Method of alpha-1-antitrypsin solution treatment

FIELD: pharmacology.

SUBSTANCE: invention concerns biotechnology. Method involves: reduction of plasmatic Cohn IV1 fraction; anion exchange chromatography of solution containing alpha-1-antitrypsin on anion exchange gel, preferably DEAE Sepharose® fast flow; concentration of obtained eluate by ultrafiltration; additional chromatography on hydrophobic carrier and processing of fraction containing alpha-1-antitrypsin by material containing immobilised heparin form; further inactivation of viruses covered with lipidic capsule by adding detergents and optional solvent; salting-out of detergents by further salt concentration increase to ≥ 0.5 M; separation of detergents and virus particles by nanofiltration with pore grade of 15-20 nm.

EFFECT: enhanced purity and safety of obtained product.

17 cl, 2 ex

 

The present invention relates to a method for producing a solution containing alpha-1-antitripsin (A1AT), and A1AT.

A1AT is a glycoprotein with a molecular mass of approximately 55000 and belongs to the family of inhibitors of serine proteases. A1AT is able to inhibit the activity of many proteases, such as trypsin, which is where the title of this inhibitor. From the point of view of physiology elastase is a protease directed action, which involved, in particular, in the processes of reconstruction and destruction of tissue and matrix and secreted by cells, such as granulocytes, and therefore involved in inflammatory processes. The activity of elastase is limited in time and in space and is regulated mainly inhibitor of A1AT. The dysregulation of such activity leads to rapid tissue destruction and may have pathophysiological consequences. In addition, arise and/or are activated inflammatory processes. A famous example of reduced regulation of the activity of elastase or its absence is a progressive local destruction of lung tissue with concomitant symptoms of inflammation, which gradually leads to emphysema, and so accompanied, sometimes significantly, pulmonary functional disorders. At the final stage it can lead to the shift of the t of the patient, avoid where possible only by means of transplantation of the lung.

These patients suffer from a lack of activity or decreased A1AT activity of A1AT. Normally, the inhibitor is produced in the liver and secreted it in relatively large quantities and circulates in plasma at relatively high concentrations (typical concentration is 1.3 mg/ml). In addition, physiologically effective and sufficient concentrations of A1AT are observed in the bodies of healthy people, specifically in the lung fluid (liquid epithelial lining). If the concentration of A1AT significantly reduced or functional activity available A1AT is reduced or it is inactive (inactivated), is an uncontrolled destruction of lung tissue with all the above-mentioned consequences.

The reasons for the lack A1AT or lack of inhibitory activity of A1AT are mainly genetic defects. The so-called Z-mutation, especially in homozygous individuals (PiZZ), leads to the polymerization of molecules A1AT right in synthesizing its cells. Accordingly, A1AT can no longer get into the bloodstream or may enter the bloodstream in very small amounts. On the one hand, this leads to a loss of inhibitory activity, which is particularly evident in the lungs after a long time, and with the other hand to the accumulation of polymers in the cell p is Cheney and, therefore, to the respective functional disorders. Heterozygous individuals (PiZ) have a correspondingly reduced inhibitory potential. Known additional mutations with similar defects.

In accordance with the latest assessment of the U.S. population, the incidence PiZZ-mutation is about 1/1600. Accordingly, the number of carriers of the mutation is much higher, and presumably found only 10%.

Currently, not all patients suffering from pulmonary functional disorders (progressive emphysema), the cause of which is the A1AT deficiency or reduced functional activity of A1AT, can be treated as the use of A1AT in the form approved drugs for the treatment and/or prevention is not enough available. Accepted treatment is based on intravenous injection containing A1AT solutions obtained from the donor drugs in blood plasma. Recognized and the currently recommended dosage A1AT is 60 mg/kg of body weight per week, which corresponds to the average patient 16-20 g of A1AT in the month. This, in turn, corresponds to the number, which on average contained 15 l of blood plasma. Taking into account that only part of the inhibitor contained in used as source material plasmas the blood, receive in the form of pure drug in the month for one patient as the source of the material required is many times more plasma than 15 HP Total volume of blood plasma, required as starting material for the extraction of A1AT and, therefore, for the continuous treatment of patients, respectively, is very large.

