Methods and systems for producing aav particles

A method for producing a recombinant adeno-associated virus (rAAV), comprising:

• (a) introducing at least one viral production cell (VPC) into a bioreactor and expanding the number of VPCs in the bioreactor to a target VPC cell density;
• (b) introducing into the bioreactor at least one expression baculovirus infected insect cell (BIIC) which comprises an AAV viral expression construct and at least one payload BIIC which comprises an AAV payload construct;
• (c) incubating the mixture of VPCs, expression BIICs and payload BIICs in the bioreactor under conditions which result in the production of one or more rAAVs within one or more of the VPCs;
• (d) harvesting a viral production pool from the bioreactor, wherein the viral production pool comprises a liquid media and the one or more VPCs containing the one or more rAAVs;
• (e) exposing the one or more VPCs within the viral production pool to chemical lysis using a chemical lysis solution under chemical lysis conditions, wherein the chemical lysis releases the one or more rAAVs from the VPCs into the liquid media of the viral production pool;
• (f) processing the viral production pool through one or more clarification filtration steps in which the viral production pool is processed through one or more clarification filtration systems;
• (g) processing the viral production pool through one or more affinity chromatography steps in which the viral production pool is processed through one or more affinity chromatography systems;
• (h) processing the viral production pool through one or more ion exchange chromatography steps in which the viral production pool is processed through one or more ion exchange chromatography systems;
• (i) processing the viral production pool through one or more tangential flow filtration (TFF) steps in which the viral production pool is processed through one or more tangential flow filtration (TFF) systems;
• and
• (j) processing the viral production pool through one or more virus retentive filtration (VRF) steps in which the viral production pool is processed through one or more virus retentive filtration (VRF) systems.