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 (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) 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 (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.
摘要：
外泌体作为药物递送的载体越来越突出，疫苗接种,和再生医学。由于它们的表面生物化学，它反映了亲本细胞膜，这些纳米级生物制剂具有低免疫原性，可调的组织嗜性，以及携带各种有效载荷穿越生物屏障的能力。外来体大小和组成的异质性，然而,使他们的净化具有挑战性。传统技术,比如超速离心和过滤，提供低的产品收率和纯度，并危及粒子的完整性。亲和层析代表了外泌体纯化的极好途径。然而，当前亲和介质依赖于抗体配体，其选择性赋予高产品纯度，但是要求定制具有不同表面生物化学的外泌体的吸附剂，而它们的结合强度施加了可能损害产品活性的洗脱条件。解决这些问题,这项研究介绍了第一个肽亲和配体，用于从重组原料中普遍纯化外泌体。肽被设计为（1）具有外来体标记的混杂生物识别，没有结合过程相关的污染物和（2）在保护产品稳定性的条件下洗脱产品。选定的配体SNGFKKHI和TAHFKKKH证明了捕获由14种细胞来源分泌的外泌体和源自HEK293，PC3，MM1，U87和COLO1细胞的纯化外泌体的能力，产量高达80％，并且在pH梯度从7.4到10.5洗脱时，宿主细胞蛋白（HCP）减少了50倍，推荐用于外泌体稳定性。SNGFKKHI-Toyopearl树脂最终用于两步纯化过程中，从HEK293细胞液中分离外泌体，提供68%的产率并将HCP的滴度降低至68ng/mL。分离的外泌体的生物分子和形态特征通过分析色谱法证实，蛋白质印迹分析,透射电子显微镜,纳米粒子跟踪分析。
