A novel screening method to assess developability of antibody-like molecules

Kohli, Neeraj; Jain, Nidhi; Geddie, Melissa L.; Razlog, Maja; Xu, Lihui; Lugovskoy, Alexey A.

doi:10.1080/19420862.2015.1048410

Cited by 59 publications

(62 citation statements)

References 32 publications

(25 reference statements)

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“…34 The HPSEC analysis of mAb-C showed a retention time of 8.3 minutes and an asymmetric, tailing peak (Fig. S2).…”

Section: High Performance Size Exclusion Chromatographymentioning

confidence: 99%

“…Historically, a number of computational and analytical screening methods have been used to predict aggregation [28][29][30] and high concentration formulation risks. 17,[31][32][33][34][35][36] While these approaches can provide valuable information and help screening of potential lead candidates during discovery and early stage development, they do not necessarily provide insight into the underlying molecular mechanisms involved. With regard to high concentration challenges, the availability of methodologies that can probe native intermolecular interactions at the structural and molecular level can effectively guide corresponding protein engineering and formulation efforts designed to mitigate the development risks.…”

Section: Introductionmentioning

confidence: 99%

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Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Geoghegan¹,

Fleming²,

Damschroder³

et al. 2016

mAbs

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Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible proteinprotein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.

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“…34 The HPSEC analysis of mAb-C showed a retention time of 8.3 minutes and an asymmetric, tailing peak (Fig. S2).…”

Section: High Performance Size Exclusion Chromatographymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Geoghegan¹,

Fleming²,

Damschroder³

et al. 2016

mAbs

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“…The observed correlation to Tm, consistent with literature precedent, supports the notion that this assumption is reasonable. A cluster is observed among SGAC100 (18), SMAC (13), and HIC, which are assays that measure the tendency of antibodies to either self-associate, or associate with a column matrix, under salt-stress conditions. The multiantigen nonspecificity ELISAs closely correlated with BVP assays that use the same ELISA plate readout-indicating a potential common plate-binding aspect to these assays.…”

Section: Significancementioning

confidence: 99%

“…Assays performed in this study included assessment of antibody selfinteraction by AC-SINS (affinity-capture self-interaction nanoparticle spectroscopy) (3, 9, 11) and CSI-BLI (clone self-interaction by biolayer interferometry) (7), a variety of metrics of cross-interaction such as binding PSR (poly-specificity reagent) (8), or BVP (baculovirus particle) (4), CIC (cross-interaction chromatography) (1), and classic ELISA with a panel of commonly used antigens (17). Data on expression titer in HEK cells, melting temperature (Tm) of the Fab, hydrophobic interaction chromatography (HIC) and a related assay, SGAC-SINS (salt-gradient affinity-capture self-interaction nanoparticle spectroscopy) were also collected (18), as were data on standup monolayer adsorption chromatography (SMAC) (13). Finally, the percentage of monomeric species assessed by size-exclusion chromatography (SEC) in the context of an accelerated stability (AS) study completes the panel of assays.…”

Section: Significancementioning

confidence: 99%

“…For monoclonal antibodies, these properties include high-level expression, high solubility, covalent integrity, conformational and colloidal stability, low polyspecificity, and low immunogenicity. The high cost of failing any of these criteria at a late stage in drug development has led to considerable efforts at predicting developability on the basis of sequence motifs and experimentally determined biophysical properties (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

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Biophysical properties of the clinical-stage antibody landscape

Jain

Sun

Durand

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

470

824

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Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotypematched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.monoclonal antibody | developability | biophysical properties | manufacturability | nonspecificity T arget binding is the predominant first concern in development of any drug. However, once a lead molecule attains the desired potency of biological modification, a suite of characteristics termed "developability" assumes critical importance. For monoclonal antibodies, these properties include high-level expression, high solubility, covalent integrity, conformational and colloidal stability, low polyspecificity, and low immunogenicity. The high cost of failing any of these criteria at a late stage in drug development has led to considerable efforts at predicting developability on the basis of sequence motifs and experimentally determined biophysical properties (1-15).In a landmark study of small-molecule drugs over 2,000 molecules with United States Adopted Names (USAN) designations and known to have oral availability were collected and computationally analyzed (16). A simple set of thresholds, encapsulated as the "Lipinski rule of fives," was formulated and has been used by many to prioritize small molecules for entry into clinical development. To date, analogous guiding principles for antibody drugs have not emerged-we therefore endeavor here to do so. By analogy to the Lipinski effort, we first collected the sequences of antibodies that had reached at least phase-2 trials and had USAN or WHO International Nonproprietary Names (INN) designations (137 in total as of the start of this project). As a common basis for comparison of intrinsic variable domain phenotypes we expressed each antibody as the human IgG1 isotype and formulated them in simple Hepes-buffered saline. Each antibody was then subjected to a battery of 12 different biophysical assays in common use for developability assessment.Unexpectedly, for many of the measures the distribution of values was not symmetrically Gaussian, but instead was lon...

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The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

Willis

Kumar

Jain

et al. 2020

Engineering Reports

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The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime, which can illicit immunogenic responses and render mAb manufacturing unfeasible. Consequently, there is a need to identify mAbs with desirable thermodynamic stability, solubility, and lack of self-association. These behaviors are assessed using an array of in silico and in vitro assays, as no single assay can predict aggregation and developability. We have developed an extensional and shear flow device (EFD), which subjects proteins to defined hydrodynamic forces which mimic those experienced in bioprocessing. Here, we utilize the EFD to explore the aggregation propensity of 33 IgG1 mAbs, whose variable domains are derived from clinical antibodies. Using submilligram quantities of material per replicate, wide-ranging EFD-induced aggregation (9-81% protein in pellet) was observed for these mAbs, highlighting the EFD as a sensitive method to assess aggregation propensity. By comparing the EFD-induced aggregation data to those obtained previously from 12 other biophysical assays, we show that the EFD provides distinct information compared with current measures of adverse biophysical behavior. Assessing a candidate's liability to hydrodynamic force thus adds novel insight into the rational selection of developable mAbs that complements other assays. K E Y W O R D S aggregation, developability, extensional flow, monoclonal antibody, shear flowThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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A novel screening method to assess developability of antibody-like molecules

Cited by 59 publications

References 32 publications

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

Biophysical properties of the clinical-stage antibody landscape

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

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