Abstract: Analytical ultracentrifugation （AUC） is a classical biophysical technique that enables precise analysis of the physicochemical properties of biomacromolecules in solution without the need for labeling. By monitoring the sedimentation behavior of molecules under a high centrifugal field, AUC provides rich information including molecular weight, sedimentation coefficient, purity, aggregation state, interactions, and particle density. This article systematically elaborated on the basic principles, instrument composition, fundamental operations, and data analysis methods of AUC. It highlighted the applications of AUC in quality assessment of biopharmaceuticals such as monoclonal antibodies, viral vectors, and nucleic acid drugs, including higher-order structure characterization, aggregation state and fragment analysis, determination of viral empty capsid ratio, and process consistency evaluation. Finally, it looked forward to the future development prospects of AUC in terms of automation, high-throughput capabilities, and applications in emergingtherapies.

Key words: analytical ultracentrifugation, biopharmaceuticals, characterization, quality control, sedimentation velocity