Standards for the Optical Detection of Protein Particles

Particles in protein therapeutics, composed of aggregated protein monomers, may cause an immunogenic response in patients. Consequently, industry and the FDA desire more accurate methods for counting and characterizing particles. Unlike many manufacturing impurities, protein particles have a refractive index very close to that of the matrix solution. In this paper, we describe some of the measurement challenges for the common optical methods of brightfield microscopy and light obscuration. The particulate refractive index is a key unknown parameter in the effort to standardize counting of protein particles. At NIST, we are developing two types of reference materials that mimic the properties of protein particles to support the validation of these two optical methods. We have developed a polymer-based protein surrogate based on the partially fluorinated polymer ethylene tetrafluoroethylene (ETFE). Mechanical abrasion of the ETFE followed by size filtration produces a polydisperse suspension of very rugged particles with a morphology quite similar to protein particles. In another approach, we have created artificial polymer particles as small as 3 μm x 4 μm x 40 μm in large quantities using photolithographic methods developed for the semiconductor industry. Future work will focus on reducing the optical contrast of these particles in order to better mimic typical protein particles. These reference materials will provide a stringent test of the sensitivity and morphological measurements of optical particle-counting and particle-characterization instruments. Product of the U.S. government; not subject to U. S. copyright