Materials & Methods

Materials

Perdeuterated 15N-labeled IgG1 FCdomain (residues 1-228 corresponding to residues 220-447 in the IgG1) expressed in E. Coli was provided by Merck & Co., Inc. (Kenilworth, NJ, 07033, USA). Disposable Zeba™ spin desalting columns (7K MWCO, 0.5 mL) were purchased from Thermo Fisher Scientific (Waltham, MA, 02451, USA). 1-propane sulfonic acid, 4-amino hippuric acid, sodium acetate, acetic acid, sodium azide and hydrochloric acid were purchased from Sigma-Aldrich (St. Louis, MO, 63103, USA). N-Benzoyl-dl-Methionine was purchased from Bachem Americas Inc. (Torrance, CA, 90505, USA). Capto™ MMC chromatographic resin was purchased from Cytiva (Uppsala, Sweden). Nuvia™ cPrime™ chromatographic resin was donated by Bio-Rad Laboratories (Hercules, CA, 94547, USA). BrukerTMmicrobore NMR sample tubes were purchased from Norell (Morganton, NC, 28680, USA). D2O was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, 01876, USA).

Chromatography Experiments

Chromatographic media were packed into a 5 x 50 mm column and experiments were carried out on an ÄKTA™ Explorer 100 (Cytiva, Upsella, Sweden) controlled by UNICORN™ 5.1 software. The IgG1 FCdomain was buffer-exchanged into buffer A (20 mM sodium acetate, pH 5) using Zeba spin desalting columns and diluted with buffer A to obtain a final protein concentration of 1 mg/mL. The chromatographic columns were equilibrated with buffer A followed by a 100 μL pulse injection of the buffer-exchanged FC solution. Bound protein was eluted with a linear salt gradient from 100% buffer A to 100% buffer B (20 mM sodium acetate, 1 M NaCl, pH 5) over 40 column volumes (CV) at a flowrate of 1 CV/min. The column effluent was monitored at 280 nm. The average of the first moments from duplicate runs was used to determine the elution salt concentration.

Nuclear Magnetic Resonance Experiments

NMR spectra were obtained at 30°C using a Bruker 800-MHz NMR spectrometer equipped with a1H/15N/13C cryoprobe with z-axis gradients. Data were acquired and processed using TopSpin™ 3.2 software and the Sparky 3 software package (Goddard and Kneller, University of California, San Francisco). Confirmation of the backbone assignments was guided using published chemical shift values (BMRB accession number 15514). Each sample had a constant protein concentration of 0.12 mM in the NMR buffer (10 mM sodium acetate, pH 5.0, 10% D2O and 0.02% sodium azide). The ligands employed in the NMR experiments to represent the SP Sepharose, Capto MMC and Nuvia cPrime chromatographic resins are shown in Figure 1. A 1.0 M stock solution of the SP Sepharose ligand (1-propane sulfonic acid) was prepared in the NMR buffer. The Nuvia cPrime and Capto MMC ligands were available as lyophilized powders and were dissolved in 0.12 mM perdeuterated 15N-labeled protein solution prior to titration. Due to the limited solubility of these ligands, the maximum solution concentration of Nuvia cPrime and Capto MMC ligands was 60 and 3.2 mM, respectively. Since the commercial material for Capto MMC is composed of immobilized enantiomers, a similar racemic mixture was used in these solution-based NMR experiments.
Ligand-induced changes in chemical shift were recorded in a series of15N-TROSY experiments at a fixed FCconcentration (0.12 mM) and various FC-ligand ratios (SP Sepharose, 1:1 to 1:800; Capto MMC, 1:0.5 to 1:25; and Nuvia cPrime, 1:0.5 to 1:320). Ligand-induced changes in chemical shift were in fast exchange and at a population- weighted average of the unbound and bound chemical shifts. The changes in combined chemical shift (∆δNH) upon ligand addition were calculated using equation 1.