Variables Impacting Silicone Oil Migration and Biologics in Prefilled Syringes.

Prefilled syringes (PFS) as a primary container for parenteral drug products offer significant advantages, such as fast delivery time, ease of self-administration and fewer dosing errors. Despite the benefits that PFS can provide to patients, the silicone oil pre-coated on the glass barrels has shown migration into the drug product, which can impact particle formation and syringe functionality. Health authorities have urged product developers to better understand the susceptibility of drug products to particle formation in PFS due to silicone oil. In the market, there are multiple syringe sources provided by various PFS suppliers. Due to current supply chain shortages and procurement preferences for commercial products, the PFS source may change in the middle of development. Additionally, health authorities require establishing source duality. Therefore, it is crucial to understand how different syringe sources and formulation compositions impact the drug product quality. Here, several design of experiments (DOE) are executed that focus on the risk of silicone oil migration induced by syringe sources, surfactants, protein types, stress, etc. We utilized Resonant Mass Measurement (RMM) and Micro Flow Imaging (MFI) to characterize silicone oil and proteinaceous particle distribution in both micron and submicron size ranges, as well as ICP-MS to quantify silicon content. The protein aggregation and PFS functionality were also monitored in the stability study. The results show that silicone oil migration is impacted more by syringe source, siliconization process and surfactant (type & concentration). The break loose force and extrusion force across all syringe sources increase significantly as protein concentration and storage temperature increase. Protein stability is found to be impacted by its molecular properties and is less impacted by the presence of silicone oil, which is the same inference drawn in other literatures. A detailed evaluation described in this paper enables a thorough and optimal selection of primary container closure and de-risks the impact of silicone oil on drug product stability.

Self-Generated Convective Flows Enhance the Rates of Chemical Reactions.

In chemical solutions, the products of catalytic reactions can occupy different volumes compared to the reactants and thus give rise to local density variations in the fluid. These density variations generate solutal buoyancy forces, which are exerted on the fluid and thus "pump" the fluid to flow. Herein, we examine if the reaction-induced pumping accelerates the chemical reaction by transporting the reactants to the catalyst at a rate faster than passive diffusion. Using both simulations and experiments, we show a significant increase in reaction rate when reaction-generated convective flow is present. In effect, through a feedback loop, catalysts speed up reactions not only by lowering the energy barrier but also by increasing the collision frequency between the reactants and the catalyst.

Enzyme aggregation and fragmentation induced by catalysis relevant species.

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

Silver-Based Self-Powered pH-Sensitive Pump and Sensor.

Nonmechanical nano/microscale pumps that provide precise control over flow rate without the aid of an external power source and that are capable of turning on in response to specific analytes in solution are needed for the next generation of smart micro- and nanoscale devices. Herein, a self-powered chemically driven silver micropump is reported that is based on the two-step catalytic decomposition of hydrogen peroxide, H2O2. The pumping direction and speed can be controlled by modulating the solution pH, and modeling and theory allow for the kinetics of the reaction steps to be connected to the fluid velocity. In addition, by changing the pH dynamically using glucose oxidase (GOx)-catalyzed oxidation of glucose to gluconic acid, the direction of fluid pumping can be altered in situ, allowing for the design of a glucose sensor. This work underscores the versatility of catalytic pumps and their ability to function as sensors.

Powering Motion with Enzymes.

Enzymes are ubiquitous in living systems. Apart from traditional motor proteins, the function of enzymes was assumed to be confined to the promotion of biochemical reactions. Recent work shows that free swimming enzymes, when catalyzing reactions, generate enough mechanical force to cause their own movement, typically observed as substrate-concentration-dependent enhanced diffusion. Preliminary indication is that the impulsive force generated per turnover is comparable to the force produced by motor proteins and is within the range to activate biological adhesion molecules responsible for mechanosensation by cells, making force generation by enzymatic catalysis a novel mechanobiology-relevant event. Furthermore, when exposed to a gradient in substrate concentration, enzymes move up the gradient: an example of chemotaxis at the molecular level. The driving force for molecular chemotaxis appears to be the lowering of chemical potential due to thermodynamically favorable enzyme-substrate interactions and we suggest that chemotaxis promotes enzymatic catalysis by directing the motion of the catalyst and substrates toward each other. Enzymes that are part of a reaction cascade have been shown to assemble through sequential chemotaxis; each enzyme follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Thus, sequential chemotaxis in catalytic cascades allows time-dependent, self-assembly of specific catalyst particles. This is an example of how information can arise from chemical gradients, and it is tempting to suggest that similar mechanisms underlie the organization of living systems. On a practical level, chemotaxis can be used to separate out active catalysts from their less active or inactive counterparts in the presence of their respective substrates and should, therefore, find wide applicability. When attached to bigger particles, enzyme ensembles act as "engines", imparting motility to the particles and moving them directionally in a substrate gradient. The impulsive force generated by enzyme catalysis can also be transmitted to the surrounding fluid and molecular and colloidal tracers, resulting in convective fluid pumping and enhanced tracer diffusion. Enzyme-powered pumps that transport fluid directionally can be fabricated by anchoring enzymes onto a solid support and supplying the substrate. Thus, enzyme pumps constitute a novel platform that combines sensing and microfluidic pumping into a single self-powered microdevice. Taken in its entirety, force generation by active enzymes has potential applications ranging from nanomachinery, nanoscale assembly, cargo transport, drug delivery, micro- and nanofluidics, and chemical/biochemical sensing. We also hypothesize that, in vivo, enzymes may be responsible for the stochastic motion of the cytoplasm, the organization of metabolons and signaling complexes, and the convective transport of fluid in cells. A detailed understanding of how enzymes convert chemical energy to directional mechanical force can lead us to the basic principles of fabrication, development, and monitoring of biological and biomimetic molecular machines.

Salt-bridging effects on short amphiphilic helical structure and introducing sequence-based short beta-turn motifs.

Determining the minimal sequence necessary to induce protein folding is beneficial in understanding the role of protein-protein interactions in biological systems, as their three-dimensional structures often dictate their activity. Proteins are generally comprised of discrete secondary structures, from α-helices to ß-turns and larger ß-sheets, each of which is influenced by its primary structure. Manipulating the sequence of short, moderately helical peptides can help elucidate the influences on folding. We created two new scaffolds based on a modestly helical eight-residue peptide, PT3, we previously published. Using circular dichroism (CD) spectroscopy and changing the possible salt-bridging residues to new combinations of Lys, Arg, Glu, and Asp, we found that our most helical improvements came from the Arg-Glu combination, whereas the Lys-Asp was not significantly different from the Lys-Glu of the parent scaffold, PT3. The marked 310-helical contributions in PT3 were lessened in the Arg-Glu-containing peptide with the beginning of cooperative unfolding seen through a thermal denaturation. However, a unique and unexpected signature was seen for the denaturation of the Lys-Asp peptide which could help elucidate the stages of folding between the 310 and α-helix. In addition, we developed a short six-residue peptide with ß-turn/sheet CD signature, again to help study minimal sequences needed for folding. Overall, the results indicate that improvements made to short peptide scaffolds by fine-tuning the salt-bridging residues can enhance scaffold structure. Likewise, with the results from the new, short ß-turn motif, these can help impact future peptidomimetic designs in creating biologically useful, short, structured ß-sheet-forming peptides.

Head-to-Tail Cyclic Peptide Inhibitors of the Interaction between Human von†Willebrand Factor and Collagen.

The development of peptide-based therapeutics is on the rise, with macrocyclic compounds providing the added stability and drug-like characteristics sought after. Currently, therapies and preventatives for pathogenic thrombosis target platelet interactions at the site of the clot and have many complications. Herein we describe novel cyclic peptides as moderate inhibitors of the protein-protein interaction between von Willebrand factor (vWF) and collagen that initiates blood clot formation. We based our designs on two known disulfide-containing, peptide-based inhibitors of the vWF-collagen interaction. Replacing the disulfide with a head-to-tail cyclization strategy confers remarkable stability to the peptides when treated with a panel of proteases. Our peptides also showed moderate activity in our developed fluorescently linked immunosorbent assay (FLISA), similar to the most active disulfide-containing peptide. These peptides provide a springboard for future advances in exceptionally stable, active cyclic peptides as drugs.