# US 2021/0277416 A1 · Use of tonicifying agents to enhance recombinant adeno-associated virus yield

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- Number: US 2021/0277416 A1
- Document ID: US20210277416A1
- Status: Published application
- Kind: A1
- Application number: 16/340,758
- Filed: 2017-10-13
- Published: 2021-09-09
- Earliest claimed: 2016-10-14
- Assignees: Dimension Therapeutics, Inc.
- Applicants: Dimension Therapeutics, Inc.
- Inventors: Ying Jing; Jan Panteli
- Web page: https://patentgrep.com/patent/US20210277416A1

## CPC

- C12N 15/86: Viral vectors
- 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 2750/14152: relating to complementing cells and packaging systems for producing virus or viral particles
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides methods for the production of recombinant adeno-associated virus vectors (rAAV), comprising culturing producer cells in media with increased osmolality. Also provided are methods for decreasing the production of helper virus by a rAAV producer cell, comprising culturing the producer cell in media with increased osmolality.

## Claims

### Claim 1

A method for producing a recombinant adeno-associated virus vector (rAAV) using a helper virus, the method comprising incubating a host cell capable of producing rAAV in the presence of helper virus for an incubation period in a cell culture medium containing helper virus and one or more tonicifying agents and having an osmolality of 360 mOsm/kg or higher at the start of the incubation period.

### Claim 2 (depends on 1)

The method of claim 1, wherein the host cell is also capable of producing helper virus.

### Claim 3

The method of any preceding claim, wherein the host cell comprises a genome, said genome comprising one or more AAV genes stably integrated therein.

### Claim 4

The method of any preceding claim, wherein the cell culture medium has an osmolality of 375 mOsm/kg or higher at the start of the incubation period.

### Claim 5

The method of any preceding claim, wherein the cell culture medium has an osmolality of 400 mOsm/kg or higher at the start of the incubation period.

### Claim 6

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 20% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 7

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 30% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 8

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 40% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 9

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce a 50% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 10

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 50% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 11

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 100% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 12

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 150% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 13

The method of any preceding claim, wherein the cell culture medium has an osmolality at the start of the incubation period sufficient to produce at least a 200% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 14

The method of any preceding claim, wherein at least one tonicifying agent is an ionic tonicifying agent.

### Claim 15

The method of any preceding claim, wherein at least one tonicifying agent is selected from the group comprising: NaCl, KCl, NaNO3, NaHCO3, Na2SO4, Na2HPO4, NaH2PO4, NaNO3, KNO3, K2SO4, K2HPO4, KH2PO4, and KNO3.

### Claim 16 (depends on 15)

The method of claim 15, wherein one tonicifying agent is NaCl.

### Claim 17 (depends on 16)

The method of claim 16, wherein at the start of the incubation period the concentration of NaCl in the cell culture medium is 4.5 g/L or higher.

### Claim 18 (depends on 16)

The method of claim 16, wherein at the start of the incubation period the concentration of NaCl in the cell culture medium is 6.5 g/L or higher.

### Claim 19 (depends on 16)

The method of claim 16, wherein at the start of the incubation period the concentration of NaCl in the cell culture medium is 7 g/L or higher.

### Claim 20 (depends on 16)

The method of claim 16, wherein at the start of the incubation period the concentration of NaCl in the cell culture medium is 7.5 g/L or higher.

### Claim 21 (depends on 14, 15, 16, 17, 18, 19, 20)

The method of any one of claims 14-20, wherein at the start of the incubation period the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 50% increase in total rAAV production and a 20% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 22 (depends on 21)

The method of claim 21, wherein at the start of the incubation period the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 100% increase in total rAAV production and a 30% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 23 (depends on 22)

The method of claim 22, wherein at the start of the incubation period the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 150% increase in total rAAV production and a 40% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 24 (depends on 23)

The method of claim 23, wherein at the start of the incubation period the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 200% increase in total rAAV production and a 50% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method of any one of claims 1-13, wherein the tonicifying agent is a non-ionic tonicifying agent.

### Claim 26 (depends on 25)

The method of claim 25, wherein the tonicifying agent is a sugar.

### Claim 27 (depends on 26)

The method of claim 26, wherein the tonicifying agent is a disaccharide.

### Claim 28 (depends on 26)

The method of claim 26, wherein the tonicifying agent is selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.

### Claim 29 (depends on 28)

Then method of claim 28, wherein the tonicifying agent is sucrose.

### Claim 30 (depends on 29)

The method of claim 29, wherein at the start of the incubation period the concentration of sucrose in the cell culture medium is 6.8 g/L or higher.

