Method for preparing human c-1 esterase inhibitor amd product for using in medicine

FIELD: biotechnology, medicine, pharmacy, veterinary science.

SUBSTANCE: method involves addition of DEAE-Sephadex A-50 to cryosupernatant from human blood plasma, incubation, filtration and addition of QAE-Sephadex to filtrate followed by incubation. Filtered off precipitate of QAE-Sephadex is subjected for successive step-by-step washing out with buffer solution at pH 5.5 and 7.5, elution at pH 7.7 and dialysis. Then PEG-6000 is added to dialyzed solution to the concentration 12%, solution is incubated and centrifuged. To the prepared supernatant glycine is added to the final concentration 100 mM and lysine is added to the final concentration 10 mM at pH 7.2, then Twin-80 is added and pH value is corrected to 6.8-7.2 followed by addition of tri-n-butyl phosphate to the final concentration 0.3%. Prepared suspension is stirred, subjected for chromatography on DEAE-Sepharose FF at pH 7.0, chromatography on Zn-chelating Sepharose FF at pH 7.5 and the end product with specific activity from 7.5 ± 0.5 U/mg of protein and above, and with the final concentration of lysine 10 mM, not less, and with the final concentration of glycine 100 mM, not less. Method provides safety of activity in antiviral treatment and preparing product containing the natural C-1 esterase inhibitor from blood plasma with high specific activity.

EFFECT: improved method for preparing.

6 cl, 2 dwg, 1 ex

 

The invention relates to the field of biotechnology, pharmacy and medicine and relates to a new method of obtaining Cl-astaranga inhibitor, pharmaceutical product which in recent times has been increasingly used in medicine.

Cl-Esterhazy inhibitor is a protein in the blood (the molecule is a chain of 478 amino acid residues, a molecular mass of 105 kDa) and the main inhibitor of the classical pathway of the complement system, an indirect inhibitor kallickrein-kinin, coagulation and fibrinolytic systems, which, in turn, produce biologically active proteins. That is, Cl-Esterhazy inhibitor is one of the regulators development of inflammatory reactions in the body and inhibits activated factor XI and can serve as an inhibitor of coagulation, and tissue plasminogen activator and plasmin (A. Groner et al., US 6242239).

This role of Cl-astaranga inhibitor in the body determined the possibility of its use in substitution therapy in angioneurotic edema, for the prevention and treatment of sepsis, including heavy, and septic shock, for the prevention and treatment of multiple organ dysfunction syndrome increased capillary permeability in sepsis and septic shock, for the prevention and treatment of acute respiratory distress syndrome, adjuvant therapy is ri atherosclerotic lesion of vessels of any localization, for the prevention and treatment of ischemic stroke and its complications, recurrent acute disorders of cerebral circulation, prevention and treatment of shock in the operative and postoperative periods during coronary artery bypass surgery, for the prevention and treatment of shock when transluminal angioplasty for prevention and treatment of reperfusion syndrome after coronary artery bypass surgery and transluminal angioplasty in combination with indirect anticoagulants for the prevention and treatment of rejection reactions by transplantation of organs and tissues (including bone marrow), for the treatment or prevention of systemic rheumatic diseases associated with activation of complement (systemic lupus erythematosus, systemic sclerodermia, dermatomyositis, rheumatoid arthritis etc), for the prevention and treatment of complications in patients with trauma injuries of different localization, for adjuvant therapy of any state, including neoplasms that occur with activation of the complement system.

An important indicator of Cl-astaranga inhibitor is its specific activity expressed as u/mg protein or IU/mg protein (hereinafter - international units) or U/mg (IU/mg).

However, proven research diversity Cl-astaranga inhibitor does not allow wide use in medical practice because of low purity is still the drug: the best results shown W.Schoenhofel et al. (EP 1244706) for transgenic Cl-astaranga inhibitor as a result of complex and expensive purification systems, do not exceed 6 ME on 1 mg of protein when declared theoretically possible (but not confirmed) activity up to 8 IU/mg (activity determined by generally accepted in the present method using the test system Berichrom Cl-INH, Dade-Behring Diagnostics). He should be ignored and unwanted side effects caused by the introduction into the organism of foreign proteins (allergic reactions, thrombosis, etc.) (Caiezi S. et al. Pharmacological Reviews 2000, v.52 (1): 91-112).

