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.