### Claim 31 (depends on 29)

The method of claim 29, wherein at the start of the incubation period the concentration of sucrose in the cell culture medium is 13.7 g/L or higher.

### Claim 32 (depends on 29)

The method of claim 29, wherein at the start of the incubation period the concentration of sucrose in the cell culture medium is 29.4 g/L or higher.

### Claim 33 (depends on 29)

The method of claim 29, wherein at the start of the incubation period the concentration of sucrose in the cell culture medium is 41.1 g/L or higher.

### Claim 34

The method of any preceding claim, wherein the cell culture medium is a serum-free cell culture medium.

### Claim 35

The method of any preceding claim, wherein the cell culture medium is a protein-free cell culture medium.

### Claim 36

The method of any preceding claim, wherein the cell culture medium is selected from the group consisting of MEM, DMEM, RPMI, Ham's F-12 medium, Leibovitz's L-15 medium, and mixtures thereof, said medium being supplemented with one or more tonicifying agents.

### Claim 37

The method of any preceding claim, wherein the cell culture medium consists essentially of DMEM supplemented with one or more tonicifying agents.

### Claim 38

The method of any preceding claim, wherein the host cell is a mammalian cell.

### Claim 39

The method of any preceding claim, wherein the host cell is selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.

### Claim 40 (depends on 39)

The method of claim 39, wherein the host cell is a HeLa cell.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The method of any one of claims 1-37, wherein the host cell is an insect cell.

### Claim 42 (depends on 41)

The method of claim 41 wherein the host cell is selected from the group consisting of Sf9, Sf-21, Tn-368, and BTI-Tn-5B1-4 (High-Five) cells.

### Claim 43

The method of any preceding claim, wherein the helper virus is selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses.

### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The method of any one of claims 1 to 40, wherein the helper virus is an adenovirus (AV).

### Claim 45 (depends on 44)

The method of claim 44 wherein the helper virus is Ad5.

### Claim 46

The method of any preceding claim, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats.

### Claim 47

The method of any preceding claim, wherein the host cell comprises rep and cap genes.

### Claim 48

The method of any preceding claim, wherein the host cell comprises helper virus genes.

### Claim 49

The method of any preceding claim, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes.

### Claim 50

The method of any preceding claim, wherein the host cell is incubated in the cell culture medium for a period of at least 2 days.

### Claim 51 (depends on 50)

The method of claim 50, wherein the incubation period is at least 3 days.

### Claim 52 (depends on 51)

The method of claim 51, wherein the incubation period is about 4 days.

### Claim 53

The method of any preceding claim, further comprising the steps of harvesting and purifying the rAAV.

### Claim 54

A cell culture system comprising:
a) a host cell capable of producing rAAV;
b) a helper virus; and
c) a cell culture medium with an osmolality of 360 mOsm/kg or higher when measured immediately after the host cell is introduced into the cell culture medium.

### Claim 55 (depends on 54)

The cell culture system of claim 54, wherein the cell culture medium has an osmolality of 375 mOsm/kg or higher.

### Claim 56 (depends on 55)

The cell culture system of claim 55, wherein the cell culture medium has an osmolality of 400 mOsm/kg or higher.

### Claim 57 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce a 20% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 58 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce a 30% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 59 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce a 40% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 60 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce a 50% reduction in total helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 61 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce at least a 50% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 62 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce at least a 100% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 63 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce at least a 150% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 64 (depends on 54, 55, 56)

The cell culture system of any one of claims 54-56, wherein the cell culture medium has an osmolality sufficient to produce at least a 200% increase in total rAAV production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 65 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)

The cell culture system of any one of claims 54-64, wherein at least one tonicifying agent is an ionic tonicifying agent.

### Claim 66 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)

T The cell culture system of any one of claims 54-64, wherein at least one tonicifying agent is selected from the group comprising: NaCl, KCl, NaNO3, Na2SO4, Na2HPO4, NaH2PO4, NaNO3, KNO3, K2SO4, K2HPO4, KH2PO4, and KNO3.

### Claim 67 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)

The cell culture system of any one of claims 54-64, wherein one tonicifying agent is NaCl.

### Claim 68 (depends on 67)

The cell culture system of claim 67, wherein the concentration of NaCl in the cell culture medium is 4.5 g/L or higher.

### Claim 69 (depends on 67)

The cell culture system of claim 67, wherein the concentration of NaCl in the cell culture medium is 6.5 g/L or higher.

### Claim 70 (depends on 67)

The cell culture system of claim 67, wherein the concentration of NaCl in the cell culture medium is 7 g/L or higher.