The main objective of the invention is a product containing natural Cl-Esterhazy inhibitor from plasma with initially high activity, 7.5±0.5 IU/mg protein and more.

Prior art

Known methods for producing Cl-astaranga inhibitor from human blood plasma. They all boil down to a combination of pre-treatment of the protein with the use of polyethylene glycol (PEG) and two or three chromatographic steps. However, either these were laboratory approaches that are difficult to scale to a wide industrial production (Harrison R. Biochemistry, 1983, 22, 5001-5007)or uses expensive affine media (Pilatte Y.M. et al., US 5030578), either method gave a low yield of the final product and had a number of technical and ek the economic disadvantages (Poulle, M. et al., US 5681750), or allocated inactive Cl-inhibitor (Rosina mathematical SCIENCES. and other RF 2068693), or the proposed approach did not give a good yield of the final product with a high specific activity in the absence of effective stabilizers on the stages of viral inactivation (Schoenhofer W. et al. EP 1244706).

Selection of the effective stabilizers plays an important role during viral inactivation, such as processing solvent/detergent or heat treatment, since it is known that these factors can lead to irreversible conformational changes Sechenov (Mast, A.E. et al., Blood 1999, 94, 11, 3922-3927), which, in turn, adversely affects their activity.

To stabilize proteins are widely used sugar, polyols, polymers, amino acids (See, for example: the Structure and stability of biological macromolecules under the editorship of Timasheff S.N. and Fasman GD M.: Mir, 1973; Privalov PS Biophysics 1987, 32, 742-759; Introduction to applied etymology Ed. by I.V. Berezin and Martinek K. M.: Moscow state University, 1982; Aisina RB and other Bioorganic chemistry 1994, 20, 2, 182-189). The choice of stabilizer is determined by both the nature of the stabilized protein and the nature of the impact.

In particular, the above sugars, amino acids, organic acid salts (sodium kaprilat, citrates of potassium, sodium or ammonium) was previously used to stabilize Cl-esterase the inhibitor in the process of getting it (Craigenne A.W. US 4379085; J Eibl et al US 5733885).

Because the greatest efficiency when antiviral treatment plasma and its components is a combination of methods, selection of the most effective protection for the target protein is a daunting task. For Cl-astaranga inhibitor this aspect is poorly known. Apparently, this is one of the main reasons for low yield of the target protein for most of the reported methods for its isolation and purification. The increasing quality of the final drug along with the obvious benefits can significantly extend the scope of its application.

The closest to the essence of the present invention is a method for Cl-astaranga inhibitor described Poulle, M. et al. in the US 5681750.

In the method-prototype (Poulle, M. et al. US 5681750) features of Cl-astaranga inhibitor was that free cryoprecipitate plasma were subjected to preliminary purification of vitamin K-dependent proteins on the column with DEAE-separate and anti-thrombin III - heparin-sepharose. Thus obtained fraction containing Cl-Esterhazy inhibitor, was applied to a weak aminoalkenes (DMAE-type) for exemption from related proteins (albumin, immunoglobulins, alpha-antitrypsin, alpha-2-macroglobulin and so on).

After antiviral treatment solvent/detergent (about changed and the activity of the target protein during this procedure, the authors do not report) the mixture is applied to a strong cation-exchanger for removal of anti-virus agents. Fractionation on this media allows us to be freed from the IgM. After concentration and dialysis by ultrafiltration membrane are responsible for final procedure for removal of viruses. To do this, the authors suggest the use of nanofiltration consistently on filters with a pore diameter of 35 nm and 15 nm. The finished product had a specific activity of about 6 u/mg protein.

It is evident that used a combination of methods in the method prototype has several disadvantages. First, this method can only exist by itself, and it cannot be implemented as a module in the process of fractionating plasma at Stake, which makes it uneconomical. Secondly, in the method prototype is not taken into account the fact that serpine, which include Cl-Esterhazy inhibitor, can significantly lose its activity during the anti-virus processing of solvent-detergent (Mast, A.E. et al., Blood 1999, 94, 11, 3922-3927).

Disclosure of inventions

In this regard, the task was to develop a method of obtaining a concentrate of CL-astaranga inhibitor devoid of these disadvantages, using easily scalable stage.

