Process for production and purification of recombinant lysosomal alpha-mannosidase
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.
1. A process for purification of a recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising said 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.
2. The process according to claim 1 , wherein said fraction of the cell culture comprising the recombinant lysosomal human alpha-mannosidase is a clarified undiluted harvest.
3. The process according to claim 1 , 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):
4. The process according to claim 1 , wherein the fraction of said cell culture loaded onto the resin comprising a multimodal ligand, is subjected to at least one washing step with a solution comprising isopropanol.
5. The process according to claim 1 , wherein a first eluate comprising the 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.
6. The process according to claim 1 , wherein a first eluate comprising the human lysosomal alpha mannosidase obtained from the resin comprising a multi-modal ligand is further subjected to a process comprising:
i) applying a fraction comprising the 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 the 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 the recombinant human lysosomal alpha-mannosidase to chromatography on an anion exchange resin to provide an eluate comprising the recombinant human lysosomal alpha-mannosidase.
7. The process according to claim 6 , wherein the hydrophobic interaction chromatography resin of step i) is an alkyl or phenyl sepharose resin.
8. The process according to claim 6 , wherein the mixed-mode ion exchange resin of step ii) is a ceramic hydroxyapatite or flouroapatite resin.
9. The process according to claim 6 , wherein the anion exchange resin of step iii) is a quaternary ammonium strong anion exchange resin.
10. The process according to claim 1 , wherein a virus inactivation step is performed.
11. The process according to claim 1 , wherein a virus reduction step is performed.
12. The process according to claim 1 , wherein the fraction of said cell culture loaded onto the resin comprising a multimodal ligand, is subjected to at least one washing step with a solution comprising ethanol, isopropanol, n-propanol or n-butanol.
13. A process for purification of a recombinant human lysosomal alpha-mannosidase from a cell culture, wherein a fraction of said cell culture comprising the 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 and, wherein the recombinant human lysosomal alpha-mannosidase has a sequence selected from the group consisting of:
A) the sequence set forth in SEQ ID NO 2, and
B) a sequence having at least 90% sequence identity to SEQ ID NO 2, wherein said sequence has lysosomal alpha-mannosidase activity.
14. The process according to claim 13 , wherein the recombinant human lysosomal alpha-mannosidase comprises a sequence that has at least 95% sequence identity to SEQ ID NO 2 and, wherein said sequence has lysosomal alpha-mannosidase activity.
15. The process according to claim 13 , wherein the recombinant human lysosomal alpha-mannosidase comprises a sequence that has at least 96% sequence identity to SEQ ID NO 2 and, wherein said sequence has lysosomal alpha-mannosidase activity.
16. The process according to claim 13 , wherein the recombinant human lysosomal alpha-mannosidase comprises a sequence that has at least 97% sequence identity to SEQ ID NO 2 and, wherein said sequence has lysosomal alpha-mannosidase activity.