Peptide Ligands for the Universal Purification of Exosomes by Affinity
Chromatography
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.