3.3. Fingerprinting elution profile of FMDV in three stage purification strategy
In this study, according to previous reports (Martin & Synge, 1941; Rodrigues et al., 1991; Ruthven, 1984), HP-SEC on Ultra hydrogel linear analytical column was applied for comparing fingerprinting elution profiles in three stages of purification strategy (Fig. 1) , and the output results are depicted in Fig. 4 .
As shown in Fig. 4, significant differences are observed between elution profiles of primary sample (Solid line), intermediate concentration step (Dash line), and final polishing step (Dotted line) by moving from capturing step toward final polishing step. After the capturing step, most of the low molecular weight impurities like HCP and majority of rDNA were removed from pooled virus fraction. Considering, the elution profile of concentrated sample obtained from intermediate concentration step on Sup-200 (Fig. 3B ), the perfect resolution between the signals related to virus particles and NSPs marker (BSA) can provide clear evidence for completely elimination of NSPs from final product. Accordingly, residual observed impurities in the purified virus fraction (Fig. 4 ) are probably related to negligible high molecular weight proteins like immunoglobulins. Furthermore, the elution profile of purified virus fraction obtained from polishing step demonstrated that final product has more than 85% purity.
Fig. 5 displays SDS-PAGE result for confirming the identity of the purified FMDV product in the final polishing step. Electrophoresis process shows three bands related to VP1, VP2 and VP3 structural proteins between 25-35 kD while the fourth structural protein, VP4, was removed from the gel for obtaining higher resolution between high molecular weight counterparts. Similar to the previous report, observed bands in molecular weight higher than related amounts for virus structural proteins can be attributed to VP1–VP2 dimer or VP3multimers (Harmsen, Jansen, Westra, & Coco-Martin, 2010).
Characterization of purified FMDV
The size distribution profile and structure of purified FMDV particles were analyzed by DLS and TEM, Fig. 6 , approaches in order to provide more evidence for preserving intact virus structure during purification procedure as the most imprtant criteria in the devolopment downstream processing. DLS analysis demonstrated a single peak with hydrodynamic diameter about 30 nm and image indicated that the most of the purified virus particles are spherical in shape and structually intact. Therefore, the obtained results confirmed that the final product in 2D-AEC×SEC is qualified for production fomulated FMD vaccine.
MALDI-TOF MS analysis
Currently, high throughput technology like MALDI-TOF MS is becoming a common methodology in clinical microbiology or virus research and diagnostics labs (Amexis et al., 2001; Giebel et al., 2010; Ilina et al. 2005; Ruelle et al. 2004; Sjöholm et al. 2008; Soleimani et al. 2020). In this work for the first time application of MALDI-TOF MS for direct investigation of purified FMDV particles was explored and obtained results were compared with the previous report of application SELDI-TOF in FMDV study (Harmsen et al., 2010). Thus, the fractions obtained from 2D-AEC×SEC (purified virus) were analyzed by MALDI-TOF MS in low (2–20 kD) (Fig. 6A ), medium (20–50 kD) (Fig. 6B ), and high (50-100 kD) (Fig. 6C ) molecular weight. Regarding the low molecular weight, there is a high intense ion in m/z = 8.7138kD (Fig. 6A ), which is in line with the previous report related to myristoylated form of VP4, a structural protein did not appear on SDS-PAGE. In the medium molecular weight (Fig. 6B ), there is a low intense and a high intense ion in m/z = 24 kD andm/z = 27.9873 kD, respectively, which are probably correlated to the VP3 and VP1 structural proteins. As observed from Fig. 6C , in the high molecular weight, there is a low intense signal in m/z = 54.0321 kD, possibly corresponding to the dimer of VP1.