The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.
Phoneutria nigriventer spider can cause severe envenomation in humans principally due to its venom toxin δ-ctenitoxin-Pn2a. Current low yielding antivenom production is extremely complicated and dangerous. Furthermore, δ-ctenitoxin-Pn2a cystine-knot motif provides exceptional stability hampering immune response activation. Here, epitopes from δ-ctenitoxin-Pn2a were identi ed, and antigenic peptides were designed for their potential use in antivenom production. The Immune Epitope Database Analysis Resource was used to identify the G 34 YFWIAWYKLANCKK 48 epitope and used to design antigenic peptides. The Cys was replaced by α-aminobutyric acid (Abu) to avoid disul de bonds formation. To increase their immunogenicity, branched and N-palmitoylated peptides were synthesized.Ac-GYFWIAWYKLAN-Abu-KKG-NH 2 (A), (Ac-GYFWIAWYKLAN-Abu-KK) 2 -KG-NH 2 (B), Palm-GYFWIAWYKLAN-Abu-KKG-NH 2 (C) and (Palm-GYFWIAWYKLAN-Abu-KK) 2 -KG-NH 2 (D) were synthesized using solid-phase peptide synthesis (SPPS) techniques and analyzed by ESI-MS demonstrating their identity. Also, they were evaluated by RP-HPLC, and all the chromatograms showed only one principal peak except that of the N-palmitoylated branched peptide which showed two principal peaks probably due to the presence of two conformations in slow interconversion. Cytotoxicity was evaluated on the murine macrophage cell line RAW264.7 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in the presence of increasing doses of each peptide (0.25-10.0 µM). Peptide A did not exhibit cytotoxicity between 0.25-10.0 µM, while B, C and D showed cytotoxicity over 10.0, 5.0 and 2.5 µM respectively. NF-κB cellular distribution was evaluated by immuno uorescence, after exposing macrophages to 0.5 µM of each peptide. An early activation was observed for all the assayed peptides demonstrating that they are promising candidates for their in vivo evaluation as immunogens in antivenom production.
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