Production of recombinant proteins with simple glycoforms

• US 9,670,271 B2
• Filed: 06/02/2016
• Issued: 06/06/2017
• Est. Priority Date: 01/11/2012
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Lin, Nan, Sealover, Natalie, George, Henry, Kayser, Kevin
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/171,977
• Publication Number:
  US 20160272696A1
• Time in Patent Office: 369 Days
• Field of Search: None
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/10 from RNA viruses
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 204/01101 Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (2.4.1.101)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention provides cell lines deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I (Mgat1). Also provided are methods for producing the Mga1 deficient cell lines and methods for using the Mgat1 deficient cell lines for the production of recombinant proteins having simple glycoforms.

Claims

1. A genetically modified cell line deficient in mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I (Mgat1) comprising a deletion of less than 200 bp in the coding region of a chromosomal sequence encoding Mgat1.

2. The genetically modified cell line of claim 1, wherein the cell line comprises an insertion in addition to the deletion in the coding region of the chromosomal sequence encoding Mgat1.

3. The genetically modified cell line of claim 1, wherein the cell line produces a reduced level of Mgat1 protein.

4. The genetically modified cell line of claim 1, wherein the cell line produces no Mgat1 protein.

5. The genetically modified cell line of claim 1, wherein the chromosomal sequence encoding Mgat1 is modified using a targeting endonuclease-mediated genome editing technology.

6. The genetically modified cell line of claim 5, wherein the targeting endonuclease is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a meganuclease, or a site-specific endonuclease.

7. The genetically modified cell line of claim 5, wherein the targeting endonuclease is a zinc finger nuclease.

8. The genetically modified cell line of claim 1, wherein the cell line further comprises a deficiency in dihydrofolate reductase (DHFR), hypoxanthine-guanine phosphoribosyltransferase (HPRT), or a combination thereof.

9. The genetically modified cell line of claim 1, wherein the cell line is a Chinese Hamster Ovary (CHO) cell line.

10. The genetically modified cell line of claim 9, wherein the CHO cell line has a genotype of Mgat1 (−

/0).

11. The genetically modified cell line of claim 1, wherein the cell line expresses at least one glycoprotein comprising one or more terminal mannose residues.

12. The genetically modified cell line of claim 1, wherein the cell line has a growth rate comparable to that of a cell line having no deficiency in Mgat1.

13. The genetically modified cell line of claim 1, wherein the cell line produces a level of protein comparable to that of a cell line having no deficiency in Mgat1.

14. A method for producing a recombinant protein having one or more terminal mannose residues, the method comprising:

a) introducing a nucleic acid encoding the recombinant protein into the cell line of claim 1; and

b) expressing the recombinant protein.

15. The method of claim 14, wherein the cell line produces no Mgat1 protein.

16. The method of claim 14, wherein the cell line is a Chinese Hamster Ovary (CHO) cell line.