Influence of connection tubing in modern size exclusion chromatography and its impact on the characterization of mAbs

The goal of the study was to evaluate the impact of connection tubing in modern size exclusion chromatography (SEC), since it may strongly impact the apparent column efficiency, as the compounds are not retained in SEC. For this purpose, a reference SEC column of 150 × 4.6 mm, 1.8 μm was considered, and various proteins were tested as model compounds. Different tube geometries (lengths and internal diameters) and materials (stainless steel and PEEK) were evaluated in a systematic way. Large proteins always showed larger tube dispersion vs. small molecules, especially when the residence time in the tube was long (at low flow rate). This confirms the need to drastically reduce the tube volume (using the shortest and narrowest connector tubing) to attain the full benefits of UHPSEC columns. In addition, PEEK tubing were found to be more appropriate than stainless steel tubing, since adsorption of proteins was less pronounced, and higher plate count can be obtained. Finally, after a careful system optimization, up to 40% increase of apparent column efficiency can be achieved compared to a regular UHPLC system, when using a 150 × 4.6 mm UHPSEC columns packed with sub-3 μm particles.