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Raman spectroscopy has been used for decades in academic environments to characterize secondary and tertiary structure, as well as disulfide bond conformations of proteins. Also, although FTIR is capable of providing secondary structural information of high concentration samples, it is not sensitive to amino acid side chain environments that enable tertiary structure to be monitored, and cannot characterize size changes. Particularly for Fourier Transform Infrared (FTIR) spectroscopy, interference from water presents a significant analytical challenge. Interference from buffer components is another common limitation, and requires that samples be prepared differently for analysis. Another workhorse characterization technique, DSC is not always able to determine the onset of soluble aggregate formation. Circular dichroism (CD) and fluorescence (both intrinsic and extrinsic), for example, require low concentration and as a result, samples that have been diluted for measurement purposes may have different stability characteristics than the high concentrations at which they are formulated, and information obtained may not be directly relevant. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.Īlthough there are a number of instruments which have traditionally been utilized to characterize molecular level structure and conformation in aggregating proteins, many of these suffer limitations. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS) and Raman spectroscopy, respectively. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Analytical challenges are compounded for materials: (1) that are formulated at high concentration, (2) that are formulated with a variety of excipients, and (3) that are available only in small volumes.

Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability.