Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands

Kish, William S.; Roach, Matthew K.; Sachi, Hiroyuki; Naik, Amith D.; Menegatti, Stefano; Carbonell, Ruben G.

doi:10.1016/j.jchromb.2018.03.039

Cited by 24 publications

(14 citation statements)

References 43 publications

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“…Amination of Workbeads™ ACT 40 resin was carried out using a protocol established in previously by Kish et al The resin, originally in 20% ( v / v ) ethanol, was first rinsed extensively in deionized water and 50 mM carbonate buffer (pH 9.8). A solution of 25% ( v / v ) ammonium hydroxide (NH 4 OH) in 100 mM carbonate buffer, pH 12.0 was then added to the resin (in 1:1 volume ratio) and incubated overnight at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Design of peptide ligands for affinity purification of human growth hormone

Chandra

Timmick

Goodwine

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND: In this study, we have demonstrated the design, screening and selection of peptide ligands for the affinity capture of human growth hormone (hGH) from yeast cell cultures. RESULTS:Ligand design was carried out using multiple approaches based on primary sequence and structures of natural binding partners of hGH. Screening of potential affinity peptides was conducted using high throughput microarray platforms followed by assessment of in-solution binding to hGH using fluorescence polarization. Peptide leads were subsequently immobilized on chromatographic resins and the binding and desorption behavior was examined using batch adsorption studies. A lead candidate was examined in further details in column chromatography studies which indicated that while high purity was attained, further refinement was necessary for improved product recovery. Histidine scanning was employed to successfully improve the recovery of hGH from cell culture fluid while still maintaining high purity. Finally, proof-of-concept was demonstrated in the column format using complex feed stock where a product purity of 95% was attained at 80% yield. CONCLUSION: The approaches presented here can be translated to other biologics of interest for the rapid development of affinity based purification processes.

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Section: Methodsmentioning

confidence: 99%

Design of peptide ligands for affinity purification of human growth hormone

Chandra

Timmick

Goodwine

et al. 2019

J of Chemical Tech & Biotech

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“…The adsorption isotherms of anti-IgG, anti-HSA, and anti-ErbB2 affibodies on IGKQRI-GSG-Toyopearl resin were performed as described in prior work [37,51]. Toyopearl resins functionalized with human IgG and serum albumin were utilized as corresponding positive controls, while Toyopearl ® HW-40F was utilized as a negative control.…”

Section: Binding Isotherms Of Model Affibodies On Peptide-based Adsormentioning

confidence: 99%

Affibody-Binding Ligands

Barozzi

Lavoie

Day

et al. 2020

IJMS

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While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody–peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 μM), recovery and purity (64–71% and 86–91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.

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“…Additionally, they serve as surfactants or tags promoting solubility of recombinant intrinsic membrane proteins [16][17][18][19][20], increasing their yield, activity, and aiding protein structural studies. Peptides are even replacing enzymes in catalytic reactions [21] and substituting proteins as ligands in affinity chromatography [22,23].…”

Section: Introductionmentioning

confidence: 99%

Evolving a Peptide: Library Platforms and Diversification Strategies

Bozovičar

Bratkovič

2019

IJMS

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Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

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Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands

Cited by 24 publications

References 43 publications

Design of peptide ligands for affinity purification of human growth hormone

Design of peptide ligands for affinity purification of human growth hormone

Affibody-Binding Ligands

Evolving a Peptide: Library Platforms and Diversification Strategies

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