Method of monomerisation of recombinant antibody molecules

Patent metadata

Field	Value
Publication number	US-10828366-B2
Application number	US-201615566307-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 of 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 conversion step of treating the composition with a denaturant selected from urea and/or Guanidine hydrochloride; b) 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 of increasing the percentage of Fab-dsFv monomer in a composition of recombinantly expressed antibody molecules characterised in that Fab-dsFv comprises at least one Fv comprising one VII and one VL and having specificity for an antigen of interest wherein said VII and VL are connected directly or indirectly via one or more linkers and are stabilised by a disulfide bond therebetween, said method comprising
a conversion step comprising treating the composition with a denaturant selected from urea, guanidine hydrochloride, or a combination thereof, wherein the urea is at a concentration of 1M to 5M and/or guanidine hydrochloride is at a concentration of 1M to 2M, wherein the conversion step is
performed in the presence of a reducing agent or after treatment with a reducing agent, wherein the reducing agent is 2-mercaptoethylamine at a concentration of 10 to 150 millimolar.

2. The method according to claim 1 , wherein the 2-mercaptoethylamine is at a concentration of 50 to 150 millimolar.

3. The method according to claim 2 , wherein the 2-mercaptoethylamine is at a concentration of 95 to 135 millimolar.

4. The method according to claim 1 , wherein the urea is at a concentration of 3M to 5M.

5. The method according to claim 4 , wherein the urea is at a concentration of 4.5M to 4.9M.

6. The method according to claim 1 , wherein the conversion step is carried out for a period of 2 to 70 hours.

7. The method according to claim 1 , wherein the method is performed at room temperature.

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

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

10. The method according to claim 9 , wherein the stirring is at a stirring rate between 100 and 1200 rpm.

11. The method according to claim 1 , comprising a further step of downstream processing.

12. The method according to claim 11 , wherein downstream processing comprises chromatography.

13. The method according to claim 1 , wherein the 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, 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.

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

15. The method according to claim 1 , wherein, prior to the 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
• Hanif Razwan
• Le Page Christopher John

Classifications

• C07K2317/31

  multispecific · CPC title

• C07K16/2878

  against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 · CPC title

• C07K16/18 Primary

  against material from animals or humans · CPC title

• C07K1/1136

  by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents · CPC title

• C07K1/1133 Primary

  by redox-reactions involving cystein/cystin side chains · CPC title