PROCESS FOR PRODUCTION AND PURIFICATION OF RECOMBINANT LYSOSOMAL ALPHA-MANNOSIDASE

1 . (canceled)

2 . A composition comprising alpha-mannosidase obtainable by 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.

3 . A process for fed batch or continuous production of recombinant alpha-mannosidase, comprising the following steps:

a. inoculating a production reactor comprising a base medium with cells capable of producing recombinant 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; and

d. applying a process for purification of the recombinant human lysosomal alpha-mannosidase from the 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.

4 . The process according to claim 3 , wherein the cell culture is free of any supplements derived from animals.

5 . The process according to claim 3 , wherein the process for fed batch or continuous production is performed at a volume of at least 30 L.

6 . The process according to claim 3 , 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.

7 . The composition comprising alpha-mannosidase obtainable by the production process according to claim 3 .

8 . A composition comprising purified recombinant alpha-mannosidase, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein.

9 . The composition according to claim 8 , 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.

10 . The composition according to claim 8 , 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.

11 . A method of treating alpha-mannosidosis and/or reducing or alleviating the symptoms associated with alpha-mannosidosis, said method comprising a step of administering a composition comprising:

A) a composition comprising alpha-mannosidase obtainable by 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;

B) a composition comprising alpha-mannosidase obtainable by a process for fed batch or continuous production of recombinant alpha-mannosidase, comprising the following steps:

i. inoculating a production reactor comprising a base medium with cells capable of producing recombinant alpha-mannosidase on day 0, to provide a cell culture;

ii. adding a feed medium to said cell culture at least once from day 1;

iii. 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; and

iv. applying a process for purification of the recombinant human lysosomal alpha-mannosidase from the 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; or

C) a composition comprising purified recombinant alpha-mannosidase, wherein at least 80% of the alpha-mannosidase is present as a 130 kDa glycoprotein.