POL Scientific / JBM / Volume 11 / Issue 2 / DOI: 10.14440/jbm.2024.0006
Cite this article
Journal Browser
Volume | Year
News and Announcements
View All

Calcium chloride and arginine show diametrically opposite effects on antibody elution in Protein A and Protein L chromatography

Ju Qu1 Yan Wan1 Yifeng Li1*
Show Less
1 Downstream Process Development, WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China
JBM 2024 , 11(2), e99010016; https://doi.org/10.14440/jbm.2024.0006
Submitted: 23 May 2024 | Revised: 20 June 2024 | Accepted: 25 July 2024 | Published: 10 July 2024
© 2024 by the Journal of Biological Methods published by POL Scientific. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )

Protein A and Protein L affinity chromatographies are extensively used in mAb and bispecific antibody (bsAb) purification. In addition to product capture, they are both capable of separating certain product-related by-products and aggregates under appropriate conditions. For both types of chromatography, previous studies suggested that adding a salt additive to the mobile phase can significantly improve the resolution between product and by-products/aggregates. Nevertheless, the effects of different salt additives on antibody elution in Protein A and Protein L chromatography have not been compared. In the current study, we compared the effects of three salt additives, sodium chloride (NaCl), calcium chloride (CaCl2), and arginine hydrochloride (Arg·HCl), on antibody elution in Protein A and Protein L chromatography. Interestingly, while NaCl suppressed antibody elution in both types of chromatography, CaCl2, and Arg·HCl promoted antibody elution in Protein A chromatography but suppressed antibody elution in Protein L chromatography. In addition, we evaluated the effect of each salt gradient on aggregate removal by Protein L chromatography. The information provided by the current study should be useful to the selection of conditions/additives for improving by-product removal by Protein A and Protein L chromatography.

Calcium chloride
Protein A
Protein L
Retention volume (time)
Salt additive
Sodium chloride
  1. Fahrner RL, Knudsen HL, Basey CD, et al. Industrial purification of pharmaceutical antibodies: Development, operation, and validation of chromatography processes. Biotechnol Genet Eng Rev. 2001;18:301-327. doi: 10.1080/02648725.2001.10648017


  1. Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies--application of platform approaches. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;848:28-39. doi: 10.1016/j.jchromb.2006.09.026


  1. Ramos-de-la-Peña AM, González-Valdez J, Aguilar O. Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies. J Sep Sci. 2019;42:1816-1827. doi: 10.1002/jssc.201800963


  1. Deisenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9-and 2.8-a resolution. Biochemistry. 1981;20:2361-2370.


  1. Li R, Dowd V, Stewart DJ, Burton SJ, Lowe CR. Design, synthesis, and application of a Protein A mimetic. Nat Biotechnol. 1998;16:190-195. doi: 10.1038/nbt0298-190


  1. Zarrineh M, Mashhadi IS, Farhadpour M, Ghassempour A. Mechanism of antibodies purification by protein A. Anal Biochem. 2020;609:113909. doi: 10.1016/j.ab.2020.113909


  1. Björck L. Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol. 1988;140:1194-1197.


  1. Nilson BH, Solomon A, Björck L, Akerström B. Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain. J Biol Chem. 1992;267:2234-2239.


  1. Graille M, Stura EA, Housden NG, et al. Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins. Structure. 2001;9:679-687. doi: 10.1016/s0969-2126(01)00630-x


  1. Li Y. Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture. Protein Expr Purif. 2021;188:105976. doi: 10.1016/j.pep.2021.105976


  1. Chen X, Wang Y, Li Y. Removing half antibody byproduct by Protein A chromatography during the purification of a bispecific antibody. Protein Expr Purif. 2020;172:105635. doi: 10.1016/j.pep.2020.105635


  1. Chen W, Zhang T, Wan Y, Li Y. Assessing four subdomain-specific affinity resins’ capability to separate half-antibody from intact bispecific antibody. Protein Expr Purif. 2022;198:106124. doi: 10.1016/j.pep.2022.106124


  1. Zhang Y, Wang Y, Li Y. A method for improving protein A chromatography’s aggregate removal capability. Protein Expr Purif. 2019;158:65-73. doi: 10.1016/j.pep.2019.02.017


