Method for purification of interferon proteins by cation-exchange chromatography

FIELD: preparative biochemistry, medicine, pharmacology.

SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.

EFFECT: improved purifying method.

8 cl, 1 tbl, 6 ex

 

Background of invention

A large part of the biomedical Sciences based on the use of pharmacologically active proteins of natural origin, obtained by means of extraction technology and synthetic origin, derived from recombinant DNA using the developed methods. The purity level of interest products is important in both cases, because understanding their activity is determined by the ability to accurately link the biological action with the presence of a fixed amount of protein.

In this context, the cleaning methods of pharmacologically active proteins have a major role, because the purity of the obtained protein acquires a special significance when drugs are included active principle or fillers. The possibility of toxic and/or occurrence of adverse effects during treatment really makes the administration responsible for registration and issuance of marketing approval of drugs based on proteins, to introduce more stringent rules for determining the quality and industrial compliance began active protein contained in the selling medicines.

From the foregoing clearly demonstrates the importance of cleaning methods (1-5), 135-200 (1999), Mullick A. and Flickingr M.C., Biotechnol. Bioeng., 65(3), 282-290 (1999) and Mc Creath G.E. et al., J. Chromatogr., 597(1-2), 189-196 (1992).

Finally, Yang L. et al., Sheng Wu Cheng Kung Hsueh Pao, 16(1), 74-77 (2000) also described are methods for removal of albumin on heavy metals and methods affinity for the matrix, with associated molecules of dyes, such as Zibarro blue F3G, were described Compagnini A. et al., J. Chromatogr. A, 736(1-2), 115 (1996).

In the patent US 6194553 disclosed a method of purification of an inhibitor of alpha-1 proteinase using cation-exchange chromatography on a solid matrix, where cation exchange chromatography is carried out at a pH value of more largely than the pH corresponding to the isoelectric point.

In document EP 0203382 disclosed a method of purification of alpha-interferon using cation-exchange chromatography at pH 4.5 to 5.0.

All these methods show different ways of problem complexity and high cost, so the problem of individualization of new cleaning methods pharmacologically active proteins as light and easy industrial feasible and cost-effective remains unsolved.

The following invention provides an answer to these important requirements by developing a method of purification of interferon proteins, which is easy in operation and low in cost, with significant economic benefits.

DISCLOSURE of INVENTIONS

Present from retina relates to a method of purification of interferon proteins, which includes conducting cation-exchange chromatography on a solid matrix at a more basic pH than the pH corresponding to the isoelectric point, pI, purified proteins, however, when the pH value of proteins still remain absorbed, and elution of the proteins by increasing ionic strength and/or pH of the aqueous buffer solutions.

Effective implementation of the present invention requires individualization right combination used chromatographic matrix, pH values above the pI and ionic strength used chromatographic eluents as defined chromatographic matrix, effective cleaning can be achieved by conducting limited changes in pH and/or ionic strength, often in tenths of units pH and/or changes in ionic strength for a few hundredths of S.

You can use all functionalityand solid matrix, usually used as stationary phases for cation exchange chromatography, however, in particular, stationary phase, called strong kationoobmennikom have a preference when pI protein intended for cleaning, below 6, while the stationary phase with both strong and weak cation exchange can be used without exception for proteins having a pI above 6. These stationary phases can be PU glue the first or polymeric matrix, functionalized using sulfanilic or carboxyl groups, as in the proton form and in the form of alkali salts.

With success, you can use commercially available stationary phases, such as, for example, Source® S (Pharmacia Biotech), Sepharosa® SP-Fast Flow Sepharose® SP-High Performance, Sp Sepharose® XL (Pharmacia Biotech), Fractogel® S (Merck. Darmstadt), Mustang® S (Pall Corporate), CM Sepharose® FF (Pharmacia Biotech), Dowex®Bio-Rad® AG (Bio-Rad), Poros® S (PerSeptive Biosystems), Shodex®-S, Toyopearl® SP (Tosohass).

The pH range in which the present invention can be effectively carried out, is very wide, depending on the isoelectric points intended for treatment of interferon proteins, and is between 2 and 11, preferably between 4 and 8.5.

A wider range of pH values greater than pI, which can be described in the present invention the method may vary from pH values corresponding to pI interferon proteins to the pH value, per unit pH in excess of specified pI, demonstrating a striking difference from protein to protein.

For example, it was found that in the case of recombinant alpha-2b-interferon (rIFNα-2b) can be absorbed protein on a cation exchange matrix to values larger than 0.2 pH units of its pI component of 5.9, and, consequently, it is possible to clean it with Katie obmennoi chromatography while it was found that in the case of serum albumin human protein remains absorbed to pH values greater than one pH unit of its pI.

