Host cells for producing a target protein

Abstract

A eukaryotic host cell designed to produce a heterologous protein of interest (POI), wherein the eukaryotic host cell has been genetically modified to reduce the production of at least one of three different endogenous host cell proteins (HCPs), and its use in a method for producing a POI.

Description

Technical Field

[0001] The present invention relates to protein production in eukaryotic host cells designed to reduce impurities in eukaryotic host cell cultures.

Background Art

[0002] Proteins produced by eukaryotic cell culture are becoming increasingly important as diagnostic and therapeutic agents. For this purpose, cells are designed and / or selected to produce recombinant or heterologous target proteins at very high levels. Optimization of cell culture conditions is important for the successful commercial production of recombinant or heterologous proteins. By-products released from host cells as host cell proteins (HCPs) and accumulated in the culture can make the purification of recombinant or heterologous proteins difficult or pose a risk to the efficacy of the product or the safety of the patient.

[0003] In addition to mammalian host cells, yeasts and filamentous fungi are commonly used as production hosts for bulk chemicals as well as biopharmaceutical proteins. Methylotrophic yeasts, such as Pichia pastoris, are well known for their ability to efficiently secrete heterologous proteins. Pichia pastoris has been reclassified into a new genus, Komagataella, and is divided into three species: Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris. Strains commonly used in biotechnology belong to two proposed species, Komagataella pastoris and Komagataella phaffii. The GS115, X-33, CBS2612, and CBS7435 strains are Komagataella phaffii, and SMD series protease-deficient strains (e.g., SMD1168) are classified as Komagataella pastoris, which is the reference strain for all available Pichia pastoris strains (Mattanovich et al. 2009, Microb Cell Fact. 8: 29; Kurtzman 2009, J Ind Microbiol Biotechnol. 36(11): 1435-8).

[0004] The biotechnology strain of Komagataella (Pichia) pastoris is Komagataella phaffii as determined by multiple gene sequence analysis.

[0005] Mattanovich et al. (Microbial Cell Factories 2009, 8:29 doi:10.1186 / 1475-2859-8-29) showed the genome sequencing of the reference strain DSMZ70382 of Komagataella pastoris and analyzed its secretome and sugar transporters.

[0006] Huang et al. (Appl Microbiol Biotechnol. 2011 Apr; 90(1): 235-47. doi:10.1007 / s00253-011-3118-5. Epub 2011 Feb 9) showed a proteomic analysis of the secretome of Pichia pastoris in methanol-induced cultures, identifying proteins secreted or released into the medium of methanol-induced fermentation cultures of Pichia pastoris X-33.

[0007] Heiss et al. (Appl Microbiol Biotechnol. 2013 Feb; 97(3): 1241-9. doi:10.1007 / s00253-012-4260-4. Epub 2012 Jul 17) identified extracellular protein X1 (Epx1) as a major contaminating host cell protein in Pichia pastoris producing antibody Fab fragments. EPX1 was generally not upregulated but was found to be upregulated only in different stress situations. Each deletion strain (Δepx1) was produced and found to be more sensitive than the wild type to the cell wall-damaging agents Calcofluor white and Congo red, indicating that Epx1 may have a protective role against the cell wall. No obvious differences in growth and product production were observed between the wild type and the Δepx1 strain.

[0008] However, the inventors found that Epx1p is not a highly produced protein and is less than about 3% (mol / mol) of the total HCP in Pichia pastoris cell cultures.

[0009] HCPs are proteins produced or encoded by cells or organisms used in the production process and are unrelated to the product of interest. Some are necessary for growth, survival, and normal cell processing, while others are not essential. HCPs are generally undesirable in the final drug substance, regardless of their usefulness. They are generally present in small amounts (ppm, expressed as nanograms per milligram of the protein of interest), and a great deal of effort and cost is expended to remove them.

[0010] There is a need to develop improved host cells suitable for the production and / or purification of heterologous and / or recombinant proteins.

Summary of the Invention

[0011] The object of the present invention is to provide a host cell modified to reduce HCP impurities when producing recombinant or heterologous proteins. Another object of the present invention is to provide a method for producing a recombinant or heterologous protein in a host cell with a reduced risk of contamination by HCP impurities of the recombinant or heterologous protein.

[0012] This object is solved by the subject matter recited in the claims.

[0013] According to the present invention, a eukaryotic host cell designed to produce a heterologous protein of interest (POI), wherein said cell is genetically modified to reduce the production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first host cell protein (HCP1), a second host cell protein (HCP2), and a third host cell protein (HCP3), a) HCP1 comprises the amino acid sequence shown in SEQ ID NO: 1 when the host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species, b) HCP2 contains the amino acid sequence shown in SEQ ID NO: 3 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species. c) HCP3 contains the amino acid sequence shown in SEQ ID NO: 5 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species, and a eukaryotic host cell is provided.

[0014] According to a particular aspect, such cells are further genetically modified to reduce the production of HCP4, which is a further HCP, where d) HCP4 contains the amino acid sequence shown in SEQ ID NO: 7 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species, and a eukaryotic host cell is provided.

[0015] Specifically, said at least one HCP comprises any one, two or three of HCP1, HCP2, HCP3. Also specifically, said at least one HCP comprises HCP4.

[0016] Specifically, said at least one HCP is HCP1, optionally or additionally HCP2 and / or HCP3 and / or HCP4. A particular embodiment relates to the reduction of two or more HCPs including HCP1. Specifically, said at least one HCP comprises HCP1 and any one, two or three of HCP2, HCP3 or HCP4.

[0017] More specifically, said at least one HCP is HCP2, optionally HCP1 and / or HCP3 and / or HCP4.

[0018] More specifically, said at least one HCP is HCP3, optionally HCP1 and / or HCP2 and / or HCP4.

[0019] Certain embodiments relate to the reduction of two or more HCPs, e.g., HCP1 and HCP2, or HCP1 and HCP3, or HCP2 and HCP3.

[0020] Certain embodiments relate to the reduction of two or more HCPs and include the reduction of any of HCP1 and HCP4, or HCP2 and HCP4, or HCP3 and HCP4.

[0021] Specifically, the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, and 7 are the wild-type (native) endogenous sequences of Komagataella phaffii, respectively.

[0022] Specifically, each homologous sequence is from a species other than Komagataella phaffii, e.g., from yeast or filamentous fungi, preferably from yeast of the genus Komagataella, Pichia, or Saccharomyces, or from any methylotrophic yeast.

[0023] According to certain aspects, the host cell is a) a yeast cell of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia, and Geotrichum, e.g., Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha, or b) It is a filamentous fungal cell, for example, Aspergillus awamori or Trichoderma reesei.

[0024] However, for the purposes described herein, each HCP can be reduced in a host cell that is an animal cell, a vertebrate cell, a mammalian cell, a human cell, a plant cell, a nematode cell, an invertebrate cell, such as an insect cell or a mollusk cell, or a stem cell derived from any of the foregoing, particularly any fungal cell or yeast cell.

[0025] Each homologous sequence is understood to be endogenous to the host cell used as the host cell for producing the POI as described herein.

[0026] For example, when the host cell is Komagataella phaffii, HCP1 is characterized by the amino acid sequence shown in SEQ ID NO: 1. However, when the host cell is a different species (other than Komagataella phaffii), the sequence of HCP1 that is endogenous to the host cell is a homologous sequence to the amino acid sequence shown in SEQ ID NO: 1, for example, an ortholog sequence of the amino acid sequence shown in SEQ ID NO: 1.

[0027] Similarly, when the host cell is Komagataella phaffii, HCP2 is characterized by the amino acid sequence shown in SEQ ID NO: 3. However, when the host cell is a different species (other than Komagataella phaffii), the sequence of HCP2 that is endogenous to the host cell is a homologous sequence to the amino acid sequence shown in SEQ ID NO: 3, for example, an ortholog sequence of the amino acid sequence shown in SEQ ID NO: 3.

[0028] Similarly, when the host cell is Komagataella phaffii, HCP3 is characterized by the amino acid sequence shown in SEQ ID NO: 5. However, when the host cell is a different species (other than Komagataella phaffii), the sequence of HCP3 that is endogenous to the host cell is a homologous sequence to the amino acid sequence shown in SEQ ID NO: 5, for example, an ortholog sequence of the amino acid sequence shown in SEQ ID NO: 5.

[0029] Similarly, when the host cell is Komagataella phaffii, HCP4 is characterized by the amino acid sequence shown in SEQ ID NO: 7. However, when the host cell is a different species (other than Komagataella phaffii), the sequence of HCP4 that is endogenous to the host cell is a homologous sequence to the amino acid sequence shown in SEQ ID NO: 7, for example, an ortholog sequence of the amino acid sequence shown in SEQ ID NO: 7.

[0030] Specifically, the homologous sequence is characterized by at least 50% sequence identity to each amino acid sequence in Komagataella phaffii, specifically, at least any one of at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity.

[0031] Specifically, the homologous sequence may have a different quantitative activity compared to each amino acid sequence in Komagataella phaffii, but is characterized by the same function, for example, a function as a structural protein or an enzyme.

[0032] Specifically, the host cell is genetically modified by one or more genetic modifications, including genomic mutations that reduce the expression of the polynucleotide encoding the at least one HCP.

[0033] Specifically, the one or more genetic modifications include genomic mutations that reduce the expression of the first and / or second and / or third endogenous polynucleotides, - The first endogenous polynucleotide encodes HCP1, - The second endogenous polynucleotide encodes HCP2, - The third endogenous polynucleotide encodes HCP3.

[0034] Optionally and additionally, the host cell is further modified to introduce a genomic mutation that reduces the expression of a further endogenous polynucleotide, - The further endogenous polynucleotide encodes HCP4.

[0035] Specifically, the endogenous polynucleotide encoding each HCP is a wild-type (native) endogenous polynucleotide having a sequence that occurs naturally in the host cell.

[0036] Specifically, the endogenous polynucleotide encoding HCP1 in Komagataella phaffii contains the nucleotide sequence shown in SEQ ID NO: 2. However, when the host cell is a different species (other than Komagataella phaffii), the coding sequence of HCP1 that is endogenous to the host cell is a homologous sequence to the nucleotide sequence shown in SEQ ID NO: 2, for example, an ortholog sequence of the nucleotide sequence shown in SEQ ID NO: 2.

[0037] Specifically, the endogenous polynucleotide encoding HCP2 in Komagataella phaffii contains the nucleotide sequence shown in SEQ ID NO: 4. However, when the host cell is a different species (other than Komagataella phaffii), the coding sequence of HCP2 that is endogenous to the host cell is a homologous sequence to the nucleotide sequence shown in SEQ ID NO: 4, for example, an ortholog sequence of the nucleotide sequence shown in SEQ ID NO: 4.

[0038] Specifically, the endogenous polynucleotide encoding HCP3 in Komagataella phaffii contains the nucleotide sequence shown in SEQ ID NO: 6. However, when the host cell is a different species (other than Komagataella phaffii), the coding sequence of HCP3 that is endogenous to the host cell is a homologous sequence to the nucleotide sequence shown in SEQ ID NO: 6, for example, an ortholog sequence of the nucleotide sequence shown in SEQ ID NO: 6.

[0039] Specifically, the endogenous polynucleotide encoding HCP4 in Komagataeibacter phaffii contains the nucleotide sequence shown in SEQ ID NO: 8. However, when the host cell is a different species (other than Komagataeibacter phaffii), the coding sequence of HCP4 that is endogenous to the host cell is a homologous sequence to the nucleotide sequence shown in SEQ ID NO: 8, for example, an ortholog sequence of the nucleotide sequence shown in SEQ ID NO: 8.

[0040] Specifically, the host cell is (i) one or more endogenous polynucleotides or a part thereof, or (ii) preferably, one or more genetic modifications including disruption, substitution, deletion or knockout of the expression control sequences of the one or more endogenous polynucleotides selected from the group consisting of promoters, ribosome binding sites, transcription or translation start and stop sequences, enhancers and activator sequences, by which the host cell is genetically modified.

[0041] Specifically, the genetic modification or knockout modification includes one or more genomic mutations including deletion or inactivation of a gene or genomic sequence that reduces the expression of a gene or a part of the gene by at least 50%, 60%, 70%, 80%, 90% or 95% or terminates its expression, as compared to each host without such genetic modification.

[0042] Specifically, the genetic modification includes at least one modification of an expression control sequence, for example, deletion or inactivation of a promoter, enhancer, signal, leader or any other regulatory sequence, particularly a sequence that controls the expression and / or secretion of a protein. Specifically, the expression control sequence is operably linked to the coding gene of the relevant protein.

[0043] Specifically, the one or more genetic modifications include genomic mutations that constitutively impair or otherwise reduce the expression of the one or more endogenous polynucleotides.

[0044] Specifically, one or more genetic modifications include genomic mutations that conditionally impair or otherwise reduce the expression of one or more endogenous polynucleotides by introducing one or more inducible or repressive regulatory sequences. Such conditionally active modifications target these regulatory elements and genes that are activated and / or expressed specifically depending on cell culture conditions.

[0045] Specifically, the gene encoding the at least one HCP is knocked out by the one or more genetic modifications.

[0046] Specifically, the expression of the one or more endogenous polynucleotides is reduced when producing the POI. Specifically, during genetic modification, the expression of the one or more endogenous polynucleotides encoding the at least one HCP is reduced under the conditions of host cell culture in which the POI is produced.

[0047] Specifically, the host cell is genetically modified to reduce the at least one HCP by at least 50%, 60%, 70%, 80%, 90% or 95% (mol / mol), or 100% compared to an unmodified host cell, thereby ending the production of the HCP. Specifically, the amount of one, two or three of HCP1 and HCP2, HCP3 or HCP4 is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 100% (mol / mol) compared to an unmodified host cell. According to certain embodiments, such reduction is achieved by knockout of the gene encoding the at least one HCP.

