AccScience Publishing / JBM / Online First / DOI: 10.14440/jbm.0138
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BRIEF REPORT

Advantages of MabSelect VH3, a novel VH3-specific Protein A resin, over its two conventional counterparts in purifying a VHH-based trispecific antibody

Dongdong Fang1† ,  Wenjing Qi1† ,  Yifeng Li1*
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1 Downstream Process Development, WuXi Biologics, Shanghai 200131, China
JBM, e99010078 https://doi.org/10.14440/jbm.0138
Submitted: 20 May 2025 | Revised: 18 August 2025 | Accepted: 26 August 2025 | Published: 19 September 2025
© 2025 by the Author(s). 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/ )
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Abstract

Background: Protein A affinity chromatography represents the most extensively used technique for initial product capture in antibody purification. The ligands of commercial Protein A resins from different, or even the same, vendors may exhibit distinct binding specificity. For example, MabSelect SuRe LX and MabSelect PrismA bind the antibody’s fragment crystallizable region and both the fragment crystallizable and variable heavy chain 3 (VH3) regions, respectively, while MabSelect VH3, a newly launched Protein A resin, binds the VH3 region exclusively. Objective: This study aimed to compare the capabilities of three Protein A resins—namely, MabSelect SuRe LX, MabSelect PrismA, and MabSelect VH3—in removing byproducts associated with a variable domain of heavy chain-only antibody-based trispecific antibody (TsAb). Methods: Clarified culture harvest containing the TsAb was processed separately using MabSelect SuRe LX, MabSelect PrismA, and MabSelect VH3 columns. For each column, byproduct separation was monitored by analyzing relevant elution fractions with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography-high-performance liquid chromatography. Results: MabSelect VH3 demonstrated superior byproduct removal performance compared with the other two Protein A resins. Conclusion: MabSelect VH3 is particularly suitable for the purification of bispecific antibodies and TsAbs, where the product and byproducts contain different numbers of VH3 domains. For multispecific antibody purification, screening different affinity resins with distinct binding specificity is recommended, as this can help identify the most effective option for separation.

Keywords
Aggregate
Half-antibody
Homodimer
MabSelect VH3
Protein A
Trispecific antibody
Funding
None.
Conflict of interest
Dongdong Fang, Wenjing Qi, and Yifeng Li are employees at WuXi Biologics. The company played no role in the study design and the writing of this manuscript.
References
  1. Hober S, Nord K, Linhult, M. Protein A chromatography for antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;848(1):40-47. doi: 10.1016/j.jchromb.2006.09.030

 

  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(1):28-39. doi: 10.1016/j.jchromb.2006.09.026

 

  1. Bolton GR, Mehta KK. The role of more than 40 years of improvement in protein A chromatography in the growth of the therapeutic antibody industry. Biotechnol Prog. 2016;32(5):1193-1202. doi: 10.1002/btpr.2324

 

  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(9):1816-1827. doi: 10.1002/jssc.201800963

 

  1. Hjelm H, Hjelm K, Sjöquist J. Protein A from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins. FEBS Lett. 1972;28(1):73-76. doi: 10.1016/0014-5793(72)80680-x

 

  1. Hjelm H, Sjödahl J, Sjöquist J. Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus. Eur J Biochem. 1975;57(2):395-403. doi: 10.1111/j.1432-1033.1975.tb02313.x

 

  1. Sjodahl J. Repetitive sequences in protein A from Staphylococcus aureus. Arrangement of five regions within the protein, four being highly homologous and Fc-binding. Eur J Biochem. 1977;73(2):343-351. doi: 10.1111/j.1432-1033.1977.tb11324.x

 

  1. Uhlén M, Guss B, Nilsson B, Gatenbeck S, Philipson L, Lindberg M. Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J Biol Chem. 1984;259(3):1695-1702.

 

  1. Lancet D, Isenman D, Sjödahl J, Sjöquist J, Pecht I. Interactions between staphylococcal protein A and immunoglobulin domains. Biochem Biophys Res Commun. 1978;85(2):608-614. doi: 10.1016/0006-291x(78)91206-8

 

  1. Hillson JL, Karr NS, Oppliger IR, Mannik M, Sasso EH. The structural basis of germline-encoded VH3 immunoglobulin binding to staphylococcal protein A. J Exp Med. 1993;178(1):331-336. doi: 10.1084/jem.178.1.331

 

  1. Potter KN, Li Y, Pascual V, Capra JD. Staphylococcal protein A binding to VH3 encoded immunoglobulins. Int Rev Immunol. 1997;14(4):291-308. doi: 10.3109/08830189709116521

 

