Pd miditrap g25 units

The IgGs were purified by protein A chromatography using a 1-mL pre-packed HiTrap™ MabSelect SuRe™ column (GE Healthcare, no. 29-0491-04) on an ÄKTA start system (GE Healthcare) according to the manufacturer’s recommendations. IgG samples were re-buffered by PD MidiTrap G25 units (GE Healthcare, no. 17-0851-01) in 30 mM phosphate buffer, 150 mM NaCl, and pH 6 and adjusted to 2–3 μM. Heat energy uptake was measured by a VP-DSC MicroCal LLC equipment (GE Healthcare). The heat capacity was monitored between 20 and 100 °C applying a scan rate of 1 °C/min. The baseline correction was performed by subtraction of a re-scan of the unfolded protein and the result was fitted with the Origin 7.0 software (OriginLab, Northampton, MA). For fitting, a non-two-state unfolding model was applied.

2F5 and 3D6 antibody fragments were purified by Protein A chromatography from pooled culture supernatants from all clones (n = 12) using a 1 mL pre‐packed MabSelect SuRe resin on an ÄKTA Purifier (both GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Eluates were desalted and buffer‐exchanged to 40 mM phosphate, 150 mM NaCl, pH 6.0 using PD MidiTrap G‐25 units (GE Healthcare) according to the manufacturer's instructions. Finally, sample quantification was performed on a NanoDrop spectrophotometer (Thermo Fisher Scientific) by applying the scFv‐Fc respective theoretical extinction coefficients. Thermal denaturation of the scFv‐Fc samples was monitored using automated differential scanning calorimetry (DSC). All DSC measurements were performed in duplicates on a VP‐DSC MicroCal LLC equipment (GE Healthcare). Protein solutions were sampled from 96‐well plates using the robotic attachment. The protein concentration of all samples was 2–3 µM. The temperature profile was recorded between 20 and 100°C with a scan rate of 1°C/min. The results were evaluated and fitted with the Origin 7.0 software (OriginLab, Northampton, MA). The unfolding states of the antibodies were fit using the non‐two state unfolding model within the software.