Claims for Patent: 8,357,535

Summary for Patent: 8,357,535

Title:	PURO-DHFR quadrifunctional marker and its use in protein production
Abstract:	This invention relates to industrial production of proteins. More specifically, the invention relates to the res-DHFR surrogate marker, which corresponds to a fusion between DHFR and a protein conferring resistance to a toxic compound or conferring a metabolic advantage. The invention further relates to the use of res-DHFR for screening cells for high expression of a protein of interest. The invention is illustrated by the Puro-DHFR surrogate marker, which corresponds to a fusion between the puromycin N-acetyltransferase and dihydrofolate reductase (DHFR).
Inventor(s):	Kobr; Michel (Echandens, CH), Dupraz; Philippe (Crissier, CH)
Assignee:	Merck Serono SA (Coinsins, Vaud, CH)
Application Number:	12/601,553
Patent Claims:	1. A method of screening cells for expression of a protein of interest, said method comprising the steps of: a) transfecting cells by an expression vector encoding: (i) a Puro-DHFR chimeric protein comprising: SEQ ID NO: 2; or a functional polypeptide fragment of dihydrofolate reductase (DHFR) fused to a functional polypeptide fragment conferring resistance to puromycin (Puro), wherein said functional polypeptide fragment of dihydrofolate reductase comprises SEQ ID NO: 6, and said functional polypeptide fragment of puromycin N-acetyl transferase comprises SEQ ID NO:4; and (ii) a protein of interest; b) selecting cells being resistant to puromycin; and c) assaying the fluorescence of the cells selected in step (b) with a fluorescent compound binding to DHFR. 2. The method of claim 1, further comprising the step of amplifying said protein of interest before performing step (c). 3. The method of claim 2, wherein said amplifying step is performed by growing the cells in the presence of methotrexate (MTX). 4. The method of claim 2, wherein said fluorescent compound binding to said functional polypeptide fragment of DI IFR is fluorescent methotrexate (f-MTX) or fluorescent trimethoprim (f-TMP). 5. The method of claim 1, wherein said functional polypeptide fragment conferring resistance to puromycin is fused to the amino terminus of said functional polypeptide fragment of DHFR. 6. The method of claim 1, wherein said functional polypeptide fragment of DHFR is fused to the amino terminus of said functional polypeptide fragment conferring resistance to puromycin. 7. The method of claim 1, wherein said chimeric protein comprises the sequence of SEQ ID NO: 2. 8. The method of claim 1, wherein said protein of interest is selected from the group consisting of human chorionic gonadotropin (hCG), human follicle-stimulating hormone (FSH), human luteinizing hormone (r-hLH), interferon beta-1a (IFN-1a) and human growth hormone (rhGHm). 9. The method of claim 1, wherein the fluorescence is measured either using a fluorescence microscope or a fluorescence-activated cell sorter (FACS). 10. The method of claim 1, further comprising the step of: d) selecting about 1% to about 20% of the cells exhibiting the highest fluorescence activity in step c). 11. The method of claim 10, further comprising the step of repeating steps b), c) and d) at least 2, 3, 5 or 10 times. 12. The method of claim 10, further comprising the step of: e) assaying the expression level of the protein of interest in the cells selected at the end of the last step d). 13. The method of claim 12, further comprising the step of: f) selecting about 1% to about 20% of the cells exhibiting the highest expression of said protein of interest. 14. A method of obtaining a cell line expressing a protein of interest, said method comprising the step of: a) screening cells according to the method of claim 12; and b) establishing a cell line from said cells. 15. A method of producing a protein of interest, said method comprising the steps of: a) culturing a cell line obtained according to the method of claim 14 under conditions which permit expression of said protein of interest; and b) collecting said protein of interest. 16. The method of claim 15, further comprising the step of purifying said protein of interest. 17. The method of claim 16, further comprising the step of formulating said protein of interest into a pharmaceutical composition. 18. The method of claim 1, wherein said Puro-DHFR chimeric protein comprises SEQ ID NO: 4 fused to SEQ ID NO: 6.

Details for Patent 8,357,535

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	01/15/1974		2027-06-07
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	12/27/1984		2027-06-07
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/15/1985		2027-06-07
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/16/1990		2027-06-07