k_a

Binding

Rate Constant of Association (kon)

1/M•s

Surface Plasmon Resonance (SPR)

HBS-EP+ with 0.1 mg/ mL BSA as running buffer

25 C

None

None

None

None

Surface plasmon resonance (Biacore) measurements. Affinity measurements were performed on Biacore model 2000 or T100 (Biacore, GE Healthcare) at 25°C using HBS-EP+ with 0.1 mg/ mL BSA as running buffer. A CM5 sensor chip with goat anti- human Fc amine-coupled in all 4 flow cells at ~15,000 RU was used to capture test antibodies with EGFR in the mobile phase. Between 50 and 75 RUs of hu225 variants were captured on the CM5 chip. A 3-fold dilution series of EGFR from 90 to 0.37 nM was injected and dissociation was monitored for 15 min. Kinetic analysis was done by simultaneously fitting the association and dissociation phases of the sensorgram using the 1:1 Langmuir binding model in BIAevaluate software as supplied by the manufacturer. Double referencing was applied in each analysis to eliminate background responses from the reference surface and buffer only control.