title: Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.
abstract: With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.
authors.affiliation: Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Piras
authors.affiliation: Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Drury
authors.affiliation: Department of Surgery, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Morton
authors.affiliation: Department of Surgery, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Yunyu
authors.initials: Y
authors.lastname: Spence
authors.affiliation: Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Lockey
authors.affiliation: UCL Katharine Dormandy Haemophilia and Thrombosis Centre, Royal Free Hospital , London, UK.
authors.forename: Amit C
authors.initials: AC
authors.lastname: Nathwani
authors.affiliation: Department of Surgery, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Davidoff
authors.affiliation: Department of Therapeutics Production & Quality, St. Jude Children's Research Hospital , Memphis, Tennessee, USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher