An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

Estep, Patricia A.; Caffry, Isabelle; Yu, Yao; Sun, Tingwan; Cao, Yuan; Lynaugh, Heather; Jain, Tushar; Vásquez, Maximiliano; Tessier, Peter M.; Xu, Yingda

doi:10.1080/19420862.2015.1016694

Cited by 52 publications

(53 citation statements)

References 23 publications

(37 reference statements)

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“…In earlier publications from our laboratories we had described and emphasized the importance of a subset of high-throughput assays applicable to very large numbers of candidate antibodies (7,8,11,18,19). It is thus both practical and instructive to see how these assays, in particular, delineate the space of antibody drugs actually in clinical development.…”

Section: Discussionmentioning

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%

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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.

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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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Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Biophysical properties of the clinical-stage antibody landscape

Jain

Sun

Durand

et al. 2017

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

Self Cite

470

824

View full text Add to dashboard Cite

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“…Although CIC retention assay gives insight about poly‐reactivity (retention times with polyclonal antibody immobilized column), it is mainly used for solubility determination of antibodies . Among hydrophobic interactions, although SGAC‐SINS assay gave a significant P ‐value of 0.024, other two assays did not give any correlation. Overall, biophysical assay results showed higher self‐interaction and poly‐specificity profiles of phage display derived therapeutic antibodies.…”

Section: Resultsmentioning

confidence: 99%

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

2019

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Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self‐interaction and polyspecificity compared to non‐phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non‐phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation‐prone natures of antibodies which should be avoided in early development phases.

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“…Gp2 aggregation resulted in a shift in the wavelength of maximum absorbance of the gold particles. This general approach has been validated as a high throughput and robust tool to evaluate the aggregation propensity of antibodies (Estep et al, ; Liu et al, ; Sule, Dickinson, Lu, Chow, & Tessier, ; Sule et al, ). The parental Gp2 exhibited substantial aggregation at mildly acidic pH under reduced salt (60 mM) and slight aggregation under higher salt (300 mM) conditions (Figure a).…”

Section: Resultsmentioning

confidence: 99%

“…Protein yield was first quantified by light absorbance at 280 nm (Pace et al, 1995) The self-aggregation propensity of Gp2 was evaluated using affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS) via a modified protocol of the procedures published previously by Tessier et al (Estep et al, 2015;Liu et al, 2014;Sule et al, 2011Sule et al, , 2013. The mixture of His 6 -tagged antibody (320 µg/ml; Proteintech, Chicago, IL) and rabbit IgG (80 µg/ml; Rockland, Limerick, PA) in 78 mM acetate buffer (pH 4.3) was incubated with 20 nm diameter gold particles (4.6×10 11 particles/ml; Ted Pella, Redding, CA) at a volume ratio of 1:9 at room temperature for 2 hours.…”

Section: Protein Verification and Quantificationmentioning

confidence: 99%

Engineering an EGFR‐binding Gp2 domain for increased hydrophilicity

Kruziki

Zudock

et al. 2018

Biotech & Bioengineering

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The Gp2 domain is a 45 amino‐acid scaffold that has been evolved for specific, high‐affinity binding towards multiple targets and was proven useful in molecular imaging and biological antagonism. It was hypothesized that Gp2 may benefit from increased hydrophilicity for improved physiological distribution as well as for physicochemical robustness. We identified seven exposed hydrophobic sites for hydrophilic mutations and experimentally evaluated single mutants, which yielded six mutations that do not substantially hinder expression, binding affinity or specificity (to epidermal growth factor receptor), and thermal stability. Eight combinations of these mutations improved hydrophilicity relative to the parental Gp2 clone as assessed by reverse‐phase high‐performance liquid chromatography (p < 0.05). Secondary structures and refolding abilities of the selected single mutants and all multimutants were unchanged relative to the parental ligand. A variant with five hydrophobic‐to‐hydrophilic mutations was identified with enhanced solubility as well as reasonable binding affinity ( K d = 53–63 nM), recombinant yield (1.3 ± 0.8 mg/L), and thermal stability ( T m = 53 ± 3°C). An alternative variant with a cluster of three leucine‐to‐hydrophilic mutations was identified with increased solubility, nominally increased binding affinity ( K d = 13–28 nM) and reasonable thermal stability ( T m = 54.0 ± 0.6°C) but reduced yield (0.4 ± 0.3 mg/L). In addition, a ≥7°C increase in the midpoint of thermal denaturation was observed in one of the single mutants (T21N). These mutants highlight the physicochemical tradeoffs associated with hydrophobic‐to‐hydrophilic mutation within a small protein, improve the solubility and hydrophilicity of an existent molecular imaging probe, and provide a more hydrophilic starting point for discovery of new Gp2 ligands towards additional targets.

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An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies

Cited by 52 publications

References 23 publications

Biophysical properties of the clinical-stage antibody landscape

Biophysical properties of the clinical-stage antibody landscape

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

Engineering an EGFR‐binding Gp2 domain for increased hydrophilicity

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