Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

This work elucidates the molecular interactions that are responsible for protein unfolding and aggregation on the surface of certain cation exchanger resins. The chromatographic behavior of a monoclonal antibody (mAb) that exhibits a two-peak elution behavior is studied for a range of strong cation exchange resins and with varying load buffer pH and composition. The two-peak elution behavior is very pronounced for the tentacle and polymer-grafted resins, but is essentially absent for hydrophilic macroporous resins. Confocal Laser Scanning Microscopy (CLSM) shows that this behavior is related to the unique kinetics of protein binding in the tentacle-type resin Fractogel. Hydrogen-Deuterium Exchange Mass Spectrometry (HXMS) proves that the two-peak elution behavior is tied to conformational changes that occur when the mAb binds. Circular dichroism suggests that the propensity of different mAbs to form stabilizing intermolecular structures can be related to their chromatographic behaviors. Another mAb exhibiting a three-peak elution behavior on the CEX resin POROS XS was also investigated. Dynamic Light Scattering (DLS) shows that the third peak contains significant levels of aggregates formed in the column that only slowly revert to monomeric species after elution. Circular dichroism and HXMS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HXMS analysis indicate that the aggregates are generated by a destabilized, unfolded intermediate that is slowly formed on the resin. The two early eluting peaks observed regardless of hold time are shown to comprise exclusively monomeric species and form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics. This work has important practical implications in downstream processing for the industrial production of biopharmaceuticals as well as broad scientific value from a biomolecular perspective.