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.