Method for production of alpha-1-antitrypsin and product useful in medicine

FIELD: pharmaceuticals industry, in particular new method for production of alpha-1-antitrypsin and pharmaceutical product containing the same.

SUBSTANCE: alpha-1-antitrypsin is isolated from Cohn fraction IV-1 by solubilization. Then protein admixtures and eventual viral particles are removed by polyethylene glycol, target protein is precipitated with zinc salt, viral inactivation using solvent/detergent is carried out, product is fractionated using Q-sepharose, and non-active alpha-1-antitrypsin is removed with S-sepharose to produced target product. Said product represents concentrate of human serum active alpha-1-antitrypsin having purity more than 98 % and specific activity not less than 40 IU/mg in 0.15 M sodium chloride solution.

EFFECT: increased yield of high pure active alpha-1-antitrypsin.

4 cl

 

The invention relates to the field of biotechnology, pharmacy and medicine and relates to a new method of producing alpha-1-antitrypsin, pharmaceutical product which can be used in medicine.

Alpha-1-antitripsin (AAT) is a glycoprotein with a molecular mass of about 55 kDa. The main function of the inhibitor of serine proteases, such as trypsin, elastase, chymotrypsin and other currently AAT is used for chronic replacement therapy in hereditary deficiency of alpha-1-proteasome inhibitor with clinical signs panacinar emphysema or decline of forced expiratory volume 1 (FEV1) less than 50%, for the prevention of chronic lung disease in preterm infants, including those requiring mechanical ventilation. The low purity of drugs (drug Prolastin", Bayer Corp. contains not only about 40% of foreign proteins and inactive AAT - Lomas DA, et al Eur.Respir.J. 1997, 10, 672-675; Lebing WR et al. US Patents 5610285 and 6462180) restricts the use of AAT in other areas. Other restrictive point of use products containing alpha-1-antitripsin, is their high price, which is connected with the cost of raw materials and imperfect technology. One of the main problems in creating effective method of obtaining AAT - stabilizationand serpina on all critical process steps. The solution to this problem and this invention is directed.

Prior art

Serpina belong to the group of proteins, the native conformation which is in a metastable condition. The transition is energetically favorable condition occurs during execution of serpine its main function is the inhibition of serine proteases. Artificial translation of native AAT from the metastable state to a more stable leads to the loss of protein in its functional activity. This raises the problem of stabilization of conformational States of native AAT in the process of separation, purification and storage. This will allow you to obtain not only high-level end product, but will increase its output in the cleanup process.

Large-scale conformational rearrangement characteristic of Sechenov, are determined by the characteristics of their spatial organization and interaction with the surrounding solvent. So, for drug AAT, obtained by recombinant DNA technology offered compared with dextran, which increases the stability of the finished product when heated and the change in pH (Gautam Mitra, US Patent 4496689).

A well-known fact of stabilization of protein structure by introducing into the aqueous solution of additional compounds: sugars, amino acids, and the like (see, e.g. the measures S.N. Timasheff Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 9721-9726 ). Although this fact is widely known, completely unclear mechanism of their influence. At the same time, the same connection can protect protein from the same influences (e.g. temperature increase) and no (or even negative) impact on the stability of the protein to other influences. Therefore, in each concrete case it is necessary to solve the problem on its own. So, it is known that such a pitch as trimethylamine-oxide, protects from AAT polymerization (Delvin GL et al. Am. J. Respir. Cell Mol. Biol. 2001, 24, 727-732), and sucrose and citrate protects the dried product AAT from thermal denaturation (Coan VY, Mitra G, Vox Sang 1984, 46, 142-148; Glaser CB, et al. Am. Rev. Respir. Div. 1983, 128, 77-81). However, in the case of citrate there is evidence and on a completely opposite effect - facilitating the transition to the latent form (Koloczek H et al. Protein. Sci. 1996, 5, 2226-2235; Bottomley SP Biochem. Biophys. Acta 2000, 1481, 11-17). Moreover, it is unclear whether this protection is reliable when processing the solvent/detergent. It is known that this technique is widely used for viral inactivation of plasma preparations, but significantly inactivate AAT (Mast AE et al Blood 1999, 94, 3922-3927). However, how to ensure the stabilization of the AAT under this treatment? The resolution of this question will not only significantly increase the output of the active AAT, but also to make the whole process more efficient.

Most the similar sequence of processes of the present invention is a method for AAT, described in the patent Lebing WR et al. US Patent 6462180.