The range of concentrations of saline aqueous solutions used as effectively eluents used depends on the type of the purified interferon proteins and, as shown, includes values between 1 mm and 100 mm, preferably between 1 mm and 30 mm.

For example, in the case of purification of recombinant alpha-2b-interferon (rIFNα-2b) the concentration of the aqueous salt solution is between 1 mm and 30 mm, preferably between 5 and 15 mm.

The need to have a fixed and stable pH eluents used for chromatographic objectives of the present invention, makes it very useful, if not absolutely necessary, the use of appropriately buffered aqueous solutions containing from 5 to 100 mm, preferably from 10 to 20 mm buffer mixtures. Mainly you can use any chemical or mixture of chemicals having a buffering capacity in the pH range between 2 and 11, since the pH of the eluents that can be applied are between 2 and 11.

Mainly in the implementation of the present invention can be used lots of water buffer solutions, including those that contain the at: glycine and sodium chloride, maleic acid and sodium hydroxide, malonic acid and sodium hydroxide, lactic acid and sodium hydroxide, formic acid and sodium hydroxide or lithium, succinic acid and sodium hydroxide, N-methylpiperazin and chloride-hydrogen acid, piperazine and chloride-hydrogen or acetic acid, L-histidine and chloride-hydrogen acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and sodium hydroxide or lithium, N-methyldiethanolamine and sulfuric acid, N-methyldiethanolamine and chloride-hydrogen or acetic acid, pyridine and formic acid, dibasic sodium citrate and sodium hydroxide, monotony phthalate and potassium chloride-hydrogen acid, monotony phthalate potassium and sodium hydroxide, monotony potassium phosphate and dibasic sodium phosphate, bitin and sodium hydroxide, barbitala sodium and chloride-hydrogen acid, sodium borate and chloride-hydrogen acid, sodium borate and sodium hydroxide, 1,3-diaminopropan and chloride-hydrogen acid, citric acid and dibasic sodium phosphate, sodium acetate and acetic acid, imidazole and chloride-hydrogen acid, triethanolamine and chloride-hydrogen or acetic acid, Tris(hydroxyethylaminomethyl) and chloride-hydrogen acid, sodium carbonate and acid sodium carbonate, ethanolamine and chloride-hydrogen acid, piperidine and chlorine is a hundred-hydrogen acid, trimethylamine and formic acid, pyridine and acetic acid, trimethylamine and acetic acid, trimethylamine and chloride-hydrogen acid, ammonium hydroxide and formic acid, ammonium hydroxide and acetic acid, trimethylamine and sodium carbonate, ammonium carbonate and ammonium hydroxide.

In particular, in the case of purification of recombinant alpha-2b-interferon (rIFNα-2b) you can use all the buffer solutions that have a buffering capacity at the pH found between 5.9 and 6.1, preferably a buffer solution at pH between 5.9 and 6.1, containing monotony potassium phosphate and dibasic sodium phosphate, monotony phthalate potassium and sodium hydroxide, dibasic sodium citrate and sodium hydroxide, citric acid and dibasic sodium phosphate, imidazole and chloride-hydrogen acid, although in the case of purification of serum albumin human, you can use buffer solutions containing the same mixture of chemical compounds having a buffer capacity between 4.9 and 6.0.

Used as eluents aqueous solutions may also contain, in addition to the chemical substances used for buffering pH, chemical substances, which is a modification of the ionic strength of the solution. This mainly can be used as organic salts, such as, for example, carboxylates, alcalali the courses, phthalates and inorganic salts such as, for example, sulfates, chlorides, phosphates, which may form a salt with sodium, potassium, ammonium, primary, secondary, tertiary or aromatic amines.

These compounds mainly can be used in a concentration in the range from 1 mm to 100 mm, preferably between 1 mm and 30 mm.

For example, in the case of purification of recombinant alpha-2b-interferon (rIFNα-2b), the concentration of such compounds can vary between 1 mm and 30 mm, preferably between 2 and 20 mm.

The cleaning efficiency can be increased using elution interferon proteins of one or more leaching suenami having a suitable pH and ionic strength, so that the column is always at pH greater than pI.

In the case of recombinant alpha-2b-interferon (rIFNα-2b), pI which is 5.9, the leaching can be carried out in buffer solutions at pH lies between 6.0 and 6.1.

The amount of solvent passing through the column during such leaching is variable, typically accounting for between 5 and 100 volumes of column (C), in the case of recombinant alpha-2b-interferon (rIFNα-2b), the amount of solvent is from 10 to 80 OK.

The number intended for cleaning product that can be loaded into the column and depends on the used chromatographic matrix and can reach a maximum of 100 milligrams total number of proteins in each ml of the stationary phase, even if you normally use lower amounts ranging from 5 to 20 mg/ml

Eluent can pass through the column with a linear speed that is compatible with the stationary phase, up to a maximum value equal to 10 cm/min

Illustrated by the above purification method can be used for all interferon proteins, in particular related to interferons alpha, beta, gamma, Delta, omega, Tau, natural alpha-interferon from leukocytes, recombinant alpha-2b, and consensus interferon, both natural and recombinant origin.

