Characterization of Nanoparticle Tracking Analysis for Quantification and Sizing of Submicron Particles of Therapeutic Proteins.

Submicron particles may play important roles in therapeutic protein product quality, stability, and adverse effects in patients. However, quantitation of these particles has been challenging. Nanoparticle tracking analysis (NTA) is capable of both sizing and counting submicron particles. We investigated the effects of product and instrument parameters on NTA results for nanoparticle standards and therapeutic protein samples. To obtain proper particle size distributions, complete tracking numbers of at least 200 and 400 were required for latex nanobeads and protein nanoparticles, respectively. In addition, when set at suboptimal values, the minimum expected particle size parameter led to inaccurate sizing and counting for all particles types investigated. A syringe pump allowed for higher sampling volumes, and results were reproducible for nanoparticle sizing and counts at flow rates ≤7 µL/min. Finally, because therapeutic protein products are being formulated at relatively high protein concentrations, we investigated the effects of protein concentration on nanoparticle characterization. With high protein concentrations, nanoparticle sizing was not affected, whereas particle concentrations were significantly reduced. Linear relationships between particle count and dilution factor were obtained when a high protein concentration formulation was diluted into particle-free solutions at the same protein concentrations, but not when dilutions were made into buffer.

Investigations into the fouling mechanism of parvovirus filters during filtration of freeze-thawed mAb drug substance solutions.

Filtration to remove viruses is one of the single most expensive steps in the production of mAb drug products. Therefore, virus filtration steps should be fully optimized, and any decline in flow rates warrants investigation into the causes of such membrane fouling. In the current study, it was found that freezing and thawing of a mAb bulk drug solution caused a substantial decrease in viral filter membrane flow rate. Freezing and thawing also caused formation of aggregates and particles across a broad size range, including particles that could be detected by microflow imaging (≥1 µm in size). However, removal of these particles offered little protection against flow rate decline during viral filtration. Further investigation revealed that trace amounts of aggregates (ca. 10⁻6 of the total mass of protein in solution) approximately 20-40 nm in size were primarily responsible for the observed membrane fouling.

Characterization and quantitation of aggregates and particles in interferon-ß products: potential links between product quality attributes and immunogenicity.

Interferon-ß (IFN-ß) products have been used for many years in the treatment of multiple sclerosis and include recombinant IFN-ß-1b (Betaseron®) and IFN-ß-1a (Avonex® and Rebif®). All three products lead to the formation of neutralizing antibodies (NAbs) and resulting loss of efficacy in patients but to different extents. Across several clinical trials, the reported rates of neutralizing-antibody formation were 22%-47% (Betaseron®), 5%-35% (Rebif®), and 2%-13% (Avonex®). In the current study, all products were purchased from the pharmacy and aggregates were characterized and/or quantified using size-exclusion chromatography (SEC), analytical ultracentrifugation, gel electrophoresis, and dot-blotting immunoassays. Particle characterization and counting were performed using microflow imaging, particle tracking analysis, and resonant mass measurement. Betaseron® and Rebif®, which are formulated with human serum albumin, had the greatest amount of aggregated protein and particles (e.g., 9%-15% high molecular weight species by SEC and >100,000 particles/mL by flow imaging). Avonex® was found to have the least amount of aggregated protein, with >95% monomer content by both SEC and analytical ultracentrifugation, and the particles detected in Avonex® were determined to be primarily silicone oil droplets. These results strongly suggest that protein aggregate and particle contents are key product quality attributes in a given product's propensity to elicit the production of NAbs in patients.

Critical evaluation and guidance for using the Coulter method for counting subvisible particles in protein solutions.

In this paper, we evaluate the suitability of Coulter method for detecting and quantifying subvisible particles in protein solutions and compare results with other particle-counting technologies. The effects of key instrument and operational parameters such as aperture diameter, solution conductivity, and cleaning procedures are demonstrated. Degraded and nondegraded intravenous immunoglobulin and human serum albumin were chosen as model proteins and sample types for this evaluation. Multisizer™4 was able to obtain reproducible and linear particle counts; however, customized analysis and cleaning procedures are needed depending on the protein analyzed and the sample type (degraded or nondegraded). The Coulter method consistently detected more particles than micro-flow imaging and light obscuration. The presence of translucent particles likely accounts for this observation because detection by the Coulter method does not depend on the optical properties of the particles or solution.

Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway.

The objective of this study was to evaluate microflow imaging (MFI) as a sensitive tool to detect and quantify subvisible particle formation during freeze-thawing of an IgG(2) monoclonal antibody (mAb). Solutions of the protein formulated in 20 mM of histidine buffer (pH 5.5) were subjected to three freeze-thaw cycles and analyzed by MFI and size-exclusion chromatography (SEC). MFI showed increased particle numbers after each freeze-thaw cycle, whereas aggregates were not detected by SEC. Estimates of the total mass of particles formed revealed that monitoring of particle formation allows for the detection of protein aggregates comprising only hundredths of a percent of the total protein mass. Furthermore, differences in protein aggregation levels due to different formulations or different freeze-thawing protocols were resolved, even though protein aggregation could not be detected by SEC. To examine whether SEC and MFI-based estimations of total aggregate mass were in quantitative agreement, mAb was freeze-thawed in phosphate-buffered saline. This process created sufficient level of insoluble aggregates to be detected by SEC as a reduction in the monomer peak area in the chromatogram. There was good agreement between the loss of monomer detected by SEC and the total mass of subvisible particles detected by MFI.