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

Kotaro Sakamoto,Yuji Ito,Takaaki Hatanaka,Preeti Brijiral Soni,Toshiyuki Mori,Kazuhisa Sugimura

doi:10.1074/jbc.m807618200

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 degrees 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 degrees 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.

Highlights

Protein A is widely used as an affinity ligand for the purification of human antibodies because it binds to the Fc region of IgG (1)
To identify the target species for our peptides, we examined the binding of our peptides to human IgG treated with a purification process and found that our peptides targeted particular conformational species, which was induced by acid treatment of human IgG
Binding Specificity of Type II Synthetic Peptide to Human IgG—The peptide sequences displayed on the phage clones shared a high consensus [C (T/–) (G) YW (P/A) (R) (E) WGLC; Ն80% of the preserved amino acids are indicated outside parentheses, while those preserved at Ն30% are within] and were named the type II motif

Summary

EXPERIMENTAL PROCEDURES

Materials—Polyclonal human IgG was purchased from ICN/Cappel Biomedicals, Aurora, OH. Detection of Human IgG Interaction with Phages or Synthetic Peptides by ELISA—Each microplate well (Nunc Maxisorp) was coated with human IgG, other control proteins (100 ng/50 ␮l/well), or 100-fold diluted human serum in PBS, and blocked with 0.5% BSA in PBS. Immunoprecipitation of Human IgG by Magnetic Beads— Immobilization of biotinylated IMG1, IMG1E6Q, IMG4, IMG4K6R, and Fc-III peptides (200 pmol) on Dynabeads M-280 SA magnetic beads (0.2 mg) (Dynal Biotech, Oslo, Norway) was performed according to the manufacturer’s protocols. The column was washed with 20 ml PBS, and the bound proteins were eluted with 1 ml of 0.1 M glycine-HCl (pH 2.7), neutralized immediately by adding 100 ␮l of 1 M Tris-HCl buffer (pH 9.1), and stored at 4 °C until use. To estimate the binding activity of the acid conformer to the antigen or Fc␥Rs, the acid-treated MRA (100 ng/␮l, 660 nM) was injected into the IMG4K6R peptide-immobilized flow cell. Human IL-6R (antigen) or human Fc␥R (FcRIa, IIb, or IIIa; 5–20 ng/␮l) was injected to monitor the association and dissociation reactions

RESULTS

Characterization of the Acid

DISCUSSION