Methods for enhancing yield of recombinant adeno-associated virus
AssigneeUltragenyx Pharmaceutical Inc.
InventorsYing Jing, Jan Panteli, Kelly Reed Clark
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Abstract
The invention provides methods for the production of recombinant adeno-associated virus vectors (rAAV), e.g., comprising the addition of a surfactant, e.g., Triton X-100, to a producer cell culture medium.
Claims42
A method of producing a clarified recombinant adeno-associated virus vector (rAAV) product comprising the steps of:
- (a) incubating a plurality of producer cells in a cell culture medium under conditions that promote release of rAAV particles, whereby rAAV particles are released from the producer cells into the culture medium, and the producer cells are not substantially lysed;
- (b) recovering the cell culture medium from the producer cells, so that the recovered cell culture medium is substantially free of intact producer cells and contains rAAV particles;
- (c) adding a surfactant to the recovered cell culture medium, wherein the surfactant is a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) between 12 and 15;
- and
- (d) passing the recovered cell culture medium containing the surfactant through a first filter;
- wherein the method does not comprise a step of lysing the producer cells.
The method of any preceding claim, wherein the step of recovering the cell culture medium from the producer cells, so that the recovered cell culture medium is substantially free of intact producer cells and contains rAAV particles, comprises the sub-step of aspirating the cell culture medium into a vessel.
The method of any preceding claim, wherein the step of recovering the cell culture medium from the producer cells, so that the recovered cell culture medium is substantially free of intact producer cells and contains rAAV particles, comprises the sub-step of changing the pH of the cell culture medium.
A method of producing a clarified recombinant adeno-associated virus vector (rAAV) product comprising the steps of:
- (a) incubating a plurality of producer cells in a cell culture medium under conditions that promote release of rAAV particles, whereby rAAV particles are released from the producer cells into the culture medium, and the producer cells are not substantially lysed;
- (b) adding a surfactant to the cell culture medium, wherein the surfactant is a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) between 12 and 15;
- (c) incubating the producer cells in the surfactant-containing cell culture medium for a time, the time being insufficient to cause the lysis of 50% of the producer cells;
- and
- (d) passing the cell culture medium containing the surfactant through a first filter.
The method of any preceding claim, wherein release of the rAAV particles from the producer cells into the culture medium results in lysis of fewer than 50% of the producer cells.
The method of any preceding claim, wherein release of the rAAV particles from the producer cells into the culture medium results in lysis of fewer than 30% of the producer cells.
The method of any preceding claim, wherein release of the rAAV particles from the producer cells into the culture medium results in lysis of fewer than 10% of the producer cells.
The method of any preceding claim, wherein release of the rAAV particles from the producer cells into the culture medium results in lysis of fewer than 1% of the producer cells.
The method of any preceding claim, wherein the surfactant is added to the cell culture medium at a final concentration of about 1% to about 0.01%.
The method of any preceding claim, further comprising step (e), passing the cell culture medium containing the surfactant through a second filter.
The method of any preceding claim, wherein one or more of the filters are selected from the group consisting of a PES membrane filter, a PVDF membrane filter, a nylon membrane filter, and a MCE membrane filter.
The method of any preceding claim, wherein the filter has a pore size of less than 1 μm.
The method of any preceding claim, wherein the concentration of rAAV particles in the rAAV-containing cell culture medium after the first filtration step is greater than 80% of the concentration of rAAV particles in the rAAV-containing cell culture medium immediately prior to the first filtration step.
The method of any preceding claim, wherein the surfactant is an octylphenol ethoxylate.
The method of any preceding claim, wherein the producer cells are mammalian cells.
A rAAV produced by the method of any preceding claim.
Dependencies are inferred from the claim text.