Reducing residual DNA in microbial fermentation products

Abstract

The present invention provides a method for reducing the amount of DNA in a microbial fermentation product by adding a fungal DNase derived from Aspergillus oryzae to the resulting microbial fermentation product during the recovery process.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to the reduction of residual recombinant DNA in microbial fermentation products using a fungal DNase. [Background technology]

[0003] The production of protein products by fermentation is a well-known process and is used for the industrial-scale production of many different proteins of interest. During fermentation, some host cells producing the protein product of interest are disrupted and their cellular contents, including DNA, are released into the fermentation broth. In addition, in some fermentations the protein of interest is produced as an intracellular product. This means that cells are inevitably disrupted / lysed, for example by homogenization, before the post-fermentation harvesting and purification process, which inevitably results in the release of a significant amount of DNA into the fermentation broth, which may then be present as residual DNA in the final protein product.

[0004] For example, due to environmental or health concerns, it may be desirable to avoid residual DNA from the host cell producing the protein of interest, which is a particular concern with recombinant DNA. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need to reduce residual DNA in fermentation products. [Means for solving the problem]

[0006] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (a) providing a fermentation broth comprising microbial host cells, recombinant DNA from the microbial host cells, and a protein of interest produced by the microbial host cells; (b) subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; (c) subjecting the fermentation broth supernatant to a membrane filtration step, the membrane having a size exclusion limit of less than 100 kDa or less than 1 μm, to provide a fermentation product. A method for reducing the amount of DNA in a microbial fermentation product, comprising: The method further comprises adding a fungal DNase or a variant thereof to the fermentation broth before, during or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c).

[0007] In another aspect, the present invention provides a microbial fermentation product containing less than 10 ng / g, less than 5 ng / g, less than 1 ng / g, or less than 0.1 ng / g of recombinant DNA.

[0008] Other aspects and embodiments of the invention will become apparent from the description and examples.

[0009] Unless otherwise stated, or unless the context makes it clear that something else is meant, all percentages are percentages by weight (% w / w).

[0010] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, it is to be understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the present specification and the related art, and should not be interpreted as such unless expressly defined in this specification in an ideal or overly formal sense. For the sake of brevity and / or clarity, well-known functions or structures may not be described in detail.

[0011] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0012] definition Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0013] In the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 6.6.0 or later, as the "longest identity" output. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version: BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified on the command line. The output of Needle, labeled "longest identity", is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0014] Cell debris: The term "cell debris" refers to cell walls and other insoluble cellular components that are released after disruption of the cell wall / membrane, e.g., after disruption / lysis / homogenization of microbial cells. Even without intentional disruption of the cell wall / membrane, some microbial cells may disintegrate and release cell debris during the fermentation process.

[0015] array SEQ ID NO: 1: Amino acid sequence of DNase from Bacillus cibi. SEQ ID NO: 2: Amino acid sequence of DNase derived from Aspergillus oryzae. SEQ ID NO: 3: Amino acid sequence of the NUC3 DNase motif. SEQ ID NO: 4: Amino acid sequence of the NUC3 DNase motif. SEQ ID NOs: 5 to 15: Nucleotide sequences of primers and probes used in dPCR (Example 1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The inventors have discovered that fungal DNases, in particular NUC3 nuclease, are highly efficient at reducing or eliminating residual DNA in fermentation products.

[0017] The present invention provides methods for reducing the amount of residual DNA in a product, including a protein of interest, produced by fermentation of microbial host cells, particularly recombinant host cells, that express the protein of interest and either secrete it into the fermentation broth or accumulate it as an intracellular product. When the protein of interest is produced as an intracellular product, the microbial host cells may be homogenized prior to recovery and purification.

[0018] After fermentation and optional homogenization, the fermentation broth is subjected to a flocculation or precipitation step to provide a fermentation broth supernatant, which is effectively a solid-liquid separation that removes most of the (insoluble) host cells and cell debris, retaining an aqueous solution of the protein of interest and some soluble host cell components, such as residual host cell DNA.

[0019] Finally, the fermentation broth supernatant is filtered through a membrane filtration process to enhance the purity of the protein of interest and provide a liquid fermentation product. Membrane filtration can remove higher and / or lower molecular weight components depending on the type of membrane and filtration process. Some membrane filtration processes retain the filtrate (microfiltration) while others retain the retentate / permeate (ultrafiltration).

[0020] The method of the invention comprises the step of adding a fungal DNase or a variant thereof to the fermentation medium before or during fermentation, to the fermentation broth before flocculation / precipitation, to the fermentation broth supernatant before membrane filtration, or to the fermentation product after membrane filtration.

[0021] The inventors have discovered that it may be advantageous to apply fungal DNase to the fermentation product after membrane filtration to reduce the amount of water and other low molecular weight compounds (ultrafiltration), thereby increasing the concentration of the protein of interest and residual host cell DNA.

[0022] According to the present invention, when fungal DNase or a variant thereof is "added" or "applied", this excludes the (endogenous) production / expression of DNase by the microbial host cells in the fermentation broth. The fungal DNase is an isolated or recovered enzyme that is added or applied from an external source. Applying DNase from an external source is advantageous, because then the productive capacity of the microbial host cells is exclusively used to produce the protein of (commercial) interest.

[0023] DNA is considered to be removed when it is degraded to single nucleotides or oligonucleotides less than 150 bp as measured, for example, by digital PCR.

[0024] microbial host cell The microbial host cell can be of any genus. The desired protein of interest can be homologous or heterologous to the host cell capable of producing the protein of interest.

[0025] The term "homologous protein" or "native protein" refers to a protein that is encoded by a gene derived from the host cell in which it is produced.

[0026] The term "heterologous protein" means a protein encoded by a gene that is foreign to the host cell in which it is produced.

[0027] The term "recombinant host cell," as used herein, means a host cell that harbors a gene encoding a desired protein and is capable of expressing said gene to produce the desired protein. The gene encoding the desired protein may be transformed, transfected, transduced, etc. into the recombinant host cell using techniques well known in the art.

[0028] If the desired protein is a heterologous protein, the recombinant host cell capable of producing the desired protein is preferably of fungal or bacterial origin. The choice of recombinant host cell will largely depend on the gene encoding the desired protein and the source of said protein.

