Manganese supplementation for control of glycosylation in mammalian cell culture process

Abstract

The present invention pertains to a cell culture medium comprising manganese as a media supplement, which was shown to control recombinant protein glycosylation and methods of using thereof. The present invention further pertains to a method of controlling or manipulating glycosylation of a recombinant protein of interest in a large scale cell culture.

1 . A method for achieving a predetermined glycosylation profile of an anti-α4-integrin antibody comprising providing manganese to a cell culture at a concentration that falls within a target manganese concentration range, wherein the cell culture comprises host cells producing the anti-α4-integrin antibody. 
     
     
         2 . The method of  claim 1 , comprising supplementing the cell culture with manganese if the manganese concentration in the cell culture is below the target manganese concentration range. 
     
     
         3 . A method for achieving a predetermined glycosylation profile of an anti-α4-integrin antibody comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, supplementing the cell culture medium with the component to achieve a manganese concentration within the target manganese concentration range, and (iii) culturing a recombinant host cell producing an anti-α4-integrin antibody in the cell culture medium comprising the cell culture medium component. 
     
     
         4 . A method for achieving a predetermined glycosylation profile of an anti-α4-integrin antibody comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, adding manganese to the component of the cell culture medium to achieve a manganese concentration within the target manganese concentration range, (iii) producing a cell culture medium using the component of cell culture medium with the target manganese concentration, and (iv) culturing a recombinant host cell producing an anti-α4-integrin antibody in the cell culture medium comprising the cell culture medium component with the target manganese concentration. 
     
     
         5 . A method for optimizing a cell culture medium for the production of an anti-α4-integrin antibody comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is below a target range, supplementing the cell culture medium or the component of the cell culture medium with manganese to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce anti-α4-integrin antibody with a predetermined glycosylation profile. 
     
     
         6 . A method for optimizing a cell culture medium for the production of an anti-α4-integrin antibody comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is above a target range, removing manganese from the cell culture medium or the component of the cell culture medium to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce anti-α4-integrin antibody with a predetermined glycosylation profile. 
     
     
         7 . The method of  claim 6 , wherein the predetermined glycosylation profile of the anti-α4-integrin antibody comprises 13 to 32% galactosylation. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 6 , wherein the predetermined glycosylation profile of the anti-α4-integrin antibody comprises 0.7 to 3.6% sialylation. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The method of  claim 6 , wherein the target manganese concentration range in the cell culture is 0.025 μM to 10 μM. 
     
     
         14 - 19 . (canceled) 
     
     
         20 . The method of  claim 6 , wherein the anti-α4-integrin antibody is produced by a eukaryotic host cell. 
     
     
         21 - 24 . (canceled) 
     
     
         25 . A method for achieving a predetermined glycosylation profile of an interferon beta-1a polypeptide comprising providing manganese to a cell culture at a concentration that falls within a target manganese concentration range, wherein the cell culture comprises host cells producing the interferon beta-1a polypeptide. 
     
     
         26 . The method of  claim 25 , comprising supplementing the cell culture with manganese if the manganese concentration in the cell culture is below the target manganese concentration range. 
     
     
         27 . A method for achieving a predetermined glycosylation profile of an interferon beta-1a polypeptide comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, supplementing the cell culture medium with the component to achieve a manganese concentration within the target manganese concentration range, and (iii) culturing a recombinant host cell producing an interferon beta-1a polypeptide in the cell culture medium comprising the cell culture medium component. 
     
     
         28 . A method for achieving a predetermined glycosylation profile of an interferon beta-1a polypeptide comprising (i) determining a manganese concentration in a component of a cell culture medium, (ii) if the manganese concentration is below a target manganese concentration range, adding manganese to the component of the cell culture medium to achieve a manganese concentration within the target manganese concentration range, (iii) producing a cell culture medium using the component of cell culture medium with the target manganese concentration, and (iv) culturing a recombinant host cell producing an interferon beta-1a polypeptide in the cell culture medium comprising the cell culture medium component with the target manganese concentration. 
     
     
         29 . A method for optimizing a cell culture medium for the production of an interferon beta-1a polypeptide comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is below a target range, supplementing the cell culture medium or the component of the cell culture medium with manganese to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce interferon beta-1a polypeptide with a predetermined glycosylation profile. 
     
     
         30 . A method for optimizing a cell culture medium for the production of an interferon beta-1a polypeptide comprising (i) determining the amount of manganese in a cell culture medium or a component used to produce a cell culture medium, and (ii) if the amount of manganese is above a target range, removing manganese from the cell culture medium or the component of the cell culture medium to achieve an amount of manganese within the target range, wherein the target range is sufficient to produce interferon beta-1a polypeptide with a predetermined glycosylation profile. 
     
     
         31 . The method of  claim 30 , wherein the predetermined glycosylation profile of the interferon beta-1a polypeptide comprises 91 to 100% sialylation. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . The method of  claim 30 , wherein the predetermined glycosylation profile of the interferon beta-1a polypeptide comprises 55 to 85% biantennary glycoproteins. 
     
     
         35 - 42 . (canceled) 
     
     
         43 . The method of  claim 30 , wherein the target manganese concentration range in the cell culture is 0.1 μM to 5 μM. 
     
     
         44 - 48 . (canceled) 
     
     
         49 . The method of  claim 30 , wherein the interferon beta-1a polypeptide is produced by a eukaryotic host cell. 
     
     
         50 - 77 . (canceled)