### Claim 71 (depends on 67)

The cell culture system of claim 67, wherein the concentration of NaCl in the cell culture medium is 7.5 g/L or higher.

### Claim 72 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)

The cell culture system of any one of claims 54-71, wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 50% increase in total rAAV production and a 20% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 73 (depends on 72)

The cell culture system of claim 72, wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 100% increase in total rAAV production and a 30% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 74 (depends on 72)

The cell culture system of claim 72, wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 150% increase in total rAAV production and a 40% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 75 (depends on 72)

The cell culture system of claim 72, wherein the cell culture medium contains an ionic tonicifying agent at a concentration sufficient to produce at least a 200% increase in total rAAV production and a 50% decrease in helper virus production compared to a host cell incubated in a medium with an osmolality of 266 mOsm/kg.

### Claim 76 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)

The cell culture system of any one of claims 54-64, wherein the tonicifying agent is a non-ionic tonicifying agent.

### Claim 77 (depends on 76)

The cell culture system of claim 76, wherein the tonicifying agent is a sugar.

### Claim 78 (depends on 77)

Then cell culture system of claim 77, wherein the tonicifying agent is a disaccharide.

### Claim 79 (depends on 78)

Then cell culture system of claim 78, wherein the tonicifying agent is selected from the group consisting of sucrose, fructose, glucose, galactose, mannose, maltose, and trehalose.

### Claim 80 (depends on 79)

The cell culture system of claim 79, wherein the tonicifying agent is sucrose.

### Claim 81 (depends on 80)

The cell culture system of claim 80, wherein the concentration of sucrose in the cell culture medium is 6.8 g/L or higher.

### Claim 82 (depends on 80)

The cell culture system of claim 80, wherein the concentration of sucrose in the cell culture medium is 13.7 g/L or higher.

### Claim 83 (depends on 80)

The cell culture system of claim 80, wherein the concentration of sucrose in the cell culture medium is 29.4 g/L or higher.

### Claim 84 (depends on 80)

The cell culture system of claim 80, wherein the concentration of sucrose in the cell culture medium is 41.1 g/L or higher.

### Claim 85 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84)

The cell culture system of any one of claims 54-84, wherein the cell culture medium is a serum-free cell culture medium.

### Claim 86 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85)

The cell culture system of any one of claims 54-85, wherein the cell culture medium is a protein-free cell culture medium.

### Claim 87 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

The cell culture system of any one of claims 54-86, wherein the cell culture medium is selected from the group consisting of MEM, DMEM, RPMI, Ham's F-12 medium, Leibovitz's L-15 medium, and mixtures thereof, said medium being supplemented with one or more tonicifying agents.

### Claim 88 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86)

The cell culture system of any one of claims 54-86, wherein the cell culture medium consists essentially of DMEM supplemented with one or more tonicifying agents

### Claim 89 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88)

The cell culture system of any one of claims 54-88, wherein the host cell is a mammalian cell.

### Claim 90 (depends on 89)

The cell culture system of claim 89, wherein the host cell is selected from the group consisting of HeLa, HEK293, COS, A549, BHK, and Vero cells.

### Claim 91 (depends on 90)

The cell culture system of claim 90, wherein the host cell is a HeLa cell.

### Claim 92 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88)

The cell culture system of any one of claims 54-88, wherein the host cell is an insect cell.

### Claim 93 (depends on 92)

The cell culture system of claim 92, wherein the host cell is selected from the group consisting of Sf9, Sf-21, Tn-368, and BTI-Tn-5B1-4 (High-Five) cells.

### Claim 94 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93)

The cell culture system of any one of claims 54-93, wherein the helper virus is selected from the group consisting of adenovirus, herpes virus, baculovirus, and recombinant forms of any of the foregoing viruses.

### Claim 95 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)

The cell culture system of any one of claims 54-91, wherein the helper virus is an adenovirus (AV).

### Claim 96 (depends on 95)

The cell culture system of claim 95 wherein the helper virus is Ad5.

### Claim 97 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)

The cell culture system of any one of claims 54-96, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats.

### Claim 98 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)

The cell culture system of any one of claims 54-96, wherein the host cell comprises rep and cap genes.

### Claim 99 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)

The cell culture system of any one of claims 54-96, wherein the host cell comprises helper virus genes.

### Claim 100 (depends on 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96)

The cell culture system of any one of claims 54-96, wherein the host cell comprises a heterologous nucleotide sequence flanked by AAV inverted terminal repeats, rep and cap genes, and helper virus genes.