# US 9,517,274 B2 · Glycoconjugation processes and compositions

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- Number: US 9,517,274 B2
- Document ID: US9517274B2
- Status: Granted
- Kind: B2
- Application number: 14/420,822
- Filed: 2013-08-12
- Published: 2015-08-06
- Granted: 2016-12-13
- Earliest claimed: 2012-08-16
- Assignees: Pfizer Inc.
- Applicants: Pfizer Inc.
- Inventors: Jianxin Gu; Jin-hwan Kim; A. Krishna Prasad; Yu-ying Yang
- Web page: https://patentgrep.com/patent/US9517274B2

## CPC

- A61K 39/095: Neisseria
- A61K 39/092: {Streptococcus}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6415: {Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins}
- A61K 47/646: {the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 37/04: Immunostimulants
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/6068: {Other bacterial proteins, e.g. OMP}
- A61K 2039/627: {characterised by the linker}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The invention provides eTEC linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, immunogenic compositions comprising such glycoconjugates, and methods for the preparation and use of such glycoconjugates and immunogenic compositions.

## Claims

### Claim 1

A method of making a glycoconjugate comprising a saccharide conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, comprising the steps of:
a) reacting a saccharide with 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1′-carbonyldiimidazole (CDI), in an organic solvent to produce an activated saccharide;
b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof, to produce a thiolated saccharide;
c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues;
d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups, to produce a thiolated saccharide-carrier protein conjugate; and
e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues;
whereby an eTEC linked glycoconjugate is produced.

### Claim 2 (depends on 1)

The method of claim 1, wherein the capping step e) comprises reacting the thiolated saccharide-carrier protein conjugate with (i) N-acetyl-L-cysteine as a first capping reagent, and/or (ii) iodoacetamide as a second capping reagent.

### Claim 3 (depends on 1)

The method of claim 1, further comprising a step of compounding the saccharide by reaction with triazole or imidazole to provide a compounded saccharide, wherein the compounded saccharide is shell frozen, lyophilized and reconstituted in an organic solvent prior to step a).

### Claim 4 (depends on 1)

The method of claim 1, further comprising purification of the thiolated polysaccharide produced in step c), wherein the purification step comprises diafiltration.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method further comprises purification of the eTEC linked glycoconjugate by diafiltration.

### Claim 6 (depends on 1)

The method of claim 1, wherein the organic solvent in step a) is a polar aprotic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) and hexamethylphosphoramide (HMPA), or a mixture thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein the saccharide:carrier protein ratio (w/w) is between 0.2 and 4, or between 0.4 and 1.7.

### Claim 8 (depends on 1)

The method of claim 1, wherein the saccharide is a capsular polysaccharide derived from S. pneumonia and said capsular polysaccharide is selected from the group consisting of pneumococcal (Pn) serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides.

### Claim 9 (depends on 1)

The method of claim 1, wherein the saccharide is a capsular polysaccharide derived from N. meningitidis.

### Claim 10 (depends on 1)

The method of claim 1, wherein the carrier protein is CRM197.

### Claim 11 (depends on 1)

An immunogenic composition comprising the glycoconjugate of claim 1 and a pharmaceutically acceptable excipient, carrier or diluent.

### Claim 12

A glycoconjugate comprising a bacterial capsular polysaccharide conjugated to a carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer through a carbamate linkage, and wherein the carrier protein is covalently linked to the eTEC spacer through an amide linkage, as shown by formula (I)

### Claim 13 (depends on 12)

The glycoconjugate of claim 12, wherein the capsular polysaccharide is selected from the group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides.

### Claim 14 (depends on 12)

The glycoconjugate of claim 12, wherein the capsular polysaccharide is derived from N. meningitidis.

### Claim 15 (depends on 12)

The glycoconjugate of claim 12, wherein the polysaccharide has a molecular weight of between 10 kDa and 2,000 kDa, or between 50 kDa and 2,000 kDa.

### Claim 16 (depends on 12)

The glycoconjugate of claim 12, wherein the polysaccharide has a degree of O-acetylation between 75-100%.

### Claim 17 (depends on 12)

The glycoconjugate of claim 12, wherein the carrier protein is CRM197.

### Claim 18 (depends on 17)

The glycoconjugate of claim 17, wherein the CRM197 comprises 2 to 20, or 4 to 16, lysine residues covalently linked to the polysaccharide through an eTEC spacer.

### Claim 19 (depends on 12)

The glycoconjugate of claim 12, wherein the saccharide:carrier protein ratio (w/w) is between 0.2 and 4, or between 0.4 and 1.7.

### Claim 20 (depends on 12)

An immunogenic composition comprising the glycoconjugate of claim 12 and a pharmaceutically acceptable excipient, carrier or diluent.

### Claim 21 (depends on 20)

The immunogenic composition of claim 20, further comprising an additional antigen.

### Claim 22 (depends on 21)

The immunogenic composition of claim 21, wherein the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from the group consisting of Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides.

### Claim 23 (depends on 20)

The immunogenic composition of claim 20, further comprising an adjuvant.

### Claim 24 (depends on 23)

The immunogenic composition of claim 23, wherein the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.