Established methods of obtaining drugs A1AT as the source material is so-called faction Cohen (Cohn) IV1. This fraction is obtained by well-known specialists of the method according to Cohn-Oncley, or in accordance with its modification, which is based on a fractional separation of blood plasma proteins by varying mainly the concentration of added ethanol, adjustment of the pH and temperature of solution. Together with A1AT so-called fraction IV1 usually contains a wide range of other proteins of blood plasma, which is partly reduced by the previous stages of deposition. A modification of this method of production is a method of Kistler-Nitschmann. Accordingly, as the source material can be applied faction, similar to the fraction Cohen IV1, and the other containing A1AT fractions, such as the so-called supernatant I+II+III.

Previously described various ways more or less pure preparation A1AT. It was repeatedly reported on applied and ion-exchange chromatography for enrichment of A1AT, especially when using the-exchangers (Gray et al., 1960; Crawford et al., 1973; Chan et al., 1973; etc.). However, one only this stage is not allowed to get the drug A1AT with purity, which corresponds to the existing prior art. Therefore, in combination with the use of ion exchangers partly used other stages of receipt. For example, using methods of adsorption or precipitation, such as incubation with polyethylene glycol (US patent # US-A-4379087), incubation with the combined zinc or heparin adsorbents (US patent # US-A-4629567), or others. These methods apply to the (additional) cleaning A1AT, but in the case of using each of them have to put up with greater or lesser reduction of product yield. Essentially, the loss of a product increases with the number of stages of receipt. In addition, it is often accompanied by an increase in spend time, which may reduce the integrity and activity of A1AT, and increase production costs.

In addition to the stages of obtaining protein, an important part of ways to get protein products from blood plasma is the so-called stage of inactivation or stage of elimination of viruses. In addition to the so-called method SD (solvent/detergent treatment with solvent/detergent), in which the respective viruses are inactivated by destroying the x protective lipid membrane, to increase viral safety methods for thermoinactivation, such as pasteurization (heating at 60°C for 10 hours). Filtration through nanofilter delays viruses with a lipid envelope or without it depending on the size of viruses. The objective of the prior art is the Union of the two based on different principles stages of the method, each of which by itself is effective, together in one method of getting with maximum viral safety.

Depending on the type of protein during these stages of the method for its stabilization to add stabilizers such as amino acids or sugar. Accordingly, they must be removed from the containing A1AT solution. In the case of applying the method SD-processing as an active inactivating viruses agents add detergents, which must be removed by suitable means at a subsequent step of the method of receipt. For these purposes, introduced the use of adsorption on hydrophobic media, such as chromatography media with immobilized C18 chains. This chromatography is also accompanied by a decrease in product yield and includes the above-mentioned disadvantages of each (additional) chromatographic stage of the way. In addition, these carriers are used, as a rule is, repeatedly, i.e. require costly and time-consuming stage of regeneration of the media.

Accordingly, there has been described an alternative effective and fast way of quality removal of detergents, which dispenses with the stage of carrying out chromatography (international publication WO 94/26287, US patent # US-A-5817765). Thus, to obtain containing detergent particles that can be separated, for example by simple filtration, the salt, such as sodium citrate, containing detergent, the protein solution was brought to sverhtehnologichny concentrations (≥0,5M). In the future, this method is called "detergent/vysalivaniya".

In the example in international publication WO 94/26287 examples of method detergent/vysalivaniya" was applied to the solution of three separate proteins, which were transferrin, antithrombin III and albumin. In these examples, the application of the method led to the recovery of protein at 95%, respectively, and to reduce the concentration of detergent. If the method used in the conditions under which the yield of the target protein was not changed too much, the concentration of Triton X-100 in product often remained high. In example 4, international publication WO 94/26287 the inventors were able to restore the activity of albumin is 95%, but the resulting product contained 250 ppm Triton X-100 and 35 ppm TnBP. Concentrations Trito X-100 in excess of 50 ppm, preferably more than 10 ppm, should be avoided, especially in the manufacture of medical preparations, and, as a rule, it is desirable to reduce the amount of detergent as possible.

