Fig. 1: No differences in FcRn affinity using SPR and elution pH using FcRn-affinity chromatography between full-size IgG1 and Fc only.

a Examples of sensorgrams of binding of IgG variants and indicated Fc fragments to FcRn as measured by SPR at pH 7.4 or 6.0. IgG variants were coupled to the SPR-array at a range of concentration (7.5, 15, 30, and 60 nM; shown is 60 nM for anti-biotin IHH and 15 nM for all other variants), with soluble FcRn injected from 0.49 to 1000 nM, represented by the different traces. The numbers indicate averaged mean K_D values for three independent experiments, and the corresponding standard deviations in (nM) were calculated by performing an equilibrium analysis and fitting a 1:1 Langmuir binding model (Supplementary Fig. 2). “n.d.” indicates that affinities were non-determinable. b Mean K_D values of measurements at pH 6.0 as mentioned above shown in histograms. Error bars indicate standard deviations. c Chromatograms obtained from comparing elution volume and elution pH (pH-gradient elution from pH 5.5 to pH 8.8) on a FcRn-affinity column for Fc-WT, Fc-MST-HN, anti-HEL IgG1-WT and anti-HEL IgG1-MST-HN using FcRn-affinity chromatography with relative UV 280 nm signal normalized to maximum response. Numbers indicate elution pH averaged from at least two independent experiments. Elution pH and chromatograms originate from independent experiments. Statistical analysis was performed by a two-sided Wald test, using a linear model with restricted maximum likelihood for the logarithm of the affinities with antibody as a fixed effect and incorporating heterogeneity of the residual variability for the different antibodies. P values were corrected by applying Hommel’s procedure for multiple testing. Data in (a) are representative of three independent SPR runs, the results of which is summarized in (b). Statistical differences are indicated with asterisks: *<0.05, ***<0.001.