[0029] The term "wild-type host cell," as used herein, refers to a host cell that naturally harbors a gene encoding a desired protein and is capable of expressing said gene.

[0030] The mutant can be, for example, a wild-type host cell in which one or more genes have been deleted to enhance preparation of a desired protein.

[0031] In a preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium or a fungus.

[0032] Microbial host cells may be yeast cells, such as strains of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia. In another embodiment, the strain is a strain of Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0033] Microbial host cells include those of the genera Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, and the like. obolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Fusarium, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillia Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus,The fungal strain may be a filamentous fungal strain, such as a strain of the genera Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella or Xylaria.

[0034] In another embodiment, the strain is selected from the group consisting of Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, and the like. keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete Chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia phimetii, fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spedoniumspededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0035] In one embodiment, the fungal host cell is a strain selected from the group consisting of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium and Trichoderma.

[0036] In a more preferred embodiment, the filamentous fungal host cell is a host cell of the genera Trichoderma and Aspergillus, in particular Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viridel, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans ... nidulans, Aspergillus niger or Aspergillus oryzae, in particular a strain of Trichoderma reesei.

[0037] In another preferred embodiment, the recombinant or wild-type microbial host cell is a bacterium.

[0038] The recombinant host cell may contain a single copy or at least two copies, for example, three, four, five or more copies, of a polynucleotide of the invention.

[0039] The host cell can be any gram-positive or gram-negative bacteria, including but not limited to Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0040] Host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus The Bacillus cell may be any Bacillus cell, including a Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cell. In one embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0041] In one embodiment, the Bacillus cell is a Bacillus subtilis cell.

[0042] In another embodiment, the Bacillus cell is a Bacillus licheniformis cell.

[0043] For purposes of the present invention, the class / genus / species Bacillus is defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0044] A bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0045] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0046] Methods for introducing DNA into a prokaryotic host cell are well known in the art and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, where the DNA is introduced as a linear polynucleotide or as a circular polynucleotide. A person skilled in the art will be able to readily identify a suitable method for introducing DNA into a given prokaryotic cell, for example depending on the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294, Choi et al., 2006, J. Microbiol. Methods 64:391-397, and Donald et al., 2013, J. Bacteriol. 195(11):2612-2620.

[0047] Fungal DNase The fungal DNase used in the present invention is a deoxyribonuclease or variant thereof derived from a fungal microorganism. DNase is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. There are two main classifications based on the locus of activity: exonucleases digest nucleic acids from the ends; endonucleases act on the central region of the DNA molecule.

[0048] Examples of fungal DNases can be found, for example, in patent applications WO 2017 / 059802 and WO 2017 / 059801 (incorporated by reference), which disclose amino acid sequences encoding fungal DNases. Fungal DNases are wild-type DNases originating from fungal strains. A particularly preferred fungal DNase is Aspergillus oryzae DNase, shown as SEQ ID NO:2.

[0049] A variant of a fungal DNase may have greater than 60%, greater than 70%, greater than 80% or greater than 90% amino acid sequence identity to the wild-type fungal DNase.

[0050] In certain embodiments, the fungal DNase or variants thereof have more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% amino acid sequence identity to SEQ ID NO: 2. Alternatively, the fungal DNase or variants thereof have one, two, three, four or five amino acid substitutions, deletions or insertions, preferably one, two, three, four or five conservative amino acid substitutions, compared to SEQ ID NO: 2.

[0051] In a preferred embodiment, the fungal DNase or variant thereof is a NUC3 nuclease.

[0052] A subgroup of DNase_NucA_NucB (Pfam domain id PF14040, Pfam version 31.0 Finn (2016). Nucleic Acids Research, Database Issue 44:D279-D285) has been named NUC3. The NUC3 nuclease contains a DNase_NucA_NucB domain and includes one of the motifs [LV][PTA][FY][DE][VAGPH]D[CFY][WY][AT][IM]L[CYQ] corresponding to amino acids 24 to 35 of Aspergillus oryzae DNase having the amino acid sequence shown in SEQ ID NO:2, and / or GPYCK (SEQ ID NO:3) motifs corresponding to amino acids 157 to 161 of Aspergillus oryzae DNase having the amino acid sequence shown in SEQ ID NO:2, or WF[QE]IT (SEQ ID NO:4) corresponding to amino acids 146 to 150 of Aspergillus oryzae DNase having the amino acid sequence shown in SEQ ID NO:2.

[0053] Thus, the Aspergillus oryzae DNase shown as SEQ ID NO:2 is a NUC3 DNase.

[0054] The amino acid changes in DNase variants may be of a minor nature, being conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein, such as small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function such as a polyhistidine tract, an antigenic epitope or a binding module, as described above.

[0055] Essential amino acids in a polypeptide (protein, enzyme) can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule and the resulting molecules are tested for DNase activity to identify amino acid residues essential for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of the structure, as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of essential amino acids can also be inferred from alignment with related polypeptides and / or from sequence homology and conserved catalytic mechanism with related polypeptides or with polypeptides / proteins (typically having similar three-dimensional structure, function and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally or alternatively, protein structure prediction tools can be used to model protein structure to identify essential amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0056] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known methods of mutagenesis, recombination and / or shuffling followed by associated screening procedures (such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625). Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0057] Protein of interest Proteins of interest are produced by microbial host cells. Such proteins can be small (peptides; <50 amino acids) or large (polypeptide; >50 amino acids) biomolecules that perform a wide range of functions in living organisms, including catalyzing reactions, replicating DNA, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins are composed of chains of polymerized amino acids that are folded into very specific three-dimensional structures. The three-dimensional structure is essential to maintain the function of the protein. Some chemicals can alter the folding or even unravel (denaturing) the three-dimensional structure, resulting in loss of function, e.g., loss of enzymatic activity.

[0058] In one embodiment, the protein is a polypeptide, preferably a globular protein / polypeptide, hi another embodiment, the protein is soluble under physiological conditions.

[0059] Proteins are classified into at least four distinct groups: enzymes, cell signaling proteins, ligand-binding proteins and structural proteins.

