EA026659B1 - Method for producing affinity sorbent, and affinity sorbent - Google Patents

Abstract

The invention is related to the field of biotechnology, in particular, to a method for producing a new affinity sorbent that interacts specifically with the recombinant interferon contained in E.Coli biomass homogenate, and to the affinity sorbent produced by said method. The objective of the invention is development of a simple method for production of a ball-shaped sorbent on the basis of an insoluble calcium tartarate salt. The sorbent production method according to the invention comprises mixing equal volumes of 0.3-0.7 M solution of a tartaric acid salt selected from a group including potassium-sodium tartarate and sodium tartarate, and aqueous 0.5-0.9 solution of calcium chloride, and stirring at a temperature of 20-22°C, which yields crystals that are left to precipitate. After that, the supernatant is decanted, and the sediment is heat treated, which is followed by at least one washing and balancing with a buffer solution until the target sorbent is produced.

Description

The invention relates to the field of biotechnology, and in particular to a method for producing a sorbent for the isolation and purification of a genetically engineered preparation of recombinant interferon (IFN).

IFN has a wide spectrum of biological effects: it exhibits antiviral and antitumor activity, inhibits cell growth, and has an immunoregulatory effect. Recombinant interferons are proteins with a molecular weight of 15-25 kDa. The main stages in the isolation of recombinant IFN are the cultivation of microorganism cells, their mechanical destruction, clarification of the cell homogenate. Further isolation and purification of IFN is carried out in various ways, in particular by chromatography on various sorbents.

Thus, a method is known for further isolation and purification of IFN from protein material [1], including chromatography on ΟΛΟ-γ gel and two subsequent chromatography on CM-cellulose.

There is also a method of purification of IFN [2], including chromatography on a sorbent Sje1aypd 8ryatok Time! P1o \ y with immobilized Cu ^{2+ ions} , ion-exchange chromatography on CM-8perOate5e and gel filtration on a sorbent of type 8regLex 75.

For purification of IFN, chromatography on SM-Touoreag1, IEAE-Touoreat1, gel filtration on Touoreag1 H \ U-55 and protein concentration using PEG 3000 can be used (approximately 6.5 mg of protein is isolated from 1 g of biomass) [3].

There is also a method for producing recombinant human IFN [4], which includes treating cells after their destruction with polyethyleneimine, separating nucleic acids, salting out proteins with ammonium sulfate, three-stage chromatography on a cation-hydrophobic sorbent of Solosa KG, on anion exchanger Spherocell-OAE and on hydrophobic sorbent Lent Sorbent M, concentration by ultrafiltration and gel filtration on 8pLaLex 0-100.

The disadvantages of these methods are the duration of the process, multi-stage and the inability to scale, which is largely determined by the properties of the sorbents used in chromatography.

At the same time, affinity chromatography is known to be characterized by the extremely high selectivity inherent in biological interactions. Often, one chromatographic procedure allows you to purify the desired protein thousands of times. However, the implementation of such chromatography is possible only on affinity sorbents, which is not always an easy task because of the danger of biological molecules losing the ability to interact specifically during their covalent attachment to the matrix. In addition, all sorbents known from the prior art for purification and isolation of IFN are not suitable for use as affinity sorbents.

The prior art use of calcium tartaric acid to obtain specific sorbents. So, there is a known method of producing a sorbent based on calcium tartaric acid and heparin, which is used to purify blood plasma from lipoproteins by column chromatography [5]. In this method, equal volumes of an aqueous solution containing 6.85 wt.% Calcium chloride and an aqueous solution containing 8.98 wt.% Potassium sodium or tartaric acid sodium and 0.09 wt.% Heparin are mixed, mixed and washed formed sorbent crystals with saline. However, this sorbent is used only as a liposorbent, has a complex (complex) composition and, in addition, does not have the properties of an affinity sorbent.

Thus, the sorbents for the purification and isolation of IFN, which would provide a significant simplification of the technology of purification and isolation, are not known from the prior art. Given this prototype for the proposed method is not identified.

The objective of the invention is to develop a method for producing a new affinity sorbent for the isolation and purification of recombinant IFN. The sorbent should have the simplest possible composition and at the same time provide a significant simplification of the process of isolation and purification of IFN by means of single-stage chromatography and, accordingly, a reduction in the time required to obtain IFN in significant quantities.

The present invention is based on the preparation of a sorbent for chromatography based on an insoluble salt of tartaric acid (calcium tartrate, KB).

