Ktaexplorer hplc system

Affinities were determined as described previously23 (link). Briefly, serial four-fold dilutions of TNF (0.01–500 ng/ml) were incubated with a fixed concentration of 50–200 pg/ml of Fab-488 (certolizumab, golimumab) or Fab’-488 (adalimumab, infliximab) in PBS-I. Samples (1000 μl) were applied using a thermostatted autosampler (25 °C) to a Superdex 200 HR 10/300 column at 0.5 ml/min (GE Healthcare, Uppsala Sweden), which was connected to an ÄKTAexplorer HPLC system (GE Healthcare, Uppsala Sweden). Elution profiles of Fab-488 were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs on-line fluorescence detector (Shimadzu, Kyoto, Japan). To calculate dissociation constants, fluorescence at 17 ml, corresponding to the peak maxima of free Fab-488, respectively, were plotted against the concentration of TNF (molar concentration of the number of binding sites) and a 1:1 binding model was fitted to the data using Microcal Origin software. Alternatively, TNF-488 (DOL of 0.6) was incubated with serial fourfold dilutions of etanercept and analyzed likewise.

Stability of TNF was assessed by incubating TNF-488 at 0.3–3 ng/mL in PBS-I for various times between 0 and 5 days, alone or in the presence of unlabeled TNF (30 or 300 ng/mL), adalimumab, certolizumab pegol, or etanercept (1 μg/mL) in the dark at 37 °C. Samples were subsequently analyzed applying 500 μl to a Superdex 200 HR 10/300 column (GE Healthcare, Uppsala Sweden) at 0.5 ml/min using a thermostatted autosampler (4 °C), which was connected to an ÄKTAexplorer HPLC system (GE Healthcare, Uppsala Sweden). Elution profiles of TNF-488 were monitored by measuring the fluorescence (excitation/emission 488/525 nm) with a Prominence RF-20Axs on-line fluorescence detector (Shimadzu, Kyoto, Japan).