“Small is beautiful”-examining reliable determination of low-abundant therapeutic antibody glycovariants

Katharina Böttinger; Christof Regl; Veronika Schäpertöns; Erdmann Rapp; Therese Wohlschlager; Christian G. Huber

doi:10.1016/j.jpha.2024.100982

Article Contents

Article Navigation > Journal of Pharmaceutical Analysis > 2024 > Accepted Manu

Katharina Böttinger, Christof Regl, Veronika Schäpertöns, Erdmann Rapp, Therese Wohlschlager, Christian G. Huber. “Small is beautiful”-examining reliable determination of low-abundant therapeutic antibody glycovariants[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2024.100982

Citation:

Katharina Böttinger, Christof Regl, Veronika Schäpertöns, Erdmann Rapp, Therese Wohlschlager, Christian G. Huber. “Small is beautiful”-examining reliable determination of low-abundant therapeutic antibody glycovariants[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2024.100982

Citation:

Katharina Böttinger, Christof Regl, Veronika Schäpertöns, Erdmann Rapp, Therese Wohlschlager, Christian G. Huber. “Small is beautiful”-examining reliable determination of low-abundant therapeutic antibody glycovariants[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2024.100982

PDF( 14623 KB)

“Small is beautiful”-examining reliable determination of low-abundant therapeutic antibody glycovariants

doi: 10.1016/j.jpha.2024.100982

a Department of Biosciences and Medical Biology, Bioanalytical Research Labs, University of Salzburg, Salzburg, 5020, Salzburg, Austria;
b Center for Tumorbiology and Immunology(CTBI), University of Salzburg, 5020, Salzburg, Austria;
c glyXera GmbH, Magdeburg, 39014, Sachsen-Anhalt, Germany;
d Max Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany

Received Date: Jan. 09, 2024
Rev Recd Date: Mar. 21, 2024
Available Online: Apr. 29, 2024

Abstract

Abstract

Glycans associated with biopharmaceutical drugs play crucial roles in drug safety and efficacy, and therefore, their reliable detection and quantification is essential. Our study introduces a multi-level quantification approach for glycosylation analysis in monoclonal antibodies, focusing on minor abundant glycovariants. Mass spectrometric data is evaluated mainly employing open-source software tools. Released N-glycan and glycopeptide data form the basis for integrating information across different structural levels up to intact glycoproteins. Comprehensive comparison showed that indeed, variations across structural levels were observed especially for minor abundant species. Utilizing MoFi (short for modification finder), a tool for annotating mass spectra of intact proteins, we quantify isobaric glycosylation variants at the intact protein level. Our workflow's utility is demonstrated on NISTmAb, rituximab and adalimumab, profiling their minor abundant variants for the first time across diverse structural levels. This study enhances understanding and accessibility in glycosylation analysis, spotlighting minor abundant glycovariants in therapeutic antibodies.
- Glycosylation,
- Mass spectrometry,
- Monoclonal antibodies,
- Abundance profiling,
- Minor glycovariants,
- Multi-level analysis,
- Data integration

FullText(HTML)

References(66)

