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In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

Song T, Ozcan S, Becker A, Lebrilla CB - Anal. Chem. (2014)

Bottom Line: The complete structures were obtained through exoglycosidase sequencing.The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances.The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , One Shields Avenue, Davis, California 95616, United States.

ABSTRACT
The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs' many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

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Chromatograms of 13 overlaid injections showing the majorN-glycansfrom Panitumumab acquired on nanoLC-Chip-TOF. Injections were performedconsecutively. The largest variation in retention time correspondedto 0.10 min.
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fig2: Chromatograms of 13 overlaid injections showing the majorN-glycansfrom Panitumumab acquired on nanoLC-Chip-TOF. Injections were performedconsecutively. The largest variation in retention time correspondedto 0.10 min.

Mentions: In order to further evaluate the robustness of using LC retentiontimes to identify isomers from the N-glycan library, the retentiontime shift between different runs within 1 day of rMab N-glycans wasexamined. The retention time repeatability was high with the largestshift in retention time correspond to approximately 0.1 min in a 60min gradient (Figure 2); however, the shiftsare generally much less. This corresponding CV was calculated to be∼0.6%.


In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs.

Song T, Ozcan S, Becker A, Lebrilla CB - Anal. Chem. (2014)

Chromatograms of 13 overlaid injections showing the majorN-glycansfrom Panitumumab acquired on nanoLC-Chip-TOF. Injections were performedconsecutively. The largest variation in retention time correspondedto 0.10 min.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4066919&req=5

fig2: Chromatograms of 13 overlaid injections showing the majorN-glycansfrom Panitumumab acquired on nanoLC-Chip-TOF. Injections were performedconsecutively. The largest variation in retention time correspondedto 0.10 min.
Mentions: In order to further evaluate the robustness of using LC retentiontimes to identify isomers from the N-glycan library, the retentiontime shift between different runs within 1 day of rMab N-glycans wasexamined. The retention time repeatability was high with the largestshift in retention time correspond to approximately 0.1 min in a 60min gradient (Figure 2); however, the shiftsare generally much less. This corresponding CV was calculated to be∼0.6%.

Bottom Line: The complete structures were obtained through exoglycosidase sequencing.The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances.The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California , One Shields Avenue, Davis, California 95616, United States.

ABSTRACT
The glycosylation in recombinant monoclonal antibody (rMab) drugs is a major concern in the biopharmaceutical industry as it impacts the drugs' many attributes. Characterization is important but complicated by the intricate structures, microheterogeneity, and the limitations of current tools for structural analysis. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) N-glycan library based on eight commercial rMab drugs. A library of over 70 structures was developed for the rapid characterization of rMab. N-Glycans were separated on a porous graphitized carbon (PGC) column incorporated on a chip and then analyzed by an electrospray ionization hybrid quadrupole time-of-flight (ESI-Q-TOF) MS. The retention time and accurate mass for each N-glycan were recorded in the library. The complete structures were obtained through exoglycosidase sequencing. The results showed that most of the N-glycans between different antibodies are nearly the same with different abundances. The utility of this library enables one to identify structures in a rapid manner by matching LC retention times and accurate masses.

Show MeSH