Comparison of various columns used in anion exchange chromatography method for mAb purification

Year 2025, Volume: 34 Issue: SI, 73 - 82, 21.08.2025

Fatih Ozturk Mehmet Emin Uslu

https://doi.org/10.38042/biotechstudies.1733671

Abstract

The impact of resins with varying ligands and pH levels on human IgG4 protein was analysed using anion exchange chromatography. Initially, a resin screening study involving five different positively charged ligands from four different brands was conducted on largely purified monoclonal antibodies, following Protein A capture. Subsequently, the influence of pH levels (6, 7, and 8.1) on an IgG4 protein with an isoelectric point of 6.9 was assessed using a single resin. Throughout the resin screening, all protein quality analyses were performed to identify the resin with the most compatible ligand. The study on pH effects revealed that when the pH exceeded 6.9, various protein fragments were removed, directly affecting the protein charge variant. When the protein pH was at or below the isoelectric point, the anion exchange chromatography flow-through method achieved a maximum protein recovery of 92-98%.

Keywords

Anion exchange chromatography , Monoclonal antibody , Igg , Resin screening , Downstream

References

• Becerra, A., & Buyel, J. (2022). Optimizing downstream purification of high-quality plasmid DNA with POROS™ Chromatography Resins. Cell & Gene Therapy Insight, 8(1), 87. https://doi.org/10.18609/cgti.2022.029
• Castro-Forero, A. A., Jokondo, Z., Voloshin, A., Hester, J. F. (2015). Anion-exchange chromatographic clarification: Bringing simplification, robustness, and savings to MAb purification. BioProcess Int., 13(6), 52-57.
• Cataldo, A. L., Burgstaller, D., Hribar, G., Jungbauer, A., & Satzer, P. (2020). Economics and ecology: Modelling of continuous primary recovery and capture scenarios for recombinant antibody production. J Biotechnol, 308, 87-95. https://doi.org/10.1016/j.jbiotec.2019.12.001
• Chen, S.-T., Xu, W., Cai, K., Ferreira, G., Wickramasinghe, R., & Qian, X. (2022). Factors affecting robustness of anion exchange chromatography: Selective retention of minute virus of mice using membrane media. J Chromatogr B Analyt Technol Biomed Life Sci. https://doi.org/10.1016/j.jchromb.2022.123449
• Du, Y., Walsh, A., Ehrick, R., Xu, W., May, K., & Liu, H. (2012). Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs, 4(5), 578-585. https://doi.org/10.4161/mabs.21328
• Gomis-Fons, J., Andersson, N., & Nilsson, B. (2020). Optimization study on periodic counter-current chromatography integrated in a monoclonal antibody downstream process. J Chromatogr A. https://doi.org/10.1016/j.chroma.2020.461055
• Green, W., & Andersen, O. (1991). Surface Charges And Ion Channel Functıon. Annual Review of Physiology, 53, pp. 341-359. https://doi.org/10.1146/annurev.ph.53.030191.002013
• Han, X., Hewig, A., & Vedantham, G. (2011). Recovery and Purification of Antibody. Antibody Expression and Production Cell Engineering, 305-40. https://doi.org/10.1007/978-94-007-1257-7_14
• Jackobek, R., Herrick-Wagman, S., Zhu, L., Francis, C., Solbrand, A., Eriksson, C., D'Antona, A. M. (2020). Automated pH and conductivity conditioning using feedback control to support a two-step continuous purification process. Journal of Chromatography A, 1630. https://doi.org/10.1016/j.chroma.2020.461537
• Jeon, H. J., Choi, B. K., Hwang, S. I., Kim, S. H., Kim, G. J., Park, J. C., Yang, Z. Y., & Hwang, K. Y. (2022). Optimization for Simultaneous Removal of Product/Process-Related Impurities of Peptide Fc-Fusion Protein Using Cation Exchange Chromatography. Processes, 10(11), 2359. https://doi.org/10.3390/pr10112359
• Koehler, K.C., Jokondo, Z., Narayan, J., Voloshin, A.M., Castro-Forero, A.A. (2019). Enhancing Protein A performance in mAb processing: A method to reduce and rapidly evaluate host cell DNA levels during primary clarification. Biotechnol Progress., 35: e2882. https://doi.org/10.1002/btpr.2882
