Table 2. Biophysical properties and off-rate screening of design mutants.
| Name | Starting Concentration (mg/ml) | Final High Concentration (mg/ml) | Retention Time (min) | Peak Width | % HMMS | % Main | % LMMS | Tm1 (°C) | Tm2 (°C) | Tm3 (°C) | Apparent Fab Tm (°C) | kd (1/s) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AB-001 | 4.63 | 98.8 | 9.35 | 0.39 | 0.73 | 99.26 | 0.01 | 69.16 ± 0.09 | 80.89 ± 0.53 | 83.49 ± 0.17 | 82.72 | 3.19E-04 |
| R1-002 | 4.94 | 107.6 | 9.41 | 0.43 | 0.91 | 99.03 | 0.06 | 69.22 ± 0.12 | 81.73 ± 0.89 | 83.90 ± 0.25 | 83.30 | 3.80E-04 |
| R1-003 | 3.92 | 88.9 | 9.37 | 0.40 | 0.61 | 99.32 | 0.07 | 69.34 ± 0.13 | 82.43 ± 1.00 | 84.62 ± 0.26 | 84.21 | 4.43E-04 |
| R1-004 | 4.74 | 104.3 | 9.37 | 0.40 | 0.53 | 99.39 | 0.08 | 69.32 ± 0.13 | 82.15 ± 1.00 | 84.44 ± 0.20 | 83.84 | 2.58E-04 |
| R1-005 | 5.25 | 130.7 | 9.53 | 0.43 | 3.28 | 96.69 | 0.04 | 69.45 ± 0.13 | 82.21 ± 1.10 | 84.53 ± 0.21 | 83.95 | 3.81E-04 |
| R1-006 | 5.13 | 124.3 | 9.43 | 0.40 | 0.59 | 98.52 | 0.04 | 68.11 ± 0.13 | 82.54 ± 0.75 | 84.72 ± 0.29 | 83.81 | 3.14E-04 |
| R1-007 | 5.38 | 156.9 | 9.34 | 0.40 | 0.33 | 99.04 | 0.02 | 68.17 ± 0.12 | 82.40 ± 0.74 | 84.41 ± 0.29 | 83.50 | 2.82E-04 |
| R1-008 | 5.19 | 111.1 | 9.36 | 0.39 | 1.69 | 98.26 | 0.05 | 69.46 ± 0.13 | 81.64 ± 0.60 | 84.34 ± 0.15 | 83.62 | 5.33E-04 |
| R1-009 | 4.70 | 102.2 | 9.38 | 0.40 | 0.59 | 99.34 | 0.07 | 69.42 ± 0.03 | 79.33 ± 0.47 | 81.48 ± 0.13 | 80.95 | 3.93E-04 |
| R1-010 | 4.74 | 121.0 | 9.44 | 0.44 | 2.39 | 97.59 | 0.02 | 69.42 ± 0.12 | 82.81 ± 0.73 | 85.63 ± 0.19 | 85.04 | 3.72E-04 |
| R1-011 | 4.99 | 110.4 | 9.33 | 0.38 | 1.35 | 98.61 | 0.04 | 69.37 ± 0.11 | 81.09 ± 0.67 | 83.47 ± 0.19 | 82.60 | 8.60E-04 |
| R1-012 | 4.94 | 109.0 | 9.35 | 0.40 | 2.05 | 97.79 | 0.17 | 68.31 ± 0.12 | 83.09 ± 0.67 | 85.30 ± 0.26 | 84.25 | 3.04E-04 |
| R1-013 | 4.70 | 100.8 | 9.42 | 0.44 | 0.24 | 99.63 | 0.13 | 69.66 ± 0.05 | 80.17 ± 1.20 | 82.12 ± 0.18 | 81.94 | 2.97E-04 |
| R1-014 | 4.62 | 123.5 | 9.39 | 0.40 | 0.33 | 99.57 | 0.10 | 69.28 ± 0.06 | 80.84 ± 1.50 | 82.56 ± 0.31 | 82.20 | 4.05E-04 |
| R1-015 | 5.08 | 104.1 | 9.35 | 0.41 | 0.92 | 99.07 | 0.01 | 67.98 ± 0.11 | 82.41 ± 1.00 | 84.22 ± 0.34 | 83.76 | 7.52E-04 |
| R1-016 | 4.93 | 156.6 | 9.41 | 0.41 | 0.65 | 99.23 | 0.12 | 69.38 ± 0.08 | 84.48 ± 0.55 | 86.83 ± 0.24 | 85.51 | 2.89E-04 |
| R1-017 | 5.15 | 89.4 | 9.4 | 0.41 | 0.14 | 99.79 | 0.07 | 69.48 ± 0.09 | 81.18 ± 1.40 | 83.07 ± 0.31 | 82.57 | 4.71E-04 |
| R1-018 | 5.17 | 109.7 | 9.46 | 0.44 | 0.75 | 99.15 | 0.10 | 68.56 ± 0.10 | 81.15 ± 1.00 | 83.12 ± 0.28 | 82.64 | 4.19E-04 |
| R2-001 | 5.38 | 153.2 | 10.72 | 0.41 | 0.28 | 99.62 | 0.11 | 69.47 ± 0.11 | 85.16 ± 0.36 | 88.11 ± 0.21 | 86.7 | 4.17E-04 |
| R2-002 | 5.46 | 119.9 | 10.61 | 0.41 | 0.34 | 99.71 | 0.00 | 69.58 ± 0.08 | 85.21 ± 0.31 | 88.15 ± 0.16 | 86.5 | 4.40E-04 |
| R2-003 | 5.02 | 135.6 | 10.45 | 0.39 | 0.60 | 99.32 | 0.06 | 70.21 ± 0.21 | 74.88 ± 0.24 | 80.03 ± 0.05 | 79.8 | 1.10E-03 |
