On-line analysis of molar mass and size utilizes multi-angle light scattering (MALS), dynamic light scattering (DLS), UV/Vis absorption ( UV), and differential refractometry ( dRI), but also additional information such as stoichiometry of protein conjugates, root mean square radius ( Rg, when greater than 10nm), or number density data for aggregate particles, among other parameters. Importantly, the addition of a MALS detector to SEC or FFF provides absolute MW (i.e., independently of geometry, density, and Separation of protein monomers and aggregates is accomplished by size-exclusion chromatography or asymmetric flow field-flow fractionation ). MALS detectors can be used in both batch mode and in combination with SEC or FFF separation. On-line analysis of molar mass and size utilizes multi-angle light scattering (MALS), dynamic light scattering (DLS), UV/Vis absorption (UV), and differential refractometry (dRI). The separation of protein monomers and aggregates is accomplished by size-exclusion chromatography (SEC) or asymmetric flow field-flow fractionation (AF4). Static light scattering can include low angle (LALS), right angle (RALS), and multi-angle LS (MALS), and MALS is, by far, the most accurate, versatile, and widely utilized detector for polymer and biopolymer characterization. SEC is common in most labs and has been utilized more widely than FFF to separate and quantify the stable protein isoforms from fragments and aggregates. Furthermore, modern SLS and DLS detectors can be coupled to online fractionation techniques such as size exclusion chromatography (SEC) and field flow fractionation (FFF), thus allowing excellent resolution and extensive characterization of the different sub-species present in protein formulations.Įven though both separate molecules are based on their hydrodynamic volume, SEC is typically used for protein separation whereas, FFF can be used for both proteins and nanoparticles. The intensity of the light scattering signal is proportional to both molar mass and concentration of the species in solution (or stable suspension).Ĭonsequently, light scattering detectors are highly sensitive in detecting large aggregates, even in small quantities. Light scattering (LS) techniques, both static light scattering (SLS) and dynamic (DLS) have been used for the characterization of protein aggregates from several nanometres (monomer/low-order oligomer) to micrometers (high-order aggregate and aggregate particles) in diameter. Static and Dynamic Light Scattering Techniques Generally, the sizes of protein aggregates vary from a few nanometres to hundreds of micrometers. As protein aggregates can considerably impact the efficacy, stability, and safety of the therapeutic agent, their sizes and abundance must be characterized at each step of the production process. Protein aggregation is a phenomenon that takes place during storage, manufacturing, administration, and shipping of a therapeutic protein product. Several light scattering methodologies have been developed for its characterization, enabling the determination of protein aggregate composition in therapeutic formulations through the application of orthogonal approaches. Protein aggregation may take place at any stage during the development of protein-based therapeutic compounds.
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