Simplified rAAV lysate purification for complex analytics of upstream samples
Bioprocess experts from Sartorius BIA Separations and BridgeBio Gene Therapy illustrate an approach with potential to expedite AAV upstream analytics and lower overall process development costs.
Optimising upstream production of recombinant adeno-associated viruses (AAV) is essential for reducing costs, improving quality and enhancing efficiency. Producing full capsids—vectors containing complete therapeutic genes—is crucial for rAAV therapeutics. This process occurs during the complex upstream phase, necessitating precise analytical methods and relatively pure samples to determine capsid ratios.
A rapid and efficient high-throughput protocol
The production of rAAV vectors requires a streamlined approach to ensure the generation of full capsids. This paper introduces a rapid and efficient high-throughput protocol for purifying crude rAAV lysates using the CIM® SO3 0.2 mL Monolithic 96-Well Plate (2 μm channels). This method is designed to purify low-volume rAAV upstream samples, suitable for various analytics requiring specific purity and concentration levels.
The purification protocol involves preparing the sample and the 96-well plate. The plate is flushed with deionised water, equilbrated with elution buffer, and washed with binding buffer. rAAV samples are acidified, vortexed, incubated, centrifuged, and loaded onto the plate. Elution is performed using either high salt and acidic-neutral pH or neutral pH without a salt gradient. The protocol was tested in-house with AAV8, AAV2, and AAV9, and externally by BridgeBio with AAV5 and AAV9 samples, both with single-step AAV elution (Figure 1) and with two different GOI.
