Figure 2.

Effect of resin regeneration and temperature on purification efficiency

(A) Overview of experimental design of figures (B) and (C). Five small-scale AAV1 preps were produced and purified sequentially on HPLC with AAVX resin without changing the resin between purifications. One prep contained AAV from one and a half 15-cm dishes. Preps 2–5 were identical except for a 100-bp barcode region. Vector genomes were quantified across all purifications. For the fifth prep, the barcode region was PCR amplified and next-generation sequenced, and the unique barcodes corresponding to each prep were quantified to estimate carry-over contamination from preps 2–4. AAV was applied to a column packed with 1 mL of AAVX resin at 1 mL/min flow rate at room temperature. (B) Purification efficiency with repeated resin use. Vector genomes in lysate, flow-through, and elution. Hash mark indicates that some of the sample was lost due to handling error. (C) Estimation of carry-over contamination. Barcode counts from preps 2–5, in the fifth prep estimated via next-generation sequencing. (D) Effect of purification temperature on the percentage of vector genomes found in flow-through for AAV9 and PHP.eB. Difference was assessed using two-way ANOVA with Šídák’s post hoc tests. (E) Stability of AAV (PHP.eB) in clarified lysate at 24°C over 96 h. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars denote standard deviation.