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RU2368619C2 - 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 - Google Patents

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 Download PDF

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RU2368619C2
RU2368619C2 RU2005113152/13A RU2005113152A RU2368619C2 RU 2368619 C2 RU2368619 C2 RU 2368619C2 RU 2005113152/13 A RU2005113152/13 A RU 2005113152/13A RU 2005113152 A RU2005113152 A RU 2005113152A RU 2368619 C2 RU2368619 C2 RU 2368619C2
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Russia
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peg
aggregate
anion exchange
isoforms
sth
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RU2005113152/13A
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RU2005113152A (en
Inventor
Деннис М. БОЙЛ (US)
Деннис М. БОЙЛ
Джон Дж. БАКЛИ (US)
Джон Дж. БАКЛИ
Гэри В. ДЖОНСОН (US)
Гэри В. ДЖОНСОН
Дейвид И. СТЕЙНМЕЙЕР (US)
Дейвид И. СТЕЙНМЕЙЕР
Майкл ТОЭЛ (US)
Майкл ТОЭЛ
Сердар ЭЙКЕНТ (US)
Сердар ЭЙКЕНТ
Энурэг С. РЭТОР (US)
Энурэг С. РЭТОР
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Фармация Корпорейшн
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Abstract

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

Description

Текст описания представлен в факсимильном виде.

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Claims (8)

