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Stronger together: Analytical techniques for recombinant adeno associated virus
Author(s) -
Hutanu Andrei,
Boelsterli David,
Schmidli Claudio,
Montealegre Cristina,
Dang Thai Mike H. N.,
Bobaly Balazs,
Koch Marius,
Schwarz Maria A.
Publication year - 2022
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.202100302
Subject(s) - capillary electrophoresis , adeno associated virus , recombinant dna , computational biology , titer , genome , sizing , chromatography , gel electrophoresis , computer science , virus , biology , chemistry , virology , microbiology and biotechnology , gene , genetics , vector (molecular biology) , organic chemistry
Abstract 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.