[0048] Thus, according to certain embodiments, when the host cells described herein are cultured in a cell culture, the amount of total HCP in the cell culture supernatant is reduced by at least 5% or 10%, or even at least 15% (mol / mol) compared to the amount of total HCP in the culture supernatant when culturing unmodified host cells.

[0049] Total host cell proteins (HCPs) in a cell culture refer to the sum of all proteins other than the protein of interest (POI) that are derived from the cells expressing the POI and are present, for example, in the cell culture supernatant from which the cells have been separated from the medium by centrifugation.

[0050] Specifically, the host cell is genetically modified to reduce the amount of said at least one HCP to less than 10%, 5% or 3% (mol / mol) of the total HCPs in the cell culture supernatant.

[0051] Specifically, the amount of said at least one HCP described herein is reduced to less than 1% (mol / mol) of the total HCPs in the cell culture supernatant, or discarded, for example, when measured by mass spectrometry.

[0052] Specifically, the amount of total HCPs in the cell culture supernatant is reduced by at least 5% or at least 10% (mol / mol) compared to the cell culture of host cells without said genetic modification.

[0053] By reducing the production of said at least one HCP, the amount (e.g., level or concentration, particularly the amount relative to a reference or total HCPs) of said at least one HCP obtained in the cell culture supernatant is reduced.

[0054] When comparing the host cells described herein with respect to the effect of said genetic modification for reducing the production of said at least one HCP, typically, it is compared with host cells without such genetic modification. The comparison is typically made between host cells of the same type that are designed to produce the POI when cultured under conditions for producing a recombinant or heterologous POI, particularly, and host cells without such genetic modification. Alternatively, it is compared with host cells of the same type that have not been further designed to produce a recombinant or heterologous POI.

[0055] According to a particular aspect, the reduction of said at least one HCP results in a reduction in the amount of said at least one HCP in the cell culture supernatant (e.g., the level or concentration, or the relative amount with respect to a reference or total HCP). Specifically, the amount of an individual HCP or the total amount of HCPs is measured by suitable methods, such as ELISA assay, HPLC, capillary electrophoresis such as SDS-PAGE or mass spectrometry, in particular mass spectrometry is, for example, the liquid chromatography mass spectrometry (LC-MS) or liquid chromatography tandem mass spectrometry (LC-MS / MS) shown by Doneanu et al. (MAbs. 2012; 4(1): 24-44).

[0056] Specifically, host cells are provided that can reduce the amount of HCPs as by-products other than the POI. HCPs include endogenous proteins that are present independently of a particular POI production process or proteins specific to a process, and such proteins are impurities or contaminants in a POI preparation, for example, a preparation of a cell culture supernatant containing the POI or a preparation obtained when purifying the POI from the cell culture supernatant.

[0057] According to a particular embodiment, the host cell is genetically modified to contain one or more deletions in one or more (of the) genomic sequences. Such host cells are typically provided as deletion strains.

[0058] According to a particular aspect, the host cells described herein contain an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to a nucleotide sequence encoding a POI, in particular, said one or more operably linked sequences are not naturally associated with the sequence encoding the POI.

[0059] Specifically, the expression cassette contains a promoter operably linked to the gene encoding the POI and, optionally, a signal sequence and a leader sequence for expressing and producing the POI as a secreted protein.

[0060] Specifically, the expression cassette includes a constitutive, inducible or repressible promoter.

[0061] Specific examples of the constitutive promoter include, for example, pGAP and its functional variants, any of the constitutive promoters disclosed in International Publication No. WO 2014 / 139608, such as pCS1.

[0062] Specific examples of the inducible or repressible promoter include, for example, the natural pAOX1 or pAOX2 and their functional variants, regulatory promoters such as pG1-pG8 and its fragments disclosed in International Publication No. WO 2013 / 050551, and regulatory promoters such as pG1 and pG1-x disclosed in International Publication No. WO 2017 / 021541.

[0063] Promoter sequences suitable for use in yeast host cells have been shown by Mattanovich et al. (Methods Mol. Biol. (2012) 824: 329-58) and include glycolytic enzymes such as triose phosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and their variants, lactase (LAC) and galactosidase (GAL), the glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (PPGK), glyceraldehyde phosphate dehydrogenase promoter (pGAP), translation elongation factor promoter (PTEF), and the promoter of enolase 1 (PEN01) of Pichia pastoris, triose phosphate isomerase (PTPI), ribosomal subunit proteins (PRPS2, PRPS7, PRPS31, PRPL1), alcohol oxidase promoters (PAOX1, PAOX2) or variants thereof having modified characteristics, formaldehyde dehydrogenase promoter (PFLD), isocitrate lyase promoter (PICL), α-ketoisocaproate decarboxylase promoter (PTHI), promoters of heat shock protein family members (PSSA1, PHSP90, PKAR2), 6-phosphogluconate dehydrogenase (PGND1), phosphoglycerate mutase (PGPM1), transketolase (PTKL1), phosphatidylinositol synthase (PPIS1), ferro-02-oxidoreductase (PFET3), high affinity iron permease (PFTR1), repressible alkaline phosphatase (PPH08), N-myristoyltransferase (PNMT1), pheromone response transcription factor (PMCM1), ubiquitin (PUBI4), single-stranded DNA endonuclease (PRAD2), the promoter of the major ADP / ATP carrier of the inner mitochondrial membrane (PPET9) (International Publication No. WO 2008 / 128701) and the formate dehydrogenase (FMD) promoter. The GAP promoter, AOX1 or AOX2 promoter, or a promoter derived from the GAP, AOX1 or AOX2 promoter is particularly preferred.The AOX promoter can be induced by methanol and is repressed by glucose.

[0064] Further examples of suitable promoters include Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase (PGK), as well as the maltase gene promoter (MAL).

[0065] According to certain aspects, the expression cassette is integrated into the host cell's chromosome or plasmid.

[0066] The expression cassette is introduced into the host cell and may be integrated into the host cell genome as an intrachromosomal element, for example, at a specific integration site or randomly, and then high-producing host cell lines are selected. Alternatively, the expression cassette may be integrated into an episomal genetic element, such as a plasmid or YAC. According to certain examples, the expression cassette is introduced into the host cell by a vector, particularly an expression vector, and such a vector is introduced into the host cell by a suitable transfection method. For this purpose, the polynucleotide encoding the POI may be ligated to the expression vector.

[0067] Preferred yeast expression vectors (preferably used for expression in yeast) are selected from the group consisting of plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis or pPUZZLE.

[0068] Methods for transfecting or transforming eukaryotic cells into which a vector or plasmid has been introduced are well known in the art. These methods include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA co-precipitation), viral infection, and, in particular, may include the use of modified viruses such as modified adenoviruses, microinjection, and electroporation.

[0069] According to certain aspects, the host cells described herein may be further genetically modified, for example, with one or more additional genes to improve protein production.

[0070] Specifically, the host cell is further designed to modify one or more genes that affect proteolytic activity, which are used to create protease-deficient strains, particularly strains lacking carboxypeptidase Y activity. Specific examples are described in WO 92 / 07595. Another example of a protease-deficient Pichia strain with a functional deficiency of vacuolar proteases, such as proteinase A or proteinase B, is described in US Pat. No. 6,153,424. Further examples are Pichia strains with an ade2 deletion and / or a deletion of one or both of the protease genes PEP4 and PRB1, provided, for example, by ThermoFisher Scientific.

[0071] Specifically, the host cell is designed to modify at least one nucleic acid sequence encoding a protease selected from the group consisting of functional gene products such as PEP4, PRB1, YPS1, YPS2, YMP1, YMP2, YMP1, DAP2, GRH1, PRD1, YSP3, and PRB3, as disclosed in WO 2010 / 099195.

[0072] The POI can be any one of a eukaryotic, prokaryotic, or synthetic peptide, polypeptide, protein, or host cell metabolite.

[0073] Specifically, the POI is heterologous to the species of the host cell.

[0074] Specifically, the POI is a secreted peptide, polypeptide or protein that is secreted from the host cell into the cell culture supernatant.

[0075] Specifically, the POI is a eukaryotic protein, preferably a protein derived from or related to a mammal, such as a human protein or a protein containing a human protein sequence, or a bacterial protein or a protein derived from bacteria.

[0076] Preferably, the POI is a therapeutic protein that functions in mammals.

[0077] In certain cases, the POI is a multimeric protein, specifically a dimer or a tetramer.

[0078] According to certain aspects, 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.

[0079] A particular POI is an antigen-binding molecule, such as an antibody or a fragment thereof, particularly an antibody fragment containing an antigen-binding domain. Among the particular POIs are antibodies such as monoclonal antibodies (mAbs), immunoglobulins (Igs), immunoglobulin class G (IgG) or heavy-chain antibodies (HcAb’s), or antibody fragments such as fragment-antigen binding (Fab), Fd, single-chain variable fragments (scFv), or Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers, or minibodies and single-domain antibodies, such as variants thereof like VH, VHH, IgNAR or V-NAR, or any protein containing an immunoglobulin fold domain. Further antigen-binding molecules may be selected from antibody mimetics, or (alternative) scaffold proteins, such as designed knotted domains, adnectins, affibodies, affilins, anticalins, or DARPins.

[0080] According to certain aspects, POIs include, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, corifollitropin alfa, filgrastim, cetrorelix, interleukin-2, Aldesleukin, teceleulin, denileukin diftitox, interferon alfa-n3 (injectable), interferon alfa-n1, DL-8234, interferon, Suntory (γ-1a), interferon gamma, thymosin alfa1, tasnelmin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, reviparin, eptoterminalfa, teriparatide (osteoporosis), calcitonin injection (bone disease), calcitonin (nasal, osteoporosis), etanercept, hemoglobin glutamer 250 (bovine), drotrecogin alfa, collagenase, carperitide, recombinant human epidermal growth factor (topical gel, wound healing), DWP401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alfa, desirudin, repirudin, bivalirudin, nonacog alfa, mononine, eptacog alfa (activated), recombinant factor VIII + VWF, riconestat, recombinant factor VIII, factor VIII (recombinant), Alphnmate, octocog alfa, factor VIII, paricalcitol, indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alfa, rFSH, hpFSH, micafungin, pegylated filgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, imiglucerase, galsulfase, leukotriene, molgramostirn, tryptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained release depot (ATRIGEL), leuprolide implant (DUROS), goserelin, Eutropin, KP-102 program, somatropin, mecasermin (dwarfism),Enfuvirtide, Org-33408, Inslrralgin, Inslrulinlisin, Insulin (for inhalation), Insulin lispro, Insulin detemir, Insulin (buccal, RapidMist), Mecasermin rinfabate, Anakinra, Sermolukin, 99mTc-apicitide injection, myelopid, betaseron, glatiramer acetate, gepon, sargramostim, oprelvekin, human leukocyte-derived α-interferon, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasenin, roferon-A, interferon-α2, α-feron, interferon alphacon-1, interferon alpha, avonex recombinant human luteinizing hormone, dornase alfa, trafermin, diconucleotide, taltrimine, dibotermin α, atosiban, becaplermin, eptifibatide, zemaira, CTC-111, Shanvac-B, HPV vaccine (quadrivalent), octreotide, lanreotide, ancestirn, agalsidase beta, agalsidase alpha, laronidase, copper prezatide acetate (topical gel), rasburicase, ranibizumab, actimmune, PEG-intron, Tricomin, recombinant chironomus thummi allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta, transgenic antithrombin III, Granditropin, vitrase, recombinant insulin, interferon-α (oral lozenge), GEM-21S, bapreotide, idursulfase, omapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor (angioedema), lanoteplase, recombinant human growth hormone, enfuvirtide (needle-free injection, Biojector2000), VGV-1, interferon (α), lucinactant, abicipadil (inhalation, lung disease), icatibant, ecallantide, omiganan, Aurograb, pexiganan acetate, ADI-PEG-20, LDI-200, degarelix, sintredekin besdotox, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide (osteoporosis), tiphacogin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amediprase, corifollitropin α, TH-9507, teduglutide, Diamyd, DWP-412,Growth hormone (sustained-release injection), recombinant G-CSF, insulin (inhalation, AIR), insulin (inhalation, Technosphere), insulin (inhalation, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C virus infection (HCV)), interferon alpha-n3 (oral), belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, opaganib, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, luceptide, MP52 (β-tricalcium phosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Incedia, bitespin, human thrombin (frozen, surgical bleeding), thrombin, TransMID, alfimeprase, purpura, terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-1 (injectable, vascular disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman-Birk inhibitor concentrate, XMP-629, 99m Tc-Hynic-annexin V, kahalalide F, CTCE-9908, teverelix (sustained-release), ostarerix, romidepsin, BAY-504798, interleukin 4, PRX-321, Pepscan, iboctadekin, rh lactoferrin, TRU-015, IL-21, ATN-161, sirendide, albferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, ovarian cancer immunotherapy vaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase), nemifitide, rAAT (inhalation), rAAT (dermatology), CGRP (inhalation, asthma), pegsunercept, thymosin β4, pridicepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), eximorelin, capromorelin, Cardeva, velafelim, 131I-TM-601, KK-220, T-10, Ularitide, Depelestat, Hemastat, Chrysalin (for local use), rNAPc2, Recombinant Factor V111 (PEGylated liposome), bFGF, PEGylated recombinant staphylokinase mutant, V-10153, SonLysis Prolyse, NeuroVax, CZEN-002, Pancreatic islet cell neogenesis therapy, rGLP-1, BIM-51077, LY-548806, Exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, Aborelin, ACM-9604, Linaclotide acetate, CETi-1, Hemospan, VAL (injectable), Rapid-acting insulin (injectable, Viadel), Nasal insulin, Insulin (inhalable), Insulin (oral, eligen), Recombinant methionyl human leptin, Pitrakina subcutaneous injection drug, Eczema), Pitrakina (inhaled dry powder, Asthma), Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune disease / inflammation), talactoferrin (for local use), rEV-131 (ophthalmic), rEV-131 (respiratory disease), Oral recombinant human insulin (diabetes), RPI-78M, Oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001, Baricitinib, Interferon α-n3 (for local use), IRX-3, RDP-58, Tauferrin, Bile salt-stimulated lipase, Merispase, Alarin phosphatase, EP-2104R, Melanotan-II, Bremelanotide, ATL-104, Recombinant human microp lasmin, AX-200, Semax, ACV-1, Xen-2174, CJC-1008, Dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, Malaria vaccine (virosome, PeviPRO), ALTU-135, Parvovirus B19 vaccine, Influenza vaccine (recombinant neuraminidase), Malaria / HBV vaccine, Anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat toxoid, YSPSL, CHS-13340, PTH(1-34) liposome cream (Novasome), Ostabolin-C,PTH analog (for local use, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FARA04, BA-210, recombinant pest FIV vaccine, AG-702, OxSODrol, rBetV1, Der-p1 / Der-p2 / Der-p7 allergen-targeted vaccine (house dust mite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16E7 lipopeptide vaccine, maze vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, iclocapecid, tervermin (skin ulcer, diabetic foot ulcer), lupintribil, rethiculose, rGRF, HA, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin therapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin (anti-cancer), DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-B / gastrin receptor-binding peptide, 111In-hEGF, AE-37, trastuzumab (trasnizumab)-DM1, antagonist G, IL-12 (recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (for local use), L-19-based radioimmunotherapy drug (cancer), Re-188-P-2045, AMG-386, DC / 1540 / KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptide), NA17.A2 peptide, melanoma vaccine (pulse antigen therapeutic agent), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injectable), ACP-HIP, SUN-11031, peptide YY[3-36] (obesity, nasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal, osteoporosis), F-18-CCR1, AT-1100 (celiac disease / diabetes), JPD-003,PTH(7-34) Liposome Cream (Novasome), Duramycin (ophthalmic, dry eye), CAB-2, CTCE-0214, GlycoPEGylated Erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, Aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, Teverelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, Sipuleucel-T, TV4710, ALG-889, Org-41259, rhCC10, F-991, Thymopentin (lung disease), r(m)CRP, liver-selective insulin, Subalin, L19-IL-2 fusion protein, Elafin, NMK-150, ALTU-139, EN-122004, rhTPO, Thrombopoietin Receptor Agonist (thrombocytopenia), AL-108, AL-208, Nerve Growth Factor Antagonist (pain), SLV-317, CGX-1007, INNO-105, Oral Teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 Fusion Vaccine (Therapore), EP-1043, Pneumococcal Vaccine for Pediatrics, Malaria Vaccine, Meningococcal Group B Vaccine, Neonatal Group B Streptococcal Vaccine, Anthrax Vaccine, HCV Vaccine (gpE1+gpE2+MF-59), Otitis Media Therapeutic, HCV Vaccine (core antigen+ISCOMATRIX), hPTH(1-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, Aviscumnine, BIM-23190, Tuberculosis Vaccine, Multi-epitope Tyrosinase Peptide, Cancer Vaccine, Enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, Vascular Targeted TNF (solid tumor), Desmopressin (oral controlled release), Onasept, or TP-9201, Adalimumab (Humira), Infliximab (Remicade®), Rituximab (Rituxan® / MAB THERA®), Etanercept (Enbrel®), Bevacizumab (Avastin®), Trastuzumab (Herceptin®), Pegfilgrastim (Neulasta®),or any other suitable POI including biosimilars and biobetters.

