# US 2019/0169236 A1 · Purification of virus like particles

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- Number: US 2019/0169236 A1
- Document ID: US20190169236A1
- Status: Published application
- Kind: A1
- Application number: 16/201,073
- Filed: 2018-11-27
- Published: 2019-06-06
- Earliest claimed: 2012-06-22
- Assignees: Takeda Vaccines, Inc.
- Applicants: Takeda Vaccines, Inc.
- Inventors: Ross TAYLOR
- Web page: https://patentgrep.com/patent/US20190169236A1

## CPC

- C07K 14/005: from viruses
- A61P 31/12: Antivirals
- A61P 31/20: for DNA viruses
- 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 2710/14143: viral genome or elements thereof as genetic vector
- C12N 2770/16022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/16023: Virus like particles [VLP]
- C12N 2770/16051: Methods of production or purification of viral material
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods of purifying virus-like particles (VLPs) that are substantially free of process contaminants and infectious agents. The methods incorporate, for example, low-pH treatment during harvest and/or inactivation by a solvent and/or detergent during VLP capture.

## Claims

### Claim 1

A method for purifying virus like particles (VLPs), comprising:
generating or obtaining a cell lysate, culture supernatant, or filtrate containing said VLPs;
adjusting the pH of the lysate, supernatant, or filtrate to a pH value less than about 5 to generate a pH-adjusted solution; and
removing non-VLP particulates/aggregates from the pH-adjusted solution to generate a purified solution comprising the VLPs.

### Claim 2 (depends on 1)

The method of claim 1, wherein the VLPs comprise at least a first subpopulation and a second subpopulation of the VLPs.

### Claim 3 (depends on 2)

The method of claim 2, wherein said removing the non-VLP particulates/aggregates comprises removing the second subpopulation of VLPs from the pH-adjusted solution, wherein the purified solution substantially retains the first subpopulation of VLPs.

### Claim 4 (depends on 3)

The method of claim 3, further comprising removing, after removing the second subpopulation of VLPs from the pH-adjusted solution to generate the purified solution, the first population of VLPs from the purified solution.

### Claim 5 (depends on 4)

The method of claim 4, wherein the VLPs of the cell lysate or culture supernatant/filtrate are norovirus VLPs, wherein the first subpopulation comprises norovirus VLPs with a full length VP1 subunit, and wherein the second subpopulation comprises norovirus VLPs with a truncated VP1 subunit.

### Claim at position 6

6-7. (canceled)

### Claim 8 (depends on 1)

The method of claim 1, wherein said adjusting the pH is carried out for a duration between about 30 minutes and about 48 hours.

### Claim 9 (depends on 1)

The method of claim 1, wherein the VLPs comprise acid-stable VLPs.

### Claim at position 9

10-11. (canceled)

### Claim 12 (depends on 1)

The method of claim 1, wherein said removing is done via one or more of the following types of processes: centrifugation, precipitation, flocculation, settling, and filtration.

### Claim 13 (depends on 12)

The method of claim 12, wherein said removing includes clarifying the pH-adjusted solution by depth filtration followed by additional filtration to generate the purified solution.

### Claim 14

(canceled)

### Claim 15 (depends on 1)

The method of claim 1, wherein at least about 98% of residual contaminating nucleic acid is removed from the lysate, supernatant, or filtrate as a result of said purifying.

### Claim 16

(canceled)

### Claim 17 (depends on 1)

The method of claim 1 further comprising:
purifying the VLPs using a multistep chromatographic process, wherein at least one chromatographic process of the multistep chromatographic process comprises:
contacting the lysate, supernatant or filtrate with a chromatographic material, wherein the VLPs bind to said chromatographic material;
treating the chromatographic material with a solvent and/or detergent; and
eluting, after said treating, the VLPs from the chromatographic material.

### Claim 18 (depends on 17)

The method of claim 17, wherein the solvent and/or detergent comprises one or more of the following: TnBP, octylphenol, ethyleneoxide condensate, polyoxyethylene sorbitan monooleate, and sodium cholate, Triton X-100, and tributyl phosphate.

### Claim 19 (depends on 15)

The method of claim 15, wherein the VLPs are one or more of: norovirus genogroup I VLPs, norovirus genogroup II VLPs, norovirus genogroup IV VLPs, chimeric norovirus VLPs, and sapovirus VLPs.

### Claim 20 (depends on 17)

The method of claim 17, wherein the ratio of the VLPs to residual contaminating protein is increased at least about ten-fold as compared to the lysate, supernatant or filtrate.

### Claim 21

(canceled)

### Claim 22 (depends on 17)

The method of claim 17, wherein at least about 50% of residual contaminating protein is removed from the lysate, supernatant or filtrate as a result of said purifying.

### Claim 23

(canceled)

### Claim 24 (depends on 17)

The method of claim 17, wherein no more than about 50% of the VLPs are lost during said purification.

### Claim 25

(canceled)

### Claim 26 (depends on 17)

The method of claim 17, further comprising separating a first subpopulation of VLPs from the purified solution using the one or more chromatographic processes, wherein each chromatographic process is independently selected from: hydroxyapatite chromatography, hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, mixed mode chromatography, membrane-based chromatography, and affinity chromatography.

### Claim at position 25

27-31. (canceled)

### Claim 32 (depends on 17)

The method of claim 17, wherein the VLPs are produced in bacterial cells, insect cells, yeast cells, plant, or mammalian cells.

### Claim 33 (depends on 3)

The method of claim 3, wherein
the removing of the second subpopulation of VLPs from the pH-adjusted solution comprising using a filtration process to generate a filtered, purified solution, wherein the filtered, purified solution substantially retains the first subpopulation of VLPs.

### Claim at position 28

34-45. (canceled)

### Claim 46 (depends on 1)

A vaccine comprising the first subpopulation of VLPs prepared by the method of claim 1.

### Claim 47 (depends on 1)

The method of claim 1, wherein
the removing non-VLP particulates/aggregates from the pH-adjusted solution, comprises clarifying the pH-adjusted solution by a depth filtration process to generate a purified, depth-filtered solution.

### Claim at position 31

48-53. (canceled)

### Claim 54 (depends on 47)

The method of claim 47, wherein at least about 75% of the VLPs are recovered from the pH-adjusted solution as compared to the lysate, supernatant or filtrate.

### Claim at position 33

55-72. (canceled)

### Claim 73

A solution comprising a purified population of VLPs, wherein said solution contains at least 50% VLPs with a full length VP1 subunit.

### Claim at position 35

74-80. (canceled)

### Claim 81

A method of clearing and/or inactivating live virus in a solution containing the live virus and further containing VLPs generated by the live virus, the method comprising adjusting the pH of the solution containing the live virus to a pH value less than about 5 to generate a pH adjusted solution.

### Claim at position 37

82-96. (canceled)