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Formulation stability is a critical attribute of any protein-based biopharmaceutical drug due to a protein's inherent tendency to aggregate. Advanced analytical techniques currently used for characterization of protein aggregates are prone to a number of limitations and usually require additional manipulations with the sample, such as dilution, separation, labeling, and use of special cuvettes. In the present work, we compared conventional techniques for the analysis of protein aggregates with a novel approach that employs the water proton transverse relaxation rate R(HO). We explored differences in the sensitivity of conventional techniques, size-exclusion chromatography (SEC), microflow imaging (MFI), and dynamic light scattering (DLS), and water NMR (wNMR) toward the presence of monoclonal antibody aggregates generated by different stresses. We demonstrate that wNMR outperformed SEC, DLS, and MFI in that it was most consistently sensitive to increases in both soluble and insoluble aggregates, including subvisible particles. The simplicity of wNMR, its sensitivity, and possibility of noninvasive measurements are unique advantages that would permit its application for more efficient and higher throughput optimization of protein formulations.

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One of the most significant challenges in developing therapeutic monoclonal antibodies (mAbs) is their unpredictable solubilities and viscosities at the high concentrations required for subcutaneous delivery. This challenge has motivated the development of screening assays that rapidly identify mAb variants with minimal self-association propensities and/or formulation conditions that suppress mAb self-association. Here we report an improved version of self-interaction nanoparticle spectroscopy (SINS)capable of characterizing both repulsive and attractive self-interactions between diverse mAbs. The basis of SINS is that self-interactions between mAbs immobilized on gold nanoparticles increase (repulsion) or decrease (attraction)interparticle distances, which shift the wavelength of maximum absorbance (plasmon wavelength) in opposite directions.We find that the robustness of SINS is improved by varying the amount of immobilized mAb by co-adsorbing a polyclonal antibody. The slopes of the plasmon wavelength shifts as a function of the amount of immobilized mAb (0.01–0.1 mg/mL) are correlated with diffusion interaction parameters measured at two to three orders of magnitude higher antibody concentrations. The ability of SINS to rapidly screen mAb self-association in a microplate format using dilute mAb solutions makes it well suited for use in diverse settings ranging from antibody discovery to formulation.