FIG 11.

Size exclusion chromatography anomaly resulted from slow conformational isomerization, which an engineered disulfide resolves. (A) Size exclusion chromatography showed 10E8v4 to elute as three peaks (top chromatogram); when each fraction was immediately reinjected, it eluted as a mostly single peak (left chromatograms). However, if each of the separated peaks was allowed to equilibrate for 22 h, each now isomerized into three peaks (right chromatograms). (B) Multiangle light scattering (SEC-MALS) analysis. MW, molecular weight in thousands. (C) Neutralization potency of 10E8v4 and 10E8v4-DS. (D) An engineered disulfide between residue 100e on the heavy chain and residue 30 on the light chain to prevent isomerization. (E) 10E8v4 with an interchain disulfide, 100eC-30C (DS), eluted as a single peak in size exclusion chromatography.