Table 1.
Comprehensive characterization of HEV p239 VLPs of antigen in aqueous solutions versus post-adsorption onto aluminum containing adjuvant and dissolution treatment to demonstrate comparable product attributes
| Parameters of p239 in different conditions | |||||
|---|---|---|---|---|---|
| Sample a | Sample b | Sample c | Sample c0 | ||
| Method | Charateristics | Native p239 VLPs | Adsorbed p239 VLPs | HEV vaccine dissolution | p239 in dissolution mixture |
| Biophysical and biochemical methods | |||||
| HPSECa | size ( reflected by retention time, min ) | 13.7 | — | 13.8 (20.0*) | 13.8 (20.1*) |
| TEM | morphology | particulate antigen ∼(20–30 nm in diameters) | |||
| DSCb | thermal stability (Tm, °C) | 75.0 ± 0.1 | 77.2 ± 0.2 | 75.2 ± 0.1 | 75.4 ± 0.4 |
| AUC | sedimentation coefficient (S) | 21.3 ± 0.5 | — | 20.8 ± 0.8 | 20.6 ± 0.5 |
| DLS | hydrodynamic diameter (nm) | 27.2 | — | 27.2 | 26.1 |
| SLSc | hydrodynamic diameter (μm) | 10.29 | |||
| icIEF | isoelectric point (pI) | 5.85 ± 0.03 (n = 3) | — | 5.80 ± 0.04 (n = 3) | 5.83 ± 0.03 (n = 3) |
| Immunochemical methods | |||||
| Cross-inhibitoryassaye | inhibitory capability | inhibitory capability by 9 mAbs from each other for different samples (Sample a, c and c0) showed by heat map (Fig. 3). | |||
| SPRe | binding to mAbs | binding activity to 6 mAbs for different samples (Sample a, c and c0) with relative antigenicity (Fig. 4). | |||
| Sandwich ELISAd | antigenicity (rEC50) | 1.00 (n = 3) | — | 1.21 ± 0.01 (n = 3) | 1.14 ± 0.07 (n = 3) |
An analytical TSK Gel PW5000xl (7.8 mm × 300 nm) column (TOSOH, Tokyo, Japan) was used in the HPSEC analysis.“*” 20 min is a buffer peak.
DSC was applied to the recombinant protein-based vaccine characterization, which reflects the thermal stability of sample.33 During the process of heating, protein in the solution tended to aggregate. The melting temperature was detected 3 times, and it is shown as an average.
The aluminum hydroxide was 11.3 ± 0.1 μm (n=3), the size of the mix compound (p239 adsorbed to aluminum hydroxide) was similar to the aluminum hydroxide with no statistically significant differences (Fig. 1C and Table 1).
Sandwich ELISA was used to detect the antigenicity of the different antigens (a, c and c0) in this assay. The capture antibody was 3A11 and the detection antibody was 8C11-HRP.
Although no numeric data are being reported here, the methods are listed for the completeness of the analytical toolbox and full results are presented in Figures 3 and 4.