The purpose of the present invention relates to an efficient and fast method of obtaining drug A1AT, which yields a product with high purity and safety. Preferably, the method of obtaining the activity and/or quality of A1AT should not change for the worse, and the content of detergents should be reduced to a level acceptable for medical products.

Another objective of the present invention relates to a method of cleaning containing A1AT solutions, which are removed by other protein components. Preferably from a solution A1AT should also be removed other components, such as lipids or viruses.

The objective is achieved by the method of obtaining the A1AT of containing A1AT solutions, which includes stages:

(a) carrying out ion-exchange chromatography containing A1AT solution;

(b) adding detergent and optionally a solvent for inactivation are covered with a lipid membrane of the virus;

(c) further increasing the salt concentration to vysalivaniya detergents.

In addition, the present invention is solved in embodiments, the implementation of this is subramania, defined in claims 1 to 19 claims. The method of obtaining of A1AT-containing A1AT solutions, for example, from recovered plasma fraction Cohen IV1, in principle, consists of only two stages, it is extremely efficient from the point of view of the enrichment of A1AT. Namely, carrying out chromatography using anion-exchanger and inactivation of viruses by SD-processing Triton X-100 and TnBP, followed by salting out inactivating viruses agents.

It was found that the last-mentioned stage can also very effectively used in containing A1AT solutions.

The method according to the present invention is particularly applicable in those cases when containing A1AT solution contains substantial amounts of non-A1AT protein. Unexpectedly, it was found that under conditions of the method according to the present invention, this stage can be used not only to remove the inactivating viruses detergents, but also for significant additional purification of any protein present, lipoproteine and lipid impurities, without negative influence on the yield of A1AT. In combination with the above-mentioned anion-exchange chromatography essentially received the product A1AT, which had a purity >90%, preferably >95% and contained A1AT in its active form. Stage treatment with solvent/detergent and salting out extracts active is th A1AT with output > 80%. If the source material is recovered fraction IV1, the method according to the present invention allows cleaning of A1AT due to the removal of containing A1AT solutions of α-2-macroglobulin, haptoglobin, α-1 acid glikoproteid, IgG, IgA and IgM. In specific embodiments, the implementation of the present invention in comparison with a solution of A1AT before treatment with solvent/detergent, which represents, for example, the eluate after the earlier anion-exchange chromatography, after the stage of vysalivaniya in containing A1AT solution remains <10% α-2-macroglobulin, <40% haptoglobin, <10% of α-1 acid glikoproteid, <10% IgG, <10% IgA and/or <10% IgM. In preferred embodiments of implementing the present invention, the content of other proteins in the source containing A1AT solution is up to 50, up to 20 or up to 10 wt.%. In the original solution preferably contains at least 1, 2, or 5 wt.% other proteins.

In addition, it has been unexpectedly found that the method according to the present invention is also applicable as a phase of inactivation and/or elimination of viruses. Despite the fact that removing A1AT occurs with high outputs, the product is the elimination or reduction of not only the content of protein components, but also viruses. This is particularly true for the uncovered lipid membrane of virus is, as covered with a lipid envelope viruses are inactivated earlier after treatment with detergent. Therefore, the method according to the present invention is also a method for the inactivation of viruses, particularly uncovered the lipid membrane of the virus, containing A1AT solutions.

Unexpected is the fact that described in international publication WO 94/26287 way "detergent/vysalivaniya" is applicable in this case, leads to decreases in the amount of detergent to acceptable medication level and, in addition, leads to highly specific enrichment of A1AT. It can be assumed that the method according to international publication WO 94/26287 can lead to the formation of products with a reduced, but, nevertheless, still too high for pharmaceutical products, detergents, and in which all the proteins and other components, with the exception of detergents, are extracted in a similar degree. Therefore, we can assume that the method according to international publication WO 94/26287 can be used for purification of the protein from other proteins.