[0060] In another embodiment, the at least one polypeptide of interest comprises a therapeutic polypeptide selected from the group consisting of an antibody, an antibody fragment, an antibody-based drug, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, a growth factor, a blood clotting factor, a hormone, an interferon (such as interferon alpha-2b), an interleukin, lactoferrin, alpha-lactalbumin, beta-lactalbumin, ovomucoid, ovostatin, a cytokine, obestatin, a human galactosidase (such as human alpha-galactosidase A), a vaccine, a protein vaccine, and a thrombolytic agent.

[0061] The enzymes are described below.

[0062] Cell signaling and ligand binding proteins include, for example, proteins such as receptors, membrane proteins, ion channels, antibodies (e.g., single domain antibodies), hormones, hemoglobin and hemoglobin-like molecules, etc. Heme-containing enzymes, such as peroxygenases and peroxidases, may contain enzymatic activity or may be inactivated variants of the heme-containing enzymes.

[0063] Structural proteins impart rigidity and stiffness to otherwise fluid biological components.

[0064] Preferably, the protein is an enzyme, a cell signaling protein or a ligand binding protein, more preferably the protein is an enzyme.

[0065] enzyme The protein of interest can be an enzyme (a catalytic protein). If the protein is an enzyme, the amount of protein is active enzyme protein.

[0066] The term "active enzyme protein" is defined herein as the amount of catalytic protein, which represents the enzyme activity. This can be determined using activity-based analytical enzyme assays. In such assays, the enzyme usually catalyzes a reaction that produces a colored compound. The amount of the colored compound can be measured and correlated to the concentration of active enzyme protein. This technique is well known in the art.

[0067] The enzyme may be, for example, one or more enzymes selected from the group consisting of hydrolases, lyases, transferases, proteases, amylases, glucoamylases, pectinases, pectate lyases, cellulases, xylanases, arabinases, arbinofuranosidases, mannanases, carrageenanases, xanthanases, endoglucanases, chitinases, asparaginases, lipases, phospholipases, cutinases, lysozymes, phytases, deamidases, transglutaminases, oxidoreductases (such as sugar oxidases, laccases, peroxidases, catalases), lactases, glucose isomerases, xylose isomerases, and esterases.

[0068] The enzymes may be naturally occurring enzymes of bacterial or fungal origin, or the enzymes may be variants derived from one or more naturally occurring enzymes by gene shuffling and / or by substitution, deletion or insertion of one or more amino acids, including chemically modified or protein engineered variants.

[0069] Fermentation Broth The present invention may be useful for any industrial scale fermentation, for example any fermentation having a culture medium of at least 50 liters, preferably at least 500 liters, more preferably at least 5,000 liters, even more preferably at least 50,000 liters.

[0070] The microorganism producing the protein of interest can be fermented by any method known in the art. The fermentation medium can be a minimal medium, for example as described in WO 98 / 37179, or the fermentation medium can be a complex medium comprising a complex nitrogen source and a carbon source, the complex nitrogen source may be partially hydrolyzed, as described in WO 2004 / 003216.

[0071] The fermentation may be carried out as a batch, repeated batch, fed-batch, repeated fed-batch or continuous fermentation process.

[0072] In a fed-batch process, before the start of fermentation, none or a portion of the compound containing one or more nutrients is added to the medium, and during the fermentation process, all or the remainder of the compound containing one or more nutrients, respectively, is fed. The compounds selected for feeding can be fed to the fermentation process together or separately.

[0073] In a repeated fed-batch or continuous fermentation process, complete starting medium is further fed during the fermentation. The starting medium can be fed together with or separately from the feed of structural elements. In a repeated fed-batch process, a portion of the fermentation broth containing the biomass is removed at regular time intervals, whereas in a continuous process, the removal of a portion of the fermentation broth occurs continuously. This allows the fermentation process to be replenished with a portion of fresh medium equivalent to the amount of fermentation broth removed.

[0074] In a preferred embodiment of the present invention, fermentation broth from a fed-batch fermentation process is preferred.

[0075] In one embodiment, the fermentation broth is fed after a culture time of at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

[0076] In certain embodiments, the fermentation broth is fed after a culture time of at least 120 hours.

[0077] According to the present invention, the fermentation broth may be diluted with water up to 2000% (w / w), preferably, the fermentation broth may be diluted with water 10-2000% (w / w), more preferably, the fermentation broth may be diluted with water 100-1500% (w / w), more preferably, the fermentation broth may be diluted with water 100-1000% (w / w), more preferably, the fermentation broth may be diluted with water 200-800% (w / w).

[0078] Dilution with water means, according to the present invention, that the dilution medium can be water, or the ultrafiltration permeate from the production of the protein of interest, or the water recycle from the production of the protein quality of interest, or the condensate from the heater, or any combination of the above, e.g. a mixture of water and ultrafiltration permeate.

[0079] The fermentation broth comprises host cells (including host cells containing a gene encoding a polypeptide of interest used to produce the polypeptide of interest), cell debris, biomass, recombinant DNA from bacterial host cells, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed cell whole broth containing organic acids, dead cells and / or cell debris, and culture medium.

[0080] For example, a fermentation broth is produced when a cultured microorganism is grown to saturation and incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by the host cells) and secretion into the cell culture medium. The fermentation broth may contain the unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, the fermentation broth is unfractionated and includes spent culture medium and cellular debris present after the microbial cells (e.g., Bacillus cells) have been removed, for example, by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0081] The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation material obtained at the end of fermentation. Typically, the cell-killed whole broth or cell composition contains spent culture medium and cell debris present after microbial cells (e.g., Bacillus cells) have been grown to saturation and incubated under carbon-limited conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains spent cell culture medium, recombinant DNA from microbial host cells, extracellular enzymes, and dead microbial cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and / or lysed using methods known in the art.

[0082] The whole broth or cell compositions described herein are typically liquid, but may contain insoluble components, such as dead cells, recombinant DNA from microbial host cells, cell debris, culture medium components, and / or insoluble enzymes, etc. In some embodiments, the insoluble components can be removed to obtain a clarified liquid composition.

