A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Area of Science:

• Biotechnology
• Gene Therapy
• Viral Vector Production

Background:

• Adeno-associated viral vectors (AAVs) are key for gene therapy.
• Separating full (gene-containing) and empty AAV capsids is crucial but challenging.
• Current methods are difficult to scale and reduce product yield.

Purpose of the Study:

• To demonstrate the feasibility of ultrafiltration for separating full and empty AAV capsids.
• To establish a scalable and efficient purification method for AAV vectors.

Main Methods:

• Ultrafiltration using polycarbonate track-etched membranes (30 nm pore size).
• Quantification of separation using ELISA, qPCR, and infectivity assays.
• Measurement of sieving coefficients for full and empty capsids.

Main Results:

• Selective permeation of empty capsids over full capsids was achieved.
• High recovery yield of 89% for full capsids.
• Empty capsids showed approximately twice the permeability of full capsids (sieving coefficients 0.49 vs. 0.25).

Conclusions:

• Ultrafiltration is a viable unit operation for AAV capsid separation.
• This method supports the development of scalable, continuous AAV purification.
• Improves efficiency and yield in gene therapy vector manufacturing.