Application of novel techniques for characterization of subvisible and sub-micron particles in biopharmaceutical preparations

A large number of protein-based medicines have successfully improved the treatment and quality life of patients in diverse therapeutic areas. From a pharmaceutical technology perspective, the main associated implications are high viscosity and increased protein aggregation tendency that typically characterize protein formulations. Despite the proven safety and efficacy of biotechnological products, some publications have suggested that the particulate matter present in liquid formulations has immunogenic properties. Thus, there is an increasing interest to research and characterize protein particles beyond the limits of current USP applications of Chapter had appeared in the recent years. A number of analytical techniques have emerged for the characterization of particles size between the upper size limit of chromatography techniques and the lower size limit of the compendial light obscuration method. Although this represents an instrumentation achievement, the analytical performance of such tools is unknown. This present Doctoral research aims to evaluate the applicability of the new techniques in the analysis of biotechnological products including deeper understanding of their principles as well as the comparative evaluation with the compendial light obscuration method

Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities

Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements

Factors Governing the Precision of Subvisible Particle Measurement Methods - A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe

PURPOSE: The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance. METHODS: The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions. RESULTS: The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results. CONCLUSIONS: The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control

Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance

A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model