[0081] According to certain aspects, the host cell can be any animal cell, vertebrate cell, mammalian cell, human cell, plant cell, nematode cell, invertebrate cell, such as an insect cell or a mollusk cell, a stem cell derived from any of the foregoing, or a fungal cell or yeast cell. Specifically, the host cell can be a cell of a genus selected from the group consisting of the genera Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia, and Geotrichum, specifically, Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta or Hansenula polymorpha, or a cell of a filamentous fungus, such as Aspergillus awamori or Trichoderma reesei. Preferably, the host cell is a methylotrophic yeast, preferably Pichia pastoris. As used herein, Pichia pastoris is used as a synonym for all of Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris.

[0082] According to certain aspects, the host cell is a yeast cell or a filamentous fungal cell selected from the group consisting of Pichia pastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia methanolica, Candida boidinii, Komagataella phaffii, Komagataella pastoris, and Schizosaccharomyces pombe.

[0083] According to certain aspects, the host cell is a lower eukaryotic cell, such as a yeast cell (e.g., Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), Komagataella (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), Kluyveromyces (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida (e.g., Candida utilis, Candida cacaoi, Candida boidinii), Geotrichum (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe). Preferably, it is the Pichia pastoris species. Examples of Pichia pastoris strains include X33, GS115, KM71, KM71H; CBS2612, and CBS7435.

[0084] Specifically, the host cell is a Pichia pastoris strain selected from the group consisting of CBS704, CBS2612, CBS7435, CBS9173 - 9189, DSMZ70877, X - 33, GS115, KM71, KM71H, and SMD1168.

[0085] Deposited strains: CBS704 (NRRL Y-1603, DSMZ70382), CBS2612 (NRRL Y-7556), CBS7435 (NRRL Y-11430), CBS9173-9189 (CBS strains: CBS-KNAW Fungal Biodiversity Centre, Centraalbureau voor Schimmelculturen, Utrecht, The Netherlands) and DSMZ70877 (German Collection of Microorganisms and Cell Cultures); strains provided by Invitrogen, such as X-33, GS115, KM71, KM71H and SMD1168. Examples of Saccharomyces cerevisiae strains include W303, CEN.PK and BY strains (EUROSCARF collection). All of the above strains have been successfully used to produce transformants and express heterologous genes.

[0086] Eukaryotic host cells include fungal cells (e.g., Aspergillus (e.g., Aspergillus niger, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus nidulans), Acremonium (e.g., Acremonium thermophilum), Chaetomium (e.g., Chaetomium thermophilum), Chrysosporium (e.g., Chrysosporium thermophile), Cordyceps (e.g., Cordyceps militaris), Corynascus, Ctenomyces, Fusarium (e.g., Fusarium oxysporum), Glomerella (e.g., Glomerella graminicola), Hypocrea (e.g., Hypocrea jecorina), Magnaporthe (e.g., Magnaporthe oryzae), Myceliophthora (e.g., Myceliophthora thermophile), Nectria (e.g., Nectria haematococca), Neurospora (e.g., Neurospora crassa), Penicillium, Sporotrichum (e.g., Sporotrichum thermophile), Thielavia (e.g., Thielavia terrestris, Thielavia heterothallicaIt can be Heterothallica, Trichoderma (e.g., Trichoderma reesei), or Verticillium (e.g., Verticillium dahlia (V. dahlia)).

[0087] According to certain aspects, the mammalian cell is a cell, a cell line, or a cell strain of human, rodent, or bovine. Examples of certain mammalian cells suitable as host cells described herein include mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK Jurkat, MDCK, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLa, EBl, EB2, EB3, tumor cell-lytic cell line, or hybridoma cell line. Preferably, the mammalian cell is a CHO cell line. In one aspect, the cell is a CHO cell. In one aspect, the cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44CHO cell, a DUXB11CHO cell, a DUKX CHO cell, a CHO-S, a CHO FUT8 knockout CHO GS knockout cell, a CHO FUT8GS knockout cell, a CHOZN, or a cell derived from CHO. The CHO GS knockout cell (e.g., GSKO cell) is, for example, a CHO-K1SV GS knockout cell. The CHO FUT8 knockout cell is, for example, Potelligent® CHOK1SV (manufactured by Lonza Biologics, Inc.). Eukaryotic cells also include cells, cell lines, or cell strains of avian, e.g., EBx® cells, EB14, EB24, EB26, EB66, or EBvl3.

[0088] According to another specific aspect, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic® Sf9, Sf21, High Five® (BT1-TN-5B1-4), or BT1-Ea88 cell), an algal cell (e.g., Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas), or a plant cell (e.g., a cell derived from a monocotyledon (e.g., maize, rice, wheat, or Setaria) or a dicotyledon (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis)).

[0089] Suitable host cells are commercially available from a culture collection, such as the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the ATCC (American Type Culture Collection).

[0090] According to a particular embodiment, the present invention provides a method for producing a protein of interest (POI) in a eukaryotic host cell, comprising: i) genetically modifying the host cell to reduce the production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first host cell protein (HCP1), a second host cell protein (HCP2), and a third host cell protein (HCP3); a) HCP1 contains the amino acid sequence shown in SEQ ID NO: 1 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species. b) HCP2 contains the amino acid sequence shown in SEQ ID NO: 3 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species. c) HCP3 contains the amino acid sequence shown in SEQ ID NO: 5 when the host cell is Komagataella phaffii, or contains a homologous sequence of said sequence that is endogenous to the host cell when the host cell is another species. ii) Introducing into a host cell an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to a nucleotide sequence encoding a POI. iii) Culturing the host cell under conditions that produce the POI, and optionally, iv) Isolating the POI from the cell culture, particularly from the cell culture supernatant, and optionally, v) Providing a method comprising purifying the POI.

[0091] Specifically, the POI can be produced by culturing the host cell in a suitable medium, isolating the POI expressed from the culture, particularly from the cell culture supernatant, and purifying it by a method suitable for the expressed product for separating the cells and the POI. Thereby, a purified POI is produced.

[0092] According to a further specific embodiment, the present invention provides a method for producing a eukaryotic host cell capable of producing a protein of interest (POI) in a host cell culture.

[0093] Specifically, step i) of the method described herein is performed before, after, or simultaneously with step ii). According to a particular aspect, the host cell is first genetically modified to reduce said at least one HCP before being designed to produce a heterologous or recombinant POI. According to a particular example, a wild-type host cell is genetically modified according to step i) of the method described herein. Specifically, the host cell is provided by introducing said one or more genetic modifications for HCP reduction into a wild-type host cell line.

[0094] According to a further aspect, the host cell is first designed to produce a heterologous or recombinant POI before being further genetically modified to reduce said at least one HCP. According to a particular example, a wild-type host cell may first be designed to contain an expression cassette for POI production. Then, the host cell thus designed may be further modified to reduce HCP as described herein.

[0095] According to a further embodiment, the host cell is designed for POI production and genetically modified for HCP reduction in one step, for example, using respective expression cassettes, reagents, and tools in one or more reaction mixtures.

[0096] Specifically, the host cell is a cell line cultured in a cell culture, particularly a cell line of a production host.

[0097] According to a further particular embodiment, the present invention provides a method for producing a POI by culturing the host cell described herein, or a host cell obtainable by the method described herein, under conditions for producing the protein of interest (POI).

[0098] According to a further particular embodiment, the present invention provides the use of the host cell described herein for the production of a POI.

[0099] According to certain embodiments, the cell line is cultured under batch, fed-batch or continuous culture conditions. The culture may be carried out in a microtiter plate, shake flask, or bioreactor, starting with a batch phase as the first step, followed by a fed-batch phase or a continuous culture phase as the next step.

[0100] Specifically, the methods described herein include at least one genetic modification of the host cell that reduces the amount of at least one of the HCPs. In particular, the at least one genetic modification introduces one or more of the following characteristics of HCP reduction in the host cell, especially when cultured under conditions that express the POI, as compared to a host cell without the genetic modification for HCP reduction: · The amount of the at least one HCP produced by the host cell is reduced to less than 10%, 5%, or 3% (mol / mol) of the total HCP; · The amount of the at least one HCP produced by the host cell is reduced by at least any one of 50%, 60%, 70%, 80%, 90%, 95% (mol / mol), or even up to 100%, thereby stopping the production of each HCP; · The amount of each of the at least one HCP produced by the host cell, especially any one, two, or three of HCP1 and HCP2, HCP3, or HCP4, is reduced by at least any one of 50%, 60%, 70%, 80%, 90%, 95%, or 100% (mol / mol); · The amount of each of the at least one HCP produced by the host cell, especially any one, two, or three of HCP1 and HCP2, HCP3, or HCP4, is reduced to less than 1% (mol / mol) of the total HCP; · The amount of total HCP in the cell culture supernatant is reduced by at least any one of 5%, 10%, or 15% (mol / mol).

[0101] According to a further specific embodiment, the present invention provides a method for reducing the risk of contamination of an endogenous host cell protein (HCP) of a protein of interest (POI) produced in a host cell culture by culturing the host cell described herein under conditions for producing the POI and isolating the POI from the cell culture. By reducing the amount of HCP, the purity or fraction of the POI in the host cell culture can be effectively increased.

Brief Description of the Drawings

[0102]

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 2

Figure 3

Figure 4

Figure 5

[0103] The specific terms used throughout this specification have the following meanings.

[0104] The term "host cell" as used herein relates to a single cell, a single cell clone, or a cell line of host cells.

[0105] As used herein, the term "established cell line" means an established clone of a particular cell type that has acquired the ability to proliferate over a long period of time. Established cell lines are typically used to express endogenous or recombinant genes, or the products of metabolic pathways that produce cell metabolites mediated by such polypeptides or cell metabolites. "Production host established cell line" or "production established cell line" is generally understood to be an established cell line that can be immediately used for cell culture in a bioreactor to obtain the product of a production process, such as a POI.

[0106] The host cells producing the POI described herein are also referred to as "production host cells", and each established cell line is referred to as a "production established cell line".

[0107] Certain embodiments described herein relate to production host established cell lines characterized by low HCP expression.

