Identification and Quantitative Study on Glycosylation Chain Modification of Antibody Drugs by Mass Spectrometry

doi:10.13523/j.cb.20180104

China Biotechnology

2018, Vol. 38

Issue (1): 32-41 DOI: 10.13523/j.cb.20180104

Orginal Article

Identification and Quantitative Study on Glycosylation Chain Modification of Antibody Drugs by Mass Spectrometry

Yi HUANG^1,²,Xiao-yu LI²,Fang TIAN²,Xiao-hong QIAN²,Wan-tao YING^1,²(

)

1 Anhui Medical University,Hefei 230032,China
2 State Key Laboratory of Proteomics, Beijing Proteome Research Center,National Center for Protein Science,Beijing Institute of Radiation Medicine, Beijing 102206,China

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Abstract Objective:

To establish a qualitative and quantitative method for the determination of antibody glycosylation from the level of intact glycopeptides based on LC-MS, and to apply it to qualitative and quantitative analysis of biomimetic drugs.

Methods:

The qualitative and quantitative analysis of glycoforms was carried out from the level of intact glycopeptides, and compared with the results from UPLC-FLR, the traditional glycan analysis approach. The optimal analysis strategy was established and used for the analysis of N-glycans on antibody biosimilars.

Result:

The UPLC-FLR methods obtained the quantitative result of seven glycoforms from bevacizumab; While the method of mass spectrometry quantified 19 glycoforms. After enrichment by hydrophilic interaction liquid chromatography (HILIC), 22 glycan forms were quantified. The quantitative results of the glycosylation mass of the trastuzumab showed good agreement.

Conclusion:

Based on the method of LC-MS, it has the advantages of fast speed, high sensitivity and high detection of glycoforms, and it is expected to be a stable quantitative strategy for site - specific glycosylation analysis of biopharmaceuticals.

Key words： UPLC-FLR LC-MS Antibody drugs Intact glycopeptides quantification

Received: 18 July 2017 Published: 31 January 2018

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Cite this article:

Yi HUANG,Xiao-yu LI,Fang TIAN,Xiao-hong QIAN,Wan-tao YING. Identification and Quantitative Study on Glycosylation Chain Modification of Antibody Drugs by Mass Spectrometry. China Biotechnology, 2018, 38(1): 32-41.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180104 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I1/32

Fig.1 Experimental design

Fig.2 Chromatograms of N-glycans from Bevacizumab after fluorescent labeling

Fig.3 MS/MS spectrum of glycopeptide HN4H3F1_EEQYNSTYR from Bevacizumab

Fig.4 Quantitative result of glycoforms from UPLC-FLR analysis

Fig.5 Quantitative results of glycoforms after trypsin digestion

Fig.6 Quantitative results of glycoforms after HILIC enrichment

Fig.7 Correlative analysis of three methods for glycoforms identification results

Table 1 Comparison of three methods for the quantitatiion of glycans

Table 2 Comparison of three methods of parallel experimental RSD values

Table 3 Comparison of the quantitative results of the mixture of trastuzumab monoclonal antibody between three batches


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