The problem is solved by the claimed method of producing a sorbent based on calcium tartrate for the isolation and purification of recombinant interferon, in which equal volumes of an aqueous 0.3-0.7 M solution of tartaric acid salt selected from the group consisting of potassium tartaric acid and sodium are mixed tartaric acid, and an aqueous 0.5-0.9 M calcium chloride solution, stirred at a temperature of 20-22 ° C, the crystals formed were allowed to settle, after which the supernatant was decanted and the precipitate was subjected to heat treatment, followed by at least m with a single wash and equilibration with a buffer solution until the desired sorbent is obtained.

In preferred embodiments of the inventive method for heat treatment, the supernatant previously decanted and heated to 80-90 ° C is poured into the precipitate and stirred for 20-30 minutes, followed by precipitation of the precipitate and decantation of the supernatant.

Also preferred are implementation forms in which, to balance and rinse, a 0.75 M sodium chloride solution prepared on a Tris-HC1 buffer, pH 8.0-8.3, is added to the heat-treated precipitate, and mixed for 10-15 min and let the precipitate settle, the resulting precipitate is washed and added (1.05 M Tris-HCl, pH 8.0-8.3, containing

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0.03 M Ναί'Ί. to obtain the target sorbent.

The problem is also solved by the claimed sorbent based on calcium tartrate for the isolation and purification of recombinant interferon obtained by the above method.

Studies and experiments showed that the sorbent obtained by the claimed method specifically interacts with IFN and exhibits the properties of an affinity sorbent. The sorption capacity of the sorbent is 2.0-2.5 mg / ml. The claimed method with its simplicity allows for a short time and in preparative quantities to obtain a sorbent, which, in turn, for almost 1 chromatography cycle allows you to get purified biologically active protein (activity of at least 1.6 x 10 ⁷ IU / mg) - recombinant IFN.

The advantages and advantages of the proposed method for producing a sorbent based on calcium tartrate for the isolation and purification of recombinant interferon, as well as the sorbent obtained by the specified method, will be illustrated below with the help of explanatory materials, on which data obtained from experimental results are presented.

In FIG. 1 shows a micrograph of calcium crystals of tartaric acid; in FIG. 2 shows the results of δΌδ-polyacrylamide gel electrophoresis (PAGE) of the isolated protein - purified γ IFN; in FIG. 3 shows the mass spectra of the selected γ-IFN.

Mass spectrometry was carried out on a MALDI mass spectrometric system of the Mühoyeh LEP series (Vgikeg Ωαΐΐοηίοδ СшЬН, Germany) in a linear mode.

The advantages and advantages of the proposed method for producing a sorbent based on calcium tartrate for the isolation and purification of recombinant interferon, as well as the sorbent obtained by the specified method are also confirmed by some examples of the preparation and use of the sorbent.

Example 1. Obtaining a sorbent based on calcium tartaric acid (KB).

500 ml of a 0.5 M solution of potassium-tartaric acid are added to 500 ml of a 0.5 M solution of calcium chloride and stirred for 5 minutes using a mechanical stirring device at a temperature of 20 ° C. The KB spherical crystals formed are allowed to settle (particle settling time 15 min). The supernatant is decanted, heated to 80 ° C and poured to a precipitate KV. After stirring, the precipitate is allowed to settle and the superiatant is carefully decanted. To the precipitate was added 150 ml of 0.75 M sodium chloride in Tris-HCl buffer solution, pH 8.2, and stirred for 10 minutes. The result is 125 ml of a suspension of sorbent. The resulting sorbent is washed and equilibrated with a solution containing 0.05 M Tris-HCl, pH 8.0-8.3 and 0.03 M No. C1.

Example 2. Obtaining a sorbent based on KB.

500 ml of a 0.9 M solution of calcium chloride are added to 500 ml of a 0.7 M sodium tartaric acid solution and stirred for 10 minutes using a mechanical stirring device at a temperature of 22 ° C. The KB spherical crystals formed are allowed to settle (particle settling time 15 min). The supernatant is decanted, heated to 90 ° C and poured to a precipitate KV. After stirring, the precipitate was allowed to settle and the supernatant was carefully decanted. 200 ml of 0.75 M sodium chloride in Tris-HCl buffer solution, pH 8.3, is added to Osaska, stirred for 15 minutes. The result is 130 ml of a suspension of sorbent. The resulting sorbent is washed and equilibrated with a solution containing 0.05 M Tris-HCl, pH 8.0-8.3 and 0.03 M No. C1.

A micrograph of the obtained crystals of a sorbent based on calcium tartrate is shown in FIG. one.

Example 3. Isolation and purification of recombinant IFN.

Isolation and purification of recombinant INF was carried out using the example of recombinant bovine γINF with preliminary preparation of protein material as follows.

