Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution. Previous studies have suggested that they can potentially act as adjuvant resulting in potential safety risks for patients. In this paper we present several analytical case studies describing the impact of the presence of SiOPs in biotherapeutics on the analysis of the drug as well as clinical case studies examining the effect of SiOPs on patient safety. The analytical case studies demonstrate that orthogonal techniques, especially flow imaging, can help differentiate SiOPs from other types of particulate matter. The clinical case studies showed no difference in the observed patient safety profile across multiple drugs, patient populations, and routes of administration, indicating that the presence of SiOPs does not impact patient safety.

An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 µm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Transthyretin variants with improved inhibition of ß-amyloid aggregation.

Aggregation of ß-amyloid (Aß) is widely believed to cause neuronal dysfunction in Alzheimer's disease. Transthyretin (TTR) binds to Aß and inhibits its aggregation and neurotoxicity. TTR is a homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel ß-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Here we report the discovery of a TTR mutant, N98A, that was more effective at inhibiting Aß aggregation than wild-type (WT) TTR, although N98A and WT bound Aß equally. The N98A mutation is located on a flexible loop distant from the putative Aß-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. In vivo, the lone cysteine in TTR is frequently modified by S-cysteinylation or S-sulfonation. Like the N98A mutation, S-cysteinylation of TTR modestly decreased tetramer stability and increased TTR's effectiveness at inhibiting Aß aggregation. Collectively, these data indicate that a subtle change in TTR tetramer structure measurably increases TTR's ability to inhibit Aß aggregation.

Evaluation of Nanoparticle Tracking for Characterization of Fibrillar Protein Aggregates.

Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods. Nanoparticle tracking analysis (NTA) is a relatively new technique that may be uniquely suited for obtaining these data. In NTA, the two-dimensional (2-D) trajectory of individual particles is tracked, from which the diffusion coefficient, and, hence, hydrodynamic radius is obtained. Here we examine the validity of NTA in tracking number concentration and size of DNA, as a model of a fibrillar macromolecule. We use NTA to examine three amyloidogenic materials: beta-amyloid, transthyretin, and polyglutamine-containing peptides. Our results are instructive in demonstrating the advantages and some limitations of single-particle diffusion measurements for investigating aggregation in protein systems.

Transthyretin as both a sensor and a scavenger of ß-amyloid oligomers.

Transthyretin (TTR) is a homotetrameric transport protein, assembled from monomers that each contain two four-stranded ß-sheets and a short α-helix and loop. In the tetramer, the "inner" ß-sheet forms a hydrophobic pocket while the helix and loop are solvent-exposed. ß-Amyloid (Aß) aggregates bind to TTR, and the level of binding is significantly reduced in mutants L82A (on the loop) and L110A (on the inner ß-sheet). Protection against Aß toxicity was demonstrated for wild-type TTR but not L82A or L110A, providing a direct link between TTR-Aß binding and TTR-mediated cytoprotection. Protection is afforded at substoichiometric (1:100) TTR:Aß molar ratios, and the level of binding of Aß to TTR is highest for partially aggregated materials and decreased for freshly prepared or heavily aggregated Aß, suggesting that TTR binds selectively to soluble toxic Aß aggregates. A novel technique, nanoparticle tracking, is used to show that TTR arrests Aß aggregation by both preventing formation of new aggregates and inhibiting growth of existing aggregates. TTR tetramers are normally quite stable; tetrameric structure is necessary for the protein's transport functions, and mutations that decrease tetramer stability have been linked to TTR amyloid diseases. However, TTR monomers bind more Aß than do tetramers, presumably because the hydrophobic inner sheet is solvent-exposed upon tetramer disassembly. Wild-type and L110A tetramers, but not L82A, were destabilized upon being co-incubated with Aß, suggesting that binding of Aß to L82 triggers tetramer dissociation. Taken together, these results suggest a novel mechanism of action for TTR: the EF helix/loop "senses" the presence of soluble toxic Aß oligomers, triggering destabilization of TTR tetramers and exposure of the hydrophobic inner sheet, which then "scavenges" these toxic oligomers and prevents them from causing cell death.

Identification of beta-amyloid-binding sites on transthyretin.

Transthyretin (TTR) binds to the Alzheimer-related peptide beta-amyloid (Aß), and may protect against Aß-induced neurotoxicity. In this work, the specific domains on TTR involved with binding to Aß were probed. An array was constructed of peptides derived from overlapping sequences from TTR. Strong binding of Aß to TIAALLSPYSYS (residues 106-117) was detected, corresponding to strand G on the inner ß-sheet of TTR. Aß bound weakly to four contiguous peptides spanning residues 59-83, which includes strand E through the E/F helix and loop. To further pinpoint specific residues on TTR involved with Aß binding, nine alanine mutants were generated: I68A, I73A, K76A, L82A, I84A, S85A, L17A, T106A and L110A. Aß binding was significantly inhibited only in L82A and L110A, indicating that Aß binding to TTR is mediated through these bulky hydrophobic leucines. Aß binding to L17A and S85A was significantly higher than to wild-type TTR. Enhancement of binding in L17A is postulated to arise from reduced steric restriction to the interior L110 site, since these two residues are adjacent in the native protein. The S85A mutation caused a reduction in TTR tetramer stability; increased Aß binding is postulated to be a direct consequence of the reduced quaternary stability.