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US-20260124150-A1 - USE OF MONOLITHIC ANION EXCHANGE CHROMATOGRAPHY AND LIGHT SCATTERING FOR QUANTIFYING EXTRACELLULAR VESICLES

US20260124150A1US 20260124150 A1US20260124150 A1US 20260124150A1US-20260124150-A1

Abstract

The present disclosure relates to methods of detecting and purifying EVs (e.g., exosomes) present within a sample using monolithic anionic exchange chromatography and light scattering. Such methods allow for a more accurate and high-throughput method of quantifying EVs present in a sample. Some aspects of the methods provided herein further comprise analyzing one or more additional properties of EVs.

Inventors

  • Gang Gary HAO

Assignees

  • LONZA SALES AG

Dates

Publication Date
20260507
Application Date
20230713

Claims (20)

  1. 1 . A method of determining the amount of extracellular vesicle (EV) present in a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column.
  2. 2 . A method of preparing an extracellular vesicle (EV) fraction from a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, (ii) collecting an eluent from the monolithic AEX chromatography column, and (iii) then measuring a light scattering emission signal from the eluent to prepare the EV fraction.
  3. 3 . A method of reducing the amount of impurity present in a sample comprising an extracellular vesicle (EV), the method comprising (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, (ii) collecting an eluent from the monolithic AEX chromatography column, and (iii) then measuring a light scattering emission signal from the eluent wherein the amount of impurity is reduced in the sample.
  4. 4 . The method of claim 3 , wherein the amount of impurity is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to that of a reference sample (e.g., correspondent eluent collected after contacting the sample to a non-monolithic AEX chromatography column).
  5. 5 . The method of claim 3 or 4 , wherein if the purity of the EV present in the eluent is less than that of a reference value, the method further comprises subjecting the eluent to one or more purification steps.
  6. 6 . The method of claim 5 , wherein the one or more purification steps comprise a filtration, centrifugation, chromatography, or combinations thereof.
  7. 7 . The method of any one of claims 1 to 6 , wherein the monolithic AEX chromatography column comprises a porous surface with a pore size of about 6 μm or greater.
  8. 8 . The method of any one of claims 1 to 7 , wherein the monolithic column comprises a monolithic tertiary amine column.
  9. 9 . The method of any one of claims 1 to 8 , wherein the eluent is collected after contacting the AEX chromatography column with an elution buffer, wherein the contacting with the elution buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column).
  10. 10 . The method of claim 9 , wherein the elution buffer comprises tris, salt, or both.
  11. 11 . The method of claim 10 , wherein the elution buffer comprises about 50 mM tris, about 2,000 mM NaCl, with a pH of about 7.4.
  12. 12 . The method of claim 10 or 11 , wherein the elution buffer further comprises sodium azide.
  13. 13 . The method of any one of claims 1 to 12 , further comprising contacting the AEX chromatography column with a wash buffer, wherein the contacting with the wash buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column) and before (ii) (i.e., measuring a light scattering emission signal from the eluent).
  14. 14 . The method of claim 13 , wherein the wash buffer comprises tris, salt, or both.
  15. 15 . The method of claim 14 , wherein the wash buffer comprises about 50 mM tris, about 200 mM NaCl with a pH of about 7.4.
  16. 16 . The method of claim 14 or 15 , wherein the wash buffer further comprises sodium azide.
  17. 17 . The method of any one of claims 1 to 16 , wherein the light scattering emission signal is generated using an excitation wavelength of about 280 nm to about 700 nm.
  18. 18 . The method of claim 17 , wherein the light scattering emission signal is generated using an excitation wavelength of about 400 nm to about 500 nm.
  19. 19 . The method of claim 18 , wherein the light scattering emission signal is generated using an excitation wavelength of about 420 nm to about 480 nm.
  20. 20 . The method of claim 19 , wherein the light scattering emission signal is generated using an excitation wavelength of about 460 nm.

Description

FIELD OF DISCLOSURE The present disclosure relates to methods of quantifying extracellular vesicles (e.g., exosomes) in a sample (e.g., biological sample) using monolithic anion exchange chromatography and light scattering. The methods provided herein can further comprise analyzing one or more properties of the extracellular vesicles present in the sample. BACKGROUND OF DISCLOSURE Extracellular vesicles (e.g., exosomes) are being used in commercial processes, including as therapeutics, and are being produced in industrial quantities. Methods of accurately and rapidly measuring extracellular vesicle presence, purity, concentration and absolute number in complex matrices remain scarce. Current approaches for the detection, isolation and purification of biological extracellular vesicles derived from cell culture or other biological samples requires laborious and time-consuming methods. For example, current ultra-centrifugation protocols are commercially unreproducible, as they produce a heterogeneous mix of extracellular vesicles, other cellular vesicles and macromolecular complexes and can lead to vesicle aggregation. Therefore, novel methods for efficient, low-cost and reliable purification and quantification of such extracellular vesicles are needed. SUMMARY OF DISCLOSURE Provided herein is a method of determining the amount of extracellular vesicle (EV) present in a sample, the method comprising: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column. Also provided herein is a method of preparing an extracellular vesicle (EV) fraction from a sample, the method comprising determining the amount of EV present in the sample, wherein the determining comprises: (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column. The present disclosure further provides a method of reducing the amount of impurity present in a sample comprising an extracellular vesicle (EV), the method comprising (i) contacting the sample with a monolithic anion exchange (AEX) chromatography column, and (ii) then measuring a light scattering emission signal from an eluent collected from the AEX chromatography column. In some aspects, the amount of impurity is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to that of a reference sample (e.g., correspondent eluent collected after contacting the sample to a non-monolithic AEX chromatography column). In some aspects, if the purity of the EV present in the eluent is less than that of a reference value, the method further comprises subjecting the eluent to one or more purification steps. In some aspects, the one or more purification steps comprise a filtration, centrifugation, chromatography, or combinations thereof. In any of the methods provided herein (e.g., provided above), in some aspects, the monolithic AEX chromatography column comprises a porous surface with a pore size of about 6 μm or greater. In some aspects, the monolithic column comprises a monolithic tertiary amine column. In any of the methods described herein (e.g., provided above), in some aspects, the eluent is collected after contacting the AEX chromatography column with an elution buffer, wherein the contacting with the elution buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column). In some aspects, the elution buffer comprises tris, salt, or both. In some aspects, the elution buffer comprises about 50 mM tris, about 2,000 mM NaCl, with a pH of about 7.4. In some aspects, the elution buffer further comprises sodium azide. In any of the methods described herein (e.g., provided above), in some aspects, the method further comprises contacting the AEX chromatography column with a wash buffer, wherein the contacting with the wash buffer occurs after (i) (i.e., contacting the sample with the AEX chromatography column) and before (ii) (i.e., measuring a light scattering emission signal from the eluent). In some aspects, the wash buffer comprises tris, salt, or both. In some aspects, the wash buffer comprises about 50 mM tris, about 200 mM NaCl with a pH of about 7.4. In some aspects, the wash buffer further comprises sodium azide. In any of the methods described herein (e.g., provided above), in some aspects, the light scattering emission signal is generated using an excitation wavelength of about 280 nm to about 700 nm. In some aspects, the light scattering emission signal is generated using an excitation wavelength of about 400 nm to about 500 nm. In some aspects, the light scattering emission signal is gene