[0083] The whole broth formulations and cell compositions of the invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0084] Flocculation / Sedimentation To flocculate the fermentation broth, divalent salts, in particular calcium and / or magnesium salts, such as calcium chloride or magnesium chloride, may be added to the fermentation broth. A preferred embodiment is a calcium salt, in particular calcium chloride.

[0085] The salt may be added to the fermentation broth at a concentration of 0.01-10% (w / w) / kg fermentation broth (undiluted), preferably 0.5-10% (w / w) / kg fermentation broth (undiluted), more preferably 1-9% (w / w) / kg fermentation broth (undiluted), and particularly 2-8% (w / w) / kg fermentation broth (undiluted).

[0086] Polyaluminum Compounds To further improve the removal of DNA from the fermentation broth, polyaluminium compounds may be added to the fermentation broth. Many aluminium compounds, such as Al2(SO4)3, NaAlO2, K2Al2O4, AlCl3, Al(NO3)3, Al acetate and Al formate, are known to improve flocculation.

[0087] A particularly useful polyaluminium chloride is of the formula Al, having the CAS number: 1327-41-9. n (OH) m Cl( 3n-m ) and polyaluminum chloride and chlorohydroxyaluminum compounds.

[0088] An example of a useful polyaluminum chloride is chlorohydroxyaluminum, GC850™ available from Gulbrandsen (Al(OH)Cl) or the empirical formula Al(OH) 1,2 Cl 1,8 NordPac18 (available from Nordisk Aluminat A / S, Denmark), which is an aluminum complex having the formula Al(OH) 1,2 Cl 1,8 Another example of a useful polyaluminum chloride having the formula Al(OH) is PAX-XL100 (available from Kemira). Two other examples of useful polyaluminum chloride are PAC (available from Shanghai Haotian Water Treatment Equipment Co., Ltd., supplied in solid form) or PAC (available from Tianjin Kairuite technology Ltd., supplied in liquid form). 1,2 Cl 1,8Another example of a useful polyaluminum chloride having the formula: is PAX18 (available from Kemira Water Solutions).

[0089] The concentration of polyaluminum chloride is usually in the range of 0.1 to 10% (w / w) calculated per kg fermentation broth (undiluted), preferably in the range of 0.5 to 5% (w / w) calculated per kg fermentation broth (undiluted).

[0090] After addition of the polyaluminum chloride, the pH can be adjusted. The pH can be adjusted to a pH in the range of pH 2 to pH 11. The pH can be adjusted with any acid or base as known in the art.

[0091] The polyaluminum chloride can also be added after the microorganisms have been separated from the fermentation broth.

[0092] Polyaluminum chloride can also be added in two or more steps, for example, before the microorganisms are removed from the fermentation broth and then again after the microorganisms have been removed and subsequently in the downstream processing liquor.

[0093] polymer Polymers can be used for particle aggregation. Anionic and cationic polymers are preferred. Useful cationic polymers can be polyamines, and useful anionic polymers can be polyacrylamides. The concentration of useful polymers is usually in the range of 0.5-20% (w / w) calculated per kg fermentation broth (undiluted), preferably in the range of 1-10% (w / w) calculated per kg fermentation broth (undiluted).

[0094] An example of a useful anionic polymer is Superfloc™ A130 (Kemira). Examples of useful cationic polymers are Polycat™ (Kemira), C521 (Kemira) and C591 (Kemira).

[0095] Filtration and other downstream operations The aggregated cell debris can be removed by methods known in the art, including but not limited to, filtration, such as drum filtration, membrane filtration, dead-end filter press filtration, cross-flow filtration or centrifugation.

[0096] The resulting fermentation supernatant can then be further processed or purified by methods known in the art, for example, the protein can be recovered by conventional procedures including, but not limited to, further filtration, such as ultrafiltration and diafiltration, extraction, spray drying, evaporation, precipitation or crystallization.

[0097] The term "recover" or "recovery" refers to the removal of the polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, for example, recovery of the polypeptide from the whole fermentation broth or from the cell-free fermentation broth, which is carried out by collecting the polypeptide from the broth medium by filtration, for example, depth filtration (by using filter aids or packed filters, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter or similar, with seed or pad filtration in a framework or modular setup) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration, either in cross-flow, dynamic cross-flow or dead-end operation), or by centrifugation (using decanter centrifuges, disc centrifuges, hydrocyclones or similar), or by precipitating the polypeptide and using particle size classification separation using suitable solid-liquid separation methods. Recovery encompasses isolation and / or purification of the polypeptide.

[0098] The isolated proteins may then be further purified and / or modified by a variety of procedures known in the art, including, but not limited to, chromatography, such as ion exchange, affinity, hydrophobicity, chromatofocusing and size exclusion, and / or electrophoretic procedures, such as preparative isoelectric focusing, and / or absorption rate differential solubility, such as ammonium sulfate precipitation, and / or extraction.

[0099] Microfiltration may require membranes having size exclusion limits of greater than 1000 kDa, greater than 500 kDa, greater than 100 kDa, or greater than 50 kDa, and / or greater than 5 μm, greater than 1 μm, greater than 0.5 μm, greater than 0.4 μm, greater than 0.3 μm, greater than 0.2 μm, or greater than 0.1 μm, or other filters with equivalent molecular weight exclusion characteristics, and the fermentation product is the filtrate of the microfiltration.

[0100] Following microfiltration, or in a combined process, the fermentation product may be subjected to an ultrafiltration step requiring a membrane with a size exclusion limit of greater than 100 kDa, greater than 80 kDa, greater than 60 kDa, greater than 50 kDa, greater than 40 kDa, greater than 30 kDa, greater than 20 kDa, greater than 15 kDa, greater than 10 kDa, greater than 5 kDa, or greater than 1 kDa, or another filter with equivalent molecular weight exclusion characteristics.

[0101] The microbial fermentation product may be, or may be used to prepare, a liquid or solid formulation.

[0102] The liquid formulation may contain a polyol (polyhydric alcohol), for example, in an amount of at least 10% w / w, at least 25% w / w, or at least 50% w / w. A polyol is an alcohol having two or more hydroxyl groups. Useful polyols typically have a molecular weight below 500 g / mol.

