Method of obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, method of obtaining antagonist of somatotrophic hormone with decreased content of aggregate of its isoforms and total content of trisulphide impurity and/or dephenylalanine impurity

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

 

The text descriptions are presented in facsimile form.

1. The method of obtaining somatotropic hormone (STH) with a reduced content of the aggregate of its isoforms, which preduster the characteristic separation of isoforms of STH by anion-exchange chromatography was carried out using anion-exchange resin to reduce the contents of the said unit to not more than 10 wt.%, on the basis of the total mass mentioned isoforms and referred to Assembly, which carry the load mentioned STH, including diphenylalanine and/or trisulfide admixture and/or Assembly, on the anion exchange resin selected from the group comprising diethylaminoethylcellulose and Q-sepharose, and loading is carried out at the value of the conductivity download anion-exchange resin, less than or equal to 10 MS/cm, at pH download anion-exchange resin from 5 to 10 and when loaded anion exchange resin STG, including the above-mentioned impurity or the said unit constituting not more than 10 g of protein/l of volume filled with anion-exchange resin.

2. The method according to claim 1, characterized in that the STG contains one or more isoforms of STH PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, Assembly, trisulfide impurity and diphenylalanine admixture polietilenglikolya admixture STH and free PEG molecules.

3. The method according to claim 2, further comprising the stage of:
separation mentioned isoforms STH on selected isoforms; and
associations mentioned selected isoforms with obtaining enriched pool mentioned selected isoforms.

4. The method according to claim 1, additionally including the state Association in which the Association of discrete quantities mentioned isoforms STH with obtaining United STG via the method selected by the CSO from the group including the electrophoresis capillary electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate, ion exchange chromatography, hydrophobic chromatography, anion exchange chromatography, cation exchange chromatography, high performance liquid chromatography with reversed phase, high performance liquid gel chromatography, affinity chromatography, and combinations thereof.

5. The method according to claim 4, characterized in that the said United STG contains one or more PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9.

6. The method according to claim 5, characterized in that the combined fraction STH PEG-4, PEG-5, PEG-6 contains at least about 70 wt.%, on the basis of the total mass mentioned isoforms STH PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, and referred to Assembly.

7. The method of obtaining antagonist STH with a reduced content of the aggregate of its isoforms, which provides for the separation of isoforms antagonist STH on anion-exchange resin by anion-exchange chromatography was carried out to reduce the contents of the said unit to no more than 6 wt.%, on the basis of the total mass mentioned isoforms and Assembly, which are download the mentioned STH, including diphenylalanine and/or trisulfide admixture and/or Assembly, on the anion exchange resin selected from the group comprising diethylaminoethylcellulose and Q-SEARO is, moreover, the loading is carried out at the value of the conductivity download anion-exchange resin, less than or equal to 10 MS/cm, at pH download anion-exchange resin from 5 to 10 and when loaded anion exchange resin STG, including the above-mentioned impurity or the said unit constituting not more than 10 g of protein/l of volume filled with anion-exchange resin.

8. The method of obtaining antagonist STH with reduced total content trisulfide impurities, diphenylalanine impurities and Assembly of its isoforms, separating isoforms antagonist STH on anion-exchange resin by anion-exchange chromatography was carried out to reduce the total content trisulfide impurities, diphenylalanine impurities and referred to Assembly to a level smaller or equal to 15 wt.%, on the basis of the total mass mentioned isoforms and referred to Assembly, which carry the load mentioned STH, including diphenylalanine admixture and/or trisulfide admixture and/or Assembly, on the anion exchange resin selected from the group comprising diethylaminoethylcellulose and Q-sepharose, and loading is carried out at the value of the conductivity download anion-exchange resin, less than or equal to 10 MS/cm, at pH download anion-exchange resin from 5 to 10 and when loaded anion exchange resin STG, including the above-mentioned admixture or KJV is anuty unit, component is not more than 10 g of protein/l of volume filled with anion-exchange resin.



 

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