Evaluating Antibody Quality via Simultaneous Size and Charge Measurement with Single Protein Oscillators.

Detecting and characterizing aggregation of therapeutic monoclonal antibodies is critical for quality assessment, as aggregation can reduce therapeutic effectiveness and increase immunogenicity. Established methods characterize aggregation by size, providing only ensemble-averaged measurements and lacking single-molecule resolution. Here, we demonstrate the application of a label-free single protein oscillator method to simultaneously measure the size and charge of therapeutic monoclonal antibodies, including adalimumab, bevacizumab, and panitumumab, and differentiate different aggregation levels of UV-stressed adalimumab. We tethered single proteins to a sensor surface via a flexible polymer, drove them into oscillation by an alternating electric field, and imaged the process through near-field optical imaging. The results align with those from size exclusion high-performance liquid chromatography (SEC-HPLC) and imaged capillary isoelectric focusing (icIEF) methods, which are commonly employed during biopharmaceutical development. Our approach detects a broader range of molecular sizes beyond the upper limits of SEC-HPLC. Additionally, simultaneously measuring size and charge at single protein resolution enables two-dimensional mapping of the charge/size distribution of aggregates. Our results reveal heterogeneous charge distribution among adalimumab aggregates with similar size, indicating structural changes in monomers and conformers that are not readily accessible by existing methods. The method offers an integrated approach for evaluating the size and charge characteristics of therapeutic antibodies at single-molecule resolution, further supporting therapeutic optimization.