Kta purifier 10

Stocks of Rituximab and Trastuzumab (from therapeutic preparations dialyzed against PBS) were diluted at 1 mg/mL in PBS and subject—or not—to ferrous treatment (500 µM FeSO₄ final concentration) or to NaOCl treatment (200 µM NaOCl final concentration). After 30 min incubation on ice, 100 µL of native and treated Abs (not dialysed) were injected on a Superose 6 Increase column (Cytiva, Marlborough, MA, USA) mounted on an Äkta Purifier 10 (Cytiva) and previously equilibrated with at least 3 column volumes of PBS. Elution was carried out with PBS at 0.5 mL/min and monitored at 280 nm. Raw data were exported from Unicorn software (Cytiva) and elution profiles were overlaid using GraphPad Prism v.9 software.

For NMR measurements, the His-tagged hydrolase was purified from the crude cell extract by immobilized metal-ion affinity chromatography (IMAC) using a 1 mL Ni-NTA agarose (Qiagen) column. The column was washed twice with 10 column volumes PBS/10 mM imidazole and the enzyme was competitively eluted with 2 × 2.5 mL PBS/250 mM imidazole. Further purification of the hydrolase was achieved by size exclusion chromatography (SEC) in PBS using a Superdex75 column (10/300 GL, Cytiva, Marlborough, MA, USA) controlled by an automated liquid chromatography system (ÄKTA Purifier 10, Cytiva). Finally, samples of the fractions were analyzed by SDS-PAGE and Coomassie staining.