Global characterization of modifications to the charge isomers of IgG antibody

Xinling Cui; Wei Mi; Zhishang Hu; Xiaoyu Li; Bo Meng; Xinyuan Zhao; Xiaohong Qian; Tao Zhu; Wantao Ying

doi:10.1016/j.jpha.2020.11.006

Volume 12 Issue 1

Feb. 2022

Turn off MathJax

Article Contents

Article Navigation > Journal of Pharmaceutical Analysis > 2022 > 12(1): 156-163

Xinling Cui, Wei Mi, Zhishang Hu, Xiaoyu Li, Bo Meng, Xinyuan Zhao, Xiaohong Qian, Tao Zhu, Wantao Ying. Global characterization of modifications to the charge isomers of IgG antibody[J]. Journal of Pharmaceutical Analysis, 2022, 12(1): 156-163. doi: 10.1016/j.jpha.2020.11.006

Citation:

Xinling Cui, Wei Mi, Zhishang Hu, Xiaoyu Li, Bo Meng, Xinyuan Zhao, Xiaohong Qian, Tao Zhu, Wantao Ying. Global characterization of modifications to the charge isomers of IgG antibody[J]. Journal of Pharmaceutical Analysis, 2022, 12(1): 156-163. doi: 10.1016/j.jpha.2020.11.006

Citation:

Xinling Cui, Wei Mi, Zhishang Hu, Xiaoyu Li, Bo Meng, Xinyuan Zhao, Xiaohong Qian, Tao Zhu, Wantao Ying. Global characterization of modifications to the charge isomers of IgG antibody[J]. Journal of Pharmaceutical Analysis, 2022, 12(1): 156-163. doi: 10.1016/j.jpha.2020.11.006

PDF( 2420 KB)

Global characterization of modifications to the charge isomers of IgG antibody

doi: 10.1016/j.jpha.2020.11.006

a. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China;
b. National Institute of Metrology, Center for Advanced Measurement Science, Beijing, China;
c. National Institute of Metrology, Chemical Metrology & Analytical Science Division, Beijing, China;
d. CanSino Bio Corporation Tianjin Key Laboratory of Recombinant Bacterial Recombination and Bacterial Vaccines, Tianjin, China

Funds:

We are grateful for the financial support from the National Key Program for Basic Research of China (Grant Nos.: 2018YFC0910302 and 2017YFF0205400), the National Natural Science Foundation of China (Grant No.: 81530021), and Innovation Foundation of Medicine (Grant Nos.: BWS14J052 and 16CXZ027).

Received Date: Mar. 12, 2020
Accepted Date: Nov. 17, 2020
Rev Recd Date: Nov. 08, 2020
Publish Date: Nov. 25, 2020

Abstract

Abstract

Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance, but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels. First, the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach; this showed differences in glycoforms and deamidation status. Second, at the peptide level, common modifications of oxidation, deamidation, and glycosylation were identified. Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs. In total, 10 N-glycoforms were detected by peptide mapping. Finally, an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides. Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms. The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity, which is otherwise missed in peptide mapping and intact molecular weight analyses. This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.
- Antibody,
- Charge isomers,
- Mass spectrometry,
- Posttranslational modification,
- Glycopeptide

FullText(HTML)

References(37)

References

F. Torkashvand, B. Vaziri, Main quality attributes of monoclonal antibodies and effect of cell culture components. Iran. Biomed. J. 21(2017)131-141

D. Reusch, M. Haberger, B. Maier, et al., Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles--part 1:separation-based methods. mAbs. 7(2015)167-179

M. K. Parr, O. Montacir, H. Montacir, Physicochemical characterization of biopharmaceuticals, J. Pharmaceut. Biomed. Anal. 130(2016)366-389

Y. Leblanc, C. Ramon, N. Bihoreau, et al., Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry:case study after a long-term storage at +5 degrees C. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1048(2017)130-139

S. A. Berkowitz, J. R. Engen, J. R. Mazzeo, et al., Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat. Rev. Drug Discov. 11(2012)527-540

R. J. Harris, B. Kabakoff, F. D. Macchi, et al., Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B Biomed. Sci. Appl. 752(2001)233-245

L. A. Khawli, S. Goswami, R. Hutchinson, et al., Charge variants in IgG1:isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs. 2(2010)613-624

H. Liu, G. Gaza-Bulseco, D. Faldu, et al., Heterogeneity of monoclonal antibodies. J. Pharm. Sci. 97(2008)2426-2447

Y. Du, A. Walsh, R. Ehrick, et al., Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. mAbs. 4(2012)578-585

S. Fekete, A. Beck, D. Guillarme, Characterization of cation exchanger stationary phases applied for the separations of therapeutic monoclonal antibodies. J. Pharmaceut. Biomed. Anal. 111(2015)169-176

