# US 11,028,372 B2 · Scalable purification method for AAVRH10

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- Number: US 11,028,372 B2
- Document ID: US11028372B2
- Status: Granted
- Kind: B2
- Application number: 16/060,404
- Filed: 2016-12-09
- Published: 2019-01-03
- Granted: 2021-06-08
- Earliest claimed: 2015-12-11
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Martin Lock; Mauricio Alvira
- Web page: https://patentgrep.com/patent/US11028372B2

## CPC

- C12N 7/02: Recovery or purification
- B01D 15/363: {Anion-exchange}
- B01D 15/3804: {Affinity chromatography}
- B01J 20/281: Sorbents specially adapted for preparative, analytical or investigative chromatography
- 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 15/8645: {Adeno-associated virus}
- G01N 21/33: using ultraviolet light (G01N21/39 takes precedence)
- B01D 15/166: {Fluid composition conditioning, e.g. gradient}
- B01J 41/05: in the strongly basic form
- B01J 41/20: Anion exchangers for chromatographic processes
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14151: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A two-step chromatography purification scheme is described which selectively captures and isolates the genome-containing rAAV vector particles from the clarified, concentrated supernatant of a rAAV production cell culture. The process utilizes an affinity capture method performed at a high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles which are substantially free of rAAV intermediates.

## Claims

### Claim 1

A method for separating recombinant AAVrh10 (rAAVrh10) viral particles containing DNA comprising pharmacologically active genomic sequences from genome-deficient AAVrh10 capsid intermediates, said method comprising:
(a) separating rAAVrh10 viral particles and AAVrh10 capsid intermediates from non-AAV contaminants obtained from an AAV producer cell culture which comprises an expression cassette encoding an AAVrh10 capsid protein operably linked to expression control sequences which direct expression thereof in the producer cell, an expression cassette comprising sequences for packaging in the AAVrh10 capsid, and an expression cassette comprising helper viruses necessary for production and packaging of the rAAVrh10 into a viral particle, wherein the separation is performed using an AAVrh10-capsid binding affinity resin comprising an AAV specific antibody to afford purified materials of (a);
(b) forming a loading mixture comprising: a suspension which comprises (i) materials of (a) purified by the AAVrh10-capsid binding affinity resin and (ii) a buffer comprising 20 mM Bis-Tris propane (BTP) at a pH of about 10.0, whereby the mixture has a pH of about 10.0;
(c) loading the mixture of (b) onto a quaternary amine anion exchange column, said column being in a vessel having an inlet for a loading flow of a suspension and/or solution and an outlet permitting for an elution flow of eluate from the vessel;
(d) washing the loaded anion exchange column with a buffer comprising 10 mM NaCl and 20 mM BTP, wherein the buffer has a pH of about 10.0;
(e) applying an increasing salt concentration gradient to the loaded and washed anion exchange column, wherein the salt gradient is an elution gradient and ranges from about 10 mM to about 190 mM NaCl, inclusive of the endpoints, or a salt gradient having an ionic strength equivalent thereto, and wherein the ionic strength is calculatable using the following formula:
I
=
1
2
⁢
∑
i
=
1
n
⁢
c
i
⁢
z
i
2
,
where ci is the molar concentration of ion i (M, mol/L), zi is the charge number of that ion, and I is taken over all ions in the solution; and
(f) monitoring eluate for ultraviolet absorbance at about A260 and at about A280 and collecting the rAAVrh10 particles from a fraction which is eluted when the peak for A260 crosses over and exceeds the peak for A280, said rAAVrh10 particles being purified away from AAVrh10 intermediates.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the pH of the suspension of (b) and the buffer of (d) is 10.0 and at least about 90% of the collected rAAVrh10 of (f) are pharmacologically active rAAVrh10 viral particles.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the average yield of rAAVrh10 viral particles is at least about 70% of the rAAVrh10 viral particles present in the loaded material of (c) as measured by genome copy (GC) titer.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the producer cell culture is selected from a mammalian cell culture, a bacterial cell culture, and an insect cell culture.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the affinity resin separation of (a) comprises:
(i) equilibrating the affinity resin with a buffer which comprises about 200 mM to about 600 mM NaCl and a pH of about 7.5 prior to applying the material to the affinity resin;
(ii) washing the loaded resin of (i) with a buffer which comprises about 800 mM NaCl to about 1200 mM NaCl and a pH of about 7.5;
(iii) washing the resin of (ii) with the buffer of (i) to reduce salt concentration;
(iv) washing the affinity resin of (iii) with a buffer which comprises about 200 mM to about 600 mM NaCl, 20 mM Sodium Citrate, at a pH of about 2.4 to about 3; and
(v) collecting the resulting eluate of (iv) which comprises the full AAVrh10 particles and the AAVrh10 capsid intermediates for loading onto the anion exchange-column.

