Polysorbate in formulated mAb detected by 2D-LC-CAD/MS
Polysorbate in formulated mAbs were detected and identified by 2D-LC-CAD/ MS following a slightly modified method by Yi Li, et.al (Borisov, Ji, Wang, Vega, & Ling, 2011; Li et al., 2014). As this study was concerned with hydrolysis of polyoxyethylene (POE) ester bonds, the gradient was set to retain all forms of POE esters while eliminating most POE, POE sorbitan, and POE isosorbide. Additionally, mAbs were eliminated using an Oasis Max Column. Reverse phase chromatography was then used to separate the remaining POE esters based on their fatty acid content and type. The esters eluted in the order of monoesters, diesters, triesters and tetraesters (Figure 1-II-V). The structure of each ester was elucidated by mass spectrometry based on the chemical formula of the polymer and dioxolanylium ion generated by in source fragmentation. Figure 1-III is the representative total ion current (TIC) profile of PS20 with major peaks labeled, containing sorbitan monoester, isosorbide monoester and diesters with a variety of fatty acid chains while Figure 1-V is the representative total ion current (TIC) profile of PS80 with major peaks labeled. The identified esters of PS20 and PS80 are summarized in Table 1-a and 1-b. Quantitation of polysorbates was determined by charged aerosol detector (CAD). Figure 1-II and 1-IV show the recovery of PS20 and PS80 in PS20 and PS80 standard solution and a formulated mAb by using 2D-LC/CAD respectively; the corresponding peaks were identified by mass spectrometry.