2004;37:6632C9. Although EC assays should be scientifically sound, potentially they cannot be validated in accordance with ICH guidelines (ICH Q2(R1)). EC assays generally have a low(er) throughput and poor(er) robustness, i.e. they may require advanced instruments and data interpretation at an expert level.Orthogonal methodsIndependent methods that fundamentally differ from each other in the physical measuring principles that are used to investigate a certain aspect of a sample. For example, aggregates may be detected by orthogonal microscopy, chromatography or centrifugation methods.ParticlesUndissolved species (other than gas bubbles or droplets) that are unintentionally present in the product. Particles can be foreign (not intrinsic to drug substance) or protein-related (i.e. large aggregates). Particles can be further categorized as visible ( ca. 50?m) and subvisible (between ca. 0.1C50?m); submicron particles (between ca. 0.1C1?m) are a subcategory of subvisible particles.Quality control (QC) assaysAssays that are used to release clinical batches throughout product development and WEHI-539 hydrochloride commercial batches after product launch (drug substance and drug product). QC assays need to be validated in accordance with ICH guidelines (ICH Q2(R1)). QC assays generally should have a high throughput and good robustness, i.e. they require conventional instruments and data interpretation at a non-expert level. QC assays are used for formal batch release testing and stability monitoring (GMP) and may also be used for development support activities (next to EC assays). Open in a separate window The challenge in analyzing protein aggregates lies in the unknown nature of the formed aggregates as well as the wide size range of up to six orders of magnitude, from a few nm to a few mm in diameter. Since no single one of the currently available techniques is able to cover this size range, a combination of several techniques is necessary. However, each technique has its own strengths and weaknesses. Moreover, the available methods differ in the physical measuring principle and, consequently, in the results and type of information obtained. The aim of this commentary is to discuss the currently available analytical methods to characterize protein aggregates in relation to product quality and also the interpretation of data resulting from these methods. Moreover, we propose approaches to use these methods for the characterization of protein therapeutics from early product development through to commercialization. This paper is a result of discussions among the co-authors of this paper, who participate in WEHI-539 hydrochloride the protein characterization subcommittee (PCS) of the European Immunogenicity Platform (EIP; see Table?II). Within WEHI-539 hydrochloride the EIP, the EIP-PCS (Table?II) was established to discuss product-related factors associated with immunogenicity and methodologies for protein characterization. Table?II EIP-PCS asymmetrical flow field flow fractionation, analytical ultra centrifugation, high performance size exclusion chromatography, multi angle laser light scattering, not applicable, polyacryl amide gel electrophoresis, Small angle neutron scattering, Small angle X-ray scattering, sodium dodecyl sulfate polyacryl amide gel electrophoresis, size exclusion chromatography, ultraviolet-visible Table?IV Typical Use of Techniques in Industry with Respect to Aggregate Analysis (23). For instance, low solution ionic strength (e.g., 50?mM or less) may encourage hydrophobic interactions of the eluting protein with the column matrix, thereby slowing elution, affecting resolution and peak shape. Addition of arginine to the eluent may inhibit interaction between solute and column matrix (24). Detergents in the sample (as opposed to the eluent), though nominally of small molecular weight, can behave as large molecules if they form micelles (above their critical micelle concentration), appearing in the chromatogram as UV-absorbing peaks (25) and potentially also giving rise to light scattering and fluorescence signals. There is an upper limit to the size of aggregate detectable by SEC, because larger aggregates can be filtered out by frits in the system or by the column itself. As a consequence, large material (large protein aggregates) may disappear and be overlooked in the analysis. They also build up on the top of the column and gradually degrade its performance, seen as broadened peaks, poorer resolution and decreased yields (smaller peaks). Another form of aggregate that may be missed is that formed by very low affinity intermolecular association, as these may dissociate into monomers following a change in conditions from those of the sample to those experienced during chromatography (e.g., dilution or change in temperature) (26). For detection of such low affinity aggregates other methods could be used, such as AUC, or method conditions of SEC could possibly be adjusted. SDS-PAGE and Capillary Electrophoresis-SDS SDS-PAGE is a very common, fairly robust method that is easy to perform and can supply WEHI-539 hydrochloride information on approximate molecular weight and quantity, when using a suitable method of quantitative staining and gel scanning. The presence of SDS means CCNE2 that non-covalent aggregates are disrupted, so the method only detects covalent aggregates. If reducing.