### Claim 6 (depends on 5)

The method according to claim 5, wherein in (iv), the pH is about 2.5.

### Claim 7 (depends on 5)

The method according to claim 5, wherein the buffer of (i) and/or the buffer of (iv), independently have about 400 mM NaCl.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the loading flow rate equals the elution flow rate, and is about 10 mL/min to about 40 mL/min for a 8 mL monolith column.

### Claim 9 (depends on 1)

The method according to claim 1, wherein buffer supplying the salt gradient in step (e) has a pH of about 10.0.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the anion exchange column is equilibrated at a pH of about 10.0 prior to the loading step of (c).

### Claim 11 (depends on 1)

The method according to claim 1, wherein the AAVrh10 intermediates are separated from the anion exchange column when the salt gradient reaches an ionic strength equivalent to about 50 mM NaCl or greater.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the anion exchange column is a monolith column or a bead-based column.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the anion exchange column comprises trimethylamine and a support matrix comprising poly(glycidyl methacrylate-co-ethylene dimethacrylate).

### Claim 14 (depends on 1)

The method according to claim 1, wherein the elution gradient is from 1% to about 19% of a buffer comprising 1M NaCl, 20 mM Bis-Tris-Propane (BTP) and having a pH of 10.0.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the anion exchange column is a monolith column and wherein column loading, washing, and elution occur in about 60 column volumes (cv).

### Claim 16 (depends on 1)

The method according to claim 1, wherein the elution flow rate is about 35 cm/h.

### Claim 17

A method for separating recombinant AAVrh10 (rAAVrh10) viral particles containing DNA comprising pharmacologically active genomic sequences from genome-deficient AAVrh10 capsid intermediates, said method comprising:
(a) separating a mixture comprising rAAVrh10 viral particles and AAVrh10 capsid intermediates from non-AAV contaminants generated in a production cell system which comprises an expression cassette, wherein the separation is performed using an AAvrh10-capsid binding affinity capture resin comprising a ligand for AAVrh10, said affinity capture comprising:
(i) equilibrating the AAVrh10-capsid binding affinity resin with a buffer which comprises about 200 mM to about 600 mM NaCl at a pH of 7.5 prior to applying the material to the affinity resin and applying the material to the affinity resin;
(ii) washing the loaded resin with a buffer which comprises about 800 mM NaCl to about 1200 mM NaCl at a pH of 7.5;
(iii) washing the affinity resin of (a)(ii) with a buffer which comprises about 200 mM to about 600 mM NaCl, 20 mM Sodium Citrate, at a pH of 2.5; and
(iv) collecting the resulting eluate of (a)(iii) which comprises the rAAVrh10 viral particles and the AAVrh10 capsid intermediates, wherein the collected rAAVrh10 viral particles and the AAVrh10 capsid intermediates contain less than about 10% contamination from non-AAV viral and cellular proteinaceous and nucleic acid materials; and
(b) purifying the material of (a) via an anion exchange column which comprises the following:
(i) forming a loading mixture comprising: a suspension which comprises the collected materials of (a) and a buffer comprising 20 mM Bis-Tris propane (BTP) at a pH of 10.0, whereby the mixture has a pH of 10.0;
(ii) loading the mixture of (b)(i) onto a quaternary amine strong anion exchange column;
(iii) washing the loaded anion exchange column with a buffer comprising 10 mM NaCl and 20 mM BTP, wherein the buffer has a pH of 10.0;
(iv) applying an increasing salt concentration gradient to the loaded and washed anion exchange column, wherein the salt gradient generated via 1% to 19% of a buffer comprising 1M NaCl, 20 mM Bis-Tris-Propane (BTP) and having a pH of 10.0, and wherein the loading flow rate equals the elution flow rate; and
(v) collecting the rAAVrh10 viral particles from a fraction which is eluted when the peak for A260 crosses over and exceeds the peak for A280, said rAAVrh10 viral particles being purified away from AAVrh10 intermediates.

