BIAcore analysis of VWF binding of FVIII variants PEGylated with 60-kDa PEG
| Sample . | ka, 1/ms . | kd, 1/s . | Rmax, RU . | Kd, nM . |
|---|---|---|---|---|
| Full-length FVIII | 3.01 × 10−6 | 9.10 × 10−4 | 42.2 | 0.30 |
| BDD-FVIII | 5.23 × 10−6 | 8.22 × 10−4 | 39.1 | 0.16 |
| K1804C | 1.57 × 10−6 | 6.34 × 10−4 | 32.8 | 0.4 |
| F129C | 1.48 × 10−6 | 8.25 × 10−4 | 27.7 | 0.56 |
| L491C/K1804C | 1.19 × 10−6 | 6.84 × 10−4 | 26.5 | 0.57 |
| F129C/K1804C | 7.99 × 10−5 | 8.93 × 10−4 | 23.2 | 1.12 |
| Sample . | ka, 1/ms . | kd, 1/s . | Rmax, RU . | Kd, nM . |
|---|---|---|---|---|
| Full-length FVIII | 3.01 × 10−6 | 9.10 × 10−4 | 42.2 | 0.30 |
| BDD-FVIII | 5.23 × 10−6 | 8.22 × 10−4 | 39.1 | 0.16 |
| K1804C | 1.57 × 10−6 | 6.34 × 10−4 | 32.8 | 0.4 |
| F129C | 1.48 × 10−6 | 8.25 × 10−4 | 27.7 | 0.56 |
| L491C/K1804C | 1.19 × 10−6 | 6.84 × 10−4 | 26.5 | 0.57 |
| F129C/K1804C | 7.99 × 10−5 | 8.93 × 10−4 | 23.2 | 1.12 |
Approximately 250 RU of vWF (Haemtech) was immobilized on either flow cell (Fc) 2 or 4 of Sensor chip CM5 at a 10 μg/mL protein concentration in 10mM sodium acetate, pH 5.5, buffer by an amine coupling method. Flow cell 1 or 3 was used as a reference cell and had been activated and deactivated by amine coupling reagents (EDC/NHS and Ethanolamine). Full-length FVIII, BDD-FVIII, and the PEG-FVIII muteins were tested on the VWF immobilized chip at 5 different protein concentrations with at least 2 concentrations run in duplicate (running buffer, 0.45-16nM in 50mM Tris, 150mM NaCl, 0.05% Surfactant P20, pH 7.2). All samples were allowed to associate with immobilized VWF for 5 minutes at 50 μL/minute flow rate and dissociate in running buffer for 3 minutes. The sensor chip was regenerated with 200mM CaCl2.2H20 in assay running buffer. The reference subtracted (Fc 2-1 or Fc 4-3) binding curves were double referenced by subtracting blank buffer curve.