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Analytics difference contour plot shown in Figure 2. Two fin- gerprint patterns demonstrate a maximum differ- ence of 25%, peaking at an excitation of 275 nm and emission of 300 nm, which corresponds to a higher concentration of tyrosine residues in PC3. Another peak is observed at an excitation of 270 nm and emis- sion of 415 nm, indicating a shift in the tryptophan emission spectrum in MCF7 compared to PC3. Fluorescence fingerprints of pure MCF7 and PC3, as well as various ratios of these two EVs, were ana- lyzed. The PARAFAC model was used to decompose the mixture data, and Excitation Emission Matrix f iltering and norma lization were applied for pre- processing. This analysis yielded two components: component 1 cor res pond i ng to t r y ptoph a n a nd component 2 corresponding to t yrosine, as shown in Figure 3. The model clearly distinguishes between the EV types and their mixtures. Furthermore, one can achieve quantification of EV concentrations and their ratios by constructing a calibration curve using the PARAFAC component scores versus the percent- age of PC3 (or MCF7) in the mixture, enabling quan- titative predictions of the relative amounts of each species in an EV mixture. Conclusion The f luorescence finger print profiles of these EV samples indicate measurable differences in protein sig nat u res bet ween t he sa mples. T he PA R A FAC model provides insights into these differences and can clearly distinguish between the EV types as well as their mixtures. Fluorescence spectroscopy represents a promising approach for characterizing EVs, offering rapid and sensitive analysis of their concentration and molecu- lar composition. The findings of this study validate the use of fluorescence spectroscopy as a reliable method for EV analysis. This technique holds significant po- tential for advancing our understanding of EV biology and its applications in diagnostics and therapeutics. Reference 1. Yarlott, L.K.; Gilmore, A. M. A-TEEM Spectroscopic Characterization of Exosome Standards and Their Mixtures. Poster presentation at the International Society for Extracellular Vesicles (ISEV) Annual Meeting, Virtual, May 18–21, 2021. ■ Cygnus Technology, Inc ................................................................ 13 Entegris............................................................................................. 2 G-CON Manufacturing .................................................................. 21 LabVantage Solutions, Inc. ........................................................... 17 Lonza Pharma & Biotech ................................................................ 9 Thermo Fisher Scientific Production ............................................. 5 Ad Index COMPANY PAGE FIGURE 3. Parallel factor analysis (PARAFAC) decomposition results of MCF7 and PC3 dataset. Component 1 corresponds to tryptophan residues in extracellular vesicles (EVs), and component 2 corresponds to tyrosine residue in EVs. 26 BioPharm International ® Manufacturing and Facilities eBook November 2024 www.biopharminternational.com