Glycoproteins with anti-inflammatory properties

Abstract

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

1 . A method of producing a pharmaceutical preparation comprising glycoproteins comprising an Fc region, wherein the branched glycans on the Fc region are selectively sialylated on the α1-3 arm at a predetermined level comprising:
 contacting a sialyltransferase enzyme with a preparation comprising glycoproteins comprising an IgG Fc region under conditions suitable for sialylation of a plurality of said branched glycans by the enzyme; 
 measuring the level of branched glycans having a sialic acid on said α1-3 arm and/or on the α1-6 arm; 
 processing said preparation into a pharmaceutical preparation if said level is equivalent to said predetermined level; 
 thereby producing a pharmaceutical preparation comprising glycoproteins comprising an Fc region, wherein the branched glycans on the Fc region are selectively sialylated on the α1-3 arm at a predetermined level. 
 
     
     
         2 . The method of  claim 1 , wherein said predetermined level is at least 95% of branched glycans having a sialic acid on said α1-3 arm. 
     
     
         3 . The method of  claim 1 , wherein said predetermined level is 20-90% of branched glycans having a sialic acid on said α1-3 arm. 
     
     
         4 . The method of  claim 1 , wherein said sialyltransferase enzyme is a ST6Gal-I enzyme. 
     
     
         5 . The method of  claim 1 , wherein said α1,3 arm of the branched glycans are sialylated with a NeuAc-α2,6-Gal terminal linkage. 
     
     
         6 . A method of increasing anti-inflammatory activity of a reference glycoprotein preparation, comprising:
 providing a reference glycoprotein preparation comprising glycoproteins comprising an IgG Fc region; and   sialylating the branched glycans on the Fc region on the α1-3 arm of a plurality of said branched glycans to produce a sialylated glycoprotein preparation;   wherein said glycoproteins in said reference glycoprotein preparation are not IgG glycoproteins or do not consist essentially of an Fc region derived from IgG glycoproteins; and   wherein said sialylated glycoprotein preparation has an increased level of anti-inflammatory activity relative to the level of anti-inflammatory activity of said reference glycoprotein preparation.   
     
     
         7 . The method of  claim 6 , further comprising measuring in said sialylated glycoprotein preparation the level of said branched glycans having a sialic acid on the α1-3 arm and/or measuring the level of said branched glycans having a sialic acid on the α1-6 arm. 
     
     
         8 . The method of  claim 6 , further comprising processing said sialylated glycoprotein preparation into a pharmaceutical preparation if the level of branched glycans having a sialic acid on the α1-3 arm and/or the level of branched glycans having a sialic acid on the α1-6 arm meets a predetermined level. 
     
     
         9 . The method of  claim 8 , wherein said the predetermined level is at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the branched glycans having a sialic acid on the α1,3 arm. 
     
     
         10 . The method of  claim 8 , wherein said predetermined level is less than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the branched glycans having a sialic acid on the α1,6 arm. 
     
     
         11 . A method of increasing anti-inflammatory activity of a reference glycoprotein preparation, comprising:
 providing a reference glycoprotein preparation comprising glycoproteins comprising an IgG Fc region; and   sialylating the branched glycans on the Fc region on the α1-3 arm of a plurality of said branched glycans to produce a sialylated glycoprotein preparation;   measuring in said sialylated glycoprotein preparation the level of said branched glycans having a sialic acid on the α1-3 arm and/or measuring the level of said branched glycans having a sialic acid on the α1-6 arm; and   processing said sialylated glycoprotein preparation into a pharmaceutical preparation if the level of branched glycans having a sialic acid on the α1-3 arm and/or the level of branched glycans having a sialic acid on the α1-6 arm meets a predetermined level;   wherein said sialylated glycoprotein preparation has an increased level of anti-inflammatory activity relative to the level of anti-inflammatory activity of said reference glycoprotein preparation.   
     
     
         12 . The method of  claim 11 , wherein said predetermined level of branched glycans having a sialic acid on the α1-3 arm is at least 95% and said predetermined level of branched glycans having a sialic acid on the α1-6 arm is less than 5%. 
     
     
         13 . The method of  claim 11 , wherein said predetermined level of branched glycans having a sialic acid on the α1-3 arm is between 20-90%. 
     
     
         14 . The method of  claim 6 , wherein said sialylated glycoprotein preparation has a level of anti-inflammatory activity that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, or more, higher than the level of anti-inflammatory activity of said reference glycoprotein preparation. 
     
