Gator® AAVX Probes for Rapid and Label-free Quantitation of AAV Serotypes

Introduction

Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. In virus development and production, it is important to determine the virus concentration at different stages of the process, to optimize the clone used as well as the production yields. Biolayer Interferometry (BLI) using AAV specific biosensors is a rapid label-free method for quantitation of AAV serotypes.

Gator AAVX Probes

The Gator AAVX probes are a high specificity antibody-based biosensors that enable direct capture and quantitation of different serotypes of AAV in crude lysates, column eluates, cell lysates and cell culture supernatants, serving as an alternative to traditional time-consuming analytical methods, such as qPCR, ddPCR, Dot blot and ELISA. The Gator AAVX probe uses proven CaptureSelect™ (Thermo Fisher Scientific) high affinity and high specificity anti-AAVX antibody.

Performance Summary

Dynamic range: 1 x 109–1 x 1013 vp/mL for most AAV serotypes
Throughput: 8 samples in 10 minutes, 96 samples in 120 minutes
Limit of detection: typically, 5 x 108 vp/mL (serotype dependent)
Crude sample tolerant
Stable over broad pH range
Cost effective – Reusable at least 10 times by regeneration

Results

Dynamic range:

The AAV2 serotype range was tested using the Gator AAVX probes. The data shows 4 orders of magnitude dynamic range. The data was acquired using 1:3 dilution in 10 min at 1,000rpm.

Accuracy and reproducibility

AAV5 serotype was used to test the accuracy and reproducibility. The table below shows recovery close to 100% and CV ranging from around 1.5% at medium and high titers to 10.9% at low titer.

Binding rate of different AAV serotypes

The binding rate of 8 different serotypes of AAV were studied. The figure below shows binding rates of tested serotypes at 2 x 1011 vp/mL concentration.