3.2. SEC analysis
ORF8-CRM and ORF8-PEG were analyzed by a Superdex 200 column (1 cm × 30 cm). As shown in Fig. 2C, ORF8, CRM197, ORF8-CRM, and ORF8-PEG were all eluted as a single peak, indicating the high purity. The elution peaks of ORF8 and CRM197 were at 20.3 mL and 17.8 mL, respectively. As compared with ORF8 and CRM197, the elution peaks of ORF8-CRM (15.8 mL) and ORF8-PEG (16.9 mL) were both left-shifted. This indicated that conjugation with CRM197 and self-conjugation with PEG could both increase the hydrodynamic volume of ORF8.