### Claim 18 (depends on 17)

The method according to claim 17, wherein at least about 90% of the collected rAAVrh10 of (b)(v) are pharmacologically active rAAVrh10 viral particles.

### Claim 19 (depends on 17)

The method according to claim 17, wherein the production cell culture is selected from a mammalian cell culture, a bacterial cell culture, and an insect cell culture.

### Claim 20 (depends on 17)

The method according to claim 17, wherein the buffer of (a)(i) comprises 400 mM NaCl.

### Claim 21 (depends on 17)

The method according to claim 17, wherein the buffer of (a)(iii) comprises 400 mM NaCl.

### Claim 22 (depends on 17)

The method according to claim 17, wherein the loading flow rate is 35 cm/h.

### Claim 23 (depends on 17)

The method according to claim 17, wherein the anion exchange column is equilibrated at a pH of 10.0 prior to the loading step of (b)(ii).

### Claim 24 (depends on 17)

The method according to claim 17, wherein the AAVrh10 intermediates are separated from the anion exchange column when the salt gradient reaches about 50 mM NaCl or greater.

### Claim 25 (depends on 17)

The method according to claim 17, wherein the anion exchange column is a monolith column or a bead-based column.

### Claim 26 (depends on 17)

The method according to claim 17, wherein the anion exchange column comprises trimethylamine and a support matrix comprising poly(glycidyl methacrylate-co-ethylene dimethacrylate).

### Claim 27 (depends on 17)

The method according to claim 17, wherein after the harvesting step of (b) and prior to the step of (a), the production cells were lysed to release the rAAVrh10 viral particles and the AAVrh10 capsid intermediates.

### Claim 28 (depends on 17)

The method according to claim 17, wherein the rAAVrh10 viral particles and the AAVrh10 capsid intermediates are released into culture media and the media is collected in the harvesting step, wherein, prior to the step of (a), the harvested media is treated at 37° C. with Benzonase at 25 U/mL for 2 h followed by a hypertonic shock with 0.5 M NaCl for 2 h, and the treated media is filter-clarified, concentrated by tangential flow filtration (TFF) and then buffer-exchanged with the equilibration buffer of step (c)(i) via incubation overnight at 4° C.

### Claim 29 (depends on 17)

The method according to claim 17, wherein the loaded resin is washed with 1.5 mM MgCl2, 40 mM NaCl, 20 mM Tris-Cl, and 250 U/mL Benzonase at pH 7.5 prior to step (a)(2) and after step (a)(i) and then is washed with the equilibration buffer of (a)(i) after step (a)(ii) and prior to step (a)(iii).

### Claim 30 (depends on 17)

The method according to claim 17, wherein the method further comprises one or more of the following: treatment with a nuclease to digest any contaminating high molecular weight nucleic acid present in the production culture, tangential flow filtration (TFF) for concentrating the rAAVrh10 particles, heat inactivation of helper virus, rAAVrh10 capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), and nanofiltration.

### Claim 31 (depends on 17)

The method according to claim 17, wherein the step (b) is performed in a form of Fast Protein Liquid Chromatography (FPLC).

### Claim 32 (depends on 17)

The method according to claim 17, further comprising clarifying a mixture comprising rAAVrh10 viral particles and AAVrh10 capsid intermediates harvested in the production cell system by filtration through a series of depth filters, thereby removing production cell debris from the mixture prior to step (a).

### Claim 33 (depends on 8)

The method according to claim 8, wherein the loading flow rate equals the elution flow rate, and is about 15 mL/min to about 30 mL/min for a 8 mL monolith column.

### Claim 34 (depends on 8)

The method according to claim 8, wherein the loading flow rate equals the elution flow rate, and is about 20 mL/min to about 25 mL/min for a 8 mL monolith column.

### Claim 35 (depends on 8)

The method according to claim 8, wherein the loading flow rate equals the elution flow rate, and is about 10 mL/min for a 8 mL monolith column.

### Claim 36 (depends on 8)

The method according to claim 8, wherein the loading flow rate equals the elution flow rate, and is about 20 mL/min for a 8 mL monolith column.

### Claim 37 (depends on 19)

The method according to claim 19, wherein the production cell system is a suspension cell culture.