Antibodies are a group of immunoglobulins found in animal blood and tissue fluids that play a role in immunity, where monoclonal antibodies (mAbs) are currently the most important class of antibody drugs known for their high specificity, strong targeting ability and low toxicity [1]. They play a significant role in the treatment of diseases such as cancer and autoimmune diseases [2]. Since the approval of the first chimeric antibody drug for lymphoma treatment in 1997, therapeutic antibody drugs have been continuously approved, with over 100 antibody-related drugs currently available. The global monoclonal antibody drug market exceeds hundreds of billions of dollars, and it maintains a growth rate of more than 10% over the past decades [3].

With the continuous expansion of the downstream scale, the rationality of the preparation process and the economy of the process attracts more and more attention, and it is urgent to improve production capacity and reduce production costs [4,5]. For instance, in order to enhance resin capacity utilization and productivity, Sun et al. [6] investigated a model-based approach to improve process development of twin-column continuous capture with Protein A resins. Protein A is currently the most widely used technique in antibody capture. However, it still has inevitable defects, such as high cost and leakage of ligands [7], [8], [9]. Therefore, the search for new cost-effective and high binding capacity resins as alternatives is still necessary. For example, hydrophobic charge-induction chromatography (HCIC) is a mixed mode chromatography that was developed for applications in various separation stages [10]. HCIC ligands such as 4-thioethylpyridine (MEP), 5-aminobenzimidazole (ABI), 2-mercaptoyl-1-methylimidazole (MMI), 2-mercaptobenzimidazole (MBI) and 5-aminopindole (AI) [11], [12], [13], [14], [15] were studied and used for antibody purification as commercial products or in research. Meanwhile, peptide ligands are promising candidates with advantages of high selectivity, stable structure and low cost [16,17]. Kilgore et al. [18] screened out the FRWNFHRNTFFP ligand via molecular docking simulation and other techniques. The DBC of this ligand was 6–16 mg/mL, and the yield and purity were 93–96% and 89%−96%, respectively. However, the elution conditions of peptide ligands are relatively harsh due to strong binding forces. Zou et al. [19] combined peptide groups with HCIC groups to effectively avoid the shortcomings of the two chromatographic methods and obtain hybrid biomimetic ligands with strong binding ability and mild elution conditions. However, the binging capacity was not high enough comparing to Protein A resins. It is still a common problem of most synthetic ligands, and current approaches focused on ligand design to improve binding capacity seems have limited capability.

Chromatographic resins are usually composed of ligands and porous matrices, with the later ones play a supporting structure and surface areas to ensure that resins can provide large surface for antibody binding and also withstand external pressure during separation processes. Agarose, cellulose, and dextran are commonly used as matrix materials [20], [21], [22], and their morphology including pore network, particle size and specific surface area can largely affect separation performance [23]. Qiao et al. [24] employed a chemical cross-linking strategy to fabricate porous cellulose bead, with values of 204.58 mg/g for BSA and 144.78 mg/g for BHb. Zhang et al. [25] synthesized pGMA-DVB microspheres, which were covalently adorned with dextran, achieving recovery yields of over 99.5% and high dynamic binding capacities. Zhao et al. [26] employed the membrane emulsification method to produce uniform agarose microspheres with an average particle size of approximately 8 μm, which exhibited precise control over the molecular range, excellent chromatographic selectivity, and extensive applicability in downstream processes.

The present study employed three agarose microsphere matrices with different particle sizes to fabricate antibody separation resins through coupling with a hybrid biomimetic ligand. The effect of particle size on IgG binding ability was investigated and the capability of resin for antibody separation from protein mixtures and CHO cell cultures were studied. The reusability of the resin was also tested in order to achieve optimal adsorption performance for IgG and enhance downstream process efficiency.