Analysis of Glycans
Intact protein UHPLC–MS and peptide mapping provided information about the heterogeneity of MAb glycosylation and indicated
that there are differences in the glycosylation patterns of the investigated samples. To gather more detailed and quantitative
information about the glycosylation patterns, we performed LC analysis using a hydrophilic interaction chromatography (HILIC)
column of released glycans using fluorescent detection (FLR). Glycans were cleaved from MAbs by PNGase F enzyme, labeled with
2-AB fluorescent dye, and analyzed using an Acquity UHPLC HILIC glycan column. Chromatograms featuring well resolved glycans
including isomers of G1 a/b and G1F a/b are shown in Figure 6.
Figure 6. LC-FLR profiling of released and 2-AB labeled glycans. Peak assignment was verified by reference standards and
by MALDI–MS analysis. Relative quantitation values shown are based on fluorescence signal.
The HILIC method quantified differences in the glycan profiles of the innovator and biosimilar MAbs. Whereas G1F was the most
abundant glycan structure in the innovator product, G0F was the most abundant in the biosimilar sample. The sensitivity of
fluorescent detection permits the relative quantitation of minor glycans. Glycan identity was confirmed by LC–MS–MS.
Comprehensive investigation of an innovator MAb (trastuzumab) and a biosimilar candidate MAb with LC–MS and LC–FLR techniques
revealed unexpected differences in their primary sequence. Intact protein analysis showed mass differences and heterogeneity
in the glycosylation patterns. To further elucidate whether the differences result from sequence variations or post-translation
modifications, a peptide mapping experiment was performed, revealing an unexpected peptide sequence. The UHPLC–MS data analysis
of the peptides in BiopharmaLynx and PepSeq software, in conjunction with information about possible allotype sequences from
DrugBank, allowed the identification of the biosimilar candidate drug sequence in a single UHPLC–MS experiment. Clear differences
in the levels of glycosylation observed in LC–MS experiment of intact and denatured MAbs were confirmed by an LC–FLR experiment
with released labeled glycans detected by fluorescence. The proposed set of experiments is robust and suitable for characterization
and quality monitoring of biopharmaceutical proteins at all stages of drug development.
MARTIN GILAR, PHD, is a principal researcher, HONGWEI XIE, PHD, is a senior research scientist, ASISH CHAKRABORTY, PHD, is a senior chemist, JOOMI AHN is a senior research chemist, YING QING YU, PHD, is a principal chemist, WEIBIN CHEN, PHD, is a principal chemist, ST. JOHN SKILTON, PHD, is the senior marketing manager, and JEFFERY
R. MAZZEO, PHD, is the biopharmaceutical business director, all at Waters Corporation, Milford, MA, 508.482.3119, firstname.lastname@example.org
DEEPALAKSHMI P. DAKSHINAMOORTHY is a senior applications specialist at Waters India Pvt Ltd, Bangalore, India.