Gf 250

N-terminal PEG conjugation of _TrastFab (see Figure 1) was obtained by reductive alkylation using 20 kDa MeO-PEG-propionaldehyde.
To a 1.0 mg/mL _TrastFab solution in 0.1 M acetate buffer, pH 4.5, MeO-PEG-propionaldehyde was added in order to achieve a 1:2 Fab/PEG molar ratio. Under these conditions, PEG-aldehyde forms a Schiff base with the N-terminal amino group; the Shiff base is subsequently transformed into a stable secondary amine by mild reduction with sodium cyanoborohydride (NaBH₃CN) [21 (link)]. NaBH₃CN was added to the solution up to a final concentration of 0.04 M. The PEGylation reaction was carried out at room temperature for 16 h under gentle stirring. The conjugated Fab, hereafter _TrastFab-(N-Term)-PEG_{20 kDa}, was first dialyzed against 0.02 M acetate buffer pH 4.5 and then purified by cation exchange chromatography on a Macrocap SP chromatography column operating at a flow rate of 1.5 mL/min. The conjugated Fab was eluted applying a 10 column volumes linear gradient of NaCl from 0 to 0.2 M. Fractions containing the purified _TrastFab-(N-Term)-PEG_{20 kDa} were analyzed by SE-HPLC and 12% SDS-PAGE. SE chromatography analyses were performed on a Zorbax GF-250 (4.6 mm × 250 mm) column in 0.063 M phosphate buffer pH 7.3, containing 3% (v/v) Isopropanol, at 25 °C, UV detection at 215 nm and applying a flow rate of 0.3 mL/min.

Aggregation propensity of the reformatted VNAR test articles was analyzed using analytical size-exclusion chromatography (SEC) and an Agilent 1200 series HPLC system with a ZORBAX GF 250 9.4 × 250 mm 4 μm column containing phosphate buffered saline pH 7.4 as the mobile phase. VNAR D1-BA11-C4 and Quad-X™ were loaded onto columns at 6 mg/ml from stock concentrations of 40 mg/ml. Purity of the test VNAR protein samples was also assessed by SDS-PAGE electrophoresis of the reduced VNAR protein samples in a MES buffer system (Invitrogen).