Determination of Interactions Between Antibody Biotherapeutics and Copper by Size Exclusion Chromatography (SEC) Coupled with Inductively Coupled Plasma Mass Spectrometry (ICP/MS)

The concept of coupling of size-exclusion HPLC with ICP/MS (SEC-ICP/MS) was first applied in this work as a novel approach in the biotechnology field to assess metal binding to Immunoglobulin G (IgG) mAbs. This method can be used to probe the mechanism and biophysical properties of metal-protein interactions to gain a deeper understanding of the potential impact of metals during drug product manufacturing. Two IgG1s and one IgG2 drugs were investigated. Cu was selected as the metal of interest due to its known ability to form strong complexes with organic molecules and to bind and enhance the degradation of mAbs. Instrument and separation conditions (interface, columns, and mobile phase) were studied for the separation of the protein-metal bound and unbound fractions of a bovine superoxide dismutase (SOD) standard prior to on-line detection of the mAb-metal (Cu) binding. The SEC-ICP/MS method was used to show copper binding by biotherapeutics by comparing the retention times of the protein by SEC and the metal by ICP/MS, to see if they co-elute at the same time. The approach developed offers considerable advantages over methods based on ultrafiltration followed by off-line metal determination in terms of speed, simplicity, precision and selectivity regarding the molecular weight of the complexes involved. In conjunction with other techniques, this method may provide in-depth knowledge of metal-induced mAb degradation mechanisms in biologics process development, be used as an analytical tool for mAb manufacturing in the cell culture process, and be applied during various stages of drug product manufacturing to gain a deeper understanding of the potential impact of metals during biotherapeutic development.