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.