Process for preparing extracellular vesicles
The present disclosure relates to multistep chromatographic methods for preparing extracellular vesicles (EVs). The methods were demonstrated to be effective in preparing highquality EVs in a large scale. The methods enable preparation of EVs for therapeutic and diagnostic applications, and isolation and/or sub-fractionation of EVs with desired properties for specific use.
1. A method of preparing EVs comprising:
(i) subjecting a sample comprising the EVs to a depth filtration (depth-filtration processed filtrate sample);
(ii) contacting the depth-filtration processed filtrate sample to a nuclease (nuclease processed sample);
iii) contacting the nuclease processed sample to ultrafiltration and/or diafiltration (UF/DF processed filtrate sample);
(iv) contacting the UF/DF processed filtrate sample with a cation exchange chromatography (CEX) resin (CEX-processed sample);
(v) contacting the CEX-processed sample with an anion exchange chromatography (AEX) resin (AEX-processed sample);
(vi) contacting the AEX-processed sample with mixed-mode chromatography (MMC) resin (MMC processed sample); and
(vii) subjecting the MMC processed sample to a UF/DF.
2. The method of claim 1 , wherein the EVs are originated from cells comprising a transgene.
3. The method of claim 2 , wherein the transgene encodes a protein comprising an EV protein.
4. The method of claim 3 , wherein the EV protein is Prostaglandin F2 Receptor Negative Regulator (PTGFRN), Basigin (BSG), Immunoglobulin superfamily member 3 (IGSF3), Immunoglobulin superfamily member 2 (IGSF2), Integrin beta-1 (ITGB1), Integrin alpha-4 (ITGA4), 4F2 cell-surface antigen heavy chain (SLC3A2), ATP transporter, or BASP1, or a fragment or a modification thereof.
5. The method of claim 4 , wherein the EV protein is linked to a biologically active molecule.
6. The method of claim 5 , wherein the CEX resin, the AEX resin, and/or the MMC resin comprises a base matrix, wherein the base matrix is a membrane, a monolith, a hydrogel, a porous device, a nanofiber, a composite resin, or a beaded resin.
7. The method of claim 6 , wherein the base matrix comprises cellulose, agarose, polystyrene derivatives, polyvinylether, silica, methacrylate derivatives, glass, ceramic hydroxyapatite, or acrylamide.
8. The method of claim 1 , wherein the sample is obtained from perfusion cell culture, fed batch cell culture, or batch cell culture.
9. The method of claim 1 , further comprising contacting the sample with magnesium and/or EDTA.
10. The method of claim 1 , wherein the pH of the CEX loading buffer is lower than the pH of the AEX loading buffer.