Stronger together: Analytical techniques for recombinant adeno associated virus

TY - JOUR

T1 - Stronger together: Analytical techniques for recombinant adeno associated virus

AU - Hutanu, Andrei

AU - Boelsterli, David

AU - Schmidli, Claudio

AU - Montealegre, Cristina

AU - Dang Thai, Mike H N

AU - Bobaly, Balazs

AU - Koch, Marius

AU - Schwarz, Maria A

N1 - © 2021 The Authors. Electrophoresis published by Wiley-VCH GmbH.

PY - 2022/5

Y1 - 2022/5

N2 - With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

AB - With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

KW - Dependovirus/genetics

KW - Electrophoresis, Capillary

KW - Genetic Vectors

KW - Ultracentrifugation

U2 - 10.1002/elps.202100302

DO - 10.1002/elps.202100302

M3 - SCORING: Journal article

C2 - 34821392

VL - 43

SP - 1107

EP - 1117

JO - ELECTROPHORESIS

JF - ELECTROPHORESIS

SN - 0173-0835

IS - 9-10

ER -