Mab select sure protein a

ActRIIA-Fc was purchased from R&D (Cat. No. 340-RC2-100, Minneapolis, MN). ActRIIB-Fc was expressed and purified from Chinese hamster ovary (CHO) cells as previously described (Sako et al., 2010 (link); Cadena et al., 2010 (link)). Briefly, ActRIIB-Fc was isolated using affinity chromatography with Mab Select Sure Protein A (GE Healthcare, Waukesha, WI), followed by dialysis into 10 mM Tris, 137 mM NaCl, and 2.7 mM KCl, pH 7.2. ActRIIB-ALK7-Fc and ActRIIB-ALK4-Fc were designed and expressed as previously described in CHO DUKX cells through the coexpression of two plasmids, each containing a receptor ECD (ActRIIB or ALK4) fused to a modified human IgG1 Fc domain (Li et al., 2021 (link); Kumar et al., 2021 (link)). Purification was performed through protein A MabSelect SuRe chromatography (Cytiva), then eluted with glycine at low pH. The resulting sample was further purified over a Ni Sepharose 6 fast flow column (Cytiva) followed by an imidazole elution gradient, an ActRIIB affinity column and ultimately, a Q Sepharose column (Cytiva). ALK7-Fc production was performed as described previously (Kumar et al., 2021 (link)).

KTN3379, cetuximab and pertuzumab were cloned, expressed and purified in-house as described below. Plasmids encoding the light and heavy chain of each antibody were transiently co-expressed in Expi293 cells (Thermo Fisher Scientific) using polyethylenimine. Conditioned media containing the antibodies was flowed over MabSelect SuRe Protein A (GE Healthcare). Bound antibody was washed with PBS and eluted in 0.1 M glycine pH 2.7 and immediately neutralized with 1 M Tris pH 7.4. Purified antibody was buffer exchanged into PBS by tangential flow filtration using a Sartorius Slice ECO with a 30 KDa cutoff Hydrosart^® cassette. Antibodies were purified with low endotoxin (<1 EU/mg), and were >95% pure and >95% monomeric as assessed by SDS-PAGE, and ultra-high pressure size exclusion chromatography (SEC-UPLC) using a UPLC BEH 200 column in a Waters Acquity instrument.