In the method-prototype (Lebing WR et al. US Patent 6462180) method of obtaining AAT was that after solubilization fraction Cohn IV-1 in a buffer solution with a pH of about 9.5 (4.5 hours at a temperature of 8-20°and 1-1 .5 hours at a temperature of about 40° (C)to the suspension was added polyethylene glycol 3350 (PEG) to a final concentration of 11.5%, summing the pH to 5.1 to 5.35 units. After that they used a specific sorbent (Hyflo Supercel. RTM) to remove lipoproteins. After incubation, the precipitate was discarded. To the supernatant was added tween-20 and, after bringing the pH to 7.0, incubated for 8-10 hours at 20-30°C. thereafter, the mixture was applied on aminoalkenes (Q-sepharose). Bound peroxidase AAT was suirable and was passed through a strong cation-exchanger, pre-adjusted pH to 5.5. Under these conditions, the target protein not contacted with the carrier. The resulting solution was subjected diafiltration with simultaneous ultrafiltration. After the final sterile filtration of the preparation is freeze-dried. The authors argue that in this way it is possible to obtain the product of about 95-98% purity (according to immunonephelometry) output target product 50-75% of the starting material. Cleaning time is plotted in 40 hours. If this is not provided data on the activity of the final product and its change during selection. In this regard, it is difficult aanitiedot a key aspect in the processing chain, moreover, at the stage of viral inactivation was not introduced to protect the target product, although it is well known (Mast AE et al Blood 1999, 94, 3922-3927)that AAT sensitive to this treatment.

The specific activity of the alpha-1-antitrypsin deficiency is determined by its ability to inhibit the elastase using, typically, a chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-alanyl-PMA (see, for example, Gautam Mitra US patent 4496689). Elastase it pNA from the substrate that is logged to increase the absorption at 405 nm. In the presence of excess elastase specific activity of alpha-1-antitrypsin was measured spectrophotometrically at 405 nm using a calibration graph constructed using serum containing certified amounts of alpha-1-antitrypsin (for example, Protein-Standard-Serum LC-V (Human) Behring containing 5,6 IU/ml). There are many discrepancies in the determination of the specific activity, as in the majority of works remains uncertain method for determining the amount of protein. In addition, when the calculations, as a rule, does not take account of the fact that alpha-1-antitripsin - glycoprotein. All this leads to discrepancies in the comparison of the specific activity electrophoretic pure drugs within 20-25%.

Disclosure of inventions

As a source of raw materials used fraction Cohn IV-1, which is the by-product of the fractionation is lazmi blood. After its solubilization was performed processing suspension of polyethylene glycol. The precipitate was discarded, and the supernatant was added zinc salts, such as chloride, zinc sulfate, etc, and after adjusting the pH incubated for one hour. The precipitate was dissolved in phosphate buffer containing EDTA. After dialysis against a solution containing glycine (0.9%) and lysine (0,3%), was added Triton X-100 and tri-n-butyl phosphate, adjust pH to 7.3 units and incubated for 6 hours at room temperature. Then, after bringing the pH to 6.5, was applied on a column of Q-separate. After elution of the target product associated with the medium, the solution was subjected to diafiltration against 20 mm phosphate buffer pH 5.5 and was passed through a column of S-separate to remove inactive AAT. In the fractions obtained were adjusting pH and adding ODM sodium chloride, centrifuged and sterile filtered. The activity of the drug is controlled at all stages of its purification using a chromogenic substrate. The purity of the final preparation was more than 98% of the active AAT, the yield of active protein - more than 50% compared to active AAT in the faction Cohn IV-1, specific activity was not less than 40 IU/mg 0,15M solution of sodium chloride at 37°C.

The AAT activity was assessed by its ability to inhibit the elastase as chromogenic substra is and used the N-succinyl-(L-alanyl) 3-paranitroaniline. The reaction course was monitored by the change in optical density at 405 nm. Quantitative determination of antigen AAT was performed using plates Partigen company Data-Behring.

The best option of disclosing inventions

To 100 g of fraction Cohn IV-1 was added 1500 ml of 50 mm sodium chloride, pH 6.0 and was stirred for one hour at a temperature of +4°C. After 15 minutes of centrifugation at 4500g supernatant was discarded and to the precipitate was added 1500 ml of 10 mm Tris buffer, pH 8.5. After 2 hours incubation at +23°the temperature was lowered to +4°and to the solution was added sodium chloride to a concentration of 0.11 M and polyethylene glycol to a concentration of 11.5%, the pH was brought to 5.25 0.5 M acetic acid, and incubated for one hour. The precipitate was discarded after 15 minutes of centrifugation at 4500g. To the supernatant volume 1810 ml) was added 8 ml of 1M sodium hydroxide to bring the pH to 7.1. Then add 90 ml of 100 mm of zinc chloride, summed pH 0.5 M Tris to 8,09 and incubated for one hour. After 15 minutes of centrifugation at 4000g, the supernatant in the volume of 1900 ml was discarded, and 24 g of sediment was added 200 ml of a solution containing 10 mm EDTA, 20 mm sodium phosphate, pH 7.4. Then the solution were dialyzed against 20 mm sodium phosphate, pH 7,2, the conductivity of the solution, 2.4 Simens/see then to 230 ml of the resulting solution was added glycine to 0.9% concentration, lizondo of 0.3% concentration, 23 ml of 11% Triton X-100 and 0.72 ml of tri-n-butyl phosphate, adjust pH to 7.3 units with 0.5 M Tris and incubated for 6 hours at room temperature. Then brought the pH to 6.5 units using 1 M acetic acid and applied to a column containing Q-sepharose (2,6×14 cm). The column was equilibrated to 20 mm sodium phosphate pH 6.5, conductivity of a solution of 2.4 Simens/see After washing the column elution was performed with 100 mm sodium chloride in 20 mm phosphate buffer, pH 7.0. 260 ml of the eluate was subjected to diafiltration against 20 mm phosphate buffer, pH 5.5, containing 5 mm sodium chloride, conductivity 2.4 Simens/see the resulting solution was applied onto a column of S-separate (1×15 cm), equilibrated with the same buffer. The breakthrough was collected, adjust the pH to 7,05 using 0.5 M Tris. Drove the concentration of sodium chloride to 0,15M, centrifuged for 15 minutes at 4500 g and sterile filtered. The result has been of 0.82 g of the target protein purity more than 98% and the yield of active product at 51.4%, the activity of elastase inhibitor was 40 IU/mg of the Obtained product was pyrogen-free, did not show toxicity in experiments on laboratory animals (rats, rabbits), did not cause allergic reactions when intradermal or intravenous injection.

