Separation of full and empty adeno-associated virus particles using a novel multi-modal anion exchange membrane

A novel multi-modal anion exchange (MMAEX) membrane with both electrostatic and hydrophobic interaction moieties was developed for the separation of full and empty AAV2 capsids with only slight differences in surface charge and hydrophobicity. Both gradient and two-step elution have been able to separate the full and empty capsids effectively. During gradient elution with slight increase in conductivity coupled with weakening electrostatic and enhanced hydrophobic interactions between the ligand and the capsids, two distinctive elution peaks representing empty and full capsids were resolved. Full capsid recovery of 94 % at 89 % purity has been achieved with a loading density of ∼10¹² virus particles per milliliter of membrane volume. During the two-step elution process, the functionalized membrane can achieve 88 % full capsid recovery at 75 % purity, or 67 % recovery at 89 % purity, or 59 % recovery at 93 % purity at first-step conductivity of 7.0, 7.5 and 8.0 mS/cm respectively and a loading density of ∼10¹³ particles per milliliter of membrane volume. Our results indicate that our MMAEX membrane coupled with careful modulation of capsid-ligand interaction has superior performance for separating the full and empty AAV capsids.