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.