CS264100B1 - The mode of production of immunomodulase thymosin peptides of fraction 5 - Google Patents

Abstract

The solution concerns the production of immunomodulatory peptides from thymus fractions by a simplified procedure, which consists in the fact that the extract from calf thymus after thermal denaturation of ballast proteins and their simultaneous removal from thymus tissue is delipidated by extraction into ethyl acetate or lower alkanol. From the separated aqueous phase, ballast proteins are extracted together with solvent residues and separated as the upper liquid phase. Then salting out:.-. Crude thymosin is obtained, which is purified by ultrafiltration and from the ultrafiltrate, after concentration and acidification, partially deoiled thymosin fraction 5 is re-precipitated. This is converted by washing with acetone 1' into a stabilized, dry intermediate product, which can be further processed as needed after desalting by gel filtration, cation exchange chromatography and lyophilization to the final product. Thymosin peptides of fraction 5 are used for the treatment of immune system disorders.

Description

The invention relates to a method for producing immunomodulatory peptides of thymosin fraction 5 from calf thymus.

Thymosin fraction 5 is a complex of peptide hormones secreted by the thymus gland, which enable the maturation and differentiation of lymphocytes into immunocompetent cells and participate in the proper functioning of the immune system.

Of the entire range of immunomodulatory peptides of the thymus, thymosin fraction 5 is currently the best studied and also clinically used in the world. It is prepared by extraction of frozen calf thymuses and its composition is determined both by the extraction method and by subsequent isolation and purification steps.

*l

The original method of preparation (A.L. Goldstein et al., Proc. Hati. Acad. Sci. USA 69, 1800, 1972) was further developed (J.A. Kooper et al., Ann. Η.Ϊ. Acad. Sci. 24§, 125, 1975). According to this procedure, a homogenate of calf thymus in 3 parts of 0.15 M sodium chloride solution is centrifuged, the supernatant is heated to 80 °C for 15 minutes and the precipitated denatured ballast proteins are filtered off. The filtrate is precipitated by dropping into a four-fold volume of acetone cooled to -10 °C, the filtered precipitate washed with acetone is dried to a powder and this is dissolved in 10 parts of 0.1 M phosphate buffer with a pH of 7.0. The undissolved residue is centrifuged and additional ballast proteins are first salted out of the supernatant with ammonium sulfate added until 25% saturation is reached. After centrifugation of the salted-out proteins, the pH of the supernatant is adjusted to 4.0 and the crude thymo-,, sin is salted out by further addition of ammonium sulfate until 50% saturation is reached. The centrifuged sediment of active peptides is dissolved in 0.01 M tris(hydroxymethyl)-aminomethane buffer (hereinafter TRIS buffer) at pH 6.0 to a concentration of 10 mg peptides/ml

- 2,264 ICO and after clarification the solution is ultrafiltered through a membrane with a cut-off limit of 10,800. The ultrafiltrate containing thymosins of fraction 5 is desalted on a dextran gel and the desired fraction, eluted in the _VQ region, is lyophilized. In an attempt to transfer the isolation of thymosin of fraction 5 to a production scale, the described procedure was further modified. According to US patent specification No. 4,128,637, the proteins in the extract are denatured by steam injection, another US patent specification No. 4,082,737 describes the removal of endotoxins and pyrogens by ultrafiltration.

The methods described so far have two common disadvantages. From the colloidal thymus homogenate it is necessary to separate the extract by centrifugation at high values of gravitational acceleration, furthermore the methods require considerable volumes of chilled acetone for precipitation and delipidation of crude thymosin. These technological operations greatly complicate the industrial use of the methods. The mentioned disadvantages are eliminated by the method described in δη» AO No. 250631, according to which the remains of thymus tissue and ballast proteins are simultaneously removed by hot filtration after heating the homogenate, the lipid layer is separated from the cooled filtrate and the thymosin peptides are directly salted out of the solution with ammonium sulfate. Furthermore, the inactive polypeptide /3 -j ⁰ isoelectric point value pl 6.7 is removed by chromatography on a weakly acidic cation exchanger column in a volatile buffer with a pH of 5.0 to 7.0. However, the current process still brings difficulties, such as turbidity of solutions after lipid separation, relatively high consumption of ammonium sulfate, the necessity of double centrifugation during salting out, and difficult storage of intermediate products in the event of production interruption.