The conduct of hydrophobic interaction chromatography (HIC), for example, on a Phenyl-Sepharose®, is desirable if you want to achieve an even higher degree of purification of the product A1AT. The implementation of this stage is especially obvious after carrying out the detergent/vysalivaniya, as has been the W protein with a hydrophobic matrix or ligands usually occurs in the presence of sverkhfizicheskoe the salt concentration. Then carry out the elution related proteins, for example, by lowering the salt concentration. Accordingly, after removal subjected to salting out of the detergent directly after a suitable reduction of the salt concentration (by dilution or ultrafiltration/diafiltration (UF/DF)), we can realize the interaction containing A1AT solution with a hydrophobic matrix and conducting chromatography known to the specialist way.

In addition to SD-processing containing A1AT solution method may include at least one additional stage of inactivation of viruses, elimination of viruses and/or elimination of prions, for example, thermoinactivation viruses containing A1AT solution. These stages can be carried out in solution, in particular directly after the stage of vysalivaniya, as contained in the solution of salt, especially sodium citrate, serve as stabilizers during the heat treatment. As another possibility suggests the use of heat treatment of liofilizirovannogo product. Alternatively, or additionally, for elimination of viruses or prions may be any suitable filtering method. For elimination of viruses and/or prion the method according to the present invention may in particular include well-known experts nanofiltration, preferably used is eating commercially available filters with pore sizes in the range from 15 to 20 nm, or any suitable filtration method. Elimination of viruses can be improved by adding amino acids, in particular at a concentration of 0,1M for each amino acid. In a preferred embodiment of the present invention add glycine in a concentration of more than 0,2M. The preferred method described by Yokoyama et al. (2004, Vox Sanguinis 86, 225-229).

Containing A1AT drug of the present invention can be obtained, in principle, in a way that is characterized by a combination of the following stages:

(a) conducting anion-exchange chromatography containing A1AT solution;

(b) optional carrying out chromatography on affinity to heparin and/or hydrophobic interaction chromatography (HIC);

(c) adding detergent and, optionally, a solvent for inactivation are covered with a lipid membrane of the virus;

(d) subsequent salting out these reagents and protein impurities;

(e) carrying out at least one stage of inactivation and/or removal of viruses.

As a rule, are suitable containing A1AT solutions obtained from blood plasma or its fractions, or recombinant or transgenic downregulation by A1AT.

In a preferred embodiment of the method according to the present invention faction Cohen IV1 restore water or buffer solution, more preferably 20 mm T is IP-buffer for ratio solution/fraction≥3/1 (preferably > 10:1), then carry out the interaction with the ion-exchange gel, preferably anion exchange gel, in the preferred embodiment, with DEAE Sepharose® (Amersham), more preferably DEAE-Sepharose® Fast Flow gel wash and elute A1AT by increasing the ionic strength of the solvent. Viral inactivation is carried out, for example, in accordance with the method according to European patent EP-A-0131740. After the optional addition of stabilizing agents add inactivating virus agents, preferably Triton X-100, Polysorbate 80 (Tween 80), TnBP and/or Caprylic acid/kaprilat, preferably to a final concentration of ≥0.1 wt.% Triton and Tween 80, ≥0.03 wt.% TnBP, ≥0.1 mm Caprylic acid/kaprilat. After an appropriate time of incubation, preferably ≥0.1 hours, more preferably ≥1 hour at ≥4°C, more preferably at ≥15°C, the salt concentration increases, in particular to the concentration of 0,5M, in particular by the addition of citrate. Thus obtained particles are removed, in particular by filtration. Repeated washing of filters or separated particles can lead to an increase in the output of A1AT. This may be followed by additional stage of inactivation of viruses, preferably pasteurization in the presence of 0,5M sodium citrate, amino acids, sugars or mixtures of these substances. The subsequent decrease in the concentration of added substances predpochtitel is but is carried out by ultrafiltration/diafiltration. More preferably, subsequent separation of the particles that carry viruses using nanofilters, preferably by means of a filter with a pore size of 15-20 nm. Thus obtained A1AT may be stored in the form of liquid or frozen drug or may be liofilizovane in accordance with methods known in the art.

