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.