Results and Discussion
Chromatographic retention of the
FCdomain
The linear salt gradient elution behavior of the IgG1 FCdomain was evaluated on a CEX resin (SP Sepharose) and two MM CEX
systems (Capto MMC and Nuvia cPrime) and the resulting elution salt
concentrations are presented in Figure 3. As can be seen, the
FC domain was weakly retained on the CEX resin, eluting
at 0.12 M NaCl in the gradient. In contrast, the FCdomain exhibited significantly stronger retention on the two MM CEX
systems with 0.63 and 0.9 M NaCl required for the elution from the Nuvia
cPrime and Capto MMC resins, respectively. The higher retention on Capto
MMC as compared to Nuvia cPrime could be due to the more solvent exposed
aromatic moiety on the Capto ligand (Robinson, Roush, et al., 2018; J.
Woo et al., 2015). Further, the elevated salt elution concentration seen
in the MM resins as compared to the single mode CEX system is likely due
to more complex interactions occurring with the FCdomain. In order to gain further insight into the interaction sites and
binding mechanisms between these ligands and the FC,
TROSY titration experiments and MD simulations were carried out.
NMR Chemical Shift Perturbation Experiments
The binding of individual chromatographic ligands to the
FC in solution was evaluated using NMR spectroscopy with
a perdeuterated 15N-labeled FC. The
ligands employed to represent the SP Sepharose, Capto MMC and Nuvia
cPrime resins are shown in Figure 1. As described in the Methods
section, ligands were titrated against a fixed concentration of the
labeled FC and the binding was monitored via the
resulting 15N-TROSY spectra. In the NMR spectra, the
amide groups on the 15N-labeled amide backbone of the
FC that were in close proximity to ligands were observed
to experience a change in the local electronic environment resulting in
chemical shift perturbations (CSPs). In the resulting15N-TROSY spectra, a single resonance peak was
observed for both the unbound and bound state of the protein for each
amide group. Thus, the observed peak was a population weighted average
of the two states and is defined by equation 3.