Therapeutic proteins are miracle drugs for millions of patients globally. Unfortunately, with many of these products, a relatively large fraction of patients experiences loss of efficacy due to adverse immunogenicity. Studies for more than 50 years in humans and in animal models have documented that aggregates and particles are important causes of immunogenicity. Also, aggregate and particle levels are critical quality attributes for protein products. Every step in the manufacturing, shipping and use of a protein product can cause aggregation and particle formation, from initial fermentation to final delivery to patients. Developing effective control strategies requires insights into mechanisms for aggregate and particle formation, as well as sensitive and robust methods for characterizing and quantifying soluble aggregates, nanoparticles and microparticles. In bulk solution, aggregates are formed from partially unfolded protein molecules, and aggregation rate is modulated by protein conformational and colloidal stabilities. Often, choosing an optimum pH will help to minimize aggregation by favoring native state stability and increasing charge-charge repulsion between protein molecules.

However, even with an optimized formulation, which minimizes aggregation in bulk solution, interfacial stresses can readily cause protein particle formation. Protein molecules adsorb to interfaces (e.g., air-water, ice-water and water-solid), aggregate and form films. Mechanical rupture of these films releases protein particles into the bulk solution. Such film formation and rupture is common throughout a product’s life history. Fully understanding the causes and control of surface-mediated particle formation requires robust characterization of nano- and microparticles. As an example, this presentation will show results for commercial filling pump operation using a peristaltic pump and three different brands of commercially-used tubing. One highlight from the study was that pumping a protein solution through Pharmed tubing resulted in much lower microparticles levels than pumping through Accusil or Masterflex tubing. But nanoparticle levels in samples pumped through Pharmed tubing were much higher than those observed in samples pumped through the other tubing brands. Furthermore, with an accelerated degradation method of post-pumping agitation, it was seen that the high level of nanoparticles resulted in very high concentrations of microparticles. Effects of formulation pH (affecting conformational and colloidal stabilities) and surfactants were also tested, and results indicated that both nano- and microparticle measurements are crucial for understanding fully the impact of these solution conditions. Finally, light obscuration was also used to measure microparticles, and it was found that this method was not suitable to detect and quantify increases in protein particles resulting from filling pump operation or post-pumping agitation.