Rapid Online Desalting Using a Supermacroporous Reversed Phase Cartridge for Mass Spectrometry Analysis of a Monoclonal Antibody

摘要

Here, we present mass spectrometry (MS) analysis of the monoclonal antibody (mAb) rituximab following sample cleanup using a reversed phase desalting cartridge in two approaches. First, we evaluate the high throughput analysis of an intact mAb over 100 runs using an optimized 4 minute gradient for loading/desalting, elution/MS analysis, and cartridge regeneration. MS detection enabled accurate mass characterization and relative quantification of the different mAb glycoforms. Using high mass loading, we were able to detect 8 different glycans, which combined to give 13 glycoforms. Sample carryover was reduced to 0.8% by area following one blank run. Second, we evaluated the analysis of the same mAb following IdeS digestion and TCEP reduction to separate the mAb into Fc/2, Fd', and LC fragments. Using an extended gradient, the different components were separated on the desalting cartridge into separate peaks. Deconvolution of the resulting mass spectra provided exact determination of the molecular weight of the separated fragments.