Protein Characterization Using Dynamic Light Scattering

Qualitative and quantitative analysis of proteins using a combination of light scattering techniques in your workflow Light scattering and chromatography are key tools in the lab as part of protein formulation and characterization studies. In this webinar we will show how different light scattering techniques give complimentary information to better understand the condition of a protein sample. Dynamic light scattering can monitor sample stability, detect early aggregates in protein formulations and study the thermal stability of proteins. GPC with static light scattering and intrinsic viscosity can quantify the proportion of aggregates present, measure molecular weight and oligomeric state, and also study conjugation, and structural changes.

On demand presentation on Oligomers & Quaternary structure. Dynamic light scattering may be used to study the aggregation process. The term ‘aggregates’ can encompass oligomers, assembly or agglomeration. Typically, the molecular weight is measured using static light scattering. A recent, more rapid approach is the estimate from dynamic light scattering using a calibration technique. This method, for example, shows a clear difference between insulin in dimeric form at pH2 and a preparation in physiological conditions. The ‘active’ hexamer is significantly increased in size compared to the inactive dimer. Bovine serum albumin (BSA) is typically present as an equilibrium of dimer/monomer and Dynamic Light Scattering is used as a first estimate of the ratio of the two contributions.

Application note on using static and dynamic light scattering for protein characterization The stability of a protein formulation is critical to its success as a pharmaceutical product. Non-invasive techniques such as light scattering are ideal to characterize proteins in a variety of solution conditions and obtain a profile of their state in solution. Protein solutions may exhibit thermal denaturation, changes in quaternary structure, aggregation and virial coefficient. Molecular weight and shape estimates complete the picture of the molecule. This note provides an overview of the information obtainable from light scattering studies.

Application note on using dynamic light scattering to characterize monoclonal antibodies Antibodies are our natural defense bodies against bacteria, viruses, and other blood borne foreign substances. Antibodies are proteins and as such prone to aggregation under perturbing solution conditions. Because of the sensitivity to small amounts of aggregates, light scattering is ideally suited to characterize antibody formulations. This application note presents measurements of the hydrodynamic size, zeta potential, absolute molecular weight and second virial coefficient of an antibody fragment (of approximately 20 kDa).

Application note on Measurement of Proteins in solution at low concentration: Lysozyme monomer at 0.1 mg/mL Measuring small, poorly scattering molecules in solution has been a challenge for dynamic light scattering systems. Lysozyme is a popular ‘standard protein’ used in many protein test assays. This enzyme is found for example in tears, and is responsible for killing bacterial cells. Lysozyme is relatively inexpensive and easily available. A preparation of hen egg lysozyme in sodium acetate buffer (at pH4.25) is measured at a concentration of only 0.1 mg/mL. The hydrodynamic size of the monomer is 1.9nm radius, in agreement with its crystallographic structure. Larger molecules increase light scattering and are detectable at even smaller concentrations.

Application note on using dynamic light scattering to evaluate the of β-lactoglobulin. This application note provides a study of the electrostatic aggregation of β-lactoglobulin at low ionic strength and pH below the isoelectric point. Here, aggregation is slow enough to be evaluated by dynamic light scattering (DLS).

At the onset of the experiment the beta lactoglobulin is primarily dimeric as seen from the hydrodynamic size, consistent with its expected molecular weight. Over time, the system then aggregates and the average size stabilizes at an aggregation/dimer equilibrium.

Application note on Measurements of lipoproteins using dynamic light scattering. The particle size of low-density lipoproteins (LDL) is a significant indicator of cardiovascular disease. Dynamic light scattering is a fast and easy method to determine particle size in solution. Measurements show good agreement between light scattering and diameters obtained from gradient gel electrophoresis (GGE).

Application note on The study of antibody - antigen interactions using dynamic light scattering. Dynamic light scattering may be used to study antibodies in solution. When the antibody binds to the antigen of a virus the z-average hydrodynamic size increases with time.

Application note on Pharmaceutical drug development: Using dynamic light scattering to screen for promiscuous inhibitors. New lead compounds for drug development are typically discovered using high throughput combinatorial techniques that utilize screening databases of known compounds (libraries). Some of these small molecules have been shown to exhibit drug-like traits. Studies indicate that their concentration dependent inhibitory nature is a consequence of non-specific aggregation. This application note summarizes measurements performed on a candidate drug that exhibits inhibitory behavior at certain concentrations. The aggregation of the compound was examined with dynamic light scattering.