The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

BackgroundABP 501 is being developed as a biosimilar to adalimumab. Comprehensive comparative analytical characterization studies have been conducted and completed.ObjectiveThe objective of this study was to assess analytical similarity between ABP 501 and two adalimumab reference products (RPs), licensed by the United States Food and Drug Administration (adalimumab [US]) and authorized by the European Union (adalimumab [EU]), using state-of-the-art analytical methods.MethodsComprehensive analytical characterization incorporating orthogonal analytical techniques was used to compare products. Physicochemical property comparisons comprised the primary structure related to amino acid sequence and post-translational modifications including glycans; higher-order structure; primary biological properties mediated by target and receptor binding; product-related substances and impurities; host-cell impurities; general properties of the finished drug product, including strength and formulation; subvisible and submicron particles and aggregates; and forced thermal degradation.ResultsABP 501 had the same amino acid sequence and similar post-translational modification profiles compared with adalimumab RPs. Primary structure, higher-order structure, and biological activities were similar for the three products. Product-related size and charge variants and aggregate and particle levels were also similar. ABP 501 had very low residual host-cell protein and DNA. The finished ABP 501 drug product has the same strength with regard to protein concentration and fill volume as adalimumab RPs. ABP 501 and the RPs had a similar stability profile both in normal storage and thermal stress conditions.ConclusionBased on the comprehensive analytical similarity assessment, ABP 501 was found to be similar to adalimumab with respect to physicochemical and biological properties.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing changes.

Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.