[0108] The term "eukaryotic host cell" shall mean any eukaryotic cell or organism that may be cultured to produce a POI or a host cell metabolite. It is well understood that this term does not include humans.

[0109] As used herein, the term "cell culture", with respect to host cells, means maintaining cells in an active or resting state under conditions favorable for cell growth, differentiation, or continued viability in an artificial environment, such as an in vitro environment, and in particular, maintaining cells in a controlled bioreactor according to methods known in the art.

[0110] When culturing cells using an appropriate medium, the cells are contacted with the medium or substrate in a culture vessel under conditions suitable for maintaining the cell culture. As described herein, culture media that can be used for the growth of eukaryotic cells, particularly yeast or filamentous fungi, are provided. Standard cell culture techniques are well known in the art.

[0111] The cell cultures described herein employ, for example, techniques for preparing to produce a POI in a cell culture medium, which is separable from the cell biomass, referred to herein as the "cell culture supernatant" and may be purified to obtain the POI at a higher purity. When a protein (e.g., HCP or POI) is produced and secreted by host cells in a cell culture, such a protein is secreted into the cell culture supernatant, and it is understood herein that it can be obtained by separating the cell culture supernatant from the host cell biomass and optionally further purifying the protein to produce a purified protein.

[0112] Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled artificial in vitro environment. The characteristics and composition of cell culture media vary depending on the particular cells. Important parameters include osmotic pressure, pH, and nutrient formulation. The supply of nutrients may be carried out in a continuous or discontinuous manner according to methods known in the art.

[0113] A batch process is a cell culture mode in which all the nutrients required to culture cells are contained in the initial culture medium and no additional nutrients are supplied during fermentation, whereas in a fed-batch process, after the batch stage, a fed-batch stage is taken in which one or more nutrients are supplied to the culture by feeding. In most processes, the mode of feeding is critical and important, but the host cells and methods described herein are not limited to a particular mode of cell culture.

[0114] In certain embodiments, the cell culture process is a fed-batch process. Specifically, host cells transformed with a nucleic acid construct encoding the desired recombinant POI are cultured in the growth phase and transferred to the production phase to produce the desired recombinant POI.

[0115] In another aspect, the host cells described herein are cultured in a continuous mode, such as in a chemostat. A continuous fermentation process is characterized by the addition of fresh culture medium to the bioreactor at a predetermined, constant and continuous rate, whereby, simultaneously, the culture broth is removed from the bioreactor at a similar, predetermined, constant and continuous removal rate. By maintaining the culture medium, the flow rate and the removal rate the same and constant, the cell culture parameters and conditions in the bioreactor are maintained constant.

[0116] The recombinant POI can be produced by culturing the host cells and their respective clonal cells described herein in a suitable medium, isolating the expression product or metabolite from the culture, and optionally purifying it by suitable methods.

[0117] Several different approaches for the production of the POI described herein are preferred. By transforming eukaryotic host cells with an expression vector carrying recombinant DNA encoding the relevant protein, preparing a culture of the transformed cells, growing the culture, inducing transcription and POI production, and recovering the POI, the POI will be expressed, processed, and optionally secreted.

[0118] The term "host cell protein (HCP)" as used herein means individual secreted proteins produced by a host cell. When the host cell is expressing a POI, the HCP is understood as a by-product of the POI. Thus, the POI is not understood as an HCP. Typically, HCPs are present in the cell culture medium or cell culture supernatant from which the cells have been separated, for example by centrifugation. The sum of all HCPs is referred to as "total HCP". For example, host cell products containing a POI or a POI preparation are at risk of being contaminated by HCPs. Current analytical methods for assaying the presence of HCPs in a POI product include ELISA, HPLC, capillary electrophoresis, SDS-PAGE, or mass spectrometry, and in particular, mass spectrometry is liquid chromatography-mass spectrometry (LC-MS), or preferably, liquid chromatography tandem mass spectrometry (LC-MS / MS), as known in the art and / or further described in the examples.

[0119] The host cells described herein are typically tested for their expression ability, HCP content or POI yield by any of the following tests: ELISA, activity assay, HPLC, or other suitable tests, such as SDS-PAGE and Western blotting, or mass spectrometry.

[0120] To determine the effect of genetic modification on the reduction of HCP in a cell culture and, for example, on the amount of impurities in a POI produced in this way, host cell lines may be cultured in microtiter plates, shake flasks, or bioreactors using fed-batch fermentation or continuous fermentation, comparing each cell to a strain without such genetic modification in that cell.

[0121] The production methods described herein enable fermentation, in particular, on a pilot or industrial scale. The industrial process scale is preferably at least 10 L, specifically at least 50 L, preferably at least 1 m 3 preferably at least 10 m 3 most preferably at least 100 m3 It is.

[0122] Industrial-scale production conditions are preferred, and these conditions, for example, typically employ a process number over several days in a reactor of 100 L to 10 m 3 or more for fed-batch culture, or a continuous process in a fermenter of about 50 to 1000 L or more, and the dilution rate is about 0.02 to 0.15 h -1 It is.

[0123] The apparatuses, facilities, and methods used for the purposes described herein are particularly suitable for use and co-use with culturing in the culture of any desired cultured cells including prokaryotic and / or eukaryotic cultured cells. Further, in certain embodiments, such apparatuses, facilities, and methods are suitable for culturing any cell type including suspension cells or substrate-dependent (adherent) cells, and are suitable for production operations configured to be suitable for the production of pharmaceuticals and biopharmaceuticals, for example, polypeptide products (POIs), nucleic acid products (e.g., DNA or RNA), or cells and / or viruses, for example, cells and / or viruses used in cell and / or virus therapies.

[0124] In one aspect, such cells express or produce a product, for example, a recombinant therapeutic or diagnostic product. As described in more detail herein, examples of products produced by cells include POIs as exemplified herein, which are antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, e.g., DARPins, affibodies, adnectins, or IgNAR), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones) or viral therapeutics (e.g., oncolytic anti-cancer viruses, gene therapy viral vectors and viral immunotherapies), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells and adult stem cells), vaccines or lipid-coated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics or amino acids, including but not limited to these. In one aspect, such devices, facilities and methods can be used to produce biosimilars.

[0125] As described above, in one aspect, the devices, facilities and methods enable the production of eukaryotic cells, such as mammalian cells or lower eukaryotic cells, such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as Gram-positive or Gram-negative cells, and / or enable the large-scale synthesis by such cells of products of eukaryotic or prokaryotic cells, such as POIs comprising proteins, peptides or antibiotics, amino acids, nucleic acids (e.g., DNA or RNA). Unless otherwise specified herein, such devices, facilities and methods can include any desired volume or production capacity, including but not limited to bench-scale, pilot-scale and full production-scale production capabilities.

[0126] Furthermore, unless otherwise specified herein, such devices, equipment, and methods may include any suitable reactor, including but not limited to stirred tanks, air lifts, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds, and / or jet fluidized bed bioreactors. As used herein, "reactor" may include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used interchangeably with "fermenter". For example, in some aspects, an exemplary bioreactor unit can perform one or more or all of the following: fed-batch addition of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inflow and outflow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or washing / sterilization. An exemplary reactor unit, e.g., a fermentation unit, may include multiple reactors within the unit. For example, this unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and / or the equipment may include multiple units having a single or multiple reactors within the equipment. In various aspects, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In one aspect, the bioreactor may have a volume of about 100 mL to about 50,000 L. Non-limiting examples include volumes of 100 mL, 250 mL, 500 mL, 750 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, 500 L, 550 L, 600 L, 650 L, 700 L, 750 L, 800 L, 850 L, 900 L, 950 L, 1000 L, 1500 L, 2000 L, 2500 L, 3000 L, 3500 L, 4000 L, 4500 L, 5000 L, 6000 L, 7000 L, 8000 L, 9000 L, 10,000 L, 15,000 L, 20,000 L, and / or 50,000 L.In addition, suitable reactors can be multi-purpose, single-purpose, disposable, or non-disposable and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and / or glass.

[0127] In certain embodiments, and unless otherwise specified herein, the apparatuses, facilities, and methods described herein may also include, although not otherwise specified, any suitable unit operations and / or equipment, such as operations and / or equipment for separating, purifying, and isolating such products. Any suitable facilities and environments can be used, such as conventional site-built facilities, modular, mobile, and temporary facilities, or any other suitable assembly, equipment, and / or layout. For example, in some embodiments, a modular clean room can be used. In addition, unless otherwise specified, the apparatuses, systems, and methods described herein can be housed and / or executed in a single location or facility or can be housed and / or executed in separate or multiple locations and / or facilities.

[0128] Suitable methods may include culturing in a bioreactor that begins with a batch phase, followed by a short exponential fed-batch phase at a high specific growth rate, and then followed by a fed-batch phase at a low specific growth rate. Another suitable culturing method may include a batch phase followed by a fed-batch phase at any suitable specific growth rate or combination of specific growth rates, such as a combination of growth rates that proceed from a high growth rate to a low growth rate or from a low growth rate to a high growth rate over the POI production time. Another suitable culturing method may include a batch phase followed by a continuous culture phase at a low dilution rate.

[0129] Preferred embodiments include a batch culture to provide biomass, followed by a fed-batch culture for high-yield POI production.

[0130] The host cells described herein are preferably cultured in a bioreactor under growth conditions to obtain a cell density of at least 1 g / L cell dry weight, more preferably at least 10 g / L cell dry weight, preferably at least 20 g / L cell dry weight, preferably at least 30, 40, 50, 60, 70 or 80 g / L cell dry weight. It is advantageous to provide for such yields of biomass production on a pilot or industrial scale.

[0131] The growth medium that enables biomass accumulation, specifically the basal growth medium, typically contains a carbon source, a nitrogen source, a sulfur source and a phosphate source. Typically, such a medium further contains trace elements and vitamins, and may further contain amino acids, peptone or yeast extract.

[0132] Preferred nitrogen sources include NH4H2PO4, NH3 or (NH4)2SO4.

[0133] Preferred sulfur sources include MgSO4, (NH4)2SO4 or K2SO4.

[0134] Preferred phosphate sources include NH4H2PO4, H3PO4, NaH2PO4, KH2PO4, Na2HPO4 or K2HPO4.

[0135] Further typical medium components include KCl, CaCl2, and trace elements such as: Fe, Co, Cu, Ni, Zn, Mo, Mn, I, B.

[0136] Preferably, the medium is supplemented with vitamin B7.

[0137] A typical growth medium for Pichia pastoris contains glycerol, sorbitol or glucose, NH4H2PO4, MgSO4, KCl, CaCl2, biotin and trace elements.

[0138] During the production phase, the production medium is specifically used only with a small amount of supplementary carbon source.

[0139] Preferably, the host cell line is cultured in an inorganic medium containing a suitable carbon source, thereby significantly further simplifying the isolation process. Examples of preferred inorganic media are salts containing available carbon sources (e.g., glucose, glycerol, sorbitol or methanol), macro elements (potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate) and trace elements (copper, iodide, manganese, molybdate, cobalt, zinc, iron salts and boric acid), and optionally those containing vitamins or amino acids, for example to complement auxotrophic strains.

[0140] Specifically, depending on the expression system and the nature of the expressed protein, e.g., whether the protein is fused to a signal peptide and whether the protein is soluble or membrane-bound, the cells are cultured under conditions suitable to bring about the expression of the desired POI that can be purified from the cells or the culture medium. As will be understood by those skilled in the art, the culture conditions vary depending on factors including the type of host cell and the specific expression vector employed.

[0141] Typical production media contain a supplemented carbon source, and further NH4H2PO4, MgSO4, KCl, CaCl2, biotin and trace elements.

[0142] For example, the feed of the supplemented carbon source added to the fermentation may contain a carbon source having up to 50% by weight of available sugar.

[0143] The fermentation is preferably carried out at a pH of 3 to 8.

[0144] Typical fermentation times are about 24 to 120 hours at a temperature of 20°C to 35°C, preferably 22 to 30°C.

[0145] Preferably, conditions are employed that result in a yield of at least 1 mg / L, preferably at least 10 mg / L, preferably at least 100 mg / L, and most preferably at least 1 g / L of the POI.

[0146] As used herein, the terms "expression" or "expression cassette" mean a nucleic acid molecule containing a desired coding sequence and control sequences operably linked such that a host transformed or transfected with these sequences can produce the encoded protein or host cell metabolite. To effect transformation, an expression system may be included in a vector or the relevant DNA may be integrated into the host chromosome. Expression may mean a secreted or non-secreted expression product, including a polypeptide or metabolite.

[0147] Expression cassettes are conveniently provided as expression constructs, for example, in the form of "vectors" or "plasmids", which typically contain cloned recombinant nucleotide sequences, i.e., DNA sequences necessary for transcription of recombinant genes and translation of their mRNAs in a suitable host organism. Expression vectors or plasmids usually contain an origin of replication for autonomous replication in a host cell, a selectable marker (e.g., a gene conferring resistance to an amino acid synthetic gene or an antibiotic, e.g., zeocin, kanamycin, G418 or hygromycin, nourseothricin), a number of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, and these components are operably linked together. As used herein, the terms "plasmid" and "vector" include autonomously replicating nucleotide sequences and genome-integrating nucleotide sequences, e.g., artificial chromosomes, e.g., yeast artificial chromosomes (YACs).

[0148] The expression vector may include, but is not limited to, a cloning vector, a modified cloning vector, and a specifically designed plasmid. The preferred expression vectors described herein are expression vectors suitable for the expression of recombinant genes in eukaryotic host cells and are selected according to the host organism. Suitable expression vectors typically contain regulatory sequences suitable for the expression of the DNA encoding the POI in eukaryotic host cells. Examples of regulatory sequences include operators, enhancers, ribosome binding sites, and sequences that control the initiation and termination of transcription and translation. The regulatory sequences may be operably linked to the DNA sequence to be expressed.

