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 ).