Antibody drugs represent a fast-growing class of biopharmaceuticals due to their effectiveness in the treatment of cancer, autoimmune diseases and infectious diseases [1,2]. To date, the upstream processing technology for antibody drugs has made great progress, with their expression levels reaching 5–-13 g/L [3], while the downstream purification process remains a critical bottleneck due to its high cost and complexity [4,5]. Protein A/G affinity chromatography, the gold standard for monoclonal antibody (mAb) purification, accounts for over 50 % of production cost, prompting the need for more efficient and cost-effective alternatives [6]. Peptide-based affinity materials, such as Fc- or Fab-peptide ligands, are promising substitutes for conventional protein A/G/L resins in mAb purification, due to their wide diversity, good chemical stability, low cost and reduced immunogenicity [7]. In recent years, peptide-based affinity materials have been developed to enrich antibodies from complex biological fluids with satisfactory selectivity and specificity, including affinity membranes [[8], [9], [10]], nanoparticles [11], resins [12], affinity columns [13], and polymer monoliths [14]. Among these materials, affinity membranes have attracted great attention because of their advantages, such as high mass transfer efficiency, high speed, cost-effectiveness and low energy consumption [15]. For instance, Boi et al. developed an A2P-DES-Sartoepoxy affinity membrane, which demonstrated good purification efficiency for IgG, good stability across repeated cycles and negligible ligand leakage [16]. However, traditional affinity membranes still face some challenges, such as lower binding capacity compared to affinity resins and susceptibility to fouling [17,18]. It is of great interest to develop novel affinity membranes with high binding capacity and good anti-fouling properties for efficient antibody purification [19,20].

Mixed matrix membranes (MMMs), which disperse organic/inorganic materials as fillers into polymeric materials as matrixes, could significantly improve the anti-fouling properties and enhance the ligand density of membranes [21]. For example, Gayatri et al. developed a novel MMM by adding hydrophilic titanium dioxide (TiO2) and pore-forming agents to hydrophobic polyvinylidene fluoride (PVDF). The resultant PVDF-TiO2 MMM showed excellent BSA rejection ability (rejection rate > 97 %) because of the high hydrophilicity of the TiO2 filler [22]. It has also been reported that the anti-bacterial activity of TiO2 is beneficial to the storage time and service life of MMMs [[23], [24]]. Covalent organic frameworks (COFs) are a class of promising organic fillers with high thermal and chemical stabilities, and high specific surface area, providing significant benefits in improving interfacial affinity, compatibility and ligand density of MMMs [[25], [26], [27]]. However, the high hydrophobicity of COFs can reduce the anti-fouling ability of modified MMMs [28]. One method to address this limitation is to prepare dual-filler MMM by coupling different nano fillers [29]. For instance, Cheng et al. prepared MOF@COF hybrid MMMs with enhanced permeability and selectivity for target molecule due to the synergistic effects of MOF (selectivity) and COF (permeability) [30]. Pang et al. fabricated ZnO/MWCNT dual-nanofiller polyether sulfone MMMs, with superior membrane filtration performance [31]. Notably, the graphene oxide (rGO) ZnO/PES hybrid membrane prepared by Kusworo et al. demonstrated that the presence of rGO in the ZnO PES matrix enhanced the uniformity of nanoparticle distribution in the rGO/ZnO membrane and increased the average flux by 4-fold [32]. These advancements underscore the significant potential of dual-filler MMMs in bio-separation applications.

Inspired by the above, a novel affinity peptide-functionalized dual-filler MMM was designed and prepared by introducing two fillers (COF and TiO2) into PES for highly efficient antibody purification. Firstly, a highly acid-resistant organic COF (TpPa) and an inorganic semiconductor TiO2 were covalently bonded to construct a hybrid heterostructure filler, with abundant carbon-carbon double bond and good hydrophilicity (Scheme 1). Then, the hybrid heterostructure filler was added to a PES solution and the dual-filler COF/TiO2/PES MMMs were fabricated by nonsolvent induced phase separation (NIPS). To improve their selectivity, a Fab-specific affinity peptide of Trastuzumab (m-EDPW, Kd = 1.91 μM) was selected as the recognition ligand and grafted onto the dual-filler MMM surface through atom-transfer radical-polymerization (ATRP) method [33]. Finally, the m-EDPW@COF@TiO2/PES membrane was rolled up and inserted into a 1 mL syringe for practical application (Scheme 1). The physical and chemical properties, selectivity, binding ability, storage stability and resistance to non-specific adsorption of the obtained dual-filler MMMs were systematically evaluated. Moreover, their performances in purifying trastuzumab from HCC1937 cell culture medium was thoroughly investigated.