Quantifying orthogonality and separability: A method for optimizing resin selection and design

Bilodeau, Camille L.; Vecchiarello, Nicholas; Altern, Scott Howard; Cramer, Steven M.

doi:10.1016/j.chroma.2020.461429

Cited by 8 publications

(2 citation statements)

References 25 publications

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“…Many studies have attempted to simplify this problem by assuming that weak multimodal interactions are additive in nature. , While this assumption can prove useful in specific circumstances, many studies have shown that even most commonly used multimodal ligands display nonadditive behavior when interacting with therapeutically relevant proteins . In fact, Woo et al , and Robinson et al , illustrated that multimodal resins with different chemistries and architectures lead to changes in selectivity and Bilodeau et al showed that multimodal resins are generally orthogonal to ion exchange resins. Thus, this nonadditive behavior is not an exception but rather the norm …”

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confidence: 99%

Behavior of Water Near Multimodal Chromatography Ligands and Its Consequences for Modulating Protein–Ligand Interactions

et al. 2021

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Multimodal chromatography is a powerful approach for purifying proteins that uses ligands containing multiple modes of interaction. Recent studies have shown that selectivity in multimodal chromatographic separations is a function of the ligand structure and geometry. Here, we performed molecular dynamics simulations to explore how the ligand structure and geometry affect ligand−water interactions and how these differences in solution affect the nature of protein−ligand interactions. Our investigation focused on three chromatography ligands: Capto MMC, Nuvia cPrime, and Prototype 4, a structural variant of Nuvia cPrime. First, the solvation characteristics of each ligand were quantified via three metrics: average water density, fluctuations, and residence time. We then explored how solvation was perturbed when the ligand was bound to the protein surface and found that the probability of the phenyl ring dewetting followed the order: Capto MMC > Prototype 4 > Nuvia cPrime. To explore how these differences in dewetting affect protein−ligand interactions, we calculated the probability of each ligand binding to different types of residues on the protein surface and found that the probability of binding to a hydrophobic residue followed the same order as the dewetting behavior. This study illustrates the role that wetting and dewetting play in modulating protein−ligand interactions.

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Section: Introductionmentioning

confidence: 99%

Behavior of Water Near Multimodal Chromatography Ligands and Its Consequences for Modulating Protein–Ligand Interactions

et al. 2021

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“…Although useful as a process development tool, the sets of orthogonal multimodal resins identified in this prior work were specific to the products being purified. To identify sets of multimodal resins which exhibit orthogonality for a broad range of products, our group has recently developed a graph-based mathematical framework for quantifying orthogonality and product separability in a product-agnostic manner (Bilodeau, Vecchiarello, et al, 2020). One limitation of this approach is that this product-agnostic definition of orthogonality requires retention time tracking of a large number of proteins (e.g.…”

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A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid

Vecchiarello

Timmick

Cramer

2021

Biotech & Bioengineering

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This paper presents a straightforward approach for measuring and quantifying orthogonality directly in complex cell culture fluids (CCFs) without the requirement for tracking the retention behaviors of large sets of proteins. Nullproducing CCFs were fractionated using linear salt gradients at constant pH on a set of multimodal resins. Fractions were then analyzed by ultraperformancereversed phase liquid chromatography and the resulting chromatograms provided host cell protein (HCP) "fingerprints." Using these fingerprints, an inner product vector-based approach was employed to quantify the degree of orthogonality between pairs of resins and operating conditions for these large HCP protein sets. To compare resin orthogonality behavior in different expression systems, the Chinese hamster ovary and Pichia pastoris null-producing CCFs were examined. Orthogonality in multimodal systems was found to strongly depend on the expression system and the HCPs being screened. We also identified several unexpected pairs of multimodal resins within the same family that exhibited significant orthogonality. Furthermore, "self-orthogonality" was evaluated between resins operated at different pHs, and important operating regimes were identified for maximizing orthogonal selectivities. The framework developed in this paper for calculating orthogonality without the need for labor-intensive HCP tracking has important implications for efficient process development and resin/ operating condition selection for both monoclonal antibody (mAb) polishing steps and non-mAb processes. In addition, this study provides a tool to unlock the untapped potential of multimodal resins by aiding in their rational selection and incorporation. Finally, the orthogonality framework here can facilitate the development of sets of next-generation multimodal resins specifically designed to provide highly orthogonal and efficient separations tailored for different expression systems.

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Engineered selective biotoxin‐binding hydrogels for toxin sequestration

Morris,

Yang,

Mai

et al. 2024

Journal of Polymer Science

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The development of synthetic selective membranes that separate materials of similar sizes, charges, and/or polarities remains a difficult challenge, and looking towards biology provides inspiration for new designs. In this work, a series of cholera toxin binding peptides (CTBPs) are identified, spanning a range of binding inhibitions, and integrated into chemically cross‐linked cholera toxin binding gels (CTBGs) via thiol‐Michael polycondensation reactions. All gels demonstrate rheological profiles consistent with elastic solids. The CTBGs are probed via small‐angle neutron scattering and exhibit a correlation length, ξ, smaller than most proteins (1.3–2.5 nm). Thus, an effective entropic mesh is formed to block non‐targeted proteins. However, the CTBGs have a dynamic mesh size, Ξ, that is larger than cholera toxin (CT) to allow the transport of target proteins. The CTBGs with the highest binding inhibitions both show high selectivity and permeation of CT, rejecting all other tested proteins. In total, two new highly selective CTBGs are synthesized and validated for use in cholera toxin remediation. Together, this platform demonstrates the wide applicability of selectively‐diffusive materials for difficult separations.

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Quantifying orthogonality and separability: A method for optimizing resin selection and design

Cited by 8 publications

References 25 publications

Behavior of Water Near Multimodal Chromatography Ligands and Its Consequences for Modulating Protein–Ligand Interactions

Behavior of Water Near Multimodal Chromatography Ligands and Its Consequences for Modulating Protein–Ligand Interactions

A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid

Engineered selective biotoxin‐binding hydrogels for toxin sequestration

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