K. Takeo, T. Tanaka, K. Nakamura, et al., Studies on the heterogeneity of anti-hapten antibodies by means of two-dimensional affinity electrophoresis. Electrophoresis. 10(1989)818-824

S. Fekete, A. Beck, J. L. Veuthey, et al., Ion-exchange chromatography for the characterization of biopharmaceuticals. J. Pharmaceut. Biomed. Anal. 113(2015)43-55

N. Lingg, M. Berndtsson, B. Hintersteiner, et al., Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range:validation of the method parameters. J. Chromatogr., A 1373(2014)124-130

S. Fekete, A. Beck, J. Fekete, et al., Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I:salt gradient approach. J. Pharmaceut. Biomed. Anal. 102(2015)33-44

S. Fekete, A. Beck, J. Fekete, et al., Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II:pH gradient approach. J. Pharmaceut. Biomed. Anal. 102(2015)282-289

F. Fussl, K. Cook, K. Scheffler, et al., Charge variant analysis of monoclonal antibodies using direct coupled pH gradient cation exchange chromatography to high-resolution native mass spectrometry. Anal. Chem. 90(2018)4669-4676

F. Fussl, Trappe A., Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. mAbs. 11(2019)116-128

A. Goyon, M. Excoffier, M. C. Janin-Bussat, et al., Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1065-1066(2017)119-128

A. Hirayama, H. Abe, N. Yamaguchi, et al., Development of a sheathless CE-ESI-MS interface. Electrophoresis. 39(2018)1382-1389

N. A. Kulak, G. Pichler, I. Paron, et al., Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11(2014)319-324

A. Makarov, E. Denisov, Dynamics of ions of intact proteins in the Orbitrap mass analyzer. J. Am. Soc. Mass Spectrom. 20(2009)1486-1495

M. W. Senko, S. C. Beu, F. W. McLaffertycor, Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J. Am. Soc. Mass Spectrom. 6(1995)229-233

D. Chelius, D. S. Rehder, P. V. Bondarenko, Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal. Chem. 77(2005)6004-6011

I. Sokolowska, J. Mo, J. Dong, et al., Subunit mass analysis for monitoring antibody oxidation. mAbs. 9(2017)498-505

R. Jefferis, Glyco-engineering of human IgG-fc to modulate biologic activities. Curr. Pharmaceut. Biotechnol. 17(2016)1333-1347

Y. Gavrilov, D. Shental-Bechor, H. M. Greenblatt, et al., Glycosylation may reduce protein thermodynamic stability by inducing a conformational distortion. J. Phys. Chem. Lett. 6(2015)3572-3577

Y. Lyubarskaya, D. Houde, J. Woodard, et al., Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity. Anal. Biochem. 348(2006)24-39

J. Vlasak, M. C. Bussat, S. Wang, et al., Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal. Biochem. 392(2009)145-154

H. Chi, C. Liu, H. Yang, et al., Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine. Nat. Biotechnol. 10(2018)1038

H. Chi, K. He, B. Yang, et al., pFind-Alioth:A novel unrestricted database search algorithm to improve the interpretation of high-resolution MS/MS data. J. Proteomics. 125(2015)89-97

S. Tyanova, T. Temu, J. Cox, The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11(2016)2301-2319

V. Timm, P. Gruber, M. Wasiliu, et al., Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies. J. Chromatog.r B Analyt. Technol. Biomed. Life Sci. 878(2010)777-784

L. Y. Lee, E. S. Moh, B. L. Parker, et al., Toward automated N-glycopeptide identification in glycoproteomics. J. Proteome Res. 15(2016)3904-3915

M. Bern, Y. J. Kil, C. Becker, Byonic:advanced peptide and protein identification software. Curr. Protoc. Bioinformatics. 13(2012)13-20

N. M. Riley, A. S. Hebert, M. S. Westphall, et al., Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis. Nat. Commun. 10(2019)1311

P. H. Jensen, S. Mysling, P. Hojrup, et al., Glycopeptide enrichment for MALDI-TOF mass spectrometry analysis by hydrophilic interaction liquid chromatography solid phase extraction (HILIC SPE). Methods Mol. Biol. 951(2013)131-144

Q. Dong, X. Yan, Y. Liang, et al., In-depth characterization and spectral library building of glycopeptides in the tryptic digest of a monoclonal antibody using 1D and 2D LC-MS/MS. J. Proteome Res. 15(2016)1472-1486

Relative Articles

Supplements(0)

Cited By

Proportional views