Table 1.
Biochemical characterization of the different Fc variants engineered for reduced PG binding.
| No | Fc proteins | PG binding | CH2 Tm (°C) | CH3 Tm (°C) |
Monomer (%) | FcRn nor. aff.* |
Expression (mg/l) |
|---|---|---|---|---|---|---|---|
| IgG1 | + | 72.6 | 85.5 | 98.7 | 1.00 | 52 | |
| 1 | M252L/E380R/E382L/S426M/M428G | - | 55.9 | 84.7 | 96.2 | NB | 30 |
| 2 | E380R/M428G/N434A/Q438K | - | 57.1 | 80.6 | 96.7 | 0.20 | 11 |
| 3 | E380A/E382A/N434A | - | 62.5 | 81.6 | 96.5 | 2.97 | 32 |
| 4 | E380R/N434A/Q438K | - | 60.5 | 83.9 | 97.2 | 2.24 | 31 |
| 5 | E380R/N434A | - | 60.8 | 82.8 | 94.3 | 2.87 | 46 |
| 6 | N434A/Q438K | + | 69.4 | 85.4 | 99.1 | ND | 63 |
| 7 | E380R/Q438K | + | 60.9 | 85.9 | 94.3 | ND | 50 |
| 8 | M428G/N434A | - | 65.3 | 79.5 | 98 | 0.95 | 41 |
| 9 | E380R/M428G | + | 58.0 | 81.1 | 92.8 | ND | 33 |
| 10 | M428G/Q438K | + | 65.1 | 85.1 | 97.9 | ND | 42 |
| 11 | E380A/N434A | -/+ | 64.4 | 82.8 | 97.9 | 4.59 | 50 |
| 12 | M252G/N434A | - | 65.1 | 82.6 | 94.5 | NB | 16 |
| 13 | M428G | + | 66.0 | 82.3 | 96.3 | 0.23 | 36 |
| 14 | N434A | + | 70.7 | 82.4 | 98.5 | 4.61 | 46 |
SPR binding affinities to FcRn were normalized against human IgG1 Fc. (ND) not done. (NB) no binding. (*) for each measurement, the relative KD value of the test antibody was calculated vs. IgG1 control wherein the control was measured on the same day with the same experimental setup (KD value of the test antibody/average KD value of control), the normalized affinity (nor. aff.) shown here corresponds to the 1/average of the different relative KD values (n ≥ 3).