The above disadvantages are eliminated by the method of producing immunomodulatory peptides thymosin fraction 5, obtained from homogenate of calf thymus after heat denaturation, according to the invention, the essence of which consists in extracting lipids from the warm filtrate with ethyl acetate or alkanol with a number of carbon atoms of 3 to 5. After cooling, ballast proteins and organic solvent, which are in the upper phase, are salted out with ammonium sulfate in a separate aqueous phase. The clear lower phase is subsequently salted out again with ammonium sulfate and the obtained crude thymosin is separated and ultra-filtered. By concentrating and acidifying the ultrafiltrate to a pH of 3.8 to 4.2, the pre-printed partially desalted thymosin is salted back, which is converted with acetone into a stable powdery product. This is processed directly into the final complex of immunomodulatory peptides of thymosin fraction 5 by gel filtration and chromatography on a cation exchanger. The method according to the invention is preferably carried out in such a way that the lipids are extracted with 2-propanol or 1-butanol and the ultrafiltrate is acidified with acetic acid to a pH of 4.0. The authors of the invention have thus found that it is possible to use and preserve the essence of the Czechoslovak AO No.

250631 to simplify and facilitate the production of thymosin fraction 5, as depicted in Scheme I.

The method of the invention is carried out by heating the homogenized gel-like suspension of thymus tissue in a 0.15 M sodium chloride solution to a temperature of 50 °C, filtering off the denatured ballast proteins together with the precipitated thymus pulp while hot, and adding ethyl acetate or a lower alkanol, preferably 2-propanol or 1-butanol, to the extract while stirring at a temperature of about 50 ^° C. After cooling, the defatted aqueous phase is separated, from which additional ballast proteins and dissolved organic solvent are salted out by adding ammonium sulfate to 25% saturation of the solution. An upper liquid phase is formed, also containing a protein precipitate with a part of the unwanted peptides and lipid residues. The lower clear phase containing thymosin can be separated very easily, unlike the previous procedure, when it was necessary to centrifuge the salted out ballasts on a flow separator. From the aqueous, completely delipidated phase, the crude thymosin is further salted out at a pH of 4.0 by adding ammonium sulfate until the solution is 40 to 45% saturated. This time, the precipitate sediments and is separated by centrifugation on a flow separator. After dissolving in a buffer with a pH of 8.0, the crude thymosin is freed from high-molecular impurities by ultrafiltration through a membrane with a cut-off limit of 10,000, and the ultrafiltrate, which still contains a significant amount of salt, is preferably purified and converted into a stable intermediate product by concentrating and acidifying to a pH of 4.0. This resalts out the thymosin, which can be easily filtered off and washed with cooled acetone. Washing has an additional cleaning effect,

- 4 264 χΟΟ because colored impurities will pass into acetone. The obtained powdery intermediate product contains about 4 times less salt than the centrifuged precipitate. After desalting by gel filtration, the effluent from the molecular sieve column is freed from the inactive polypeptide fb according to the procedure specified in Czechoslovak AO No. 250631 by flowing through a column of a weakly acidic cation exchanger in a volatile buffer environment with a pH value of 5.0 to 7j0, e.g. ammonium acetate. The active peptides of thymosin fraction 5 pass through the column and can be directly lyophilized, the captured polypeptide ý3 is washed out of the ion exchanger by increasing the pH value of the elution solution and thereby the ion exchanger is regenerated at the same time,

The process according to the invention has the following main advantages; delipidation of the acetate with ethyl acetate or alkanol added to the filtered extract immediately after thermal denaturation is very fast and effective. The use of the mentioned organic solvents results in a change in the specific gravity of the precipitate of ballast proteins compared to existing processes and allows their separation from the upper liquid phase, thereby eliminating one centrifugation on a flow separator from the technological process. Crude thymosin is salted out from a perfectly clear solution, which facilitates the dissolution of the centrifuged precipitate and preliminary clarification before ultrafiltration. The organic solvent has a beneficial effect on the salting out of ballast substances and thymosin. This is manifested by a reduction in the required amount of ammonium sulfate and further by the fact that the final product contains fewer ineffective peptides. Precipitation of thymosin fraction 5 20 of the concentrated ultrafiltrate and its conversion to acetone powder allows for the collection of a stable intermediate product from individual batches with regard to the needs for subsequent gel filtration and lyophilization. Conversion to acetone powder has an additional refining effect with minimal acetone consumption compared to previous procedures. Partial desalting of the ultrafiltrate reduces the salt to thymosin ratio several times, thereby reducing the demands on gel filtration equipment. The method according to the invention significantly speeds up the production process, reduces the demands on equipment and energy consumption. It also reduces the consumption of raw materials, such as ammonium sulfate and dextran gel. This also results in economic savings. .

264 ICO

In the following, the invention is explained in more detail by means of examples of embodiments, without being limited thereto, and by means of Scheme I.

