Downstream processing and analytics of adeno-associated viral vectors

Abstract

Viruses are crucial in biopharmaceutical applications, including their use as vaccines, oncolytic agents for cancer therapy, and gene therapy vectors to deliver therapeutic genes into cells. Among the viruses used in gene therapy, adeno‑associated viruses (AAV) are particularly promising and frequently used vectors. The growing demand for AAV, especially due to the high doses required in certain applications (≥10^14 viral genomes / kg body weight), requires advances in AAV production and purification. Efficient and simplified methods for both the production of AAV in cell culture and their purification, referred to as downstream processing (DSP), and for the corresponding analytics are essential. Furthermore, the diversity of AAV serotypes, which differ in their capsid protein composition, underscores the need for serotype‑independent purification strategies. This thesis focuses on the development of an alternative, efficient, and serotype‑independent AAV DSP, complemented by the development of advanced analytical methods. The work centers on three key process steps: clarification, chromatographic purification and isolation, as well as chromatographic polishing. Each step has been comprehensively investigated and solutions have been proposed to address identified limitations. In addition, advanced analytical methods for AAV titer determination were developed and evaluated. The individual chapters of this thesis describe the development of analytical methods, the optimization of individual process steps, and their combination into an integrated process. The first part of the thesis evaluates current methods for AAV titer determination and presents advanced alternatives to increase sample throughput and accelerate process development. The second part focuses on the development of a clarification process using diatomaceous earth as a filter aid. This approach improved filtration speed, simplified handling, and increased filter capacity. The third section focuses on the separation of genome‑containing AAV (full) and genome‑deficient AAV (empty) particles by anion exchange chromatography, while the purification and isolation step is addressed in the fourth section. At that time, immunoaffinity chromatography was the only available purification and isolation method. Consequently, the development and optimization of the novel separation method described in this section relied on AAV material that was pre‑purified by immunoaffinity chromatography. In this novel approach, empty particles remain in the flow‑through during sample application and do not bind to the stationary phase. This approach reduces the required column volume and eliminates the need for precise resolution of elution peaks since empty particles are already removed during sample application. The final chapter presents the development of a steric exclusion chromatography (SXC) method for the purification and isolation of AAV, as well as the combination of the investigated process steps into an integrated DSP. SXC proved to be a serotype‑independent and effective alternative to conventional immunoaffinity chromatography, while also eliminating intermediate steps such as concentration and buffer exchange. The entire DSP was reduced to only three steps and achieved a high AAV recovery along with a high removal of impurities.