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.