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Sensitivity of a combined DLS/Raman technique to estimate the conformational and colloidal stability of protein therapeutics
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Malvern Instruments Limited

Combining dynamic light scattering (DLS) and Raman spectroscopy provides the ability to extract a wealth of chemical, structural, and physical parameters from proteins at both low and formulation concentrations. DLS, used to study protein size, and Raman, used to determine protein conformation, is combined into a single instrument to monitor stressed protein samples under identical conditions.

DLS is ideally suited to measuring the size of proteins at low concentrations (down to 0.1 mg/mL), while Raman spectroscopy is ideally suited to deriving secondary and tertiary structural markers from higher concentration protein samples (50 mg/mL or greater). One practical area of interest is understanding the lowest concentration of a protein that can be studied using these combined analytical techniques. Because DLS is known to be appropriate for analyzing low concentration samples, this note will outline the lowest practical protein concentration limit for acquiring Raman spectra, and determine if the spectra provide useful secondary and tertiary structure information. Results using lysozyme indicate that useful information from concentrations as low as 3 mg/mL can be acquired, while monoclonal antibody samples (mAbs) may require a higher concentration to obtain correct structural and thermal information.

The ability of a combined DLS/Raman instrument to study various concentrations of protein samples is explored. DLS, with its capability to measure protein size, is ideal for low concentration samples, while Raman, with its ability to interrogate protein secondary and tertiary structure, is ideal for high concentration samples. Joining the techniques into a single technology allows for the determination of size and structure on a single small volume sample. However, because the complementary techniques have differing ideal concentrations, it is important to first establish the upper and lower practical concentration boundaries for a protein sample. This note focuses on determining the lowest concentration limit for studying proteins with combined DLS/ Raman. 

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