Monoclonal antibodies (mAbs) are established as a critical therapeutic modality for a range of diseases. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo.
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