AAV Purification Book Notes (1)
AAV2, -3, -6, and -13 are natural heparin-binding serotypes¹
Heparin affinity chromatography has been used to purify clinical AAV2
Process-related impurities in AAV vector manufacturing may include residual host cell protein, host cell DNA/RNA, plasmid DNA, helper viruses, cell culture medium components, purification buffers, chromatography medium ligands, centrifugation media, detergents, and enzymes, etc. ²
Product-related impurities in AAV vector manufacturing may include AAV empty capsids, encapsidated host cell nucleic acids, encapsidated helper component DNA, replication-competent AAV, noninfectious AAV particles, aggregated, degraded and oxidized AAV vectors. ²
AAV-encapsidated host cell DNA impurities correspond to heterogeneous fragments of host cellular DNA unintentionally packaged within AAV capsid particles. ²
Encapsidated host cell DNA presents significant challenges in downstream purification, since it cannot be removed by benzonase treatment or through affinity purification. ³
rAAV9 does not efficiently bind to some AEX or CEX. ⁴
It is recommended to keep residual cell-substrate DNA ≤10 ng per dose, with DNA size below 200 base pairs.⁵
Purilogics® develops novel membrane adsorbers for biologics purification. Unlike other high capacity membrane products, Purexa™ membrane products are differentiated by their tentacle based ion-exchange ligands.
Two such products, Purilogics weak anion-exchange membrane adsorbers (Purexa™ NAEX Plus) and multimodal anion-exchange membrane adsorbers (Purexa™ MQ), provide high protein and very high DNA binding capacity (e.g. >40 mg/mL). In addition, tentacle based membrane provides higher affinity towards HCP and HCDNA.
We also offer a Purexa™-Screen program (for both NAEX Plus and MQ) to provide downstream scientists membranes adsorbers with different ligand densities and structures to more quickly and easily optimize their purification schemes.
July 6, 2020
References:
1. Mietzsch, M., Broecker, F., Reinhardt, A., Seeberger, P. H., Heilbronn, R. (2014). Differential adeno-associated virus serotype-specific interaction patterns with synthetic heparins and other glycans. Journal of virology, 88(5), 2991-3003.
2. Wright, J. F. (2014). Product-related impurities in clinical-grade recombinant AAV vectors: characterization and risk assessment. Biomedicines, 2(1), 80-97.
3. Wang, Y., Cooper, R., Kiladjian, A., Bergelson, S., Feschenko, M. (2019). A digestion-free method for quantification of residual host cell DNA in rAAV gene therapy products. Molecular Therapy-Methods & Clinical Development, 13, 526-531.
4. Zhou, J., Yang, X., Wright, J. F., High, K. A., Couto, L., Qu, G. (2011). PEG-modulated column chromatography for purification of recombinant adeno-associated virus serotype 9. Journal of virological methods, 173(1), 99-107.
5. Geigert, J. (2013). The challenge of CMC regulatory compliance for biopharmaceuticals and other biologics (pp. 242). New York, NY: Springer.