[0103] Polyols include non-sugar polyols such as glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), etc., as well as sugar alcohols such as sorbitol, mannitol, erythritol, dulcitol, inositol, xylitol, and adonitol, etc. Polyols also include sugar polyols, such as monosaccharides and disaccharides, such as glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

[0104] The solid formulation can be granulated, for example, prepared by high shear granulation or fluid bed granulation or a combination. Coatings with salts or polymers can also be applied.

[0105] Detection of residual host cell DNA The term residual host cell DNA includes genomic DNA from the production strain as well as fragments of DNA encoding the protein of interest.

[0106] Detection and quantification of trace amounts of residual host cell DNA can be achieved by various methods known in the art. Many methods have been developed to measure specific single target sequences. Examples of methods are as follows: (a) Hybridization-based methods to detect specific DNA of defined origin using dot blots and hybridization of radiolabeled DNA probes using random hexamers to generate representative probes covering the entire genome of the host cell; (b) A quantitative PCR-based method to detect specific DNA of defined origin, targeting specific gene sequences for amplification and calibration using purified, species-matched genomic DNA; and (c) A qualitative PCR method to detect specific DNA of defined origin, targeting specific gene sequences for amplification and calibration using purified, species-matched genomic DNA. A threshold is determined based on the minimum amount of genomic DNA that can be detected using this method.

[0107] According to the present invention, purification of minute amounts of DNA was achieved using FastDNA™ Spin Kit (MP Biomedicals). The eluted sample was then subjected to PCR reaction using primers specific for the host cell chromosomal locus. A positive control was included where a known amount of host DNA was added to the PCR reaction at different concentrations. After the PCR reaction, the sample was subjected to gel electrophoresis, and the intensity of the DNA bands was compared to estimate the concentration of host DNA in the original sample. The detailed protocol of PCR is described in lnnis et al. (1990) PCR Protocols, A Guide to methods and applications, Academic Press inc., NY.

[0108] Treatment of fermentation broth or other protein preparations using the present methods results in a significant reduction in the amount of DNA present in the fermentation broth, preferably reducing the DNA content to undetectable levels, which are considered undetectable when any segment of genomic DNA present in a single copy in a haploid genome is PCR amplified and subsequently stained with ethidium bromide reveals no visible bands.

[0109] Preferably, the DNA level in the fermentation broth is reduced to a level of less than 1 μg / ml, preferably less than 500 ng / ml, preferably less than 200 ng / ml, preferably less than 100 ng / ml, preferably less than 50 ng / ml, preferably less than 20 ng / ml, preferably less than 10 ng / ml, preferably less than 5 ng / ml, preferably less than 2 ng / ml, preferably less than 1 ng / ml, most preferably to a level of less than 500 pg / ml.

[0110] In one embodiment, the microbial fermentation product contains less than 10 ng / g, less than 5 ng / g, less than 1 ng / g, less than 0.5 ng / g, less than 0.1 ng / g, less than 0.05 ng / g, or less than 0.01 ng / g of recombinant DNA.

[0111] In a typical regulatory environment, a PCR-based assay with a detection limit of, for example, 1, 5, 10 or 20 ng / mL of an enzyme preparation can be used to confirm the absence of detectable DNA.

[0112] Additionally, the use of the improvised method in combination with conventional methods for extracting DNA from fermentation broths or other protein preparations is also contemplated.

[0113] Some further embodiments of the invention include the following.

[0114] Embodiment 1. (a) providing a fermentation broth comprising microbial host cells, recombinant DNA from the microbial host cells, and a protein of interest produced by the microbial host cells; (b) subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; (c) subjecting the fermentation broth supernatant to a membrane filtration step, the membrane having a size exclusion limit of less than 100 kDa or less than 1 μm, to provide a fermentation product. A method for reducing the amount of DNA in a microbial fermentation product, comprising: The method, wherein a fungal DNase or a variant thereof is added to the fermentation medium before or during fermentation, to the fermentation broth in or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c).

[0115] Embodiment 2. The method of embodiment 1, wherein a fungal DNase or a variant thereof is added to the fermentation medium before or during fermentation.

[0116] Embodiment 3. The method of embodiment 1, wherein a fungal DNase or a variant thereof is added to the fermentation broth in step (a).

[0117] Embodiment 4. The method of embodiment 1, wherein a fungal DNase or a variant thereof is added to the fermentation broth after step (a).

[0118] Embodiment 5. The method of embodiment 1, wherein a fungal DNase or a variant thereof is added to the fermentation broth after step (b).

[0119] Embodiment 6 The method of embodiment 1, wherein a fungal DNase or a variant thereof is added to the fermentation broth after step (c).

[0120] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the protein of interest is heterologous to the microbial host cell.

[0121] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the protein of interest is secreted by the microbial host cell into the fermentation broth.

[0122] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the protein of interest is not secreted into the fermentation broth by the microbial host cell.

[0123] Embodiment 10. The method according to any one of embodiments 1 to 9, wherein the fermentation broth of step (a) comprises the protein of interest in an amount of at least 0.1% w / w.

[0124] Embodiment 11. The method according to any one of embodiments 1 to 10, wherein the fermentation broth of step (a) comprises the protein of interest in an amount of at least 0.5% w / w.

[0125] Embodiment 12. The method according to any one of embodiments 1 to 11, wherein the fermentation broth of step (a) comprises the protein of interest in an amount of at least 1% w / w.

[0126] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the protein of interest is an enzyme, a heme-containing protein, a cell signaling protein, or a ligand-binding protein.

[0127] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the protein of interest is an enzyme.

[0128] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the membrane filtration of step (c) comprises a microfiltration step.

[0129] Embodiment 16. The method of embodiment 15, wherein the microfiltration membrane has a size exclusion limit of 0.1 μm to 10 μm.

[0130] Embodiment 17. The method of embodiment 16, wherein the microfiltration membrane has a size exclusion limit of 0.5 μm to 5 μm.

[0131] Embodiment 18. The method according to any one of embodiments 1 to 17, wherein the membrane filtration of step (c) comprises an ultrafiltration step.

[0132] Embodiment 19. The method of embodiment 18, wherein the ultrafiltration membrane has a size exclusion limit of less than 100 kDa.

[0133] Embodiment 20. The method of embodiment 19, wherein the ultrafiltration membrane has a size exclusion limit of less than 50 kDa.