Defined in the PP. 12-17 formulas of the present invention A1AT of the present invention differs from the drugs, known from the prior art. The preparations in accordance with the patents EP436086 and DE4407837 not have a high purity of the preparation according to the present invention and IgA content ≤1 mg/ml

Thus obtained product A1AT may be in the form of a solution subcutaneously, intramuscularly, topically or as an aerosol, preferably intravenously. In the form of dried material it can also be used for inhalation in powdered form. Can be used with the addition of other solutions, for example, for intravenous administration. Possible dosage is, for example, 60 mg/kg of body weight per week or 250 mg/kg in a month.

Another preferred method involves performing HIC in addition to the above-mentioned stages of the method, preferably, the holding of HIC on the phenyl matrix, preferably after SD treatment and vysalivaniya virucidal agents and other additives, as described above. predpochtitelno perform negative clearance, i.e. the desired substance A1AT passes through the chromatographic matrix without binding and unwanted substances are linked and thus are removed from the working solution.

In another preferred method, which may include the above-mentioned HIC, spend chromatography on immobilized heparin, preferably through the use of heparin-sepharose or heparin-fractogel. Thus, containing A1AT solution lead in the interaction with heparin gel in the column or the periodic process. Enriched A1AT passes through the column without binding or detected in the supernatant after the separation gel. This stage of the method is preferably carried out before or after the above-mentioned ion-exchange chromatography, or after vysalivaniya detergent and reducing the ionic strength of the solution, for example by dialysis.

The present invention also relates to a medicinal product containing A1AT of the present invention as the sole active ingredient or in combination with anti-inflammatory drugs, preferably with steroids, non-steroidal anti-inflammatory drugs, and to the use of A1AT of the present invention to obtain drugs for the treatment of A1AT deficiency, degenerative processes in the lungs, such as FIB the lake of the lungs and emphysema.

Suitable forms of introduction containing A1AT medicines, as such, are known in the art. Specifically, suitable are all forms of introduction proteins, for example, parenteral forms of administration, intravenous and inhalation administration.

The method in accordance with the present invention is additionally illustrated by the following example.

Example 1

To restore the frozen fraction Cohen IV1 was dissolved in 20 mm Tris-buffer to weight ratio of 1:9 with stirring for 3 hours at an alkaline pH.

Anion-exchange chromatography

The preferred chromatographic material for this stage of the method is DEAE-Sepharose FF (Fast Flow), although the specialist may find conditions suitable for each anion-exchange material. Filled with DEAE-Sepharose FF chromatography column was balanced 20 mm Tris buffer (pH 8.0). After that, at pH 8.0 was applied dissolved fraction Cohen IV1. Washed with equilibrating buffer solution, and then washed with buffer solution. Contacting matrix A1AT may be blueraven by washing the column 20 mm Tris-buffer (0,075M NaCl, pH 8.0). The specific activity of the thus obtained solution A1AT was approximately 0.5 PEU/mg protein (PEU: equivalent unit plasma corresponds to the activity of A1AT detected in the tournament in 1 ml of human blood plasma). The column was suirable concentrated salt buffer (e.g., 2M NaCl) and subsequently could regenerate chromatographic gel known per se methods.

Treatment with solvent/detergent

Thus obtained eluate was concentrated by ultrafiltration. Then added a pre-mixed solution of Triton X-100, TnBP and water for use in the pharmaceutical industry (WFI: water for injection) to a final concentration of 1 wt.% Triton X-100 and 0.3 wt.% TnBP. SD-processing was carried out for 4 hours at 20°C under moderate stirring.

Salting out

To remove the SD reagents and loose sediments unwanted associated protein solution containing A1AT, was diluted with a solution containing 1,5M sodium citrate and 20 mm Tris buffer at pH 7.0. When mixing was carried out by adding citrate at a concentration of 1M for at least 15 minutes. Then the working solution were incubated for at least 1 hour under moderate stirring. The resulting whitish precipitate was then filtered. This led to a decrease in the concentration SD reagents to below 10 ppm and to the separation of undesirable accompanying proteins and denatured A1AT.

