Immobilizing Calcium-dependent Affinity Ligand Onto Iron Oxide Nanoparticles for Mild Magnetic MAb Separation

Overview

Journal Biotechnol Rep (Amst)

Specialty Biotechnology

Date 2024 Dec 18

PMID 39691100

Authors

Ines Zimmermann

Friederike Eilts

Anna-Sophia Galler

Jonas Bayer

Sophia Hober

Sonja Berensmeier

Affiliations

Soon will be listed here.

Abstract

Current downstream processing of monoclonal antibodies (mAbs) is limited in throughput and requires harsh pH conditions for mAb elution from Protein A affinity ligands. The use of an engineered calcium-dependent ligand (Z) in magnetic separation applications promises improvements due to mild elution conditions, fast processability, and process integration prospects. In this work, we synthesized and evaluated three magnetic nanoparticle types immobilized with the cysteine-tagged ligand Z-cys. Ligand homodimers were physically immobilized onto bare iron oxide nanoparticles (MNP) and MNP coated with tetraethyl orthosilicate (MNP@TEOS). In contrast, Z-cys was covalently and more site-directedly immobilized onto MNP coated with (3-glycidyloxypropyl)trimethoxysilane (MNP@GPTMS) via a preferential cysteine-mediated epoxy ring opening reaction. Both coated MNP showed suitable characteristics, with MNP@TEOS@Z-cys demonstrating larger immunoglobulin G (IgG) capacity (196 mg ) and the GPTMS-coated particles showing faster magnetic attraction and higher IgG recovery (88 %). The particles pave the way for the development of calcium-dependent magnetic separation processes.

References

Yuan Y, Rende D, Altan C, Bucak S, Ozisik R, Borca-Tasciuc D . Effect of surface modification on magnetization of iron oxide nanoparticle colloids. Langmuir. 2012; 28(36):13051-9. DOI: 10.1021/la3022479. View

Kim S, Sung D, Chang J . Highly efficient antibody purification with controlled orientation of protein A on magnetic nanoparticles. Medchemcomm. 2018; 9(1):108-112. PMC: 6072530. DOI: 10.1039/c7md00468k. View

Lu R, Hwang Y, Liu I, Lee C, Tsai H, Li H . Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci. 2020; 27(1):1. PMC: 6939334. DOI: 10.1186/s12929-019-0592-z. View

Schwaminger S, Surya R, Filser S, Wimmer A, Weigl F, Fraga-Garcia P . Formation of iron oxide nanoparticles for the photooxidation of water: Alteration of finite size effects from ferrihydrite to hematite. Sci Rep. 2017; 7(1):12609. PMC: 5626691. DOI: 10.1038/s41598-017-12791-9. View

Rosen J, Gu F . Surface functionalization of silica nanoparticles with cysteine: a low-fouling zwitterionic surface. Langmuir. 2011; 27(17):10507-13. DOI: 10.1021/la201940r. View

Rabe M, Verdes D, Seeger S . Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci. 2011; 162(1-2):87-106. DOI: 10.1016/j.cis.2010.12.007. View

Walchli R, Ressurreicao M, Vogg S, Feidl F, Angelo J, Xu X . Understanding mAb aggregation during low pH viral inactivation and subsequent neutralization. Biotechnol Bioeng. 2019; 117(3):687-700. DOI: 10.1002/bit.27237. View

Brechmann N, Eriksson P, Eriksson K, Oscarsson S, Buijs J, Shokri A . Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture. Biotechnol Prog. 2019; 35(3):e2775. PMC: 6617771. DOI: 10.1002/btpr.2775. View

Scheffel J, Isaksson M, Gomis-Fons J, Schwarz H, Andersson N, Noren B . Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand. J Chromatogr A. 2022; 1664:462806. DOI: 10.1016/j.chroma.2022.462806. View

10.

Mateo , Abian , GUISAN . Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*. Enzyme Microb Technol. 2000; 26(7):509-515. DOI: 10.1016/s0141-0229(99)00188-x. View

11.

GULICH S, Uhlen M, Hober S . Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography. J Biotechnol. 2000; 76(2-3):233-44. DOI: 10.1016/s0168-1656(99)00197-2. View

12.

Zimmermann I, Kaveh-Baghbaderani Y, Eilts F, Kohn N, Fraga-Garcia P, Berensmeier S . Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies. ACS Appl Bio Mater. 2024; 7(6):3942-3952. PMC: 11190986. DOI: 10.1021/acsabm.4c00280. View

13.

Khawli L, Goswami S, Hutchinson R, Kwong Z, Yang J, Wang X . Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. MAbs. 2010; 2(6):613-24. PMC: 3011216. DOI: 10.4161/mabs.2.6.13333. View

14.

Swinnen K, Krul A, Van Goidsenhoven I, Van Tichelt N, Roosen A, Van Houdt K . Performance comparison of protein A affinity resins for the purification of monoclonal antibodies. J Chromatogr B Analyt Technol Biomed Life Sci. 2006; 848(1):97-107. DOI: 10.1016/j.jchromb.2006.04.050. View

15.

Salimi K, Usta D, Kocer I, Celik E, Tuncel A . Protein A and protein A/G coupled magnetic SiO microspheres for affinity purification of immunoglobulin G. Int J Biol Macromol. 2018; 111:178-185. DOI: 10.1016/j.ijbiomac.2018.01.019. View

16.

Kaplon H, Crescioli S, Chenoweth A, Visweswaraiah J, Reichert J . Antibodies to watch in 2023. MAbs. 2022; 15(1):2153410. PMC: 9728470. DOI: 10.1080/19420862.2022.2153410. View

17.

Filipe V, Kukrer B, Hawe A, Jiskoot W . Transient molten globules and metastable aggregates induced by brief exposure of a monoclonal IgG to low pH. J Pharm Sci. 2012; 101(7):2327-39. DOI: 10.1002/jps.23157. View

18.

Yang Q, Dong Y, Qiu Y, Yang X, Cao H, Wu Y . Design of Functional Magnetic Nanocomposites for Bioseparation. Colloids Surf B Biointerfaces. 2020; 191:111014. DOI: 10.1016/j.colsurfb.2020.111014. View

19.

von Roman M, Berensmeier S . Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization. J Chromatogr A. 2014; 1347:80-6. DOI: 10.1016/j.chroma.2014.04.063. View

20.

Warner C, Addleman R, Cinson A, Droubay T, Engelhard M, Nash M . High-performance, superparamagnetic, nanoparticle-based heavy metal sorbents for removal of contaminants from natural waters. ChemSusChem. 2010; 3(6):749-57. DOI: 10.1002/cssc.201000027. View