# US 11,944,658 B2 · Methods of heat inactivation of adenovirus

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- Number: US 11,944,658 B2
- Document ID: US11944658B2
- Status: Granted
- Kind: B2
- Application number: 17/238,743
- Filed: 2021-04-23
- Published: 2022-03-10
- Granted: 2024-04-02
- Earliest claimed: 2016-03-28
- Assignees: Ultragenyx Pharmaceutical Inc.
- Applicants: Ultragenyx Pharmaceutical Inc.
- Inventors: Christopher J. Morrison; James D. Maratt
- Web page: https://patentgrep.com/patent/US11944658B2

## CPC

- A61K 35/761: Adenovirus
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- C12N 2523/00: Culture process characterised by temperature
- C12N 2710/10361: Methods of inactivation or attenuation
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14151: Methods of production or purification of viral material
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure generally relates to methods of protecting the genomic integrity and/or biological activity of AAV viral particles in a sample containing both AAV particles and helper virus particles during heat inactivation. The methods include heating, to a temperature greater than or equal to 45′C, a sample containing helper virus particles, AAV particles, and a buffer. The buffer includes a concentration of 10 mM or greater kosmotropic salts and/or a concentration of 10 mM or greater of divalent or trivalent cations.

## Claims

### Claim 1

A method of inactivating helper virus in a sample containing helper virus, adeno-associated virus particles, and a buffer comprising a concentration of 25 mM to 500 mM of divalent or trivalent cations, or a concentration of 0.5 M to 0.6 M of kosmotropic salts, wherein the method comprises:
heating the sample to a temperature of 45° C. to 55° C.,
and maintaining the sample at the temperature for a time period of 1 minute to 6 hours, thereby inactivating helper virus.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample is maintained at the temperature for a time period of 10 to 180 minutes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method results in a log reduction of helper virus of 6.3 or greater.

### Claim 4 (depends on 1)

The method of claim 1, wherein the adeno-associated virus particle comprises a genome of approximately or more than 4.7 kb of DNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the buffer further comprises a chaotropic salt.

### Claim 6 (depends on 1)

The method of claim 1, wherein the buffer further comprises a polyol selected from the group consisting of: glycerol, propylene glycol, and 1,6-Hexanediol.

### Claim 7 (depends on 1)

The method of claim 1, wherein the buffer maintains a pH of 3.0 to 10.0 at temperatures of 4° C. to 70° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the buffer further comprises: 40 mM bis-tris propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, and 0.001% (w/v) Pluronic F68.

### Claim 9 (depends on 7)

The method of claim 7, wherein the buffer is a Tris buffer, a phosphate buffer, or a triazolamine buffer.

### Claim 10 (depends on 1)

The method of claim 1, wherein the helper virus is an adenovirus.

### Claim 11 (depends on 10)

The method of claim 10, where the adenovirus is Ad5.

### Claim 12 (depends on 1)

The method of claim 1, wherein the concentration of divalent or trivalent cations is 25 mM to 400 mM.

### Claim 13 (depends on 1)

The method of claim 1, wherein the buffer-comprises divalent or trivalent cations of a metal selected from the group consisting of: Mg, Ca, Mn, Ni, Zn, Co, Sr, Cu, Cr, Fe, and Sc.

### Claim 14 (depends on 13)

The method of claim 13, wherein the cations are selected from the group consisting of: Mg2+, Ca2+, Mn2+, Ni2+, Zn2+, Co2+, Sr2+, Cu2+ and Cr2+.

### Claim 15 (depends on 14)

The method of claim 14 wherein the cations are Ca2+.

### Claim 16 (depends on 14)

The method of claim 14 wherein the cations are Mg2+.

### Claim 17 (depends on 13)

The method of claim 13, wherein the cations are Sc3+.

### Claim 18 (depends on 1)

The method of claim 1, wherein the buffer comprises a kosmotropic salt selected from the group consisting of ammonium sulfate, ammonium acetate, sodium citrate, sodium acetate, sodium sulfate, potassium phosphate, and cesium chloride.

### Claim 19 (depends on 18)

The method of claim 18, wherein the buffer comprises ammonium sulfate.

### Claim 20 (depends on 18)

The method of claim 18, wherein the concentration of kosmotropic salts is 0.5 M.