Subvisible Particles in Solutions of Remicade in Intravenous Saline Activate Immune System Pathways in In Vitro Human Cell Systems.

Among patients that receive Remicade® therapy, more than 20% have adverse infusion related reactions and approximately 50% have immunogenic responses.1-3 Upon characterization of initial Remicade®-IV solution we observed a high concentration of subvisible particles that could inadvertently be delivered to patients. This solution was processed through the IV infusion system, mimicking the typical clinical administration setup - either with or without an in-line filter connected to the IV line. The samples generated thereafter were tested using various in vitro assays for activation of the innate immune system via cytokine release in whole blood and in peripheral blood mononuclear cell (PBMC) cultures, and activation of the Toll like receptors (TLRs). Activation of the adaptive immune system was evaluated by monitoring upregulation of surface receptors on dendritic cells (DCs) and CD4+ T cell proliferation in response to IV solution of Remicade®. Our results indicate that subvisible particles in Remicade®-saline solution have a significant role in activation of the immune system but there are extrinsic factors potentially contributed by the in-line filters or other process parameters that also contribute to immune system activation.

Protein Nanoparticles Promote Microparticle Formation in Intravenous Immunoglobulin Solutions During Freeze-Thawing and Agitation Stresses.

In this study, we investigated the potential roles of nanoparticles (<100 nm) and submicron (100-1000 nm) particles in the formation of microparticles (>1000 nm) in protein formulations under some pharmaceutically relevant stress conditions. Exposure of intravenous immunoglobulin solutions to the interface-associated stresses of freeze-thawing or agitation resulted in relatively large increases in microparticle concentrations, which depended directly on the levels of pre-existing nano- and submicron particles. Thus, agglomeration of nanoparticles and submicron particles appears to play a role in microparticle formation under these stresses. In contrast, increases in microparticle concentrations during quiescent incubation at elevated temperatures were independent of the initial nano- and submicron particle concentrations in solution.

Microparticles and Nanoparticles Delivered in Intravenous Saline and in an Intravenous Solution of a Therapeutic Antibody Product.

Intravenous (IV) infusion is used for administration of a large proportion of biologic therapeutics, including most monoclonal antibody products. In this study, we determined the subvisible particle levels in IV solutions and after the solutions were processed with an IV administration setup that mimicked the typical clinical method of administration. IV saline in bags manufactured by both Hospira and Baxter contained 1600-8000 microparticles/mL and 4-73 × 106 nanoparticles/mL in solution. When IV immunoglobulin was diluted into the IV saline, 3700-23,000 microparticles/mL and 18-240 × 106 nanoparticles/mL were detected. During processing of the solution through the IV system, in-line filters removed most microparticles. However, there were still 1-21 × 106 nanoparticles/mL in IV saline and 7-83 × 106 nanoparticles/mL in IV immunoglobulin diluted in saline. Finally, in samples processed through in-line filters, we found relatively large microparticles (20-60 µm) that were composed of protein or polycarbonate. These particles resulted from shedding of polycarbonate and sloughing off of protein films downstream from the filter membrane. Overall, the results document that even with in-line filters in place, high levels of subvisible particles are delivered to patients and there is a need for improved, more effective filters and IV solutions with lower particle levels.

Transmission electron microscopy as an orthogonal method to characterize protein aggregates.

Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety because of potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size-exclusion chromatography, dynamic light scattering, microflow imaging, and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three-dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semiquantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis.

Pharmacokinetics of DS-96, an alkylpolyamine lipopolysaccharide sequestrant, in rodents.

The pharmacokinetics of DS-96, an N-alkylhomospermine analog designed to sequester bacterial lipopolysaccharides, has been determined in rodent species. The elimination half-life in mice and rats are about 400 and 500 min, respectively, with other PK parameters being quite similar in the two rodent species. Interestingly, the mouse intravenous plasma concentration time curves exhibit an apparent absorption phase. While the rat intravenous data did not exhibit a pronounced apparent absorption phase immediately following injection, plasma levels did increase between 10 and 30 min following an expected drop from time 0 to 5 min. The data are consistent with first-pass uptake, possibly by the lung, with back diffusion as a function of time. The observed C(max) values of 1.36 microg/mL in the mouse intraperitoneal model suggest that a plasma concentration of 0.5-1 microg/mL corresponds to complete protection for a 200 ng/animal dose of intraperitoneally administered LPS in the D-galactosamine-primed model of endotoxin-induced lethality.