Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

Abstract

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. 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. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. 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. 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. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

“…The effects in the Ab wt Fab upon FcRn binding were reproducible and were observed in replicate HDX-MS experiments (n ϭ 3). This result provides strong experimental evidence of a conformational origin in line with earlier biochemical studies that indicate a role of IgG Fab in FcRn binding (34,50,51). The sequence of the antibody used in this study (Ab wt ) has standard human constant regions (hIgHG1 and light chain), and a comparison of V region sequences of Ab wt with the closest human germlines showed that the V and J elements has 90% identity with the closest human germline (data not shown).…”

“…Recombinant human FcRn expressed in HEK293F cells was purified as described previously (34) and buffered in 50 mM Na phosphate, 50 mM NaCl, pH 6.5 (Roche Diagnostics). All other chemicals were of the highest grade commercially available.…”

“…FcRn Affinity Chromatography of Antibodies-FcRn affinity chromatography was performed as described previously (34,39) for Ab wt , Ab degly , and ustekinumab using buffer A (20 mM MES, 140 mM NaCl, pH 5.5) and buffer B (20 mM TrisHCl, 140 mM NaCl, pH 8.8) and a linear gradient from 20% to 100% buffer B in 70 min.…”

“…FcRn Surface Plasmon Resonance Analysis-The FcRn binding properties of Ab wt and Ab degly were analyzed via surface plasmon resonance using a T200 instrument as described previously (34). FcRn was immobilized onto a Biacore CM5-Biosensor chip at a concentration of 10 g/ml and 400 RU.…”

Target-independent variable region mediated effects on antibody clearance can be FcRn independent

“…37 No negative control experiment was reported determining retention time on an uncoated column to gauge the contribution of nonspecific interactions with the streptavidin sepharose itself (e.g., as reported here in Fig. 2D).…”