[0149] To enable the expression of the recombinant nucleotide sequence in the host cell, the expression vector may have a promoter provided at a position adjacent to the 5' end of the coding sequence, for example, upstream of the signal peptide gene that enables the secretion of the gene of interest (GOI) or POI. Transcription is regulated and initiated by this promoter sequence.

[0150] The expression constructs described herein specifically include a promoter operably linked to the nucleotide sequence encoding the POI and are under the transcriptional control of the promoter. Specifically, the promoter is not naturally associated with the coding sequence of the POI.

[0151] A multi-cloning vector, which is a vector having a multi-cloning site, can also be used as described herein, and a desired heterologous gene can be incorporated into the multi-cloning site to provide an expression vector. In the case of a multi-cloning vector, since the gene of the POI is introduced into the multi-cloning site, the promoter is typically located upstream of the multi-cloning site.

[0152] As used herein, the term "endogenous" means a molecule and sequence that is present in a wild-type (natural) host cell prior to modification to reduce an endogenous protein, particularly including an endogenous protein. In particular, an endogenous nucleic acid molecule (e.g., a gene) or protein that is present in (and can be obtained from) a particular host cell found in nature is understood to be "endogenous to the host cell" or "endogenous to the host cell." Further, a cell that "endogenously expresses" a nucleic acid or protein expresses that nucleic acid or protein in the same manner as a particular type of host found in nature. Further, a host cell that "endogenously produces" or "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound in the same manner as a particular type of host cell found in nature.

[0153] Thus, even if an endogenous protein is no longer produced by a host cell, as in a knockout mutant of the host cell in which the gene encoding the protein is inactivated or deleted, this protein is still considered "endogenous" herein.

[0154] As used herein, the term "heterologous" with respect to a nucleotide or amino acid sequence or a protein means a compound that is foreign, i.e., "exogenous" to a given host cell (not found in nature), or a compound that is found in nature in a given host cell, e.g., in the context of a heterologous construct using a heterologous nucleic acid, but which means a "compound that is endogenous". A heterologous nucleotide sequence as found endogenously may also be produced in a cell in an unnatural amount (e.g., an amount greater than expected or an amount greater than that found in nature). A heterologous nucleotide sequence, or a nucleic acid containing a heterologous nucleotide sequence, will probably have a sequence different from the endogenous nucleotide sequence, but will encode the same protein as found endogenously. Specifically, a heterologous nucleotide sequence is one that is not found in nature in the same relationship to the host cell. Any recombinant or artificial nucleotide sequence is understood to be heterologous. Examples of heterologous polynucleotides include, for example, nucleotide sequences that are not naturally associated with a promoter or are not operably linked to a coding sequence as described herein in order to obtain a hybrid promoter. As a result, a hybrid or chimeric polynucleotide may be obtained. Further examples of heterologous compounds include transcriptional regulators, e.g., a POI-encoding polynucleotide operably linked to a promoter, to which an endogenous, naturally occurring POI-encoding sequence is not normally operably linked.

[0155] As used herein, the term "host cell" specifically means, in this context, derivatives of artificial organisms and natural (wild-type) host cells. It is to be fully understood that the host cells, methods and uses described herein (e.g., specifically referring to one or more genetic modifications, expression constructs, transformed host cells and recombinant proteins) do not occur in nature and are "artificial" or synthetic and are thus not considered the result of "natural laws".

[0156] The host cell is specifically a recombinant host cell that is designed to reduce the amount of endogenous host cell protein (HCP) produced by the host cell and obtained in the cell culture supernatant. Specifically, one or more proteins that are abundantly present in the culture of wild-type host cells are targets for genetic modification to reduce their expression. A protein is considered particularly abundant if it is present at high levels in the cell culture supernatant, for example, if it reaches at least 10% or at least 5% (mol / mol) of the total HCP. According to certain embodiments, the host cell is designed to knockdown or knockout (due to inactivation or deletion of the gene or a part thereof) the host cell gene encoding at least one, two, or three of the most abundantly secreted endogenous proteins.

[0157] Specifically, a deletion strain in which the gene is disrupted is provided.

[0158] As used herein, the term "disrupt" means a significant reduction towards complete removal of the expression of one or more endogenous proteins in the host cell, for example, knockdown or knockout. This may be measured, for example, by mass spectrometry as the presence of this one or more endogenous proteins in the culture medium of the host cell, and the total content of the endogenous protein may be below a threshold or undetectable.

[0159] The term "disrupted" specifically means the result of genetic engineering by at least one step selected from the group consisting of gene silencing, gene knockdown, gene knockout, delivery of a dominant negative construct, conditional gene knockout, and / or genetic modification of a specific gene.

[0160] The terms "knockdown", "reduction", or "attenuation" in the context of gene expression, as used herein, mean an experimental approach that results in a reduction of the expression of a given gene as compared to its expression in control cells. Gene knockdown can be achieved by various experimental means, such as introducing into the cell a nucleic acid molecule (e.g., shRNA, RNAi, miRNA) that hybridizes to a portion of the gene's mRNA and causes its degradation, or by altering the gene's sequence so as to result in a reduction of transcription, a reduction of mRNA stability, or an attenuation of mRNA translation.

[0161] Complete inhibition of the expression of a given gene is called "knockout". Gene knockout means that no functional transcript is synthesized from the gene, resulting in the loss of the function normally provided by this gene. Gene knockout is achieved by altering the DNA sequence to cause disruption or deletion of the gene or its regulatory sequences, or a portion of such gene or regulatory sequences. Knockout techniques include the use of homologous recombination methods to replace, interrupt, or delete important parts or the entire gene sequence, or the use of DNA modifying enzymes such as zinc fingers or meganucleases to introduce double-strand breaks into the DNA of the target gene, as shown, for example, by Gaj et al. (Trends Biotechnol. 2013; 31(7): 397-405).

[0162] Certain embodiments use one or more knockout plasmids that are transfected into a host cell. By homologous recombination, the target gene in the host cell can be disrupted. This procedure is typically repeated until all alleles of the target gene are stably removed.

[0163] One particular method for knocking out a specific gene described herein is the CRISPR-Cas9 method, as shown, for example, by Weninger et al. (J. Biotechnol. 2016, 235: 139-49).

[0164] Another aspect relates to the degradation of target mRNA by using small interfering RNA (siRNA) to transfect a host cell and targeting the mRNA encoding a target protein that is endogenously expressed by the host cell.

[0165] As used herein, the term "gene expression" means encompassing at least one step selected from the group consisting of DNA transcription into mRNA, mRNA processing, maturation of non-coding mRNA, mRNA export from the nucleus, translation, protein folding, and / or protein transport.

[0166] Gene expression of a gene may be inhibited or reduced by a method that directly interferes with gene expression, which includes, but is not limited to, inhibition or reduction of DNA transcription (e.g., by using a repressor associated with a specific promoter, by site-specific mutagenesis of a given promoter, by promoter replacement), or inhibition or reduction of translation (e.g., by post-transcriptional gene silencing induced by RNAi). Expression of a gene product with reduced activity, dysfunctional, or inactive can be achieved, for example, by site-specific or random mutagenesis, insertion, or deletion within the coding gene.

[0167] Inhibition or reduction of the activity of a gene product can be achieved, for example, by administering or incubating an inhibitor for each enzyme before or simultaneously with protein expression. Examples of such inhibitors include, but are not limited to, inhibitory peptides, antibodies, aptamers, fusion proteins or antibody mimetics for the enzyme, or its ligand or receptor, or inhibitory peptides or nucleic acids, or small molecules having similar binding activity. Other methods of inhibiting an enzyme are reduction of specific cofactors of the enzyme in the medium, such as copper, which is a PAM-specific ionic cofactor (e.g., in the form of CuSO4), ascorbate, which acts as an electron donor for PAM, oxygen molecules, catalase, and other specific cofactors known today or discovered in the future by those skilled in the art.

[0168] Gene silencing, gene knockdown, and gene knockout refer to techniques for reducing gene expression by genetic modification or by treatment with oligonucleotides having sequences complementary to either mRNA transcripts or genes. When genetic modification of DNA is performed, the result is a knockdown or knockout organism. When the change in gene expression is caused by oligonucleotides that bind to mRNA or transiently to the gene, this is a transient change in gene expression without modification of chromosomal DNA and is called a transient knockdown.

[0169] In transient knockdowns, which are also encompassed by the above terms, the binding of this oligonucleotide to the active gene or its transcript causes a decrease in expression by blocking transcription (in the case of gene binding), degradation of the mRNA transcript (e.g., by small interfering RNA (siRNA) or RNase-H-dependent antisense), or blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for the maturation of other functional RNAs, such as miRNAs (e.g., by morpholino oligos or other RNase-H-independent antisense). Other approaches include the use of shRNA (small hairpin RNA, an RNA sequence that forms a tight hairpin turn that can be used to silence gene expression via RNA interference), esiRNA (endoribonuclease-prepared siRNA, a mixture of siRNA oligos resulting from endoribonuclease cleavage of long double-stranded RNA (dsRNA)), or activation of the RNA-induced silencing complex (RISC).

[0170] Other approaches for performing gene silencing, knockdown or knockout are known to those skilled in the art from the respective literature, and their application in the context of the present invention is considered routine. Gene knockout means a technique that completely blocks the expression of a gene, i.e., inactivates or removes each gene. The methodological approaches for achieving this purpose are diverse and known to those skilled in the art. Examples include the production of mutants that are dominant negative to a given gene. Such mutants can be produced by site-directed mutagenesis (e.g., deletions, partial deletions, insertions or nucleic acid substitutions), by the use of suitable transposons, or by other approaches known to those skilled in the art from the respective literature, and thus their application in the context of the present invention is considered routine. One example is knockout by the use of targeted zinc finger nucleases. Each kit is provided by Sigma Aldrich as "CompoZR knockout ZFN". Another approach involves the use of transcription activator-like effector nucleases (TALENs).

[0171] Delivery of dominant negative constructs involves, for example, transfection and the introduction of a sequence encoding a dysfunctional enzyme. The coding sequence is functionally linked to a strong promoter such that the gene expression of the dysfunctional enzyme overrides the native expression of the wild-type enzyme, and thus results in an effective physiological defect in each enzyme activity.

[0172] Conditional gene knockout enables the blocking of gene expression in a tissue-specific or time-specific manner. This is done, for example, by introducing a short sequence called the loxP site near the gene of interest. Also, other approaches are known to those skilled in the art from the respective literature, and their application in the context of the present invention is considered routine.

[0173] Another approach is genetic alteration that can result in a dysfunctional gene product or a gene product with reduced activity. This approach involves the introduction of mutations that result in frameshift mutations, nonsense mutations (i.e., the introduction of premature stop codons), or amino acid substitutions that render the entire gene product dysfunctional or cause a reduction in activity. Such genetic alterations can be caused, for example, by either non-specific (random) mutagenesis or site-directed mutagenesis (e.g., deletions, partial deletions, insertions, or nucleic acid substitutions). Protocols describing gene silencing, gene knockdown, gene knockout, delivery of dominant negative constructs, conditional gene knockout, and / or the actual application of genetic alterations are generally available to those of skill in the art and are within the routine operations of those of skill in the art. Accordingly, the technical teachings provided herein are fully practicable with respect to all contemplated methods that result in the inhibition or reduction of gene expression of a gene product, or the expression of a dysfunctional or inactive gene product, or a reduction in activity.

[0174] The genetic modifications described herein may employ tools, methods, and techniques known in the art, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001).

[0175] As used herein, the term "operably linked" means that the function of one or more nucleotide sequences is affected by at least one other nucleotide sequence present on the nucleic acid molecule, such that the nucleotide sequences are associated on a single nucleic acid molecule, such as a vector or expression cassette. For example, a promoter is operably linked to a coding sequence when it can effect the expression of the coding sequence of the recombinant gene. As a further example, a nucleic acid encoding a signal peptide is operably linked to a nucleic acid sequence encoding a POI when it can express the protein in a secreted form, such as a preform of the mature protein or the mature protein. Specifically, such nucleic acids operably linked to each other may be linked immediately, i.e., without the presence of additional elements or nucleic acid sequences between the nucleic acid encoding the signal peptide and the nucleic acid sequence encoding the POI.

[0176] A promoter sequence is typically understood to be operably linked to a coding sequence when the promoter controls the transcription of the coding sequence. If the promoter sequence is not naturally associated with the coding sequence, either its transcription is not controlled by the promoter in a native (wild-type) cell, or these sequences are recombined with a different contiguous sequence.

[0177] The term "constitutive" with respect to a regulatory element, such as a promoter, relates to an element that is active under different cell culture conditions using different media or substrates. Among the constitutive promoters of yeast cells, in particular the GAP and TEF promoters are strong and have been evaluated as useful for recombinant protein production.

[0178] A promoter is particularly understood as a constitutive promoter when it can control expression without the need for induction or the possibility of repression. Thus, there is continuous and stable expression at a certain level. Preferably, the promoter has a high promoter strength at all growth phases or growth rates of the host cell.

[0179] The term "regulatable" with respect to an inducible or repressive regulatory element, e.g., a promoter, refers to an element that is repressed in a host cell in the presence of an excess of a substance (e.g., a nutrient in a cell culture medium), e.g., during the growth phase of a batch culture, according to a fed-batch strategy, and is derepressed to induce strong activity, e.g., during a production phase (e.g., when reducing the amount of a nutrient or supplying a supplemental substrate). A regulatory element can also be designed to be regulatable such that the element is inactive without the addition of a cell culture additive and is active in the presence of such an additive. Thus, expression of a POI under the control of such a regulatory element can be induced upon addition of such an additive.