  1. Chen C, Wakabayashi T, Muraoka M, et al. Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency. MAbs. 2019;11:632-638. doi: 10.1080/19420862.2019.1583996


  1. Chen SW, Tan D, Yang YS, Zhang W. Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies. MAbs. 2020;12:1718440. doi: 10.1080/19420862.2020.1718440


  1. Tustian AD, Endicott C, Adams B, Mattila J, Bak H. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. MAbs. 2016;8:828-838. doi: 10.1080/19420862.2016.1160192


  1. Chen X, Wang Y, Wang Y, Li Y. Protein L chromatography: A useful tool for monitoring/separating homodimers during the purification of IgG-like asymmetric bispecific antibodies. Protein Expr Purif. 2020;175:105711. doi: 10.1016/j.pep.2020.105711


  1. Koehnlein W, Kastenmueller E, Meier T, Treu T, Falkenstein R. The beneficial impact of kosmotropic salts on the resolution and selectivity of Protein A chromatography. J Chromatogr A. 2024;1715:464585. doi: 10.1016/j.chroma.2023.464585


  1. Huang B, Liu FF, Dong XY, Sun Y. Molecular mechanism of the effects of salt and pH on the affinity between Protein A and human immunoglobulin G1 revealed by molecular simulations. J Phys Chem B. 2012;116:424-433. doi: 10.1021/jp205770p


  1. Dong W, Zhang D, Li Y. CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability. Protein Expr Purif. 2024;220:106503. doi: 10.1016/j.pep.2024.106503


  1. Ejima D, Yumioka R, Tsumoto K, Arakawa T. Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography. Anal Biochem. 2005;345:250-257. doi: 10.1016/j.ab.2005.07.004


  1. Arakawa T, Philo JS, Tsumoto K, Yumioka R, Ejima D. Elution of antibodies from a Protein-A column by aqueous arginine solutions. Protein Expr Purif. 2004;36:244-248. doi: 10.1016/j.pep.2004.04.009


  1. Shukla D, Zamolo L, Cavallotti C, Trout BL. Understanding the role of arginine as an eluent in affinity chromatography via molecular computations. J Phys Chem B. 2011;115:2645-2654. doi: 10.1021/jp111156z


  1. Arakawa T, Tsumoto K, Nagase K, Ejima D. The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. Protein Expr Purif. 2007;54:110-116. doi: 10.1016/j.pep.2007.02.010


  1. Tsumoto K, Ejima D, Nagase K, Arakawa T. Arginine improves protein elution in hydrophobic interaction chromatography. The cases of human interleukin-6 and activin-A. J Chromatogr A. 2007;1154:81-86. doi: 10.1016/j.chroma.2007.02.061


  1. Hirano A, Arakawa T, Kameda T. Interaction of arginine with Capto MMC in multimodal chromatography. J Chromatogr A. 2014;1338:58-66. doi: 10.1016/j.chroma.2014.02.053


  1. Hirano A, Maruyama T, Shiraki K, Arakawa T, Kameda T. Mechanism of protein desorption from 4-mercaptoethylpyridine resins by arginine solutions. J Chromatogr A. 2014;1373:141-148. doi: 10.1016/j.chroma.2014.11.032


  1. Hirano A, Arakawa T, Kameda T. Effects of arginine on multimodal anion exchange chromatography. Protein Expr Purif. 2015;116:105-112. doi: 10.1016/j.pep.2015.07.013


  1. Parimal S, Garde S, Cramer SM. Effect of guanidine and arginine on protein-ligand interactions in multimodal cation-exchange chromatography. Biotechnol Prog. 2017;33:435-447. doi: 10.1002/btpr.2419


  1. Hirano A, Shiraki K, Kameda T. Effects of arginine on multimodal chromatography: Experiments and simulations. Curr Protein Pept Sci. 2019;20:40-48. doi: 10.2174/1389203718666171024115407


  1. Zhang D, Wu L, Koch KJ, Cooks RG. Arginine clusters generated by electrospray ionization and identified by tandem mass spectrometry. Eur Mass Spectrom. 1999;5:353-361. doi: 10.1255/ejms.295
Conflict of interest
The authors declare no conflicts of interest.
Back to top
Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TAB, Published by POL Scientific