# US 2020/0399325 A1 · Host cell for producing a protein of interest > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399325 A1 - Document ID: US20200399325A1 - Status: Published application - Kind: A1 - Application number: 16/968,719 - Filed: 2019-01-30 - Published: 2020-12-24 - Earliest claimed: 2018-02-12 - Assignees: Lonza Ltd. - Applicants: Lonza Ltd - Inventors: Katrien CLAES; James GRAHAM; Sylwia JOZWIAK; Christoph KIZIAK - Web page: https://patentgrep.com/patent/US20200399325A1 ## CPC - C07K 14/39: from yeasts - C07K 14/37: from fungi - C12N 15/815: {for yeasts other than Saccharomyces} - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - 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)} ## Abstract A eukaryotic host cell engineered to produce a heterologous protein of interest (POI), which cell is genetically modified to reduce production of at least one of three different endogenous host cell protein (HCP), and its use in a method of producing the POI. ## Claims ### Claim 1 A eukaryotic host cell engineered to produce a heterologous protein of interest (POI), which cell is genetically modified to reduce production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first HCP (HCP1), a second HCP (HCP2), and a third HCP (HCP3), wherein a) HCP1 comprises the amino acid sequence identified as SEQ ID NO:1, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species; b) HCP2 comprises the amino acid sequence identified as SEQ ID NO:3, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species; and c) HCP3 comprises the amino acid sequence identified as SEQ ID NO:5, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species. ### Claim 2 (depends on 1) The host cell of claim 1, wherein said at least one HCP is HCP1, and optionally HCP2 and/or HCP3. ### Claim 3 (depends on 1, 2) The host cell of claim 1 or 2, which cell is further genetically modified to reduce production of a further HCP which is HCP4, wherein a) HCP4 comprises the amino acid sequence identified as SEQ ID NO:7, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species. ### Claim 4 (depends on 1, 2, 3) The host cell of any of claims 1 to 3, wherein each of the homologous sequences is characterized by at least 50% sequence identity to the respective amino acid sequence. ### Claim 5 (depends on 1, 2, 3, 4) The host cell of any of claims 1 to 4, wherein the host cell is genetically modified by one or more genetic modifications of the host cell genome comprising a disruption, substitution, deletion or knockout of (i) one or more endogenous polynucleotides, or a part thereof; or (ii) an expression control sequence, preferably wherein said expression control sequence is selected from the group consisting of a promoter, a ribosomal binding site, transcriptional or translational start and stop sequences, an enhancer and activator sequence. ### Claim 6 (depends on 5) The host cell of claim 5, wherein a gene encoding any of said at least one HCP is knocked out by said one or more genetic modifications. ### Claim 7 (depends on 1, 2, 3, 4, 5, 6) The host cell of any one of claims 1 to 6, which is genetically modified to reduce the amount of any of said at least one HCP by at least 50% (mol/mol) compared to the host cell without said modification, preferably by a knockout of a gene encoding any of said at least one HCP. ### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7) The host cell of any one of claims 1 to 7, which comprises an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to a nucleotide sequence encoding the POI, wherein said one or more operably linked sequences are not naturally associated with the POI encoding sequence. ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) The host cell of any one of claims 1 to 8, wherein the POI is a peptide or protein selected from the group consisting of an antigen-binding protein, a therapeutic protein, an enzyme, a peptide, a protein antibiotic, a toxin fusion protein, a carbohydrate—protein conjugate, a structural protein, a regulatory protein, a vaccine antigen, a growth factor, a hormone, a cytokine, a process enzyme, and a metabolic enzyme. ### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9) The host cell of any one of claims 1 to 9, which is any one of an animal cell, a vertebrate cell, a mammalian cell, a human cell, a plant cell, a nematodal cell, an invertebrate cell, an insect cell, a mollusc cell, a stem cell derived of any of the foregoing, or a yeast or fungal cell. ### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) The host cell of any one of claims 1 to 10, which is a) a yeast cell of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia, and Geotrichum, such as Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica or Hansenula polymorpha; or b) a cell of filamentous fungi, such as Aspergillus awamori or Trichoderma reesei. ### Claim 12 A method for producing a protein of interest (POI) in a eukaryotic host cell, comprising the steps: i) genetically modifying the host cell to reduce production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first HCP (HCP1), a second HCP (HCP2), and a third HCP (HCP3), wherein a) HCP1 comprises the amino acid sequence identified as SEQ ID NO:1, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species; b) HCP2 comprises the amino acid sequence identified as SEQ ID NO:3, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species; and c) HCP3 comprises the amino acid sequence identified as SEQ ID NO:5, if the host cell is Komagataella phaffii, or its homologous sequence that is endogenous to the host cell if of another species; ii) introducing into the host cell an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to a nucleotide sequence encoding the POI; iii) culturing said host cell under conditions to produce said POI; and optionally iv) isolating said POI from the cell culture; and optionally v) purifying said POI. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) A method for producing a protein of interest (POI) by culturing the host cell of any one of claims 1 to 11 under conditions to produce said POI. ### Claim 14 (depends on 12, 13) The method of claim 12 or 13, wherein the amount of any of said at least one HCP is reduced by at least 50% (mol/mol) compared to the host cell without said modification, preferably by a knockout of a gene encoding any of said at least one HCP. ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) A method of reducing the risk of endogenous host cell protein (HCP) contaminations of a protein of interest (POI) produced in a host cell culture, by culturing the host cell of any one of claims 1 to 10 under conditions to produce said POI and isolating the POI from said cell culture.