Mass spectrometry and HPLC with fluorescent detection-based orthogonal approaches to characterize N-linked oligosaccharides of recombinant monoclonal antibodies.
A number of HPLC and mass spectrometric techniques are used to characterize post-translational modification in recombinant monoclonal antibodies (MAbs) using the intact glycoprotein and free glycans. LC separation utilizing fluorescent detection technique allows tentative structural assignment of MAb oligosaccharides. Intact molecular weight analysis via electrospray allows for an accurate mass determination and observation of the native glycoform mass envelope. N-linked oligosaccharides are then analyzed by MALDI-ToF. Their structures are further confirmed by analyzing the fragmentation patterns formed by MS/MS. All these techniques provide useful information when performed in isolation. However, the combined information allows for definitive and robust characterization of the N-linked glycans from recombinant MAbs.
Lucka
Adam W
AW
Alexion Pharmaceuticals, Cheshire, CT, USA.
Kilgore
Bruce R
BR
Patel
Rekha
R
Andrien
Bruce A
BA
Dhume
Shirish T
ST
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009844:Oligosaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D009844:Oligosaccharides; D011994:Recombinant Proteins