Superdex 200 analytical column

Fabs were expressed by transient transfection of HEK 293F suspension cells, using linear PEI following the manufacturer’s suggested protocol. After 5 d, supernatants were clarified by centrifugation and diluted twofold with 1x PBS buffer, and the protein isolated from the diluted spernatant using CaptureSelect LC-Kappa (Hu) affinity matrix (Thermo Fisher Scientific, Waltham, MA), according to manufacturer’s protocols. Fractions containing the protein of interest were pooled, concentrated, and further purified by gel filtration chromatography using a Superdex 200 analytical column (GE Healthcare Life Sciences, Pittsburgh, PA) in a buffer of 2.5mM Tris, pH 7.5, 350mM NaCl, and 0.02% sodium azide.

The heavy- and light-chain variable and constant domains of the DH270.3 and DH270.6 Fabs were cloned into a modified pVRC-8400 expression vector using Not1 and Nhe1 restriction sites and the tissue plasminogen activator signal sequence. The DH270.6 scFv was cloned into the same vector as above. The C terminus of the heavy-chain constructs and scFv contained a noncleavable 6× histidine tag. Site-directed mutagenesis was performed using manufacturer’s protocols (Stratagene) to introduce mutations into the CDR regions of DH270.3 and DH270.6 Fabs. Fabs and the DH270.6 scFv were expressed and purified as described previously^{12 (link), 37 (link)}. Briefly, Fabs and the DH270.6 scFv were expressed using transient transfection of HEK 293T cells (ATCC: CRL-3216) using linear polyethylenimine (PEI) following the manufacturer’s suggested protocol. After 5 days of expression, supernatants were clarified by centrifugation. His-tagged Fabs were loaded onto Ni-NTA superflow resin (Qiagen) preequilibrated with Buffer A (10 mM Tris, pH 7.5, 100 mM NaCl), washed with Buffer A + 10 mM imidazole, and eluted with Buffer A + 350 mM imidazole. Fabs were then purified by gel filtration chromatography in Buffer A using a superdex 200 analytical column (GE Healthcare).