2.1. Monoclonal antibody
The mAb used in this study was provided by Manufacturing Technology
Association of Biologics, Japan (MAB). The mAb (pI 8.2) was grown in CHO
cell culture by fed batch method, separated from cells and applied to a
two-column process. The first column was a bind-elute affinity
chromatography step using Protein A resin (KanCapA, Kaneka Corporation,
Tokyo, Japan). The second column consisted of a cation exchange
chromatography step using Cellufine MAX S-h resin (JNC Corporation) and
yielded 40 mg/mL mAb. Purified mAb was diluted with buffer containing 20
mM Tris-Acetate, pH 7 adjusted to 15 mS/cm with NaCl, which is a
relevant column chromatography flow through buffer condition for mAb, to
produce 15 mg/mL mAb solution for use in aggregate spiking experiments
and 11 mg/mL for use as a reference solution for filtration. Purified
mAb solution at 10 mg/mL was used to prepare mAb aggregate solution.