Synthetic peptides to produce antivenoms against the Cys-rich toxins of arachnids.

Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures. Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized. This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.

Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose.

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. •Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification.

Bevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein A affinity chromatography (AC). However, protein A is a very expensive ligand, thereby increasing the cost of purification. Furthermore, the harsh elution conditions required to recover bevacizumab from the AC column can damage both the mAb and protein A. In contrast, short peptides show higher stability, easier synthesis and lower cost and are therefore ideal ligands for AC. In the present study, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment that binds bevacizumab, was synthesized and immobilized on agarose. The peptidyl-agarose showed affinity for bevacizumab, with an equilibrium dissociation constant value of 2.2±0.5 x 10-7 M under optimal conditions. Samples of CHO cell filtrate producing bevacizumab were loaded on the peptidyl-agarose chromatography column. Bevacizumab was recovered from the elution fraction with a yield of 94% and a purity of 98%. The maximum capacity (qm) 38±2 mg of bevacizumab per mL of matrix was comparable to that of commercial protein A matrices. Moreover, the peptide ligand showed greater stability and a lower cost than protein A. Unlike peptides previously reported for IgG purification, the ligand described herein allows mAb elution under mild conditions, thereby favoring the integrity of bevacizumab. The lack of Trp, Met or Cys in the peptide prevents its oxidation and extends the useful life of the chromatographic matrix.