Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG1).

Cation exchange chromatography of an aglycosylated IgG1 resulted in two distinct peaks during gradient elution. The early eluting peak contained <1% high molecular weight (HMW) species, while the later peak contained 23% HMW species. Analysis by hydrogen-deuterium exchange and Fourier transform infrared spectroscopy (FTIR) indicated that aggregate formation and generation of the second peak were caused by antibody denaturation on the resin surface. Denaturation and HMW generation was increased by the use of strong cation exchange media, by increasing antibody residence time on the exchanger, or increasing temperature. Denaturation and HMW generation was reduced by increasing pH or ionic strength, by the use of preferentially excluded solutes such as citrate or glycine and controlled entirely by addition of 125 mM arginine to the process buffers. This leads to the hypothesis that denaturation and HMW generation of this antibody can be managed by reducing the strength of binding, by increasing its conformational stability, or by suppressing non-native protein-protein interactions. The glycosylated version of this antibody exhibited less than 2% denatured form, suggesting that glycosylation contributes significantly to the stability of this antibody. These findings may be helpful in managing aggregation in other antibodies, and particularly useful in developing purification processes for aglycosylated antibodies.