AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations

Abstract

Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

“…The melting temperature (T m ) of 82DI is 5 C lower than that of Anc82 (Figure 4E), a primary indication of a biophysically distinct entity (Pacouret et al, 2017). Considering the marked changes in Anc82 versus 82DI’s T m and AAP phenotypes, we were compelled to test the infectivity of both variants.…”

“…Thermostability of purified vector was assayed by AAV-ID (Pacouret et al, 2017). Briefly, a 500-μL sample of SYPRO Orange 50× was prepared using PBS 2+ (21-030-CV, Corning Life Sciences, Corning, NY) as a solvent.…”

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

“…The DSF method using SO was previously described in Pacouret et al and Rayaprolu et al 10,22 SYPRO Orange 5000X (Thermo Fisher Scientific, Dreieich, Germany) was aliquoted (5-10 mL) and stored at À30 C for up to 18 months. For each new experiment, a 500 mL sample of SYPRO Orange 50X was prepared using PBS2þ (Corning) as a solvent.…”

“…Fluorescence signals F were normalized between 0% and 100%; melting temperatures were defined as the temperature for which the numerical derivative dF/dT reached its maximum. 10 Size Exclusion Chromatography A Dionex Summit 2 system (Thermo Fisher) and an Acclaim 1000, 7.8 Â 300 mm, 7 mm column (Thermo Fisher Scientific, Dreieich, Germany) were used for the size exclusion chromatography (SEC). AAV elution was detected at 280 nm and 260 nm with a UV detector (Dionex UVD170U) and at excitations wavelength 280 nm and emission wavelength 330 nm with a fluorescence detector (Dionex RF2000).…”

“…So far, the established method for T m measurement is based on differential scanning fluorimetry (DSF) with AAV samples treated with a fluorescent dye-like SYPRO Orange (SO), which binds to hydrophobic regions of unfolding proteins and enables monitoring of capsid unfolding in response to a temperature ramp. 9,10 The fluorescence of SO is quenched by solvent molecules and increases upon binding to the hydrophobic sites that are externalized during thermally induced protein unfolding. 11 We present a simple method with excellent precision, utilizing DSF without using SO.…”