Expi293f human embryonic kidney cells

The heavy and light chain sequences of mAb45D8^{23 (link)} were separately cloned into pcDNA3.4 and the resulting vectors were transfected into Expi293F Human Embryonic Kidney cells (unauthenticated and untested for mycoplasma contamination, Life Technologies) using a 1:2 mass ratio of light and heavy chain DNA with the Expifectamine transfection kit (Life Technologies) as per the manufacturer’s instructions. Supernatant containing mAb45 was harvested 136 h after transfection and loaded on homemade Protein G Sepharose beads and extensively washed with a buffer comprising 20 mM HEPES pH 7.50, and 100 mM NaCl. mAb45D8 was eluted with 100 mM glycine (pH 3.0) and fractions were immediately neutralized with 200 mM HEPES pH 7.50. To generate the Fab fragment, 10 mg of purified mAb45D8.1 was diluted into 9.5 ml freshly prepared cleavage buffer (20 mM sodium phosphate pH 7.00, 10 mM EDTA, and 10 mM cysteine) and treated with 0.5 ml agarose immobilized papain (Thermo Scientific) at 37°C. After 16 h the cleaved Fab45D8 fragment was purified by reverse Protein A affinity chromatography, followed by SEC into buffer comprising 20 mM HEPES pH 7.50 and 100 mM NaCl. Fab45D8 was concentrated on a Vivaspin 10-kDa MWCO concentrator, and aliquots were flash frozen in liquid nitrogen and stored at −80°C for later use.