References

[1]	C. Reily, T.J. Stewart, M.B. Renfrow, J. Novak, Glycosylation in health and disease, Nat. Rev. Nephrol. 2019156. 15(2019) 346-366. https://doi.org/10.1038/s41581-019- 0129-4.
[2]	M. Schiestl, T. Stangler, C. Torella, T. Čepeljnik, H. Toll, R. Grau, Acceptable changes in quality attributes of glycosylated biopharmaceuticals, Nat. Biotechnol. 29(2011) 310- 312. https://doi.org/10.1038/nbt.1839.
[3]	T. Wohlschlager, K. Scheffler, I.C. Forstenlehner, W. Skala, S. Senn, E. Damoc, J. Holzmann, C.G. Huber, Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals, Nat. Commun. 9(2018) 1-9. https://doi.org/10.1038/s41467-018-04061-7.
[4]	A. Beck, H. Liu, Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies, Antibodies. 8(2019) 18. https://doi.org/10.3390/antib8010018.
[5]	M. Thomann, K. Reckermann, D. Reusch, J. Prasser, M.L. Tejada, Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies, Mol. Immunol. 73(2016) 69-75. https://doi.org/10.1016/j.molimm.2016.03.002.
[6]	Y. Kaneko, F. Nimmerjahn, J. V. Ravetch, Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation, Science (80-.). 313(2006) 670-673. https://doi.org/10.1126/SCIENCE.1129594/SUPPL_FILE/KANEKO.SOM.PDF.
[7]	V.S. Shivatare, P.K. Chuang, T.H. Tseng, Y.F. Zeng, H.W. Huang, G. Veeranjaneyulu, H.C. Wu, C.H. Wong, Study on antibody Fc-glycosylation for optimal effector functions, Chem. Commun. (2023). https://doi.org/10.1039/d3cc00672g.
[8]	S.A. Berkowitz, J.R. Engen, J.R. Mazzeo, G.B. Jones, Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars, Nat. Rev. Drug Discov. 11(2012) 527-540. https://doi.org/10.1038/nrd3746.
[9]	ICH HARMONISED TRIPARTITE GUIDELINE:Specifications:Test Procedures and Acceptance Criteria for Biotechnological/Biological Products-Q6B, (n.d.). https://database.ich.org/sites/default/files/Q6B Guideline.pdf.
[10]	S. Carillo, R. Pérez-Robles, C. Jakes, M. Ribeiro da Silva, S. Millán Martín, A. Farrell, N. Navas, J. Bones, Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies, J. Pharm. Anal. 10(2020) 23-34. https://doi.org/10.1016/j.jpha.2019.11.008.
[11]	N. De Haan, M. Pučić-Baković, M. Novokmet, D. Falck, G. Lageveen-Kammeijer, G. Razdorov, F. Vučković, I. Trbojević-Akmačić, O. Gornik, M. Hanić, M. Wuhrer, G. Lauc, A. Guttman, R. Cummings, S. Mora, Y. Rombouts, A. Mehta, Developments and perspectives in high-throughput protein glycomics:enabling the analysis of thousands of samples, Glycobiology. 32(2022) 651-663. https://doi.org/10.1093/glycob/cwac026.
[12]	X. Liu, Z. Sun, Z. Li, Y. Zhang, H. Lu, Mass spectrometry-based analysis of IgG glycosylation and its applications, Int. J. Mass Spectrom. 474(2022) 116799. https://doi.org/10.1016/j.ijms.2022.116799.
[13]	W. Skala, T. Wohlschlager, S. Senn, G.E. Huber, C.G. Huber, MoFi:A Software Tool for Annotating Glycoprotein Mass Spectra by Integrating Hybrid Data from the Intact Protein and Glycopeptide Level, Anal. Chem. 90(2018) 5728-5736. https://doi.org/10.1021/acs.analchem.8b00019.
[14]	S. Millán-Martín, C. Jakes, S. Carillo, T. Buchanan, M. Guender, D.B. Kristensen, T.M. Sloth, M. Ørgaard, K. Cook, J. Bones, Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes, Anal. Bioanal. Chem. (2020) 1-16. https://doi.org/10.1007/s00216-020-02809-z.
[15]	C.I. Butré, V. D'Atri, H. Diemer, O. Colas, E. Wagner, A. Beck, S. Cianferani, D. Guillarme, A. Delobel, Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody, Mol. 2023, Vol. 28, Page 2855. 28(2023) 2855. https://doi.org/10.3390/MOLECULES28062855.
