Fig 5. Isolation and characterization of the heterodimeric Fc variants from the LibA2 and LibB2 libraries.

(a) Enrichment profiles of diploid cells with higher Fc heterodimerization levels during 4 sequential rounds of FACS with each library, as shown by the relative MFI (%) of each sample compared with that of the EW-RVT Fc variant (set as 100%). (b) Comparison of the heterodimerization levels of isolated Fc variants on the yeast cell surface expressed as a percentage of the MFI relative to that of the EW-RVT Fc variant (set as 100%). (c) SDS-PAGE analyses under non-reducing conditions of the purified coexpressed scFv-Fc_CH3A/Fc_CH3B proteins (5 μg) carrying the indicated CH3 variant pair. The newly introduced mutations in the CH3A or CH3B domain of isolated the Fc variants are shown in red font. The EW-RVT, A107_{w/o W-VT}, and B168_{w/o W-VT} variants are included for comparison. (d) Heterodimer yield of the indicated scFv-Fc_CH3A/Fc_CH3B proteins, estimated as described in Fig 3e.