Abstract
This chapter focuses on the application of capillary zone electrophoresis hyphenated with mass spectrometry (CZE-MS) for the characterization of monoclonal antibodies (mAbs). mAbs are complex molecules comprising different glycoforms and many other posttranslational modifications. In addition to this inherent microheterogeneity, misassembling of antibodies can take place during production contributing to their macroheterogeneity. CZE-MS is a versatile and powerful technique which has demonstrated high potential for the assessment of both micro- and macroheterogeneity of mAbs. In this chapter, technical and practical considerations for the characterization of mAbs by CZE-MS are described. CE-MS interfacing, capillary coatings for the prevention of mAb adsorption, and sample preparation considerations are covered in detail. The assessment of the macro- and microheterogeneity is discussed and exemplified through three different approaches involving analysis of intact, enzymatically digested, and reduced antibodies. The examples also illustrate the use of two commercially available interfacing techniques (i.e., sheath liquid and sheathless) as well as different types of capillary coatings (positively charged and neutral coatings).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC (2005) Monoclonal antibody successes in the clinic. Nat Biotechnol 23(9):1073–1078
Kaplon H, Reichert JM (2019) Antibodies to watch in 2019. MAbs 11(2):219–238
Schirrmacher V (2019) From chemotherapy to biological therapy: a review of novel concepts to reduce the side effects of systemic cancer treatment (review). Int J Oncol 54(2):407–419
Chames P, Van Regenmortel M, Weiss E, Baty D (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157(2):220–233
Liu H, Ponniah G, Zhang H-M, Nowak C, Neill A, Gonzalez-Lopez N, Patel R, Cheng G, Kita AZ, Andrien B (2014) In vitro and in vivo modifications of recombinant and human IgG antibodies. MAbs 6(5):1145–1154
Gstottner C, Reusch D, Haberger M, Dragan I, Van Veelen P, Kilgour DPA, Tsybin YO, van der Burgt YEM, Wuhrer M, Nicolardi S (2020) Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry. MAbs 12(1):1682403
Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Iida S, Nakano R, Wakitani M, Niwa R, Sakurada M, Uchida K, Shitara K, Satoh M (2004) Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 87(5):614–622
Reusch D, Tejada ML (2015) Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 25(12):1325–1334
Mouchahoir T, Schiel JE (2018) Development of an LC-MS/MS peptide mapping protocol for the NISTmAb. Anal Bioanal Chem 410(8):2111–2126
Dick LW, Mahon D, Qiu D, Cheng K-C (2009) Peptide mapping of therapeutic monoclonal antibodies: improvements for increased speed and fewer artifacts. J Chromatogr B 877(3):230–236
Haselberg R, De Vijlder T, Heukers R, Smit MJ, Romijn EP, Somsen GW, Dominguez-Vega E (2018) Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry. Anal Chim Acta 1044:181–190
Haberger M, Leiss M, Heidenreich AK, Pester O, Hafenmair G, Hook M, Bonnington L, Wegele H, Haindl M, Reusch D, Bulau P (2016) Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry. Mabs 8(2):331–339
Turner A, Yandrofski K, Telikepalli S, King J, Heckert A, Filliben J, Ripple D, Schiel JE (2018) Development of orthogonal NISTmAb size heterogeneity control methods. Anal Bioanal Chem 410(8):2095–2110
Woods RJ, Xie MH, Von Kreudenstein TS, Ng GY, Dixit SB (2013) LC-MS characterization and purity assessment of a prototype bispecific antibody. MAbs 5(5):711–722
Ding W, Qiu D, Bolgar MS, Miller SA (2018) Improving mass spectral quality of monoclonal antibody middle-up LC-MS analysis by shifting the protein charge state distribution. Anal Chem 90(3):1560–1565
Sokolowska I, Mo J, Dong J, Lewis MJ, Hu P (2017) Subunit mass analysis for monitoring antibody oxidation. Mabs 9(3):498–505
Wang C, Vemulapalli B, Cao M, Gadre D, Wang J, Hunter A, Wang X, Liu D (2018) A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. Mabs 10(8):1226–1235
