2.5.4. Anion Exchange Chromatography—AEX—pH Gradient

The CIMmultus PrimaS (AAV) column (1 mL, 2-µm pores; BIA separations) was evaluated as an additional option for separating empty from full rAAVs. Buffer A contained 10-mM Tris, 10-mM bis-tris-propane (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 2-mM MgCl₂, and 0.1% poloxamer 188 (Kolliphor P 188, BASF, Ludwigshafen, Germany) at pH 8.0, and buffer B had the same composition but at pH 10.0. To achieve a conductivity below 4 mS/cm, the samples from CEX or AC were diluted 30-fold with dilution buffer (buffer A diluted 2-fold with water), and the pH was adjusted to 8.0. Loading and elution were performed at a flow rate of 4 mL/min and a linear pH gradient ranging from 0% to 100% B within 100 CVs. In some cases, a step gradient was used.