# US 9,518,100 B2 · Methods for increasing N-glycan occupancy and reducing production of hybrid N-glycans in Pichia pastoris strains lacking Alg3 expression

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- Number: US 9,518,100 B2
- Document ID: US9518100B2
- Status: Granted
- Kind: B2
- Application number: 14/764,993
- Filed: 2014-02-07
- Published: 2015-12-31
- Granted: 2016-12-13
- Earliest claimed: 2013-02-13
- Assignees: Merck Sharp & Dohme Corp.
- Applicants: Merck Sharp & Dohme Corp.
- Inventors: Byung-Kwon Choi
- Web page: https://patentgrep.com/patent/US9518100B2

## CPC

- C07K 14/44: from protozoa
- C07K 14/39: from yeasts
- C07K 14/62: Insulins
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12P 21/005: {Glycopeptides, glycoproteins}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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)
- 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)}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Methods are disclosed for increasing the yield and N-glycosylation site occupancy of paucimannose or complex N-glycans of recombinant glycoproteins produced in a recombinant host cell lacking dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (Alg3p) activity. In particular, the present invention provides recombinant host cells for producing recombinant glycoproteins that comprise a disruption of the expression of an OS-9 family gene in the host cell and which overexpress one or more Trypanosoma brucei STT3 proteins.

## Claims

### Claim 1

A host cell that does not display dolichyl —P—Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (alg3p) activity, Att1p activity, and osteosarcoma 9 (OS-9) protein activity comprising a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein integrated into the genome of the host cell, wherein the host cell is a yeast or filamentous fungus and the OS-9 protein activity is Yos9p activity.

### Claim 2 (depends on 1)

The host cell of claim 1, wherein the host cell further includes a nucleic acid molecule encoding a heterologous protein.

### Claim 3 (depends on 1)

The host cell of claim 1, wherein the host cell is genetically engineered to produce glycoproteins comprising one or more mammalian- or human-like N-glycans.

### Claim 4

A host cell comprising:
(a) a disruption in expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) gene;
(b) a disruption in the expression of endogenous osteosarcoma 9 (OS-9) family gene;
(c) disruption in expression of an endogenous gene encoding ATT; and
(d) a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein integrated into the genome of the host cell, wherein the host cell is a yeast or filamentous fungus and the OS-9 family gene is a YOS9 gene.

### Claim 5 (depends on 4)

The host cell of claim 4, wherein the disruption in the expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) and endogenous osteosarcoma 9 (OS-9) family gene or homolog is achieved by deleting or disrupting the gene.

### Claim 6 (depends on 4)

The host cell of claim 4, wherein the host cell further includes a nucleic acid molecule encoding a heterologous protein.

### Claim 7 (depends on 4)

The host cell of claim 4, wherein the host cell is genetically engineered to produce glycoproteins comprising one or more mammalian- or human-like N-glycans.

### Claim 8

A method for producing a heterologous glycoprotein, comprising;
(a) providing a host cell that does not display dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (Alg3p) activity, osteosarcoma 9 (OS-9) protein activity, Att1p activity, and having integrated into the genome of the host cell a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein and a nucleic acid molecule encoding the heterologous glycoprotein; and
(b) culturing the host cell under conditions for expressing the heterologous glycoprotein to produce the heterologous glycoprotein, wherein the host cell is a yeast or filamentous fungus and the OS-9 family gene is a YOS9 gene.

### Claim 9 (depends on 8)

The method of claim 8, wherein the host cell is genetically engineered to produce glycoproteins comprising one or more mammalian- or human-like N-glycans.