Optimization of rAAV capture step purification using SO3 monolith chromatography.

Area of Science:

• Biotechnology
• Gene Therapy
• Downstream Processing

Background:

• Adeno-associated virus (AAV) vectors are essential for gene therapy.
• Stringent purity is required for in vivo AAV administration.
• Downstream processing must adhere to regulatory guidelines.

Purpose of the Study:

• To optimize the non-affinity, serotype-independent capture of recombinant AAV (rAAV).
• To evaluate Convective Interaction Media (CIM) cation-exchange SO3 monoliths for rAAV capture.
• To identify optimal binding mobile phase conditions for rAAV capture.

Main Methods:

• Screening of buffer pH, NaCl concentration, and poloxamer 188 using CIM SO3 96-well plates.
• Analysis of differentially pretreated viral samples from Sf9 cell lines.
• Determination of dynamic binding capacity and retesting on CIMmultus industrial lines.

Main Results:

• High overall vector recovery of 51% was achieved.
• Significant impurity reduction: 99.98% for protein and 99.25% for DNA.
• Optimized capture step parameters confirmed on an industrial scale.

Conclusions:

• The cation-exchange SO3 monolith capture step was successfully optimized.
• This optimization enhances the downstream processing of rAAV for gene therapy.
• The developed method meets stringent purity requirements for in vivo applications.