Evaluation of an Fcrn Affinity Chromatographic Method for Igg1-Type Antibodies and Evaluation of Igg Variants

Abstract

Aim:The neonatal Fc-receptor (FcRn) mediates long serum half-life of therapeutic IgG-type antibodies. This interaction represents a critical quality attribute in terms of pharmacokinetics and should be covered by respective quality control strategies. Antibodies are taken up by cells unspecifically and can bind to FcRn in early endosomes preventing lysosomal degradation and allowing release back into circulation. Reflecting this complex cycle in an in vitro assay strategy represents a challenging task. Methodology: We report the qualification of an FcRn affinity chromatographic method and, for the first time, establish a noncriticality window. We analyzed different IgG-type antibodies, subtypes, glycoforms as well as mutants. Conclusion: The FcRn affinity chromatographic method allows the assessment of mAb samples with respect to their pH-dependent FcRn interaction. Furthermore, the method's capabilities and current limitations are discussed.

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

“…Enhanced affinity from longer retention by terminal galactosylation and sialylation observed agrees with previous reports, 30,31 but our results were able to discern for the first time retention differences between partial deglycosylation to core GlcNAc and no effect due to core fucosylation.…”

“…For pH gradient human FcRn affinity chromatography, sialylated and galactosylated glycoforms showed longer retention time compared to terminal agalactosylated species (mAb-FA 2G0 and mAb-A2G0) which is consistent with previous reports. 30,31 Partially deglycosylated glycoforms showed shorter retention time on the column, and the terminally mannosylated mAb-HM showed further shortened retention time compared to the other glycoforms (Figure 9a,b).…”

Radiolabeled IgG antibodies: Impact of various labels on neonatal Fc receptor binding

“…Absorbance at 280 nm was used for detection. Our samples were eluted by a linear pH gradient from pH 6 to 8.8 within 103 minutes using 20 mM 2‐(N‐morpholino)ethanesulfonic acid, 140 mM NaCl at pH 5.5, and 20 mM Tris (hydroxymethyl)aminomethane, 140 mM NaCl at pH 8.8 as eluents …”

“…In this study, we used an affinity chromatography method, which can sensitively detect differences in the pH‐dependent interaction between IgGs and FcRn . The chromatography column contains biotinylated heterodimeric FcRn, immobilized onto sepharose streptavidin beads .…”

“…In this study, we used an affinity chromatography method, which can sensitively detect differences in the pHdependent interaction between IgGs and FcRn. 8,9 The chromatography column contains biotinylated heterodimeric FcRn, immobilized onto sepharose streptavidin beads. 10 The protein elution, driven by a pH gradient, resolves protein species according to their pH-dependent affinity to FcRn in a process that mimics the events, which take place when recycling endosomes emerge at the cell surface.…”