| R2-004 | 5.01 | 164.4 | 10.64 | 0.42 | 0.45 | 99.43 | 0.09 | 69.64 ± 0.03 | 81.48 ± 1.60 | 82.92 ± 0.49 | 82.5 | 4.39E-04 |
| R2-005 | 5.08 | 168.8 | 10.61 | 0.42 | 0.76 | 99.11 | 0.11 | 69.89 ± 0.06 | 80.17 ± 0.02 | 80.22 ± 0.09 | 80.3 | 3.48E-04 |
| R2-006 | 5.35 | 173.5 | 10.72 | 0.41 | 0.28 | 99.61 | 0.10 | 69.53 ± 0.09 | 84.20 ± 0.52 | 86.54 ± 0.26 | 85.6 | 3.66E-04 |
| R2-007 | 5.00 | 177.2 | 10.75 | 0.44 | 0.24 | 99.78 | 0.00 | 69.77 ± 0.07 | 84.13 ± 0.54 | 86.49 ± 0.26 | 85.4 | 4.01E-04 |
| R2-008 | 5.09 | 179.7 | 10.72 | 0.45 | 0.47 | 99.54 | 0.00 | 69.58 ± 0.10 | 84.86 ± 0.44 | 87.53 ± 0.25 | 86.1 | 3.96E-04 |
| R2-009 | 5.37 | 168.1 | 10.69 | 0.42 | 0.36 | 99.59 | 0.09 | 69.71 ± 0.06 | 83.79 ± 0.51 | 86.05 ± 0.24 | 85.3 | 2.65E-04 |
| R2-010 | 5.00 | 184.5 | 10.59 | 0.40 | 0.40 | 99.50 | 0.10 | 69.54 ± 0.12 | 85.01 ± 0.45 | 87.76 ± 0.27 | 86.5 | weak |
| R2-011 | 5.32 | 168.5 | 10.45 | 0.38 | 0.52 | 99.42 | 0.09 | 69.72 ± 0.04 | 81.43 ± 1.10 | 83.09 ± 0.37 | 82.2 | 4.14E-04 |
| R2-012 | 6.79 | 186.2 | 11.40 | 0.58 | 0.44 | 99.56 | 0.00 | 68.75 ± 0.08 | 85.17 ± 0.29 | 88.26 ± 0.14 | 87.0 | 3.47E-04 |
| R2-013 | 5.30 | 187.3 | 11.25 | 0.49 | 0.33 | 99.83 | 0.00 | 69.66 ± 0.07 | 85.39 ± 0.27 | 88.25 ± 0.12 | 86.6 | 3.44E-04 |
| R2-014 | 4.58 | 187.0 | 13.01 | 0.70 | 2.92 | 97.08 | 0.00 | 68.54 ± 0.11 | 85.00 ± 0.32 | 88.22 ± 0.15 | 87.0 | 3.96E-04 |
| R2-015 | 5.03 | 159.7 | 11.56 | 0.54 | 0.41 | 99.59 | 0.00 | 68.54 ± 0.11 | 84.23 ± 0.50 | 86.83 ± 0.27 | 85.7 | 8.34E-04 |
| R2-016 | 6.47 | 146.7 | 11.44 | 0.52 | 0.20 | 99.80 | 0.00 | 68.61 ± 0.12 | 82.45 ± 0.64 | 84.99 ± 0.19 | 84.4 | 2.66E-04 |
| R2-017 | 5.21 | 154.2 | 11.53 | 0.53 | 0.17 | 99.83 | 0.00 | 68.55 ± 0.08 | 79.42 ± 0.01 | 84.66 ± 0.15 | 79.3 | 6.73E-04 |
| R2-018 | 5.10 | 171.7 | 10.84 | 0.43 | 0.33 | 99.66 | 0.00 | 69.59 ± 0.07 | 84.54 ± 0.46 | 87.10 ± 0.23 | 85.8 | 3.86E-04 |
| R2-019 | 5.20 | 163.2 | 10.44 | 0.59 | 1.45 | 98.59 | 0.00 | 69.47 ± 0.07 | 82.53 ± 0.64 | 84.56 ± 0.31 | 83.4 | 6.73E-04 |
| R2-020 | 12.69 | 200.5 | 12.06 | 0.59 | 0.15 | 99.85 | 0.00 | 68.65 ± 0.11 | 85.57 ± 0.21 | 90.00 ± 0.10 | 89.2 | 3.86E-04 |
| R2-021 | 8.23 | 174.0 | 11.40 | 0.51 | 0.30 | 99.71 | 0.00 | 68.05 ± 0.14 | 84.69 ± 0.50 | 87.33 ± 0.29 | 86.1 | weak |
| R2-022 | 6.94 | 160.8 | 11.44 | 0.53 | 0.55 | 99.45 | 0.00 | 68.55 ± 0.14 | 84.97 ± 0.46 | 87.78 ± 0.25 | 86.2 | 3.61E-04 |
This table includes the biophysical properties of AB-001 and two rounds of design mutants. This includes the initial concentration and final high concentration for viscosity measurements, analytical size exclusion chromatography (aSEC) retention time, peak width and species fractions (main, high molecular mass species (HMMS), low molecular mass species (LMMS)), and differential scanning calorimetry (DSC) thermal stability properties. Additionally, the off-rate (kd) screening measurements are also presented where R2-010 and R2-021 showed “weak” binding and the curve was not fit.