Orientation of monoclonal antibodies in ion-exchange chromatography: A predictive quantitative structure–activity relationship modeling approach

Kittelmann, Jörg; Lang, K.; Ottens, Marcel; Hubbuch, Jürgen

doi:10.1016/j.chroma.2017.06.047

Cited by 20 publications

(11 citation statements)

References 36 publications

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“…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 CEX system (Chung, Evans, et al, 2010; Chung, Holstein, et al, 2010). Kittelmann et al (2017) recently developed QSAR models for predicting the binding orientation of antibodies on CEX chromatographic surfaces.…”

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

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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: 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

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“…Such consideration of the molecular electrostatic surface potential provides insight into the sensitivity of our CEX method to changes in this region of the molecule and is consistent with the strategy used by other authors to rationalize the preferred binding orientations of proteins exposed to ion exchange resins. 34,[47][48][49][50] A similar argument can be made for HIC (Figure 7), where the HC-CDR3 loop itself would provide a hydrophobic contact surface, while the elution order of less hydrophobic open conformation/more hydrophobic closed conformation can be rationalized by noting the relative deprotonated/protonated state of Asp-100 and Asp-118 in the proposed opened/closed conformations. In the case of RPLC (Figure 3a), the identical retention times of the two Fab peaks results from the aggressively denaturing method conditions.…”

Section: Discussionmentioning

confidence: 59%

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb

Lan

Valente

Ilott

et al. 2020

mAbs

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During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatographymass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementaritydetermining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

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“…While useful for R&D studies, such non-native sequence modifications, however, run the risk of increasing immunogenicity and anti-drug antibodies. Detailed computational analysis and modelling of the antibodies and fragments thereof (Fab, Fc) can reveal insights into their surface charge properties, which can be used to infer binding strength and orientations with chromatography ligands through QSAR modelling approaches in the case of ion exchange [ 11 ] or experimentally using NMR combined with MD simulations with mixed-mode chromatography ligands [ 12 ]. These kinds of studies can provide deeper mechanistic insight into molecular level binding between residues of the mAb and the purification resin functional group.…”

Section: Developability Assessment For Lead Antibody Moleculesmentioning

confidence: 99%

Recent Advances and Future Directions in Downstream Processing of Therapeutic Antibodies

Matte¹

2022

IJMS

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Despite the advent of many new therapies, therapeutic monoclonal antibodies remain a prominent biologics product, with a market value of billions of dollars annually. A variety of downstream processing technological advances have led to a paradigm shift in how therapeutic antibodies are developed and manufactured. A key driver of change has been the increased adoption of single-use technologies for process development and manufacturing. An early-stage developability assessment of potential lead antibodies, using both in silico and high-throughput experimental approaches, is critical to de-risk development and identify molecules amenable to manufacturing. Both statistical and mechanistic modelling approaches are being increasingly applied to downstream process development, allowing for deeper process understanding of chromatographic unit operations. Given the greater adoption of perfusion processes for antibody production, continuous and semi-continuous downstream processes are being increasingly explored as alternatives to batch processes. As part of the Quality by Design (QbD) paradigm, ever more sophisticated process analytical technologies play a key role in understanding antibody product quality in real-time. We should expect that computational prediction and modelling approaches will continue to be advanced and exploited, given the increasing sophistication and robustness of predictive methods compared to the costs, time, and resources required for experimental studies.

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Orientation of monoclonal antibodies in ion-exchange chromatography: A predictive quantitative structure–activity relationship modeling approach

Cited by 20 publications

References 36 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

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb

Recent Advances and Future Directions in Downstream Processing of Therapeutic Antibodies

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Orientation of monoclonal antibodies in ion-exchange chromatography: A predictive quantitative structure–activity relationship modeling approach

Cited by 20 publications

References 36 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

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb

Recent Advances and Future Directions in Downstream Processing of Therapeutic Antibodies

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About

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