Introduction

A range of multimodal (MM) chromatographic systems have been developed which exhibit enhanced selectivity as compared to single mode interaction resins due to a combination of electrostatic, hydrophobic, aromatic and/or hydrogen bonding interactions within a single ligand (S. C. Burton, Haggarty, & Harding, 1997; Simon Christopher Burton & Harding, 1997; Cramer & Holstein, 2011; Ghose, Hubbard, & Cramer, 2005; Holstein, Parimal, McCallum, & Cramer, 2012; Johansson et al., 2003; Melander, El Rassi, & Horváth, 1989). Further, new prototypes of MM resins that vary in solvent exposure and presentation of functional groups as well as those having differences in linker length and chemistry have been shown to have unique windows of selectivity (Robinson, Snyder, et al., 2018; J. A. Woo et al., 2015; J. Woo, Parimal, Brown, Heden, & Cramer, 2015). The utility of MM chromatography has also been demonstrated for important industrial applications such as the capture of mAbs and related formats (Arakawa, Kita, Sato, & Ejima, 2009; Gagnon et al., 2010; Kaleas, Tripodi, Revelli, Sharma, & Pizarro, 2014; Pezzini et al., 2011) as well as for challenging polishing steps such as removal of product related variants, fragments and aggregates (Chen et al., 2010; Liu, Ma, Winter, & Bayer, 2010; O’Connor et al., 2017).
In order to design novel chromatographic ligands and to facilitate process development for downstream bioprocessing, a deeper understanding of protein binding regions with chromatographic systems is important. Several groups have contributed to the understanding of protein interactions in single mode interaction systems and some representative examples are presented here. Roush et al. identified patches on rat cytochrome b5 for interaction with an anion exchange surface using a series of computational and experimental studies (Roush, Gill, & Willson, 1994). Yao et al. investigated the distribution of charges on the surface of cytochrome c and its variants and explained the retention behavior on cation exchange systems (Yao & Lenhoff, 2004). Sun et al. employed a computational approach to predict preferred binding orientations in both cation exchange (CEX) and hydrophobic interaction systems (Sun, Welsh, & Latour, 2005). Dismer et al. utilized a lysine specific fluorescent dye (Cy5) to identify the binding orientation of lysozyme on a cation exchange surface under various mobile phase conditions (Dismer & Hubbuch, 2007; Dismer, Petzold, & Hubbuch, 2008). Our group has previously employed amino acid specific covalent labeling in combination with mass spectrometry (MS) to examine the binding regions of protein libraries generated from lysozyme and cytochrome C in a cation exchange system (Chung, Evans, et al., 2010; Chung, Holstein, et al., 2010). Kittelmann et al. recently developed QSAR models for predicting binding orientation of antibodies on CEX chromatographic surfaces (Kittelmann, Lang, Ottens, & Hubbuch, 2017).
Studies have also been carried out on understanding preferred binding regions of proteins in more complex systems such as hydrophobic charge induction (HCIC) and MM CEX. Zhang et al. performed a series of molecular dynamics (MD) simulations to study the changes in binding orientation of a β- barrel protein on HCIC surface as a function of salt concentration (Zhang, Zhao, & Sun, 2009). Yu et al. employed coarse-grained simulations to investigate the preferred binding orientation of lysozyme on a HCIC surface at different ligand densities under a range of salt concentrations (Yu, Liu, & Zhou, 2015). Our lab has been actively involved in studying the preferred binding regions of small proteins in MM CEX systems by employing protein libraries (Chung, Hou, et al., 2010), MD simulations (Banerjee, Parimal, & Cramer, 2017; Freed, Garde, & Cramer, 2011; Parimal, Garde, & Cramer, 2015, 2017), Atomic Force Microscopy (AFM) (Srinivasan et al., 2017) and Nuclear Magnetic Resonance (NMR) (Chung, Freed, et al., 2010; Holstein, Chung, et al., 2012; Holstein, Parimal, McCallum, & Cramer, 2013; Srinivasan, Parimal, Lopez, McCallum, & Cramer, 2014).
Ligand-induced chemical shift perturbations (CSP) have been widely used to identify binding regions on proteins (Clarkson & Campbell, 2003). The sensitivity of the chemical shifts to changes in the local environment enables the identification of ligand complexation at an atomic level, while also being able to accurately determine the residue level binding affinity (Hong et al., 2009; Lu, Guo, Jin, & Xiao, 2009; Williamson, 2013). Previous work in our group employed 2D heteronuclear single quantum correlation (HSQC) NMR experiments to identify binding sites of single mode and MM CEX ligands on a small model protein, ubiquitin, and its mutants (Chung, Freed, et al., 2010; Holstein, Chung, et al., 2012). NMR was also employed to evaluate the effects of urea on preferred binding regions in MM systems (Holstein et al., 2013). Further, we have employed MM functionalized gold nanoparticles in solution to identify binding regions on ubiquitin using NMR (Srinivasan et al., 2014). We have also employed NMR in concert with MD simulations to obtain more detailed molecular level information on protein interactions in multimodal systems (Holstein, Chung, et al., 2012).
While the understanding of small protein binding in multimodal chromatographic systems is valuable, it is important to extend these studies to more complex and industrially relevant biomolecules. One such protein is the FC domain, which is highly conserved across a given class of mAbs and which plays an important role in a number of biotherapeutics such as bispecific antibodies and fusion proteins (Shukla, Hubbard, Tressel, Guhan, & Low, 2007). Robinson et al. has examined the domain contributions of mAb binding using a strategic set of chromatographic experiments and shown a shift in domain dominance with pH and resin type (Robinson, Roush, & Cramer, 2018, 2020). Gagnon et al. demonstrated that the binding of the FC domain was driven by calcium affinity interactions in hydroxyapatite (HA) chromatography (Gagnon, Cheung, & Yazaki, 2009). Lin et al. employed molecular simulations to identify preferred binding sites of an HCIC ligand (MEP Hypercel) on single-chain FC fragments (Lin, Tong, Wang, & Yao, 2012). Even though these studies have improved our understanding of FC binding in MM systems, there is clearly a need to develop a deeper understanding of these interactions at the molecular level.
In the present study, we employ a combination of NMR and molecular simulations to examine the interactions of chromatographic ligands with the Fc. Transverse relaxation optimized spectroscopy (15N-TROSY) NMR titration experiments are carried out using perdeuterated 15N-labeled FCwith both single mode and MM CEX ligands in free solution to identify primary binding sites on the protein and to obtain residue specific binding affinities to the ligands. We then examine the NMR results as they compare to the protein surface property maps to develop a deeper understanding of the relation between the binding regions and patches on the protein surface. Finally, we employ MD simulations to provide further insights into the intermolecular interactions and binding mechanisms occurring with the FC in MM systems.