Isolation of Extracellular Vesicles using a Weak Anion Exchange Functionalized Nanofiber Matrix Sujeong Yang, Emma Burman, Ian Scanlon Astrea Bioseparations, Horizon Park, Barton Road, Comberton, Cambridge, CB23 7AJ, UK
4
Abstract
Extracellular vesicles (EV), including exosomes, have a promising role in regenerative medicine, diagnostics, and delivery of gene therapies. Mild processing conditions for EV isolation are crucial to maintain the integrity and functionality of these nanosized membrane-bound structures. There is a need for scalable isolation techniques that achieve high recovery and high purity at scale, while minimizing EV aggregation or damage.
Breakthrough study on EVs from HEK 293 cells: ExoHERO® Weak Anion Exchange adsorbent chemistry Breakthrough of particles ininflowthrough Breakthrough of particles flowthrough by by nanoFCM NTA
In this study, EVs were isolated from mesenchymal stem cell (MSCs) and from HEK 293 cell culture, using ExoHERO®, a novel chromatography capsule containing a unique electrospun composite nanofiber, AstreAdept®.
1.40E+10
1.00E+10
Methods and materials
Extracellular vesicles EVs were produced from two different cell lines, human mesenchymal stem cells (EVerZom, France) and HEK 293 cells (EchoBiotech, China).
Chromatography downstream purification EV isolation was performed at each CDMO facilities using ExoHERO®, a novel chromatography capsule containing a unique electrospun composite nanofiber, AstreAdept®.
Feedstock preparation In both cases EVs were harvested, clarified and diafiltered into Phosphate Buffered Saline (PBS), concentrated, and loaded directly onto the fiber. The diafiltered concentrate was then loaded directly onto the chromatography capsule
Particles/ mL
1
Load EV solution from HEK 293 cells Diafiltrated into PBS Concentrated 10x Load concentration: 2.17E+10 particles/ mL, measured by nanoFCM ~2.17E+10 particles/mL Adsorbent volume 0.12mL Results shown normalised to Particles loaded/ mL adsorbent
8.00E+09
6.00E+09
4.00E+09
Analysis Particle load/mL was assessed by a nanoparticle tracking analysis (NTA) or nanoFCM, and purity of the load and elution pools were assessed for total protein content.
10% breakthrough
2.00E+09
0.00E+00
-2.00E+09
Capacity of the capsule, as well as recovery and purity of the EV purification, are reported for each EV feedstock.
0
20
40
60
80
100
mL of feed/ mL of adsorbent Particle breakthrough by NTA
2
METHOD Buffers A: Equilibration buffer PBS B: Elution buffer PBS 0.55 M NaCl C: Elution buffer PBS, 0.85 M NaCl
1.20E+10
OUTCOME
QB10
Capacity for HEK 293 EVs ~1.5 E+12/ mL of adsorbent
Experiments kindly performed by EchoBiotech
AstreAdept® Novel composite electrospun nanofiber adsorbent with expansive flowpath
5
• Average mean flowpath diameter 860 nm • Giving excellent flow characteristics for ATMP targets
Purification of EVs by total protein analysis: ExoHERO® - Weak Anion Exchange adsorbent chemistry Recovery and purity in elution pool
TARGET
DIAMETER (nm)
EXTRACELLULAR VESICLES
30-150
OUTCOME Weak AEX fiber can be used to separate EVs from protein impurities, with greater efficiency than either SEC or strong AEX
EV
Recovery
Fold increase in purity
MsC EV single step PBS +0.4M NaCl elution
50%
1.8*
HEK 293 EV elution 1 PBS 0.4M NaCl
37%
0.7875
*Purity ratio of 108 particles/ µg of protein. These values are comparable to the ratio achieved by current SEC method.
HEK 293 EV Elution II PBS 0.85M NaCl
22%
5.23#
#significantly improved ratio compared to current strong AEX method used for EV isolation (2 fold increase reported)
Experimental data kindly provided by EchoBiotech and by EVerZom
3
Primary Capture of EVs from Mesenchymal Stem Cells: ExoHERO® Weak Anion Exchange adsorbent chemistry Primary fromMesenchymal Mesenchymal cells Primarycapture capture of of EVs EVs from cells
METHOD
300
Buffers A: Equilibration buffer PBS B: Elution buffer PBS +1M NaCl (elution at 40% B ~400 mM NaCl in PBS)
100 90
250
200
70 60
UV
150
50 100
40 30
50
20 0
-50
Conductivity (mS/cm)
80
Load EV solution from MSCs cells Diafiltrated into PBS Concentrated 10x Load concentration measured by NTA ~4E+10 particles/mL 5 mL load Adsorbent volume 0.12mL
6
ExoView data from load and elution MINIMAL POPULATIONS
LOAD ELUTION
METHOD
CD63+
CD81+
CD9+
91%
63%
50%
88%
72%
CD63
CD81
CD9
CD63/CD81 CD63/CD9
LOAD
25%
0%
2%
25%
ELUTION
14%
1%
3%
25%
60%
CD81/CD9
Triple+
10%
6%
31%
10%
8%
36%
“The tetraspanin colocalizations remain identical before and after chromatography. This indicates that the technology doesn’t select an EV sub-population.” EVerZom
Biophysical characterization using ExoView based on fluorescent antibody detection of tetraspanins using Single Particle interferometric reflectance imagining sensor (SP-IRIS) Results shown normalised to Particles loaded/ mL adsorbent Main Populations: • Triple positive EV • CD63/CD81+ EV • CD63+ EV
Experiments kindly performed by EVerZom
10
0 0
5
10
15
20
25
Volume (mL) UV
Conductivity
Experiments kindly performed by Everzom
OUTCOME Recovery ~50% Capacity for MSCs EVs : 1.7E+12/ mL of adsorbent
Conclusions
The data from two different EV expression systems shows the applicability of the weak anion exchange nanofiber material for the purification of EVs, with recoveries comparable to those reported by our partners for platform processes. Purity results from these initial test are already promising, and with additional optimisation of the elution conditions it may be possible to achieve greater clearance or impurities and recovery of the particles. The weak anion exchange functionality allows an additional optimisation parameter of the buffer pH to achieve separation of impurities. This could be utilised in the load conditioning to prevent binding of proteins, or during elution to selectively remove targeted impurities.
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