Isotype dependent on-column non-reversible aggregation of monoclonal antibodies

Monoclonal antibodies of the IgG2 and IgG4 isotype were found to exhibit an increased propensity for displaying two-peak elution profiles during cation exchange chromatography. In some cases, this two-peak elution profile also resulted in the formation of non-reversible mAb aggregates. Comparison of IgG1, IgG2, and IgG4 molecules with the same variable region reveals that the two-peak behaviour is predominantly mediated by the constant region and most likely the lower CH1, hinge and upper CH2 regions of the mAb. Furthermore, comparison of the behaviour of two different IgG4 molecules, reveals that the degree of non-reversible aggregate formation, whilst facilitated by the isotype format, is mediated primarily by the variable region of the molecule. As well as the properties of the mAb molecule itself, the chemistry and structure of the cation exchange resin was also found to have an effect, with the two-peak elution profile being more pronounced with polymer-grafted resins such as Capto S Impact and Eshmuno CPX. These results combined support the theory that binding of IgG2 and IgG4 mAbs to cation exchange resins usually occurs through at least two mechanisms mediated by the structural features of the constant region of IgG2s and IgG4s. One of these mechanisms is not only stronger than the other, but also can lead to a conformational change in the molecule. This conformational change can occur in both variable and constant domains of the antibody. This transitory unfolded state can in turn lead to non-reversible aggregation of some mAb molecules.