# US 2021/0340585 A1 · Cmp-dependent sialidase activity

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- Number: US 2021/0340585 A1
- Document ID: US20210340585A1
- Status: Published application
- Kind: A1
- Application number: 17/355,962
- Filed: 2021-06-23
- Published: 2021-11-04
- Earliest claimed: 2014-12-22
- Assignees: Roche Diagnostics Operations, Inc.
- Applicants: Roche Diagnostics Operations, Inc.
- Inventors: Harald SOBEK; Michael GREIF; Marco THOMANN; Sebastian MALIK
- Web page: https://patentgrep.com/patent/US20210340585A1

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 9/1081: {transferring other glycosyl groups (2.4.99)}
- C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)}
- C12Y 204/99001: Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1)
- C12Y 301/03: Phosphoric monoester hydrolases (3.1.3)
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that such enzyme are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

## Claims

### Claim at position 1

1-10. (canceled)

### Claim 11 (depends on 1)

A method of producing in vitro a sialylated target molecule, the method comprising the steps of
a) providing an aqueous composition according to claim 1;
(b) forming one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s) by incubating the aqueous composition of step (a), thereby reacting cytidine-5′-monophospho-N-acetylneuraminic acid as co-substrate, thereby forming 5′-cytidine-monophosphate; and
(c) hydrolyzing the phosphoester bond of the 5′-cytidine-monophosphate formed in step (b), thereby reducing 5′-cytidine-monophosphate-mediated inhibition, thereby maintaining the activity of the soluble human β-galactoside-α-2,6-sialyltransferase I; thereby producing in vitro the sialylated target molecule.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the method is performed at a temperature of 0° C. to 40° C.

### Claim 13 (depends on 11)

The method according to claim 11, wherein steps (b) and (c) are performed in the same vessel.

### Claim 14 (depends on 11)

The method according to claim 11, wherein steps (b) and (c) are performed for a period selected from the group consisting of 2 h to 96 h, 2 h to 23 h, 2 h to 6 h, and about 2 h.

### Claim 15 (depends on 11)

The method according to claim 11, wherein steps (b) and (c) are performed for a period selected from the group consisting of 6 h to 96 h, 6 h to 23 h, and about 6 h.

### Claim 16 (depends on 11)

The method according to claim 11, wherein steps (b) and (c) are performed for a period selected from the group consisting of 23 h to 96 h, and about 23 h.

### Claim 17 (depends on 11)

The method according to claim 11, wherein steps (b) and (c) are performed for a period of about 96 h.