Production of heteromultimeric proteins using mammalian cells

Patent metadata

Field	Value
Publication number	US-10941190-B2
Application number	US-201615344123-A
Country	US
Kind code	B2
Filing date	Nov 4, 2016
Priority date	May 6, 2014
Publication date	Mar 9, 2021
Grant date	Mar 9, 2021

Abstract

Described herein are methods for the efficient production of antibodies and other multimeric protein complexes (collectively referred to herein as heteromultimeric proteins) capable of specifically binding to more than one target. The targets may be, for example, different epitopes located on a single molecule or located on different molecules.

First claim

What is claimed is:

1. A method of preparing an antibody comprising i) a first heavy chain comprising a knob modification, wherein the first heavy chain is paired with a first light chain, and ii) a second heavy chain comprising a hole modification, wherein the second heavy chain is paired with a second light chain, wherein the knob modification of the first heavy chain interacts with the hole modification of the second heavy chain at an interface, and wherein the first and second heavy chains are linked by at least one interchain disulfide bond, the method comprising the steps of:
(a) culturing a first host cell and a second host cell, wherein the first host cell expresses the first heavy chain and the first light chain, wherein the second host cell expresses the second heavy chain and the second light chain, and wherein the first host cell and the second host cell are each a mammalian cell;
(b) adding a reducing agent to a combined culture comprising the first host cell and the second host cell; and
(c) harvesting a combined culture medium from the combined culture 4 hours to 24 hours after adding the reducing agent to the combined culture, wherein harvesting comprises removing the first host cell and second host cell from the combined culture medium without disrupting cell membranes of the first and second host cells, and wherein the combined culture medium comprises the antibody.

2. The method of claim 1 , wherein the combined culture medium is harvested 15 hours after the reducing agent is added.

3. The method of claim 1 , wherein the step of harvesting a combined culture medium comprises removing the first host cell and second host cell from the combined culture medium.

4. The method of claim 1 , wherein the combined culture medium is incubated for 4 hours to 7 days.

5. The method of claim 1 , wherein the first host cell and the second host cell are cultured separately before being combined in the combined culture.

6. The method of claim 1 , wherein the knob modification comprises a T366W substitution (EU numbering) and the hole modification comprises two or more amino acid substitutions selected from the group consisting of T366S, L368A, and Y407V (EU numbering).

7. The method of claim 1 , wherein the reducing agent is glutathione (GSH).

8. The method of claim 7 , wherein the GSH is added at a concentration of about 5 mM to no more than about 20 mM.

9. The method of claim 8 , wherein the GSH is added at a concentration of about 15 mM.

10. The method of claim 1 , wherein the mammalian cell is a CHO cell.

11. The method of claim 1 , wherein the mammalian cell is a stable cell line.

12. The method of claim 6 , wherein the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V (EU numbering).

Assignees

• Genentech Inc

Inventors

• Scheer Justin
• Shatz Whitney
• Ng Domingos

Classifications

• C07K2317/52

  Constant or Fc region; Isotype · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K2317/14

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

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C07K1/02

  in solution {(C07K1/003, C07K1/006 take precedence)} · CPC title