The amount of the above-described purification method consists of obtaining the industrial and economical way of pharmacologically active proteins with a high degree of purity, so that they can directly be used to obtain the containing drugs.

In particular, drugs in the scope of the present invention includes preparations containing interferon, even more preferably recombinant alpha-2b-interferon (rIFNα-2b), both natural and recombinant origin.

The following are some illustrative examples of the object of the method according to the present invention.

Example 1

Production of recombinant alpha-2b-interferon (rIFNα-2b)

Part of the cells of Escherichia coli strain BL21 DE3 transformed with 5 ng plasmid pET9aIFNα 2b, obtained by cloning the synthetic gene, reproductive gene sequence IFNα-2b human modified appropriately to match the sequence of the more frequent codons in Escherichia coli, plasmid rate (Novagen).

The sequence of the protein expressed by the cells of Escherichia coli, modified as described above, is similar to the sequence reported in Methods in Enzymology, Interferons, part C, edited by Pastka S., 119, 3-14 (1996), published by Academic Press Inc.

The Escherichia coli strain BL21 DE3 transformed with the plasmid pET9a-IFNα-2b, were placed in a culture flask with a suitable culture medium, for example, a solution containing 12 g/l of fetopathy (Phyto peptoton, BBL), 24 g/l yeast extract (Yeast extract, DIFCO), 4 g/l of glycerol (BDH) and neomycin, 3°With in a period of time sufficient to achieve the values of optical density at 600 nm, equal to 0.6-0.8, usually within 7-9 hours. Thus obtained culture was then used at a dilution of 1 to 100 for insulinopenia 5 l fermenters, which contained cultural environment similar to the previously described medium in the flask. The culture was kept for 14 hours at 37°With aeration, part of one volume of air for each minute relative to the volume of culture.

Bacterial cells were collected centrifugation is at 6000 revolutions per minute (rpm) at the end of cultivation, them suspended in a suitable aqueous solution, containing 1 mm dithiothreitol (DTT) in the amount of not more than 6 ml per gram wet weight of bacterial centrifugate. The bacterial suspension was subjected to cell lysis using established and described methods, such as the splitting of ultrasound or by using hydraulic pressure.

The resulting suspension was isolated by centrifugation and the hard part is suspended in 50 ml of saline solution containing 1 mm DTT and again centrifuged.

A solid component comprising the incorporated body, collected and suspended with vigorous stirring at room temperature to 450 ml of a solution containing 6 M of chloride guanidine, 50 mm Tris-HCl at pH 8, and 0.1 mm etc. The suspension was centrifuged and the supernatant was diluted in a ratio of from 1 to 100 to 1 : 200 in physiological solution containing 50 mm Tris-HCl at pH 8, and 0.1 mm add at pH 8, suitable for renaturation of the protein. The solution for renaturation may contain amino acids such as glycine or arginine; mixtures of compounds containing sulphides in oxidized and reduced form, formed by a disulfide bridges, such as glutathione, ethanolamine, cysteine. Renaturation carried out with vigorous stirring of the solution at 4°almost within 72 hours, then the solution is filtered is then concentrated by using method dia-filtration relative to the buffer, obtained from 40 mm Tris-HCl at pH 8 to reach the final concentration factor 5-10 times. The final concentration of the solution is typically between 0.4 and 1.0 mg/ml

Example 2.

Purification of recombinant alpha-2b-interferon (rIFNα-2b)

1 M sodium acetate solution are added to a final concentration of 20 mm to a mixture of proteins containing rIFNα-2b, obtained in example 1 and the mixture is brought to pH 5.5 with acetic acid. Thus obtained solution is loaded into a strong cation exchange column, containing commercially available chromatographic matrix Mustang® S (Pall Corporate). Before loading the protein solution cation exchange column is conditioned with 20 mm sodium acetate solution at pH 5.5 in the amount equal to 20 column volumes (OK).

Then load the protein solution in such amount, not to exceed the amount of loaded proteins, equal to 10 mg, per ml of stationary phase, preferably in a quantity amounting to between 6 and 8 mg/ml

After loading products related to stationary phase, is subjected to a first washing cycle using a salt solution at a pH of 6.1, obtained from a mixture of nonoonono potassium phosphate and dibasic phosphate in total concentration, component between 5 and 15 mm. The optimal concentration of the solution one way or another is fixed by the fact that his conduct is here should not exceed about 1800 S. Use the total amount of solution comprising from 5 to 60 OK, preferably between 25 and 35 OK.

