Supramolecular Mimotope Peptide Nanofibers Promote Antibody-Ligand Polyvalent and Instantaneous Recognition for Biopharmaceutical Analysis.

Peptide-based supramolecules exhibit great potential in various fields due to their improved target recognition ability and versatile functions. However, they still suffer from numerous challenges for the biopharmaceutical analysis, including poor self-assembly ability, undesirable ligand-antibody binding rates, and formidable target binding barriers caused by ligand crowding. To tackle these issues, a "polyvalent recognition" strategy employing the CD20 mimotope peptide derivative NBD-FFVLR-GS-WPRWLEN (acting on the CDR domains of rituximab) was proposed to develop supramolecular nanofibers for target antibody recognition. These nanofibers exhibited rapid self-assembly within only 1 min and robust stability. Their binding affinity (179 nM) for rituximab surpassed that of the monomeric peptide (7 µM) by over 38-fold, highlighting that high ligand density and potential polyvalent recognition can efficiently overcome the target binding barriers of traditional supramolecules. Moreover, these nanofibers exhibited an amazing "instantaneous capture" rate (within 15 s), a high recovery (93 ± 3%), and good specificity for the target antibody. High-efficiency enrichment of rituximab was achieved from cell culture medium with good recovery and reproducibility. Intriguingly, these peptide nanofibers combined with bottom-up proteomics were successful in tracking the deamidation of asparagine 55 (from 10 to 16%) on the rituximab heavy chain after 21 day incubation in human serum. In summary, this study may open up an avenue for the development of versatile mimotope peptide supramolecules for biorecognition and bioanalysis of biopharmaceuticals.

Mimotope peptide modified pompon mum-like magnetic microparticles for precise recognition, capture and biotransformation analysis of rituximab in biological fluids.

Due to low immobilized ligand density, limited binding capacity, and severe interference from serum proteins, developing ideal peptide-based biomaterials for precise recognition and in vivo analysis of biopharmaceuticals remains a huge challenge. In this study, mimotope peptide modified pompon mum-like biomimetic magnetic microparticles (MMPs, 3.8 µm) that mimic the specific functionalities of CD20 on malignant B cells were developed for the first time. Benefit from the numerous ligand binding sites (Ni2+) on the pompon mum-like MMPs, these novel materials achieved ≥10 times higher peptide ligand densities (>2300 mg/g) and antibody binding capacities (1380 mg/g) compared to previous reported biomaterials. Leveraging the high specificity of the mimotope peptide, rituximab can be precisely recognized and enriched from cell culture media or serum samples. We also established an LC‒MS/MS method using the MMPs for tracking rituximab biotransformation in patient serum. Intriguingly, deamidation of Asn55 and Asn33, as well as oxidation of Met81 and Met34 were observed at the key complementarity determining regions of rituximab, which could potentially influence antibody function and require careful monitoring. Overall, these versatile biomimetic MMPs demonstrate superior recognition and enrichment capabilities for target antibodies, offering interesting possibilities for biotransformation analysis of biopharmaceuticals in patient serum.