Some Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein Aggregates

There are numerous problems with size exclusion chromatography (SEC), which often lead to inaccuracies in protein aggregate characterization. Hence, this study tested sedimentation velocity analytical ultracentrifugation (SV-AUC) as an orthogonal tool to SEC for quantifying the monomer and aggregates in intravenous immunoglobulin (IVIg) formulations. IVIg samples were subjected to agitation stress and analyzed using SEC mobile phases composed of 200 mM sodium phosphate (pH 7.0) with 0, 50, 100, 200, or 400 mM of NaCl. Surprisingly, 400 mM of NaCl was required in the mobile phase to attain complete protein recovery from the SEC column. Significant discrepancies between SEC and SV-AUC were reported when SEC analysis was performed using suboptimal concentrations (e.g., 0, 50, 100, and 200 mM) of NaCl in the mobile phase. The continuous sedimentation coefficient distributions obtained with SV-AUC resolved the high molecular weight species, whereas with SEC the high molecular weight species eluted as a single peak. Only with the orthogonal use of SV-AUC, we were able to develop a robust SEC method for accurate quantitation of monomer and aggregates in unagitated and agitated IVIg samples. Additionally, this article describes a modification to an existing method of quantitating insoluble aggregates from SV-AUC boundary data.