# US 11,078,511 B2 · Aqueous composition

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- Number: US 11,078,511 B2
- Document ID: US11078511B2
- Status: Granted
- Kind: B2
- Application number: 15/629,040
- Filed: 2017-06-21
- Published: 2017-10-19
- Granted: 2021-08-03
- Earliest claimed: 2015-12-21
- 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/US11078511B2

## 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 alpha-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 these enzymes 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 1

An aqueous composition comprising:
(a) a soluble human β-galactoside-α-2,6-sialyltransferase I comprising the amino acid motif from position 90 to position 108 in SEQ ID NO:1;
(b) a cytidine-5′-monophospho-N-acetylneuraminic acid;
(c) a glycosylated target molecule selected from the group consisting of a glycoprotein and a glycolipid, the target molecule comprising one or more antenna(e), at least one antenna having as a 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;
(d) an aqueous solution permitting glycosyltransferase enzymatic activity comprising a buffer salt;
wherein the aqueous composition further comprises an enzyme selected from the group consisting of an alkaline phosphatase, an acid phosphatase, and a 5′ nucleotidase capable of hydrolyzing the phosphoester bond in 5′-cytidine-monophosphate under conditions permitting glycosyltransferase enzymatic activity.