Full-length antibody variants were compared to bD1 and hu4D5 by SEC. Equal masses (75 μg) of purified, full-length hu4D5 (gray, solid), bD1 (gray, dashed) and variant (black, solid) antibodies were analyzed by SEC using a Superdex 200 column on an Åkta Pure FPLC. The variants shown are (A) R2D, (B) R2H, (C) R2J and (D) R2S. In panel (E), SEC profiles for R2J (black, solid), R2J-S (black, dashed) and R2J-Y (black, dotted) were compared. Standards in each SEC plot are indicated by a triangle and include: ferritin (10.3 ml, 440 kDa), beta amylase (11.7 ml, 200 kDa), aldolase (12.6 ml, 158 kDa), conalbumin (14.1 ml, 75 kDa), ovalbumin (15.1 ml, 44 kDa) and carbonic Anhydrase (16.5 ml, 29 kDa). (F) The eluted protein from each of the four R2J peak fractions (Peak 1, 11.5–12.0 ml; Peak 2, 12.0–12.5 ml; Peak 3, 12.5–13.0 ml and Peak 4, 13.0–13.5 ml) and three bD1 peak fractions (Peak 1, 11.5–12.0 ml; Peak 2, 12.0–12.5 ml and Peak 3, 12.5–13.0) were precipitated and analyzed by SDS-PAGE for comparison to molecular weight standards.