Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors

Abstract

Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

Current challenges in biotherapeutic particles manufacturing

“…Besides these techniques, western blotting for protein identity matching, size exclusion chromatography HPLC, capillary zone electrophoresis, Asymmetrical-Flow Field-Flow Fractionation (A4F) and surface plasmon resonance are assays that can be used to complement biotherapeutic particles characterization [159][160][161]. Analytical ultracentrifugation (AUC) has been the method of choice for investigation of the empty capsids content [162]. This technique distinguishes and quantifies the different species (empty and full particles) by either mass or density without the need for standard material for quantification comparison.…”

Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS

“…46 The vector preparation was substantially free of contaminants, as assessed by SDS-PAGE analysis with SYPRO staining; additionally the fractional content of empty capsids in the vector preparation was low at 13%, as determined by analytical ultracentrifugation ( Figure S4). 47…”

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes

“…However, the differences were too small to clearly distinguish between full and empty AAVs in a complex sample mixture. For this purpose, other analytical methods can be used such as anion-exchange-chromatography, 33,34 analytical ultracentrifugation, 35,36 and transmission-or cryo-electron microscopy. 37 In summary, DSF offers a rapid, cost-effective, and robust method for AAV serotype identification due to their unique thermal stability profiles in the commonly utilized formulation and storage buffers and detergents.…”