Example 1

A batch of 50 kg of frozen calf thymus is ground and homogenized in 125 liters of 0.15 M sodium chloride solution at a temperature of 15 to 20 °C. The suspension is stirred in a boiler equipped with a heating jacket at this temperature for 1 hour. Then, while stirring, it is heated to a temperature of 80 ^U C within 30 minutes. At this temperature, it is stirred for 10 minutes and immediately filtered while hot under reduced pressure through a cloth. The precipitate is washed with 20 liters of water. The warm filtrate is pumped into a container equipped with a powerful stirrer and immediately, while hot, 1/4 of the volume, i.e. about 30 liters of ethyl acetate, is added while stirring. After establishing the extraction equilibrium, the liquid phases are pumped into a glass container equipped with a bottom outlet. After cooling to a temperature of 20 -°C, the aqueous delipidated phase is separated, to which such an amount of solid ammonium sulfate is added with stirring to achieve a saturation of ζ 2zo / livr and the roe is stirred for 100, 30 minutes. The resulting suspension is pumped back into an inclined container, where the upper phase is separated as a layer of the salted-out solvent together with the ballast proteins. The separated clear aqueous phase is acidified with glacial acetic acid to a pH of 4.0 and by further addition of ammonium sulfate to 40 to 45% saturation (185 g / liter), crude thymosin is separated under the same conditions as in the first salting-out. This time, the precipitate is sedimented and centrifuged at 5,000 to 10,000 g on a flow separator.

The centrifuged precipitate of crude thymosin weighing about 800 g and containing about 49% dry matter is dissolved in 2,500 ml of 0.01 M TKIS buffer with a pH of 8.0, the pH being maintained at the stated value by the addition of 25% aqueous ammonia. The resulting turbid solution is roughly filtered through a sinter, then clarified on an asbestos-cellulose filter and ultrafiltered through a membrane with a cut-off limit of 10.0U0. The ultrafiltrate is concentrated in vacuo at an external temperature of up to 50°C to a content of about 10% dry matter and the chromium is desalted.

264 ΐΟΟ matography on dextran gel. Immunomodulatory peptides of thymosin fraction 5» eluted in the νθ region, are collected. directly lyophilized, or freed from polypeptide /3^ by pre-filtration of the eluate through a weakly acidic cation exchanger column. The yield is 5.0 to 7.5 g of thymosin fraction 5.

Example 2

The ultrafiltrate of thymosin fraction 5 prepared according to the procedure of Example 1 is concentrated to 1/15 of the volume, thereby achieving the ammonium sulfate concentration required for the re-salting of the thymosin peptides. These are separated by adjusting the pH to 4.0, filtered with suction and washed with acetone cooled to -10 °C. The obtained stabilized and partially desalted powder contains about 35% of the active peptides. After dissolution in a volatile buffer, e.g. in a solution of ammonium ethane at pH 8.0, it is desalted and further processed according to the procedure described in Example 1,

Example 3

Delipidation of the extract can be carried out under the same conditions as in Example 1, except that a lower alcohol, preferably 2-propanol or 1-butanol, is used instead of ethyl acetate.

The purity of the obtained immunomodulatory peptides of thymosin fraction 5 was verified by the analytical method of isoelectric focusing in the pH range of 3.5 to 9.5 and by ultraviolet spectroscopy at 200 to 300 nm.

Scheme I calf thymus homogenization, extraction |~~~h omo q en i z & t ] heating to 80 °C, filtration precipitate -}· thymus tissue

-j filtrate precipitated extraction with hot organic solvent, cooling of the upper phase with lipids precipitated ____E salting out to 25% saturation _with phase separation.

| lower phase is expressed pH 4.0, salting out to 40 to 45% sat., centrifugation of the upper phase with the precipitate

____ _n ___'..... ...______.— < j gray ment J discharge pH 8

supernatant extracted ultrafiltered extracted procedure 1 desalting to dextran.

process 2 concentration, precipitation at pK 4.0, filtration, washing with acetone effluent acetone powder removal of polypeptide, iyoíili:

;aée dissolution, desalting on dextran. gel thymosin fraction 5

JTflixentJ remove ⁱⁿ it polypeptide /3-j, lyXXlisation thymosin fraction 5

Thymoglobin yield of fraction 5: around 0.01% of the thymus weight.

Claims (2)

BEFORE THE INVENTION 264 iCU A process for producing the iraunomodulatory peptides of thymosin fraction 5 peptides, obtained from calf worm homogenate after thermal denaturation, characterized in that the filt warm filtrate is extracted with ethyl acetate or alicanolern lipids. having a carbon number of 3 to 5, after cooling, the ballast proteins are salted out with ammonium sulphate in the separated aqueous phase and the organic solvent which is in the upper phase is subsequently salted out, and the clear lower phase is subsequently salted out again with ammonium sulphate. concentrating and acidifying the ultrafiltrate to a pE value The pre-purified and partially desaturated thymosin is then salted out of 3.8 to 4 > 2, which is converted by acetone to a stable powder intermediate which is gel-filtered and chromatographed. on the cation exchanger, it directly processes the complex of thymosin modulating peptides of fraction 5 * Method according to claim 1, characterized in that the lipids • .A ¹ - * «· O · _R \ * R *. · * - * - · 'Λ r *, n V-. f 'OICO 2. The process of claim 1, wherein the ultrafiltrate is acidified to pH with acetic acid