Showing results for 
Search instead for 
Did you mean: 

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer

Reputable Mentor II
Reputable Mentor II
Bondarenko PV, Second TP, Zabrouskov V, Makarov A, Zhang Z.
J Am Soc Mass Spectrom. 2009 Aug;20(8):1415-24
Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within +/-2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collision-induced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications.
Amgen Incorporated, Thousand Oaks, California 91320, USA.
Version history
Last update:
‎10-15-2021 12:08 PM
Updated by: