Method for increasing the percentage of monomeric antibody Fab-dsFv multimeric species

Patent metadata

Field	Value
Publication number	US-10829565-B2
Application number	US-201615568018-A
Country	US
Kind code	B2
Filing date	Apr 22, 2016
Priority date	Apr 22, 2015
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present invention provides method for increasing the percentage of monomer in a composition of recombinantly expressed antibody molecules characterised in that the antibody molecule comprises at least one Fv with specificity for an antigen of interest comprising one VH and one VL wherein said VH and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprises a) a thermal conversion step of holding the composition comprising the antibody molecule at a temperature in the range 30 to 60° C. for a period of at least 1 hour, wherein step a) is performed in the presence of a reducing agent or after treatment with a reducing agent.

First claim

The invention claimed is:

1. A method for increasing the percentage of Fab-dsFv monomer in a composition of recombinantly expressed antibody wherein the Fab-dsFv monomer comprises at least one Fv with specificity for an antigen of interest comprising one VH and one VL wherein said VH and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprising a thermal conversion step of holding the composition comprising the recombinantly expressed antibody at a temperature in the range 45 to 55° C. for a period of at least 1 hour in the presence of a reducing agent or after treatment with a reducing agent, wherein the reducing agent is selected from 2-mercaptoethanol (BME) and 2-mercaptoethylamine (BMEA) and the reducing agent is present at a concentration of 1 mM to 100 mM and wherein the intra-chain disulfide bonds in the Fab and the Fv of Fab-dsFv are not reduced.

2. The method according to claim 1 , wherein the temperature is 50° C.

3. The method according to claim 1 , wherein the period is in the range 1 to 70 hours.

4. The method according to claim 1 , wherein the method is performed at a temperature in the range 45 to 55° C. for a period of 4 to 6 hours and the reducing agent is at a concentration in the range 60 to 90 mM.

5. The method according to claim 1 , wherein the method is performed at a temperature of 50° ° C. for a period of 5 hours and the reducing agent is at a concentration 70 to 80 mM.

6. The method according to claim 1 , wherein the reducing agent is employed in the presence of lysine.

7. The method according claim 6 , wherein the concentration of amino acid is in the range 0.01 to 1.0 M.

8. The method according to claim 1 , wherein the antibody is at a concentration in the range 0.5 g/L to 5 g/L in the composition.

9. The method according to claim 1 , wherein the thermal step is performed in the presence of concomitant stirring.

10. The method according to claim 9 , wherein the stirring in the range 100 to 1200 rpm.

11. The method according to claim 1 , wherein reducing agent is added to the recombinant antibody composition before the temperature is raised to 45 to 55° C.

12. The method according to claim 1 , wherein reducing agent is added to the recombinant antibody composition after the temperature is raised to 45 to 55° C.

13. The method according to claim 1 , comprising a further step of downstream purification.

14. The method according to claim 13 , wherein said downstream purification comprises chromatography.

15. The method according to claim 14 , wherein the chromatography is hydrophobic interaction chromatography.

16. The method according to claim 14 , wherein the chromatography is ion exchange chromatography.

17. The method according to claim 1 , wherein the VH and VL which are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween are a complementary VH/VL pair which form an antigen binding site.

18. The method according to claim 1 , wherein the VH and VL are connected directly via a linker or each VH and VL comprise a linker which indirectly connects the VH and VL via a second antibody.

19. The method according to claim 18 , wherein each VH and VL comprise a linker which indirectly connects the VH and VL via a second antibody.

20. The method according to claim 1 , wherein the recombinantly expressed antibody is a bispecific antibody fusion protein which binds human OX40 and human serum albumin comprising:
a heavy chain comprising, in sequence from the N-terminal, a first heavy chain variable domain (V H 1), a C H 1 domain and a second heavy chain variable domain (V H 2),
a light chain comprising, in sequence from the N-terminal, a first light chain variable domain (V L 1), a C L domain and a second light chain variable domain (V L 2),
wherein said heavy and light chains are aligned such that V H 1 and V L 1 form a first antigen binding site and V H 2 and V L 2 form a second antigen binding site,
wherein the antigen bound by the first antigen binding site is human OX40 and the antigen bound by the second antigen binding site is human serum albumin; and
wherein the first heavy chain variable domain (V H 1) comprises the sequence given in SEQ ID NO:1 for CDR-H1, the sequence given in SEQ ID NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3 for CDR-H3 and the first light chain variable domain (V L 1) comprises the sequence given in SEQ ID NO:4 for CDR-L1, the sequence given in SEQ ID NO:5 for CDR-L2 and the sequence given in SEQ ID NO:6 for CDR-L3,
wherein the second heavy chain variable domain (V H 2) has the sequence given in SEQ ID NO:11 and the second light chain variable domain (V L 2) has the sequence given in SEQ ID NO: 12 and
the second heavy chain variable domain (V H 2) and second light chain variable domain (V L 2) are linked by a disulfide bond.

21. The method according to claim 1 , wherein the composition of recombinantly expressed antibody molecules is a clarified supernatant.

22. The method according to claim 1 , wherein, prior to the thermal conversion step, the method further comprises a step of protein A chromatography to remove impurities from the composition.

Assignees

• Ucb Biopharma Sprl

Inventors

• Heywood Sam Philip
• Wild Gavin Barry
• Le Page Christopher John
• Hanif Razwan

Classifications

• C07K2317/66

  comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1 · CPC title

• C07K2317/624

  Disulfide-stabilized antibody (dsFv) · CPC title

• C07K2317/55

  Fab or Fab' · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K16/468 Primary

  Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies · CPC title