Abstract
The removal of protein charge variants due to complex chemical and enzymatic modifications like glycosylation, fragmentation and deamidation presents a significant challenge in the purification of monoclonal antibodies (mAb) and complicates downstream processing. These protein modifications occur either in vivo or during fermentation and downstream processing. The presence of charge variants can lead to diminished biological activity, differences in pharmacokinetics, pharmacodynamics, stability and efficacy. Therefore, these different product variants should be appropriately controlled for the consistency of product quality and to ensure patient safety. This investigation focuses on the development of a chromatography step for the removal of the charge variants from a recombinant single-chain variable antibody fragment (scFv−Fc−Ab). Poly(ethyleneimine)-grafted cation-exchange resins (Poly CSX and Poly ABX) were evaluated and compared to traditional macroporous cation-exchange and tentacle cation-exchange resins. Linear salt gradient experiments were conducted to study the separation efficiency of scFv–Fc–Ab variants using different resins. A classical thermodynamic model was used to develop a mechanistic understanding of the differences in charge variant retention behaviour of different resins. High selectivity in separation of scFv−Fc−Ab charge variants is obtained in the Poly CSX resin.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Avantor (2022) JT Baker BAKERBOND—process chromatography media. https://in.vwr.com/cms/process_chromatography_media. Accessed on 21 Apr 2022
Bowes BD, Koku H, Czymmek KJ, Lenhoff AM (2009) Protein adsorption and transport in dextran-modified ion-exchange media. I. Adsorption. J Chromatogr A 1216:7774–7784. https://doi.org/10.1016/j.chroma.2009.09.014
Di Donato A, Ciardiello MA, de Nigris M, Piccoli R, Mazzarella L, D’Alessio G (1993) Selective deamidation of ribonuclease A. Isolation and characterization of the resulting Isoaspartyl and aspartyl derivatives. J Biol Chem 268:4745–4751
Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. Mabs 7:9–14
Fernandes JC (2018) Therapeutic application of antibody fragments in autoimmune diseases: Current state and prospects. Drug Discov Today 23:1996–2002
Ganesh S, Lalit K, Sudarshan R, Darshak T, Surbhi D, Bhavesh V, Amit J, Indraneel S, Hanuman M (2014) Challenges in expression and purification of an unstable peptidase and solutions. In: Proceedings of the Bioprocessing Asia Conference, Langkawi, Malaysia, Nov 3–6, 38–40
Guillarme D, Fekete S, Beck A (2015) Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography. J Pharm Biomed Anal 102:33–44. https://doi.org/10.1016/j.jpba.2014.08.035
Harris RJ (1995) Processing of C-terminal lysine and arginine residues of proteins isolated form mammalian cell culture. J Chromatogr A 705:129–134
Hsu YR, Chang WC, Mendiaz EA, Hara S, Chow DT, Mann MB, Langley KE, Lu HS (1998) Selective deamidation of recombinant human stem cell factor during in vitro aging: isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers. Biochemistry 37:2251–2262
Itoh D, Yoshimoto N, Yamamoto S (2019) Retention mechanism of proteins in Hydroxyapatite chromatography—multimodal interaction based protein separations: a model study. Curr Protein Pept Sci 20:75–81. https://doi.org/10.2174/1389203718666171024122106
Kholodenko RV, Kalinovsky DV, Doronin II, Ponomarev ED, Kholodenko I (2017) V Antibody fragments as potential biopharmaceuticals for cancer therapy: success and limitations. Curr Med Chem 26:396–426
Kluters S, Hafner M, von Hirschheydt T, Frech C (2015) Solvent modulated linear pH gradient elution for the purification of conventional and bispecific antibodies: Modeling and application. J Chromatogr A 1418:119–129
Kroon DJ, Freedy J, Burinsky DJ, Sharma B (1995) Rapid profiling of carbohydrate glycoforms in monoclonal antibodies using MALDI/TOF mass spectrometry. J Pharm Biomed Anal 13:1049–1054
Kumar V, Leweke S, Lieres EV, Rathore AS (2015) Mechanistic modelling of ion-exchange process chromatography of charge variants of monoclonal antibody products. J Chromatogr A 1426:140–153. https://doi.org/10.1016/j.chroma.2015.11.062