After this stage of obtaining the specific activity of A1AT was at least 0,8 PEU/mg (PEU: equivalent unit plasma corresponds to the activity of A1AT detected average in 1 ml of human blood plasma).

Nanofiltration

To further increase the viral safety of the product A1AT after removal of low molecular weight substances by UF/DF filter the resulting solution through a filter with a nominal size of the penetrating particles 15-20 nm, such as filters DV20 company Pall.

The result is:

After filtration, the concentration of Triton X-100 and TNBP in the product was less than 5 ppm, Determined the content of A1AT and other protein components and compared with the content recorded after treatment with solvent/detergent. The values of degree of extraction: A1AT >80%, α-2-macroglobulin <10%, haptoglobin <40%, α-1 acid glycoprotein <10%, IgG <10%, IgA <10%, IgM <10%.

The results obtained demonstrate that the product A1AT specifically extracted in large quantities, while from the product largely removed other protein components. This is further illustrated in figure 1, which presents the results of SDS-PAGE solution before treatment with solvent/detergent (lane 2) and after the stage of vysalivaniya (lane 3). Position A1AT follows an extensive line slightly above 50 kDa (compared with the molecular weight marker in lane 1). The content of various other protein components that are clearly distinguishable in the original solution, is significantly reduced.

Example 2

As the e modification described in example 1 of the method, cooperated containing A1AT of the eluate after conducting ion-exchange chromatography with heparin-separate. A1AT passes through the filled heparin-separate column without binding. When using periodic process A1AT remains in the supernatant. Additional processing of the eluate or the supernatant in the case of periodic process was carried out as described in example 1. Thus obtained product was characterized by high purity.

The drawing shows the result of SDS-PAGE in reducing conditions, the gradient 4-20%, the coloring on Kumasi, 5 g of protein/lane. Lane 1: molecular weight marker; lane 2: sample solution in accordance with example 1 before treatment with solvent/detergent; lane 3: sample solution in accordance with example 1 after stage vysalivaniya.

1. The method of purification of alpha-1-antitrypsin (A1AT) containing A1AT
solutions from other protein components, which includes stages:
(a) carrying out ion-exchange chromatography containing AAT solution, and ion-exchange chromatography performed on an anion-exchange gel;
(b) adding detergent and optionally a solvent for inactivation are covered with a lipid membrane of the virus;
(c) further increasing the salt concentration to ≥0.5 M to vysalivaniya detergent and remove the floor is built so particles by filtering.

2. The method according to claim 1, in which the mentioned containing AAT solution from the plasma of blood or blood fractions, preferably from recovered plasma fraction Cohen IV1, or produced from recombinant or transgenic downregulation by drug AAT or supernatant after fermentation.

3. The method according to claim 1, wherein the ion exchange chromatography is carried out on DEAE-Sepharose® or DEAE-Sepharose® Fast Flow.

4. The method according to claim 1, in which the inactivation of viruses in accordance with stage (b) is performed by adding Triton X-100, Polysorbate 80 (Tween 80), TnBP and/or Caprylic acid or kaprilat preferably to a final concentration of ≥0.1 wt.% Triton and Tween 80, ≥0.03 wt.% TnBP, ≥0.1 mm Caprylic acid or kaprilat with incubation for ≥0,1 h, preferably ≥1 h at ≥4°C, especially at ≥15°C.

5. The method according to claim 1, in which, after stage (a) additionally perform chromatography on hydrophobic chromatographic materials.

6. The method according to claim 1, in which, after stage (a) additional processes containing OAT fraction material, which contains immobilized form of heparin (heparin gel).

7. The method according to claim 1, in which, after stage (C) perform the additional step of inactivation of viruses, preferably pasteurization in the presence of ≥0.5 m sodium citrate, amino acids, sugars or mixtures thereof.

8. With whom persons according to claim 1, in which the ionic strength of the solution after stage (C) is preferably reduced by ultrafiltration/diafiltration.