Then carry out a second cycle of washing with the same solution as in the first cycle of washing, to which is added a quantity of potassium chloride, equal to a final concentration not exceeding 3 mm, preferably 2 mm; use the total amount of solution comprising between 10 and 100 OK, preferably between 30 and 60 OK.

After the wash cycles are phase elution with a solution similar to the one used in the first cycle of washing with a finite amount of potassium chloride in a concentration factor of 15 to 25 mm. For elution of use total quantity of a solution comprising between 15 and 40 OK, preferably between 20 and 30 OK.

All solutions and downloaded the sample is passed through the column with a linear velocity component of from 0.1 to 1 cm/min, preferably between 0.4 and 0.7 cm/min

Under these conditions rIFNα-2b elute from the column with a degree of purity greater than 98%, although in the initial solution, the degree of purity was approximately 40%, when the selection of the target product, in excess of 80%.

Chromatographic profiles before and after chromatographic purification presented on figa and 1b.

On figa shows the chromatographic profile of a solution of interferon before cleaning, and fig.1b showing the chromatographic profile after cleaning.

Chromatographic profiles were obtained using HPLC using a liquid chromatograph HP 1090, column Vydac C18 and UV detector set at 214 nm. Elution was performed at a flow rate of 1 ml/min using a mixture of two eluents, eluent And received from 700 ml of water, 298 ml of acetonitrile and 2 ml triperoxonane acid and eluent b received from 198 ml of water, 800 ml of acetonitrile and 2 ml triperoxonane acid. Two eluent was mixed during elution in accordance with the following table:

Time (minutes)%%
07228
17228
56733
206337
305743
404060
424060
502872
607228

Example 3

The method was carried out in accordance with the description of example 2 using a buffer solution obtained from nonoonono phthalate potassium and sodium hydroxide.

Example 4

The method was implemented in compliance and with the description of example 2 using a buffer solution, derived from dibasic sodium citrate and sodium hydroxide.

Example 5

The method was carried out in accordance with the description of example 2 using a buffer solution obtained from citric acid and dibasic phosphate.

Example 6

The method was carried out in accordance with the description of example 2 using a buffer solution derived from imidazole and chloride-hydrogen acid.

1. The method of purification of interferon proteins, including cation-exchange chromatography on a solid matrix when the pH value is more General, the pH corresponding to the isoelectric point, pI, purified proteins in this pH value at which these proteins are still absorbed, and elution of the proteins by increasing the ionic strength and/or pH of the aqueous buffer solutions.

2. The method according to claim 1, characterized in that the aqueous buffer solutions contain from 5 to 100 mm buffer mixtures selected from mixtures obtained from nonoonono potassium phosphate and dibasic phosphate, nonoonono phthalate potassium and sodium hydroxide, dibasic sodium citrate and sodium hydroxide, citric acid and dibasic phosphate, or imidazole and chloride-hydrogen acid.

3. The method according to claim 2, characterized in that the buffer solutions contain from 1 to 100 mm organically is or inorganic salts, is able to modify the ionic strength of the solution.

4. The method according to any one of claims 1 to 3, wherein the interferon proteins are alpha, beta, gamma, Delta, omega, Tau, alpha natural of white blood cells, recombinant alpha-2b, and consensus interferon.

5. The method according to claim 1, characterized in that carry out the purification of the recombinant alpha-2b-interferon, rIFNa-2b, which includes uploading obtained by carrying out the fermentation rIFNa-2b mixture of proteins, supplemented by adding a 1M solution of sodium acetate and brought to pH 5.5 with acetic acid, over a column filled with a strong cation exchange resin, air-conditioned at pH 5.5 with 20 mm sodium acetate solution so that each ml of the stationary phase takes from 6 to 8 mg protein; two cycles of the washing column, the first of which is a washing buffer solution at pH of 6.1 in concentrations between 5 and 15 mm, then the same buffer, supplemented with 2 mm potassium chloride, and, finally, conduct elution of pure rIFNa-2b from the column using a buffer solution at pH of 6.1, containing potassium chloride at a concentration of between 15 and 25 mm.

6. The method according to claim 5, characterized in that the resin is a strong cation exchange medium consisting of a hydrophilic polymer with sulfonic acid groups, sewn on poliey the sulfonic membrane, and buffer mixture are selected from mixtures obtained from nonoonono potassium phosphate and dibasic phosphate, nonoonono phthalate potassium and sodium hydroxide, dibasic sodium citrate and sodium hydroxide, citric acid and dibasic phosphate, or imidazole and chloride-hydrogen acid.

7. The application of the methods according to any one of the preceding paragraphs to obtain the active principles contained in the medicines on the basis of the interferon proteins.

8. The use according to claim 7, where the interferon protein is a recombinant alpha-2b-interferon.



 

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