[16]	M.L.A. De Leoz, D.L. Duewer, A. Fung, L. Liu, H.K. Yau, O. Potter, G.O. Staples, K. Furuki, R. Frenkel, Y. Hu, Z. Sosic, P. Zhang, F. Altmann, C. Grunwald-Grube, C. Shao, J. Zaia, W. Evers, S. Pengelley, D. Suckau, A. Wiechmann, A. Resemann, W. Jabs, A. Beck, J.W. Froehlich, C. Huang, Y. Li, Y. Liu, S. Sun, Y. Wang, Y. Seo, H.J. An, N.C. Reichardt, J.E. Ruiz, S. Archer-Hartmann, P. Azadi, L. Bell, Z. Lakos, Y. An, J.F. Cipollo, M. Pucic-Bakovic, J. Štambuk, G. Lauc, X. Li, P.G. Wang, A. Bock, R. Hennig, E. Rapp, M. Creskey, T.D. Cyr, M. Nakano, T. Sugiyama, P.K.A. Leung, P. LinkLenczowski, J. Jaworek, S. Yang, H. Zhang, T. Kelly, S. Klapoetke, R. Cao, J.Y. Kim, H.K. Lee, J.Y. Lee, J.S. Yoo, S.R. Kim, S.K. Suh, N. De Haan, D. Falck, G.S.M. Lageveen-Kammeijer, M. Wuhrer, R.J. Emery, R.P. Kozak, L.P. Liew, L. Royle, P.A. Urbanowicz, N.H. Packer, X. Song, A. Everest-Dass, E. Lattová, S. Cajic, K. Alagesan, D. Kolarich, T. Kasali, V. Lindo, Y. Chen, K. Goswami, B. Gau, R. Amunugama, R. Jones, C.J.M. Stroop, K. Kato, H. Yagi, S. Kondo, C.T. Yuen, A. Harazono, X. Shi, P.E. Magnelli, B.T. Kasper, L. Mahal, D.J. Harvey, R. O'Flaherty, P.M. Rudd, R. Saldova, E.S. Hecht, D.C. Muddiman, J. Kang, P. Bhoskar, D. Menard, A. Saati, C. Merle, S. Mast, S. Tep, J. Truong, T. Nishikaze, S. Sekiya, A. Shafer, S. Funaoka, M. Toyoda, P. De Vreugd, C. Caron, P. Pradhan, N.C. Tan, Y. Mechref, S. Patil, J.S. Rohrer, R. Chakrabarti, D. Dadke, M. Lahori, C. Zou, C. Cairo, B. Reiz, R.M. Whittal, C.B. Lebrilla, L. Wu, A. Guttman, M. Szigeti, B.G. Kremkow, K.H. Lee, C. Sihlbom, B. Adamczyk, C. Jin, N.G. Karlsson, J. Örnros, G. Larson, J. Nilsson, B. Meyer, A. Wiegandt, E. Komatsu, H. Perreault, E.D. Bodnar, N. Said, Y.N. Francois, E. LeizeWagner, S. Maier, A. Zeck, A.J.R. Heck, Y. Yang, R. Haselberg, Y.Q. Yu, W. Alley, J.W. Leone, H. Yuan, S.E. Stein, NIST interlaboratory study on glycosylation analysis of monoclonal antibodies:Comparison of results from diverse analytical methods, Mol. Cell. Proteomics. 19(2020) 11-30. https://doi.org/10.1074/mcp.RA119.001677.
[17]	M. Bern, T. Caval, Y.J. Kil, W. Tang, C. Becker, E. Carlson, D. Kletter, K.I. Sen, N. Galy, D. Hagemans, V. Franc, A.J.R. Heck, Parsimonious Charge Deconvolution for Native Mass Spectrometry, J. Proteome Res. 17(2018) 1216-1226. https://doi.org/10.1021/acs.jproteome.7b00839.
[18]	S. Millán-Martín, S. Carillo, F. Füssl, J. Sutton, P. Gazis, K. Cook, K. Scheffler, J. Bones, Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry, Eur. J. Pharm. Biopharm. 158(2021) 83-95. https://doi.org/10.1016/j.ejpb.2020.11.006.
[19]	K. Böttinger, W. Esser-Skala, M. Segl, C. Herwig, C.G. Huber, At-line quantitative profiling of monoclonal antibody products during bioprocessing using HPLC-MS, Anal. Chim. Acta. 1207(2022) 339813. https://doi.org/10.1016/J.ACA.2022.339813.
[20]	F. Di Marco, T. Berger, W. Esser-skala, E. Rapp, C. Regl, C.G. Huber, Simultaneous monitoring of monoclonal antibody variants by strong cation-exchange chromatography hyphenated to mass spectrometry to assess quality attributes of rituximab-based biotherapeutics, Int. J. Mol. Sci. 22(2021). https://doi.org/10.3390/ijms22169072.
[21]	B. MacLean, D.M. Tomazela, N. Shulman, M. Chambers, G.L. Finney, B. Frewen, R. Kern, D.L. Tabb, D.C. Liebler, M.J. MacCoss, Skyline:an open source document editor for creating and analyzing targeted proteomics experiments, Bioinformatics. 26(2010) 966-968. https://doi.org/10.1093/BIOINFORMATICS/BTQ054.
[22]	W. Esser-Skala, T. Wohlschlager, C. Regl, C.G. Huber, A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N-glycosylation in Biopharmaceuticals, Angew. Chemie Int. Ed. (2020) anie.202002147. https://doi.org/10.1002/anie.202002147.
