High-Throughput Counting and Sizing of Therapeutic Protein Aggregates in the Nanometer Size Range by Nano-Flow Cytometry.

Protein aggregation is one of the greatest challenges in biopharmaceuticals as it could decrease therapeutic efficacy, induce immunogenicity, and reduce shelf life of protein drugs. However, there lacks high-throughput methods than can count and size protein aggregates in the nanometer size range, especially for those smaller than 100 nm. Employing a laboratory-built nano-flow cytometer (nFCM) that enables light scattering detection of single silica nanoparticles as small as 24 nm with sizing resolution and accuracy comparable to those of electron microscopy, here, we report a new benchmark to analyze single protein aggregates as small as 40 nm. With an analysis rate of up to 10,000 particles/min, the size distribution and particle concentration of nanometer protein aggregates can be acquired in 2-3 min. Employing heat-induced aggregation of bovine serum albumin (BSA) at high concentrations as the model system, effects of different categories of excipients, including sugars, polyols, salts, and amino acids on the inhibition of protein aggregation were investigated. Strikingly enough, as high as 10 10 to 10 12 particles/mL of protein aggregates were observed in the size range of 40 to 200 nm for therapeutic proteins of human serum albumin injection, reconstituted recombinant human interieukin-2 solution, and human immunoglobulin injection. nFCM opens a new avenue to count and size nanometer protein aggregates, suggesting its future usability in the quality assessment and formulation promotion of therapeutic proteins.