Quantitative characterization of intact sialylated O-glycans with MALDI-MS for protein biotherapeutics

Cheol-Hwan Hwang; Hae-Min Park; Han-Gyu Park; Da-Hee Ahn; Seong-Min Kim; Byoung Joon Ko; Young Hwan Kim; Yung-Hun Yang; Yun-Gon Kim

Korean Journal of Chemical Engineering, Vol.35, No.7, 1462-1467, July, 2018

DOI10.1007/s11814-018-0058-0 Export Citation

Quantitative characterization of intact sialylated O-glycans with MALDI-MS for protein biotherapeutics

Cheol-Hwan Hwang, Hae-Min Park¹, Han-Gyu Park, Da-Hee Ahn, Seong-Min Kim, Byoung Joon Ko², Young Hwan Kim³, Yung-Hun Yang⁴, and Yun-Gon Kim^†

Department of Chemical Engineering, Soongsil University, Seoul 06978, Korea
¹Department of Chemistry, Northwestern University, Evanston, Illinois 60208, U.S.A., USA
²New Drug Development Center, Osong Medical Innovation Foundation, Cheongju 28160, Korea
³Biomedical Omics Team, Korea Basic Science Institute (KBSI), Cheongju 28119, Korea
⁴Department of Biological Engineering, Konkuk University, Seoul 05029, Korea

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For validating O-glycosylation of protein biotherapeutics, we presented a quantitative O-glycomics method which is based on the neutralization of sialic acids, the specific release of O-glycans, and the introduction of permanent positive charge followed by quantitative MALDI-MS analysis. This method shows excellent technical reproducibility, linearity and sensitivity. In addition, it enables the quantification of intact O-glycans with minimal degradation or loss of sialic acids on these glycans compared to a conventional HPLC-based method. We then applied this method to quantitatively characterize O-glycans present on Etanercept. The analysis showed the relative abundances of mono- and di-sialylated core 1 O-glycans - were 79.3±0.8% and 17.3±1.4%, respectively. This glycomics technology could allow for the reliable quantitative analysis of intact O-glycans from glycoproteins and may contribute to validation of O-glycosylation protein biotherapeutics in the pharmaceutical industry.

Keywords:Chemical Derivatization;MALDI-MS;O-glycan;Protein Biotherapeutics;Quantitative Analysis

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[2] Sola RJ, Griebenow K, BioDrugs, 24, 9 (2010)

[3] Sinclair AM, Elliott S, J. Pharm. Sci., 94, 1626 (2005)

[4] Sola RJ, Griebenow K, J. Pharm. Sci., 98, 1223 (2009)

[5] Kang P, Mechref Y, Kyselova Z, Goetz JA, Novotny MV, Anal. Chem., 79, 6064 (2007)

[6] Tao WA, Aebersold R, Curr. Opin. Biotechnol., 14, 110 (2003)

[7] Alvarez-Manilla G, Warren NL, Abney T, Atwood J, 3rd, Azadi P, York WS, Pierce M, Orlando R, Glycobiology, 17, 677 (2007)

[8] Wang C, Zhang P, Jin W, Li L, Qiang S, Zhang Y, Huang L, Wang Z, J. Proteomics, 150, 18 (2017)

[9] Toyoda M, Ito H, Matsuno YK, Narimatsu H, Kameyama A, Anal. Chem., 80, 5211 (2008)

[10] Gil GC, Iliff B, Cerny R, Velander WH, Van Cott KE, Anal. Chem., 82, 6613 (2010)

[11] Jeong HJ, Adhya M, Park HM, Kim YG, Kim BG, Xenotransplantation, 20, 407 (2013)

[12] Wang C, Wu Z, Yuan J, Wang B, Zhang P, Zhang Y, Wang Z, Huang L, J. Proteome. Res., 13, 372 (2014)

[13] Shinohara Y, Furukawa J, Niikura K, Miura N, Nishimura S, Anal. Chem., 76, 6989 (2004)

[14] Kim YW, Sung C, Lee S, Kim KJ, Yang YH, Kim BG, Lee YK, Ryu HW, Kim YG, Anal. Chem., 87, 858 (2015)

[15] Kim KJ, Kim HJ, Park HG, Hwang CH, Sung C, Jang KS, Sci. Rep., 6, 24489 (2016)

[16] Kim KJ, Kim YW, Hwang CH, Park HG, Yang YH, Koo M, Kim YG, Biotechnol. Lett., 37(10), 2019 (2015)

[17] Liu L, Gomathinayagam S, Hamuro L, Prueksaritanont T, Wang W, Stadheim TA, Hamilton SR, Pharm. Res., 30, 803 (2013)

[18] Pennica D, Lam VT, Weber RF, Kohr WJ, Basa LJ, Spellman MW, Ashkenazi A, Shire SJ, Goeddel DV, Biochemistry, 32, 3131 (1993)

[19] DiPaola M, Li J, Stephens E, J. Bioanal. Biomed., 5, 5 (2013)

[20] Houel S, Hilliard M, Yu YQ, McLoughlin N, Martin SM, Rudd PM, Williams JP, Chen W, Anal. Chem., 86, 576 (2014)