Efficient removal of antibody aggregates using TOYOPEARL MX-Trp-650M mixed-mode resin under salt or pH gradient elution
Background: TOYOPEARL MX-Trp-650M is a mixed-mode resin (Tosoh Bioscience, Japan), which mediates both cation exchange and hydrophobic interactions. While mixed-mode resins are generally effective at removing antibody aggregates, reports specifically evaluating the application of MX-Trp-650M for this purpose remain limited. A previous study suggested that effective separation of monomeric and aggregated antibodies using MX-Trp-650M was achieved only under dual-gradient elution with pH and salt. Objective: This study aimed to further evaluate MX-Trp-650M’s aggregate separation potential under various elution conditions. Methods: In an antibody purification case where aggregates were the predominant byproducts, both Capto MMC ImpRes and MX-Trp-650M were evaluated as the first polishing step following Protein A capture. Aggregate separation was monitored and assessed using native gel electrophoresis and size-exclusion chromatography-high-performance liquid chromatography. Results: MX-Trp-650M marginally outperformed Capto MMC ImpRes and achieved excellent aggregate clearance under either salt or pH mono-gradient elution. Conclusion: Combining the results from prior studies, the current data confirm the strong aggregate separation capability of MX-Trp-650M. The results also suggest that the optimal elution conditions for efficacious separation may vary across different antibody purification scenarios.
- Tosoh Bioscience. TOYOPEARL MX-Trp-650M. Available from: https://www.separations.eu.tosohbioscience.com/ OpenPDF.aspx?path=~/File%20Library/TBG/Products%20 Download/Flyer/f22p01a.pdf [Last accessed on 2025 Jun 09].
- Tosoh Bioscience. Development of a High Capacity Mixed- Mode Resin for High Conductivity Mab Feedstocks. Application Note. Available from: https://www.bioprocessintl. com/monoclonal-antibodies/development-of-a-high-capacity-mixed-mode-resin-for-high-conductivity-mab-feedstocks [Last accessed on 2025 Jun 09].
- Fouque N, Depoisier JF, Wilson K, Vajda J, Muller E, Dabre R. Methods of Purifying Bispecific Antibodies. US 10,457,749 B2. United States Patent; 2019.
- Arakawa T. Isoform separation by a mixed-mode resin, TOYOPEARL MX-Trp-650M. Curr Protein Pept Sci. 2019;20(1):61-64. doi: 10.2174/1389203718666171009111355
- Tang J, Zhang X, Chen T, Wang Y, Li Y. Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MMC ImpRes mixed-mode chromatography. Protein Expr Purif. 2020;167:105529. doi: 10.1016/j.pep.2019.105529
- Wan Y, Zhang T, Wang Y, Wang Y, Li Y. Removing light chain-missing byproducts and aggregates by Capto MMC ImpRes mixed-mode chromatography during the purification of two WuXiBody-based bispecific antibodies. Protein Expr Purif. 2020;175:105712. doi: 10.1016/j.pep.2020.105712
- Chen T, Guo G, Tan G, Wang Y, Li Y. Antibody aggregate removal using a mixed-mode chromatography resin. Methods Mol Biol. 2021;2178:345-354. doi: 10.1007/978-1-0716-0775-6_23
- Zhang L, Parasnavis S, Li Z, Chen J, Cramer S. Mechanistic modeling based process development for monoclonal antibody monomer-aggregate separations in multimodal cation exchange chromatography. J Chromatogr A. 2019;1602:317-325. doi: 10.1016/j.chroma.2019.05.056
- Altern SH, Lyall JY, Welsh JP, et al. High-throughput in silico workflow for optimization and characterization of multimodal chromatographic processes. Biotechnol Prog. 2024;40(6):e3483. doi: 10.1002/btpr.3483
- Gao D, Wang LL, Lin DQ, Yao SJ. Evaluating antibody monomer separation from associated aggregates using mixed-mode chromatography. J Chromatogr A. 2013;1294:70-75. doi: 10.1016/j.chroma.2013.04.018
- Robinson J, Vats M, Hartmann M. Implementation of multimodal anion exchange chromatography to address product quality challenges and downstream platform limitations: A case study. J Chromatogr A. 2025;1746:465784. doi: 10.1016/j.chroma.2025.465784
- Vajda J. Separation of monoclonal immunoglobulin G and its aggregates using TOYOPEARL MX-Trp-650M. Bioprocess Int. 2013;2013-2014:73-75.
- Vajda J, Mueller E, Bahret E. Dual salt mixtures in mixed mode chromatography with an immobilized tryptophan ligand influence the removal of aggregated monoclonal antibodies. Biotechnol J. 2014;9(4):555-565. doi: 10.1002/biot.201300230
- Cytiva. Polishing of Monoclonal Antibodies Using Capto MMC ImpRes in Bind and Elute Mode, Application Note, 2013, 29- 0373-49 AA. Available from: https://cdn.cytivalifesciences. com/api/public/content/digi-16467-pdf [Last accessed on 2025 Jun 09].
- Cytiva. Capto MMC ImpRes, Datafile, 2015, 29-035674 AB. Available from: https://gels.yilimart.com/assets/images/doc/ file/17371601_datafile_01.pdf [Last accessed on 2025 Jun 09].
- Cytiva. Capto MMC ImpRes Resin. Available from: https:// cdn.cytivalifesciences.com/api/public/content/digi-16468-pdf [Last accessed on 2025 Jun 09].
- Kramarczyk JF, Kelley BD, Coffman JL. High-throughput screening of chromatographic separations: II. Hydrophobic interaction. Biotechnol Bioeng. 2008;100(4):707-720. doi: 10.1002/bit.21907
- Wensel DL, Kelley BD, Coffman JL. High-throughput screening of chromatographic separations: III. Monoclonal antibodies on ceramic hydroxyapatite. Biotechnol Bioeng. 2008;100(5):839-854. doi: 10.1002/bit.21906
- Kelley BD, Tobler SA, Brown P, et al. Weak partitioning chromatography for anion exchange purification of monoclonal antibodies. Biotechnol Bioeng. 2008;101(3):553-566. doi: 10.1002/bit.21923
- McDonald P, Tran B, Williams CR, et al. The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resins using high-throughput screening. J Chromatogr A. 2016;1433:66-74. doi: 10.1016/j.chroma.2015.12.071
- Ram J, Snyder M, Belisle C, Koley S, Vecchiarello N. Defining operating regimes for partition coefficient measurements in protein chromatography. J Chromatogr A. 2025;1745:465730. doi: 10.1016/j.chroma.2025.465730