Exosome recovery methods with low molecular weight organic zwitterions

• US 9,927,427 B2
• Filed: 10/15/2014
• Issued: 03/27/2018
• Est. Priority Date: 10/24/2013
• Status: Expired due to Fees

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Gagnon, Peter Stanley
• Primary Examiner(s): Russel, Jeffrey E
• Application Number:
  US15/031,660
• Publication Number:
  US 20160266097A1
• Time in Patent Office: 1,259 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: B01D 15/364 Amphoteric or zwitterionic ion-exchanger
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Abstract

A method of isolating exosomes includes conducting at least one purification step in the presence of an organic zwitterion having a molecular weight of less than about 350 Daltons, a buffering pK of a negatively charged portion of the organic zwitterion is at least one full pH unit below an operating pH at which the at least one purification step is conducted, and a buffering pK of the positively charged portion of the organic zwitterion is at least one full pH unit above the operating pH.

Claims

1. A method of isolating exosomes comprising:

conducting at least one purification step in the presence of an organic zwitterion having a molecular weight of less than about 350 Daltons, wherein a buffering pK of a negatively charged portion of the organic zwitterion is at least one full pH unit below an operating pH at which the at least one purification step is conducted, and a buffering pK of a positively charged portion of the organic zwitterion is at least one full pH unit above the operating pH, wherein the organic zwitterion is an amino acid or a peptide comprising from 2 to 5 amino acids, the amino acid or peptide amino acids being selected from the group consisting of glycine, alanine, N,N,N-trimethylglycine (betaine), and taurine.

2. The method of claim 1, wherein a molar dielectric increment of the organic zwitterion under the operating conditions of the at least one purification step is greater than about 17.

3. The method of claim 1, wherein the at least one organic zwitterion is glycine or alanine.

4. The method of claim 1, wherein a concentration of the organic zwitterion is from about 20 mM to about 3 M, or saturated.

5. The method of claim 4, wherein the concentration of the organic zwitterion is selected from the group consisting of (a) from about 5 mM to about 20 mM, (b) from about 20 mM to about 50 mM, (c) from about 50 mM to about 100 mM, (d) from about 100 mM to about 300 mM, (e) from about 300 mM to about 3 M, and (f) saturated.

6. The method of claim 1, wherein before conducting the at least one purification step, the exosomes are unpurified, partially purified, or purified.

7. The method of claim 1, wherein an osmolarity during the at least one purification step is in a range from about 250 milliOsmoles/kg to about 300 milliOsmoles/kg.

8. The method of claim 1, wherein an osmolarity during the at least one purification step is less than 250 milliOsmoles/kg.

9. The method of claim 1, wherein an osmolarity during the at least one purification step is greater than 300 milliOsmoles/kg.

10. The method of claim 1, wherein the at least one purification step comprises a filtering step.

11. The method of claim 1, wherein the at least one purification step comprises a chromatography step.

12. The method of claim 1, wherein the at least one purification step comprises a field flow fractionation step.

13. The method of claim 1, wherein the at least one purification step comprises a centrifugation step.

14. The method of claim 11, wherein the chromatography step comprises a size exclusion chromatography step.

15. The method of claim 14, wherein the organic zwitterion is glycine or alanine at a concentration of 20 mM to 300 mM.

16. The method of claim 14, wherein the at least one purification step comprises contacting a sample comprising exosomes with a size exclusion chromatography media in the presence of the organic zwitterion, wherein the organic zwitterion is in a soluble form.

17. The method of claim 16, wherein the sample, prior to conducting of the at least one purification step, comprises non-exosome proteins, and the method further comprises, after the conducting of the at least one purification step, providing isolated exosomes, wherein the isolated exosomes comprise at least 95% of the exosomes in the sample and less than 5% of the of the non-exosome proteins in the sample.

18. A method of isolating exosomes comprising:

conducting at least one purification step in the presence of an organic zwitterion having a molecular weight of less than about 350 Daltons, wherein a buffering pK of a negatively charged portion of the organic zwitterion is at least one full pH unit below an operating pH at which the at least one purification step is conducted, and a buffering pK of a positively charged portion of the organic zwitterion is at least one full pH unit above the operating pH, wherein the at least one purification step comprises a filtering step, a field flow fractionation step, a centrifugation step, or a size exclusion chromatography step.

19. The method of claim 18, wherein the at least one purification step comprises contacting a sample comprising exosomes with a size exclusion chromatography media in the presence of the organic zwitterion, wherein the organic zwitterion is in a soluble form.

20. The method of claim 18, wherein the organic zwitterion comprises a positively charged nitrogen group and a negatively charged group distal to the positively charged nitrogen group on the organic zwitterion such that there is a separation by at least one carbon atom.

21. The method of claim 20, wherein the at least one carbon atom comprises an uncharged side chain.