[23]	M. Lebede, F. Di Marco, W. Esser-Skala, R. Hennig, T. Wohlschlager, C.G. Huber, Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes:An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry, Anal. Chem. 93(2021) 10424-10434. https://doi.org/10.1021/ACS.ANALCHEM.1C02199/SUPPL_FILE/AC1C02199_SI_003.ZIP.
[24]	R. Hennig, S. Cajic, M. Borowiak, M. Hoffmann, R. Kottler, U. Reichl, E. Rapp, Towards personalized diagnostics via longitudinal study of the human plasma Nglycome, Biochim. Biophys. Acta-Gen. Subj. 1860(2016) 1728-1738. https://doi.org/10.1016/J.BBAGEN.2016.03.035.
[25]	C.H. Gao, G. Yu, P. Cai, ggVennDiagram:An Intuitive, Easy-to-Use, and Highly Customizable R Package to Generate Venn Diagram, Front. Genet. 12(2021) 1598. https://doi.org/10.3389/FGENE.2021.706907/BIBTEX.
[26]	N. Hulstaert, J. Shofstahl, T. Sachsenberg, M. Walzer, H. Barsnes, L. Martens, Y. PerezRiverol, ThermoRawFileParser:Modular, Scalable, and Cross-Platform RAW File Conversion, J. Proteome Res. 19(2020) 537-542. https://doi.org/10.1021/acs.jproteome.9b00328.
[27]	H. Wickham, W. Chang, L. Henry, T.L. Pedersen, K. Takahashi, C. Wilke, K. Woo, H. Yutani, D. Dunnington, ggplot2:Create Elegant Data Visualisations Using the Grammar of Graphics, Springer-Verlag New York, 2016. https://ggplot2.tidyverse.org/(accessed February 14, 2023).
[28]	S. Xu, M. Chen, T. Feng, L. Zhan, L. Zhou, G. Yu, Use ggbreak to Effectively Utilize Plotting Space to Deal With Large Datasets and Outliers, Front. Genet. 12(2021) 2122. https://doi.org/10.3389/FGENE.2021.774846/BIBTEX.
[29]	Z. Gu, Complex heatmap visualization, IMeta. 1(2022) e43. https://doi.org/10.1002/imt2.43.
[30]	Z. Gu, R. Eils, M. Schlesner, Complex heatmaps reveal patterns and correlations in multidimensional genomic data, Bioinformatics. 32(2016) 2847-2849. https://doi.org/10.1093/bioinformatics/btw313.
[31]	S. Cajic, R. Hennig, R. Burock, E. Rapp, Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis, NLM (Medline), 2021. https://doi.org/10.1007/978-3-030-76912-3_4.
[32]	S. Cajic, R. Hennig, V. Grote, U. Reichl, E. Rapp, Removable Dyes-The Missing Link for In-Depth N-Glycan Analysis via Multi-Method Approaches, Engineering. 26(2023) 132-150. https://doi.org/10.1016/J.ENG.2023.02.016.
[33]	M. Hilliard, W.R. Alley, C.A. McManus, Y.Q. Yu, S. Hallinan, J. Gebler, P.M. Rudd, Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution:From sample preparation to data analysis, MAbs. 9(2017) 1349-1359. https://doi.org/10.1080/19420862.2017.1377381.
[34]	J. Zhao, W. Peng, X. Dong, Y. Mechref, Analysis of NIST Monoclonal Antibody Reference Material Glycosylation Using the LC-MS/MS-Based Glycoproteomic Approach, J. Proteome Res. 20(2021) 818-830. https://doi.org/10.1021/acs.jproteome.0c00659.
[35]	J.O. Kafader, R.D. Melani, L.F. Schachner, A.N. Ives, S.M. Patrie, N.L. Kelleher, P.D. Compton, Native vs Denatured:An in Depth Investigation of Charge State and Isotope Distributions, J. Am. Soc. Mass Spectrom. 31(2020) 574-581. https://doi.org/10.1021/jasms.9b00040.
[36]	M. Bi, B. Bai, Z. Tian, Structure-Specific N-Glycoproteomics Characterization of NIST Monoclonal Antibody Reference Material 8671, J. Proteome Res. 21(2022) 1276-1284. https://doi.org/10.1021/acs.jproteome.2c00027.
[37]	W. Zhu, M. Li, J. Zhang, Integrating Intact Mass Analysis and Middle-Down Mass Spectrometry Approaches to Effectively Characterize Trastuzumab and Adalimumab Structural Heterogeneity, J. Proteome Res. 20(2021) 270-278. https://doi.org/10.1021/acs.jproteome.0c00373.
