Quantitative control of sialylation

Patent metadata

Field	Value
Publication number	US-9809835-B2
Application number	US-201514950443-A
Country	US
Kind code	B2
Filing date	Nov 24, 2015
Priority date	May 29, 2013
Publication date	Nov 7, 2017
Grant date	Nov 7, 2017

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Roche Diagnostics Operations Inc

Inventors

• Engel Alfred
• Greif Michael
• Jung Christine
• Malik Sebastian
• Mueller Rainer
• Sobek Harald
• Suppmann Bernhard
• Thomann Marco

Classifications

• C12P19/18 Primary

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• C12P19/44

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title