  1. Jansson B, Uhlén M, Nygren PA. All individual domains of staphylococcal protein A show fab binding. FEMS Immunol Med Microbiol. 1998;20(1):69-78. doi: 10.1111/j.1574-695X.1998.tb01112.x

 

  1. Ghose S, Allen M, Hubbard B, Brooks C, Cramer SM. Antibody variable region interactions with protein A: implications for the development of generic purification processes. Biotechnol Bioeng. 2005;92(6):665-673. doi: 10.1002/bit.20729

 

  1. Kanje S, Scheffel J, Nilvebrant J, Hober S. Engineering of protein A for improved purification of antibodies and Fc-fused proteins, In: Matte A, editor. Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. Netherlands: Elsevier; 2020. p. 35-54.

 

  1. Cytiva. MabSelect SuRe LX, Affinity Chromatography; 2020. Available from: https://cdn.cytivalifesciences.com/api/public/ content/digi-15712-pdf [Last accessed on 12 Sep 2025].

 

  1. Cytiva. MabSelect PrismA Resin, Affinity Chromatography; 2020. Available from: https://cdn.cytivalifesciences.com/api/ public/content/digi-26546-original [Last accessed on 12 Sep 2025].

 

  1. Cytiva. Guide to Transition from MabSelect SuRe™ Based Resins to Next-Generation MabSelect™ PrismA Resin; 2021. Available from: https://cdn.cytivalifesciences.com/api/public/ content/digi-28317-pdf [Last accessed on 12 Sep 2025].

 

  1. Cytiva. MabSelect VH3 Resin, Affinity Chromatography; 2023. Available from: https://cdn.cytivalifesciences.com/api/ public/content/wzr3i-c_euafvmypt54vjq-pdf [Last accessed on 12 Sep 2025].

 

  1. Dong W, Wang R, Li Y. MabSelect VH3 protein A affinity resin effectively separates antibody species containing different numbers of VH3 domain and shows improved aggregate separation capability. Protein Expr Purif. 2025;227:106646. doi: 10.1016/j.pep.2024.10664

 

  1. Lee CV, Viadiu H, Kalamkar A, et al. Identification and engineering of potent bispecific antibodies that protect against herpes simplex virus recurrent disease. Cell Rep. 2025;44(8):116063. doi: 10.1016/j.celrep.2025.116063

 

  1. Mullin M, McClory J, Haynes W, Grace J, Robertson N, Van Heeke G. Applications and challenges in designing VHH-based bispecific antibodies: Leveraging machine learning solutions. MAbs. 2024;16(1):2341443. doi: 10.1080/19420862.2024.2341443

 

  1. Madsen AV, Kristensen P, Goletz S. IgG-VHH bispecific fusion antibodies: Challenges and opportunities as therapeutic agents. Expert Opin Biol Ther. 2024;24(4):203-206. doi: 10.1080/14712598.2024.2336068

 

  1. King LA, Veth M, Iglesias-Guimarais V, et al. Leveraging Vγ9Vδ2 T cells against prostate cancer through a VHH-based PSMA-Vδ2 bispecific T cell engager. iScience. 2024;27(12):111289. doi: 10.1016/j.isci.2024.111289

 

  1. Asanuma T, Ding P, Kato Y, Nakanishi T, Asano R, Makabe K. Evaluation of an anti-CD3 VHH and construction of an anti-CD3/ anti-EGFR bispecific tandem VHH as a cancer cell targeting drug construct. Biochem Biophys Rep. 2025;42:102015. doi: 10.1016/j.bbrep.2025.102015

 

  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(4):632-638. doi: 10.1080/19420862.2019.1583996

 

  1. Ingavat N, Wang X, Kok YJ, et al. Affinity resin selection for efficient capture of bispecific antibodies as guided by domain composition. Process Biochem. 2025;154:1-11. doi: 10.1016/j.procbio.2025.04.007

 

  1. NanoChrom Technologies. Therapeutic Proteins Analysis using Size Exclusion Chromatography. Available from: https://gooees.com/data/www.nanochrom.com/static/upload/ file/20240419/1713497302564134.pdf [Last accessed on 12 Sep 2025].

 

  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. 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. 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(4):828-838. doi: 10.1080/19420862.2016.1160192

 

  1. Tustian AD, Laurin L, Ihre H, Tran T, Stairs R, Bak H. Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity. Biotechnol Prog. 2018;34(3):650-658. doi: 10.1002/btpr.2622

 

  1. Chen SW, Zhang W. Current trends and challenges in the downstream purification of bispecific antibodies. Antib Ther. 2021;4(2):73-88. doi: 10.1093/abt/tbab007
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