Double Tangential Flow Filtration for purification of Urine
EVs provides high particle yield and removal of THP.
Andres Lõhmus, Paolo Guazzi; HansabioMed Life Sciences

Introduction:
The study of Urine EVs and their associated biomarkers requires an efficient separation of vesicles from common contaminants. In particular, the attention is paid to the presence of the Tamm-Horsfall protein (THP), a
human uromodulin that often co-isolates with the vesicles. Numerous attempts have been made to remove
the THP contamination, some using chemical reagents, such as DTT or CHAPS [1;2], or trying to raise thesolubility of the uromodulin [3] so as to avoid co-precipitation with vesicles. This application note shows theeffectiveness and versatility of Tangential Flow Filtration in obtaining high yield of urine vesicles, free from
THP contamination, in a short time and with an extremely simple, scalable and reproducible procedure.
References:[1] Fernández-Llama, Patricia, et al. "Tamm-Horsfall protein and urinary exosome isolation." Kidney international 77.8 (2010):
736-742.
[2] Musante, Luca, et al. "A simplified method to recover urinary vesicles for clinical applications and sample banking." Scientificreports 4.1 (2014): 1-11.
[3] Puhka, M., et al. "KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine." European journal ofpharmaceutical sciences 98 (2017): 30-39.

Workflow:
40 ml of human urine have been filtered through the tangential flow filter TFF-MV (200 nm pore size, HansaBioMed Life Sciences) in order to remove large particles, contaminants and debris, then the EVs containedin the flowthrough have been purified by using the tangential flow filter TFF-EVs (50 nm pore size, HansaBioMed Life sciences). EVs were finally recovered in 4 ml of PBS 1x.

1. Filtration of 40 ml of urine through TFF-MV (200 nm pore size).
2. Purification of EVs contained in the TFF-MV filtrate by TFF-EVs.
3. Washing of the TFF-EVs retentate containing EVs with PBS 1x
4. Recovery of EVs from TFF-EVs in 4 ml of PBS 1x.

Results:• Particle content analysis in purification process.
SAMPLE
Urine (40 ml)
TFF-MV-RE
TFF-MV-FT
TFF-EVs-FT
UR-EVs

CONCENTRATION St. Dev MEAN SIZE(Part/ml)(Part/ml)(nm)1.40E+10
4.20E+10
9.40E+09
3.80E+08
8.20E+10

1.50E+09
4.30E+09
9.40E+08
2.40E+07
5.10E+09

101.2
138.2
95
84.2
102.9

St. Dev(nm)

VOLUME(ml)

TOTAL
PARTICLES

40238
38
4

5.6E+11
8.4E+10
3.572E+11
1.444E+10
3.28E+119.5
16.6
8.2
8.8
9.4

• Recovery of ≈ 60% of total particles.

• Protein content analysis in purification process.
PROTEIN
TOTAL
St. Dev
VOLUMEPROTEINS
CONCENTRATION(mg/ml)(ml)(mg/ml)(mg)
Urine (40 ml)
8.28970.844540 331.5885
TFF-MV-RE
8.42490.1214
2 16.84971
TFF-MV-FT
7.02410.129138 266.9166
TFF-EVs-FT
6.68630.121438 254.078
UR-EVs
0.04500.0325
40.18
SAMPLE• Removal of more than 99% of contaminant
proteins.

• Purifiled Urine EVs; analysis of associated markers and size distribution profiling.

• EV internal markers TSG101 andALIX in purified EVs (UR-EVs) andTHP removal.

• Detection of EV surface associated markers CD9, CD63,
CD81 in purified intact EVs (UR-EVs) and size distributionprofiling.

Conclusion:
The double tangential flow filtration is a relatively simple and fast protocol, which can be carried out manuallyon the lab bench or under the safety cabinet, providing high EV yield and purity.

••••

Recovery of about 60% of the total particles
Removal of contaminant proteins, including THP.
Turnaround time of total process: less than 10 minutes.
No chemical treatment of the sample
HansaBioMed Life Sciences LTD
Mäealuse 2/1,12618 Tallinn, Estonia+372 6561996
info@hansabiomed.eu