Recombinant Antibody Expression and Purification

PGT125, PGT126, PGT128 and PGT130 Fabs were expressed in 293S (ATCC) or FreeStyle 293F cells (Life Technologies) grown in suspension. The cells were transfected at a 1:1 ratio of plasmids encoding the respective antibodies’ light and heavy chains (truncated at AspH234). Supernatants were harvested six days after transfection, and cell debris was removed by centrifugation. Antibody was purified from the clarified supernatant using an anti-human lambda affinity matrix (CaptureSelect LC-lambda (hu); Thermo Scientific) equilibrated in PBS. Fab fragments were eluted with 0.1 M glycine, pH 3, and the eluate neutralized with concentrated PBS. Recombinant IgGs were expressed in FreeStyle 293F cells and purified on a protein A resin (Thermo Scientific) as described elsewhere.^{[13 (link)]}