Figure 1 . (A) Elution profiles from AEX processes employing a linear gradient elution containing 20 mM BTP, 0.001% (w/v) Pluronic F-68, and 25–182.5 mM NaCl (top panel) or 25–340 mM TMAC (bottom panel) at pH 9 over 90 CVs. Blue and red traces correspond to A280 and A254, respectively. The dashed, green trace corresponds to the solution ionic strength. (B) Elution profiles from AEX processes employing a linear gradient elution containing 20 mM BTP, 0.001% (w/v) Pluronic F-68, and 25–340 mM QA chloride at pH 9 over 90 CVs. The tetra-alkyl chain was varied across the QA chloride runs with the red, purple, light-blue, and blue traces corresponding to tetra-methyl (TMAC), tetra-ethyl (TEAC), tetra-propyl (TPAC), and tetra-butyl (TBAC) QA-chloride salt, respectively (listed from top to bottom). The traces were normalized to the elution volume corresponding to the maximum A280 signal of the empty peak, which is indicated by the vertical dashed line. The empty peak is denoted as “Empty”, while the full peak is denoted as “Full”.
3.2 Effect of Alkyl-chain Length in Quaternary Ammonium Salt
To investigate the effect of alkyl-chain length on Empty and Full separation in preparative AEX chromatography, AEX salt LGE chromatography runs were conducted using a series of tetraalkylammonium chlorides, including tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPAC), and tetrabutylammonium chloride (TBAC). As evident in Figure 1B , the empty peak to full peak separation increases with increasing alkyl-chain length. The differential retention time of various tetraalkylammonium salt also indicated that increasing alkyl-chain length could weaken tetraalkylammonium salt ability to elute rAAV particles. Among all four tetraalkylammonium salts evaluated in this study, TBAC exhibited the highest level of empty full peaks separation, while also suffering from broadest peak width and largest peak collection volume. In addition, due to the high UV280nm absorbance of both TPAC and TBAC salt, they were not considered process feasible since their concentration will interfere with the UV280nm based peak collection criteria of rAAV product. In this case, TEAC was deemed as the optimal salt to perform further experiments in this study.
3.3 Effect ofAnionic Counter-ioninQuaternary Ammonium Salt
With solvation effects assumed to be at play, it is likely that the counter-anion in the QA compound may contribute to the separation of empty- and full-rAAV. To this end, AEX chromatography runs were performed using a series of TEA salts with different anions, including acetate (Ac), bromide (Br), and tetrafluoroborate (BF4), which were chosen on the basis of their relative affinity to quaternary ammonium ligands, with Ac, Br, and BF4 exhibiting low, moderate, and high affinity, respectively.[24] As seen in Figure 2 , among all three runs, TEA-BF4 run has the lowest degree of empty-to-full peak separation, whereas eluting with TEA-Ac resulted in the highest degree of empty-to-full peak separation. It is well known that acetate is a weak eluent in AEX chromatography. Furthermore, given the observation that elution with TEAC resulted in longer retention volumes than NaCl, the overall weak eluent property of TEA-Ac may be leveraged for separation of empty- and full-rAAV species by AEX chromatography.