# US 2014/0155290 A1 · Host cells comprising alpha 1, 2 mannosidase and culture methods thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155290 A1 - Document ID: US20140155290A1 - Status: Published application - Kind: A1 - Application number: 14/174,297 - Filed: 2014-02-06 - Published: 2014-06-05 - Earliest claimed: 2005-12-08 - Assignees: Amgen Inc. - Applicants: Amgen Inc. - Inventors: Christopher Kenyon Crowell - Web page: https://patentgrep.com/patent/US20140155290A1 ## CPC - C12P 21/005: {Glycopeptides, glycoproteins} - C07K 14/505: Erythropoietin [EPO] - C12N 5/0693: {Tumour cells; Cancer cells} - C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)} - C12N 15/85: for animal cells - C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection - C12Y 302/01024: Alpha-mannosidase (3.2.1.24) - G01N 33/746: {Erythropoetin} - C12N 2500/95: Protein-free medium and culture conditions - C12N 2501/999: Small molecules not provided for elsewhere - C12N 2510/02: Cells for production - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12Y: ENZYMES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Improved host cells and culture methods involving overexpression of MAN1C1 activity to improve protein production are provided. ## Claims ### Claim 1 A host cell engineered to overexpress (a) alpha 1,2 mannosidase (MAN1C1) native to the host cell and (b) a glycoprotein of interest. ### Claim 2 (depends on 1) The host cell of claim 1 wherein said host cell has been transfected with a vector comprising a nucleic acid encoding MAN1C1 operably linked to a heterologous expression control sequence. ### Claim 3 (depends on 1) The host cell of claim 1 wherein said host cell comprises a heterologous expression control sequence operably linked to a nucleic acid encoding MAN1C1. ### Claim 4 (depends on 1, 2, 3) The host cell of any of claims 1-3 wherein said host cell has been transfected with a vector comprising a nucleic acid encoding said glycoprotein of interest operably linked to a heterologous expression control sequence. ### Claim 5 (depends on 1, 2, 3) The host cell of any of claims 1-3 wherein said host cell comprises a heterologous expression control sequence operably linked to a nucleic acid encoding said glycoprotein of interest. ### Claim 6 (depends on 1, 2, 3, 4, 5) The host cell any of claims 1-5 wherein the glycoprotein of interest is an erythropoiesis-stimulating molecule. ### Claim 7 (depends on 6) The host cell of claim 6 wherein the glycoprotein of interest is erythropoietin of SEQ ID NO: 3. ### Claim 8 (depends on 6) The host cell of claim 6 wherein the glycoprotein of interest is darbepoetin of SEQ ID NO: 5. ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is a mammalian cell. ### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is a CHO cell. ### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is a human cell. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is a BHK cell. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is an NS/0 cell. ### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any of claims 1-8 which is an HT-1080 cell. ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) A method of producing a glycoprotein of interest comprising the steps of: culturing the host cell of any of claims 1-14 in culture medium; and recovering the glycoprotein of interest from the host cell or culture medium. ### Claim 16 (depends on 15) The method of claim 15 wherein said host is cultured under conditions that induce increased MAN1C1 expression. ### Claim 17 (depends on 15) The method of claim 15 wherein said host cell expresses MAN1C1 protein at a level that increases specific productivity (pg/cell/day) of glycoprotein of interest produced. ### Claim 18 (depends on 17) The method of claim 17 wherein at least a 2-fold increase in specific productivity is achieved by overexpression of MAN1C1. ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) A method of increasing production of a glycoprotein of interest comprising the steps of: culturing the host cell of any of claims 1-14 in culture medium under conditions that permit expression of MAN1C1 at levels that improve production of the glycoprotein of interest; and recovering the glycoprotein of interest from the host cell or culture medium. ### Claim 20 (depends on 15) The method of claim 15 comprising the step of further adding an inducer of protein production to the culture medium. ### Claim 21 A method of screening for an inducer of protein production comprising the steps of: (a) contacting a host cell with a candidate compound, (b) determining expression level of MAN1C1, and (c) identifying said candidate compound as an inducer of protein production if the expression level of MAN1C1 increases. ### Claim 22 (depends on 21) The method of claim 21 wherein MAN1C1 mRNA expression level is determined. ### Claim 23 (depends on 21) The method of claim 21 wherein MAN1C1 protein expression level is determined.