Purification of Adeno‐Associated Viral Vector Serotype 9 Using Ceramic Hydroxyapatite Chromatography and its Analysis

Adeno‐associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissues in vivo. Owing to their convenience, high efficiency, long‐term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functions in vivo, especially in non‐clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high‐quality AAV vectors. Commercial AAV vectors are typically associated with high costs. Further, in‐laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof‐of‐concept experiments. Herein, we present an optimized method for producing and purifying high‐quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed‐mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic Protocols and optional methods for evaluating purified AAV vectors are also described. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1 : Production of AAV9 vectors in 293EB cells

Basic Protocol 2 : Concentration and buffer exchange of AAV9 vectors from 293EB cell culture supernatants using tangential flow filtration

Basic Protocol 3 : Purification of AAV9 vectors from TFF samples using ceramic hydroxyapatite chromatography

Basic Protocol 4 : Analysis of the purified AAV9 vectors