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.

Simple method to assess stability of immobilized peptide ligands against proteases.

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin-bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide-ChemMatrix resin. Peptidyl-resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C-terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography.

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.

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.

Targeted anti-inflammatory peptide delivery in injured endothelial cells using dermatan sulfate/chitosan nanomaterials.

This work describes a novel delivery system for targeting egg-derived anti-inflammatory tripeptide Ile-Arg-Trp (IRW) to endothelial cells. The nanomedicine is synthesized by a simple and reproducible ionotropic gelification method that results in the efficient loading of the positively charged IRW within the dermatan sulfate/ chitosan matrix, as demonstrated by ss-NMR spectroscopy. The incorporation of IRW results in a stable nanoparticle dispersion with a single size population of 442 ±â€¯43 nm. Fluorescence microscopy studies demonstrate the capacity of the nanomaterial to distinguish between a quiescent and an injured endothelium through the interaction of dermatan sulfate with the CD44 receptor. Remarkably, no additional surface functionalization is required as dermatan sulfate mediates their internalization and the intracellular release of this natural anti-inflammatory tripeptide to modulate endothelial inflammatory response. This simple, scalable, and versatile nanotechnology platform opens new opportunities to apply in the therapy of vascular disease.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography.

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999-1005, 2018.

Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides.

Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4-hydroxymethylbenzoic acid. One strategy involves the synthesis of a one-bead-two-peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis. The other protocol is based on the synthesis of a cyclic depsipeptide library in which a glycolamidic ester group is incorporated by adding glycolic acid. After library screening, the ring is opened and the peptide is released simultaneously for subsequent MS analysis. © 2016 by John Wiley & Sons, Inc.

Latest Advances in OBOC Peptide Libraries. Improvements in Screening Strategies and Enlarging the Family From Linear to Cyclic Libraries.

Solid phase screenings of one bead one compound (OBOC) libraries have been widely used to find ligands with pharmacological and analytical uses, and to purify or detect proteins in complex mixtures. To improve library screening, in the last years various strategies have been developed to avoid the selection of false positive beads and to obtain selective ligands. Currently, there is great interest in cyclic peptides because of their resistance to enzymatic degradation and higher selectivity compared to their linear counterparts. Lots of cyclic peptide libraries protocols have been recently developed to facilitate hits analysis. The aim of this review is to summarize the latest applications of solid phase screening of OBOC combinatorial peptide libraries, the improvements in the screening methods including mass spectrometry MS/MS techniques and the strategies to synthesize OBOC cyclic peptide libraries.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.