Stronger together: Analytical techniques for recombinant adeno associated virus

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.

Distinguishing between DNA-Loaded Full and Empty Capsids of Adeno-Associated Virus with Atomic Force Microscopy Imaging

“…Several studies have reported that AAVs purified using ultracentrifuga- tion techniques contain proteasome contamination. 38 The height value observed by AFM (∼14 nm) was similar to the reported dimension of proteasomes. 39 Core−shell structures with empty inner spaces often cause morphological rupture or distortion in AFM images.…”

“…The population with the smallest height, lower than 14 nm, was considered to be proteasome contamination. Several studies have reported that AAVs purified using ultracentrifugation techniques contain proteasome contamination . The height value observed by AFM (∼14 nm) was similar to the reported dimension of proteasomes …”

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis

“…■ RESULTS AND DISCUSSION Basic Principles of Interaction between PNA and DNA Oligonucleotides. In our previous report, 33 we used orthogonal analytical techniques including transmission electron microscopy, analytical ultra centrifugation, and CGE to get a better idea of DNA heterogeneity inside rAAV capsids. The complex DNA purity profiles that were observed with CGE naturally caused the question to identify the unknown species.…”

Correlating physicochemical and biological properties to define critical quality attributes of a recombinant AAV vaccine candidate

“…Although recent reviews have highlighted potential key structural properties of rAAV vectors for use as gene therapy candidates [65][66][67] , no actual data sets with representative rAAV vectors were provided. At the same time, there are numerous recent reports employing individual analytical methods to monitor specific structural attributes of rAAV vectors including chromatography, electrophoresis, light scattering, SV-AUC, TEM, immunoassays and mass spectrometry 48,[68][69][70][71][72][73][74] , yet only a few showing a combination of comparative data sets 72,75,76 . Quality-by-design analysis for rAAV vectors for gene therapy applications have proposed the following CQAs: encapsidated capsids, empty-partially filled capsid ratios, aggregates and deamidated forms 77,78 .…”