1 IgG Charge 2

It has been known since the 1930’s that all immunoglobulins carry a weak negative 12 charge in physiological solvents. However, there has been no systematic exploration of this 13 fundamental property. Accurate charge measurements have been made using membrane 14 confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as 15 well as their F(ab’)2 and Fc fragments. The following observations were made at pH 5.0: 1) the 16 measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the 17 Fcs; 2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of 18 the F(ab)’2 and Fc charge; 3) the Fc charge is consistent within a class. In phosphate buffered 19 saline, pH 7.4: 1) the intact IgG charges ranged from 0 to -13; 2) the F(ab’)2 fragments are nearly 20 neutral for IgG1s and IgG2s, and about -5 for some of the IgG4s; 3) all Fc fragments are weakly 21 anionic, with IgG1 < IgG2 < IgG4; 4) the charge on the intact IgGs does not equal the sum of the 22 F(ab’)2 and Fc charge. In no case is the calculated charge, based on H+ binding, remotely close to 23 the measured charge. The charge on IgGs in physiological solvent is sufficiently small to 24 minimize its contribution to thermodynamic nonideality. Some of the mAbs carried a charge in 25 physiological salt that was outside the range observed for serum-purified human poly IgG. To 26 best match physiological properties, a therapeutic mAb should have a measured charge that falls 27 within the range observed for serum-derived human IgGs. 28