D’Atri V, Fekete S, Beck A, Lauber M, Guillarme D (2017) Hydrophilic interaction chromatography hyphenated with mass spectrometry: a powerful analytical tool for the comparison of originator and biosimilar therapeutic monoclonal antibodies at the middle-up level of analysis. Anal Chem 89(3):2086–2092
Domínguez-Vega E, Haselberg R, Somsen GW (2016) Capillary zone electrophoresis–mass spectrometry of intact proteins. In: Tran NT, Taverna M (eds) Capillary electrophoresis of proteins and peptides: methods and protocols. Springer, New York, pp 25–41
Bertoletti L, Schappler J, Colombo R, Rudaz S, Haselberg R, Domínguez-Vega E, Raimondi S, Somsen GW, De Lorenzi E (2016) Evaluation of capillary electrophoresis-mass spectrometry for the analysis of the conformational heterogeneity of intact proteins using beta2-microglobulin as model compound. Anal Chim Acta 945:102–109
Patrick JS, Lagu AL (2001) Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins. Electrophoresis 22(19):4179–4196
Dominguez-Vega E, De Vijlder T, Romijn EP, Somsen GW (2017) Capillary electrophoresis-tandem mass spectrometry as a highly selective tool for the compositional and site-specific assessment of multiple peptide-deamidation. Anal Chim Acta 982:122–130
Maxwell EJ, Chen DD (2008) Twenty years of interface development for capillary electrophoresis-electrospray ionization-mass spectrometry. Anal Chim Acta 627(1):25–33
Ramautar R, Heemskerk AA, Hensbergen PJ, Deelder AM, Busnel JM, Mayboroda OA (2012) CE-MS for proteomics: advances in interface development and application. J Proteome 75(13):3814–3828
Fanali S, D'Orazio G, Foret F, Kleparnik K, Aturki Z (2006) On-line CE-MS using pressurized liquid junction nanoflow electrospray interface and surface-coated capillaries. Electrophoresis 27(23):4666–4673
Moini M (2007) Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip. Anal Chem 79(11):4241–4246
Haselberg R, Ratnayake CK, de Jong GJ, Somsen GW (2010) Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins. J Chromatogr A 1217(48):7605–7611
Michalikova K, Dominguez-Vega E, Somsen GW, Haselberg R (2019) Middle-up characterization of the monoclonal antibody infliximab by capillary zone electrophoresis-mass spectrometry. LC GC Eur 32(3):130–137
Belov AM, Zang L, Sebastiano R, Santos MR, Bush DR, Karger BL, Ivanov AR (2018) Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis—mass spectrometry. Electrophoresis 39(16):2069–2082
Espinosa-de la Garza CE, Salazar-Flores RD, Perez NO, Flores-Ortiz LF, Medina-Rivero E (2017) Capillary electrophoresis separation of monoclonal antibody isoforms using a neutral capillary. JoVE 119:55082
Haselberg R, van der Sneppen L, Ariese F, Ubachs W, Gooijer C, de Jong GJ, Somsen GW (2009) Effectiveness of charged noncovalent polymer coatings against protein adsorption to silica surfaces studied by evanescent-wave cavity ring-down spectroscopy and capillary electrophoresis. Anal Chem 81(24):10172–10178
Haselberg R, de Jong GJ, Somsen GW (2010) Capillary electrophoresis-mass spectrometry of intact basic proteins using Polybrene-dextran sulfate-Polybrene-coated capillaries: system optimization and performance. Anal Chim Acta 678(1):128–134
Erim FB, Cifuentes A, Poppe H, Kraak JC (1995) Performance of a physically adsorbed high-molecular-mass Polyethyleneimine layer as coating for the separation of basic-proteins and peptides by capillary electrophoresis. J Chromatogr A 708(2):356–361
Ferrige AG, Seddon MJ, Jarvis S (1991) Maximum-entropy deconvolution in electrospray mass-spectrometry. Rapid Commun Mass Sp 5(8):374–377
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gstöttner, C., Haselberg, R., Wuhrer, M., Somsen, G.W., Domínguez-Vega, E. (2022). Assessment of Macro- and Microheterogeneity of Monoclonal Antibodies Using Capillary Zone Electrophoresis Hyphenated with Mass Spectrometry. In: Neusüß, C., Jooß, K. (eds) Capillary Electrophoresis-Mass Spectrometry . Methods in Molecular Biology, vol 2531. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2493-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2493-7_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2492-0
Online ISBN: 978-1-0716-2493-7
eBook Packages: Springer Protocols