Quantitative control of sialylation
The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. Contrary to previous findings certain truncations were found to exhibit sialidase enzymatic activity, particularly a variant of human sialyltransferase (hST6Gal-I) with a truncation deletion involving the first 89 N-terminal amino acids of the respective wild-type polypeptide. A fundamental finding documented in the present disclosure is that there exists a variant of this enzyme which is capable of catalyzing transfer of a glycosyl moiety as well as hydrolysis thereof. Thus, disclosed is a specific exemplary variant of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variant of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.
1. A method of producing in vitro a sialylated target molecule with a controlled quantity of sialyl residues, the method comprising the steps of
(a) providing a glycosylated target molecule in an aqueous solution and under conditions permitting glycosyltransferase enzymatic activity, the target molecule being selected from a glycoprotein and a glycolipid, the target molecule comprising a plurality of antennae, at least two of the antennae each having as terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue;
(b) forming one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s) [=α2,6 sialylated terminal antennal residue(s)] by incubating the target molecule of step (a) for a first pre-determined time with N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I having the amino acid sequence of SEQ ID NO:2 and in the presence of cytidine-5′-monophospho-N-acetylneuraminic acid, or a functional equivalent thereof, as donor compound thereby providing a sialylated target molecule;
(c) hydrolyzing the α2,6 glycosidic bond in one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl- 1,4-N-acetyl-β-D-glucosamine residues by incubating the sialylated target molecule of step (b) for a second pre-determined time with the N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I having the amino acid sequence of SEQ ID NO:2;
thereby producing in vitro the sialylated target molecule with a controlled quantity of sialyl residues.
2. The method according to claim 1 , wherein between the steps (b) and (c) sialylation of the target molecule is determined quantitatively.
3. The method according to claim 1 wherein after step (c) sialylation of the target molecule is determined quantitatively.
4. The method according to claim 1 , wherein steps (a), (b) and (c) are performed continuously in the same vessel.
5. The method according to claim 1 , wherein the target molecule is a purified immunoglobulin molecule of the IgG class, particularly a monoclonal antibody of an immunoglobulin class selected from IgG1, IgG2, IgG3 and IgG4.
6. The method according to claim 5 , wherein steps (a), (b) and (c) are performed continuously in the same vessel with a measured amount of target molecules, wherein step (b) is performed for 0 h to about 24 h and subsequent step (c) is performed for 0 h, and wherein the relative amount of bi-sialylated target molecules is about 35% to about 90%.
7. The method according to claim 5 , wherein steps (a), (b) and (c) are performed continuously in the same vessel with a measured amount of target molecules, wherein step (b) is performed for 24 h and subsequent step (c) is performed for 0 h to about 72 h or longer, and wherein the relative amount of mono-sialylated target molecules is about 60% to about 75%.
8. The method according to claim 6 , wherein the weight-by-weight [w/w] ratio of target (immunoglobulin) molecules : human β-galactoside-α-2,6-sialyltransferase I molecules is 10:1, wherein each has a relative purity of 80% or higher.