3.4 Effect of Magnesium Chloride in Combination with Quaternary Ammonium Salt
Previous data indicated when MgCl2 was added to AEX mobile phase, the full peak yield was increased.[16] Therefore, the TEA-Ac salt LGE runs were performed with MgCl2 presence in elution mobile phase with various concentrations, in an aim to identify the optimal AEX condition. As shown in Figure 3, the presence of MgCl2 as low as 1 mM was sufficient to observe a noticeable increase of UV280nm area for full peak. In addition, the difference in the ionic strength at which the empty and full peaks are eluted appear to taper off at 5 mM of Mg2+, suggesting that it is likely that the binding sites associated with Mg2+ may be saturated at that concentration, and long-range interactions (charge screening effects) begin to dominate afterward and consequently, the retention volumes of empty and full peaks become equally affected (e.g., the AEX run with 10 mM Mg2+ in the mobile phase). It should be mentioned that a hump observed between empty and full peaks (indicated by an asterisk in Figure 4 ) gradually disappears when mobile phase MgCl2 concentration increases. It is likely that certain charge variants of empty-rAAVs eventually merged into the major empty peak in the presence of MgCl2, which is evidenced by the increase of viral particle counts in empty peak (from 1.4 x 1015 to 2.6 x 1015) with increase of MgCl2 concentration.