title: An alternative assay to hydrophobic interaction chromatography for high-throughput characterization of monoclonal antibodies.
abstract: The effectiveness of therapeutic monoclonal antibodies (mAbs) is governed not only by their bioactivity, but also by their biophysical properties. Assays for rapidly evaluating the biophysical properties of mAbs are valuable for identifying those most likely to exhibit superior properties such as high solubility, low viscosity and slow serum clearance. Analytical hydrophobic interaction chromatography (HIC), which is performed at high salt concentrations to enhance hydrophobic interactions, is an attractive assay for identifying mAbs with low hydrophobicity. However, this assay is low throughput and thus not amenable to processing the large numbers of mAbs that are commonly generated during antibody discovery. Therefore, we investigated whether an alternative, higher throughput, assay could be developed that is based on evaluating antibody self-association at high salt concentrations using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS). Our approach is to coat gold nanoparticles with polyclonal anti-human antibodies, use these conjugates to immobilize human mAbs, and evaluate mAb self-interactions by measuring the plasmon wavelengths of the antibody conjugates as a function of ammonium sulfate concentration. We find that hydrophobic mAbs, as identified by HIC, generally show significant self-association at low to moderate ammonium sulfate concentrations, while hydrophilic mAbs typically show self-association only at high ammonium sulfate concentrations. The correlation between AC-SINS and HIC measurements suggests that our assay, which can evaluate tens to hundreds of mAbs in a parallel manner and requires only small (microgram) amounts of antibody, will enable early identification of mAb candidates with low hydrophobicity and improved biophysical properties.
authors: a Protein Analytics; Adimab ; Lebanon , NH , USA.
authors: Patricia
authors: P
authors: Estep
authors: Isabelle
authors: I
authors: Caffry
authors: Yao
authors: Y
authors: Yu
authors: Tingwan
authors: T
authors: Sun
authors: Yuan
authors: Y
authors: Cao
authors: Heather
authors: H
authors: Lynaugh
authors: Tushar
authors: T
authors: Jain
authors: Maximiliano
authors: M
authors: Vásquez
authors: Peter M
authors: PM
authors: Tessier
authors: Yingda
authors: Y
authors: Xu
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002853:Chromatography, Liquid; D006046:Gold / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D053768:Metal Nanoparticles / Q000737:chemistry*; D012995:Solubility; D013057:Spectrum Analysis
chemical_list: D000911:Antibodies, Monoclonal; D006046:Gold
keywords: HIC; aggregation; developability; high-throughput screening, solubility, viscosity; hydrophobic interaction chromatography; manufacturability; self-association; self-interaction nanoparticle spectroscopy