The inventive method differs from human blood plasma get concentrate CL-astaranga inhibitor with specific activity in the final preparation of 7.5±0,5 IU/mg protein or more, containing lysine in the final end of the ation of not less than 10 mm glycine at a final concentration of not less than 100 mm. The method includes the following mandatory steps in the sequence indicated: remove plasma proteins using ion exchangers, polyethylene glycol fractionation, adding to the resulting solution of glycine and lysine, conducting viral inactivation, destruction of its products by chromatography and thin purification of the final product from impurities.

Thus preferably the consistent use of weak and strong anion-exchanger at different pH values, and performing the fine purification of the final product by the method of metal-gelatinous chromatography; also preferably, the viral inactivation spent solvent-detergent.

The advantage of the inventive method is the ability after use of ion exchangers for the direction of the plasma in the process of its production of protein drugs alcohol by the method of Cohn.

In more detail the method shown in Fig. 1 (scheme receiving concentrate CL-astaranga inhibitor):

first, preliminary stage exempt from proteins prothrombin complex. For this purpose, fresh frozen plasma is thawed, separated by centrifugation cryoprecipitate and cryosupernatant add dry DEAE-Sephadex a-50 at the rate of 1.5 g per liter of cryosupernatant. After incubation with constant stirring, the precipitate was separated by filtration. Sediment OTP is ablaut on regeneration or receiving components of the prothrombin complex. To the filtrate after summarizing the pH to 5.5 with acetic acid add dry Q-Sephadex based 2.5 g per liter of plasma. After incubation the Sephadex adsorbed thereon proteins separated by filtration and subjected to successive washing, the pH of the filtrate adjusted to the original level and return for further fractionation at Stake.

The filter cake containing Q-Sephadex and sorbed thereon proteins, including Cl-Esterhazy inhibitor, is subjected to successive washing first with 100 mm sodium acetate buffer pH 5.5, containing 50 mm sodium chloride, then with 20 mm Tris-Hcl buffer pH 7.5, containing 200 mm sodium chloride. The elution spend 20 mm Tris-Hcl buffer pH of 7.7, containing 1 M sodium chloride.

The fraction containing Cl-Esterhazy inhibitor, is subjected to diafiltration against 20 mm Tris-Hcl buffer pH 7.4, containing 50 mm sodium chloride. Add PEG-6000 to a concentration of 12% and incubated with stirring in the cold for hours. By centrifugation at 4000 rpm for 10 minutes to obtain a residue blue and colorless supernatant.

The supernatant is separated and add glycine to a final concentration of 100 mm and a lysine to a final concentration of 10 mm, pH down to 7.2 with 0.5 M Tris. To the resulting solution was added tween 80 to a final concentration of 1% and three(n-butyl)phosphate to a final concentration of 0.3% DL is holding inactivation of viruses. The process is carried out at room temperature for 6 hours. Loss of activity when this is not observed. In the absence of stabilizers activity fell by about 50% (Fig. 2: the Stabilizing role of glycine and lysine. And the activity of the Cl-astaranga inhibitor before the procedure viral inactivation, B - activity of Cl-astaranga inhibitor when conducting viral inactivation in the absence of glycine and lysine, In the activity of the Cl-astaranga inhibitor when conducting viral inactivation in the presence of glycine and lysine). For determining the activity of the used test system Berichrom Cl-INH, Dade-Behring Diagnostics, Germany (Plasma calibrator Plasma S Dade-Behring). After the procedure, viral inactivation adjust the conductivity to 6.0 mS/cm.

The mixture was applied to a column of DEAE-separate FF equilibrated with 10 mm Tris buffer pH 7.0, containing 50 mm sodium chloride. The column was washed with 10 mm Tris-Hcl buffer pH 7.0, containing 100 mm sodium chloride. The elution of the target protein were 10 mm Tris-Hcl buffer pH 7.0, containing 250 mm sodium chloride.

The final, fine purification of Cl-astaranga inhibitor was performed using medulloblastomas chromatography. The purpose of this step is to get rid of minor impurities. Target protein passes through the column, not Cerberus, same impurity proteins remain associated with the media. For this purpose caretcolumn, filled with Zn-chelating separate FF equilibrated with 10 mm Tris buffer containing 250 mm sodium chloride, pH 7.5, missed the protein solution obtained at the previous stage. Target protein passed through the column without delay, the column was barbirollis remains an undelimited ceruloplasmin. Regeneration of the column was performed with 50 mm sodium acetate buffer pH 4.0, containing 500 mm sodium chloride. After this column was balanced startover buffer.

