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.