In the case of proteins, combined information from DLS ( Rh) and MALS (molecular weight) can be valuable in understanding molecular conformation and folding states. In order to determine the structure of aggregate particles or measure protein radius less than 10 nm, DLS can be combined with MALS, downstream of the FFF or SEC, to give the hydrodynamic radii ( Rh) of the eluting species.
Characterization of Aggregate Particle Structure On-line analysis of size and molar mass uses DLS, MALS, differential refractometry (dRI), and UV/Vis absorption (UV), as well as information such as number density data for aggregate particles, root mean square radius ( Rg, when >10 nm), or stoichiometry of protein conjugates, among other parameters. independently of density and geometry) and SEC or asymmetric flow field-flow fractionation (AF4) is used to separate protein monomers and aggregates. Importantly, the addition of a MALS detector to FFF or SEC gives the absolute molecular weight (i.e. MALS detectors may be used either in batch mode or together with FFF or SEC separation.
On-line analysis of molar mass and size uses MALS, DLS, UV/Vis absorption (UV), and differential refractometry (dRI). Separation of protein monomers and aggregates is accomplished by SEC or asymmetric flow field-flow fractionation (AF4). Of these, MALS is the most precise, flexible and widely used method for comprehensive characterization of polymers and biopolymers (Figure 1).įigure 1. Static light scattering can be right angle, low angle, or multi-angle LS (MALS). In most laboratories, SEC is used to isolate and measure the stable protein isoforms from aggregates and fragments. In addition, contemporary DLS and SLS detectors can be combined with on-line fractionation techniques such as field-flow fractionation (FFF) and size exclusion chromatography (SEC) to allow superior resolution and excellent characterization of the various sub-species present in protein formulations.Īlthough both SEC and FFF separate molecules based on their hydrodynamic volume, SEC is generally used to separate proteins, whereas FFF can separate both proteins and nanoparticles. Light scattering detectors are therefore extremely sensitive in the detection of large aggregates, even at small quantities.
The strength of the light scattering signal is relative to both the concentration and molar mass of the species in solution. Dynamic and Static Light Scattering Techniquesĭynamic light scattering ( DLS) and static light scattering (SLS) are techniques that have been applied in the characterization of protein aggregates ranging from several nanometers (monomer and low-order oligomer) to micrometers (high-order aggregate and aggregate particles) in diameter. To this end, a number of light scattering techniques have been developed to characterize protein aggregates, enabling their composition in therapeutic formulations to be determined. The size and abundance of any protein aggregates must be determined during each step of the production of therapeutic proteins. The size of protein aggregates can range from a few nanometers to hundreds of micrometers.
This aggregation can occur during the production, administration, storage, and shipping of therapeutic protein compounds, which can compromise their stability, efficacy, and safety. Protein aggregation refers to the clumping together or accumulation of mis-folded proteins. Sponsored Content by Wyatt Technology Introduction
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