PARTITIONING ANION EXCHANGE CHROMATOGRAPHY FOR PURIFICATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV)

A method of separating full capsids and empty capsids in a preparation of recombinant adeno-associated virus (rAAV) particles that comprise a gene of interest (GOI), wherein the method comprises the steps of:

• (a) loading a preparation comprising rAAV viral particles in a load buffer adjusted to a final load conductivity of about 2 to 12 mS/cm and pH of about 6.0 to about 11 on anion exchange chromatography (AEX) column, and continue the loading beyond the AEX column binding capacity until some AAV capsids flow out of the column while load material is still being flowed in, wherein the load amount is between about 1×1015 to 6×1015 cp/ml;
• (b) eluting the column with an elution buffer, wherein the elution buffer comprises the load buffer and at least one salt to increase the conductivity relative to the load buffer;
• and
• (c) collecting separated fractions of full and not full rAAV capsids using a linear gradient elution or an isocratic elution.

A purified preparation of recombinant adeno-associated virus (rAAV) comprising a gene of interest (GOI), wherein the purified preparation is made by the method comprising the steps of:

• (a) loading a preparation comprising rAAV viral particles in a load buffer adjusted to a final load conductivity of about 2 to 12 mS/cm and pH of about 6.0 to about 11 on anion exchange chromatography (AEX) column, and continue the loading beyond the AEX column binding capacity until some AAV capsids flow out of the column while load material is still being flowed in, wherein the load amount is between about 1×1015 to 6×1015 cp/ml;
• (b) eluting the column with an elution buffer, wherein the elution buffer comprises the load buffer and at least one salt to increase the conductivity relative to the load buffer;
• and
• (c) collecting separated fractions of full and not full rAAV capsids using a linear gradient elution or an isocratic elution.