Lee YF, Johnck M, Frech C (2018) Evaluation of differences between dual salt-pH gradient elution and mono gradient elution using a thermodynamic model: simultaneous separation of six monoclonal antibody charge and size variants on preparative-scale ion exchange chromatographic resin. Biotechnol Prog 34:973–986. https://doi.org/10.1002/btpr.2626
Lenhoff AM (2011) Protein adsorption and transport in polymer functionalised ion-exchangers. J Chromatogr A 1218:8748–8759. https://doi.org/10.1016/j.chroma.2011.06.061
Liu YD, Goetze AM, Bass RB, Flynn GC (2011) N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies. J Biol Chem 13:11211–11217
Müller-Späth T, Krättli M, Aumann L, Ströhlein G, Morbidelli M (2010) Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP). Biotechnol Bioeng 107:652–662. https://doi.org/10.1002/bit.22843
Nelson AL (2010) Antibody fragments: hope and hype. Mabs 2:77–83
Neue UD (2005) Theory of peak capacity in gradient elution. J Chromatogr A 1079:153–161. https://doi.org/10.1016/j.chroma.2005.03.008
Rathore AS (2010) Setting specifications for a biotech therapeutic product in the quality by design paradigm. BioPharm Int 23(1):46–53
Rudt M, Gillet F, Heege S, Hitzler J, Kalbfuss B, Guelat B (2015) Combined Yamamoto approach for simultaneous estimation of adsorption isotherm and kinetic parameters in ion-exchange chromatography. J Chromatography a 1413:68–76. https://doi.org/10.1016/j.chroma.2015.08.025
Santora LC, Krull IS, Grant K (1999) Characterization of recombinant human monoclonal tissue necrosis factor-a antibody using cation-exchange HPLC and capillary isoelectric focusing. Anal Biochem 275:98–108
Shekhawat LK, Rathore AS (2019) An overview of mechanistic modelling of liquid chromatography. Prep Biochem Biotechnol 49:623–638
Sivanathan GT, Mallubhotla H, Suggala SV (2019) Selective removal of closely related clipped protein impurities using poly(ethylenimine)-grafted anion exchange chromatography resin. Prep Biochem Biotechnol 49:1020–1032. https://doi.org/10.1080/10826068.2019.1650373
Staby A, Rathore AS, Ahuja S (eds) (2017) Preparative chromatography for separation of proteins. Wiley, Hoboken
Stone MC, Tao Y, Carta G (2009) Protein adsorption and transport in agarose and dextran grafted agarose media for ion exchange chromatography: effect of ionic strength and protein characteristics. J Chromatogr A 1216:4465–4474. https://doi.org/10.1016/j.chroma.2009.03.044
Tao Y, Carta G, Ferreira G, Robins D (2011) Adsorption of deamidated antibody variants on microporous and dextran-grafted cation exchangers: II Adsorption kinetics. J Chromatogr A 1218:8027–8035. https://doi.org/10.1016/j.chroma.2011.09.010
Urmann M, Hafner M, Frech C (2011) Influence of protein and stationary phase properties on protein-matrix-interaction in cation exchange chromatography. J Chromatogr A 1218:5136–5145. https://doi.org/10.1016/j.chroma.2011.05.085
Vlasak J, Ionescu R (2011) Fragmentation of monoclonal antibodies. Mabs 3:253–263
Walsh G (2010) Biopharmaceutical benchmarks. Nat Biotechnol 28:917–924
Yu LL, Tao SP, Dong XY, Sun Y (2013) Protein adsorption to poly(ethylenimine)-modified Sepharose FF: I. A critical ionic capacity for drastically enhanced capacity and uptake kinetics. J Chromatogr A 1305:76–84. https://doi.org/10.1016/j.chroma.2013.07.014
Zhao G, Dong XY, Sun Y (2009) Ligands for mixed-mode protein chromatography, principles, characteristics and design. J Biotechnol 144:3–11. https://doi.org/10.1016/jbiotec.2009.04.009
Zhu M, Carta G (2014) Adsorption of polyethylene-glycolated bovine serum albumin on microporous and polymer-grafted anion exchangers. J Chromatogr A 1326:29–38. https://doi.org/10.1016/j.chroma.2013.12.007
Acknowledgements
The primary author would like to acknowledge support from Syngene International Ltd., Biopharmaceutical Development Department, Bangalore, India for partial funding and laboratory facilities to carry out the research work. Syngene Biologics DSP and AD colleagues have been generous in their support.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest in the publication.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sivanathan, G.T., Mallubhotla, H., Suggala, S.V. et al. Separation of closely related monoclonal antibody charge variant impurities using poly(ethylenimine)-grafted cation-exchange chromatography resin. 3 Biotech 12, 293 (2022). https://doi.org/10.1007/s13205-022-03350-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-022-03350-9