The yield of the target protein was 30-40%. The specific activity of Cl-astaranga inhibitor in the final product (concentrate) is 7.5±0,5 IU/mg protein; lysine not less than 10 mm glycine - not less than 100 mm.

For a more detailed presentation of the proposed method can be illustrated by an example.

Example (best option of carrying out the invention)

To 2 l of cryosupernatant blood plasma is added 3 g DEAE-Sephadex A50. After incubation for 1 hour the precipitate was separated by filtration. the pH of the thus obtained filtrate was brought 1M acetic acid to 5.5. In the specific example in 1900 ml of the filtrate was added 90 ml of acid. The temperature of the solution was brought to 5-8°and added dry QAE-Sephadex A50 based 2.5 g per 1 liter of plasma. After two hours of incubation, the sorbent with the associated Cl-astraslim inhibitor was filtered, and the pH of the filtrate is brought up to 7.3 to 7.4. (Received Rast is the PR can be used for further fractionation, in particular Stake). QAE-Sephadex with its associated proteins were left on the filter and was subjected to a washing step. The process was carried out at a temperature of 5-8°C. First washed with 1 l of 100 mm sodium acetate buffer pH 5.5, containing 50 mm sodium chloride. Then 1 liter of 20 mm Tris-Hcl buffer pH 7.5, containing 200 mm sodium chloride. The elution was performed with 300 ml of 20 mm Tris-Hcl buffer pH of 7.7, containing 1 M sodium chloride. The eluate were dialyzed against 20 mm Tris-Hcl buffer pH 7.4, containing 50 mm sodium chloride.

To a 250 ml cialisovernight solution was added 79 ml of a 50%aqueous solution of polyethylene glycol 6000. The resulting suspension was stirred for 1 hour at a temperature of 5-8°C, then centrifuged for 10 minutes at 4000 rpm/min

To the resulting 325 ml of supernatant was added glycine and lysine to a final concentration of 100 mm and 10 mm, respectively, and then adjust the pH with 0.5 M Tris, leading up to 7.25 units. Then contributed 11% of tween-80 to a final concentration of 1% and again adjust the pH, bringing it up to 6.8 to 7.2 with 0.5 M Tris. After this was added tri-n-butyl phosphate in an amount necessary to create a final concentration of 0.3%. The suspension was stirred for 6 hours at room temperature, followed by a 2 M sodium chloride drove the conductivity of the solution to a value of 6.0 to 6.2 mS/cm, a pH to 6.8-7.0 and using 0.5 M three is.

The resulting solution was applied to a column containing DEAE-sepharose, balanced 10 mm Tris-Hcl buffer with pH 7.0, containing 50 mm sodium chloride and having a conductivity of 6.0 mS/cm. The column was washed with the starting buffer and then with 10 mm Tris-Hcl buffer pH 7.0, containing 100 mm sodium chloride. The conductivity of the buffer of 11.4 mS/cm. The target protein was suirable 10 mm Tris-Hcl buffer pH 7.0, containing 250 mm sodium chloride.

100 ml of a solution containing the target protein was passed through a column filled with Zn-chelating separate and equilibrated to 10 mm Tris-Hcl buffer pH 7.5, containing 150 mm sodium chloride. Regeneration of the column was performed with 50 mm sodium acetate buffer pH of 4.0, containing 0.5 M sodium chloride.

The specific activity of Cl-astaranga inhibitor in the final product (concentrate) was 7.6 IU/mg protein, lysine, not less than 10 mm glycine - not less than 100 mm. To evaluate the activity of Cl-astaranga inhibitor used test Berichrom Cl-INH company Dade-Behring Diagnostics, Germany.

The yield of the target product was 37%. The resulting 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 pharmaceutical product for use in medicine, veterinary medicine and/or science-the nama Mala research purposes, containing concentrate Cl-astaranga inhibitor, lysine at a final concentration of not less than 10 mm glycine at a final concentration of not less than 100 mm, obtained by the method above.