[38]	J. Liu, T. Eris, C. Li, S. Cao, S. Kuhns, Assessing Analytical Similarity of Proposed Amgen Biosimilar ABP 501 to Adalimumab, BioDrugs. 30(2016) 321-338. https://doi.org/10.1007/s40259-016-0184-3.
[39]	Y. Yan, A.P. Liu, S. Wang, T.J. Daly, N. Li, Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry, Anal. Chem. 90(2018) 13013- 13020. https://doi.org/10.1021/acs.analchem.8b03773.
[40]	O. Montacir, H. Montacir, M. Eravci, A. Springer, S. Hinderlich, A. Saadati, M.K. Parr, Comparability study of Rituximab originator and follow-on biopharmaceutical, J. Pharm. Biomed. Anal. 140(2017) 239-251. https://doi.org/10.1016/j.jpba.2017.03.029.
[41]	B.L. Duivelshof, S. Denorme, K. Sandra, X. Liu, A. Beck, M.A. Lauber, D. Guillarme, V. D'atri, Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis, Pharmaceutics. 13(2021) 1744. https://doi.org/10.3390/pharmaceutics13111744.
[42]	T. Mouchahoir, J.E. Schiel, Development of an LC-MS/MS peptide mapping protocol for the NISTmAb, Anal. Bioanal. Chem. 410(2018) 2111-2126. https://doi.org/10.1007/s00216-018-0848-6.
[43]	C.-H. Chen, H. Feng, R. Guo, P. Li, A.K.C. Laserna, Y. Ji, B.H. Ng, S.F.Y. Li, S.H. Khan, A. Paulus, S.-M. Chen, A.E. Karger, M. Wenz, D.L. Ferrer, A.F. Huhmer, A. Krupke, Intact NIST monoclonal antibody characterization-Proteoforms, glycoforms-Using CE-MS and CE-LIF, Cogent Chem. 4(2018) 1480455. https://doi.org/10.1080/23312009.2018.1480455.
[44]	C. Grünwald-Gruber, A. Thader, D. Maresch, T. Dalik, F. Altmann, Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry, Anal. Bioanal. Chem. 409(2017) 2519-2530. https://doi.org/10.1007/s00216-017-0235- 8.
[45]	K. Stavenhagen, H. Hinneburg, M. Thaysen-Andersen, L. Hartmann, D.V. Silva, J. Fuchser, S. Kaspar, E. Rapp, P.H. Seeberger, D. Kolarich, Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity:an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides, J. Mass Spectrom. 48(2013) 627-639. https://doi.org/10.1002/JMS.3210.
[46]	T. Čaval, A. Buettner, M. Haberger, D. Reusch, A.J.R. Heck, Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches:A Case Study into the Glycosylation of Erythropoietin Variants, J. Am. Soc. Mass Spectrom. 32(2021) 2099-2104. https://doi.org/10.1021/jasms.1c00060.
[47]	B. Wang, Y. Tsybovsky, K. Palczewski, M.R. Chance, Reliable determination of site specific in vivo protein N-glycosylation based on collision-induced MS/MS and chromatographic retention time, J. Am. Soc. Mass Spectrom. 25(2014) 729-741. https://doi.org/10.1007/s13361-013-0823-6.
[48]	P. Kozlik, R. Goldman, M. Sanda, Study of structure-dependent chromatographic behavior of glycopeptides using reversed phase nanoLC, Electrophoresis. 38(2017) 2193-2199. https://doi.org/10.1002/elps.201600547.
[49]	F. Di Marco, C. Blöchl, W. Esser-Skala, V. Schäpertöns, T. Zhang, M. Wuhrer, K. Sandra, T. Wohlschlager, C.G. Huber, Glycoproteomics of a single protein:revealing hundreds of thousands of Myozyme^® glycoforms by hybrid HPLC-MS approaches, (2022). https://doi.org/10.26434/CHEMRXIV-2022-87TWX.
[50]	R.A. Kerr, D.A. Keire, H. Ye, The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry, MAbs. 11(2019) 930- 941. https://doi.org/10.1080/19420862.2019.1599632.
[51]	L.E. Kilpatrick, E.L. Kilpatrick, Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins, J. Proteome Res. 16(2017) 3255-3265. https://doi.org/10.1021/ACS.JPROTEOME.7B00244/ASSET/IMAGES/LARGE/PR- 2017-00244C_0004.JPEG.
[52]	H. Kaur, Characterization of glycosylation in monoclonal antibodies and its importance in therapeutic antibody development, Crit. Rev. Biotechnol. 41(2021) 300-315. https://doi.org/10.1080/07388551.2020.1869684.