     
         15 . A method of manufacturing a pharmaceutical product comprising glycoproteins comprising an IgG Fc region comprising:
 providing a preparation comprising glycoproteins comprising an IgG Fc region;   measuring the level of branched glycans on the Fc region in said preparation having a sialic acid on the α1-3 arm and/or on the α1-6 arm; and   processing the preparation into a pharmaceutical product if the level of said branched glycans having a sialic acid on the α1-3 arm and/or on the α1-6 arm is equivalent to a predetermined level,   thereby manufacturing a pharmaceutical product comprising glycoproteins comprising an IgG Fc region.   
     
     
         16 . The method of  claim 15 , wherein the predetermined level is a pharmaceutical specification of greater than 25% branched glycans having a sialic acid on the α1-3 arm and/or less than 40% branched glycans having a sialic acid on the α1-6 arm. 
     
     
         17 . The method of  claim 15 , further comprising measuring an anti-inflammatory activity of the preparation. 
     
     
         18 . The method of  claim 17 , wherein said anti-inflammatory activity is measured in vivo or in vitro. 
     
     
         19 . The method of  claim 1 , wherein said preparation is a preparation of antibodies. 
     
     
         20 . The method of  claim 1 , wherein said preparation is formulated for subcutaneous administration. 
     
     
         21 . The method of  claim 1 , wherein said glycoproteins are present in said preparation at a concentration of 50-250 mg/mL. 
     
     
         22 . The method of  claim 1 , wherein said glycoproteins consist essentially of an Fc region. 
     
     
         23 . The method of  claim 1 , wherein said glycoproteins further have a Fab region. 
     
     
         24 . The method of  claim 1 , wherein said glycoproteins are derived from plasma. 
     
     
         25 . The method of  claim 1 , wherein said glycoproteins are recombinant glycoproteins. 
     
     
         26 . The method of  claim 1 , wherein said glycoproteins are IgG glycoproteins or said glycoproteins consist essentially of an Fc region derived from IgG glycoproteins. 
     
     
         27 . A pharmaceutical preparation comprising sialylated glycoproteins produced by the method of  claim 1 . 
     
     
         28 . A pharmaceutical preparation comprising glycoproteins comprising an Fc region, wherein at least 95% of branched glycans on the Fc region have a sialic acid on the α1-3 arm and do not have a sialic acid on the α1-6 arm, and wherein said preparation has anti-inflammatory activity. 
     
     
         29 . A pharmaceutical preparation comprising glycoproteins comprising an Fc region, wherein 20-90% of branched glycans on the Fc region have a sialic acid on the α1-3 arm and do not have a sialic acid on the α1-6 arm, and wherein said preparation has anti-inflammatory activity. 
     
     
         30 . A pharmaceutical preparation comprising a plurality of glycoproteins comprising an IgG Fc region, wherein the IgG Fc region of each of the plurality of glycoproteins comprises a first branched glycan sialylated on the α1-3 arm, and wherein said pharmaceutical preparation has anti-inflammatory activity. 
     
     
         31 . The pharmaceutical preparation of  claim 30 , wherein said IgG Fc region of the plurality of glycoproteins further comprises a second branched glycan. 
     
     
         32 . The pharmaceutical preparation of  claim 30 , said IgG Fc region of the plurality of glycoproteins further comprises a high mannose glycan. 
     
     
         33 . The pharmaceutical preparation of  claim 30 , said IgG Fc region of the plurality of glycoproteins further comprises a second branched glycan sialylated on the α1-3 arm. 
     
     
         34 . The pharmaceutical preparation of  claim 30 , wherein said IgG Fc region of the plurality of glycoproteins further comprises a second branched glycan sialylated on the α1-6 arm. 
     
     
         35 . The pharmaceutical preparation of  claim 30 , wherein the plurality of glycoproteins comprising an IgG Fc region comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the glycoproteins in said preparation. 
     
     
         36 . The pharmaceutical preparation of  claim 27 , wherein said pharmaceutical preparation has a level of anti-inflammatory activity that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, or more, higher than a level of anti-inflammatory activity of a reference glycoprotein preparation. 
     
     
         37 . The pharmaceutical preparation of  claim 27 , wherein said pharmaceutical preparation is a preparation of antibodies. 
     
     
         38 . The pharmaceutical preparation of  claim 27 , wherein said pharmaceutical preparation is formulated for subcutaneous administration. 
     
     
         39 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins are present in said preparation at a concentration of 50-250 mg/mL. 
     
     
         40 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins consist essentially of an Fc region. 
     
     
         41 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins further have a Fab region. 
     
     
         42 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins are derived from plasma. 
     
     
         43 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins are recombinant glycoproteins. 
     
     
         44 . The pharmaceutical preparation of  claim 27 , wherein said glycoproteins are IgG glycoproteins or said glycoproteins consist essentially of an Fc region derived from IgG glycoproteins.