Preparation and/or purification of oligonucleotide conjugates
View Patent ↗Methods, systems and/or kits for the preparation, purification and isolation of oligonucleotide conjugates, comprising conjugation of modified antibodies or proteins with at least one modified oligonucleotide at greater than 80% efficiency to form oligonucleotide conjugates and isolating the oligonucleotide conjugates from the conjugation solution by binding the conjugates to an immobilized binder, wherein the binder may be a metal ion or an antibody.
1. A method for isolating antibody-oligonucleotide conjugates, comprising:
i) introducing a modified antibody into a buffered solution, wherein the modified antibody includes a histidine-rich region;
ii) conjugating the modified antibodies with at least one modified oligonucleotide at greater than 80% efficiency to form antibody-oligonucleotide conjugates; and
iii) isolating the antibody-oligonucleotide conjugates from the conjugation solution by binding the conjugates to an immobilized binder, removing the unconjugated oligonucleotide in a wash step followed by release of the bound conjugate from the solid support.
2. The method of claim 1 , wherein the immobilized binder comprises a metal ion.
3. The method of claim 2 , wherein the metal ion is a transition metal ion.
4. The method of claim 3 , wherein the transition metal ion is selected from the group comprising: nickel ion, zinc ion, copper ion, iron ion and cobalt ion.
5. The method of claim 1 , wherein the immobilized binder comprises an immobilized antibody.
6. The method of claim 1 , wherein conjugating efficiency is greater than 85%.
7. The method of claim 1 , wherein the modified antibody is prepared from an IgG or IgM type antibody.
8. The method of claim 1 , wherein the modified antibody comprises an antibody that has been prepared by attaching at least one moiety comprising a reactive linker capable of conjugating to a modified oligonucleotide.
9. The method of claim 1 , wherein modified antibody further comprises a succinimidyl 6-hydrazinonicotinate acetone hydrazone modification.
10. The method of claim 1 , wherein the isolated antibody-oligonucleotide conjugates comprise on average at least 2 modified oligonucleotides.
11. The method of claim 1 , wherein the isolated antibody-oligonucleotide conjugates comprises a composition of antibody-oligonucleotide conjugates having on average between 1.0 and 2.5 modified oligonucleotides conjugated to the antibody.
12. A method for isolating antibody-oligonucleotide conjugates, comprising:
i) introducing a modified antibody into a buffered solution;
ii) conjugating the modified antibodies with at least one modified oligonucleotide at greater than 80% efficiency to form antibody-oligonucleotide conjugates; and
iii) isolating the antibody-oligonucleotide conjugates from the conjugation solution by binding the conjugates to an immobilized binder, removing the unconjugated oligonucleotide in a wash step followed by release of the bound conjugate from the solid support,
wherein the immobilized binder comprises an organic chelator selected from the group comprising: iminodiacetic acid, nitrilotriacetic acid and bicinchoninic acid.
13. The method of claim 12 , wherein conjugating efficiency is greater than 85%.
14. The method of claim 12 , wherein the modified antibody is prepared from an IgG or IgM type antibody.
15. The method of claim 12 , wherein the modified antibody comprises an antibody that has been prepared by attaching at least one moiety comprising a reactive linker capable of conjugating to a modified oligonucleotide.
16. The method of claim 12 , wherein modified antibody further comprises a succinimidyl 6-hydrazinonicotinate acetone hydrazone modification.
17. The method of claim 12 , wherein the isolated antibody-oligonucleotide conjugates comprise on average at least 2 modified oligonucleotides.
18. The method of claim 12 , wherein the isolated antibody-oligonucleotide conjugates comprises a composition of antibody-oligonucleotide conjugates having on average between 1.0 and 2.5 modified oligonucleotides conjugated to the antibody.
19. A method for isolating antibody-oligonucleotide conjugates, comprising:
i) introducing a modified antibody into a buffered solution, wherein the modified antibody comprises a molecular tag comprising a poly-histidine tag, a Flag Tag, a c-Myc-Tag, an S-tag, or a peptide tag;
ii) conjugating the modified antibodies with at least one modified oligonucleotide at greater than 80% efficiency to form antibody-oligonucleotide conjugates; and
iii) isolating the antibody-oligonucleotide conjugates from the conjugation solution by binding the conjugates to an immobilized binder, removing the unconjugated oligonucleotide in a wash step followed by release of the bound conjugate from the solid support.
20. The method of claim 19 , wherein the immobilized antibody is complementary to the molecular tag that is bound to the modified antibody.
21. The method of claim 19 , wherein the immobilized binder comprises a metal ion.
22. The method of claim 21 , wherein the metal ion is a transition metal ion.
23. The method of claim 22 , wherein the transition metal ion is selected from the group comprising: nickel ion, zinc ion, copper ion, iron ion and cobalt ion.
24. The method of claim 19 , wherein the immobilized binder comprises an immobilized antibody.
25. The method of claim 19 , wherein conjugating efficiency is greater than 85%.
26. The method of claim 19 , wherein the modified antibody is prepared from an IgG or IgM type antibody.
27. The method of claim 19 , wherein the modified antibody comprises an antibody that has been prepared by attaching at least one moiety comprising a reactive linker capable of conjugating to a modified oligonucleotide.
28. The method of claim 19 , wherein modified antibody further comprises a succinimidyl 6-hydrazinonicotinate acetone hydrazone modification.
29. The method of claim 19 , wherein the isolated antibody-oligonucleotide conjugates comprise on average at least 2 modified oligonucleotides.
30. The method of claim 19 , wherein the isolated antibody-oligonucleotide conjugates comprises a composition of antibody-oligonucleotide conjugates having on average between 1.0 and 2.5 modified oligonucleotides conjugated to the antibody.