment, a prepurified fraction, for example a preparation obtained commercially, is used as prolastin. In the process according to the invention, a step is also preferentially made for the inactivation or decrease of the pathogens that may exist. This inactivation is guaranteed by a surfactant and / or heat treatment, for example a heat treatment in a solid state, especially a steam treatment according to EP-0 159 311 or EP 0 519 901 or EP 0 674 531 , but also with organic solvents and / or detergents, for example according to EP 0 131 740 0 and EP 0 50 061. Other treatments for the inactivation of viruses or pathogens also include treatment with chemical or chemical-physical methods, for example. with chaotropene materials according to WO 94/13329 or DE 44 34 538 or a photoinactivation.

A radiation or nanofiltration represent preferred physical methods for decreasing viruses within the framework of the present invention. The process according to the invention is preferably designed in such a way that also the gain of other proteins is possible, especially the gain of transferin, albumin, orosomucoid and polypoprotein, which can then be obtained in another fraction of the chromatographic treatment according to the invention. The exact conditions can be determined by the technician for each chromatographic material in light of the present invention easily or chosen as excellent. The pH in the embodiment of the process according to the invention, especially the elution, is preferably between 5.5 and 8.0, especially 6.5-6.8. The use of the buffer with an ion intensity corresponding to lOmM in phosphate has also been found to be favorable in carrying out the process according to the invention. According to another aspect, the present invention relates to the use of a carrier material for the separation of inactive al-AT from active al-AT. As the solid carrier material for adsorption and separation, an inorganic carrier material such as hydroxylapatite or an anion exchanger in the presence of a detergent, especially Q-Sepharose, in the presence of Tween is preferably used in another preferred embodiment. The present invention will be explained below by means of the following examples, which in no way limit it EXAMPLES: Example 1: Purification from a Cohn V precipitation of alAT (this according to the applicant's opinion as the best way to carry out the invention) The Cohn V precipitation is suspended with triple the weight of a buffer A (1.2 g / 1 Na2HPo4 x 2 H20 = 6.74 mM, 10 g / l NaCl = 171 mM, pH 7.0 adjusted with 96% acetic acid) 4 a 6 hours at 4 ° C with shaking. Then the AT is precipitated from this turbid suspension with 17.5% ethanol a final concentration at 6 ° C at night. The centrifuged precipitate is stirred with 24 times the weight of lOmM Tris-HCl pH 10.4 (adjusted with 6M NaOH) and for 1/2 hour at room temperature, then the pH is adjusted to 6.5 and rinsed through a CUNO filter. 50SA. 15% Tween v / v is added to the filtrate and the mixture is stirred for 2 hours at 26 ° C. The obtained solution is placed in a balanced column of chromatography filled with Q-Sepharose® Fast Flow (Pharmacia) and washed corresponding to 10 mg protein per ml gel (approximately 2 column volumes), 25 mM sodium phosphate buffer, pH 6.5 (eluted largely from Tween) and then washed with 3 column volumes 60m chloride of sodium in 25 mM sodium phosphate buffer, pH 6.5 (eluate transferrin). By elution with 3 column volumes lOOmM sodium chloride in 25 mM sodium phosphate buffer, pH 6.5 the fraction containing al-AT is obtained with approximately 0.40 to 0.60 units of plasma (PE) al-AT or per mg protein, where the activity to inhibit elastase was determined according to example 3 (1PE al-AT corresponds to approximately 1.3 mg of pure al-AT). With 3 column volumes 125 mM sodium chloride in 25 mM sodium phosphate buffer, pH 6.5 can still be eluted mainly mainly serum albumin and other plasma proteins and with 1 to 2 M sodium chloride in 25 mM sodium phosphate buffer pH 6.5 the inactivates (practically no inhibition of elastase, but immunoreactive) and the remaining impurities. The fraction containing al-AT is diafiltered with an ultrafiltration membrane (limit of exclusion 30000 dalton) concentrating it approximately 30 times and with 0.15 g / 1 citrate Na3 x2 H20 = 0.5mM. The diafiltrate obtained below is lyophilized. The lyophilized powder is wetted at a residual water content of 7.5 +/- 0.5% and subjected to S-TIM treatment according to EP-0 159 311 (10 hours at 60 ° and 1 hour at 80 ° C).

