TY - JOUR
T1 - Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System
AU - Mendes, Sónia
AU - Faria, Tiago Q.
AU - Nascimento, André
AU - Noverraz, Marc
AU - Bollmann, Franziska
AU - Nestola, Piergiuseppe
AU - Roldão, António
AU - Peixoto, Cristina
AU - Silva, Ricardo J.S.
N1 - Funding Information:
This work was funded by the Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and projects PTDC/EQU-EQU/0142/2020 and EXPL/EQU-EQU/1567/2021.
Publisher Copyright:
© 2024 by the authors.
PY - 2024/2/26
Y1 - 2024/2/26
N2 - Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.
AB - Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.
KW - adeno-associated virus
KW - downstream processing
KW - gene therapy
KW - process development
KW - tangential flow filtration
UR - http://www.scopus.com/inward/record.url?scp=85188924253&partnerID=8YFLogxK
U2 - 10.3390/separations11030073
DO - 10.3390/separations11030073
M3 - Article
AN - SCOPUS:85188924253
SN - 2297-8739
VL - 11
JO - Separations
JF - Separations
IS - 3
M1 - 73
ER -