Characterization of Antibody Drugs by Analytical Ultracentrifugation (AUC) - Introduction to AUC Technology

Analytical Ultracentrifugation (AUC) is one of the main technical means to characterize the characteristics of biomacromolecules and study the physicochemical properties of biomolecules. It is used to analyze sample heterogeneity, aggregate formation, and intermolecular interactions. The Swedish scientist Theodor Svedberg invented the technology in 1925 and built the first analytical ultracentrifuge the following year. After nearly a hundred years of application, verification, and development, this technology has been widely used in research fields such as biopharmaceuticals, life sciences, and polymer sciences.

The analytical methods of analytical ultracentrifugation mainly include sedimentation velocity (Sedimentation Velocity) and sedimentation equilibrium (Sedimentation Equilibrium).

Settling rate is a hydrodynamic technique. In the sedimentation rate experiment, the sample is rotated at a high speed, the solute molecules move toward the bottom of the sample cell, and the rate of the solute molecule movement is measured by the recorded data. The rate of movement is expressed by the sedimentation coefficient, which depends on molecular weight, molecular shape, or conformation. For samples of different components, each component is detected according to different sedimentation coefficients.

Settling equilibrium is a thermodynamic technique. The sedimentation equilibrium test was carried out at a low rotational speed, and the distribution of molecular equilibrium concentration was determined when the sedimentation and diffusion reached equilibrium. In equilibrium, the distribution of concentration depends only on the mass and not on the shape of the molecule. At the beginning of centrifugation, the molecular particles settle. After a period of time, the sedimentation results in a concentration gradient, thus resulting in the reverse diffusion of protein molecules. When the reverse diffusion and centrifugal sedimentation reach equilibrium, the concentration distribution remains unchanged.

After the sedimentation data is obtained by scanning, the sample characterization results can be obtained through subsequent software analysis. At present, SEDFIT/SEDPHAT analysis software is widely used. According to the analysis results, the aggregation state of biological macromolecules and the accurate percentage content of aggregates can be accurately detected. Analytical ultracentrifugation technology has its unique advantages in the detection of protein drug aggregates. It does not require standards and markers, and directly detects the state of the sample in the final solution, which is closer to the real physiological state of the protein.

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