3.1. Strong AEC for capturing step of FMDV vaccine purification
The art of keeping labile FMDV particles in their native conformation
can guarantee the conservation of their structure and function
throughout a chromatographic process. The sample loading is the most
important bottleneck for application SEC in downstream step. Although,
in our last work, application of 2D-SEC×SEC as an alternative strategy
could compensate this drawback with increasing sample loading to 12.5%
of total column volume but increase loadability to higher than, due to
significant reduction of virus recovery in the first dimension, was
impossible (Bagheri et al., 2018). In AEC step, selecting an appropriate
pH is considered as one of the most important parameters so that the
target protein can have the highest activity and opposite charge to bind
to the ion exchangers. The application of cation exchange chromatography
in acidic condition to bind target protein to media is impossible given
that the highest infectivity and stability for FMDV particles was
described in neutral pH (Liang et al., 2014), and early study indicated
very close p I for FMDV particles to the BSA (p I=4.8
(Rodrigues et al., 1991)) (data not shown). Thus, AEC can be considered
as another alternative for keeping the protein structure of FMDV and
compensating the low loading capacity of SEC in capturing step.
Diethylaminoethyl (DEAE) and quaternary ammonium (Q) ligands are
regarded as two most common exploited anion exchange candidates.
Comparison of DEAE and Q anion exchanger on Sepharose base matrixes such
as DEAE–Sepharose Fast Flow and Q Sepharose Fast Flow revealed there
are higher dynamic binding capacity for strong anion exchanger (Q) than
weak anion exchanger (DEAE) (Helfferich & James, 1970). Therefore, in
the prospective of sample loading capacity for capturing step Q anion
exchanger seemed more suitable for downstream step of FMDV production,
but the activity of the final product should be evaluated. Thus, the
suitability of Q Sepharose XL in capturing FMDV purification was
evaluated due to aforementioned advantages (Fig. 1 ).