[0180] The term "protein of interest (POI)" as used herein means a polypeptide or protein produced recombinantly in a host cell. More specifically, the protein may be a polypeptide that does not occur naturally in the host cell, i.e., a heterologous protein, or it may be a protein native to the host cell, i.e., a protein homologous to the host cell, but is produced, for example, by transformation with a self-replicating vector containing a nucleic acid sequence encoding the POI, or by recombinant techniques for integration of one or more copies of the nucleic acid sequence encoding the POI into the genome of the host cell, or by recombinant modification of one or more regulatory sequences, e.g., a promoter sequence, that control the expression of the gene encoding the POI. In some cases, the term POI as used herein also means any metabolite of the host cell mediated by the recombinantly expressed protein.

[0181] The term "scaffold" as used herein refers to a broad group of compact and stably folded proteins of different sizes, structures, and origins that function as a starting point for the generation of antigen-binding molecules. Inspired by the structure-function relationship of antibodies (immunoglobulins), the scaffold of such alternative proteins provides a rigid and conserved structural backbone that supports interaction sites that can be refashioned for the close and specific recognition of a given (biological) molecular target.

[0182] The term "sequence identity" of a variant, homolog or ortholog, when compared to a parental nucleotide or amino acid sequence, indicates the degree of identity of two or more sequences. Two or more amino acid sequences can have the same or conserved amino acid residues at corresponding positions to some extent up to 100%. Two or more nucleotide sequences can have the same or conserved base pairs at corresponding positions to some extent up to 100%.

[0183] Sequence similarity search is an effective and reliable strategy for identifying homologs with substantial (e.g., at least 50%) sequence identity. Frequently used sequence similarity search tools are, for example, BLAST, FASTA and HMMER.

[0184] Sequence similarity search can identify such homologous proteins or genes by detecting statistically significant similarities that reflect substantial similarity and a common ancestor. Homologs can include orthologs as understood herein as the same protein in different organisms, e.g., variants of such proteins in different organisms or species.

[0185] Homolog or ortholog sequences of the same protein from different organisms or species, particularly different organisms or species of the same genus, typically have at least about 50% sequence identity, preferably at least about 60% identity, more preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity, more preferably at least about 95% identity.

[0186] The HCP characterized by the sequences shown in SEQ ID NOs: 1-8 is of Komagataella phaffii. It is well understood that homologous sequences exist in other eukaryotic host cells. For example, yeast cells have been reclassified into a new genus, Komagataella, and divided into three species, Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris, and in particular, Pichia pastoris yeast contains respective homologous sequences. Further homologous sequences are found, for example, in Saccharomyces cerevisiae or Yarrowia lipolytica.

[0187] HCP1 (Komagataella phaffii): F2QXM5: According to NCBI, this protein is zonadhesin belonging to the lectin family, but also shows limited homology to subtilisin-like serine protease SUB2 in blast analysis. In most databases, this protein is still designated as an uncharacterized protein. 63 kDa, 587 AAs.

[0188] Each homologous sequence in Komagataella pastoris is referred to herein as an HCP1 homolog and is characterized by an amino acid sequence that contains or consists only of the amino acid sequence shown in SEQ ID NO: 20 (NCBI accession number: BA75_00021T0 [Komagataella pastoris], GenBank: ANZ74151.1).

[0189] HCP2 (Komagataella phaffii): F2QNG1: SCW10 is a cell wall protein with similarity to glucanase. It is involved in the carbohydrate metabolism process and has hydrolase activity. The homolog in Saccharomyces cerevisiae may play a role in conjugation during mating.

[0190] Each homologous sequence in Komagataella pastoris is referred to herein as an HCP2 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 21 (NCBI accession number: BA75_01624T0 [Komagataella pastoris], GenBank: ANZ73790.1).

[0191] HCP3 (Komagataella phaffii): F2QQT7: SUN4 is another protein with similarity to glucanase. According to data on the homolog in Saccharomyces cerevisiae, this protein may be involved in DNA replication and / or cell wall septum formation and is a protein of the SUN family (Sim1p, Uth1p, Nca3p, Sun4p). 45 kDa, 431 AAs.

[0192] Each homologous sequence in Komagataella pastoris is referred to herein as an HCP3 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 22 (NCBI accession number: BA75_01931T0 [Komagataella pastoris], GenBank: ANZ76017.1).

[0193] HCP4 (Komagataella phaffii): F2QXH5: EPX1, or extracellular protein 1. Although no clear function has been assigned to this protein, deletion strains (Δepx1) have been generated and found to be more sensitive to the cell wall-damaging agents Calcofluor white and Congo red than the wild type, revealing that Epx1 may have a protective role against the cell wall.

[0194] Each homologous sequence in Komagataella pastoris is referred to herein as an HCP4 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 23 (NCBI accession number: BA75_00070T0 [Komagataella pastoris], GenBank: ANZ73364.1).

[0195] Exemplary additional homologous sequences of HCPs described herein that are found in yeasts other than Komagataella phaffii are as follows.

[0196] The homologous sequence of HCP2 in Saccharomyces cerevisiae is referred to herein as an HCP2 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 24 (NCBI accession number: SCW10 [Saccharomyces cerevisiae], GenBank: KZV09161.1; >SCW10 YMR305C SGDID:S000004921).

[0197] A further homologous sequence of HCP2 in Saccharomyces cerevisiae is referred to herein as an HCP2 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 25 (NCBI accession number: SCW4 [Saccharomyces cerevisiae], GenBank: KZV11513.1; >SCW4 YGR279C SGDID:S000003511).

[0198] The homologous sequence of HCP3 in Saccharomyces cerevisiae is referred to herein as an HCP3 homolog and is characterized by an amino acid sequence that includes or consists of only the amino acid sequence shown in SEQ ID NO: 26 (NCBI accession number: SUN4 [Saccharomyces cerevisiae]; GenBank: CAA95939.1; >SUN4 YNL066W SGDID:S000005010).

[0199] As used herein, "percent (%) amino acid sequence identity" with respect to an amino acid sequence, homolog, and ortholog described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0200] For the purposes described herein, sequence identity between two amino acid sequences is determined using blastp set with the following representative parameters, using the NCBI BLAST program version 2.2.29 (Jan-06-2014). Program: blastp, Word size: 6, Expect value: 10, Hitlist size: 100, Gapcosts: 11.1, Matrix: BLOSUM62, Filter string: F, Genetic Code: 1, Window Size: 40, Threshold: 21, Composition-based stats: 2.

[0201] "Percent (%) identity" with respect to a nucleotide sequence, e.g., a nucleotide sequence of a promoter or a gene, is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in a DNA sequence after aligning the sequences and introducing gaps as needed to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignments for the purpose of determining percent nucleotide sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0202] The terms "isolated" or "isolation" as used herein with respect to a POI refer to a compound that is sufficiently separated to exist in a "purified" or "substantially pure" form from the environment that would naturally accompany it, particularly from cell culture supernatants. However, "isolated" does not necessarily mean the exclusion of an artificial or synthetic mixture with other compounds or materials, or the presence of impurities that do not interfere with the basic activity, e.g., due to incomplete purification. An isolated compound can be further formulated to make a preparation thereof and still be isolated for practical purposes (e.g., a POI can be mixed with a pharmaceutically acceptable carrier or excipient when used in a diagnosis or treatment).

[0203] The term "purified" as used herein refers to a preparation that contains at least 50% (mol / mol), preferably at least 60%, 70%, 80%, 90% or 95% of a compound (e.g., a POI). Purity is measured by methods appropriate for the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.). The isolated and purified POI described herein is obtained by purifying cell culture supernatants to reduce impurities.

[0204] As methods for isolating and purifying recombinant polypeptides or protein products, methods that utilize differences in solubility, such as salting out and solvent precipitation; methods that utilize differences in molecular weight, such as ultrafiltration and gel electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing, etc. can be used.

[0205] The following standard methods are preferred: separation and washing of cells (debris) by microfiltration, tangential flow filtration (TFF) or centrifugation, purification of POI by precipitation or heat treatment, activation of POI by enzymatic digestion, purification of POI by chromatography, such as ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion (SEC) or HPLC chromatography, concentration precipitation and washing of POI by ultrafiltration steps.

[0206] The highly purified product essentially contains no contaminating proteins, especially contaminating HCP, and preferably has a purity of at least 90%, more preferably at least 95%, or even more preferably at least 98%, up to 100%. The purified product is obtained by purification of cell culture supernatant or from cell debris.

[0207] The isolated and purified POI can be identified by conventional methods, such as Western blot, HPLC, activity assay, or ELISA.

[0208] As used herein, the term "recombinant" shall mean "prepared by genetic engineering or as a result of genetic engineering". A recombinant host may be designed to delete and / or inactivate one or more nucleotides or nucleotide sequences, and in particular, may specifically contain an expression vector or a cloning vector containing a recombinant nucleic acid sequence using a nucleotide sequence foreign to the host. A recombinant protein is produced by expressing each recombinant nucleic acid in a host. As used herein, the term "recombinant" with respect to a POI includes a POI prepared, expressed, produced or isolated by recombinant means, for example, a POI isolated from a host cell transformed to express the POI. In accordance with the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art may be used. Such techniques are well described in the literature. See, for example, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982).

[0209] The foregoing description will be more fully understood with reference to the following examples. However, such examples are merely representative of ways of carrying out one or more aspects of the invention and should not be construed as limiting the scope of the invention.

Examples

[0210] Example 1: Identification of Host Cell Protein Impurities in Pichia pastoris To identify host cell protein (HCP) impurities derived from Pichia pastoris cells (CBS2612 strain) that produce recombinant proteins as POIs, cell culture supernatant samples were analyzed for HCP composition, content, and identity as described below. Using two different expression systems, pG1-3 (SEQ ID NO: 38 of WO 2017 / 021541) and pAOX1 (e.g., Stratton et al., 1998 (High Cell-Density Fermentation. In: Higgins D.R., Cregg J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 103. Humana Press)) containing different signal peptides (POI1: SEQ ID NO: 12 of WO 2014 / 067926, POI2 and POI3: signal peptide of α-mating factor (e.g., SEQ ID NO: 1 of US Patent No. 9534039)), three different POIs were expressed by fed-batch culture under various fermentation conditions of pH and temperature shown in Table 1 each time. Each sample was tested three times for HCP composition, content, and identity.

[0211] The reporter proteins are three different proteins. POI1: an antigen-binding protein of bacterial origin, POI2: an artificial antigen-binding protein, POI3: an antigen-binding protein of human origin.

[0212]

Table 1

[0213] Preparation of LC-MS / MS Samples Samples were denatured in 6.6 M guanidine HCl, reduced with TCEP, and digested with trypsin before analysis. Three replicates were prepared for each sample. Each sample with a volume of 18.5 μl was transferred to a 96-well plate. 90 μl of 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5, 6.6 M guanidine HCl, and 10 mM tris(2-carboxyethyl)phosphine (TCEP) were added to each replicate. Incubation was carried out at 50 °C for 30 minutes. Then, all samples were buffer-exchanged using ZebaSpin desalting plates (Thermo) according to the manufacturer's instructions into 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS), 4-morpholinopropanesulfonic acid pH 7.3, 2 M urea, 2 mM CaCl2, 1 mM TCEP. Digestion was performed with mass spectrometry grade trypsin (Promega). To 75 μl aliquots of each sample after buffer exchange, 25 μl of trypsin digestion solution (4 mg / ml trypsin, 0.1 M MOPS pH 7.3, 2 M urea, 2 mM CaCl2, 1 mM TCEP) was added and mixed. The samples were incubated overnight at 30 ± 2 °C. Digestion was quenched by the addition of 2% (final) trifluoroacetic acid (TFA).

[0214] LC-MS / MS data collection Data were collected using a Dionex RSSLnano nanoLC system coupled to a Thermo Fusion Tribrid Q-OT-qIT (Quadrupole-Orbitrap-Linear Ion Trap) mass analyzer. For each sample, 1 μl of tryptic peptides was injected at 12 μl / min for 3 min onto an Acclaim PepMap100 C18 5 μm, 100 Å, 300 μm i.d. × 5 mm Nano-Trap column (Thermo) with a loading buffer consisting of water:acetonitrile (98:2) and 0.05% TFA. After 3 min, the nanoLC flow was directed in reverse through the trap column onto the analytical column (EasySpray PepMap C18 2 μm, 100 Å, 75 μm × 25 cm (Thermo)). A linear gradient was applied between 0.1% formic acid in water and 0.08% formic acid in acetonitrile:water (80:20).

[0215] The source ionization settings were static during collection at a spray voltage of 2500 V and a transfer tube temperature of 275 °C. The mass analyzer was operated in MS mode with an Orbitrap at a nominal resolution of 120,000 FWHM, a scan range of 200 - 2000 m / z, an AGC target of 2.0e5, and a maximum injection time of 50 ms. 1 It was configured in positive ionization mode to collect data. The data were mass corrected using an internal standard based on a fluoranthene ion lock mass generated from a different ion source. Only charge states of z = 2 - z = 8 were selected for MS 2 fragmentation.

[0216] Data-dependent decision methods including HCD and ETD methods were used to perform MS 2 fragmentation in the linear ion trap. HCD was performed with a collision energy of 28%, an AGC target of 1.0e4, and a maximum scan time of 100 ms at the "normal" trap scan rate. ETD was performed with an auxiliary activation collision energy of 15%, an AGC target of 1.0e4, and a maximum scan time of 100 ms at the "normal" trap scan rate.

[0217] Mass spectrometry data analysis MS 2 Protein identification based on fragmentation was performed using PEAKS Studio software. Protein identification was performed only for clarified cell culture supernatant (CCCS). The false discovery rate at the peptide level was controlled to <0.5% using the decoy fusion method (Zhang, J, et al., "PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification", Mol. Cell Proteomics 4(11), 111 (2012)). At least two specific peptides were required for the assignment of each protein. The mass tolerance was specified as <5 ppm for the parent ion and <0.3 Da for the fragment ion.

