US 12,030,926 B2
Production of heteromultimeric proteins using mammalian cells
Justin Scheer, San Francisco, CA (US); Whitney Shatz, San Francisco, CA (US); and Domingos Ng, San Francisco, CA (US)
Assigned to Genentech, Inc., South San Francisco, CA (US)
Filed by Genentech, Inc., South San Francisco, CA (US)
Filed on Feb. 9, 2021, as Appl. No. 17/171,962.
Application 17/171,962 is a continuation of application No. 15/344,123, filed on Nov. 4, 2016, granted, now 10,941,190.
Application 15/344,123 is a continuation of application No. PCT/US2015/029546, filed on May 6, 2015.
Claims priority of provisional application 61/989,509, filed on May 6, 2014.
Prior Publication US 2022/0002386 A1, Jan. 6, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C07K 16/00 (2006.01); C07K 16/46 (2006.01); C12N 15/79 (2006.01); C12N 15/90 (2006.01)
CPC C07K 16/00 (2013.01) [C07K 16/468 (2013.01); C07K 2317/14 (2013.01); C07K 2317/31 (2013.01); C07K 2317/52 (2013.01); C07K 2317/526 (2013.01); C07K 2317/53 (2013.01); C12N 15/79 (2013.01); C12N 15/907 (2013.01)] 19 Claims
 
1. A method of preparing a heteromultimeric protein comprising i) a first hinge-containing polypeptide having a first heterodimerization domain, wherein the first hinge-containing polypeptide is associated with a first light chain, and ii) a second hinge-containing polypeptide having a second heterodimerization domain, wherein the second hinge-containing polypeptide is associated with a second light chain, wherein the second heterodimerization domain interacts with the first heterodimerization domain at an interface, and wherein the first and second hinge-containing polypeptides are linked by at least one interchain disulfide bond, the method comprising the steps of:
(a) culturing a first host cell capable of expressing the first hinge-containing polypeptide and the first light chain;
(b) culturing a second host cell capable of expressing the second hinge-containing polypeptide and the second light chain;
(c) obtaining a combined culture medium from step (a) and step (b) without disrupting cell membrane of the first and second host cells;
(d) adding a reducing agent to the combined culture medium; and
(e) incubating the combined culture medium containing the reducing agent for about 4 hours to about 7 days, wherein the combined culture medium comprises the heteromultimeric protein, and wherein the first host cell and the second host cell are each a mammalian cell.