A trimeric immunoglobin G-binding domain outperforms recombinant protein G and protein L as a ligand for fragment antigen-binding purification.

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGRR (designated C) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of C for Fab purification. However, we found that monomeric C showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to C to produce C-tri. It was found that C-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by C-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and C-tri-HP column. However, unlike C-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by C-tri-HP column. These results indicate that C-tri outperforms protein G and protein L as a ligand for Fab purification, and C-tri-based affinity chromatography might be developed as a novel platform for Fab purification.