Simple method to assess stability of immobilized peptide ligands against proteases

Giudicessi, Silvana L.; Salum, María Laura; Saavedra, Soledad L.; Martínez‐Ceron, María C.; Cascone, Osvaldo; Erra‐Balsells, Rosa; Camperi, Silvia A.

doi:10.1002/psc.3012

Cited by 7 publications

(6 citation statements)

References 19 publications

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“…It was performed as per Giudicessi et al Approximately 10 mg of Ac‐KVPLTVSKAKVAG‐HMBA‐CM resin was incubated with Chinese hamster ovary (CHO) cell culture supernatant overnight at 37°C during 24 hours, and after incubation they were thoroughly washed with H 2 O, H 2 O:DMF (7:3; 5:5; 3:7 v/v), DMF, and CH 2 Cl 2 . Peptides were detached from the resin with ammonia vapor as has been previously described .…”

Section: Methodsmentioning

confidence: 99%

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Messina

Giudicessi

Ceron

et al. 2018

Journal of Peptide Science

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Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C‐termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

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Section: Methodsmentioning

confidence: 99%

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Messina

Giudicessi

Ceron

et al. 2018

Journal of Peptide Science

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“…Thereby, immobilized peptide evaluation is preferred. A solid-phase strategy using 4-(hydroxymethyl)benzamide-ChemMatrix (HMBA-CM) resin and mass spectrometry (MS) has been developed to test peptide stability and applied in the selection of ligands for rhEPO, rhFSH, rhGH and bevacizumab purification by AC (Giudicessi et al 2017 ; Saavedra et al 2018 ; Gurevich Messina et al 2018 ; Barredo et al 2019 ). The method entitles: (a) solid-phase peptide synthesis on HMBA-CM resin; (b) peptidyl-resin beads incubation in solutions containing proteases or in cell culture broth; (c) whole peptide or C-terminal degradation products detachment from solid support with ammonia vapor; (d) MS sequencing of the peptide and C-terminal fragments (Fig.…”

Section: Preliminary Peptide Ligand Evaluationmentioning

confidence: 99%

“…Different strategies have been proposed to increase ligand stability, like the use of some d -amino acids in the peptide or by using the retroinverso analog which has the chirality of the amino acid inverted from l to d and the sequence in reverse direction with respect to the natural peptide. Retroinverso peptides mimic the structure of the original l -peptide, therefore show similar affinity for the target but with increased stability (Giudicessi et al 2017 ; Rai 2019 ). Also, cyclic peptides can be used as ligands due to their reduced conformational flexibility compared to linear peptides which confers strong resistance to proteolytic degradation.…”

Section: Preliminary Peptide Ligand Evaluationmentioning

confidence: 99%

“…Thereby, immobilized peptide evaluation is preferred. A solid-phase strategy using 4-(hydroxymethyl)benzamide-ChemMatrix (HMBA-CM) resin and mass spectrometry (MS) has been developed to test peptide stability and applied in the selection of ligands for rhEPO, rhFSH, rhGH and bevacizumab purification by AC (Giudicessi et al 2017;Saavedra et al 2018;Gurevich Messina et al 2018;Barredo et al 2019) (c) SPR affinity analysis: (c 1 ) peptide is separated from the solid support with ammonia vapor. (c 2 ) interactions between peptides and antibodies can be studied in real time by SPR without labeling the analytes of the peptide and C-terminal fragments (Fig.…”

Section: Preliminary Peptide Ligand Evaluationmentioning

confidence: 99%

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Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

Barredo-Vacchelli

Giudicessi

Martínez‐Ceron

et al. 2021

Int J Pept Res Ther

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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.

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“…Peptide ligands can be tailored to avoid sequences susceptible to protease degradation or to improve their affinity and selectivity. Furthermore, they can be produced by large-scale good manufacturing practices (GMPs) . Finally, peptide ligand matrices allow the use of more astringent elution or regeneration conditions than those based on proteins. − Peptide ligands suitable for protein purification can be found by screening combinatorial peptide libraries, an approach in which thousands of molecules can be evaluated in one step.…”

Section: Introductionmentioning

confidence: 99%

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification

et al. 2021

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Peptide ligands are widely used in protein purification by affinity chromatography. Here, we applied a fully automated two-stage library screening method that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)–rhodamine conjugate. This time, fluorescent beads were isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM sodium phosphate, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a K d of 9.18 ± 0.07 nmol/L and a q m of 1.31 ± 0.029 μmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.

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Simple method to assess stability of immobilized peptide ligands against proteases

Cited by 7 publications

References 19 publications

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification

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