Envenomation caused by snakes, scorpions, and spiders represents a serious public health problem in Latin America. The antivenoms used for its treatment are produced by immunizing horses repeatedly with sublethal doses of animal venoms along with the adjuvant. However, venom availability is a bottleneck. Furthermore, toxin-neutralizing antibodies are only a few of the total produced with this classical method. Therefore, high doses of antivenom are required to achieve the neutralization power, which usually causes adverse reactions in the patient. With the aim of obtaining a higher proportion of toxin-neutralizing antibodies while reducing the dependency on venom availability, alternative immunization protocols have been explored using synthetic peptides with epitopes from clinically relevant toxins. The process to design an immunogenic peptide entitles: (a) choice of the medical relevant toxins in the venom; (b) identification of the epitopes in the selected toxins; (c) improvement of peptide immunogenicity; (d) immunogen synthesis; and e) in vitro and in vivo evaluation. The present article aims to review the advances in the design of immunogenic synthetic peptides for their application in antivenom production in Latin America during the 21st century. Epitopes have been identified from many clinically important toxins in Latin American snakes (snake venom metalloproteinases, snake venom serine proteases, crotamine, phospholipases A2, and three-finger toxins), scorpions (beta-mammal/insect toxin Ts1, alpha-mammal toxin Ts2, alpha-mammal toxin Ts3, toxin Ts4, and beta-mammal Tt1g neurotoxin), and spiders (dermonecrotic toxin and delta-ctenitoxin-Pn2a). Nevertheless, their application is still experimental, even though they are ideal for large-scale and low-cost antivenom production, factors that are necessary to meet national and regional demands.

The therapeutic monoclonal antibody bevacizumab is typically purified using Protein-A affinity chromatography, a highly effective but costly method. As a lower-cost alternative, affinity-based precipitation has been described to purify antibodies. Therefore, in this work, a precipitation protocol was developed for bevacizumab purification using the branched peptide (Ac-PHQGQHIG-Ahx₃)2-K-Ahx₃-PHQGQHIG-NH₂, which contains the epitope PHQGQHIG responsible for interaction with bevacizumab. The peptide was synthesised by microwave-assisted solid-phase peptide synthesis employing LiCl as an additive to prevent aggregation and ensure high purity and yield. Three molecules of 6-aminohexanoic acid were introduced between each epitope branch as spacer arms to promote the formation of cyclic complexes. Bevacizumab purification from the cell-free culture broth was achieved through a fractional precipitation process. First, a negative precipitation step using (NH₄)₂SO₄ 1.18 M was performed to remove contaminants. Afterwards, 5 moles of peptide per mol of bevacizumab were added to the supernatant, together with additional (NH₄)₂SO₄ to reach a final concentration of 1.20 M. Under these conditions, bevacizumab was recovered in the precipitate with 98% purity and a yield of 73%. In addition to being recyclable, the peptide´s relative low production cost may enable the development of a single-use purification process, which would be particularly advantageous for biopharmaceutical manufacturing.

Introduction: Bevacizumab, applied in cancer treatment, binds vascular endothelial growth factor (VEGF) inhibiting blood vessels growth, avoiding tumours expansion

The therapeutic monoclonal antibody bevacizumab is typically purified using protein A affinity chromatography, a highly effective but costly method. Affinity-based precipitation for antibody purification is a lower-cost approach. In this work, a precipitation protocol was developed for bevacizumab purification using a branched peptide (Ac-PHQGQHIG-Ahx₃)₂-K-Ahx₃-PHQGQHIG-NH₂, which contains the epitope PHQGQHIG that is responsible for interacting with bevacizumab. The peptide was synthesised by a microwave-assisted solid-phase peptide method, employing LiCl as an additive to prevent aggregation and ensure high purity and yield. Three molecules of 6-aminohexanoic acid were introduced between each epitope branch as spacer arms to promote the formation of cyclic complexes. Bevacizumab purification from cell-free culture broth was achieved through a fractional precipitation process. First, a negative precipitation step using (NH₄)₂SO₄ 1.18 M was performed to remove contaminants. Afterwards, 5 moles of peptide per mol of bevacizumab was added to the supernatant, together with additional (NH₄)₂SO₄, to reach a final concentration of 1.20 M. Under these conditions, bevacizumab was recovered in the precipitate with 98% purity and a yield of 73%. In addition to being recyclable, the peptide’s relatively low production cost could enable the development of a single-use purification process, which would be particularly advantageous for biopharmaceutical manufacturing.