Table 1.
Bevacizumab stabilization by single point mutation in its Fab domain. Wild-type (WT) bevacizumab and the variants were tested for their aggregation propensity by heat-induced accelerated aggregation study. The percentage of monomer and aggregates detectable by SEC-HPLC was measured at various time points up to 48 h of incubation at 52°C at 50 mg/mL antibody concentration (His 10 mM, pH 6.0). The percentage of monomer in the soluble fraction after 48 h of heat treatment is reported in the table. Data are the mean ± SD. (n=3 experiments with 3 different protein batches, *n=2 experiments with 2 different protein batches). The kinetic data were fitted to extract a second order rate constant (“Aggregation rate”). A stabilization factor was calculated and the “Fold increase stability” is the ratio of the percentage of WT aggregates to the percentage of variant aggregates at t=48 h. The thermostability of bevacizumab formulated in 10 mM histidine buffer (pH 6.0) was characterized by DSC to determine and attribute 3 transition temperatures, Tm1 for the CH2 domain, Tm2 which can be attributed to the Fab domain and Tm3 corresponding to the transition temperature of the CH3 domain. The transition temperatures in degrees Celsius for the WT and each variant were obtained by fitting Gaussians to each thermogram. Equilibrium dissociation constants, KD, for the WT bevacizumab and its variants binding to VEGF-A were extracted from Figure 2A. WT and engineered mAbs bound to the target VEGF with the same affinity as previously reported for bevacizumab.
| Variants | % monomer at 48 h | Aggregation rate (*10−2 mol−1.L.min−1) | Fold increase stability | Transition temperature (°C) Tm1 Tm2 Tm3 | KD (nM) |
|---|---|---|---|---|---|
| WT | 68 ± 2 | 31.3 ± 5.6 | 1.0 ± 0.1 | 70.4 71.8 82.4 | 0.85 ± 0.28 |
| V5K | 74 ± 2 | 13.9 ± 1.0 | 1.2 ± 0.1 | 70.4 71.5 82.9 | 2.57 ± 0.24 |
| F50D | 87 ± 5 | 9.6 ± 0.6 | 2.5 ± 1.0 | 71.0 72.9 83.2 | 1.19 ± 0.28 |
| V110K | 88 ± 5 | 6.3 ± 2.8 | 2.8 ± 1.2 | 71.3 72.7 83.0 | 1.32 ± 0.28 |
| L154D | 88 ± 3 | 5.9 ± 1.8 | 2.8 ± 0.8 | 71.4 72.6 83.3 | 0.62 ± 0.1 |
| L154K* | 92 ± 0 | 4.5 ± 1.2 | 4.0 ± 0.4 | 72.1 73.5 82.7 | 0.85 ± 0.02 |
| L180K | 82 ± 3 | 9.9 ± 1.7 | 1.8 ± 0.3 | 71.3 72.0 84.3 | 2.39 ± 0.13 |
| L201K* | 87 ± 2 | 3.1 ± 2.1 | 2.5 ± 0.5 | 72.2 72.6 82.8 | 0.68 ± 0.2 |