Spectroscopic, scattering, and imaging methods play an important role in advancing the study of pharmaceutical and biopharmaceutical therapies. The tools more familiar to scientists within industry and beyond, such as nuclear magnetic resonance and fluorescence spectroscopy, serve two functions: as simple high-throughput techniques for identification and purity analysis, and as potential tools for measuring dynamics and structures of complex biological systems, from proteins and nucleic acids to membranes and nanoparticle delivery systems. With the expansion of commercial small-angle x-ray scattering instruments into the laboratory setting and the accessibility of industrial researchers to small-angle neutron scattering facilities, scattering methods are now used more frequently in the industrial research setting, and probe-less time-resolved small-angle scattering experiments are now able to be conducted to truly probe the mechanism of reactions and the location of individual components in complex model or biological systems. The availability of atomic force microscopes in the past several decades enables measurements that are, in some ways, complementary to the spectroscopic techniques, and wholly orthogonal in others, such as those related to nanomechanics. As therapies have advanced from small molecules to protein biologics and now messenger RNA vaccines, the depth of biophysical knowledge must continue to serve in drug discovery and development to ensure quality of the drug, and the characterization toolbox must be opened up to adapt traditional spectroscopic methods and adopt new techniques for unraveling the complexities of the new modalities. The overview of the biophysical methods in this review is meant to showcase the uses of multiple techniques for different modalities and present recent applications for tackling particularly challenging situations in drug development that can be solved with the aid of fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, atomic force microscopy, and small-angle scattering.
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