Removal of Endotoxin from rAAV Samples Using a Simple Detergent-Based Protocol

Area of Science:

• Biotechnology
• Virology
• Molecular Biology

Background:

• Endotoxin is a frequent contaminant in protein and viral preparations, posing significant risks.
• Recombinant adeno-associated virus (rAAV) stocks often contain endotoxin, which can cause adverse reactions and complicate research.
• Existing endotoxin removal methods can lead to substantial loss of rAAV titer, especially with diverse serotypes.

Purpose of the Study:

• To develop a universal and efficient protocol for removing endotoxin from rAAV stocks.
• To ensure the safety and efficacy of rAAV vectors by reducing endotoxin levels.
• To maintain high viral recovery rates across different rAAV serotypes.

Main Methods:

• A simple protocol involving mild detergent treatment of endotoxin-contaminated rAAV.
• Repeated buffer-exchange washing to remove endotoxin.
• Concentration of purified viral stock using low-speed centrifugation.

Main Results:

• The protocol effectively reduces endotoxin levels from >1,000 EU/mL to <2.5 EU/mL.
• Viral titer recovery ranged from 50% to 100% after purification.
• Physical and biological equivalency of viral stocks was confirmed through multiple assays.

Conclusions:

• The described protocol offers a simple, universal solution for endotoxin removal from rAAV vectors.
• This method is effective across various rAAV serotypes and variants.
• The protocol successfully purifies rAAV stocks while maintaining high viral recovery and biological activity.