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SEC/MALS Analysis of Exosome (SB-806 HQ)


As an example of exosome (EV) analysis, the EV preparation process from cell culture supernatant was followed by a combination of polymer-based aqueous SEC (GFC) column OHpak SB-806 HQ and various detectors. UV 280 nm covers general culture-derived impurities, and fluorescence (Ex 280 nm / Em 348 nm) responds mainly to proteins via Trp residue fluorescence. In addition, MALS scattered light (LS) provides a highly sensitive response especially for large objects like nanoparticles. Also, MALS gives an estimate of the target RMS (root mean square) radius.
The fraction consisted mainly EV was separated from many culture-derived impurity components and was found around 8 minutes. While UV and fluorescence provide important insights into the progress and efficacy of the purification process and profiling of purified products, they are less sensitive to EVs mostly composed of lipid membranes and containing trace amounts of protein / nucleic acid cargo. It can be seen that LS is an effective EV tracking method, especially in the early stages of purification.
SB-806 HQ is a high-performance aqueous SEC (GFC) column suitable for bioproducts with a sufficient pore size to hold and separate EV-class nanoscale objects. Combined with a variety of detectors, it is useful for comprehensive analysis of the complex bio-nano target preparation process.

EV fraction preparation conditions
(1) Concentration step: Commercially available centrifugal ultrafiltration membrane 100 kDa (40 times concentrated)
(2) Affinity step: Commercial affinity purification kit (equivalent to 10-fold concentration)
Cooperation of sample preparation by Showa Denko Materials Co., Ltd.

Sample :
1. Cell culture supernatant 50 μL inj.
2. Concentrate 50 μL inj.
3. Crude product 15 μL inj.

Column       : Shodex OHpak SB-806 HQ (8.0 mm I.D. x 300 mm) 
Eluent       : PBS (-) 
Flow rate    : 1.0 mL/min
Detector     : UV (280 nm) 
               Fluorescence (Ex.280 nm, Em.348 nm)
               MALS (DAWN8+ produced by Wyatt Technology Corp.)
Column temp. : 25 °C

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