Adalimumab

We purchased the therapeutic mAb immunoglobulin (IgG) gamma 1 drugs adalimumab and rituximab from Eisai Co., Ltd. (Tokyo, Japan) and Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. In addition, we purchased Tris-HCl buffer (pH 8.0), 10% trifluoroacetic acid, 0.1% formic acid, acetonitrile 0.1% formic acid and 7 K Dialysis Casettes from Thermo Fisher Scientific (San Jose, CA, USA). We obtained 8 M guanidine hydrochloride from Sigma-Aldrich (St. Louis, MO), dithiothreitol, iodoacetamide, sodium acetate, 2-morpholinoethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid and deuterium oxide from FUJIFILM Wako Pure Chemical Co., Ltd. (Tokyo, Japan), and MicroSpin G-25 columns from GE Healthcare (Chicago, IL). We purchased trypsin from Promega Co. (Madison, WI); angiotensin II from Peptide Institute, Inc. (Osaka, Japan); and ¹⁸O-water, acetonitrile (LC-MS grade), and ordinary water (LC-MS grade) from Merck (Darmstadt, HE).

Ozoralizumab was generated at Ablynx by a previously described method^{21 (link)}. Adalimumab was purchased from Eisai Co., Ltd (Tokyo, Japan). Ozoralizumab and Adalimumab were de-salted on a PD-10 column (GE Healthcare, 17-0851-01, IL, USA) to remove amine-containing reagents, and labeled with Alexa Fluor 680 (SAIVI Rapid Antibody Labeling Kit, Invitrogen, S30045, MA, USA), in accordance with the manufacturer's instructions.

Ozoralizumab was generated at Wyeth Research in the manner previously described (37 (link)). Adalimumab was purchased from Eisai Co., Ltd (Japan). Recombinant human TNFα alpha was purchased from R&D Systems (United States). All samples were diluted with PBS (Thermo Fisher Scientific, United States) or saline (Otsuka, Japan). A 20.3 μM solution of ozoralizumab or Adalimumab was mixed with 2.3 μM, 6.6 or 6.8 μM, 20.3 μM, or 60.8 μM solution of TNFα trimer and incubated overnight at 4°C. Mixtures of the respective antibodies and TNF were added to mouse neutrophils to a final antibody concentration of 33-34 nM. Reactive Oxygen Species (ROS) were measured by using L-012 sodium salt (8-Amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d] pyridazine sodium salt) (38 (link)). Neutrophils (5 x 105 cells/well) in CL medium containing 50 μM L-012 (FUJIFILM Wako Pure Chemical, Japan) were transferred to white microplates (Greiner Bio-One, Austria), and ROS-dependent chemiluminescence was measured with an ARVO microplate reader (PerkinElmer, United States). ROS released by the neutrophils were monitored for 1 min at 37°C.