# US 11,896,656 B2 · Methods for providing a homogenous solution of lyophilized mutant diptheria toxin in dimethylsulfoxide

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- Number: US 11,896,656 B2
- Document ID: US11896656B2
- Status: Granted
- Kind: B2
- Application number: 17/049,266
- Filed: 2019-04-25
- Published: 2021-08-19
- Granted: 2024-02-13
- 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/US11896656B2

## CPC

- A61K 39/092: {Streptococcus}
- C07K 14/34: from Corynebacterium (G)
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- C12Y 204/02036: NAD(+)--diphthamide ADP-ribosyltransferase (2.4.2.36)
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- 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)
- 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)
- C12Y: ENZYMES

## Abstract

A method is described for reconstituting lyophilized mutant diphtheria toxin in dimethyl sulfoxide for use in producing pneumococcal capsular polysaccharide mutant diphtheria toxin conjugates.

## Claims

### Claim 1

A method for making a homogeneous solution of mutant diphtheria toxin (mDT) in anhydrous dimethyl sulfoxide (DMSO), the method comprising:
(a) providing a dried composition of mDT; and
(b) reconstituting the dried composition to a final concentration of 10 mg/mL or less in anhydrous DMSO by adding the anhydrous DMSO to the dried composition over a time period of two minutes or less and mixing for at least 10 seconds to provide a homogenous solution comprising the mDT.

### Claim 2 (depends on 1)

The method of claim 1, wherein the dried composition is prepared by sublimative drying of an aqueous solution comprising the mDT wherein the sublimative drying is selected from lyophilization and radiant energy vacuum (REV) dehydration.

### Claim 3 (depends on 2)

The method of claim 2, wherein the sublimative drying comprises freezing the aqueous solution in the form of cakes or lyosphere beads.

### Claim 4 (depends on 2)

The method of claim 2, wherein the sublimative drying process comprises bulk drying performed in a container selected from the group consisting of metal tray, plastic tray, plastic bag, and class I tubing vials.

### Claim 5 (depends on 1)

The method of claim 1, wherein the dried composition is prepared by sublimative drying of an aqueous solution comprising mDT to produce the dried composition, wherein the aqueous solution further comprises about 0.5% (w/v) or more sucrose and a buffer, and the sublimative drying is selected from lyophilization and radiant energy vacuum (REV) dehydration.

### Claim 6 (depends on 5)

The method of claim 5, wherein the buffer is a Histidine, Succinate, MES, MOPS, HEPES, or Acetate buffer in a pH range of 5.0-7.0.

### Claim 7 (depends on 5)

The method of claim 5, wherein the buffer is a Phosphate or a Citrate buffer in a pH range of 5.0-7.0.

### Claim 8 (depends on 1)

The method of claim 1, wherein the dried composition has a moisture content of less than 6%.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mixing in step (b) is performed in 120 minutes or less.

### Claim 10 (depends on 1)

The method of claim 1, wherein the homogenous solution comprising the mDT is conjugated to activated polysaccharides six hours or less following the reconstituting.

### Claim 11 (depends on 1)

The method of claim 1, wherein the homogenous solution comprising the mDT has no detectable aggregation.

### Claim 12 (depends on 1)

The method of claim 1, wherein the homogenous solution comprising the mDT has no detectable beta-sheet mediated aggregation.

### Claim 13 (depends on 1)

The method of claim 1, wherein the homogenous solution comprising the mDT has no detectable beta-sheet mediated aggregation as determined by Fourier-transform infrared spectroscopy or dynamic light scattering.