The Impact of the Metal Interface on the Stability and Quality of a Therapeutic Fusion Protein.

Subvisible particle formation, which occurs after the sterile filtration step of the fill/finish process, is a challenge that may occur during the development of biotherapeutics with complex molecular structures. Here, we show that a stainless steel pump head from a rotary piston pump produces more protein aggregates, past the limit of the acceptable quality range for subvisible particle counts, in comparison to a ceramic pump head. The quartz crystal microbalance was used to quantify the primary layer, proteins irreversibly adsorbed at the solid-liquid interface, and the secondary diffuse gel-like layer interacting on top of the primary layer. The results showed that the mass of protein irreversibly adsorbed onto stainless steel sensors is greater than on an aluminum oxide surface (ceramic pump mimic). This suggests that the amount of adsorbed protein plays a role in surface-induced protein aggregation at the solid-liquid interface.

Effects of nanoparticle size and charge on interactions with self-assembled collagen.

HYPOTHESIS: Insights into bone formation have suggested that the critical first step in the biomineralization process is the integration of small (nanometer dimension) mineral clusters into collagen fibers. Not only is such behavior of interest for understanding biomineralization but also should be important to nanotoxicology because collagen is a major component of structural tissues in the human body and accounts for more than 25% of the whole body protein content. Here, utilizing the current insights from biomineralization, we hypothesize that the binding affinity of nanoparticles to self-assembled collagen fibers is size and surface charge dependent. EXPERIMENTS: We developed a self-assembled collagen substrate compatible with Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), which is very sensitive to mechanical changes of the substrate as a consequence of nanoparticle binding. QCM-D experiments were conducted with both positively and negatively charged gold nanoparticles between 2 and 10 nm in size. Complementary ex situ imaging Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were used to confirm the QCM-D results. FINDINGS: We find that both positively and negatively charged nanoparticles of all sizes exhibited binding affinity for self-assembled collagen fibers. Furthermore, the smallest particles (2 nm) mechanically integrated with collagen fibers.