Current State of the Science
Table 1. Most commonly used characterization methods for biopharmaceutical products
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Existing characterization methods are intended to provide a detailed, comprehensive analysis of protein size, charge, purity,
activity, and structure (primary, secondary, and tertiary). These analyses also are used to examine impurity profiles, focusing
particularly on aggregates, a key concern for regulatory agencies because of their potential immunogenicity.7 A broad range of analytical methods can be used to characterize proteins on the basis of identity, purity, yield, aggregation,
specificity, and activity. These methods are used to assess comparability, form the basis for product release, determine product
stability, and guide formulation development. Table 1 summarizes the most commonly used characterization methods for biopharmaceutical
products.
Selecting the specific analytical methods to be used in characterizing a given biomolecule involves an assessment of the capabilities,
advantages, and limitations of the available options. The sensitivity of the method in question is critical to identify minor
contaminants or product variants that could potentially elicit significant immune responses in vivo. Reverse phase high performance liquid chromatography (RP-HPLC), mass spectrometry (MS), and dynamic light scattering (DLS)
provide high sensitivity and are commonly used throughout the drug discovery-development-manufacturing chain. Size exclusion
chromatography (SEC), analytical ultracentrifugation, and UV-Vis spectrophotometry are valued for their ability to accurately
quantify specific analytes. Specificity is best determined with biological assays that mimic the desired in vivo molecular interactions in vitro, such as Western blots, enzyme assays, and cell-based assays.8 From both an operational and quality perspective, the best assays are robust, relatively insensitive to changes in sample
matrix or product concentration, rapid, and easy to perform.
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