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Peptide Ligands for the Universal Purification of Exosomes by Affinity Chromatography
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  • Ryan Kilgore,
  • Brandyn Moore,
  • Sobhana Alekhya Sripada,
  • Wenning Chu,
  • Shriarjun Shastry,
  • Eduardo Barbieri,
  • Shiqi Hu,
  • Weihua Tian,
  • Heidi Petersen,
  • Mohammad Mohammadifar,
  • Aryssa Simpson,
  • Ashley Brown,
  • Joseph Lavoie,
  • Driss Elhanafi,
  • Steffen Goletz,
  • Ke Cheng,
  • Michael A. Daniele,
  • Stefano Menegatti
Ryan Kilgore
NC State University Department of Chemical and Biomolecular Engineering
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Brandyn Moore
NC State University Department of Chemical and Biomolecular Engineering
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Sobhana Alekhya Sripada
NC State University Department of Chemical and Biomolecular Engineering
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Wenning Chu
NC State University Department of Chemical and Biomolecular Engineering
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Shriarjun Shastry
NC State University Department of Chemical and Biomolecular Engineering
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Eduardo Barbieri
NC State University Department of Chemical and Biomolecular Engineering
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Shiqi Hu
NC State University Department of Molecular Biomedical Sciences
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Weihua Tian
Danmarks Tekniske Universitet Institut for Bioteknologi og Biomedicin
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Heidi Petersen
Danmarks Tekniske Universitet Fodevareinstituttet
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Mohammad Mohammadifar
Danmarks Tekniske Universitet Fodevareinstituttet
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Aryssa Simpson
UNC/NCSU Joint Department of Biomedical Engineering
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Ashley Brown
UNC/NCSU Joint Department of Biomedical Engineering
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Joseph Lavoie
Biomanufacturing Training and Education Center (BTEC
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Driss Elhanafi
Biomanufacturing Training and Education Center (BTEC
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Steffen Goletz
Danmarks Tekniske Universitet Institut for Bioteknologi og Biomedicin
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Ke Cheng
NC State University Department of Molecular Biomedical Sciences
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Michael A. Daniele
UNC/NCSU Joint Department of Biomedical Engineering
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Stefano Menegatti
NC State University Department of Chemical and Biomolecular Engineering

Corresponding Author:[email protected]

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Abstract

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes’ size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product’s activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (i) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants, and (ii) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up-to 80% and up-to 50-fold reduction of host cell proteins upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a 2-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of host cell proteins to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blotting, transmission electron microscopy, nanoparticle tracking analysis.
Submitted to Biotechnology and Bioengineering
06 Jun 2024Reviewer(s) Assigned
09 Jul 2024Editorial Decision: Revise Minor
20 Jul 20241st Revision Received
22 Jul 2024Assigned to Editor
22 Jul 2024Submission Checks Completed
22 Jul 2024Review(s) Completed, Editorial Evaluation Pending
22 Jul 2024Editorial Decision: Accept