Figure 3 . Elution profiles from AEX processes employing a linear gradient elution (LGE) containing 20 mM BTP, 0.001% (w/v) Pluronic F-68, 25–340 mM TEA-Ac at pH 9 over 90 CVs, while the MgCl2 concentration was held constant at 0, 0.2, 1, and 5 mM for the blue, light-blue, purple, and red traces, respectively (listed from bottom to top; each trace is offset by 100 mAU). Traces correspond to absorbance at 280 nm (A280) and asterisks denote a population not apparently visible in traces containing 1 and 5 mM MgCl2. The total viral particle measured in the empty peak are displayed adjacent to their corresponding UV trace. The empty peak is denoted as “Empty”, while the full peak is denoted as “Full”.
3.5 Quaternary Ammonium Salt in Wash Phase of AEX Chromatography
As the TEA-Ac salt has dose dependent toxicity,[25] it is imperative to remove it from rAAV product stream. To this end, converting AEX LGE to step elution appears to be a feasible solution, since QA salt could be placed only in the wash phase to facilitate empty-rAAVs reduction while allowing a non-QA-containing buffer to elute the full peak. In addition, to ensure maximum removal of TEA-Ac, a second wash phase (using NaCl) could be implemented to flush residual TEA-Ac off the column. To achieve similar full-rAAV enhancement through AEX chromatography, the TEA-Ac concentration in wash buffer was set referencing its concentration observed at the empty peak apex in LGE run.
In the AEX step elution run, to demonstrate the effectiveness of TEA-Ac in reduction of empty-rAAVs, the elution phase was executed using either single-buffer isocratic elution (intended for final AEX step elution method) or two-buffer LGE (intended to resolve empty and full peaks to assess empty-rAAV reduction). As shown in Figure 4A and4C , the elution phase with two-buffer linear gradient resolved only the full and third peaks, demonstrating significant amount of empty-rAAVs had been removed by TEA-Ac washes. In comparison, the elution phase with a single-buffer isocratic elution resulted in a sharp elution peak with slight tailing. For better comparison, the magnified images of wash and elution peaks from both runs were shown inFigures 4B and 4D , respectively. As linear gradient operation may encounter manufacturing facility constraints when scale-up,[26] isocratic elution is a preferred operation that enables elution volume reduction and rAAV concentration increase in AEX pool, which is beneficial for subsequent downstream operation, since less concentration factor could be used in the final concentration and buffer exchange unit operation.