3.4. Purification and characterization of the yellow fever VLPs
In parallel to the investigation of perfusion processes discussed above, the VLPs produced by stable cell pool 2 were purified and characterized in order to allow evaluation of product attributes and to establish hallmarks for future integration of upstream and downstream processing.
Steric exclusion chromatography (SXC) was selected for single-step purification of YF VLPs due to the good results reported for this technique for concentration and purification of large biomolecules, such as nucleic acids, large proteins and viruses (Lee et al., 2012; Marichal-Gallardo et al., 2017; Levanova & Poranen, 2018). SXC works by capturing the large biomolecules at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG) (Lee et al., 2012). Since the contaminants in the cell culture supernatant are much smaller than the VLPs, they are eliminated during feed in the flowthrough, whereas VLPs are subsequently eluted by reducing PEG concentration.
Figure 5A shows a typical chromatogram obtained for a single-step purification from cell culture supernatant using SXC with 8% (m/v) of PEG 6,000. The chemiluminescent immunoblot of the fractions collected during chromatographic steps shows that most VLPs were recovered as a concentrated eluate fraction and that no product was lost either in the feed flowthrough or in the wash step. Some product was detected in the fraction of the regeneration with 1M NaCl, indicating that there was some degree of adsorption of VLPs to the membrane used as stationary phase. Although adsorption is not the intended mechanism of SXC, the low intensity of the signal in the regeneration shows that it is a minor part of the VLPs that is lost in this fraction.
Panels C, D and E (Figure 5) show SDS-PAGE, Western blot and SEC-HPLC analyses performed to evaluate purity, identity and multimeric state of the VLPs, respectively. As shown in Figure 5C, sample complexity was significantly decreased when the cell culture supernatant is compared to the SXC eluate. The Western blot in Figure 5D shows that two protein bands in the supernatant, in the SXC eluate (containing 8% PEG) and in a concentrated/diafiltered sample of the eluate are recognized by the pan-flavivirus 4G2 antibody. The presence of these two bands having molecular mass (MM) around that of the envelope protein (50 kDa) correspond to two different glycoforms of the E protein, as we have shown in a previous work (Lima et al., 2019). In the SXC eluate the presence of PEG changed protein migration, but upon diafiltration the pattern is similar to the one observed in the supernatant sample. SEC-HPLC analysis of SXC eluate (Fig. 5D) further shows that contaminants, such as other proteins or eventually monomeric E protein not assembled in VLPs, were successfully removed in the purification step.
Concentrations of VLP, total proteins and DNA in the cell culture supernatant and in the SXC eluate were quantified in order to have an overview of SXC performance as a purification step for YF VLPs (Table 1). VLP recovery and DNA removal were 72.3% and 60.3% respectively, which are lower levels than usually reported for SXC, but in a range comparable to other types of chromatography (Pato et al., 2019). On the other hand, there was 99.9% total protein removal, which is comparable to values reported by other authors for SXC (Lee et al., 2012; Marichal-Gallardo et al., 2017; Levanova & Poranen, 2018).
The SXC-purified VLPs were further analyzed by transmission electron microscopy (TEM) and analytical size exclusion chromatography (A-SEC) to further investigate VLP structure and size. Negative-stained TEM confirmed the 3D structure of VLPs (Fig. 6A), whereas A-SEC runs revealed a non-symmetric and broader peak when compared to the protein standards used (Fig. 6B), which goes in line with the variability in sizes observed in the panel of micrographs (Fig. 6A).