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.

Figure 1

Immobilization of mAbs on gold nanoparticles. (A) UV-visible absorbance spectra of gold particles (black circles), gold-mAb1 conjugates (gray circles), and gold-mAb2 conjugates (white triangles). (B) DLS analysis of gold particles, antibodies (wild-type and thiolated), and gold-antibody conjugates.

Figure 2

Analysis of variable antibody self-association at near-neutral pH. (A) Plasmon wavelengths (λ_p) of mAb1 and mAb2 conjugates at pH 6.5 and 6, respectively. (B) SLS measurements of WAMW values for mAb1 and mAb2 at 6 mg/mL.

Figure 3

SINS analysis of antibody self-association as a function of pH and ionic strength. Plasmon wavelengths (λ_p) of (A) gold-mAb1 and (B) gold-mAb2 conjugates at pH 4.3 (solid circles) and pH 6.5/6 (open triangles).

Figure 4

SLS analysis of antibody self-association as a function of pH and ionic strength. WAMW measurements for (A) mAb1 and (B) mAb2 (6 mg/mL) at pH 4.3 (solid circles) and pH 6.5 (open triangles).

Figure 5

Correlation between SINS and SLS measurements of antibody self-association. Plasmon wavelengths (λ_p) for gold-antibody conjugates plotted versus WAMW values obtained at antibody concentrations of (A) 6 and (B) 42 mg/mL.

Figure 6

Comparison of self-association behavior for three mAbs. Measurements of (A) WAMW (6 mg/mL) and (B) plasmon wavelengths (λ_p) at pH 6.5 (mAb1 and mAb3) and pH 6 (mAb2). The salt concentration was 150 mM NaCl.

Figure 7

Self-association behavior of a polyclonal antibody as a function of pH and ionic strength. Plasmon wavelengths (λ_p) of polyclonal antibody-gold conjugates at pH 4.3 (black circles), pH 6 (gray squares) and pH 6.5 (white triangles).