Hitrap columns

The stability of the Z_Ca resin under continuous exposure to a CHO cell supernatant was studied over time, to obtain information about a suitable residence time in the column and the maximum number of purification cycles that can be efficiently run before the set of Z_Ca columns should be exchanged in the pilot‐scale run. Repeated purifications were carried out with the same process conditions as for the ICB but applied in small‐scale batch mode to one Z_Ca column, instead of in continuous mode using three capture columns. The different parameters were thus adjusted for the 1 ml HiTrap columns (Cytiva) that were used, previously coupled and packed by Cytiva after in‐house production. The loading time was doubled in each purification within the stability study to subject the resin to the supernatant for an equal amount of time as in the ICB, where each capture column acts as a second column of the loading zone followed by becoming the first column of the loading zone. A flow rate of 0.1 ml/min was used to minimize the volume of supernatant required, and the concentration of mAb was changed every 20 cycles. The CIP of the capture column was excluded from the procedure to limit the number of factors that could impact the resin lifetime. The purifications were continuously repeated for 114 cycles (>10 days).

For the testing of different solutions, MMC was performed with 1 mL HiTrap columns loaded with Capto Core 700 (Cytiva) and an ÄKTA pure 25 FPLC system (Cytiva) to automate the process. Each column was equilibrated with 10 column volumes (CV) of one of eight buffered solutions (see Table 1) followed by the application of 20 CV of bacterial lysate (previously dialyzed in the same solvent, see above) containing recombinant γPFD. The flow-through was collected in 1 mL volume fractions. After each run, the columns were cleaned with 10 CV of distilled water, 10 CV of a cleaning-in-place solvent (1 M sodium hydroxide in 30% (v/v) isopropanol), 10 CV of distilled water, and re-equilibrated with one of the eight buffers being tested (Table 1). All steps were performed at 0.5 mL/min flow rate. In all other experiments, MMC was performed by gravity flow using Poly-Prep Chromatography Columns (Bio-Rad) loaded with up to 2 mL of Capto Core 700 resin. The procedure was essentially the same, except all steps were performed manually.

Production of recombinant ITS01 was performed as previously described (12 (link)). Briefly, HEK293T cells in 175 mm plates were transfected with 60 μg total DNA/plate at 80% confluency with PEIpro transfection reagent (Polyplus). Cells were co-transfected with the AAV ITS01 transfer plasmid and a plasmid encoding furin at a 4:1 ratio. At 16 hrs post-transfection, 10% FBS-DMEM media was replaced with serum-free 293 Freestyle media (Invitrogen). Media was collected after 48 hrs, debris was cleared by centrifugation for 10 min at 1,500 g and filtered using 0.45 μm filter flasks (Millipore Sigma). Proteins were isolated with HiTrap columns (Cytiva) and eluted with IgG Elution Buffer (Thermo Scientific) into 1M Tris-HCl Buffer, pH 9.0 (G Biosciences, St. Louis, MO). Buffer was exchanged with PBS and protein concentrated to 1-2 mg/mL with Amicon Ultra Centrifugation Filters (Millipore Sigma). Heavy and light chains of the antibodies were assessed by Coomassie-stained SDS-PAGE. Antibodies were stored at 4°C before use and frozen at -80C for long term storage.