• Kunert, R., & Reinhart, D. (2016). Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol, 3451-61. https://doi.org/10.1007/s00253-016-7388-9
• Kurák, T., & Polakovič, M. (2022). Adsorption Performance of a Multimodal Anion-Exchange Chromatography Membrane: Effect of Liquid Phase Composition and Separation Mode. Membranes, 12(21), 1173. https://doi.org/10.3390/membranes12121173
• Li, W., Fan, Z., Lin, Y., & Wang, T.-Y. (2021). Serum-Free Medium for Recombinant Protein Expression in Chinese Hamster Ovary Cells. Front Bioeng Biotechnol, 9(64). https://doi.org/10.3389/fbioe.2021.646363
• Matos, T., Mohamed, E., & Queiroz, J. (2016). Capto™ Resins for Chromatography of DNA: A Minor Difference in Ligand Composition Greatly Influences the Separation of Guanidyl-Containing Fragments. Chromatographia, 79(19-20), 1277-1282. https://doi.org/10.1007/s10337-016-3148-3
• Matte, A. (2020). Recent Advances and Future Directions in Downstream Processing of Therapeutic Antibodies. Int J Mol Sci, 23(15). https://doi.org/10.3390/ijms23158663
• Müller-Späth, T., Aumann, L., Ströhlein, G., Kornmann, H., Valax, P., Delegrange, L., Morbidelli, M. (2010). Two step capture and purification of IgG2 using multicolumn countercurrent solvent gradient purification (MCSGP). Biotechnol Bioeng, 107(6), 974-984. https://doi.org/10.1002/bit.22887
• Nadar, S., Somasundaram, B., Charry, M., Billakanti, J., Shave, E., Baker, K., & Lua, L. (2022). Design and optimization of membrane chromatography for monoclonal antibody charge variant separation. Biotechnology Progress, 38(6). https://doi.org/10.1002/btpr.3288
• Parra, S. C., & Gebski, C. (2011). Benefits of a Revised Approach to Anion Exchange Flow-Through Polish Chromatography. BioPharm International, 11(3).
• Pergande, M., & Cologna, S. (2017). Isoelectric Point Separations of Peptides and Proteins. Proteomes, 5(1), 4. https://doi.org/10.3390/proteomes5010004
• Ramos-de-la-Peña, A., González-Valdez, J., & Aguilar, O. (2019). Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies. J Sep Sc,, 42(9), 1816-1827. https://doi.org/10.1002/jssc.201800963
• Sadeghalvad, M., & Rezaei, N. (2021). Introduction on Monoclonal Antibodies. IntechOpen. https://doi.org/10.5772/intechopen.98378
• Sánchez-Trasviña, C., Flores-Gatica, M., Enriquez-Ochoa, D., Rito-Palomares, M., Mayolo-Deloisa, K. (2021). Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies. Front. Bioeng. Biotechnol., 9, 1-25. https://doi.org/10.3389/fbioe.2021.717326
• Schroeder, H., & Cavacini, L. (2010). Structure and function of immunoglobulins. J Allergy Clin Immunol, 125(2), 41-52. https://doi.org/10.1016/j.jaci.2009.09.046
• Stone, M., Borman, J., Ferreira, G., & Robbins, D. (2018). Effects of pH, conductivity, host cell protein, and DNA size distribution on DNA clearance in anion exchange chromatography media. Biotechnol Prog., 34, pp. 141–149. https://doi.org/10.1002/btpr.2556
• Trnovec, H., Doles, T., Hribar, G., Furlan, N., & Podgornik, A. (2020). Characterization of membrane adsorbers used for impurity removal during the continuous purification of monoclonal antibodies. J Chromatogr A., 1609. https://doi.org/10.1016/j.chroma.2019.460518
• Yüce, M., Sert, F., Torabfam, M., Parlar, A., Gürel, B., Çakır, N., Çapan, Y. (2021). Fractionated charge variants of biosimilars: A review of separation methods, structural and functional analysis. Anal Chim Acta. https://doi.org/10.1016/j.aca.2020.12.064
• Zhang, X., Chen, T., & Li, Y. (2019). A parallel demonstration of different resins' antibody aggregate removing capability by a case study. Protein Expression and Purification, 153, 59-69. https://doi.org/10.1016/j.pep.2018.08.011
• Zhu, M. M., Mollet, M., Hubert, R. S., Kyung, Y. S., & Zhang, G. G. (2017). Industrial Production of Therapeutic Proteins: Cell Lines, Cell Culture, and Purification. Handbook of Industrial Chemistry and Biotechnology. (ss. 1639-1669). USA. https://doi.org/10.1007/978-3-319-52287-6_29