(EP3281636) Process for production and purification of recombinant lysosomal alpha-mannosidase 机翻标题: 暂无翻译,请尝试点击翻译按钮。

源语言标题
(EP3281636) Process for production and purification of recombinant lysosomal alpha-mannosidase
公开号/公开日
EP3281636EP3281636 / 2020-08-052018-02-14
申请号/申请日
EP17192938 / 2011-02-23
发明人
FOGH JENSANDERSSON CLAESWEIGELT CECILIAHYDÉN PIAREUTERWALL HELENANILSSON STEFAN;
申请人
CHIESI FARMACEUTICI;
主分类号
IPC分类号
A61K-038/46 C07K-001/16
摘要
(EP3281636) The present invention relates to a process for purification of recombinant alpha-mannosidase, a process for production of alpha-mannosidase, a composition comprising alpha-mannosidase, use of the composition as a medicament, use as a medicament for the treatment of alpha-mannosidosis and a method of treating alpha-mannosidosis and/or alleviating the symptoms of alpha-mannosidosis.
机翻摘要
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地址
代理人
(EP3281636) Plougmann Vingtoft a/s ([DK]) Reg. Nb: 101215840
代理机构
;
优先权号
2010DK-0070067 2010US-61307587 2011EP-0705806 2011WO-DK50054
主权利要求
(EP3281636) 1. A process for purification of recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising recombinant human lysosomal alpha-mannosidase is subjected to chromatography on a resin comprising a multi-modal ligand, wherein said resin bound multi-modal ligand is a substance having a carboxylic acid or sulphonic acid group,  wherein said process results in a composition comprising purified alpha-mannosidase characterized in that at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. 2. A process according to claim 1, wherein the cell culture comprises cells specifically designed to express the recombinant alpha-mannosidase, wherein said cells are selected from the group consisting of monkey kidney CVI line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK); Chinese hamster ovary cells/-DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV I); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). 3. A process according to any one of claims 1 or 2, wherein the recombinant alpha-mannosidase is obtained using cells transfected with a nucleic acid construct, and wherein said nucleic acid construct comprises a nucleic acid sequence selected from the group consisting of:   i) the nucleic acid sequence set forth in SEQ ID NO: 1; and   ii) a nucleic acid sequence coding for the sequence set forth in SEQ ID NO: 2 or a sub-sequence or analogue of the sequence set forth in SEQ ID NO: 2. 4. A process according to any one of claims 1-3, wherein said fraction of the cell culture comprising the recombinant human lysosomal alpha-mannosidase is a clarified undiluted harvest. 5. A process according to any of claims 1-4, wherein the resin bound multi-modal ligand is a substance of formula (I), (II) or (III):wherein R of the substances of formula (II) and (III) is a functional group of formula (IV): 6. A process according to any of any of claims 1-5, wherein a first eluate comprising recombinant human lysosomal alpha-mannosidase is eluted from the resin comprising a multi-modal ligand using an aqueous solution comprising ethylene glycol or propylene glycol. 7. A process according to any of claims 1-6, wherein a first eluate comprising recombinant human lysosomal alpha-mannosidase obtained from the resin comprising a multi-modal ligand is further subjected to a process comprising the steps of   i) applying a fraction comprising recombinant human lysosomal alpha-mannosidase to a hydrophobic interaction chromatography resin to provide an eluate comprising the recombinant human lysosomal alpha-mannosidase,   ii) passing a fraction comprising recombinant human lysosomal alpha-mannosidase through a mixed-mode ion exchange resin to allow retention of contaminates to provide a flow through comprising the recombinant human lysosomal alpha-mannosidase; and   iii) subjecting a fraction comprising recombinant human lysosomal alpha-mannosidase to chromatography on a anion exchange resin to provide an eluate comprising the recombinant human lysosomal alpha-mannosidase. 8. A process according to any of claims 1-7, wherein the recombinant human lysosomal alpha-mannosidase has a sequence selected from:   A) the sequence set forth in SEQ ID NO 2,   B) a sequence having at least 80% sequence identity to SEQ ID NO 2,   C) a subsequence of the sequence in A) or B). 9. A composition comprising purified recombinant lysosomal alpha-mannosidase obtainable by the purification process according to claim 8, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. 10. A process for fed batch or continuous production of recombinant human lysosomal alpha-mannosidase, comprising the following steps:   a. inoculating a production reactor comprising a base medium with cells capable of producing recombinant human lysosomal alpha-mannosidase on day 0, to provide a cell culture;   b. adding a feed medium to said cell culture at least once from day 1;   c. adjusting the temperature of said cell culture to at the most 35 °C, either after day 3 or when the viable cell density is higher than 2.1 MVC/mL, whichever comes first.   d. a purification process according to any of claims 1-8. 11. A process according to claim 10, wherein the cell culture is essentially free of any supplements derived from animals, such as cod liver oil supplements. 12. A process according to any of any of claims 10-11, wherein the cells are selected from the group consisting of monkey kidney CVI line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK); Chinese hamster ovary cells/-DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV I); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). 13. The process according to claim 12, wherein step d) is a purification process as defined in claim 7. 14. A composition comprising alpha-mannosidase obtainable by the production process according to claim 13, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein. 15. A composition according to claim 9 or claim 14, wherein the recombinant alpha-mannosidase remains stable in liquid solution for at least 4 days when stored at +5 °C or for at least 24 months when stored at -20 °C. 16. A composition according to any of claims 9, 14 or 15, wherein the alpha-mannosidase has a sequence selected from:   A) the sequence set forth in SEQ ID NO 2,   B) a sequence having at least 80% sequence identity to SEQ ID NO 2,   C) a subsequence of the sequence in A) or B). 17. The composition according to any of claims 9 and 14-16 for use as a medicament. 18. The composition according to any of claims 9 and 14-16 for use in the treatment of alpha-mannosidosis.
法律状态
GRANTED
专利类型码
B1A1
国别省市代码
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