Method of monomerisation of recombinant antibody molecules

Patent metadata

Field	Value
Publication number	US-2021069328-A1
Application number	US-202017078127-A
Country	US
Kind code	A1
Filing date	Oct 23, 2020
Priority date	Apr 22, 2015
Publication date	Mar 11, 2021
Grant date	—

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

1 . A 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 comprising one VH and one VL and having specificity for an antigen of interest 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.

2 . The method according to claim 1 , wherein the reducing agent is selected from the group consisting of: glutathione (GSH), ethylsulfite, 2-mercaptoethanol (BME), 2-mercaptoethylamine (BMEA), cysteine-HCl and dithiothreitol (DTT).

3 . The method according to claim 2 , wherein the reducing agent is selected from 2-mercaptoethanol (BME) and 2-mercaptoethylamine (BMEA).

4 . The method according to claim 3 , wherein the 2-mercaptoethylamine is 10 to 150 millimolar; 50 to 150 millimolar; or 95 to 135 millimolar.

5 . The method according to claim 1 , wherein the denaturant is urea and is at a concentration of 1 to 5 molar; a concentration of 3 to 5 molar; or a concentration of 4.5 to 4.9 molar.

6 . The method according to claim 1 , wherein the denaturant is Guanidine hydrochloride and is at a concentration of 1 to 2 molar.

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

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

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

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

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

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

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

14 . The method according to claim 1 , wherein the VH and VL are connected directly via a linker.

15 . The method according to claim 14 , wherein the antibody is selected from a dsscFv, a Fab-2×dsscFv, a Fab-dsscFv-dsFv, a Fab-dsscFv-sdAb, a Fab-dsscFv-scFv and a Fab-dsscFv.

16 . The method according to claim 1 , wherein each VH and VL comprise a linker which indirectly connects the VH and VL via a second antibody.

17 . The method according to claim 16 , wherein the antibody is a Fab-dsFv.

18 . The method according to claim 17 , 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.

19 . A composition comprising 2-mercaptoethylamine and a denaturant selected from urea and Guanidine hydrochloride for converting multimeric species of an antibody molecule to monomers wherein 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.

20 . The composition according to claim 19 , wherein urea is 3 to 5 molar and 2-mercaptoethylamine is 80 to 150 millimolar.

Assignees

• Ucb Biopharma Sprl

Inventors

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

Classifications

• A61K39/39591 Primary

  Stabilisation, fragmentation · CPC title

• C07K16/18 Primary

  against material from animals or humans · CPC title

• C07C323/12

  the carbon skeleton being acyclic and saturated · CPC title

• C07K1/1133 Primary

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

• C07K2317/31

  multispecific · CPC title