Novel anion-exchange chromatography methods for separation of empty from full recombinant adeno-associated virus particles
AssigneeUltragenyx Pharmaceutical Inc.
InventorsWenjun Di, Dennis Perry Chen, Kyle Koczera, Amy Medeiros, Ying Cai, Chao Huang, James C. Warren
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Abstract
Provided are methods of separating full capsid particles and empty capsid particles in a viral capsid preparation by overloading viral capsid preparations onto an anion exchange medium integrated with weak partitioning chromatography and multiple column chromatography (AEX-WPC-MCC). Also provided are viral capsid loading ranges for overloading AEX medium facilitating separating full and empty capsid particles.
Claims79
A method of separating empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of:
- a) applying an equilibrating solution to at least one anion exchange (AEX) medium;
- and
- b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flowthrough comprising empty capsid particles,
- wherein the volume of the viral capsid preparation comprises between 1×1014 to 5.5×1015 vp/ml of the AEX medium.
A method of separating full capsid particles and empty capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of:
- a) applying an equilibrating solution to at least one anion exchange (AEX) medium;
- b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flowthrough comprising empty capsid particles;
- and
- c) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluent comprising full capsid particles,
- wherein the volume of the viral capsid preparation comprises between 1×1014 to 5.5×1015 vp/ml of the AEX medium.
A method of increasing the percentage of full capsid particles in a viral capsid preparation comprising empty and full capsid particles, the method comprising the steps of:
- a) applying an equilibrating solution to at least one anion exchange (AEX) medium;
- b) passing a volume of the viral capsid preparation through the at least one AEX medium to generate a flowthrough;
- c) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluent;
- and
- d) collecting the eluent from step c),
- wherein a volume of the eluent of step d) comprises full capsid particles at a percentage that is higher than the percentage of full capsid particles in an equivalent volume of the viral capsid preparation, and
- wherein the volume of the viral capsid preparation comprises between 1×1014 to 5.5×1015 vp/ml of the AEX medium.
An elution method comprising:
- a) passing a volume of a viral capsid preparation comprising full capsid particles and empty capsid particles through at least one anion exchange (AEX) medium to generate a flowthrough comprising empty capsid particles;
- and
- b) passing an elution solution comprising a salt through the at least one AEX medium to generate an eluent comprising full capsid particles,
- wherein the volume of the viral capsid preparation comprises between 1×1014 to 5.5×1015 vp/ml of the AEX medium.
Dependencies are inferred from the claim text.