Tuning
Mobile Phase Properties to Improve Empty Full Particle Separation in
Adeno-associated Virus by Anion Exchange Chromatography
Dennis P. Chen, James C. Warren,
Chao Huang
Correspondence
should be addressed to C.H.
(chuang@ultragenyx.com)
Pharmaceutical Development, Ultragenyx Pharmaceutical Inc., 19
Presidential Way, Woburn MA 01801
Chao Huang
Director of Downstream Process Development
chuang@ultragenyx.com
19 Presidential Way
Woburn MA 01801
ABSTRACT
In the past decade, recombinant adeno-associated virus (rAAV) has gained
increased attention as a prominent
gene therapy technology to treat monogenetic disease. One of the
challenges in rAAV production is the enrichment of full-rAAV particles
containing the gene of interested (GOI) payload. Herein, we demonstrated
that by adjusting the mobile phase properties of anion-exchange
chromatography (AEX), Empty and Full separation of rAAV was improved in
monolith based preparative AEX chromatography. When compared to the
baseline method using NaCl, the presence of tetraethylammonium acetate
(TEA-Ac) in the AEX mobile phase
resulted in enhanced resolution (from 0.75 to 1.23) between Empty and
Full peaks by salt linear gradient elution, as well as increased the
percentage of full-rAAV particles from 20% to 36% and GOI genome
recovery (from 59% to 62%). Furthermore, a dual wash + step elution
AEX method was developed to harness TEA-Ac contribution on Empty and
Full separation in the first wash (wash1) step while removing TEA-Ac in
the second wash (wash2) step to ensure product safety. The resulting
optimized AEX purification method could be easily adapted in scaled-up
manufacturing and could also be applied to purification processes
involving other AAV serotypes facing similar Empty and Full rAAV
separation challenges.
Keywords: AEX Chromatography; AEX
Mobile Phase; Adeno-associated Virus; Empty Full Separation
abbreviation: rAAV , Recombinant Adeno-Associated Virus;AEX , Anion Exchange Chromatography; QA , Quaternary
Ammonium