[0134] Embodiment 21. The method of embodiment 20, wherein the ultrafiltration membrane has a size exclusion limit of less than 40 kDa.

[0135] Embodiment 22. The method of embodiment 21, wherein the ultrafiltration membrane has a size exclusion limit of less than 30 kDa.

[0136] Embodiment 23 The method of embodiment 22, wherein the ultrafiltration membrane has a size exclusion limit of less than 20 kDa.

[0137] Embodiment 24. The method of embodiment 23, wherein the ultrafiltration membrane has a size exclusion limit of less than 10 kDa.

[0138] Embodiment 25. The method of any one of embodiments 1 to 24, wherein some or all of the microbial host cells of step (a) have been mechanically or enzymatically disrupted / homogenized.

[0139] Embodiment 26. The method according to any one of embodiments 1 to 25, wherein the fungal DNase or variant thereof is a wild-type fungal DNase or a variant thereof having at least 60% amino acid sequence identity with the wild-type fungal DNase, and wherein the variant exhibits DNase activity.

[0140] Embodiment 27 The method of embodiment 25, wherein the variant has at least 70% amino acid sequence identity with the wild-type fungal DNase.

[0141] Embodiment 28 The method of embodiment 25, wherein the variant has at least 80% amino acid sequence identity with the wild-type fungal DNase.

[0142] Embodiment 29. The method of embodiment 25, wherein the variant has at least 90% amino acid sequence identity with the wild-type fungal DNase.

[0143] Embodiment 30 The method of embodiment 25, wherein the variant has at least 95% amino acid sequence identity with the wild-type fungal DNase.

[0144] Embodiment 31 The method of embodiment 25, wherein the variant has at least 96% amino acid sequence identity with the wild-type fungal DNase.

[0145] Embodiment 32 The method of embodiment 25, wherein the variant has at least 97% amino acid sequence identity with the wild-type fungal DNase.

[0146] Embodiment 33 The method of embodiment 25, wherein the variant has at least 98% amino acid sequence identity with the wild-type fungal DNase.

[0147] Embodiment 34 The method of embodiment 25, wherein the variant has at least 99% amino acid sequence identity with the wild-type fungal DNase.

[0148] Embodiment 35. The method according to any one of embodiments 1 to 34, wherein the fungal DNase or variant thereof is a wild-type fungal DNase or a variant thereof having one, two, three, four or five substitutions, deletions or insertions, preferably one, two, three, four or five conservative substitutions.

[0149] Embodiment 36. The method according to any one of embodiments 1 to 35, wherein the fungal DNase or variant thereof is a wild-type fungal DNase or a variant thereof having one, two, three, four or five conservative substitutions.

[0150] Embodiment 37. The method of any one of embodiments 25 to 35, wherein the wild-type fungal DNase has the amino acid sequence shown in SEQ ID NO:2.

[0151] Embodiment 38. The method of any one of embodiments 1 to 37, wherein the fungal DNase or a variant thereof is NUC3 nuclease.

[0152] Embodiment 39. The method according to any one of embodiments 1 to 38, wherein the fungal DNase or variant thereof comprises a DNase_NucA_NucB domain and further comprises the motifs [LV][PTA][FY][DE][VAGPH]D[CFY][WY][AT][IM]L[CYQ] and / or any of the motifs GPYCK (SEQ ID NO: 3) or WF[QE]IT (SEQ ID NO: 4).

[0153] Embodiment 40. The method according to any one of embodiments 1 to 39, wherein the fungal DNase or a variant thereof comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0154] Embodiment 41. The method of any one of embodiments 1 to 40, wherein the microbial host cell is a bacterial host cell, a fungal host cell or a yeast host cell, preferably the microbial host cell is a bacterial host cell.

[0155] Embodiment 42. The microbial host cell is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Fusarium, and the like. The method according to any one of the preceding embodiments, wherein the strain is selected from the group consisting of the genera Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium and Trichoderma.

[0156] Embodiment 43. The method of any one of embodiments 1 to 42, wherein the microbial host cell is a strain of the genus Pichia.

[0157] Embodiment 44. The method of any one of embodiments 1 to 43, wherein the microbial host cell is a strain of Pichia pastoris.

[0158] Embodiment 45. The method of any one of embodiments 1 to 44, wherein the microbial host cell is a strain selected from the group consisting of Bacillus, Streptomyces, Escherichia, Buttiauxella and Pseudomonas.

[0159] Embodiment 46. The method of any one of embodiments 1 to 45, wherein the microbial host cell is a strain of the genus Bacillus or Escherichia.

[0160] Embodiment 47. The method of any one of embodiments 1 to 46, wherein the microbial host cell is a Bacillus host cell, preferably a Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis host cell.

[0161] Embodiment 48. The method of any one of embodiments 1 to 47, wherein the microbial fermentation product contains less than 10 ng / g of recombinant DNA.

[0162] Embodiment 49. The method of any one of embodiments 1 to 48, wherein the microbial fermentation product contains less than 5 ng / g of recombinant DNA.

[0163] Embodiment 50. The method of any one of embodiments 1 to 49, wherein the microbial fermentation product contains less than 1 ng / g of recombinant DNA.

[0164] Embodiment 51. The method of any one of embodiments 1 to 50, wherein the microbial fermentation product contains less than 0.5 ng / g of recombinant DNA.

[0165] Embodiment 52. The method of any one of embodiments 1 to 51, wherein the microbial fermentation product contains less than 0.1 ng / g of recombinant DNA.

[0166] Embodiment 53. The method of any one of embodiments 1 to 52, wherein the microbial fermentation product contains less than 0.05 ng / g of recombinant DNA.

[0167] Embodiment 54. The method of any one of embodiments 1 to 53, wherein the microbial fermentation product contains less than 0.01 ng / g of recombinant DNA.

[0168] Embodiment 55. The method of any one of embodiments 1 to 54, wherein the microbial fermentation product comprises a fungal DNase or a variant thereof.

