In most likely applications, DLS at a single angle can be employed to solve for D t, and as such to attain the diameter of the highlighted protein. As displayed below for BSA, Г is subject linearly to q 2 for a particle of an established size. A configurable correlator layout improves the capacity to measure such a wide variety of decay rates. This Г is linked to the translational diffusion coefficient (D t) as follows:įor a standard medium-sized globular protein, decay rates (Г) as small as several hundred s -1 and as big as 50,000 s -1 may be seen, contingent on scattering angle. The varying elements of this purpose can be divided into a characteristic decay rate, Г, and usually additionally a polydispersity index. For an established scattering angle, θ, and refractive index, n, the scattering vector q is determined from the following expression:Īn established correlation function C(τ) is deconvoluted into either a single-exponential, stretched-exponential, or sum of exponentials. Conversely, forward-scatter, is especially sensitive to larger species and can be employed to identify the presence of even a tiny amount of aggregated protein. Backscatter, usually referring to angles much greater than 90 o, is extremely commonly employed to measure the dimensions of small, monomer-sized proteins. The convention in light scattering is to denote small scattering angles as forward-scatter, and large-scattering angles as backscatter. This expression can be simplified to a proportionality: Where the Boltzmann constant (k B), temperature (T), and bulk viscosity (η) are all established values. The relationship between D t, measured in DLS, and hydrodynamic size, d h, is inverse, and is provided by the Stokes-Einstein Equation: It should be noted that the diameter acquired from DLS, frequently known as the hydrodynamic diameter (d h), is inversely proportional to the diffusion coefficient (D t). As will be outlined below, the configurability of the correlator is critical to evaluating otherwise challenging heterogeneous biological samples. The difficulty in this instance is selecting the suitable correlator layout to completely resolve these signals. A standard sample comprising partly aggregated protein may extend over 1-2 orders of scale in hydrodynamic size. Protein aggregates can attain large sizes on the order of several hundred nanometers or more with ease. A high-speed correlator is required to measure the size of such quickly diffusing proteins. Protein monomers are usually <10 nm, with lots of them as small as 1 nm or under. Image Credit: Brookhaven Instrument CorporationĪ lot of frequently occurring globular proteins are tiny in hydrodynamic size. This is the foundation for measuring a particle size distribution. The signal that develops from the scattered intensity from the laser light is obtained and converted into an autocorrelation function. These variations are directly associated with the motion of particles. The timescale of these intensity fluctuations is at the demand of tens of nanoseconds to hundreds of milliseconds. IntroductionĭLS depends on the principle that freely diffusing material, traveling randomly because of Brownian motion, will generate fast variations in scattered laser light. To show this theory, aggregation studies performed on pharmaceutical-grade monoclonal antibody (mAb), are compared with results acquired for a common protein, serum albumin (BSA). When DLS is employed to its full potential, aggregation of proteins can be found and the size of monomeric protein may be ascertained to a very high degree of accuracy. Neglecting to follow these stringent and thorough standards compromises the processability, activity, and shelf stability of antibody-based products. In each of these applications it is critical that antibodies stay complete and monomeric. These extremely adaptable proteins are employed as the main functional component in immunoassays and other fast diagnostics, in vaccine manufacture, and as the main element of a broad range of injectable protein drugs. One of the quickest growing groups of pharmaceutically active biologics are antibodies. Sponsored Content by Brookhaven Instruments Corporation May 20 2021ĭynamic light scattering (DLS) is a formidable method that is amenable to the measurement of biological samples, especially proteins and biopolymers.
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