The effect of pH on antibody retention in multimodal cation exchange chromatographic systems

Robinson, Julie; Roush, David J.; Cramer, Steven M.

doi:10.1016/j.chroma.2019.460838

Cited by 21 publications

(19 citation statements)

References 42 publications

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“…In the C H 2–C H 3 interface (Figure 10b), ligands appeared to interact with the histidines through two mechanisms: direct positive (histidine)–negative (carboxylic acid) interactions, and π–π stacking (between the phenyl ring and histidine ring). Previous work in our group has shown that pH can be used as an effective tool for modulating the interactions of Fc with MMC supports (Robinson et al, 2020). The simulation results presented here suggest that this may be due to the strong ligand–histidine interactions, which would be significantly affected by a change in pH from 5 to 7.…”

Section: Resultsmentioning

confidence: 99%

“…One such protein is the F C 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 et al, 2007). Robinson et al (2018, 2020) have 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. Gagnon et al (2009) demonstrated that the binding of the F C domain was driven by calcium affinity interactions in hydroxyapatite(HA) chromatography.…”

Section: Introductionmentioning

confidence: 99%

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Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Gudhka

Bilodeau

McCallum

et al. 2020

Biotech & Bioengineering

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In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15N‐labeled FC domain indicated that while single‐mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the FC. The multimodal ligand‐binding sites on the FC were concentrated in the hinge region and near the interface of the CH2 and CH3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular‐level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand‐FC binding in these preferred regions was shown to be electrostatic interactions and π–π stacking of surface‐exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular‐level understanding of multimodal ligand–FC interactions and sets the stage for future analyses of even more complex biotherapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Gudhka

Bilodeau

McCallum

et al. 2020

Biotech & Bioengineering

Self Cite

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show abstract

“…In the C H 2-C H 3 interface (Figure 10b), ligands appeared to interact with the histidines through two mechanisms: direct positive (histidine)-negative (carboxylic acid) interactions, and pi-pi stacking (between the phenyl ring and histidine ring). Previous work in our group has shown that pH can be used as an effective tool for modulating the interactions of Fc with multimodal chromatographic supports (Robinson et al, 2020). The simulation results presented here suggest that this may be due to the strong ligand-histidine interactions, which would be significantly affected by a change in pH from 5 to 7.…”

Section: Molecular Dynamics Simulationsmentioning

confidence: 66%

Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations

Gudhka

Bilodeau

McCallum

et al. 2020

Preprint

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In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15N-labeled FC domain indicated that while single mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the Fc. The multimodal ligand binding sites on the FC were concentrated in the hinge region and near the interface of the CH2 and CH3 domains. Further, the multimodal binding sites were primarily composed of positively charged, polar and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-FC binding in these preferred regions was shown to be electrostatic interactions and pi-pi stacking of surface exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular level understanding of multimodal ligand-FC interactions and sets the stage for future analyses of even more complex biotherapeutics.

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“…Furthermore, J. Robinson et al (2020) showed that the domain contribution of mAbs in MM chromatography is affected by the mobile phase pH value.…”

Section: Introductionmentioning

confidence: 99%

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

Saleh

Hess

Ahlers-Hesse

et al. 2021

Biotech & Bioengineering

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A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate.While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementaritydetermining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody.Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.

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The effect of pH on antibody retention in multimodal cation exchange chromatographic systems

Cited by 21 publications

References 42 publications

Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

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The effect of pH on antibody retention in multimodal cation exchange chromatographic systems

Cited by 21 publications

References 42 publications

Identification of preferred multimodal ligand‐binding regions on IgG1 FC using nuclear magnetic resonance and molecular dynamics simulations

Identification of preferred multimodal ligand‐binding regions on IgG1 FC using nuclear magnetic resonance and molecular dynamics simulations

Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations

Modeling the impact of amino acid substitution in a monoclonal antibody on cation exchange chromatography

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Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations