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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 J+Extracell+Vesicles
2018 ; 7
(1
): 1442088
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Scalable, cGMP-compatible purification of extracellular vesicles carrying
bioactive human heterodimeric IL-15/lactadherin complexes
#MMPMID29535850
Watson DC
; Yung BC
; Bergamaschi C
; Chowdhury B
; Bear J
; Stellas D
; Morales-Kastresana A
; Jones JC
; Felber BK
; Chen X
; Pavlakis GN
J Extracell Vesicles
2018[]; 7
(1
): 1442088
PMID29535850
show ga
The development of extracellular vesicles (EV) for therapeutic applications is
contingent upon the establishment of reproducible, scalable, and high-throughput
methods for the production and purification of clinical grade EV. Methods
including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and
size-exclusion chromatography (SEC) have been employed to isolate EV, each facing
limitations such as efficiency, particle purity, lengthy processing time, and/or
sample volume. We developed a cGMP-compatible method for the scalable production,
concentration, and isolation of EV through a strategy involving bioreactor
culture, tangential flow filtration (TFF), and preparative SEC. We applied this
purification method for the isolation of engineered EV carrying multiple
complexes of a novel human immunostimulatory cytokine-fusion protein,
heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the
fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium
was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF +
SEC. SEC demonstrated comparable particle recovery, size distribution, and
hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved
a 100-fold reduction in ferritin concentration, a major protein-complex
contaminant. Comparative proteomics suggested that SEC additionally decreased the
abundance of cytoplasmic proteins not associated with EV. Combination of TFF and
SEC allowed for bulk processing of large starting volumes, and resulted in
bioactive EV, without significant loss in particle yield or changes in size,
morphology, and hetIL-15/lactadherin density. Taken together, the combination of
bioreactor culture with TFF + SEC comprises a scalable, efficient method for the
production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which
may be useful in targeted cancer immunotherapy approaches.
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