Importance of vial shape and type on the reproducibility of size exclusion chromatography measurement of monoclonal antibodies.

The goal of this work is to understand the phenomenon behind the poor injection repeatability often observed with protein biopharmaceuticals. All the measurements were carried out in size exclusion chromatography (SEC) mode, using fifteen commercially available therapeutic monoclonal antibodies (mAbs). First of all, we proved that the variation of peak areas between consecutive injections was much more critical with highly concentrated mAb samples (up to 80mg/mL), while the SEC measurements of commercial mAbs having concentrations below 5mg/mL were reliable. Second, we emphasized that the shapes, volumes and materials of the sample vial insert also contribute to the change of peak areas observed during consecutive injections. In this study, six different insert models were studied and the most critical were the ones possessing the narrowest conical shape at the bottom. Furthermore, the homogenization of samples (with pipette mixing rather than vortex) prior to analysis was of great interest and allows a significant improvement in injection repeatability. Finally, because the on-wall (on-insert) adsorption of mAbs reaches its equilibrium in around 50min, it is advised to add a specified residence time prior to injection to achieve repeatable injection.