# US 2016/0257983 A1 · N-glycosylation of peptides and proteins

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2016/0257983 A1
- Document ID: US20160257983A1
- Status: Published application
- Kind: A1
- Application number: 15/029,730
- Filed: 2014-10-15
- Published: 2016-09-08
- Earliest claimed: 2013-10-16
- Assignees: Isis Innovation Limited
- Applicants: Isis Innovation Limited; Glycovaxyn AG
- Inventors: Benjamin G. DAVIS; Balakumar VIJAYAKRISHNAN; Feng LUI; Michael WACKER; Amirreza FARIDMOAYER; Michael Thomas KOWARIK
- Web page: https://patentgrep.com/patent/US20160257983A1

## CPC

- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C07H 11/04: Phosphates; Phosphites; Polyphosphates
- C07K 9/00: Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C12P 19/28: N-glycosides
- 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)}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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)

## Abstract

A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline, wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB: (I) to produce the glycoconjugate comprising the protein or peptide having a saccharide [SI] linked to the asparagine in the sequence D/E-X—N—X—S/T. Polyisoprenylpyrophosphates used as substrates in the biocatalytic process are also provided, as well as certain glycoconjugates.

## Claims

### Claim 1

A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:
wherein n is 1, 2, 3 or 4; and
[S1] is a monosaccharide or polysaccharide;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.

### Claim 2 (depends on 1)

A process according to claim 1, wherein the protein or peptide comprising the sequence D/E-X—N—X—S/T is an antibody.

### Claim 3 (depends on 1)

A process according to claim 1, wherein n is 1, 2 or 3.

### Claim 4 (depends on 1)

A process according to claim 1, wherein [S1] is a mono- or disaccharide.

### Claim 5 (depends on 1)

A process according to claim 1, wherein the saccharide [S1] is modified at one or more positions.

### Claim 6 (depends on 1)

A process according to claim 1, wherein [S1] is GlcNAc or GlcNAc-GlcNAc, and wherein [S1] is optionally further modified at one or more additional positions.

### Claim 7 (depends on 1)

A process according to claim 1, wherein [S1] is modified to carry one or more groups R1, wherein R1 comprises a reactive group.

### Claim 8 (depends on 7)

A process according to claim 7, wherein the reactive group is an azide, alkene or alkyne group.

### Claim 9 (depends on 7)

A process according to claim 7, wherein R1 comprises the reactive group and a linker moiety L.

### Claim 10 (depends on 9)

A process according to claim 9, wherein L is selected from an alkylene moiety, an oxy-alkylene- moiety and a moiety of formula —NH—C(═O)-alkylene-.

### Claim 11 (depends on 9)

A process according to claim 9, wherein R1 is a group of formula —(C1-C6 alkylene)-Y, —O—(C1-C6 alkylene)-Y, —NH—C(═O)—(C1-C6 alkylene)-Y, —NH—C(═O)—O—(C1-C6 alkylene)-Y, —O—C(═O)—(C1-C6 alkylene)-Y or —O—C(═O)—O—(C1-C6alkylene)-Y, wherein Y is a reactive group.

### Claim 12 (depends on 1)

A process according to claim 1, which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.

### Claim 13 (depends on 12)

A process according to claim 12, wherein the saccharide [S2] is a mono- or polysaccharide modified to carry a group R2, wherein R2 comprises a reactive group capable of reacting with R1 or with OH.

### Claim 14 (depends on 13)

A process according to claim 13, wherein [S2] is further modified to carry one or more labelling groups.

### Claim 15 (depends on 14)

A process according to claim 14, wherein the labelling group is a radioactive label selected from 18-F, 64-C and 131-I.

### Claim 16

A polyisoprenyl pyrophosphate of formula (I) or a salt thereof
wherein n is 1, 2 or 3 and [S1] is a mono saccharide or polysaccharide.

### Claim 17

A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:
wherein n is 1 to 7; and
[S1] is a monosaccharide or polysaccharide which is modified to carry one or more groups R1, wherein R1 comprises a reactive group;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.

### Claim 18 (depends on 17)

A process according to claim 17, which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.

### Claim 19 (depends on 6)

A process according to claim 6, wherein [S1] is modified to carry one or more groups R1, wherein R1 is a group of formula —(C1-C6 alkylene)-Y, —O—(C1-C6 alkylene)-Y, —NH—C(═O)—(C1-C6 alkylene)-Y, —NH—C(═O)—O—(C1-C6 alkylene)-Y, —O—C(═O)—(C1-C6alkylene)-Y or —O—C(═O)—O—(C1-C6alkylene)-Y, wherein Y is a reactive group.