Date of Completion
Rayleigh light scattering, protein absorption, reversible and irreversible aggregation
Devendra S. Kalonia
Michael J. Pikal
Robin H. Bogner
Field of Study
Doctor of Philosophy
Monoclonal antibodies and their derivatives have emerged as one of the most successful class of biologics in the past few decades. Antibodies are often formulated at high concentrations in order to achieve the desired therapeutic levels on subcutaneous administration. Reversible self-association and aggregation are two of the major challenges associated with formulating high concentration antibodies. The use of static light scattering as a tool for characterizing self-association and aggregation in protein solutions is reviewed. A detailed understanding of the effect of absorption on Rayleigh light scattering intensity and turbidity was developed for wavelengths where both scattering and absorption occur simultaneously. Macromolecular solutions of tyrosine-starch mixtures, bovine serum albumin and a monoclonal antibody were used to understand the interdependence of Rayleigh scattering (at 90⁰ angle) and light absorption. Results from the studies show that at wavelengths less than 300 nm, 90⁰ scattering intensity decreases as a function of chromophore concentration and depends on molar absorptivity, molecular weight and concentration of molecules. Furthermore, at wavelengths of 300 – 360 nm, Rayleigh scattering intensity for macromolecular solutions was independent of chromophoric absorption. Additionally, turbidity of tyrosine-starch mixtures was also measured in the wavelength range of 250 – 360 nm. Turbidity of starch in the mixtures was independent of the tyrosine concentration at all wavelengths. These findings suggest that the decrease in 90⁰ scattering intensity observed at 250 – 300 nm for starch-tyrosine mixtures and proteins is because of the reabsorption of scattered light by absorbing chromophores.
A novel method called UV light scattering, was developed to detect reversible and irreversible aggregation by using static light scattering intensities in the UV range. The studies demonstrate that UV light scattering can be used as a fast and rapid tool for detecting reversible and irreversible aggregation in proteins at a wide range of concentrations. It can be used in high-throughput mode for screening different solution conditions and optimizing protein formulations at an early development stage.
Kaur, Japneet, "Application of UV Light Scattering to Detect Reversible Self-association and Aggregation of Proteins in Solution" (2017). Doctoral Dissertations. 1386.