Adeno-associated virus (AAV) is a widely used vector for gene therapies and there is an ever-growing need within the industry to meet impurity thresholds and standardize AAV sample formulation. One major limiting factor in developing a consistent AAV therapeutic is the presence of “empty” AAV capsids which lack the gene of interest and may be considered a process impurity.<br/>Here we showcase the progress in implementing our novel conductivity step elution method using anion exchange chromatography (AEX) with Mustang® Q XT membrane for full AAV capsid enrichment. We previously developed this technique using AAV5 and here describe purification of the additional serotypes AAV8 and AAV9. By comparing Absorbance measurements (260nm vs 280nm) the elution conditions for obtaining optimal amounts of full capsids from an affinity purified pool were determined. PCR and intact capsid ELISA, affirmed the majority presence of full capsids in these elution fractions. This approach to enrichment of full AAV capsids can be used as a rapid process development tool. Using information from the multistep elution method enables the implementation of a two step elution for production and manufacturing settings. This work demonstrates the promise of membrane chromatography as an AAV polishing step: It can supply full capsid enrichment, remove host cell protein and DNA contaminants and can be operated at high flow rates, up to 10 MV/min. The ability to operate polishing step at high flow rates faciltates process intensification as typically a large dilution is required after AAV affinity chromatography to prepare for the AEX polishing step.

Separation of full and empty AAV capsids using membrane chromatography

ACS Fall 2021

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