Another aspect of the claimed invention is a product, for use in medicine, veterinary medicine and/or research purposes, containing a concentrate of active alpha-1-antitrypsin in the blood plasma of a person with a specific activity of 40 IU/mg in 0.15 M solution of sodium chloride and purity more than 98%, obtained as described above.

The product may be a solution or a lyophilized powder (for which the above concentrate is transferred into vials and dried by freezing under vacuum). If necessary, it may further contain conventional pharmaceutical excipients, solvents and/or excipients that are acceptable for parenteral (e.g. intravenous bolus or infusion, intramuscular, intranasal) administration. Listed excipients are mixed with a dry concentrate or added to the solution according to conventional methods. Fillers are, for example, sugars (e.g. glucose, lactose, sucrose, maltose, or mixtures thereof), a restored sugar (aritra, mannitol and other) in a physiologically acceptable concentrations, if necessary, antioxidants, traditional injectable pharmaceutical compositions. Optionally, the product may contain inorganic and/or organic salts and their combinations to maintain an acceptable pH values.

The solvent for the lyophilized product can serve distilled pyrogen-free sterile water, f is Zoologicheskii solution buffered saline, dextrose.

You can also liofilizirovanny ready solution containing concentrate AAT and any of the listed excipients, or a combination thereof.

If necessary, the concentrate containing the AAT, it is possible to dissolve before introduction into the blood plasma, blood serum of the patient or mix with solutions and/or protein fractions (proteins).

The product can have an appropriate package and, optionally, instructions for use.

Proposed according to the invention, the product can be used in wider areas compared with previously known. In statistically and methodologically sound experimental studies (experimental animals, cell lines), it was shown that in contrast to the known analogues of the drug has demonstrated high efficiency in pathologies characterized by both local and generalized activation of proteolytic enzymes: sepsis and systemic inflammatory response of any etiology, inflammation of the skin of various origins, the treatment of rheumatologic diseases.

1. A method of obtaining a concentrate of alpha-1-antitrypsin deficiency, which consists in the fact that the fraction of Cohn IV-1 add 50 mm sodium chloride pH 6.0, stirred for one hour at 4°C, centrifuged 15 mi is at 4500 g, to the precipitate add 10 mm Tris buffer, pH 8.5, and incubated for 2 hours at 23°C, cooled to 4°add sodium chloride to 0.11 M and polyethylene glycol 11.5%, and establish a pH of 5.25 and incubated for one hour, then centrifuged 15 min at 4500 g, the supernatant add 1 m sodium hydroxide to a pH of 7.1, then add 100 mm zinc salts, establish the pH of 8.09 0.5 M solution of Tris buffer and incubated for one hour, then centrifuged 15 min at 4000 g, to the precipitate add a solution containing 10 mm EDTA, 20 mm sodium phosphate, dialist add glycine to 0.9%, lysine 0,3%Triton X-100 11%, tri-n-butylphosphate, set pH to 7.3, incubated for 6 h at room temperature, set pH 6.5 and applied to a column containing Q-sepharose, elute 100 mm sodium chloride in 20 mm phosphate buffer, the eluate is subjected to diafiltration against 20 mm phosphate buffer, pH 5.5, containing 5 mm of sodium chloride, the resulting solution was applied onto a column of S-separate, set pH 7,05, the concentration of sodium chloride 0.15 M, centrifuged 15 min at 4500 g and sterile filtered.

2. The product obtained by the method according to claim 1, represents a concentrate of active alpha-1-antitrypsin in the human blood plasma purity more than 98% with a specific activity of not less than 40 IU/mg in 0.15 M solution of sodium chloride.

3. The product according to claim 2, characterized in that it presents in the form of a solution or in lyophile the new form.

4. The product according to claim 2 or 3, characterized in that it further comprises pharmaceutical excipients, solvents and/or excipients that are acceptable for parenteral, inhalation, intranasal, and transdermal applications.



 

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