Chromaster hplc system 600

The Ab purity was verified by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (12% separation gel, 5% stacking gel) and subsequent staining of the gel with Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific GmbH, Schwerte, Germany). Western blotting (wet tank transfer system) was performed on a nitrocellulose membrane (Hybond^TM ECL^TM, VWR International GmbH, Darmstadt, Germany). After blocking, Western blots were analyzed using an alkaline phosphatase conjugated rabbit-Ab against murine immunoglobulin G (IgG) (Dianova, Berlin, Germany).
Size-exclusion (SEC)-HPLC was performed to detect potential Ab aggregates. For this purpose, an Agilent Bio SEC-3 column (3 µm, 150 Å, 7.8 ID × 150 mm; Agilent, Santa Clara, CA, USA) was run at 1.0 mL/min flow and with 0.1 M sodium phosphate buffer (pH 7.0) as mobile phase. HPLC was performed using a Chromaster HPLC-System 600 (VWR Hitachi, Darmstadt, Germany).

For size exclusion high-performance liquid chromatography (SE-HPLC) analysis 10 to 15 µl samples containing the respective TM were applied to a size exclusion column (Agilent Bio SEC-3 (3 µm, 150 Å, 7.8 × 300 mm) from Agilent Technologies, Böblingen, Germany). HPLC chromatography was performed using a Chromaster HPLC-System 600 (VWR-Hitachi, Darmstadt, Germany). As running buffer we used 100 mM Na₂HPO₄ adjusted to pH 7.0 with HCl. Proteins were detected by UV at 280 nm. The same equipment and settings were used to quantitatively separate the Nickel affinity purified anti-GD2 sample into the peaks 1 and 2 but using as running buffer 150 mM Na₂HPO₄ adjusted to pH 7.0 instead (flow rate: 1ml / min).