Two-Dimensional Liquid Chromatography- Charged Aerosol detector (CAD)/ Mass Spectrometry (MS) Assay to Analyze Polysorbate Degradation
Degradation of PS20 and PS80 in CHO cell-free media or formulated antibody were analyzed by two-dimensional HPLC-CAD/ MS system. The details of the setup were previously described by Genentech (Li et al., 2014). Polysorbates were separated from formulated mAb by using an Oasis MAX column (2.1×20 mm, 30 μm), with the initial gradient held at 99% solvent A (0.1% formic acid in water) and 1% solvent B (0.1% formic acid in acetonitrile) for 1 min. The gradient was then increased to 20% solvent B in 1.5 min and dropped back down to 1% in another 1.5 min. The up and down gradient cycle for solvent B was repeated 3 times over 10 min to achieve the complete removal of mAb from the polysorbates. By using a switch valve, polysorbates were then subjected to separation by reversed phase chromatography using Acquity BEH C4 column (2.1×50 mm, 1.7μm). During the separation, solvent B was increased to 20% from 1% in 1.5 min at the end of the 10 min gradient cycle, then gradually increased to 99% at 45 min and held for 5 min, followed by an equilibration step of 1%B for 5 min. The flow rate was kept at 0.1 mL/min and column temperature at 40°C.
The 2D-LC system was set up with Thermo UltiMate 3000 and coupled with Corona Ultra CAD detector operated under a nitrogen pressure of 75 psi for quantitation. Chromeleon 7 was used for system control and data analysis. Q-Exactive Plus with ESI source was coupled with the 2D-LC system for characterization only. The instrument was operated in a positive mode with capillary voltage at 3.8kV, capillary temperature at 350°C, sheath flow rate at 40, and aux flow rate at 10. Full scan spectra were collected over the m/z range of 150-2000. Thermo Xcalibur software was used to collect and analyze MS data.
Peak area of each ester was integrated from the CAD chromatogram and summed up to account for the total intact ester in PS20 or PS80. Because peak areas of diesters do not change during incubation, only the sum of peak areas of monoesters were used for calculations. The remaining percentage of PS20 used in this work was calculated by comparing the sum of the peak areas of monoesters eluting between 27.5 min and 33 min at each time point to the sum of peak areas at time zero. The remaining percentage of PS80 was calculated by comparing the sum of the peak areas of monoesters eluting between 30 min and 35 min at each time point to the sum of peak areas at time zero.