The treated powder is dissolved in 10 mmol of sodium phosphate buffer, pH 6.8 approximately 10 mg / ml protein and placed on a column of balanced chromatography filled with ceramic hydroxyapatite (Macropep Type I, grain size 80-μm.Bio Rad) and with 10 mM sodium phosphate buffer, pH 6.8 corresponding to 10 mg protein per ml of column bed (approximately 1 column volume). By elution with 5 column volumes 40 M sodium phosphate buffer, pH 6.8, the al-AT fraction is obtained with approximately 0.90 to 1.05 units of plasma (PE) al-AT per mg protein. With 3 volumes of 100 mM column of sodium phosphate, pH 6.8 can then be eluted mainly al- AT inactive, serum albumin and other plasma proteins and with 500 mM sodium phosphate buffer, pH 6.8, the remaining impurities. It has been shown that with this purification procedure, al-AT can be obtained with a high specific activity of 1.0 PE / mg (corresponding to 130% of the plasma activity of al-AT). The result is shown in the following table l.

TABLE The prolastin (signature Cutter) that is in the pharmaceutical market is according to the information of the manufacturer, pure between 45-80% and produces in the test of inhibition of the elastase a specific activity of 0.60 PE (corresponding to 78% of activity plasma of al-AT) Example 2: purification of al-AT (Serva) by ceramic hydroxylapatite * • al-AT Serva, with a minimum purity of 95% (depending on the manufacturer was determined by SDS gel) that presents the al-AT activity of 0.81Pe / mg, which is the highest specific activity measured so far, was also treated through ceramic hydroxyapatite. The purified fractions gave an activity of 0.92 PE / mg (corresponding approximately to 120% relative plasma activity of al-AT). In the SDS-Excel reducer gel, a clear separation of the inactive AT and other impure proteins is seen with a silver color of the protein. Example 3: Prescription for the measurement of elastase inhibition The test was done according to a prescription by J. Bieth B Spiess and CG Wermuth (1974), The synthesis and analytical use of a highly sensitive and convenient substrate of elastase, Biochem.Med. 11 (4), 350-357 or J. Travis and D. Johnson (1981) Human al-proteinase Inhibitor, Methods of Enzymology 80, 754-765. Tris: 02. tris -HCl, pH 8.0 Tris +: Tris with 0.1% human serum albumin (HSA) prepared shortly before use Elastase; 6% elastase (from pork pancreas, Boehringer Mannheim) is thinned 1: 1000 and sterile filtered by freezing at -20 ° C Substrate: 22.5 mg Succinyl- (alanine) -para-nitroanilide (Bachem) Feincehemikalien AG Switzerland) is dissolved in 5 ml dimethylformamide and stored at 4 ° C. The samples are thinned with Tris + to the corresponding concentration, 25 μl elastase is thinned with 275 μl Tris + (in a water bath at 25 °) and incubate with a sample of 100 μl 3 minutes a ° C. After the substrate 1:10 thins with Trist +, 00μl of it is mixed and immediately the increase of extinction is measured at 405 nm and 25 ° C for 5 minutes. The empty value of the inhibition is mixed instead of a sample of 100μl Tris +. The empty value occurs in the previous application between 0.05 to 0.06dA / min. The standard curve used is a 1: 100 to 1: 400 weight loss series of reference plasma (Immuno AG 1A3E). The prolastin (Cutter) is thinned from a pre-thinning 1:50 (in aliquots thawed to -20 °) 1:80 with Tris + (thinning (1: 4000) and with the thinning 1? 150 of the reference plasma is measured together with each series of measurements In a deviation too strong with respect to the standard line, this series of measurements must be discarded or the results considered with approximate directive values The stopping is calculated by:% = (l-idA / min) move / (dA / min) empty value) * 100. The stopping of the samples must be in a stop or inhibition in the range of 20 to 50% and is then calculated by means of the standard line in plasma units Example 4. Characterization of different products -AT through the inhibition of Elasüasa.

CHARACTERIZATION OF DIFFERENT A1AT PRODUCTS BY MEANS OF INHIBITION OF ELASTASE E lEF Table 2 NJ rf * > . +/- imperceptible + present - absent n.c. not calculated 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 (32)

