A pre-screening method and a methods for separating adeno-associated virus capsids

The method of any preceding claim, wherein the chromatography material is defined by:

• i) Formula I:
• wherein
• R1 is selected from C1-C3 alkyl, and R2 and R3 are independently selected from C1-C3 alkyl, CH2OH, and CH2CHOHCH3;
• optionally wherein each of R1, R2, and R3 is CH3;
• or
• ii) Formula II:
• wherein:
• m is an integer of from 1 to 3;
• R1 and R2 are independently selected from a C1-C3 alkyl;
• R3, and R4 are independently selected from C1-C3 alkyl and CH2CHOHCH3;
• and R5 is selected from hydrogen, a C1-C3 alkyl and CH2CHOHCH3;
• provided that if m is 1, the chromatography material is defined by Formula III:
• wherein n is an integer of from 0 to 3;
• provided that if n is 0, R3 and R4 are independently selected from C1-C3 alkyl, and R5 is hydrogen or CH2CHOHCH3;
• optionally wherein the chromatography material is defined by Formula III and comprises a combination of two or more of the following structures (i)-(iv):
• (i) n is 0;
• R3 and R4 are ethyl;
• and R5 is hydrogen or CH2CHOHCH3;
• (ii) n is 1;
• R1, R2, R3, R4 are ethyl;
• and R5 is hydrogen or CH2CHOHCH3;
• (iii) n is 2;
• each R1 and R2 is ethyl;
• R3 and R4 is ethyl;
• and R5 is hydrogen or CH2CHOHCH3;
• (iv) n is 3;
• each R1 and R2 is ethyl;
• R3 and R4 is ethyl;
• and R5 is hydrogen or CH2CHOHCH3.

Use of an anion exchange chromatography material comprising a support, a ligand, and a surface extender connecting the ligand to the support, and being defined by Formula IV:

• for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material,
• comprising determining elution conditions suitable for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material,
• wherein said elution conditions are determined by performing the steps:
• a. adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5%, such as 10%, of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, to the chromatography material;
• b. eluting the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material from the chromatography material by applying an elution buffer comprising a step gradient of increasing conductivity, which starts at from about 0 to about 5 mS/cm, and which increases by from about 0.5 to about 3 mS/cm per step, such as from about 1 to about 2 mS/cm per step, such as from about 1.2 to about 1.5 mS/cm per step, at least up to and including a conductivity at which the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material have been eluted from the chromatography material;
• c. based on an elution profile obtained in step (b), determining a first value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids not fully packaged with genetic material, and
• d. based on the elution profile obtained in step (b), determining a second value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids fully packaged with genetic material.