2.7. mAb with mAb aggregate spike experiments
Purified mAb solution (40 mg/mL) with 175 ppm HCP and an aggregate composition of 1.4% dimer and 0.2% trimer or larger species as determined by SEC analysis was used to prepare test solutions. mAb solution (11 mg/mL) in 20 mM Tris-Acetate, pH 7 adjusted to 15 mS/cm with NaCl and with no aggregate spike was prepared as the “reference” solution. mAb solution prepared at 15 mg/mL in the same buffer and spiked with 1.0% mAb aggregate spike was used as the “control” and was processed by one of these methods: column chromatography with an AEX resin (mixed-mode AEX1, mixed-mode AEX2 and normal AEX) or filtration with a nylon filter.
For column chromatography on 2 mL columns operated in flow-through mode, 30 mL of control solution was loaded at a flow rate of 0.25 mL/min (residence time, 8 min), resulting in a mAb loading capacity of 225 mg/mL-resin. Following a 10 mL wash with equilibration buffer (20 mM Tris-Acetate, pH 7 adjusted to 15 mS/cm with NaCl), the entire 40 mL output of 11 mg/mL mAb was collected, and protein recovery by UV absorbance was ≥95% for all runs.
For nylon filter treatment, control solution was filtered through the nylon filter, and the output was adjusted to 11 mg/mL mAb.
For the reference and control solutions (no processing), output from each chromatography columns and permeate from the nylon filter, 40 mL was prefiltered through a 0.2 µm microfilter (Minisart RC 25 mm, Sartorius) to remove potential precipitates and placed in a feed vessel. The feed solution was then filtered with a 0.0003 m2Planova BioEX filter in constant pressure mode at 0.3 MPa. Filtration runs had a throughput of 130 L/m2 or 1500 g/m2, and protein recovery of ≥99% was confirmed by UV absorbance for all runs.
2.8. Plasma IgG with IgG aggregate spike experiments
Commercially available plasma IgG (50 mg/mL) was diluted in 20 mM sodium acetate, 100 mM NaCl, pH 6 (12 mS/cm) to prepare test solutions. Plasma IgG solution at 11 mg/mL was prepared as a reference solution for virus filtration. Plasma IgG solution prepared at 15 mg/mL in the same buffer and spiked with 0.5% IgG aggregate was used as the control and was processed by one of these types of column chromatography: AEX resin (mixed-mode AEX1 and normal AEX) or sulfate ligand resin (modified CEX1 and modified CEX2).
For column chromatography on 1 mL columns operated in flow-through mode, 20 mL of aggregate-spiked plasma IgG solution was loaded at a flow rate of 0.5 mL/min (residence time, 2 min), resulting in a plasma IgG loading capacity of 300 mg/mL-resin. Following a 7 mL wash with equilibration buffer (20 mM sodium acetate, 100 mM NaCl, pH 6; 12 mS/cm), the entire 27 mL output of 11 mg/mL plasma IgG was collected. Protein recovery was ≥90% for mixed-mode AEX1 and modified CEX1 and ≥95% for normal AEX and modified CEX2.
For the reference and control solutions (no processing) and the output from each chromatography column at 11 mg/mL, 27 mL was filtered with a 0.0003 m2 Planova BioEX filter in constant pressure mode at 0.35 MPa. Filtration runs had a throughput of 90 L/m2 or 900 g/m2, and protein recovery of ≥99% was confirmed by UV absorbance for all runs.