Circular dichroism spectroscopy in protein engineering and pharmaceutical development: Applications in structural characterization and quality assessment

Circular dichroism (CD) spectroscopy is a rapid and versatile method for assessing the higher-order structures of proteins and peptides. Far-UV CD enables quantitative evaluation of secondary structures, while near-UV CD provides sensitive fingerprints of tertiary organization. With advances in recombinant protein expression, CD spectroscopy has become widely used for the characterization of novel generated proteins. This review focuses on the applications of CD spectroscopy in protein engineering and pharmaceutical sciences. Examples include strategies for reliable measurements with limited sample quantities and quality assessments such as lot-to-lot comparisons and biosimilar evaluations. CD spectroscopy also serves as a valuable tool for detecting conformational changes associated with protein-protein and protein-drug interactions, as well as for evaluating proteins and peptides in membrane-mimetic environments. Obtaining reliable CD spectra requires careful selection of buffers, water-miscible solvents, and excipients. Here, we summarize their properties and propose practical criteria for their selection to ensure high-quality CD measurements.