[0169] EMBODIMENT 56. (a) providing a fermentation broth comprising microbial host cells, recombinant DNA from the microbial host cells, and a protein of interest produced by the microbial host cells; (b) subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; (c) subjecting the fermentation broth supernatant to a membrane filtration step, the membrane having a size exclusion limit of less than 100 kDa or less than 1 μm, to provide a fermentation product. A method for reducing the amount of DNA in a microbial fermentation product, comprising: DNase is added to the fermentation medium before or during fermentation, to the fermentation broth in or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c); DNase has at least 80% amino acid sequence identity with SEQ ID NO:2, and A method wherein the microbial fermentation product contains less than 10 ng / g of recombinant DNA.

[0170] Embodiment 57. The method according to embodiment 56, wherein DNase is added to the fermentation medium before or during fermentation.

[0171] Embodiment 58. The method of embodiment 56, wherein DNase is added to the fermentation broth in step (a).

[0172] Embodiment 59. The method of embodiment 56, wherein DNase is added to the fermentation broth after step (a).

[0173] Embodiment 60. The method of embodiment 56, wherein DNase is added to the fermentation broth after step (b).

[0174] Embodiment 61 The method of embodiment 56, wherein DNase is added to the fermentation broth after step (c).

[0175] Embodiment 62. The method of any one of embodiments 1 to 61, wherein the protein of interest is heterologous to the microbial host cell.

[0176] Embodiment 63 The method of any one of embodiments 1 to 62, wherein the protein of interest is secreted by the microbial host cell into the fermentation broth.

[0177] Embodiment 64. The method of any one of embodiments 1 to 63, wherein the protein of interest is not secreted into the fermentation broth by the microbial host cell.

[0178] Embodiment 65. The method according to any one of embodiments 1 to 64, wherein the fermentation broth of step (a) contains the protein of interest in an amount of at least 0.1% w / w.

[0179] Embodiment 66. The method of any one of embodiments 1 to 65, wherein the fermentation broth of step (a) contains the protein of interest in an amount of at least 0.5% w / w.

[0180] Embodiment 67. The method according to any one of embodiments 1 to 66, wherein the fermentation broth of step (a) contains the protein of interest in an amount of at least 1% w / w.

[0181] Embodiment 68. The method of any one of embodiments 1 to 67, wherein the protein of interest is an enzyme, a heme-containing protein, a cell signaling protein, or a ligand-binding protein.

[0182] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the protein of interest is an enzyme.

[0183] Embodiment 70. The method of embodiment 69, wherein the membrane has a size exclusion limit of less than 50 kDa.

[0184] Embodiment 71. The method of embodiment 70, wherein the membrane has a size exclusion limit of less than 40 kDa.

[0185] Embodiment 72. The method of embodiment 71, wherein the membrane has a size exclusion limit of less than 30 kDa.

[0186] Embodiment 73. The method of embodiment 72, wherein the membrane has a size exclusion limit of less than 20 kDa.

[0187] Embodiment 74. The method of embodiment 73, wherein the membrane has a size exclusion limit of less than 10 kDa.

[0188] Embodiment 75. The method according to any one of embodiments 1 to 74, wherein some or all of the microbial host cells of step (a) have been mechanically or enzymatically disrupted / homogenized.

[0189] Embodiment 76. The method of any one of embodiments 1 to 75, wherein the DNase has at least 85% amino acid sequence identity with SEQ ID NO:2.

[0190] Embodiment 77. The method of any one of embodiments 1 to 76, wherein the DNase has at least 90% amino acid sequence identity with SEQ ID NO:2.

[0191] Embodiment 78. The method of any one of embodiments 1 to 77, wherein the DNase has at least 95% amino acid sequence identity with SEQ ID NO:2.

[0192] Embodiment 79. The method of any one of embodiments 1 to 78, wherein the DNase has at least 96% amino acid sequence identity with SEQ ID NO:2.

[0193] Embodiment 80. The method of any one of embodiments 1 to 79, wherein the DNase has at least 97% amino acid sequence identity with SEQ ID NO:2.

[0194] Embodiment 81. The method of any one of embodiments 1 to 80, wherein the DNase has at least 98% amino acid sequence identity with SEQ ID NO:2.

[0195] Embodiment 82. The method of any one of embodiments 1 to 81, wherein the DNase has at least 99% amino acid sequence identity with SEQ ID NO:2.

[0196] Embodiment 83. The method according to any one of embodiments 1 to 82, wherein the DNase has one, two, three, four or five substitutions, deletions or insertions, preferably one, two, three, four or five conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO:2.

[0197] Embodiment 84. The method of any one of embodiments 1 to 83, wherein the DNase is NUC3 nuclease.

[0198] Embodiment 85. The method of any one of embodiments 1 to 84, wherein the DNase comprises a DNase_NucA_NucB domain and further comprises the motifs [LV][PTA][FY][DE][VAGPH]D[CFY][WY][AT][IM]L[CYQ] and / or any of the motifs GPYCK (SEQ ID NO: 3) or WF[QE]IT (SEQ ID NO: 4).

[0199] Embodiment 86. The method of any one of embodiments 1 to 85, wherein the DNase has the amino acid sequence shown in SEQ ID NO:2.

[0200] Embodiment 87. The method of any one of embodiments 1 to 86, wherein the microbial host cell is a bacterial host cell, a fungal host cell or a yeast host cell.

[0201] Embodiment 88. The microbial host cell is selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Fusarium, and the like. The method according to any one of the preceding embodiments, wherein the strain is selected from the group consisting of Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium and Trichoderma.

[0202] Embodiment 89. The method of any one of embodiments 1 to 88, wherein the microbial host cell is a strain of the genus Pichia.

[0203] Embodiment 90. The method of any one of embodiments 1 to 89, wherein the microbial host cell is a strain of Pichia pastoris.

[0204] Embodiment 91 The method of any one of embodiments 1 to 90, wherein the microbial host cell is a bacterial host cell.

[0205] Embodiment 92. The method of any one of embodiments 1 to 91, wherein the microbial host cell is a strain selected from the group consisting of Bacillus, Streptomyces, Escherichia, Buttiauxella and Pseudomonas.

[0206] Embodiment 93. The method of any one of embodiments 1 to 92, wherein the microbial host cell is a strain of the genus Bacillus or Escherichia.