Obtaining biomass of E. soy cells accumulating recombinant IFN

The biomass of bacterial cells accumulating bovine IFN-y is obtained during controlled cultivation in a fermenter with a working volume of 10 l. A 500 ml seed crop is pre-grown on a shaker at 37 ° C, 180 rpm for 12 hours.

The culture medium has the following composition (g / l): bacteriological peptone - 10; yeast extract - 5; Ναί'Ί - 10; ΝΉ ₄ Ο - 1; glucose - 1; glycerin - 10; lactose - 10; antibiotics: kanamycin - 30 IU / ml, chloramphenicol - 15 IU / ml.

Cultivation conditions: temperature 37 ° C; pH 6.5-7.3; the concentration of dissolved oxygen is not less than 60%; mixer revolutions - 500 rpm at the beginning of cultivation and 1000 rpm at the end; pressure 0 bar at the beginning and 2 bar at the end of cultivation; cultivation time is 9 hours

Obtaining protein material

Biomass is collected by centrifugation in a flow centrifuge at 35,000 rpm. The collected biomass (8 g) was resuspended in 250 ml of buffer of the following composition: 50 mmol / L Tris-HCl, pH 10.2, 30 mmol / L No. C1, a cocktail of protease inhibitors, and destroyed on a high pressure homogenizer at 1000-1200 bar. The resulting cell homogenate is centrifuged at 20,000 d for 30 min at 4 ° C, the supernatant is passed through a filter with a pore diameter of 0.45 μm and used to isolate recombinant bovine γ-IFN.

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Isolation and purification of bovine y-IFN

To 30 ml of CV in Example 1 suspended in 50 mmol / L Tris-HCl, pH 8.0, containing 30 mmol / L sodium chloride, 250 ml of the protein material obtained by the above method are added, stirred for 20 minutes, the precipitate is allowed to settle, supernatant decanted. The sorbent suspension is transferred to a chromatographic column, washed with 0.1 M sodium chloride solution, pH 8.0, and the adsorbed protein is eluted with a 0.75 M sodium chloride solution, pH 8.0.

The results of δΌδ-polyacrylamide gel electrophoresis (PAGE) of the isolated protein — purified γ-IFN are shown in FIG. 2.

The characteristics of the obtained γ-IFN were also determined by mass spectrometry on a MALDI mass spectrometric system of the Mutoyeh LKE series (Vgikeg Ea11op1C5 STN, Germany) in a linear mode. The mass spectra of the isolated γ-IFN are shown in FIG. 3.

Similar results were obtained for recombinant INF of sheep, pig, and human.

Information sources

1. Methods for the production of recombinant cytokine proteins. P. An effective method for the isolation, purification, and renaturation of recombinant human γ-interferon A.N. Wulfson, R.V. Tikhonov, S.E. Pechenov, V.E. Klyushnichenko, A.I. Miroshnikov. Bioorganic chemistry, v. 23, No. 9, 1997, p. 721-726

2. Patent KI 2242516, C1, publ. 12/20/2004

3. Microbiological journal (IA) t. 24, No. 3, 2012

4. Patent KI 2225722, C1, publ. 03/20/2004

5. Patent No. 17337 C1, publ. 08/30/2013

Claims (4)

CLAIM 1. A method of producing a sorbent based on calcium tartaric acid for the isolation and purification of recombinant interferon, in which equal volumes of an aqueous 0.3-0.7 M solution of a tartaric acid salt selected from the group consisting of potassium-tartaric acid and sodium are mixed tartaric acid, and an aqueous 0.5-0.9 M solution of calcium chloride, stirred at a temperature of 20-22 ° C, the crystals formed were allowed to settle, after which the supernatant was decanted and the precipitate was subjected to heat treatment, followed by at least one wash and equal stallation buffer solution to obtain the desired sorbent. 2. The method according to claim 1, characterized in that the supernatant previously decanted and heated to 80-90 ° C is added to the precipitate for heat treatment and stirred for 20-30 minutes, followed by precipitation of the precipitate and decantation of the supernatant. 3. The method according to any one of claims 1 or 2, characterized in that for washing and balancing to the heat-treated precipitate, add a 0.75 M solution of sodium chloride prepared on a buffer based on Tris-HCl, pH 8.0-8, 3, stirred for 10-15 minutes and allowed to precipitate, the resulting precipitate was washed and 0.05 M Tris-HCl, pH 8.0-8.3, containing 0.03 M No. C1 was added. to obtain the target sorbent. 4. Sorbent based on calcium tartaric acid for the isolation and purification of recombinant interferon obtained by the method according to one of claims 1 to 3.