publications

2025

1. Implementation of Multimodal Anion Exchange Chromatography to Address Product Quality Challenges and Downstream Platform Limitations: A Case Study

   Julie Robinson, Mayank Vats, and Michael Hartmann

   Journal of Chromatography A, Apr 2025

   Abs DOI

   Flow through anion exchange chromatography (AEX) has provided reliable process-and product-related impurities removal as well as viral clearance for monoclonal antibodies (mAbs). The application of AEX to molecules with more complex impurity profiles or non-platform characteristics such as a low pI can become challenging because viral clearance considerations often constrain the AEX step design space. Multimodal anion exchange chromatography (MMAEX) can address the limitations of the platform AEX step while still allowing a “platform-like” manufacturing process. This work presents a case study on polishing step development for a Fc-fusion protein with pI < 6.5, high surface hydrophobicity, and aggregate content of up to 35% in the harvested cell culture fluid. An integrated computational and experimental high throughput screening (HTS) workflow was implemented to rapidly identify the MMAEX resin Capto Adhere at pH 5.5 as a viable alternative to the conventional AEX flow through polishing step. This work presents the impact of pH, conductivity, load, and load impurity level/composition on MMAEX step performance. Step performance was measured based on yield, HMW clearance, residual HCP and DNA clearance, as well as viral clearance. HMW clearance varied based on the starting load HMW level (from 5 -35%) and the highest relative clearance was obtained at a moderate HMW load level. Residual HCP and DNA showed a strong dependance on load HMW level, highlighting the competitive adsorption behavior that can impact resin-protein interactions in complex mixtures. Both residual HCP and DNA were removed to below quantification at pH 5.5. Viral clearance of up to 4 logs of XMULV and MVM was demonstrated at pH 5.5 using process relevant feed streams. The workflow presented here demonstrates how the integration of in silico modeling and high throughput screening (HTS) can streamline process development and enable rapid polishing step optimization. These results also underscore the impact of feed impurity and impurity composition on MMAEX resin performance.

2021

1. Bridging Gaussian Density Fluctuations from Microscopic to Macroscopic Volumes: Applications to Non-Polar Solute Hydration Thermodynamics

   Henry S. Ashbaugh, Mayank Vats, and Shekhar Garde

   Journal of Physical Chemistry B, Jul 2021

   Abs DOI

   The hydration of hydrophobic solutes is intimately related to the spontaneous formation of cavities in water through ambient density fluctuations. Information theory-based modeling and simulations have shown that water density fluctuations in small volumes are approximately Gaussian. For limiting cases of microscopic and macroscopic volumes, water density fluctuations are known exactly and are rigorously related to the density and isothermal compressibility of water. Here, we develop a theory—interpolated gaussian fluctuation theory (IGFT)—that builds an analytical bridge to describe water density fluctuations from microscopic to molecular scales. This theory requires no detailed information about the water structure beyond the effective size of a water molecule and quantities that are readily obtained from water’s equation-of-state—namely, the density and compressibility. Using simulations, we show that IGFT provides a good description of density fluctuations near the mean, that is, it characterizes the variance of occupancy fluctuations over all solute sizes. Moreover, when combined with the information theory, IGFT reproduces the well-known signatures of hydrophobic hydration, such as entropy convergence and solubility minima, for atomic-scale solutes smaller than the crossover length scale beyond which the Gaussian assumption breaks down. We further show that near hydrophobic and hydrophilic self-assembled monolayer surfaces in contact with water, the normalized solvent density fluctuations within observation volumes depend similarly on size as observed in the bulk, suggesting the feasibility of a modified version of IGFT for interfacial systems. Our work highlights the utility of a density fluctuation-based approach toward understanding and quantifying the solvation of non-polar solutes in water and the forces that drive them toward surfaces with different hydrophobicities.

