Chromatographic separation of full and empty Adeno-Associated Virus (AAV) particles on CIM® monoliths

Adeno-Associated Virus (AAV)-based vectors of various serotypes are considered to have high potential in human gene therapy and genetic vaccination applications. During manufacturing of AAV vectors undesired, incomplete particles are co-produced. They lack recombinant viral genomes and consist of empty capsid proteins only. Empty capsids increase the required dose of AAV virus for medical applications and are thought to cause immunological reactions against the vector capsid, leading to unwanted side effects. Removal of empty capsids during manufacturing, as well as the ability to quantify the amount of empty AAV particle content in a formulation is hence a critical requirement for any AAV production process.

Current methods for preparative separation of empty capsids (CsCl or iodixanol gradients) are challenging to scale-up and are not suitable for large-scale production. Furthermore, analytical methods for detection of empty capsids and determination of full to empty particle ratio (electron microscope (EM) assay, total particle assay [ELISA] combined with genome copy titration [qPCR]) are time- and labour consuming, influenced by operator technique or do not provide readily available reagents for different serotypes of AAV.

A new approach for separation of full and empty AAV8 particles by exploiting minor charge differences is presented in this application note. By using linear gradient elution on a CIM QA Disk Monolithic Column, a simple, rapid and reproducible assay for analysis of AAV particles is introduced. The method was successfully applied to AAV8 particles prepared by two different manufacturing processes.