9. The method according to claim 1, in which, after stage (C) perform the separation of viral particles preferably by nanofiltration preferably using filters with a pore size of 15-20 nm.

10. The method according to claim 1, in which the obtained fraction AAT stored in the form of liquid, frozen or liofilizirovannogo drug.

11. AAT obtained by the method according to claim 1, with a purity of >90%, the activity ≥0,8 PEU/mg, in its active form, with the content of IgA≤1 mg/ml, residual detergent < 50 ppm, especially < 10 ppm, and a monomer content of > 90%, based on the total content of AT.

12. EAT according to claim 11, obtained by a process comprising the following stages:
restoration of plasma fractions Cohen IV1;
conducting anion-exchange chromatography on DEAE-Sepharose® Fast Flow;
optional carrying out chromatography on a solid phase, which contains immobilized form of heparin (chromatography on affinity to heparin);
optionally, the hydrophobic interaction chromatography (HIC);
inactivation of viruses by adding ≥ 0.1 wt.% Triton/ ≥ 0.03 wt.% TnBP and/or ≥ 0.1 mm Caprylic acid or kaprilat with incubation for ≥ 1 h ≥15°C;
adding salt to increase the ionic strength of the solution and
deleting received the same, the way particles by filtering.

13. A1AT indicated in paragraph 12, where then perform the additional step of inactivation of viruses, preferably pasteurization in the presence of ≥ 0.5 m sodium citrate, amino acids, sugars or mixtures thereof.

14. A1AT indicated in paragraph 12, where the ionic strength of the solution is preferably reduced by ultrafiltration/diafiltration.

15. A1AT indicated in paragraph 12, where they perform stage of elimination or inactivation of the virus and/or prion, preferably the separation of viral particles by nanofiltration preferably by means of a filter with a pore size of 15-20 nm.

16. A1AT indicated in paragraph 12, where the resulting fraction AAT stored in the form of liquid, frozen or liofilizirovannogo drug.

17. Drug for the treatment of deficiency AAT, degenerative processes in the lung, such as pulmonary fibrosis and emphysema, containing AAT obtained by the method according to claim 1, as the sole active ingredient or in combination with anti-inflammatory drugs, preferably with steroids, non-steroidal anti-inflammatory drugs.



 

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FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention refers to the selective method for preparation of "АХЭ" inhibitor - perindopril with usage as initial reagent of the sterospecific amino acid N-/1-(S)-ethoxycarbonylbutyl/-(S)-alanine which is activated by tetramethyl-uronium salts in the presence of tertiary organic base and following interreaction with (2S,3aS,7aS)-octahydroindolo-2-carbonic acid or its ester. After completing of the reaction the protective group is removed by the hydrogenation, interphase hydrogenation or extraction.

EFFECT: obtaining of perindopril with usage of tetramethyl-uronium salts as reagents of coupling reaction.

5 cl, 3 ex

Ra antigen peptides // 2359974

FIELD: biotechnologies.

SUBSTANCE: claimed invention relates to novel class II antigen peptides of main histocompatibility copmplex, which are bound with "МНС" class II molecule.

EFFECT: claimed invention allows expanding arsenal of technical means used in early diagnostics of rheumatoid arthritis.

2 cl, 5 dwg, 4 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: extraction is done using affinity chromatography with immobilised metal. The method can be realised in native conditions. Biologically active G-CSF is obtained with purity over 95%. Two more chromatography stages are done, cation-exchange and gel filtration, to remove trace amounts of impurities. The method allows for obtaining G-CSF with high output and over 99% purity.

EFFECT: described method is especially suitable for industrial production of G-CSF.

16 cl, 5 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to field of molecular biology, virology and medicine. Claimed is application of adenovirus for manufacturing medication for tumor treatment. Said virus is replication-deficient in cells which do not contain YB-1 in nucleus and encodes oncogenic protein E1A, which transactivates at least one viral gene from group including E1B55kDa, E4orf6, E4orf6 and E3ADP.

EFFECT: invention can be used for treating tumors demonstrating resistance to many cytostatic medications.

19 cl, 19 dwg, 13 ex

FIELD: chemistry, pharmaceutics.

