Rapid Assessment of Molecular Similarity between a Candidate Biosimilar and an Innovator Monoclonal Antibody Using Complementary LC–MS Methods - Intact protein LC–MS detected a mass varian

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Rapid Assessment of Molecular Similarity between a Candidate Biosimilar and an Innovator Monoclonal Antibody Using Complementary LC–MS Methods
Intact protein LC–MS detected a mass variance of 62 Da and peptide mapping located a difference of two amino acids.


BioPharm International Supplements


Analysis of Glycans


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.
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.

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.

Conclusions

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,
DEEPALAKSHMI P. DAKSHINAMOORTHY is a senior applications specialist at Waters India Pvt Ltd, Bangalore, India.


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