2.3. Tangential flow filtration (TFF)

Harvested culture supernatants were clarified by centrifugation (4000×g, 20 min, 4 °C) followed by filtration (catalog# 12,993, Pall Corporation, Port Washington, NY). When used in column chromatography, clarified supernatants were concentrated and buffer exchanged by TFF. Specifically, a MasterFlex peristaltic pump (Vernon Hills, IL) fed the clarified supernatant to a 10-kDa MWCO cassette (catalog# SK1P003W4, MilliporeSigma, Burlington, MA). The clarified supernatant was concentrated to 10% of the initial volume, and then buffer exchanged with 5 vol of 1× PBS, pH 7.4 (Buffer A, diluted from 10× PBS, catalog #70011069 Thermo Fisher Scientific, Waltham, MA). Following buffer exchange, the TFF cassette was rinsed with 200–250 ml Buffer A, to collect any protein remaining in the cassette. Clarified supernatants for use in batch purification were not buffer exchanged.