2. Probing IgG1 F\textsubscriptC –Multimodal Nanoparticle Interactions: A Combined Nuclear Magnetic Resonance and Molecular Dynamics Simulations Approach

   Ronak B. Gudhka, Mayank Vats, Camille L. Bilodeau, and 5 more authors

   Langmuir, Oct 2021

   Abs DOI

   In this study, NMR and molecular dynamics simulations were employed to study IgG1 FC binding to multimodal surfaces. Gold nanoparticles functionalized with two multimodal cation-exchange ligands (Capto and Nuvia) were synthesized and employed to carry out solutionphase NMR experiments with the FC. Experiments with perdeuterated 15N-labeled FC and the multimodal surfaces revealed micromolar residuelevel binding affinities as compared to millimolar binding affinities with these ligands in free solution, likely due to cooperativity and avidity effects. The binding of FC with the Capto ligand nanoparticles was concentrated near an aliphatic cluster in the CH2/CH3 interface, which corresponded to a focused hydrophobic region. In contrast, binding with the Nuvia ligand nanoparticles was more diffuse and corresponded to a large contiguous positive electrostatic potential region on the side face of the FC. Results with lower-ligand-density nanoparticles indicated a decrease in binding affinity for both systems. For the Capto ligand system, several aliphatic residues on the FC that were important for binding to the higher-density surface did not interact with the lower-density nanoparticles. In contrast, no significant difference was observed in the interacting residues on the FC to the high- and low-ligand density Nuvia surfaces. The binding affinities of FC to both multimodal-functionalized nanoparticles decreased in the presence of salt due to the screening of multiple weak interactions of polar and positively charged residues. For the Capto ligand nanoparticle system, this resulted in an even more focused hydrophobic binding region in the interface of the CH2 and CH3 domains. Interestingly, for the Nuvia ligand nanoparticles, the presence of salt resulted in a large transition from a diffuse binding region to the same focused binding region determined for Capto nanoparticles at 150 mM salt. Molecular dynamics simulations corroborated the NMR results and provided important insights into the molecular basis of FC binding to these different multimodal systems containing clustered (observed at high-ligand densities) and nonclustered ligand surfaces. This combined biophysical and simulation approach provided significant insights into the interactions of FC with multimodal surfaces and sets the stage for future analyses with even more complex biotherapeutics.

3. Evaluation of Guanidine-Based Multimodal Anion Exchangers for Protein Selectivity and Orthogonality

   Sushmita Koley, Scott H. Altern, Mayank Vats, and 5 more authors

   Journal of Chromatography A, Sep 2021

   Abs DOI

   In this paper, we examined the chromatographic behavior of a new class of guanidine-based multimodal anion exchange resins. The selectivities and protein recoveries on these resins were first evaluated using linear gradient chromatography with a model acidic protein library at pH 5, 6 and 7. While a single-guanidine based resin exhibited significant recovery issues at high ligand density, a bis-guanidine based resin showed high recoveries of all but two of the proteins evaluated in the study. In addition, the bis-guanidine resin showed a more pH dependent selectivity pattern as compared to the low density single-guanidine resin. The salt elution range for the low density single-guanidine and bis-guanidine resins was also observed to vary from 0.250 to 0.621 M and 0.162 to 0.828 M NaCl, respectively. A QSAR model was then developed to predict the elution behavior of these proteins on the guanidine prototypes at multiple pH with overall training and test scores of 0.88 and 0.85, respectively. In addition, molecular dynamics simulations were performed with these ligands immobilized on a self-assembled monolayer (SAM) to characterize their conformational preferences and to gain insight into the molecular basis of their chromatographic behavior. Finally, a recently developed framework was employed to evaluate the separability of the bis-guanidine resin as well as its orthogonality to the multimodal cation exchanger, Nuvia cPrime. This evaluation was carried out using a second model protein library which included both acidic and basic proteins. The results of this analysis indicated that the bis-guanidine prototype exhibited both higher pair separability (0.73) and pair enhancement (0.42) as compared to the less hydrophobic commercial Nuvia aPrime 4A with pair separability and enhancement factors of 0.57 and 0.22, respectively. The enhanced selectivity and orthogonality of this new multimodal anion exchange ligand may offer potential opportunities for bioprocessing applications.