Process for production and purification of recombinant lysosomal alpha-mannosidase

Abstract

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 . (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.