Inline optical cell alignment inspection (IOCAI) improves data quality in sedimentation-velocity analytical ultracentrifugation experiments.

Sedimentation-velocity analytical ultracentrifugation (SV-AUC) is a biophysical method to characterize analytes in solution regarding their size, weight, density, shape and heterogeneity. Depending on the selected speed of rotation, SV-AUC can be applied for solutes, ranging from small peptides to viruses, and aggregates thereof. The correct alignment of the measurement cells in the rotor is key for the precision and accuracy of SV-AUC analysis. Alignment tools have been developed as separate instruments or devices, which are applied either to adjust or to confirm the alignment by optical means. We describe a novel approach of inline optical cell alignment inspection (IOCAI), which utilizes the UV/VIS optical detection system integrated into the centrifuge to assess the alignment of the measurement cells. IOCAI allows (i) to confirm the alignment of the measurement cells in the centrifuge, (ii) to confirm the comparability of different measurement cells, and (iii) to verify the consistency of cell positions in the rotor. These three different aspects are useful in assessing the validity of the obtained SV-AUC results. We experimentally demonstrate the benefit of the IOCAI method to improve data quality for a monoclonal antibody sample for an internally misaligned AUC measurement cell. IOCAI has the potential to improve the analytical ultracentrifugation workflow to generate reliable data and to achieve regulatory-compliant documentation, which is required for SV-AUC in quality control applications.