High-Throughput Analysis of Concentration-Dependent Antibody Self-Association

Abstract

Monoclonal antibodies are typically monomeric and nonviscous at low concentrations, yet they display highly variable associative and viscous behavior at elevated concentrations. Although measurements of antibody self-association are critical for understanding this complex behavior, traditional biophysical methods are not capable of characterizing such concentration-dependent self-association in a high-throughput manner. Here we describe a nanoparticle-based method, termed self-interaction nanoparticle spectroscopy, that is capable of rapidly measuring concentration-dependent self-interactions for three human monoclonal antibodies with unique solution behaviors. We demonstrate that gold nanoparticles conjugated with antibodies at low protein concentrations (<40 μg/mL) display self-association behavior (as measured by the interparticle distance-dependent plasmon wavelength) that is well correlated with static light-scattering measurements obtained at three orders of magnitude higher antibody concentrations. Using this methodology, we find that the antibodies display a complex pH-dependent self-association behavior that is strongly influenced by the solution ionic strength. Importantly, we find that a polyclonal human antibody is nonassociative for all solution conditions evaluated in this work, suggesting that antibody self-association is more specific than previously realized. We expect that our findings will guide rational manipulation of antibody phase behavior, and enable studies that elucidate sequence and structural determinants of antibody self-association.

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…can either increase or decrease the extent of RSA of mAbs, depending on the distinct nature of the non-covalent interactions for an individual mAb formulated under specific conditions. 18,19 A variety of analytical tools have been used to characterize the RSA of proteins, and related effects on solution properties, including dynamic light scattering (DLS), 21 composition-gradient multi-angle static light scattering, 20,21 isothermal titration calorimetry, 15 surface plasmon resonance, 22 proton magnetic relaxation dispersion, 23 nuclear magnetic resonance, 24 fluorescence resonance energy transfer, 25 mass spectrometry (MS), 26 self-interaction nanoparticle spectroscopy, 27 size-exclusion chromatography, 28 analytical ultracentrifugation, 29 small angle X-ray scattering, 30 and atomic force microscopy. 31 Most of these measurements provide reliable data only at low-to-moderate protein concentrations (»1-20 mg/mL).…”

Biophysical properties of the clinical-stage antibody landscape

“…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).…”

High throughput cross-interaction measures for human IgG1 antibodies correlate with clearance rates in mice

“…Alternatively, direct observation of mAb selfinteraction is enabled by gold nanoparticles using self-interaction nanoparticle spectroscopy (SINS). [6][7][8] The mAb of interest is loaded directly 9 or through capturing antibodies (Affinity capture SINS, AC-SINS), 10 to the surface of gold nanoparticles. The mAbs prone to self-association cause clustering of nanoparticles, which can be monitored by plasmon wavelength shift.…”