Table 1.
Comparison of Aggregate and Particle Formation in Different Size Ranges for the Three NRRV Antigens
| Size Range | Analytical Methods | P[4] | P[6] | P[8] | ||||
|---|---|---|---|---|---|---|---|---|
| T = 0 h | T = 1.5 h | T = 0 h | T = 1.5 h | T = 0 h | T = 1.5 h | |||
| >100 F020µm | Visual assessment of visible particles | – | – | – | + | + | + | |
| 1 nm-100 µm | UV-visible absorption spectroscopy | A280 | 0.35 ± 0.0 | 0.25 ± 0.01 | 0.35 ± 0.0 | 0.23 ± 0.0 | 0.37 ± 0.0 | 0.26 ± 0.0 |
| OD350 | 0.01 ± 0.0 | 0.46 ± 0.07 | 0.01 ± 0.0 | 0.35 ± 0.02 | 0.01 ± 0.0 | 0.40 ± 0.04 | ||
| Turbidimetry (NTU) | 0.5 ± 0.0 | 12.9 ± 0.8 | 0.5 ± 0.0 | 7.8 ± 0.5 | 0.5 ± 0.0 | 9.9 ± 0.3 | ||
| 2-100 µm | Micro-flow imaging (total particles/mL of 10× diluted sample) | 1.4 ± 0.4 × 103 | 1.0 ± 0.2 × 105 | 3.3 ± 0.5 × 103 | 0.8 ± 0.1 × 105 | 4.4 ± 0.7 × 103 | 0.5 ± 0.0 × 105 | |
| 0.1-2 µm | Resonant mass measurement (total Particles/mL) | 2.1 ± 0.1 × 105 | 1.2 ± 1.0 × 106 | 1.0 ± 0.1 × 106 | 3.3 ± 2.2 × 107 | 3.2 ± 1.0 × 105 | 6.3 ± 3.9 × 106 | |
| 1-100 nm | SV-AUC | Monomer (%) | 100 ± <1 | 70 ± 2 | 100 ± <1 | 74 ± 1 | 100 ± <1 | 67 ± 2 |
| Soluble aggregates (%) | 0 ± <1 | 0 ± <1 | 0 ± <1 | 0 ± <1 | 0 ± <1 | 0 ± <1 | ||
| SEC | Monomer (%) | 99.4 ± 0.1 | 71.4 ± 4.2 | 98.5 ± 0.3 | 74.7 ± 0.8 | 96.7 ± 0.1 | 65.2 ± 3.6 | |
| Fragments (%) | 0.3 ± 0.0 | 1.3 ± 0.4 | 0.2 ± 0.2 | 2.6 ± 0.4 | 3.3 ± 0.1 | 3.9 ± 0.1 | ||
| Soluble + insoluble aggregates (%) | 0.3 ± 0.0 | 27.4 ± 4.3 | 1.3 ± 0.1 | 21.9 ± 1.2 | 0.0 ± 0.0 | 30.8 ± 3.8 | ||
Similar levels of particles were generated in each NRRV antigen under forced degradation conditions of shaking stress for 1.5 h at 250 RPM at room temperature in 10 Mm sodium phosphate 150 mM NaCl pH 7.2 buffer. Error bars represent 1 SD from triplicate measurements.