The quality of the purified mAb IgGs and their fragments generated by subsequent enzymatic digestion was evaluated by analytical size-exclusion ultra-performance liquid chromatography (SE-UPLC) using a BEH200 column on the Waters Acquity UPLC system (Waters Corporation, Milford, MA, USA). The IgGs were purified by ÄKTA affinity chromatography system and MabSelect Sure resin (GE Healthcare, Chicago, IL, USA) following standard methods. Human serum derived from male AB plasma was purchased from Sigma-Aldrich, St. Twelve anti-IL13 IgGs comprising three unique variable regions, each constructed as four human IgG subclasses, IgG1, IgG2, wild-type IgG4(Ser 222), and a hinge mutant IgG4(Pro 222), were made from stable NS0 cell line at Boehringer Ingelheim. It is apparent that a systematic analysis of the charge on mAbs would be useful. Consequently, it is not known whether the charge on therapeutic mAbs falls into the rather narrow range observed for normal human IgGs. There is no published charge data for mAbs in physiological solvents. The narrow range of charge is somewhat surprising since isoelectric focusing analysis of the same sample yielded pIs covering the pH range from less than 4 to greater than 10. More recently, it was shown that freshly prepared human polyclonal IgGs have a Debye–Hückel–Henry charge, Z DHH, between −3 and −9. Furthermore, IgGs from several species are anionic in the pH 5–6 range. However, it has been known for over 80 years that all serum proteins, including the immunoglobulins, carry a net negative charge under physiological conditions. The majority of biotherapeutic mAbs exhibit pIs ≥ 8, and carry a positive charge under formulation conditions (typically pH 5–6). Based on readily measured properties, interaction curves may be generated to aid in the selection of proteins and solvent conditions. A thermodynamically rigorous, concentration-dependent protein–protein interaction parameter is introduced. To best match physiological properties, a therapeutic mAb should have a measured charge that falls within the range observed for serum-derived human IgGs. Some mAbs carried a charge in physiological salt that was outside the range observed for serum-purified human poly IgG. In no case is the calculated charge, based solely on H + binding, remotely close to the measured charge. In phosphate buffered saline, pH 7.4: (1) the intact IgG charges ranged from 0 to −13 (2) the F(ab’) 2 fragments are nearly neutral for IgG1s and IgG2s, and about −5 for some of the IgG4s (3) all Fc fragments are weakly anionic, with IgG1 < IgG2 < IgG4 (4) the charge on the intact IgGs does not equal the sum of the F(ab’) 2 and Fc charge. The following observations were made at pH 5.0: (1) the measured charge differs from the calculated charge by ~40 for the intact IgGs, and by ~20 for the Fcs (2) the intact IgG charge depends on both Fv and Fc sequences, but does not equal the sum of the F(ab)’ 2 and Fc charge (3) the Fc charge is consistent within a class.
Accurate charge measurements have been made using membrane confined electrophoresis in two solvents (pH 5.0 and pH 7.4) on a panel of twelve mAb IgGs, as well as their F(ab’) 2 and Fc fragments. However, there has been no systematic exploration of this fundamental property. It has been known since the 1930s that all immunoglobulins carry a weak negative charge in physiological solvents. Biologically, IgG charge isomers exhibit differences in clearance and potency. Practically, IgG charge can contribute significantly to thermodynamic nonideality, and hence to solubility and viscosity.
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