The product can be presented in the form of a solution, lyophilized powder (for which the above concentrate is transferred into vials and dried by freezing under vacuum), it can also optionally contain conventional pharmaceutical excipients, carriers, solvents and/or excipients that are acceptable for parenteral (e.g. intravenous bolus or infusion, intramuscular, intra-articular, coronary, subcutaneous, intradermal, intraorbital and/or intracranial infusion. Listed excipients are mixed with a dry concentrate or added to the solution according to the conventional methods in accordance with the requirements in the technology of finished dosage forms (see, for example, “the Technology of drugs” Isagain or “Technology of medicinal forms” edited Tschechei).

Fillers are, for example, sugar (specifically, glucose, lactose), proteins such as albumin, optional antioxidants, traditional injectable pharmaceutical compositions. If necessary, the product may contain inorganic and/or organic salts and/is whether their combination to maintain pH in the range of 7.2 to 7.4. Solvents can serve distilled pyrogen-free sterile water, saline, buffered saline, a solution of 1,3-butanediol, glycosaminoglycans, dextrose, the solution may contain, if necessary proteins serum - albumin and/or ceruloplasmin (the latter can also be fillers in dried finished product).

You can also liofilizirovanny ready solution containing concentrate Cl-astaranga inhibitor, glycine and lysine, and any of the listed excipients or combinations thereof.

If necessary, the concentrate containing CL-Esterhazy inhibitor, lysine at a final concentration of not less than 10 mm glycine at a final concentration of not less than 100 mm, it is possible to dissolve before introduction into the blood plasma, blood serum of the patient, or to mix with solutions and/or protein fractions (proteins).

The product can also be made in the form of a single dosage form, for example, in the form of vials or vials with a solution or with liofilizirovannam concentrate containing CL-Esterhazy inhibitor, lysine at a final concentration of not less than 10 mm glycine at a final concentration of not less than 100 mm and optional excipients or combinations thereof, while the activity of Cl-astaranga inhibitor in each of one dose of 1, 5, 10, 20, 50, 10, 200 or 500 units; the product can additionally 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: treatment of septic shock, including refractory phase of the ineffectiveness of standard antishock therapy, treatment and prevention of the syndrome of multiple organ failure, prevention and treatment of acute respiratory distress syndrome, adjuvant therapy for atherosclerotic vascular lesions of any localization, treatment and prevention of systemic rheumatic diseases associated with activation of complement (systemic lupus erythematosus, systemic scleroderma, dermatomyositis, rheumatoid arthritis etc).

1. A method of obtaining a concentrate of C1-astaranga inhibitor of man, characterized by the fact that cryosupernatant of human blood plasma add DEAE-Sephadex a-50 at the rate of 1.5 g per liter, incubated, filtered, the filtrate at pH 5.5 add QAE-Sephadex based 2.5 g per 1 liter of plasma, incubated for two hours, filtered, the filter cake QAE-Sephadex subjected to sequential manual washing buffer solution at pH 5.5 and 7.5, elution at pH of 7.7, dialysis at pH 7.4, to cialisbuynow solution add PEG-6000 to the end of the ation 12%, incubated at a temperature of 5-8°C, centrifuged, the resulting supernatant add glycine to a final concentration of 100 mm and a lysine to a final concentration of 10 mm at pH of 7.2, contribute tween 80 to a final concentration of 1%, adjusting the pH to 6.8 to 7.2, then add tri-n-butyl phosphate to a final concentration of 0.3%, the resulting suspension is stirred, subjected to chromatography on DEAE-sepharose FF at pH 7.0, chromatography on Zn-chelating sepharose FF at pH 7.5 and obtain the target product with a specific activity in the final drug 7.5±0,5 IU/mg protein or more, containing lysine at a final concentration of not less than 10 mm glycine at a final concentration of not less than 100 mm.

2. The product represents a concentrate of C1-astaranga inhibitor, characterized in that it is obtained from human blood plasma by the method according to claim 1 with a specific activity of 7.5±0,5 IU/mg protein or more and contains lysine at a final concentration of not less than 10 mm glycine at a final concentration of not less than 100 mm.

3. The product according to claim 2, which is a solution or lyophilized.

4. The product according to claim 2, which further comprises a pharmaceutically acceptable for parenteral administration fillers, carriers, solvents and/or excipients.

5. The product according to claim 2, which is intended for use in medicine, veterinary medicine and/or science issledovatel the purposes.

6. The product according to claim 2, which is intended for the treatment or prevention of conditions associated with the activation of the complement system.



 

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