Extending the performance of FcRn and Fc γ RIIIa affinity liquid chromatography for protein biopharmaceuticals

Affinity liquid chromatography using FcRn and Fc gamma RIIIa columns can provide important information on the drug effector functions and the unique PK/PD properties of therapeutic mAbs. In this study, we pro-pose a unique strategy to improve the performance of affinity chromatography by applying pH-gradient programs that incorporate multi-isocratic and negative gradient segments. These alternative gradient pro-grams are known to greatly improve the separation of large solutes that follow a bind-and-elute type retention behavior. First, judicious optimization of the mobile phase compositions was performed to ob-tain a linear pH response. Then, with the developed strategy using multi-isocratic analysis conditions, the FcRn affinity separation selectivity for the analysis of oxidized mAb species was greatly improved. Fur-thermore, the introduction of negative gradient segments after each eluted peak improved the resolution between multiple glycosylated mAb species on the Fc gamma RIIIa column. Therefore, this work provides a new strategy to improve the performance of affinity chromatography with mAb species, and could assist in the development of more accurate binding assays for important critical quality attributes related to FcRn and Fc gamma RIIIa binding.(c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license( http://creativecommons.org/licenses/by/4.0/ )

Automated summary

In this study, a new strategy to improve the performance of affinity chromatography with monoclonal antibody (mAb) species is proposed. The strategy involves the use of pH-gradient programs that incorporate multi-isocratic and negative gradient segments, which greatly improve separation and selectivity.