Membrane proteins are involved in various cellular processes. However, purification of membrane proteins has long been a challenging task, as membrane protein stability in detergent is the bottleneck for purification and subsequent analyses. Therefore, the optimization of detergent conditions is critical for the preparation of membrane proteins. Here, we utilize analytical ultracentrifugation (AUC) to examine the effects of different detergents (OG, Triton X-100, DDM), detergent concentrations, and detergent supplementation on the behavior of membrane protein TmrA. Our results suggest that DDM is more suitable for the purification of TmrA compared with OG and TritonX-100; a high concentration of DDM yields a more homogeneous protein aggregation state; supplementing TmrA purified with a low DDM concentration with DDM maintains the protein homogeneity and aggregation state, and may serve as a practical and cost-effective strategy for membrane protein purification.

When employing the fluorescence detection system for analytical ultracentrifuge (AU-FDS) with confocal optical structure, the inner filter effect (IFE) introduces a non-linear relationship between fluorescence intensity and concentration at high concentrations. This phenomenon limits the potential applications of AU-FDS. To address the challenge, we propose a correction method specifically designed for the confocal optical structure, which substituting uncorrected data with a concentration-proportional, difference invariant 1 – f. By performing real-time calculation of system parameters, this method achieve self-calibration, ensuring that the difference invariant 1 – f is not affected by systematic noise and variations in environmental factors, including fluorescent material adhering to the upper window, temperature fluctuation, excitation light power shift and so on. Experiments conducted on the rotation equipment demonstrate that, by applying our method, the linear correlation coefficient of fluorescein solutions within the concentration range of 0.86 109.9 μM is improved from 0.9166 to 0.9965.

Background: As essential biomolecules composed of proteins and carbohydrate moieties, glycoproteins play pivotal roles in numerous biological processes. The glycosylation level plays a crucial role in determining the functionality of glycoproteins. Therefore, the precise quantification of glycan components in proteins holds significant importance for research on and development of polysaccharide–protein-conjugated vaccines. Methods: In this study, a novel glycan quantification approach was developed, leveraging analytical ultracentrifugation (AUC) technology that synergistically utilizes ultraviolet wavelength absorption and interference data to directly determine glycan mass fractions in glycoproteins. Results: This methodology expands the analytical framework for glycoproteins while retaining the intrinsic advantages of AUC, enabling analysis in native states with high reproducibility as indicated by low standard deviation across replicates. Conclusions: The approach was implemented in our proprietary AUC data analysis software called AUCAgent (v1.8.8), providing a new method for glycoprotein quantification and polysaccharide ratio determination in polysaccharide-protein-conjugate vaccines.

Modern biotechnology has rapidly developed, and biotechnological drugs have become the center of global drug research and development as an important aspect of clinical treatment. These drugs have unique advantages, such as strong species specificity and prominent targeting, and they are widely used in the treatment of various intractable diseases. However, their complex molecular structure, poor stability, and significant heterogeneity make quality control much more difficult than that of traditional small-molecule drugs that require high-precision analytical methods. Analytical ultracentrifugation (AUC) technology was pioneered by Theodor Svedberg during the early 20th century, and it has become an indispensable biophysical tool through technological innovation. This technology has unique advantages for the quality control of biotechnological drugs, including non-destructive detection, high resolution, and wide applicability. Therefore, AUC has been extensively adopted for the characterization of various biological products. This review systematically summarizes AUC technology applications in major categories of biotechnological drugs, with the aim to provide technical guidance and promote the standardized application of AUC.