[53]	A.M. Goetze, Y.D. Liu, Z. Zhang, B. Shah, E. Lee, P. V. Bondarenko, G.C. Flynn, Highmannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans, Glycobiology. 21(2011) 949-959. https://doi.org/10.1093/glycob/cwr027.
[54]	M. Yu, D. Brown, C. Reed, S. Chung, J. Lutman, E. Stefanich, A. Wong, J.P. Stephan, R. Bayer, Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans, MAbs. 4(2012) 475-487. https://doi.org/10.4161/mabs.20737.
[55]	B. Wei, K. Berning, C. Quan, Y.T. Zhang, Glycation of antibodies:Modification, methods and potential effects on biological functions, MAbs. 9(2017) 586-594. https://doi.org/10.1080/19420862.2017.1300214.
[56]	B. Chi, C. Veyssier, T. Kasali, F. Uddin, C.A. Sellick, At-line high throughput sitespecific glycan profiling using targeted mass spectrometry, Biotechnol. Reports. 25(2020) e00424. https://doi.org/10.1016/j.btre.2020.e00424.
[57]	A.R. Hines, M. Edgeworth, P.W.A. Devine, S. Shepherd, N. Chatterton, C. Turner, K.S. Lilley, X. Chen, N.J. Bond, Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation, J. Am. Soc. Mass Spectrom. 34(2023) 1330-1341. https://doi.org/10.1021/jasms.3c00037.
[58]	T. Wang, L. Chu, W. Li, K. Lawson, I. Apostol, T. Eris, Application of a Quantitative LC-MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites, Anal. Chem. 89(2017) 3562-3567. https://doi.org/10.1021/acs.analchem.6b04856.
[59]	S. Luo, B. Zhang, Benchmark Glycan Profile of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems, Pharm. Res. 1(2023) 1-9. https://doi.org/10.1007/s11095-023-03628-4.
[60]	L. Alessandri, D. Ouellette, A. Acquah, M. Rieser, D. LeBlond, M. Saltarelli, C. Radziejewski, T. Fujimori, I. Correia, Increased serum clearance of oligomannose species present on a human IgG1 molecule, MAbs. 4(2012) 509-520. https://doi.org/10.4161/mabs.20450.
[61]	J.E. Huffman, M. Pučić-Baković, L. Klarić, R. Hennig, M.H.J. Selman, F. Vučković, M. Novokmet, J. Krištić, M. Borowiak, T. Muth, O. Polašek, G. Razdorov, O. Gornik, R. Plomp, E. Theodoratou, A.F. Wright, I. Rudan, C. Hayward, H. Campbell, A.M. Deelder, U. Reichl, Y.S. Aulchenko, E. Rapp, M. Wuhrer, G. Lauc, Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research, Mol. Cell. Proteomics. 13(2014) 1598-1610. https://doi.org/10.1074/MCP.M113.037465.
[62]	C. Jakes, F. Füssl, I. Zaborowska, J. Bones, Rapid Analysis of Biotherapeutics Using Protein A Chromatography Coupled to Orbitrap Mass Spectrometry, Anal. Chem. (2021). https://doi.org/10.1021/acs.analchem.1c02365.
[63]	E. Largy, F. Cantais, G. Van Vyncht, A. Beck, A. Delobel, Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level, J. Chromatogr. A. 1498(2017) 128-146. https://doi.org/10.1016/j.chroma.2017.02.072.
[64]	M. Li, W. Zhu, H. Zheng, J. Zhang, Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level, Anal. Chim. Acta. 1189(2022) 339232. https://doi.org/10.1016/j.aca.2021.339232.
[65]	J. Giorgetti, V. D'Atri, J. Canonge, A. Lechner, D. Guillarme, O. Colas, E. WagnerRousset, A. Beck, E. Leize-Wagner, Y.N. François, Monoclonal antibody Nglycosylation profiling using capillary electrophoresis-Mass spectrometry:Assessment and method validation, Talanta. 178(2018) 530-537. https://doi.org/10.1016/j.talanta.2017.09.083.
[66]	K. Groves, A. Cryar, S. Cowen, A.E. Ashcroft, M. Quaglia, Mass Spectrometry Characterization of Higher Order Structural Changes Associated with the Fc-glycan Structure of the NISTmAb Reference Material, RM 8761, J. Am. Soc. Mass Spectrom. 31(2020) 553-564. https://doi.org/10.1021/jasms.9b00022.=