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US 12,761,144 B2Granted
Systems and methods of spectrophotometry for the determination of genome content, capsid content and full/empty ratios of adeno-associated virus particles
AssigneeREGENXBIO Inc.
InventorsZhuchun Wu, Keith Webber, Brian Howie, Li Zhi
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to using spectrophotometry to estimate genome copies and full/empty ratios adeno-associated virus particles.
Claims9
A method of producing a pharmaceutical composition comprising isolated recombinant adeno-associated virus (rAAV) particles, comprising
- (1) isolating rAAV particles under non-denaturing conditions from a feed comprising an impurity by one or more of centrifugation, depth filtration, tangential flow filtration, ultrafiltration, affinity chromatography, size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography,
- (2) determining percentage vector genome copies per capsid (Vg %) of the isolated rAAV particles,
- (3) using the Vg % to determine the quality of the isolated rAAV particles, and
- (4) producing the pharmaceutical composition comprising the isolated rAAV particles and a pharmaceutically acceptable carrier when the Vg % meets a predetermined quality requirement,
- wherein the Vg % is determined by
- a) determining the absorbance of a composition comprising the rAAV particles at least at 260 nm and at 280 nm under non-denaturing conditions, and
- b) calculating the Vg %, wherein the calculating comprises using the following equations
- Vg
-
- (
- GC
- /
- mL
- )
- =
- (
- A
- 260
- -
- α
-
- A
- 280
- )
- ε
- DNA
-
- 260
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- Cp
-
- (
- capsid
- /
- mL
- )
- =
- (
- A
- 280
- -
- A
- 260
- /
- β
- )
- ε
- protein
-
- 280
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- and
- Vg %=Vg/Cp
- wherein A=Absorbance;
- ε=Extinction Coefficient (Molar absorptivity);
- C=Sample Concentration;
- L=Path length, α=εprotein260/εprotein280, β=εDNA260/εDNA280, wherein the extinction coefficients are specific for the genome and for the capsid of the isolated rAAV particles under non-denaturing conditions.
74 more elements
A method of producing a pharmaceutical composition comprising isolated recombinant adeno-associated virus (rAAV) particles, comprising
- (1) obtaining a feed sample comprising rAAV particles,
- (2) determining percentage vector genome copies per capsid (Vg %) of the rAAV particles, and
- (3) using the Vg % to determine the quality of the rAAV particles, and
- (4) isolating rAAV particles under non-denaturing conditions from the feed sample when they meet a predetermined quality requirement or removing the feed sample when they do not meet the quality requirement,
- wherein the Vg % is determined by
- a) determining the absorbance of a composition comprising the rAAV particles at least at 260 nm and at 280 nm, under non-denaturing conditions, and
- b) calculating the Vg %, wherein the calculating comprises using the following equations
- Vg
-
- (
- GC
- /
- mL
- )
- =
- (
- A
- 260
- -
- α
-
- A
- 280
- )
- ε
- DNA
-
- 260
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- Cp
-
- (
- capsid
- /
- mL
- )
- =
- (
- A
- 280
- -
- A
- 260
- /
- β
- )
- ε
- protein
-
- 280
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- and
- Vg %=Vg/Cp
- wherein A=Absorbance;
- ε=Extinction Coefficient (Molar absorptivity);
- C=Sample Concentration;
- L=Path length, α=εprotein260/εprotein280, β=εDNA260/εDNA280, wherein the extinction coefficients are specific for the genome and for the capsid of the isolated rAAV particles under non-denaturing conditions.
74 more elements
A method of producing a pharmaceutical unit dosage comprising a predetermined quantity of recombinant adeno-associated virus (rAAV) genomes, comprising
- (1) isolating rAAV particles under non-denaturing conditions from a feed comprising an impurity,
- (2) determining percentage vector genome copies per capsid (Vg %) of the isolated rAAV particles,
- (3) using the Vg % to determine the quality of the isolated rAAV particles,
- (4) producing a bulk drug substance comprising the isolated rAAV particles and a pharmaceutically acceptable carrier when the isolated rAAV particles meet a predetermined quality requirement, and
- (5) producing a pharmaceutical unit dosage comprising a physically discreet unit of the bulk drug substance comprising the predetermined quantity of the rAAV genomes,
- wherein the Vg % is determined by
- a) determining the absorbance of a composition comprising the rAAV particles at least at 260 nm and at 280 nm, under non-denaturing conditions, and
- b) calculating the Vg %, wherein the calculating comprises using the following equations
- Vg
-
- (
- GC
- /
- mL
- )
- =
- (
- A
- 260
- -
- α
-
- A
- 280
- )
- ε
- DNA
-
- 260
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- Cp
-
- (
- capsid
- /
- mL
- )
- =
- (
- A
- 280
- -
- A
- 260
- /
- β
- )
- ε
- protein
-
- 280
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- and
- Vg %=Vg/Cp
- wherein A=Absorbance;
- ε=Extinction Coefficient (Molar absorptivity);
- C=Sample Concentration;
- L=Path length, α=εprotein260/εprotein280, β=εDNA260/εDNA280, wherein the extinction coefficients are specific for the genome and for the capsid of the isolated rAAV particles under non-denaturing conditions.
75 more elements
A method of producing a pharmaceutical unit dosage comprising a predetermined quantity of recombinant adeno-associated virus (rAAV) genomes, comprising
- (1) isolating rAAV particles under non-denaturing conditions from a feed comprising an impurity,
- (2) determining percentage vector genome copies per capsid (Vg %) of the isolated rAAV particles,
- (3) using the Vg % to produce a bulk drug substance comprising a predetermined genome titer of the isolated rAAV particles and a pharmaceutically acceptable carrier, and
- (4) producing a pharmaceutical unit dosage comprising a physically discreet unit of the bulk drug substance comprising the predetermined quantity of the rAAV genomes,
- wherein the Vg % is determined by
- a) determining the absorbance of a composition comprising the rAAV particles at least at 260 nm and at 280 nm, under non-denaturing conditions, and
- b) calculating the Vg %, wherein the calculating comprises using the following equations
- Vg
-
- (
- GC
- /
- mL
- )
- =
- (
- A
- 260
- -
- α
-
- A
- 280
- )
- ε
- DNA
-
- 260
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- Cp
-
- (
- capsid
- /
- mL
- )
- =
- (
- A
- 280
- -
- A
- 260
- /
- β
- )
- ε
- protein
-
- 280
-
- (
- 1
- -
- α
- /
- β
- )
-
- L
- ,
- and
- Vg %=Vg/Cp
- wherein A=Absorbance;
- ε=Extinction Coefficient (Molar absorptivity);
- C=Sample Concentration;
- L=Path length, α=εprotein260/εprotein280, β=εDNA260/εDNA280, wherein the extinction coefficients are specific for the genome and for the capsid of the isolated rAAV particles under non-denaturing conditions.
74 more elements
Dependencies are inferred from the claim text.