Revealing Charge Heterogeneity of Stressed Trastuzumab at the Subunit Level

Overview

Journal Anal Bioanal Chem

Specialty Chemistry

Date 2023 Jan 24

PMID 36693954

Authors

Baubek Spanov

Bas Baartmans

Oladapo Olaleye

Simone Nicolardi

Natalia Govorukhina

Manfred Wuhrer

Nico C van de Merbel

Rainer Bischoff

Affiliations

Soon will be listed here.

Abstract

Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN®. The basic pI of Fab and F(ab) fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN® and papain digestion and the corresponding modifications were determined by LC-MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region.

References

Liu H, Ponniah G, Zhang H, Nowak C, Neill A, Gonzalez-Lopez N . In vitro and in vivo modifications of recombinant and human IgG antibodies. MAbs. 2014; 6(5):1145-54. PMC: 4622420. DOI: 10.4161/mabs.29883. View

Cymer F, Beck H, Rohde A, Reusch D . Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential. Biologicals. 2017; 52:1-11. DOI: 10.1016/j.biologicals.2017.11.001. View

Wagner-Rousset E, Fekete S, Morel-Chevillet L, Colas O, Corvaia N, Cianferani S . Development of a fast workflow to screen the charge variants of therapeutic antibodies. J Chromatogr A. 2017; 1498:147-154. DOI: 10.1016/j.chroma.2017.02.065. View

Haberger M, Bomans K, Diepold K, Hook M, Gassner J, Schlothauer T . Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes. MAbs. 2014; 6(2):327-39. PMC: 3984323. DOI: 10.4161/mabs.27876. View

Nowak C, Cheung J, M Dellatore S, Katiyar A, Bhat R, Sun J . Forced degradation of recombinant monoclonal antibodies: A practical guide. MAbs. 2017; 9(8):1217-1230. PMC: 5680805. DOI: 10.1080/19420862.2017.1368602. View

Fekete S, Guillarme D, Sandra P, Sandra K . Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal Chem. 2015; 88(1):480-507. DOI: 10.1021/acs.analchem.5b04561. View

Sandra K, Vandenheede I, Sandra P . Modern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization. J Chromatogr A. 2013; 1335:81-103. DOI: 10.1016/j.chroma.2013.11.057. View

Fekete S, Veuthey J, Beck A, Guillarme D . Hydrophobic interaction chromatography for the characterization of monoclonal antibodies and related products. J Pharm Biomed Anal. 2016; 130:3-18. DOI: 10.1016/j.jpba.2016.04.004. View

Haverick M, Mengisen S, Shameem M, Ambrogelly A . Separation of mAbs molecular variants by analytical hydrophobic interaction chromatography HPLC: overview and applications. MAbs. 2014; 6(4):852-8. PMC: 4171020. DOI: 10.4161/mabs.28693. View

10.

Cao M, Mulagapati S, Vemulapalli B, Wang J, Saveliev S, Urh M . Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography. Anal Biochem. 2018; 566:151-159. DOI: 10.1016/j.ab.2018.11.021. View

11.

Zhang T, Bourret J, Cano T . Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography. J Chromatogr A. 2011; 1218(31):5079-86. DOI: 10.1016/j.chroma.2011.05.061. View

12.

Khanal O, Kumar V, Westerberg K, Schlegel F, Lenhoff A . Multi-column displacement chromatography for separation of charge variants of monoclonal antibodies. J Chromatogr A. 2018; 1586:40-51. DOI: 10.1016/j.chroma.2018.11.074. View

13.

Fekete S, Beck A, Fekete J, Guillarme D . Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach. J Pharm Biomed Anal. 2014; 102:33-44. DOI: 10.1016/j.jpba.2014.08.035. View

14.

Fekete S, Beck A, Fekete J, Guillarme D . Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach. J Pharm Biomed Anal. 2014; 102:282-9. DOI: 10.1016/j.jpba.2014.09.032. View

15.

Harris R, Kabakoff B, Macchi F, Shen F, Kwong M, Andya J . Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B Biomed Sci Appl. 2001; 752(2):233-45. DOI: 10.1016/s0378-4347(00)00548-x. View

16.

Spanov B, Olaleye O, Lingg N, Bentlage A, Govorukhina N, Hermans J . Change of charge variant composition of trastuzumab upon stressing at physiological conditions. J Chromatogr A. 2021; 1655:462506. DOI: 10.1016/j.chroma.2021.462506. View

17.

Lingg N, Tan E, Hintersteiner B, Bardor M, Jungbauer A . Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range. J Chromatogr A. 2013; 1319:65-71. DOI: 10.1016/j.chroma.2013.10.028. View

18.

Lingg N, Berndtsson M, Hintersteiner B, Schuster M, Bardor M, Jungbauer A . Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range: Validation of the method parameters. J Chromatogr A. 2014; 1373:124-30. DOI: 10.1016/j.chroma.2014.11.021. View

19.

Bailey A, Han G, Phung W, Gazis P, Sutton J, Josephs J . Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis. MAbs. 2018; 10(8):1214-1225. PMC: 6284562. DOI: 10.1080/19420862.2018.1521131. View

20.

Fussl F, Cook K, Scheffler K, Farrell A, Mittermayr S, Bones J . Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry. Anal Chem. 2018; 90(7):4669-4676. DOI: 10.1021/acs.analchem.7b05241. View