AAV Purification by anion-exchange chromatography

Abstract

We describe a new optimized, scalable and reproducible method based on anion exchange chromatography to obtain high titers of rAAV vectors without empty capsids contamination. The method takes advantage of Q-sepharose matriz to development a scalable procedure. After the virus harvest from supernatant and lysate cells, virus crude was subjected to anion exchange chromatography with Q-sepharose column. Three different protocols were tested, and the elution peaks were evaluated through qPCR rAAV titration and 260/280 nm ratio determination in order to identified empty capsid-containing fractions. A 150 mM NH4Ac wash step fallowing by 1 M NaCl elution step generate a a high titer eluted fraction of rAAV with 1.334 260/280 nm ratio. The described method makes rAAV vector purification an easily adapted for a large scale GMP production format to produce empty capsid free rAAV for clinics.

Approaches to purification and concentration of rAAV vectors for gene therapy

“…One type of ion-exchange matrix is rarely used to obtain pure rAAV virions ready for preclinical and clinical studies. Most of the studies are based on the initial purification of rAAV by cation exchange chromatography (CX), [10,30,31] UC in a density gradient, [32][33][34][35] preliminary precipitation with PEG 8000, [36] followed by purification using a weak anionic exchange resin [8] or affinity chromatography (AC), [37,38] and then directly using anion exchange resins to effectively remove empty capsids (Table 1). Cation exchange resins are usually used as the first stage of viral purification from the cell lysate, since in a certain pH range these resins interact with cellular proteins and other contaminating components, but not with viral particles.…”

“…[10] Similar results with a minimum content of empty capsids were obtained using AX on a Q Sepharose matrix with preliminary precipitation in 40% PEG 8000. [36] Kaludov et al [8] demonstrated the efficient removal of empty capsids of rAAV2, rAAV4, and rAAV5 using POROS PI and POROS HQ adsorbents. At the same time, the content of empty virions in the final viral preparation was less than 10%.…”

“…>90% purity [36] IAC (AVB Sepharose HP) AX (Poros 50HQ) 50%-90% particle recovery (depending on serotype). Empty particles:…”