[0218] The generated LC-MS / MS data was analyzed separately for each of the three POIs to enable better alignment of the data during processing. Database searches were performed against the proteome of Komagataella phaffii (ATCC76273 / CBS7435 / CECT11047 / NRRL Y-11430 / Wegner21-1) (yeast) (Pichia pastoris) from the UniProt database, and the existing proteins were identified using PEAKS studio7. All samples were processed using Progenesis QI for Proteomics. Identifications from PEAKS were imported into Progenesis throughout the experiment for quantification. The data for each POI was processed separately. Quantification was performed using the Hi5 method. Proteins showing significant changes in the expression profile (q-value <0.01) and fold change >2 were evaluated for similarity in the expression profile.

[0219] Total HCP is determined as follows. For each identified protein, the peak areas of the five strongest peptide signals derived from that protein are determined and then added together. The resulting number enables comparison of the abundance of the protein on a molar basis (Silva et al., 2006: "Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition." Mol Cell Proteomics 5(1): 144-56.). The values thus obtained for all individual HCPs in the test sample were summed. The resulting total value is directly proportional to the amount (mol) of total HCP in the test sample. Therefore, comparisons can be made between samples with respect to differences in percentage or fold change in HCP.

[0220] Results Samples using the pAOX1 expression system showed significant increases in proteins specific to methanol metabolism (alcohol oxidase, formate dehydrogenase, alcohol dehydrogenase), as well as in the metabolism of reactive oxygen species generated during methanol metabolism (superoxide dismutase, peroxiredoxin PMP, protein disulfide isomerase, thioredoxin). Further changes correlating with the high titers of samples 9 and 11 were observed within this experiment compared to samples 7, 8, and 10. These changes included proteins such as an ATPase involved in protein folding, a GPI-anchored cell surface glycoprotein, peptidyl-prolyl cis-trans isomerase, and an uncharacterized protein F2QUJ0 showing homology to translation elongation factor EF-1γ (Komagataella phaffii) (C4R6E8). Samples using the pG1-3 expression system showed increased expression of enzymes involved in carbohydrate processing (glucanase, glucosidase) and structural proteins. Various fermentation conditions (temperature, pH) have been shown to result in changes in the HCP expression profile, for example, an increase in the amount of chaperone protein HSP90 was observed when fermentation was carried out at high temperature and pH.

[0221] Surprisingly, it has been found that a small number of different HCPs constitute the most abundant proteins present in all Pichia pastoris cell culture supernatants expressing different POIs under different expression conditions (as described above).

[0222] The most abundant HCPs identified are summarized in Table 2 below.

[0223] POI3 was expressed under the control of the AOX1 promoter in Samples 7 and 8 and under the control of the G1.3 promoter in Samples 9, 10, and 11. Some HCPs specific to one of the two induction systems could be observed, but their abundances were negligible compared to the major HCP1 and less than those of HCP2 and 3.

[0224]

Table 2

[0225] Together, HCP1 and HCP2, which represent approximately 56 - 81% of the total HCP content and on average approximately 68% of the total HCP content, were experiment - dependent. Interestingly, the Epx1 (F2QXH5) protein shown by Heiss et al. (Appl Microbiol Biotechnol. 2013 Feb; 97(3): 1241 - 9. doi:10.1007 / s00253 - 012 - 4260 - 4. Epub 2012 Jul 17) was found to be far less than HCP1, HCP2, or any combination thereof, i.e., it accounted for 3% of the total HCPs in Pichia pastoris.

[0226] Example 2: Generation of an HCP1 knockout strain in Pichia HCP1 (F2QXM5: represented by SEQ ID NOs: 1 and 2) was identified in the host cell protein identification analysis of Example 1 and accounted for 26 - 64% and on average approximately 50% of the total HCP load in different Pichia pastoris strains expressing three different POIs using different expression systems and fermentation conditions (Example 1).

[0227] For disruption of the gene encoding HCP1 in Pichia pastoris (CBS2612 strain), the split marker cassette approach shown by Heiss et al. (Appl Microbiol Biotechnol. 2013; 97(3): 1241-9) was used.

[0228] The primers used for disruption of the gene encoding HCP1 are listed in Table 3 below (two overlapping split marker cassettes are used per knockout target).

[0229] [Table 3]

[0230] Using primer pairs of A_forward / A_reverse, B_forward / B_reverse, C_forward / C_reverse, and D_forward / D_reverse, fragments A, B, C, and D were amplified by PCR (Q5 High-Fidelity 2X Master Mix, New England Biolabs). Fragment A for knocking out the gene encoding HCP1 was amplified from the genomic DNA of Pichia pastoris, starting from 1500 bp in the 5-prime direction of each ATG (of the gene encoding HCP1) and ending at 1 bp in the 5-prime direction of the ATG. Fragment D for knocking out the gene encoding HCP1 was amplified from the genomic DNA of Pichia pastoris, starting from 500 bp in the 3-prime direction of each ATG (of the target gene) and ending at 2000 bp in the 3-prime direction of the ATG. Fragment B consists of the first two-thirds of the KanMX selection marker cassette and was amplified from a plasmid containing the KanMX cassette vector DNA template. Fragment C consists of the last two-thirds of the KanMX selection marker cassette and was also amplified from a plasmid containing the KanMX cassette vector DNA template. Fragments A and B were annealed together by overlap PCR using the primers A_forward and B_reverse (Fragment AB). Fragments C and D were annealed together by overlap PCR using the primers C_forward and D_reverse (Fragment CD).

[0231] To generate knockout strains, four host strains (KO strain 1: CBS2612 strain containing HCP1 knockout (CBS2612 KO HCP1); KO strain 2: CBS2612 strain containing a heterologous gene encoding POI1 and HCP1 knockout (CBS2612 KO HCP1+POI1); KO strain 3: CBS2612 strain containing a heterologous gene encoding POI2 and HCP1 knockout (CBS2612 KO HCP1+POI2); and KO strain 4: CBS2612 strain containing a heterologous gene encoding POI3 and HCP1 knockout (CBS2612 KO HCP1+POI3)) were transformed with a total of 0.5 μg of fragments AB and CD. Cells were selected on YPD agar plates containing 500 μg / mL of Geneticin. Positive knockout clones were confirmed by PCR using primer pairs of control_forward (binding upstream of fragment A) and control_reverse (binding downstream of fragment D). By replacing the region near the start codon with the KanMX cassette, the PCR product band of the positive knockout strain is larger than that of the wild-type sequence (Figure 2). Either EcoR1 or Nco1 was used to perform restriction enzyme digestion on the PCR product to further examine the PCR amplification product. The PCR product of the positive knockout strain should not be cleaved by EcoR1 (4602 bp fragment), but should be digested by Nco1 (1955 bp and 2647 bp fragments).

[0232] To analyze the total host cell protein (HCP) content of the HCP1 knockout strain and compare it with the strain containing the intact HCP1 locus, the CBS2612 strain expressing POI3 (CBS2612 POI3) and the strain further containing the HCP3 knockout (CBS2612 KO HCP1+POI3) were cultured in fed-batch culture, and the total HCP in each culture supernatant was as described in Example 1 using the promoter pG1.3 and the signal peptide aMF_EAEA (the α mating factor signal peptide of Saccharomyces cerevisiae having the tetrapeptide EAEA, and the tetrapeptide EAEA is shown as SEQ ID NO: 19 for the production of POI3). The analysis results are shown in Table 4.

[0233]

Table 4

[0234] As can be seen from Table 4, the strain containing the knockout of the gene encoding HCP1 produced approximately 50% (mol / mol) less total HCP compared to each strain containing the gene encoding the HCP1 protein.

[0235] Example 3: Preparation of Multiple Recombinant Protein Expression Strains in Pichia HCP1 Knockout Strains To further demonstrate the strong reduction in the total amount of HCP upon knockout of HCP1, additional strains were prepared. In contrast to Example 2, first, a knockout strain was prepared, and then this strain was transformed with one of three plasmids expressing the protein of interest.

[0236] In the case of Pichia pastoris (CBS2612 strain), the same approach described in Example 2 was used for the disruption of the gene encoding HCP1. The HCP1KO strain was confirmed by four different PCR reactions (Figure 3). Using PCR1 (primer pair of control forward and control reverse 2, Table 3), the 5'-side of the integration of the knockout cassette at the correct genomic position was confirmed. No amplification product should be obtained in the wild-type strain, but a 1687 bp amplification product should be obtained for the HCP1KO strain. Using PCR2 (primer pair of control forward 2 and control reverse), the 3'-side of the integration of the knockout cassette at the correct genomic position was confirmed. No amplification product should be obtained in the wild-type strain, but a 1723 bp amplification product should be obtained for the HCP1KO strain. Using PCR3 (primer pair of control forward 3 and control reverse), the HCP1 genomic position was confirmed. A 2172 bp amplification product should be obtained in the wild-type strain, but no amplification product should be obtained for the HCP1KO strain. Using PCR4 (primer pair of control forward 4 and control reverse 3), it was confirmed that the knockout fragment of the HCP1 gene was not re-integrated anywhere else in the genome. A 115 bp amplification product should be obtained in the wild-type strain, but no amplification product should be obtained for the HCP1KO strain.

[0237] In the second step, both the wild-type (CBS2612) strain and the HCP1KO strain were transformed with 1 - 5 μg of one of three plasmids encoding POI1, POI2, or POI3, respectively, under the control of the G1-3 promoter (SEQ ID NO: 38 of WO 2017 / 021541). Cells were selected on YPD agar plates containing 100 - 1000 μg / mL of Zeocin. Positive clones were slightly adapted, i.e., the medium was exchanged to the medium from the Media Development Kit (M-KIT-100, m2pLabs, DE), and analyzed for expression as described in WO 2017 / 021541. Furthermore, the gene copy number (GCN) was analyzed using methods known to those skilled in the art (e.g., Abad et al., 2010). Based on similar POI titers and GCN in the wild-type and HCP1KO backgrounds, eight strains were selected.

[0238] Example 4: Characterization of host cell protein impurities in the culture supernatant of the strains of Example 3 To characterize the HCP impurities derived from the eight selected strains (Example 3), the strains were cultured in fed-batch fermentation as described in Example 1. The end-fermentation samples were used for HCP identification.

[0239] Samples were prepared, MS data were collected, and the data were analyzed using a workflow similar to that described in Example 1 with minor modifications. The analysis was performed as follows.

[0240] Preparation of LC-MS / MS samples The samples were denatured in 6.6 M guanidine HCl, reduced with TCEP, and digested with trypsin before analysis. Three replicates were prepared for each sample. Each 60 μl sample was transferred to a 96-well plate. 90 μl of 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5, 6.6 M guanidine HCl, and 10 mM tris(2-carboxyethyl)phosphine (TCEP) were added to each replicate. Incubation was carried out at 50 °C for 30 minutes. Subsequently, all samples were buffer-exchanged using ZebaSpin desalting plates (Thermo) according to the manufacturer's instructions into 0.1 M 3-(N-morpholino)propanesulfonic acid (MOPS), 4-morpholinopropanesulfonic acid) pH 7.3, 2 M urea, 2 mM CaCl2, 1 mM TCEP. Digestion was performed with mass spectrometry-grade trypsin (Promega). To 50 μl aliquots of each sample after buffer exchange, 16.6 μl of trypsin digestion solution (4 mg / ml trypsin, 0.1 M MOPS pH 7.3, 2 M urea, 2 mM CaCl2, 1 mM TCEP) was added and mixed. The samples were incubated overnight at 30 ± 2 °C. Digestion was quenched by the addition of 2% (final) trifluoroacetic acid (TFA). The samples were diluted 1:10 with digestion buffer.

[0241] LC-MS / MS data collection Data were collected using a Dionex RSSL nano nanoLC system coupled to a Thermo Fusion Tribrid Q-OT-qIT (Quadrupole-Orbitrap-Linear Ion Trap) mass analyzer. For each sample, 1 μl of tryptic peptides was injected at 12 μl / min for 3 min onto an Acclaim PepMap100 C18 5 μm, 100 Å, 300 μm i.d. × 5 mm Nano-Trap column (Thermo) with a loading buffer consisting of water:acetonitrile (98:2) and 0.05% TFA. After 3 min, the nanoLC flow was directed in reverse onto the analytical column (EasySpray PepMap C18 2 μm, 100 Å, 75 μm × 25 cm (Thermo)) through the trap column. A linear gradient was applied between 0.1% formic acid in water and 0.08% formic acid in acetonitrile:water (80:20). The source ionization settings were static during collection at a spray voltage of 2500 V and a transfer tube temperature of 275 °C. The mass analyzer was run in MS in the Orbitrap at a nominal resolution of 120,000 FWHM with a scan range of 350 - 1500 m / z, an AGC target of 5.0e5, and a maximum injection time of 150 ms. 1 It was configured in positive ionization mode to collect data. The data were mass corrected using an internal standard based on the fluoranthene ion lock mass generated from a different ion source. Only charge states of z = 2 to z = 8 were selected for MS 2 fragmentation. The most intense data-dependent mode of TopN using the HCD method was used for MS 2 fragmentation in the linear ion trap. HCD was performed with a collision energy of 28%, an AGC target of 1.0e4, and a maximum scan time of 100 ms at a "fast" trap scan speed.

