Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography

Figure 3

AAV vector genome and capsid protein characterization using SDS-PAGE (7.5% gel) and qPCR probe-based titration. POROS-AAVX affinity column purified (POR) vectors are purer than their iodixanol (IOD) purified counterparts (a). With the same titer of vg loaded, POR vectors show darker bands which indicate more empty capsids than IOD vectors (b). Using qPCR probe-based analysis, vg recovered by both purification methods are comparably similar ((c) top: from 50 mL of triple-plasmid-transfected cell culture; bottom: from 200 mL). Backbone contaminations were determined and multiplexed using an ampicillin-primers/probe, which indicated a similar amount of the plasmid backbone packaged by AAV8 purified by both methods (d). 0 U = no pretreatment of DNase I before qPCR titration; 5 U = 5 units of DNase I pretreatment were used prior to qPCR.

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(b)

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(d)