Biosensing Probe for Quality Control Monitoring of the Structural Integrity of Therapeutic Antibodies

Transcription

1 Supporting Information Biosensing Probe for Quality Control Monitoring of the Structural Integrity of Therapeutic Antibodies Hideki Watanabe, Seiki Yageta, Hiroshi Imamura, and Shinya Honda *,, Biomedical Research Institute, the National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki , Japan Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwanoha, Kashiwa, Chiba , Japan * For correspondence, s.honda@aist.go.jp This file includes -Additional EXPERIMENTAL PROCEDURES -Supplementary Figure S1-S3 S-0

2 EXPERIMENTAL PROCEDURES Surface Plasmon Resonance (SPR) Assays for the Binding of AF.2A1 to Acid-Treated Fab and Fc Fragments The Fab and Fc fragments of humanized monoclonal IgG1 was prepared by using a Fab Preparation Kit (Thermo SCIENTIFIC) followed by affinity chromatography with MabSelect SuRe (GE Healthcare), anion exchange chromatography with diethylaminoethyl Sepharose (GE Healthcare), and size-exclusion chromatography with a Superdex 200 (10/300) column (GE Healthcare). The Fab and Fc fragments were dialyzed for 14 hours against each of the following buffers: 10 mm sodium acetate, 150 mm NaCl, ph4.0; 10 mm sodium phosphate, 150 mm NaCl, ph 3.0; 10 mm sodium phosphate, 150 mm NaCl, ph 2.0. After the acid treatment, the samples were neutralized by dialysis against HBS-T (10 mm HEPES, 150 mm NaCl, 0.05% (w/v) Tween20, ph 7.4). SPR assays were performed with a Biacore T100 (GE Healthcare) RU of AF.2A1 was immobilized on a Series S CM5 sensor chip (GE Healthcare). Another flow cell was blocked with 1 M ethanolamine to provide a reference cell. Binding assays were performed in HBS-T. The obtained sensorgrams were processed using Biacore T100 evaluation software (GE Healthcare). SPR Assays for Cross-Reactivity of AF.2A1 and Non-Native IgG Unrelated proteins for negative control experiments (human serum albumin, E. coli β-galactosidase, and bovine pancreas ribonuclease A) were purchased from Sigma-Aldrich. SPR tests for non-specific binding of AF.2A1 were performed by using an AF.2A1-immobilized sensor chip and the unrelated protein that was prepared at concentrations of 10, 1, 0.1, and 0.01 µg/ml in HBS-T. SPR tests for non-specific binding of non-native IgG were performed by using ph 2.0-treated non-native IgG and the unrelated protein immobilized on a sensor chip. The SPR assays were performed in accordance with the procedures described above. SPR Assays for the Binding of AF.2A1 to Enzyme-Digested Fragments of IgG Humanized IgG1 was digested by using immobilized pepsin (Thermo SCIENTIFIC) in accordance with manufacture s protocol. Briefly, the digestion reaction was performed in 20 mm sodium acetate, ph 4.4 at 37 C for 4 hours. The digest products were purified in HBS-T buffer using a Superdex 200 (10/300) column equipped with an AKTA purifier (GE Healthcare). For SPR assays, fractions of F(ab )2, pfc, and digested products of antibody domains were collected. SPR assays were performed as described above. S-1

3 Figure S1. SPR assays for the binding of AF.2A1 to acid-treated Fab and Fc fragments. (a)-(f) show sensorgrams for the binding of AF.2A1 to the ph 4.0-treated Fc fragment (a), the ph 4.0-treated Fab fragment (b), the ph 3.0-treated Fc fragment (c), the ph 3.0-treated Fab fragment (d), the ph 2.0-treated Fc fragment (e), and the ph 2.0-treated Fab fragment (f), respectively. The sample concentrations were 2000, 1000, 500, 250, 125 nm. S-2

4 Figure S2. Cross-reactivity of AF.2A1 and non-native IgG. (a)-(d) SPR assays for the binding of an AF.2A1-immobilized sensor chip to unrelated proteins and non-native IgG: human serum albumin (a), bovine pancreas ribonuclease A (b), E. coli β-galactosidase (c), and non-native IgG (d). The sample concentrations were 10, 1, 0.1, and 0.01 µg/ml. (e)-(f) SPR assays for the binding of non-native IgG to unrelated proteins immobilized on a sensor chip: human serum albumin (e) and bovine pancreas ribonuclease A (f). The sample concentrations were 100, 10, 1, 0.1, and 0.01 µg/ml. S-3

5 Figure S3. The binding of AF.2A1 to enzyme-digested fragments of IgG. (a) Size-exclusion chromatography for fractionation of a digested sample. The red horizontal bar indicates collected fractions. An arrow shows a molecular weight of a fraction. Fraction 11 (F11) and 17 (F17) correspond to F(ab ) 2 and pfc, respectively. Fraction 22 (F22) and 25 (F25) correspond to digestion products of antibody domains. (b)-(c) SPR sensorgrams for the binding of AF.2A1 to the fractionated fragments: F11 (blue), F17 (red), F22 (orange), and F25 (green). The sample concentration was 25 µg/ml. Whereas F(ab ) 2 showed no binding to AF.2A1, pfc showed a high binding response to AF.2A1 probably because pfc consisted of the CH3 domain that was acid-treated at ph 4.4 in the digestion reaction. F22 and F25 also showed binding responses to AF.2A1, indicating that the digestion products of antibody domains were recognized by AF.2A1. S-4