1. Having described the invention as above, the content of the following claims is claimed as property: i.- Chromatographically purified native preparation of al-AT, characterized in that it contains at least one isomer with a pl value between 4.3 and 4.4, which it has a purity of at least 0.7 PE / mg protein and an aiAT plasma activity of at least 120% and because the inactive al-AT fraction is less than 10%.
2. 2. Preparation according to claim 1, characterized in that it is presented as a pharmaceutical preparation.
3. 3. Preparation according to claim 16 2, characterized in that it is purified from a plasma sample.
4. 4. Preparation according to claim 1 to 3, characterized in that it has a purity of more than 0.9 PE / mg, preferably more than 1.0 PE / mg, especially more than 1.1 PE / mg.
5. 5. Preparation according to claim 4, characterized in that it has a purity greater than 1.2 PE / mg.
6. 6. - Preparation according to one of claims 1 to 5, characterized in that the inactive AT-al fraction is less than 5%, especially less than 2%.
7. 7. - Preparation according to one of claims 1-6, characterized in that it is free of inactive AT.
8. 8. Preparation according to one of the claims 1 to 7, characterized in that AL-AT is a preservative.
9. 9. Preparation according to one of claims 1 to 8, characterized in that the isomer distribution of al-AT corresponds to that of the native protein, especially a visible quadruplet band sample by focusing.
10. 10. Preparation according to one of claims 1 to 9, characterized in that it is used for the inactivation, if necessary, of existing pathogens.
11. 11. Preparation according to one of claims 1 to 10, characterized in that it is presented in a storage-stable form, preferably in the form of a lyophilisate or as a solution, especially as a suitable solution to be applied as an aerosol or as a spray.
12. 12. -Preparation according to one of claims 1 to 11, characterized in that it is associated with liposomes, phospholipids or other forms of micro or nano particles.
13. 13. Process for the manufacture of a preparation according to claim 1, characterized by the purification of a fraction containing al-AT, obtainable by means of adsorption chromatography in such a way, that the alAT is absorbed and the AT active in a fraction, is obtained by elution.
14. 14. - Process for the manufacture of a preparation according to claim 13, characterized in that plasma or a plasma fraction is used as the starting material, especially a fraction dispersed in albumin, preferably precipitated Cohn V.
15. 15. Process for the manufacture of a preparation according to claim 13 or 14, characterized in that it starts from a pre-purified fraction
16. 16. Process for manufacturing a preparation according to claim 13 to 15, characterized in that it is used for adsorption chromatography of an inorganic chromatography material, preferably hydroxylapatite > much better ceramic hydroxylapatite.
17. 17. Process for the manufacture of a preparation according to claim 13 to 16, characterized in that it is used for the adsorption chromatography of an anion exchanger, preferably in the presence of a detergent.
18. 18. Process for the manufacture of a preparation according to claim 13 to 16, characterized in that the anion exchanger is Q-Sepharose.
19. 19. Process for the manufacture of a preparation according to claim 13 to 18, characterized in that the adsorption chromatography is carried out using a buffer, which is free of mercaptoethanol.
20. 20. Process for the manufacture of a preparation according to claim 13 to 18, characterized in that when eluting the absorber used has an H between 5.5 and 8.0 preferably 6.5-6.8
21. 21. Method according to one of claims 13 to 16 and 20, characterized in that the buffer used to elute contains a salt with an ionic strength corresponding to 60mM, preferably 40mM ^ e phosphate
22. 22.- Process according to one of claims 13 to 15 and 17 to 20, characterized in that the buffer used to elute contains a salt with an ionic strength corresponding to 50 to 130 mM sodium chloride.
23. 23. Process according to one of claims 13 to 22, characterized in that in a further step a treatment is selected from the precipitation group, filtration, gel filtration, with a carrier material and purification is carried out by chromatography.
24. 24. Process according to one of claims 13 to 22, characterized in that the adsorption chromatography is combined in an anion exchanger, preferably in the presence of a detergent with a hydroxylapatite adsorption.
25. 25. - Process according to one of claims 13 to 22, characterized in that only a single adsorption chromatography is carried out.
26. 26. Method according to claim 25, characterized in that another purification step is provided, which does not include any adsorption chromatography.
27. 27. Method according to one of claims 13 to 26, characterized in that a protein selected from the transferrin group, albumin, orosomucoid and polyprotein is obtained in another fraction.
28. 28. Method according to one of claims 13 to 27, characterized in that a step is carried out for the inactivation of the pathogens, which if any exist.
29. 29. Method according to one of claims 13 to 28, characterized in that a treatment with detergents, solvents and / or heat is carried out as inactivation.
30. 30. Process according to one of claims 13 to 29, characterized in that the elution is carried out in such a way that the fraction containing al-AT also contains the isomer with a pl value between 4.3 and 4.4.
31. 31.- Use of a carrier material for the separation of active al-AT from inactive al-AT.
32. 32. Use according to claim 31, characterized in that the carrier material is an inorganic carrier material, preferably hydroxylapatite, better than all ceramic hydroxylapatite.