Practical in vitro sialylation of recombinant glycoproteins

Abstract

This invention provides methods for practical in vitro sialylation of glycoproteins, including recombinantly produced glycoproteins. The methods are useful for large-scale modification of sialylation patterns.

What is claimed is:  
     
         1 . A method of sialylating a saccharide group on a recombinant glycoprotein, the method comprising contacting a saccharide group which comprises a galactose or N-acetylgalactosamine acceptor moiety on a recombinant glycoprotein with a sialic acid donor moiety and a recombinant sialyltransferase in a reaction mixture which provides reactants required for sialyltransferase activity for a sufficient time and under appropriate conditions to transfer sialic acid from said sialic acid donor moiety to said saccharide group.  
     
     
         2 . The method of  claim 1 , wherein the sialic acid donor moiety is CMP-sialic acid.  
     
     
         3 . The method of  claim 2 , wherein the CMP-sialic acid is enzymatically generated in situ.  
     
     
         4 . The method of  claim 1 , wherein the sialyltransferase is a recombinant eukaryotic sialyltransferase which substantially lacks a membrane-spanning domain.  
     
     
         5 . The method of  claim 1 , wherein the sialyltransferase includes a sialyl motif which has an amino acid sequence that is at least about 40% identical to a sialyl motif from a sialyltransferase selected from the group consisting of ST3Gal I, ST6Gal I, and ST3Gal III.  
     
     
         6 . The method of  claim 1 , wherein the sialyltransferase is a recombinant ST3Gal III.  
     
     
         7 . The method of  claim 6 , wherein the sialyltransferase is a recombinant rat ST3Gal III.  
     
     
         8 . The method of  claim 1 , wherein the sialyltransferase is a recombinant ST3Gal IV.  
     
     
         9 . The method of  claim 1 , wherein the sialyltransferase is a recombinant ST6Gal I.  
     
     
         10 . The method of  claim 1 , wherein the sialyltransferase is a recombinant ST3Gal I.  
     
     
         11 . The method of  claim 10 , wherein the reaction mixture comprises a second recombinant sialyltransferase, which second recombinant sialyltransferase is an ST3Gal III.  
     
     
         12 . The method of  claim 1 , wherein the sialyltransferase is a recombinant bacterial sialyltransferase.  
     
     
         13 . The method of  claim 12 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         14 . The method of  claim 13 , wherein the bacterial sialyltransferase is a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         15 . The method of  claim 12 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         16 . The method of  claim 15 , wherein the bacterial sialyltransferase is a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         17 . The method of  claim 12 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a Haemophilus 2,3-sialyltransferase.  
     
     
         18 . The method of  claim 17 , wherein the sialyltransferase is a Haemophilus 2,3-sialyltransferase.  
     
     
         19 . The method of  claim 12 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         20 . The method of  claim 19 , wherein the sialyltransferase is a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         21 . The method of  claim 1 , wherein the sialyltransferase is produced by recombinant expression of a sialyltransferase in a host cell selected from the group consisting of an insect cell, a mammalian cell, and a fungal cell.  
     
     
         22 . The method of  claim 21 , wherein the host cell is an  Aspergillus niger  cell.  
     
     
         23 . A method of sialylating a saccharide group on a recombinant glycoprotein, the method comprising contacting a saccharide group which comprises a galactose or an N-acetylgalactosamine acceptor moiety on a recombinant glycoprotein with a sialic acid donor moiety and a bacterial sialyltransferase in a reaction mixture which provides reactants required for sialyltransferase activity for a sufficient time and under appropriate conditions to transfer sialic acid from said sialic acid donor moiety to said saccharide group.  
     
     
         24 . The method of  claim 23 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         25 . The method of  claim 24 , wherein the bacterial sialyltransferase is a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         26 . The method of  claim 23 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         27 . The method of  claim 26 , wherein the sialyltransferase is a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         28 . The method of  claim 23 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         29 . The method of  claim 28 , wherein the sialyltransferase is a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         30 . The method of  claim 23 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a Haemophilus 2,3-sialyltransferase.  
     
     
         31 . The method of  claim 30 , wherein the sialyltransferase is a Haemophilus 2,3-sialyltransferase.  
     
