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.