# US 11,992,521 B2 · Methods for producing Streptococcus pneumoniae capsular polysaccharide carrier protein conjugates

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- Number: US 11,992,521 B2
- Document ID: US11992521B2
- Status: Granted
- Kind: B2
- Application number: 18/049,507
- Filed: 2022-10-25
- Published: 2023-03-16
- Granted: 2024-05-28
- Earliest claimed: 2018-04-30
- Assignees: Merck Sharp & Dohme Corp.
- Applicants: Merck Sharp & Dohme LLC
- Inventors: Patrick Leonard Ahl; Akhilesh Bhambhani; Christopher Jon Farrell; Patrick McHugh; Morrisa C. Jones; Daniel D. Roth; Jessica R. Sinacola; Justin Stanbro; Matthew P. Watson; Emily Wen; Michael A. Winters
- Web page: https://patentgrep.com/patent/US11992521B2

## CPC

- A61K 39/092: {Streptococcus}
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61K 47/62: the modifying agent being a protein, peptide or polyamino acid
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/19: lyophilised {, i.e. freeze-dried, solutions or dispersions (lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00)}
- A61K 2039/55505: {Inorganic adjuvants}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- A61K 2039/70: {Multivalent vaccine}
- 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)

## Abstract

A method is described for producing a pneumococcal capsular polysaccharide protein conjugate in which one or more activated pneumococcal polysaccharides of particular pneumococcal serotypes and carrier protein are separately lyophilized, the separately lyophilized polysaccharides and carrier protein are separately reconstituted in an organic solvent, and the reconstituted polysaccharide and carrier protein are then combined together by Tee-mixing and conjugated together to produce polysaccharide carrier protein conjugates. A plurality of conjugates, each comprising polysaccharides of a particular serotype, may be used to produce multivalent pneumococcal immunogenic compositions having a combination of conjugates for use in vaccines.

## Claims

### Claim 1

A method for making a composition comprising a Streptococcus pneumoniae polysaccharide covalently linked to a carrier protein, the method comprising:
(a) providing a first dried composition comprising activated Streptococcus pneumoniae polysaccharides from one or more Streptococcus pneumoniae serotypes and a second dried composition comprising carrier protein;
(b) separately reconstituting the first dried composition and the second dried composition in an organic solvent and mixing to provide a first homogenous solution comprising the one or more activated polysaccharides and a second homogenous solution comprising the carrier protein, wherein the reconstituting comprises eight minutes or less and the mixing comprises 120 minutes or less;
(c) combining the first homogenous solution with the second homogenous solution by Tee-mixing to produce a mixture; and
(d) adding a reducing agent to the mixture to produce a conjugate solution comprising a carrier protein conjugated to one or more polysaccharides of the Streptococcus pneumoniae serotype;
wherein the first and second dried compositions are prepared by a sublimative drying process comprising lyophilization or radiant energy vacuum (REV) dehydration, and wherein the sublimative drying process comprises freezing the first and second aqueous solutions in the form of cakes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sublimative drying is bulk drying performed in a container selected from the group consisting of metal tray, plastic tray, plastic bag, and class I tubing vials.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first and second dried compositions are prepared by a sublimative drying process comprising lyophilization-or radiant energy vacuum (REV) dehydration comprising providing a first aqueous solution comprising activated Streptococcus pneumoniae polysaccharides from one or more Streptococcus pneumoniae serotypes and a second aqueous solution comprising a carrier protein and a buffer, wherein the first and second aqueous solutions comprise about 0.5% (w/v) or more sucrose, and subjecting the first and second aqueous solutions to the sublimative drying process to produce the first and second dried compositions.

### Claim 4 (depends on 3)

The method of claim 3, wherein the buffer is a Histidine, Succinate, MES, MOPS, HEPES, or Acetate buffer in a pH range of 5.0-7.0.

### Claim 5 (depends on 3)

The method of claim 3, wherein the buffer is a Phosphate or Citrate buffer in a pH range of 5.0-7.0.

### Claim 6 (depends on 1)

The method of claim 1, wherein the first and second dried compositions have a moisture content of less than 6%.

### Claim 7 (depends on 1)

The method of claim 1, wherein the organic solvent is dimethylsulfoxide (DMSO).

### Claim 8 (depends on 1)

The method of claim 1, wherein each conjugate solution comprises polysaccharide conjugated to the carrier protein at a ratio from about 0.6 to about 1.3 polysaccharide to carrier protein on a weight to weight basis.

### Claim 9 (depends on 1)

The method of claim 1, wherein the conjugate solution comprises a free polysaccharide amount that is less than about 15% of the total polysaccharide in the solution.

### Claim 10 (depends on 1)

The method of claim 1, wherein the one or more polysaccharides are obtained from a Streptococcus pneumoniae serotype selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3.

### Claim 11 (depends on 1)

The method of claim 1, wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysins or pneumolysin.

### Claim 12 (depends on 9)

The method of claim 9, wherein the inactivated bacterial toxoid is CRM197.

### Claim 13 (depends on 1)

The method of claim 1, wherein the conjugate solution is sterile filtered.

### Claim 14 (depends on 1)

The method of claim 1, wherein the Streptococcus pneumoniae polysaccharide is activated by reacting with an oxidizing agent.