US 9,809,835 B2
Quantitative control of sialylation
Alfred Engel, Weilheim (DE); Michael Greif, Penzberg (DE); Christine Jung, Iffeldorf (DE); Sebastian Malik, Antdorf (DE); Rainer Mueller, Penzberg (DE); Harald Sobek, Biberach (DE); Bernhard Suppmann, Weilheim (DE); and Marco Thomann, Penzberg (DE)
Assigned to Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed by Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed on Nov. 24, 2015, as Appl. No. 14/950,443.
Application 14/950,443 is a continuation of application No. PCT/EP2014/060101, filed on May 16, 2014.
Claims priority of application No. 13169714 (EP), filed on May 29, 2013; and application No. 13175390 (EP), filed on Jul. 5, 2013.
Prior Publication US 2016/0076068 A1, Mar. 17, 2016
Int. Cl. C12P 19/44 (2006.01); C12P 21/00 (2006.01); C12N 9/10 (2006.01); C12P 19/02 (2006.01); C12P 19/18 (2006.01)
CPC C12P 19/18 (2013.01) [C12N 9/1081 (2013.01); C12P 19/02 (2013.01); C12P 19/44 (2013.01); C12P 21/005 (2013.01)] 8 Claims
 
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.