Variable light (VL) and heavy (VH) chains of Fabs used in these studies were gene synthesized and cloned (GeneArt) into custom pcDNA3.4 expression vectors immediately upstream of human Igk and Igg1-CH1 domains. Fab heavy chain and Fab light chain plasmids were co-transfected at a 2:1 ratio into FreeStyle 293-F cells (Thermo Fisher Scientific) at a cell density of 0.8 x 106 cells/ml for transient expression using FectoPRO DNA transfection reagent (Polyplus). Cells were cultured in GIBCO FreeStyle 293 Expression Medium for 7 days, and supernatants were isolated by centrifugation and filtered through a 0.22-mm membrane. Fabs were purified following a scheme of HiTrap KappaSelect affinity chromatography (Cytiva), cation exchange chromatography (MonoS, Cytiva), and size-exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva). The Pfs230 D1+ amino acid sequence (552–731), containing an N585Q mutation to remove a potential N-glycosylation site,71 was back-translated, codon-optimized for expression in human cells, appended to a C-terminal TEV cleavage site followed by a Strep-tag II sequence, and cloned in a pcDNA3.4 expression vector. Pfs230 D1+ plasmid was transfected into FreeStyle 293-F cells (Thermo Fisher Scientific) at a cell density of 0.8 x 106 cells/ml for transient expression using FectoPRO DNA transfection reagent (Polyplus). Cells were cultured in GIBCO FreeStyle 293 Expression Medium for 7 days, and supernatants were isolated by centrifugation and filtered through a 0.22-mm membrane. Pfs230 D1+ was purified using StrepTrap HP affinity chromatography (Cytiva) and size-exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva) into a final buffer of 20 mM Tris and 150 mM sodium chloride at pH 8.0. Purified Pfs230 D1+ protein was concentrated to 1 mg/ml and immediately frozen and stored at -80 o C.