Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Kinetic analysis of immobilized human FcRn and a human IgG1 (mAb1) in solution at pH 6.0 utilizing a medium FcRn density on a switchSENSE biosensor chip. (a) shows the schematic assay configuration allowing affinity and avidity to occur simultaneously. mAb1 was injected in five different concentrations as two-fold dilution series with a highest concentration of 300 nM for hIgg1 Fc WT (b) and 60 nM for hIgg1 Fc YTE (c). Each plot (b,c) shows the measured raw data (gray) and the global fit analysis as solid lines (blue fading). The sensorgram display a monophasic association phase and biphasic dissociation phase reflecting affinity and avidity binding mode. The determined kinetic parameters are described in . Illustration a is created with BioRender.com. Table 2. Summary of the affinity and avidity measurements of immobilized human FcRn and an mAb1 Fc variants in solution using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error. Sample Affinity Avidity k ON (x10 6 M −1 s −1 ) k OFF,AFFINITY (x10 −2 s −1 ) K D,AFFINITY (nM) k OFF,AVIDITY (x10 −2 s −1 ) K D,AVIDITY (nM) hIgG1 Fc WT 7.53 ± 0.38 34.2 ± 1.50 45.4 ± 3.0 4.70 ± 0.30 6.24 ± 0.51 hIgG1 Fc YTE 8.42 ± 0.15 2.60 ± 0.06 3.09 ± 0.09 0.25 ± 0.01 0.29 ± 0.01

Article Snippet: Real-time binding kinetic experiments were performed with a switchSENSE DRX2 instrument (Dynamic Biosensors, Germany) on a switchSENSE chip (MPC-96-2-G1R1-S, Dynamic Biosensors, Germany) using the fluorescence proximity sensing (FPS) mode.

Techniques: Injection, Concentration Assay, Binding Assay

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Summary of the affinity and avidity measurements of immobilized human FcRn and an mAb1 Fc variants in solution using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in Figure 2 . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error.

Article Snippet: Real-time binding kinetic experiments were performed with a switchSENSE DRX2 instrument (Dynamic Biosensors, Germany) on a switchSENSE chip (MPC-96-2-G1R1-S, Dynamic Biosensors, Germany) using the fluorescence proximity sensing (FPS) mode.

Techniques:

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Kinetic analysis of human FcRn immobilized and a human IgG1 (mAb1) Fc YTE mutant in solution at nine different pH values on switchSENSE biosensor chip. mAb1 Fc YTE was injected in five different concentration as two-fold dilution series with a highest concentration of (a) 60 nM at pH 5.8, (b) 60 nM at pH 6.0, (c) 100 nM at pH 6.2, (d) 150 nM at pH 6.4, (e) 200 nM at pH 6.6, (f) 300 nM at pH 6.8, (g) 400 nM at pH 7.0, (h) 800 nM at pH 7.2 and (i) 800 nM at pH 7.4. Note that the x axis for each sensorgram (a – i) has a different time scale. Each plot (a – i) shows the measured raw data (grey) and the global fit analysis as solid lines (blue fading). For (a – h) the dissociation phase is biphasic characterized by two different dissociation rate constants reflecting the affinity (1:1) and the avidity (2:1) binding mode. The interaction displays a biphasic dissociation curve reflecting affinity and avidity binding where one or two hFcrn are engaged with one mAb1 Fc YTE. The dissociation of mAb1 Fc YTE and hFcrn at pH 7.4 (I) is described by a monophasic fit model reflecting the affinity binding mode (1:1) where one hFcrn is engaged with one mAb1 Fc YTE. Panel (J) shows the applied, exemplary biphasic fit model for FcRn with mAb1 Fc YTE mutant injecting 300 nM at pH 6.0. The adequacy of the fit model is confirmed by the minimal residuals, indicating no significant deviation. The association phase occurred to be monophasic while the dissociation phase is biphasic. The overall dissociation curve is superposition of two exponential time-courses, namely the affinity binding mode (fast dissociation) and the avidity binding mode (slow dissociation). The measured data is shown in blue and the fit in black solid lines, whereas the two deconvoluted exponential time-courses are shown in grey as dashed lines. The contribution of fast and slow dissociation to the overall signal change is shown as Amplitude A fast or A slow . The determined kinetic parameters are described in .

Article Snippet: Real-time binding kinetic experiments were performed with a switchSENSE DRX2 instrument (Dynamic Biosensors, Germany) on a switchSENSE chip (MPC-96-2-G1R1-S, Dynamic Biosensors, Germany) using the fluorescence proximity sensing (FPS) mode.

Techniques: Mutagenesis, Injection, Concentration Assay, Binding Assay

Journal: mAbs

Article Title: Insight into the avidity–affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn

doi: 10.1080/19420862.2024.2361585

Figure Lengend Snippet: Summary of the affinity and avidity measurements of immobilized human FcRn and mAb1 Fc YTE mutant as solute using a switchSENSE biosensor chip having a medium ligand density. The kinetic rate parameters are determined from analyzing the sensorgrams shown in Figure 3 . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error.

Article Snippet: Real-time binding kinetic experiments were performed with a switchSENSE DRX2 instrument (Dynamic Biosensors, Germany) on a switchSENSE chip (MPC-96-2-G1R1-S, Dynamic Biosensors, Germany) using the fluorescence proximity sensing (FPS) mode.

Techniques: Mutagenesis