     
         32 . A method for in vitro sialylation of saccharide groups present on a glycoprotein, said method comprising contacting said saccharide groups with a sialyltransferase, a sialic acid donor moiety, and other reactants required for sialyltransferase activity for a sufficient time and under appropriate conditions to transfer sialic acid from said sialic acid donor moiety to said saccharide group, wherein said sialyltransferase is present at a concentration about 50 mU per mg of glycoprotein or less.  
     
     
         33 . The method of  claim 32 , wherein the sialyltransferase is present at a concentration of between about 5-25 mU per mg of glycoprotein.  
     
     
         34 . The method of  claim 32 , wherein the sialyltransferase is present at a concentration of between about 10-50 mU/ml of reaction mixture and the glycoprotein is present in the reaction mixture at a concentration of at least about 2 mg/ml.  
     
     
         35 . The method of  claim 32 , wherein the method yields a glycoprotein having sialylation of at least about 80% of terminal galactose residues present on the saccharide groups.  
     
     
         36 . The method of  claim 32 , wherein the sialyltransferase is a recombinant sialyltransferase.  
     
     
         37 . The method of  claim 36 , wherein the sialyltransferase substantially lacks a membrane-spanning domain.  
     
     
         38 . The method of  claim 32 , wherein the sialyltransferase includes a sialyl motif which has an amino acid sequence that is at least about 40% identical to a sialyl motif from a sialyltransferase selected from the group consisting of ST3Gal I, ST6Gal I, and ST3Gal III.  
     
     
         39 . The method of  claim 32 , wherein the sialyltransferase is an ST3Gal III.  
     
     
         40 . The method of  claim 39 , wherein the ST3Gal III is a rat ST3Gal III.  
     
     
         41 . The method of  claim 32 , wherein the sialyltransferase is an ST3Gal IV.  
     
     
         42 . The method of  claim 32 , wherein the sialyltransferase is an ST3Gal I.  
     
     
         43 . The method of  claim 42 , wherein the reaction mixture comprises a second recombinant sialyltransferase, which second recombinant sialyltransferase is an ST3Gal III.  
     
     
         44 . The method of  claim 32 , wherein the sialyltransferase is a bacterial sialyltransferase.  
     
     
         45 . The method of  claim 44 , wherein the bacterial sialyltransferase is a recombinant sialyltransferase.  
     
     
         46 . The method of  claim 44 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         47 . The method of  claim 46 , wherein the bacterial sialyltransferase is a  Neisseria meningitidis  2,3-sialyltransferase.  
     
     
         48 . The method of  claim 44 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         49 . The method of  claim 48 , wherein the bacterial sialyltransferase is a  Photobacterium damsela  2,6-sialyltransferase.  
     
     
         50 . The method of  claim 44 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         51 . The method of  claim 50 , wherein the sialyltransferase is a  Campylobacter jejuni  2,3-sialyltransferase.  
     
     
         52 . The method of  claim 44 , wherein the bacterial sialyltransferase has an amino acid sequence which is at least 50% identical to an amino acid sequence of a Haemophilus 2,3-sialyltransferase.  
     
     
         53 . The method of  claim 52 , wherein the sialyltransferase is a Haemophilus 2,3-sialyltransferase.  
     
     
         54 . The method of  claim 32 , wherein the sialic acid donor moiety is CMP-sialic acid.  
     
     
         55 . The method of  claim 54 , wherein the CMP-sialic acid is enzymatically generated in situ.  
     
     
         56 . The method of  claim 32 , wherein the sialic acid is selected from the group consisting of NeuAc and NeuGc.  
     
     
         57 . A method for in vitro sialylation of saccharide groups present on a glycoprotein, the method comprising contacting the saccharide groups with an ST3Gal III sialyltransferase, a sialic acid donor moiety, and other reactants required for sialyltransferase activity for a sufficient time and under conditions to transfer sialic acid from said sialic acid donor moiety to said saccharide group, wherein said ST3Gal III sialyltransferase is present at a concentration of less than about 50 mU per mg of glycoprotein.  
     
     
         58 . The method of  claim 57 , wherein the method further comprises contacting the saccharide groups with an ST6GalI sialyltransferase.