Enhanced Separation of AAV Capsids Using Analytical Anion Exchange Monolithic Columns: Emphasis on Organic Modifiers.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Anion exchange chromatography (AEX) is crucial for separating empty (E) and full (F) adeno-associated virus (AAV) capsids.
• Current AEX methods struggle with baseline separation, especially in complex samples and are serotype-dependent.
• Challenges include incomplete separation of E/F capsids and the need for serotype-specific methods.

Purpose of the Study:

• To develop strategies for significantly enhancing the separation of empty and full AAV capsids using AEX.
• To achieve baseline separation and identify additional AAV capsid subpopulations.
• To provide insights into optimizing AEX for diverse AAV serotypes and sample complexities.

Main Methods:

• Utilized an AEX analytical Convective Interaction Media (CIM) monolithic column.
• Investigated the impact of organic solvents, concentrations, Mg2+ counterions, and buffer pH on AAV capsid separation.
• Integrated findings into an enhanced chromatographic AEX method.

Main Results:

• Demonstrated how buffer composition and column choice influence E/F AAV capsid separation.
• Achieved enhanced separation, including baseline resolution in some cases.
• Identified strategies applicable across various AAV serotypes and sample purities.

Conclusions:

• The enhanced AEX method provides rapid and comprehensive characterization of AAV capsid subpopulations.
• This advancement aids in developing safer and more effective gene therapies.
• The method's versatility across serotypes and purities adds significant value to AAV research.