1. Способ получения соматотропного гормона (СТГ) со сниженным содержанием агрегата его изоформ, который предусматривает отделение изоформ СТГ посредством анионообменного хроматографирования с применением анионообменной смолы для снижения содержания упомянутого агрегата до не более чем 10 мас.%, исходя из общей массы упомянутых изоформ и упомянутого агрегата, для чего осуществляют загрузку упомянутого СТГ, включающего дефенилаланиновую и/или трисульфидную примесь и/или его агрегат, на анионообменную смолу, выбранную из группы, включающей диэтиламиноэтилцеллюлозу и Q-сефарозу, причем загрузку осуществляют при значении проводимости загрузки анионообменной смолы, меньшем либо равном 10 мСм/см, при pH загрузки анионообменной смолы от 5 до 10 и при загрузке анионообменной смолы СТГ, включающим упомянутую примесь или упомянутый агрегат, составляющей не более чем 10 г белка/л объема, заполненного анионообменной смолой.1. A method of producing somatotropic hormone (STH) with a reduced content of its isoform aggregate, which involves the separation of STH isoforms by anion exchange chromatography using anion exchange resin to reduce the content of said aggregate to no more than 10 wt.%, Based on the total weight of the said isoforms and the aforementioned unit, for which they carry out the loading of the above-mentioned STH, including the phenylalanine and / or trisulfide impurity and / or its aggregate, on an anion exchange resin selected from the group including diethylaminoethyl cellulose and Q-sepharose, the loading being carried out at an anion exchange resin loading conductivity of less than or equal to 10 mS / cm, at an anion exchange resin loading pH of 5 to 10, and at loading an STH anion exchange resin comprising the above impurity or said aggregate that does not constitute more than 10 g of protein / l volume filled with anion exchange resin. 2. Способ по п.1, отличающийся тем, что СТГ содержит одну или несколько изоформ СТГ PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8 и PEG-9, агрегат, трисульфидную примесь и дефенилаланиновую примесь, неполиэтиленгликолизированную примесь СТГ и свободные молекулы PEG.2. The method according to claim 1, characterized in that the STH contains one or more isoforms of STG PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8 and PEG-9, an aggregate, a trisulfide impurity and a phenylalanine impurity, an unpolyethylene glycolized impurity of STH, and free PEG molecules. 3. Способ по п.2, дополнительно включающий стадии:
разделения упомянутых изоформ СТГ на избранные изоформы; и
объединения упомянутых избранных изоформ с получением обогащенного пула упомянутых избранных изоформ.3. The method according to claim 2, further comprising the steps of:
separating said isoforms of STH into selected isoforms; and
combining said selected isoforms to obtain an enriched pool of said selected isoforms. 4. Способ по п.1, дополнительно включающий стадию объединения, на которой осуществляется объединение дискретных количеств упомянутых изоформ СТГ с получением объединенного СТГ посредством метода, выбранного из группы, включающей электрофорез в капиллярах, электрофорез в полиакриламидном геле в присутствии додецилсульфата натрия, ионообменную хроматографию, гидрофобную хроматографию, анионообменную хроматографию, катионообменную хроматографию, высокоэффективную жидкостную хроматографию с обращенной фазой, высокоэффективную жидкостную гель-хроматографию, афинную хроматографию и их комбинации.4. The method according to claim 1, further comprising combining the discrete amounts of the said STH isoforms to obtain the combined STH by a method selected from the group comprising electrophoresis in capillaries, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, ion-exchange chromatography, hydrophobic chromatography, anion exchange chromatography, cation exchange chromatography, high-performance liquid chromatography with reverse phase, high-performance liquid gel chromatography, affinity chromatography, and combinations thereof. 5. Способ по п.4, отличающийся тем, что упомянутый объединенный СТГ содержит одну или несколько PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8 и PEG-9.5. The method according to claim 4, characterized in that said combined STH comprises one or more PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8 and PEG-9. 6. Способ по п.5, отличающийся тем, что объединенная фракция СТГ PEG-4, PEG-5 и PEG-6 содержит по меньшей мере около 70 мас.%, исходя из общей массы упомянутых изоформ СТГ PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8 и PEG-9 и упомянутого агрегата.6. The method according to claim 5, characterized in that the combined fraction of STG PEG-4, PEG-5 and PEG-6 contains at least about 70 wt.%, Based on the total weight of the said isoforms of STG PEG-1, PEG-2 , PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, and PEG-9, and said aggregate. 7. Способ получения антагониста СТГ со сниженным содержанием агрегата его изоформ, который предусматривает отделение изоформ антагониста СТГ на анионообменной смоле посредством анионообменного хроматографирования для снижения содержания упомянутого агрегата до не более чем 6 мас.%, исходя из общей массы упомянутых изоформ и агрегата, для чего осуществляют загрузку упомянутого СТГ, включающего дефенилаланиновую и/или трисульфидную примесь и/или его агрегат, на анионообменную смолу, выбранную из группы, включающей диэтиламиноэтилцеллюлозу и Q-сефарозу, причем загрузку осуществляют при значении проводимости загрузки анионообменной смолы, меньшем либо равном 10 мСм/см, при pH загрузки анионообменной смолы от 5 до 10 и при загрузке анионообменной смолы СТГ, включающим упомянутую примесь или упомянутый агрегат, составляющей не более чем 10 г белка/л объема, заполненного анионообменной смолой.7. A method of producing an STH antagonist with a reduced aggregate content of its isoforms, which involves the separation of isoforms of an STH antagonist on an anion exchange resin by anion exchange chromatography to reduce the content of said aggregate to no more than 6 wt.%, Based on the total weight of said isoforms and aggregate, for which loading said STH, including the phenylalanine and / or trisulfide impurity and / or its aggregate, onto an anion exchange resin selected from the group consisting of diethylaminoethyl cellulose oz and Q-Sepharose, moreover, the loading is carried out at an anion exchange resin loading conductivity value less than or equal to 10 mS / cm, at an anion exchange resin loading pH of 5 to 10 and when loading an STH anion exchange resin comprising the above impurity or said aggregate of not more than than 10 g protein / l volume filled with anion exchange resin. 8. Способ получения антагониста СТГ со сниженным общим суммарным содержанием трисульфидной примеси, дефенилаланиновой примеси и агрегата его изоформ, предусматривающий отделение изоформ антагониста СТГ на анионообменной смоле посредством анионообменного хроматографирования для снижения общего содержания трисульфидной примеси, дефенилаланиновой примеси и упомянутого агрегата до уровня, меньшего либо равного 15 мас.%, исходя из общей массы упомянутых изоформ и упомянутого агрегата, для чего осуществляют загрузку упомянутого СТГ, включающего дефенилаланиновую примесь и/или трисульфидную примесь и/или его агрегат, на анионообменную смолу, выбранную из группы, включающей диэтиламиноэтилцеллюлозу и Q-сефарозу, причем загрузку осуществляют при значении проводимости загрузки анионообменной смолы, меньшем либо равном 10 мСм/см, при pH загрузки анионообменной смолы от 5 до 10 и при загрузке анионообменной смолы СТГ, включающим упомянутую примесь или упомянутый агрегат, составляющей не более чем 10 г белка/л объема, заполненного анионообменной смолой. 8. A method for producing an STH antagonist with a reduced total content of trisulfide impurity, a phenylalanine impurity and its isoform aggregate, comprising separating isoforms of an STH antagonist on an anion exchange resin by anion exchange chromatography to reduce the total content of trisulfide impurity, defenylalanine impurity and the said aggregate to a level lower than 15 wt.%, Based on the total mass of the said isoforms and the said aggregate, for which they carry out the loading of the mentioned STH, including a defenylalanine impurity and / or a trisulfide impurity and / or its aggregate onto an anion exchange resin selected from the group consisting of diethylaminoethyl cellulose and Q-sepharose, the loading being carried out at an anion exchange resin loading conductivity of less than or equal to 10 mS / cm, at a pH of the loading anion exchange resin from 5 to 10; and when loading an STG anion exchange resin comprising said impurity or said aggregate comprising not more than 10 g of protein / l of the volume filled with the anion exchange resin.
RU2005113152/13A 2002-09-20 2003-09-22 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 RU2368619C2 (en)

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US41222702P 2002-09-20 2002-09-20
US60/412,227 2002-09-20
US10/646,798 2003-08-25
US10/646,798 US20040048315A1 (en) 2002-08-28 2003-08-25 Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
US10/662,884 2003-09-16

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4880913A (en) * 1986-12-02 1989-11-14 Schwab & Co. Ges.M.B.H. Process for the preparation of an immunoglobulin which can be administered intravenously and is stable in liquid form
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
RU2180595C2 (en) * 1996-05-31 2002-03-20 Ф. Хоффманн-Ля Рош Аг Interferon conjugates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4880913A (en) * 1986-12-02 1989-11-14 Schwab & Co. Ges.M.B.H. Process for the preparation of an immunoglobulin which can be administered intravenously and is stable in liquid form
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
RU2180595C2 (en) * 1996-05-31 2002-03-20 Ф. Хоффманн-Ля Рош Аг Interferon conjugates

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TWI318629B (en) 2009-12-21

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