Cell lines

Patent metadata

Field	Value
Publication number	US-9732367-B2
Application number	US-201314430542-A
Country	US
Kind code	B2
Filing date	Sep 24, 2013
Priority date	Sep 24, 2012
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

First claim

The invention claimed is:

1. A process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl, wherein the cell line is capable of incorporating a gene encoding a target protein and wherein said target protein comprises one or more non-natural amino acids encoded by an amber codon, wherein said process comprises culturing said cell line in the presence of a decoy amino acid of:
(a) formula VIIA:

wherein K is CO or SO 2 ;
Q=H, C 1-6 alkyl, aryl, heteroaryl, -OC 1-6 alkyl, -OCH 2 aryl, -OCH 2 heteroaryl, C 2-6 alkenyl or -OC 2-6 alkenyl; or
(b) formula VIIB:

wherein G=H;
a=4 or 5; and
R=C 1-6 alkyl, C 2-6 alkenyl, CH 2 aryl, C 2-6 alkynyl, C 1-6 haloalkyl or C 1-6 azidoalkyl,
wherein the viability of said eukaryotic cell line is enhanced in comparison to the viability of said eukaryotic cell line cultured in the absence of said decoy amino acid.

2. A process according to claim 1 wherein the decoy amino acid is selected from:

3. A process according to claim 1 wherein the expression of tRNAPyl occurs under the control of a repressible promoter.

4. A process according to claim 1 wherein the eukaryotic cell line is a mammalian cell line is selected from CHO, HEK293, PERC6, COS-1, COS-7, HeLa, VERO, mouse hybridoma and mouse myeloma cell lines.

5. A process according to claim 1 wherein the nonsense codon is an amber codon.

6. A process according to claim 1 wherein the compound of formula VII is a compound of formula VIIA:

wherein K is CO or SO2;
Q=—H, C1-6alkyl, aryl, heteroaryl OC1-6alkyl, —OCH2aryl, —OCH2heteroaryl, —C2-6alkenyl or —OC2-6alkenyl.

Assignees

• Medimmune Ltd

Inventors

• Grabstein Kenneth H
• Van Brunt Michael
• Marelli Marcello
• Brady William
• Johnson Jeffrey C

Classifications

• C07K17/08

  the carrier being a synthetic polymer · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2317/31

  multispecific · CPC title

• C07K2317/40

  characterized by post-translational modification · CPC title

• C07C271/20

  to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups · CPC title