Chromatography resin and uses thereof

Patent metadata

Field	Value
Publication number	US-12187763-B2
Application number	US-202117173767-A
Country	US
Kind code	B2
Filing date	Feb 11, 2021
Priority date	Feb 11, 2020
Publication date	Jan 7, 2025
Grant date	Jan 7, 2025

Abstract

An affinity chromatography resin comprising an anti-tissue factor antibody or antigen-binding fragment thereof attached to a base resin, and methods of using the same.

First claim

What is claimed is:

1. A method of purifying a multi-chain chimeric polypeptide, the method comprising:
loading an affinity chromatography resin with a liquid comprising the multi-chain chimeric polypeptide;
washing the affinity chromatography resin using one or more wash buffer(s); and
eluting the multi-chain chimeric polypeptide using an elution buffer, wherein:
the affinity chromatography resin comprises an anti-tissue factor antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 or 9, and a CDR3 of SEQ ID NO: 3, and a light chain variable domain comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO: 5, and a CDR3 of SEQ ID NO: 6, attached to a base resin;
the multi-chain chimeric polypeptide comprises:
(a) a first chimeric polypeptide comprising:
(i) a first target-binding domain comprising a sequence that is at least 80% identical to amino acids 19 to 305 of SEQ ID NO: 70;
(ii) a soluble tissue factor domain comprising a sequence that is at least 80% identical to SEQ ID NO: 10; and
(iii) a first domain of a pair of affinity domains comprising a sequence that is at least 80% identical to SEQ ID NO: 125; and

(b) a second chimeric polypeptide comprising:
(i) a second domain of a pair of affinity domains comprising a sequence that is at least 80% identical to SEQ ID NO: 123; and
(ii) a second target-binding domain comprising a sequence that is at least 80% identical to amino acids 19 to 305 of SEQ ID NO: 70;

the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and
the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

2. The method of claim 1 , wherein the liquid comprising the multi-chain chimeric polypeptide is a clarified liquid culture medium.

3. The method of claim 1 , wherein the liquid comprising the multi-chain chimeric polypeptide comprises a cell lysate.

4. The method of claim 1 , wherein the one or more wash buffer(s) are:
(i) a first wash buffer comprising phosphate buffered saline; and
(ii) a second wash buffer comprising 0.01 M to 0.2 M citrate and having a pH of 4.5 to 5.5.

5. The method of claim 4 , wherein:
(i) the first wash buffer is phosphate buffered saline; and
(ii) the second wash buffer is 0.1 M citrate, pH 5.0.

6. The method of claim 1 , wherein the elution buffer comprises 0.01 M to 0.2 M acetate and has a pH of 2.5 to 3.5.

7. The method of claim 6 , wherein the elution buffer comprises 0.1 M acetate and has a pH of 2.9.

8. The method of claim 1 , wherein the method further comprises:
performing one or more additional unit operations on an eluate obtained from the step of eluting the multi-chain chimeric polypeptide.

9. The method of claim 8 , wherein the one or more additional unit operations comprises, in sequential order:
performing low pH viral inactivation;
performing depth filtration;
performing polishing chromatography;
performing nanofiltration; and
performing ultrafiltration and diafiltration (UF/DF).

10. The method of claim 1 , wherein the heavy chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO: 8.

11. The method of claim 10 , wherein the heavy chain variable domain comprises SEQ ID NO: 7, and the light chain variable domain comprises SEQ ID NO: 8.

12. The method of claim 1 , wherein the base resin is sepharose.

13. The method of claim 1 , wherein the base resin is any support material.

14. The method of claim 13 , wherein the support material is agarose or a capto resin.

15. The method of claim 1 , wherein the anti-tissue factor antibody or antigen-binding fragment thereof is non-covalently attached to the base resin.

16. The method of claim 1 , wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin.

17. The method of claim 16 , wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin through the formation of a disulfide bond between a cysteine in the anti-tissue factor antibody or antigen-binding fragment thereof and a chemical group on the base resin.

18. The method of claim 16 , wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin through the formation of a covalent bond between a free amine of the anti-tissue factor antibody or antigen-binding domain and a chemical group on the base resin.

19. The method of claim 18 , wherein the base resin is CNBr-activated or N-hydroxysuccinimide (NHS)-activated solid support material.

20. The method of claim 18 , wherein the covalent bond is represented by Formula I below:

wherein R represents the anti-tissue factor antibody or antigen-binding fragment thereof.

21. The method of claim 1 , wherein the first target-binding domain and the soluble tissue factor domain directly abut each other in the first chimeric polypeptide.

22. The method of claim 1 , wherein the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

23. The method of claim 1 , wherein the soluble tissue factor domain and the first domain of the pair of affinity domains directly abut each other in the first chimeric polypeptide.

24. The method of claim 1 , wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide.

25. The method of claim 1 , wherein the second domain of the pair of affinity domains and the second target-binding domain directly abut each other in the second chimeric polypeptide.

26. The method of claim 1 , wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide.

27. The method of claim 1 , wherein the soluble tissue factor domain is a soluble human tissue factor domain.

28. The method of claim 1 , wherein:
the first target-binding domain comprises a sequence that is at least 90% identical to amino acids 19 to 305 of SEQ ID NO: 70;
the soluble tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10;
the first domain of the pair of affinity domains comprises a sequence that is at least 90% identical to SEQ ID NO: 125;
the second target-binding domain comprises a sequence that is at least 90% identical to amino acids 19 to 305 of SEQ ID NO: 70; and
the second domain of the pair of affinity domains comprises a sequence that is at least 90% identical to SEQ ID NO: 123.

29. The method of claim 28 , wherein:
the first target-binding domain comprises a sequence that is at least 95% identical to amino acids 19 to 305 of SEQ ID NO: 70;
the soluble tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO

Assignees

• Immunitybio Inc

Inventors

• Wong Hing C
• Jiao Jin-An
• Prendes Caitlin

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title

• C07K16/36 Primary

  against blood coagulation factors · CPC title

• C07K14/745

  Blood coagulation or fibrinolysis factors · CPC title

• C07K14/7155

  for interleukins [IL] · CPC title