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.

Keywords: HIC; aggregation; developability; high-throughput screening, solubility, viscosity; hydrophobic interaction chromatography; manufacturability; self-association; self-interaction nanoparticle spectroscopy.

Figure 1.

(A) Representative chromatograms for 5 mAbs with variable regions identical to clinical-stage molecules on a hydrophobic interaction chromatography (HIC) column. (B) HIC retention times for 32 IgGs with variable region sequences identical to those of the indicated clinical-stage mAbs. Four mAbs (*) did not elute.

Figure 2.

(A) Plasmon wavelengths for 32 IgGs with variable region sequences identical to the indicated clinical-stage human mAbs that were obtained using AC-SINS in PBS (pH 7.4). (B) Comparison between AC-SINS and HIC results.

Figure 3.

Evaluation of AC-SINS measurements as a function of ammonium sulfate concentration. The control is for gold particles coated only with polyclonal (anti-human Fc) capture antibody and the Fc fragment is from human IgG1 polyclonal antibody.

Figure 4.

AC-SINS reveals unique sensitivities of human mAbs to ammonium sulfate. Examples of mAbs that display significant plasmon shifts at (A) high, (B) intermediate and (C) low ammonium sulfate concentrations, as well as (D) mAbs with more complex and difficult-to-classify behavior.

Figure 5.

Comparison of AC-SINS and HIC results for 32 clinical stage mAbs. HIC retention times are plotted against (A) average plasmon wavelengths for ammonium sulfate concentrations of 300–1000 mM, (B) average plasmon wavelengths for ammonium sulfate concentrations of 700–900 mM ammonium sulfate, and (C) plasmon wavelengths at 800 mM ammonium sulfate. In B and C, the cutoff values are 550 nm for AC-SINS and 24 min for HIC.