SUBSTANCE: claimed invention relates to method of acidifying of one or several amino groups of peptide which is selected from group including exendin-3, exendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), des(B30)-human insulin and their analogues, in which reaction of acidifying is carried out in water mixture containing less than 10% wt/wt aprotonic polar solvent, and interaction of peptide with acidifying agent of general formula I is carried out, where n is 0-8; R1 represents COOR4; R2 represents lipophilic part of molecule; R3 together with carboxyl group, to which R3 is bound, represents reaction-able ester or reaction-able N-hydroxyimidoesther; and R4 is selected from group including hydrogen, C1-12-alkyl and benzyl; in base conditions in water solution, acidifying agent being added to reaction mixture in form of solution stabilised by adding acid.

EFFECT: obtaining efficient method of peptide acidifying.

17 cl, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and namely to oncourology and can be used for treatment of bladder tumours. The method is implemented in the following way: during the period prior to the operation there created is depot of cytostatic incubated with autoplasma in regional lymphatic nodes. For that purpose 50 ml of blood is taken from the patient, centrifugated at the speed of 1500 rpm during 40 min; 15 ml of the obtained autoplasma is added to cytostatic diluted in the proportion of 0.9% NaCl per 5 ml, and incubated during 1 hour at t=37°C. Then puncture of lymphatic vessel on the back of the foot is performed and cytostatic with autoplasma is injected; repeated injection in one and the same limb is carried out in 12 days.

EFFECT: use of invention provides the effect on tumour and its regional metastases owing to keeping the remedy in lymphatic bed for a long time and its being deposited in regional lymphatic nodes at low probability of complications.

1 ex

FIELD: medicine.

SUBSTANCE: method for recovering and purifying trophoblastic beta-1-glycoprotein (TBG) from retroplacental blood serum by ammonium sulphate fractionation, washing and centrifugation of the solution residual matter, affinity sepharose chromatography followed by elution and lyophilisation of an end product. The retroplacental blood is centrifugated at 10000 rpm within one hour. Sodium chloride is added to a supernatant to end concentration 0.1-1.0 mol/l, while triton X-100 is added to end concentration 0.01-0.9%. Centrifugation is performed once again. The prepared serum is saturated with ammonium sulphate to 30%, incubated within a night in stirring, centrifugated. The deposit is threefold washed in 30% ammonium sulphate, suspended in 0.15M phosphate buffered saline pH 7.0-7.5, containing 0.5 M sodium chloride and 0.05% triton X-100, dialysed in an ultrafiltration cell, with tenfold amount of the buffered saline passed, five-fold diluted and ultrafiltered at filtration limit 100000 D. The prepared permeate is processed with single affinity sepharose chromatography with immobilised monoclonal TBG antibodies. The affinity-bound TBG is eluted with 0.1M glycine-Hcl buffer solution pH 2.55. The eluate is immediately neutralised, concentrated, dialysed against physiological saline and lyophilised.

EFFECT: higher yield and purity of the end product.

2 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to ophthalmology and can be used in surgery of subretinal haemorrhages combined with age-specific macular dystrophy with subretinal neovascular membrane. The technique involves subtotal vitrectomy and removal of posterior hyaloid membrane. Bevacizumab and Ranibizumab are injected intravitreally. It is followed with intravitreal injection of tissue plasminogen activator and expanding gas while a patient is pronated.

EFFECT: method allows deploying blood from under retina, ensuring pathogenetic action on haemorrhage source, preventing proliferative processes in vitreal cavity.

2 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns biology and medicine. There is disclosed method of biotreatment blood serum production involving electrostimulation of an animal, other than a human, blood drawing of specified animal, serum separation from specified blood and gamma irradiation in a dose 10 to 40 kGy. There is offered blood serum product and pharmaceutical composition containing said blood serum product, and also their application for treatment of various diseases.

EFFECT: invention provides production of biotreatment animal's blood serum applied for treatment of wide range of diseases, including epileptic attacks and apoplexy.

24 cl, 4 ex, 7 tbl, 11 dwg

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