Exosomes are extracellular vesicles (EVs) produced by all types of cells and secreted into most body fluids, such as blood, urine, saliva, and cerebrospinal fluid. Exosomes are possible targets of liquid biopsy because they contain active biomolecules e.g., protein, RNA, DNA, and phospholipids, which are involved in cell-cell communications and reportedly associated with various disease conditions such as cancer progression and metastasis. Three major ways to isolate exosomes from specimens are: ultracentrifugation, polymer precipitation, and immunoaffinity purification (IP) using surface marker antibodies. Of these, IP outperforms the other two as it enables specific isolation, but it still has some issues to be solved for clinical application. One of the issues is that purity of exosomes samples after purification is not enough for clinical experiments. Another problem is the low reproducibility of exosome isolation, which leads to unclear experimental results. In this study, we have developed antibodies and a condition that enable efficient antigen-antibody reaction to isolate high-purity exosomes from serum and plasma. Then, the whole recovery process, including IP, was automated to achieve better isolation reproducibility with respect to quality and quantity. First, antibodies against CD9 and CD63, which localize on the membrane surface of exosomes, were established and used in a customized condition for IP of exosome from serum, plasma and urine. Purity of the obtained exosome samples were assessed by protein measuring and nanoparticle tracking analysis. As a result, 2.4 times more exosome particles were isolated from serum samples using our method than conventional IP. Second, to identify the variety of proteins found in the exosome, proteomic analysis was performed with the obtained samples. As a result, greater variety (1.7-fold) of proteins was detected from the samples obtained using our antibodies in the customized condition. Interestingly, EML4-ALK fusion gene was identified 10 times more frequently from the exosomes obtained from lung cancer cell line using our method than the conventional method. This result is consistent with the result that our new antibodies and condition enable exosome isolation with higher purity. Third, to increase the reproducibility of exosome isolation, we have developed a mechanically controlled, automated process. The reproducibility of exosome isolation by the automated device was evaluated by measuring the amount of miR-223-3p in the obtained samples by qPCR. The result showed that the reproducibility has been significantly improved, compared to isolation by manual handling. In summary, immunoprecipitation using our new antibodies with the customized condition was useful in the isolation of highly pure exosomes efficiently. Our method is expected to contribute to the detection of new biomarkers and molecular targets for therapeutics and practical use for liquid biopsy test at the clinical site. Furthermore, our newly developed automated device can save the time and labor in experiments involving exosome isolation, and is expected to minimize discrepancy of results in exosome studies caused by unstandardized isolation method.

Citation Format: Tatsutoshi Inuzuka, Ayako Kurimoto, Yuki Kawasaki. High-purity purification of exosome from multiple specimens and automation of the process [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A045. doi:10.1158/1535-7163.TARG-19-A045