Methods for increasing N-glycan occupancy and reducing production of hybrid N-glycans in pichia pastoris strains lacking ALG3 expression

Patent metadata

Field	Value
Publication number	US-9428784-B2
Application number	US-201214354160-A
Country	US
Kind code	B2
Filing date	Oct 23, 2012
Priority date	Oct 28, 2011
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

Methods 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 are disclosed. In particular, recombinant host cells are provided that comprise a disruption of the expression of an OS-9 family gene in the host cell. These recombinant host cells may then be used for producing recombinant glycoproteins. In further embodiments, the recombinant host cells further overexpress at least one heterologous single-subunit oligosaccharyltransferase, which in particular embodiments is capable of functionally suppressing the lethal phenotype of a mutation of at least one essential protein of the yeast oligosaccharyltransferase (OTase) complex.

First claim

What is claimed:

1. A host cell comprising:
(a) a disruption in the expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) gene; and
(b) a disruption in the expression of the endogenous YOS9 gene; and
(c) a nucleic acid molecule encoding a heterologous protein having one or more N-linked glycosylation sites,
wherein the host cell is a mutant of P. pastoris having a deletion or disruption of the OCH1 gene.

2. The host cell of claim 1 , wherein the disruption in the expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) and endogenous YOS9 gene is achieved by deleting or disrupting the gene.

3. The host cell of claim 1 , wherein the host cell further includes at least one nucleic acid molecule encoding a heterologous single-subunit oligosaccharyltransferase.

4. The host cell of claim 3 , wherein the single-subunit oligosaccharyltransferase is the Leishmania sp. STT3A protein, STT3B protein, STT3C protein, STT3D protein or combinations thereof.

5. The host cell of claim 3 , wherein the single-subunit oligosaccharyltransferase is the Leishmania major STT3D protein.

6. 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.

7. The host cell of claim 1 , wherein the host cell further expresses a protein that has endomannosidase activity.

8. A method for producing a heterologous glycoprotein, comprising providing a host cell comprising:
(a) a disruption in the expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) gene,
(b) a disruption in the expression of the endogenous YOS9 gene, and
(c) a nucleic acid molecule encoding the heterologous protein having one or more N-linked glycosylation sites, and
culturing the host cell under conditions for expressing the heterologous protein to produce the heterologous glycoprotein,
wherein the host cell is a mutant of P. pastoris having a deletion or disruption of the OCH1 gene.

9. The host cell of claim 8 , wherein the disruption in the expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) and endogenous YOS9 gene is achieved by deleting or disrupting the gene.

10. The method of claims 8 , wherein the host cell further includes at least one nucleic acid molecule encoding a heterologous single-subunit oligosaccharyltransferase.

11. The method of claim 10 , wherein the single-subunit oligosaccharyltransferase is the Leishmania sp. STT3A protein, STT3B protein, STT3C protein, STT3D protein or combinations thereof.

12. The method of claim 10 , wherein the single-subunit oligosaccharyltransferase is the Leishmania major STT3D protein.

13. 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.

14. The method of claim 8 , wherein the host cell further expresses a protein that has endomannosidase activity.

Assignees

• Merck Sharp & Dohme

Inventors

• Choi Byung-Kwon
• Hamilton Stephen R
• Sethuraman Natarajan

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K16/11

  Paramyxoviridae (F); Pneumoviridae (F), e.g. respiratory syncytial virus [RSV] · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title