Despite its ubiquitous use, the mechanistic details of PrA chromatography have not yet been characterized fully, and some factors may help in achieving an optimal balance between yield and purity. The investigation here was prompted by a significant reduction in mAb yield during a high-pH wash to improve clearance of host-cell proteins, which raises questions about similar behavior that may arise for other Fc-based structures, such as Fc fusion proteins. The loss of yield was found to result from an appreciable dependence of both the static and dynamic capacities on pH, including a significant decrease at high pH. We investigated further by seeking to distinguish the effects of pH on mAb affinity to the PrA ligand from effects that altered the capacity directly. We prepared Fc fragments by papain digestion and measured the dependence of their static capacity on pH. Similarly to the intact mAb, the capacity of the Fc fragment also drops at high pH, indicating that the high pH yield loss is a property intrinsic to the Fc fragment, with little contribution from the Fab fragments. Next, we fitted the general rate model to the breakthrough and elution data generated using the intact mAb and the Fc fragments to identify and distinguish between changes in mAb-PrA (or Fc-PrA) interactions and mAb-mAb (or Fc-Fc) interactions. The model parameters for the intact mAb and its Fc fragment will be compared, and possible mechanistic interpretations will be discussed.

Determinants of capacity of Fc-based antibody structures in protein A chromatography

ACS Fall 2021

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