Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions

Abstract

In therapeutic antibody preparation, acidic pH conditions are generally used for elution from Protein A affinity column of IgG or for its viral inactivation. Exposing IgG to low pH conditions induces conformational changes, leading to its functional damage or loss, although the mechanisms have not been fully elucidated. In this study using random peptide T7 phage display libraries, we isolated a unique and novel peptide motif that specifically recognized the non-native conformer (acid conformer) of human IgG that was generated by the low pH treatment, but not the native conformer. We examined the generation conditions and biochemical properties of acid conformer using the peptide motif as an affinity ligand. The acid conformer was easily generated at acidic pH (<pH 3.0) and at moderate temperatures (20 -40°C). The conformer was present in a monomeric form functionally maintaining antigen or Fc receptor binding, but showed a tendency to aggregate with a long incubation time at neutral pH (>25°C). The peptides isolated here could contribute to the elucidation of the mechanisms of antibody dysfunction or aggregation during acid exposure as well as storage of human IgG.

The identification of affinity peptide ligands specific to the variable region ofhuman antibodies

“…Krumpe et al reported a T7 phage-displayed peptide library system that showed less bias for amino acids in the displayed peptides and an increased peptide diversity compared to the M13 filamentous phage display (18). Using the T7 phage display system, Sakamoto et al identified a novel IgG-binding peptide ligand which interestingly, recognized acid conformers but not the normal conformation of human IgG (28). The variable domain of the heavy chain (VHH) fragment antibody, derived from the original heavy chain antibodies found in Camelidae, may be the smallest known functional antigen-binding fragment (26).…”

Survey of the 2009 commercial optical biosensor literature

“…Full references for the 316 papers that were given the thumbs up are provided in the reference section. To make this list useful as a tool for identifying examples of the various assay formats, the citations are subgrouped by reviews/protocols dedicated to biosensor studies, kinetic, equilibrium/competition, or concentration analyses, qualitative formats (e.g., yes/no, screening, epitope mapping),…”

Progress in phage display: evolution of the technique and its applications

“…While trying to isolate ligands of human IgG-Fc region from T7-displayed library of random peptides, Sakamoto et al [148] accidentally stumbled upon a series of peptides selectively recognizing a conformation that was induced by acidic treatment of IgG during the isolation process. This non-native conformer, which maintained the ability to bind antigen and the Fc receptor, but was shown to be prone to aggregation, was effectively depleted from IgG preparations using one of the synthetic peptides as affinity matrix in column chromatography.…”