Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated <i>N</i>-glycan linkage isomers in therapeutic glycoproteins

Chi Soo Park; Minju Kang; Ahyeon Kim; Chulmin Moon; Mirae Kim; Jieun Kim; Subin Yang; Leeseul Jang; Ji Yeon Jang; Ha Hyung Kim

doi:10.1016/j.jpha.2023.01.001

Volume 13 Issue 3

Mar. 2023

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Chi Soo Park, Minju Kang, Ahyeon Kim, Chulmin Moon, Mirae Kim, Jieun Kim, Subin Yang, Leeseul Jang, Ji Yeon Jang, Ha Hyung Kim. Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins[J]. Journal of Pharmaceutical Analysis, 2023, 13(3): 305-314. doi: 10.1016/j.jpha.2023.01.001

Citation:

Chi Soo Park, Minju Kang, Ahyeon Kim, Chulmin Moon, Mirae Kim, Jieun Kim, Subin Yang, Leeseul Jang, Ji Yeon Jang, Ha Hyung Kim. Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins[J]. Journal of Pharmaceutical Analysis, 2023, 13(3): 305-314. doi: 10.1016/j.jpha.2023.01.001

Citation:

Chi Soo Park, Minju Kang, Ahyeon Kim, Chulmin Moon, Mirae Kim, Jieun Kim, Subin Yang, Leeseul Jang, Ji Yeon Jang, Ha Hyung Kim. Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins[J]. Journal of Pharmaceutical Analysis, 2023, 13(3): 305-314. doi: 10.1016/j.jpha.2023.01.001

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Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins

doi: 10.1016/j.jpha.2023.01.001

Department of Global Innovative Drugs, Graduate School of Chung-Ang University, Seoul, 06974, Republic of Korea

Funds:

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Korea (Grant No.: 2021R1A6A1A03044296). This study was supported by the Chung-Ang University Graduate Research Scholarship in 2022.

Received Date: Oct. 13, 2022
Accepted Date: Jan. 14, 2023
Rev Recd Date: Dec. 29, 2022
Publish Date: Jan. 20, 2023

Abstract

Abstract

Sialylated N-glycan isomers with α2-3 or α2-6 linkage(s) have distinctive roles in glycoproteins, but are difficult to distinguish. Wild-type (WT) and glycoengineered (mutant) therapeutic glycoproteins, cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig), were produced in Chinese hamster ovary cell lines; however, their linkage isomers have not been reported. In this study, N-glycans of CTLA4-Igs were released, labeled with procainamide, and analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) to identify and quantify sialylated N-glycan linkage isomers. The linkage isomers were distinguished by comparison of 1) intensity of the N-acetylglucosamine ion to the sialic acid ion (Ln/Nn) using different fragmentation stability in MS/MS spectra and 2) retention time-shift for a selective m/z value in the extracted ion chromatogram. Each isomer was distinctively identified, and each quantity (>0.1%) was obtained relative to the total N-glycans (100%) for all observed ionization states. Twenty sialylated N-glycan isomers with only α2-3 linkage(s) in WT were identified, and each isomer's sum of quantities was 50.4%. Furthermore, 39 sialylated N-glycan isomers (58.8%) in mono- (3 N-glycans; 0.9%), bi- (18; 48.3%), tri- (14; 8.9%), and tetra- (4; 0.7%) antennary structures of mutant were obtained, which comprised mono- (15 N-glycans; 25.4%), di- (15; 28.4%), tri- (8; 4.8%), and tetra- (1; 0.2%) sialylation, respectively, with only α2-3 (10 N-glycans; 4.8%), both α2-3 and α2-6 (14; 18.4%), and only α2-6 (15; 35.6%) linkage(s). These results are consistent with those for α2-3 neuraminidase-treated N-glycans. This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.
- Therapeutic glycoprotein,
- Sialylation,
- Linkage isomer,
- LC-MS/MS

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