[0242] Mass spectrometry data analysis MS 2Protein identification based on fragmentation was performed using PEAKS Studio software. Protein identification was performed only for clarified cell culture supernatant (CCCS). The false discovery rate at the peptide level was controlled to <0.1% using the decoy fusion method (Zhang, J, et al., "PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification", Mol. Cell Proteomics 4(11), 111 (2012)). At least two specific peptides were required for the assignment of each protein. The mass tolerance was specified as <5 ppm for the parent ion and <0.3 Da for the fragment ion. The generated LC-MS / MS data were analyzed separately for each of the three POIs to enable better alignment of the data during processing. Database searches were performed against the proteome of Komagataella phaffii (ATCC76273 / CBS7435 / CECT11047 / NRRL Y-11430 / Wegner21-1) (yeast) (Pichia pastoris) from the UniProt database, and the existing proteins were identified using PEAKS studio7. All samples were processed using Progenesis QI for Proteomics. Identifications from PEAKS were imported into Progenesis throughout the experiment for quantification. The data for each POI were processed separately. Quantification was performed using the Hi3 method. Proteins showing significant changes in expression profiles (p-value <0.05) and fold change >2 were evaluated for similarity in expression profiles. Total HCP was determined as follows. For each identified protein, the peak areas of the three strongest peptide signals derived from that protein were determined and then added together. The resulting number enables comparison of the abundance of the protein on a molar basis (Silva et al., 2006: "Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition." (Mol Cell Proteomics 5(1): 144-56). The values thus obtained for all individual HCPs in the test sample were summed. The total value obtained is directly proportional to the amount (mol) of total HCP in the test sample. Therefore, comparisons can be made between samples with respect to the difference in percentage or fold change in HCP.

[0243] Results As seen in Example 1, a surprisingly high abundance of HCP1 in the total HCP pool of the non-engineered strain was confirmed in this experiment (Figure 4). HCP1 constitutes 43-70% of the total HCP in the supernatant of wild-type strains with or without recombinant protein expression.

[0244] Comparing the total amount of HCP in wild-type and HCP1 knockout strains, a 20-79% reduction was obtained in total HCP (Figure 5). The HCP1 content of the wild-type strain expressing POI1 exceeded 40% of total HCP, while the amount of total HCP was only reduced by 20%. This is due to a slight upregulation of other HCPs in the background of this strain. Nevertheless, for all strains, knockout of HCP1 had a substantial positive impact on the impurity profile of the recombinant-produced protein in cell-free medium, which was unexpected. The invention described in the claims of the original application is appended below. [1] A eukaryotic host cell designed to produce a heterologous protein of interest (POI), wherein said cell is genetically modified to reduce the production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first host cell protein (HCP1), a second host cell protein (HCP2), and a third host cell protein (HCP3), a) HCP1 comprises the amino acid sequence shown in SEQ ID NO: 1 when said host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence endogenous to said host cell when said host cell is another species, b) HCP2 comprises the amino acid sequence shown in SEQ ID NO: 3 when said host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence endogenous to said host cell when said host cell is another species, c) HCP3 comprises the amino acid sequence shown in SEQ ID NO: 5 when said host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence endogenous to said host cell when said host cell is another species, Eukaryotic host cell. [2] The host cell according to [1], wherein said at least one HCP is HCP1, optionally HCP2 and / or HCP3. [3] The cell is further genetically modified to reduce the production of HCP4, which is a further HCP, a) HCP4 comprises the amino acid sequence shown in SEQ ID NO: 7 when said host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence endogenous to the host cell when said host cell is another species, the host cell according to [1] or [2]. [4] The host cell according to any one of [1] to [3], wherein said homologous sequence is characterized by at least 50% sequence identity to each amino acid sequence. [5] The host cell is of the host cell genome, (i) one or more endogenous polynucleotides or a part thereof, or (ii) Preferably, by one or more genetic modifications including disruption, substitution, deletion or knockout of an expression control sequence selected from the group consisting of a promoter, ribosome binding site, transcription or translation start and stop sequences, enhancer and activator sequences, The host cell according to any one of [1] to [4], which is genetically modified by one or more genetic modifications including disruption, substitution, deletion or knockout of an expression control sequence selected from the group consisting of a promoter, ribosome binding site, transcription or translation start and stop sequences, enhancer and activator sequences. [6] The host cell according to [5], wherein the gene encoding any one of the at least one HCP is knocked out by the one or more genetic modifications. [7] The host cell according to any one of [1] to [6], which is genetically modified such that the amount of the at least one HCP is reduced by at least 50% (mol / mol) as compared to the host cell without modification, preferably by knockout of the gene encoding the at least one HCP. [8] The host cell according to any one of [1] to [7], comprising an expression cassette containing one or more regulatory nucleic acid sequences operably linked to the nucleotide sequence encoding the POI, wherein the one or more operably linked sequences are not naturally associated with the sequence encoding the POI. [9] The host cell according to any one of [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.

[10] The host cell according to any one of [1] to [9], which is any one of an animal cell, a vertebrate cell, a mammalian cell, a human cell, a plant cell, a nematode cell, an invertebrate cell, an insect cell, a mollusk cell, a stem cell derived from any of the foregoing, or a yeast or fungal cell.

[11] a) Yeast cells of a genus selected from the group consisting of the genus Pichia, the genus Hansenula, the genus Komagataella, the genus Saccharomyces, the genus Kluyveromyces, the genus Candida, the genus Ogataea, the genus Yarrowia, and the genus Geotrichum, for example, Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica or Hansenula polymorpha, or b) Cells of filamentous fungi, for example, Aspergillus awamori or Trichoderma reesei, the host cell according to any one of [1] to

[10] .

[12] A method for producing a protein of interest (POI) in a eukaryotic host cell, comprising: i) genetically modifying the host cell to reduce the production of at least one endogenous host cell protein (HCP) selected from the group consisting of a first host cell protein (HCP1), a second host cell protein (HCP2), and a third host cell protein (HCP3); a) HCP1 comprises the amino acid sequence shown in SEQ ID NO: 1 when the host cell is Komagataella phaffii, or comprises a homologous sequence of the sequence endogenous to the host cell when the host cell is another species; b) HCP2 comprises the amino acid sequence shown in SEQ ID NO: 3 when the host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence that is endogenous to said host cell when the host cell is another species, c) HCP3 comprises the amino acid sequence shown in SEQ ID NO: 5 when the host cell is Komagataella phaffii, or comprises a homologous sequence of said sequence that is endogenous to said host cell when the host cell is another species, ii) introducing into the host cell an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to the nucleotide sequence encoding the POI, iii) culturing the host cell under conditions to produce the POI, and optionally, iv) isolating the POI from the cell culture, and optionally v) purifying the POI A method comprising the steps of:

[13] A method for producing a POI by culturing the host cell according to any one of [1] to

[11] under conditions for producing the protein of interest (POI).

[14] The method according to

[12] or

[13] , wherein the amount of the at least one HCP is preferably reduced by at least 50% (mol / mol) compared to the host cell without modification, by knockout of the gene encoding the at least one HCP.

[15] A method for reducing the risk of contamination by endogenous host cell proteins (HCPs) of the POI produced in a host cell culture by culturing the host cell according to any one of [1] to

[10] under conditions for producing the POI, and isolating the POI from the cell culture.

Claims

1. A eukaryotic host cell designed to produce a heterologous protein of interest (POI), wherein said cell is genetically modified to reduce the production of at least one endogenous host cell protein (HCP), and said at least one HCP comprises any one, two or three of a first host cell protein (HCP1), a second host cell protein (HCP2) and a third host cell protein (HCP3), and one of said at least one HCP is HCP1, a) HCP1 comprises the amino acid sequence shown in SEQ ID NO: 1, or an HCP1 homologous sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, i) when said host cell is Komagataella phaffii, said HCP1 homologous sequence is the wild-type sequence in Komagataella phaffii, or, ii) when said host cell is Komagataella pastoris, said HCP1 homologous sequence is the HCP1 wild-type sequence in Komagataella pastoris, or, iii) when said host cell is Komagataella pseudopastoris, said HCP1 homologous sequence is the HCP1 wild-type sequence in Komagataella pseudopastoris, b) HCP2 comprises the amino acid sequence shown in SEQ ID NO: 3, or an HCP2 homologous sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, i) when said host cell is Komagataella phaffii, said HCP2 homologous sequence is the wild-type sequence in Komagataella phaffii, or, ii) when said host cell is Komagataella pastoris, said HCP2 homologous sequence is the HCP2 wild-type sequence in Komagataella pastoris, or, iii) when said host cell is Komagataella pseudopastoris, said HCP2 homologous sequence is the HCP2 wild-type sequence in Komagataella pseudopastoris, c) HCP3 comprises the amino acid sequence shown in SEQ ID NO: 5, or an HCP3 homologous sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, i) when said host cell is Komagataella phaffii, said HCP3 homologous sequence is the wild-type sequence in Komagataella phaffii, or, ii) when the host cell is Komagataella pastoris, the HCP3 homologous sequence is the wild-type HCP3 sequence in Komagataella pastoris, or, iii) when the host cell is Komagataella pseudopastoris, the HCP3 homologous sequence is the wild-type HCP3 sequence in Komagataella pseudopastoris, the eukaryotic host cell is Pichia pastoris selected from the group consisting of Komagataella phaffii, Komagataella pastoris and Komagataella pseudopastoris, eukaryotic host cell.

2. the cell is further genetically modified to reduce the production of HCP4, which is a further HCP, d) HCP4 comprises the amino acid sequence shown in SEQ ID NO: 7, or an HCP4 homologous sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, i) when the host cell is Komagataella phaffii, the HCP4 homologous sequence is the wild-type sequence in Komagataella phaffii, or, ii) when the host cell is Komagataella pastoris, the HCP4 homologous sequence is the wild-type HCP4 sequence in Komagataella pastoris, or, iii) when the host cell is Komagataella pseudopastoris, the HCP4 homologous sequence is the wild-type HCP4 sequence in Komagataella pseudopastoris, the host cell according to claim 1.

3. the host cell is of the host cell genome, (i) one or more endogenous polynucleotides or a part thereof, or (ii) an expression control sequence, genetically modified by one or more genetic modifications including disruption, substitution, deletion or knockout, the host cell according to claim 1 or 2.

4. the expression control sequence is selected from the group consisting of a promoter, a ribosome binding site, transcription or translation start and stop sequences, an enhancer and an activator sequence, the host cell according to claim 3.

5. the gene encoding any of the at least one HCP is knocked out by the one or more genetic modifications, the host cell according to claim 3 or 4.

6. The host cell according to any one of claims 1 to 5, which is genetically modified such that the amount of HCP1 is reduced by at least 50% (mol / mol) as compared to the host cell without modification.

7. The host cell according to claim 6, wherein the reduction is achieved by knockout of the gene encoding HCP1.

8. The host cell according to any one of claims 1 to 7, comprising an expression cassette containing one or more regulatory nucleic acid sequences operably linked to the nucleotide sequence encoding the POI, wherein the one or more operably linked sequences are not naturally associated with the sequence encoding the POI.

9. 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. The host cell according to any one of claims 1 to 8.

10. A method for producing a protein of interest (POI) in a eukaryotic host cell, comprising: i) genetically modifying the host cell to reduce the production of at least one endogenous host cell protein (HCP), wherein the at least one HCP comprises any one, two, or three of a first host cell protein (HCP1), a second host cell protein (HCP2), and a third host cell protein (HCP3), and one of the at least one HCP is HCP1. a) HCP1 comprises the amino acid sequence represented by SEQ ID NO: 1, or an HCP1 homologous sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO:

1. a) When the host cell is Komagataella phaffii, the HCP1 homologous sequence is a wild-type sequence in Komagataella phaffii, or i) When the host cell is Komagataella pastoris, the HCP1 homologous sequence is an HCP1 wild-type sequence in Komagataella pastoris, or (c) When the host cell is Komagataella pseudopastoris, the HCP1 homologous sequence is the wild-type HCP1 sequence in Komagataella pseudopastoris, b) HCP2 comprises the amino acid sequence represented by SEQ ID NO: 3, or an HCP2 homologous sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 3, a) When the host cell is Komagataella phaffii, the HCP2 homologous sequence is the wild-type sequence in Komagataella phaffii, or i) When the host cell is Komagataella pastoris, the HCP2 homologous sequence is the wild-type HCP2 sequence in Komagataella pastoris, or c) When the host cell is Komagataella pseudopastoris, the HCP2 homologous sequence is the wild-type HCP2 sequence in Komagataella pseudopastoris, c) HCP3 comprises the amino acid sequence represented by SEQ ID NO: 5, or an HCP3 homologous sequence having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 5, a) When the host cell is Komagataella phaffii, the HCP3 homologous sequence is the wild-type sequence in Komagataella phaffii, or i) When the host cell is Komagataella pastoris, the HCP2 homologous sequence is the wild-type HCP3 sequence in Komagataella pastoris, or c) When the host cell is Komagataella pseudopastoris, the HCP3 homologous sequence is the wild-type HCP3 sequence in Komagataella pseudopastoris, ii) introducing into the host cell an expression cassette comprising one or more regulatory nucleic acid sequences operably linked to the nucleotide sequence encoding the POI, iii) preparing a cell culture by culturing the host cell under conditions for producing the POI, and optionally, iv) isolating the POI from the cell culture, and optionally v) purifying the POI comprising, wherein the eukaryotic host cell is Pichia pastoris selected from the group consisting of Komagataella phaffii, Komagataella pastoris and Komagataella pseudopastoris, method. Claim 11 A method for producing a POI by culturing the host cell according to any one of claims 1 to 9 under conditions for producing the target protein (POI).

12. The method according to claim 10 or 11, wherein the amount of HCP1 is reduced by at least 50% (mol / mol) compared to the host cell without modification.

13. The method according to claim 12, wherein the reduction is achieved by knockout of the gene encoding HCP1.

14. A method for reducing the risk of contamination by endogenous host cell proteins (HCPs) of POI produced in a host cell culture by preparing a cell culture by culturing the host cell according to any one of claims 1 to 9 under conditions for producing the target protein (POI), and isolating the POI from the cell culture.

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