Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV)

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Adeno-associated virus (AAV) is a human parvovirus utilized in gene therapy.
• Traditional purification methods like CsCl gradients are unsuitable for large-scale manufacturing and yield contaminated products.
• A need exists for efficient, scalable purification of recombinant AAV (rAAV) vectors.

Purpose of the Study:

• To develop a process-scale method for rapid and efficient purification of rAAV from cellular lysates.
• To establish a scalable purification strategy for clinical-grade rAAV vectors.

Main Methods:

• Purification involves treating infected cell lysates with trypsin and nuclease.
• Utilizes a combination of ion exchange chromatography (ceramic hydroxyapatite, DEAE-Sepharose) and affinity chromatography (cellufine sulphate).
• Exclusively employs column chromatography for purification from crude cellular lysates co-infected with adenovirus.

Main Results:

• Achieved purification of rAAV particles solely through column chromatography.
• Demonstrated >90% purity of column-purified rAAV.
• Confirmed rAAV was free of detectable adenovirus contamination, biologically active, and capable of efficient gene transfer in vivo.

Conclusions:

• Column chromatography alone is feasible for isolating highly purified rAAV vectors.
• The described methods represent advancements in rAAV purification.
• These purification procedures are adaptable for commercial production of clinical-grade rAAV vectors.