Capture of fully assembled secretory immunoglobulin A by affinity chromatography with nanobodies as ligands

Abstract

Secretory immunoglobulin A (sIgA) has strongpotential for passive immunization and other therapeutic applications. This complex molecule with a molecular mass of 410 kDa consists of two IgA monomers linked by a connecting chain (J) and a secretory component (SC). Fully assembled sIgA was overexpressed in CHO cells. In order to exploit the therapeutic potential of recombinant sIgA expressed in CHO cells, a platform process for capture of the antibody directly from the clarified culture supernatant using available affinity chromatography material was investigated. The hydrodynamic radius was 10.2 ± 0.9 nm determined by dynamic light scattering and the isoelectric point 5.5 by electrophoretic mobility using the Malvern Zetasizer. Capture Select IgA and Capture Select IgA-CH1 chromatography media with camelid-derived single-domain antibody fragments comprising the 3 CDR domain antibody fragment with a 13 kD or 14 kDa as ligand were used to purify the antibody. sIgA was directly captured from the clarified culture supernatant and a highly purified, full-length, correctly assembled sIgA was obtained. The residence time must be in the range of 5–10 min to achieve a high binding capacity. The effective diffusivity of sIgA was in the range of 10−9 cm2 s−1 and the dynamic binding capacity was in the range of 10 mg/ml packed bed. We conclude that the current version of the resin has a pore size that is too small for this type of molecule to achieve substantial resin utilization and productivity, although affinity ligand-based purification led to high degree of purity suited for an industrial capture process.