2.3. HFn Endotoxin Removal with EndotrapHD Resins

Endotrap^® HD prepacked columns containing 1 mL of resin (LET0010, Lionex GmbH, Braunschweig Germany) were regenerated and equilibrated according to the manufacturer protocol, through three cycles of centrifugation (3000× g, 2′) in 1× regeneration and equilibration buffers, using 15 mL pyrogen free certified sterile tubes. HFn was suspended in equilibration buffer at 1 mg/mL, and 3 mL of protein suspension were loaded into the column. To elute HFn and maximize ETX-resin contact time, up to 6 centrifugation steps were included to the manufacturer protocol, testing different centrifugation speed (3000× g for 2′ or 100× g for 10′), to facilitate HFn passage and increase protein recovery, as reported in Table 1. Centrifuge was initiated as soon as the resin volume was filled with the protein. The centrifugation time of 2′ was chosen according to the instructions by the manufacturer, while 10′ were selected as it was the shortest time that allowed us to recover all the protein volume loaded in the column at that given speed (100 g).

Protocol description for column mode purification with Endotrap HD 1 mL columns.

At the end of the protocols, protein recovery has been evaluated by A280, and endotoxin levels have been measured by the kinetic turbidimetric LAL test (for details see below).

Endotrap^® HD resin (Lionex GmbH, Braunschweig Germany) was incubated with HFn in batch mode. New resin was regenerated and equilibrated in 15 mL pyrogen-free certified sterile tubes, as previously described for the column mode. HFn was resuspended in equilibration buffer at 1 mg/mL and the amount of ETX was determined by LAL test, as reported above. Since the resin is able to bind 5 × 10⁶ EU/ mL, HFn solution (1 mg/mL) was incubated with an excess of resin one hundred-fold more than ETX contamination. For instance, one milliliter of HFn solution (1 mg/mL) containing 5 × 10⁴ EU has been incubated with 1 mL of Endotrap HD resin. Indeed, an excess of resin was considered necessary in order to be sure that all the ETX content was bound and removed from the sample. The protein-resin mix was incubated on an orbital shaker at 180 rpm at room temperature (RT) for 2 h. At the end of the incubation the whole protein-resin mix was transferred to an empty column to separate the resin (trapped in the column) from the protein, that was collected into pyrogen-free certified sterile tubes. The resins were washed twice with an amount of equilibration buffer equal to 50% of the recovered protein volume. Protein concentration and endotoxin content of all the collected fractions were measured by A280 and LAL test, respectively.