[0207] Embodiment 94. The method of any one of embodiments 1 to 93, wherein the microbial host cell is a Bacillus host cell, preferably a Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis host cell.

[0208] Embodiment 95. The method of any one of embodiments 1 to 94, wherein the microbial fermentation product contains less than 5 ng / g of recombinant DNA.

[0209] Embodiment 96. The method of any one of embodiments 1 to 95, wherein the microbial fermentation product contains less than 1 ng / g of recombinant DNA.

[0210] Embodiment 97. The method of any one of embodiments 1 to 96, wherein the microbial fermentation product contains less than 0.5 ng / g of recombinant DNA.

[0211] Embodiment 98. The method of any one of embodiments 1 to 97, wherein the microbial fermentation product contains less than 0.1 ng / g of recombinant DNA.

[0212] Embodiment 99. The method of any one of embodiments 1 to 98, wherein the microbial fermentation product contains less than 0.05 ng / g of recombinant DNA.

[0213] Embodiment 100. The method of any one of embodiments 1 to 99, wherein the microbial fermentation product contains less than 0.01 ng / g of recombinant DNA.

[0214] Embodiment 101. The method of any one of embodiments 1 to 100, wherein the microbial fermentation product comprises DNase.

[0215] EMBODIMENT 102. (a) providing a fermentation broth comprising a Bacillus host cell, recombinant DNA from the Bacillus host cell, and an enzyme produced by the Bacillus host cell; (b) subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; (c) subjecting the fermentation broth supernatant to a membrane filtration step, the membrane having a size exclusion limit of less than 100 kDa or less than 1 μm, to provide a fermentation product. A method for reducing the amount of DNA in a microbial fermentation product, comprising: DNase is added to the fermentation medium before or during fermentation, to the fermentation broth in or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c); DNase has at least 80% amino acid sequence identity with SEQ ID NO:2, and A method wherein the microbial fermentation product contains less than 10 ng / g of recombinant DNA.

[0216] Embodiment 103. The method according to embodiment 102, wherein DNase is added to the fermentation medium before or during fermentation.

[0217] Embodiment 104. The method of embodiment 102, wherein DNase is added to the fermentation broth in step (a).

[0218] Embodiment 105. The method of embodiment 102, wherein DNase is added to the fermentation broth after step (a).

[0219] Embodiment 106. The method of embodiment 102, wherein DNase is added to the fermentation broth after step (b).

[0220] Embodiment 107. The method of embodiment 102, wherein DNase is added to the fermentation broth after step (c).

[0221] Embodiment 108. The method of any one of embodiments 1 to 107, wherein the enzyme is heterologous to the Bacillus host cell.

[0222] Embodiment 109. The method of any one of embodiments 1 to 108, wherein the enzyme is secreted into the fermentation broth by the Bacillus host cell.

[0223] Embodiment 110. The method of any one of embodiments 1 to 109, wherein the fermentation broth of step (a) comprises the enzyme in an amount of at least 0.1% w / w.

[0224] Embodiment 111. The method according to any one of embodiments 1 to 110, wherein the fermentation broth of step (a) comprises the enzyme in an amount of at least 0.5% w / w.

[0225] Embodiment 112. The method according to any one of embodiments 1 to 111, wherein the fermentation broth of step (a) comprises the enzyme in an amount of at least 1% w / w.

[0226] Embodiment 113. The method of embodiment 112, wherein the membrane has a size exclusion limit of less than 50 kDa.

[0227] Embodiment 114. The method of embodiment 113, wherein the membrane has a size exclusion limit of less than 40 kDa.

[0228] Embodiment 115. The method of embodiment 114, wherein the membrane has a size exclusion limit of less than 30 kDa.

[0229] Embodiment 116. The method of embodiment 115, wherein the membrane has a size exclusion limit of less than 20 kDa.

[0230] Embodiment 117. The method of embodiment 116, wherein the membrane has a size exclusion limit of less than 10 kDa.

[0231] Embodiment 118. The method of any one of embodiments 1 to 117, wherein the DNase has at least 85% amino acid sequence identity with SEQ ID NO:2.

[0232] Embodiment 119. The method of any one of embodiments 1 to 118, wherein the DNase has at least 90% amino acid sequence identity with SEQ ID NO:2.

[0233] Embodiment 120. The method of any one of embodiments 1 to 119, wherein the DNase has at least 95% amino acid sequence identity with SEQ ID NO:2.

[0234] Embodiment 121. The method of any one of embodiments 1 to 120, wherein the DNase has at least 96% amino acid sequence identity with SEQ ID NO:2.

[0235] Embodiment 122. The method of any one of embodiments 1 to 121, wherein the DNase has at least 97% amino acid sequence identity with SEQ ID NO:2.

[0236] Embodiment 123. The method of any one of embodiments 1 to 122, wherein the DNase has at least 98% amino acid sequence identity with SEQ ID NO:2.

[0237] Embodiment 124. The method of any one of embodiments 1 to 123, wherein the DNase has at least 99% amino acid sequence identity with SEQ ID NO:2.

[0238] Embodiment 125. The method according to any one of embodiments 1 to 124, wherein the DNase has one, two, three, four or five substitutions, deletions or insertions, preferably one, two, three, four or five conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO:2.

[0239] Embodiment 126. The method of any one of embodiments 1 to 125, wherein the DNase is NUC3 nuclease.

[0240] Embodiment 127. The method according to any one of embodiments 1 to 126, wherein the DNase comprises a DNase_NucA_NucB domain and further comprises the motifs [LV][PTA][FY][DE][VAGPH]D[CFY][WY][AT][IM]L[CYQ] and / or any of the motifs GPYCK (SEQ ID NO: 3) or WF[QE]IT (SEQ ID NO: 4).

[0241] Embodiment 128. The method according to any one of embodiments 1 to 127, wherein the DNase has the amino acid sequence shown in SEQ ID NO:2.

[0242] Embodiment 129. The method of any one of embodiments 1 to 128, wherein the Bacillus host cell is a Bacillus amyloliquefaciens, Bacillus licheniformis or Bacillus subtilis host cell.

[0243] Embodiment 130. The method of any one of embodiments 1 to 129, wherein the microbial fermentation product contains less than 5 ng / g of recombinant DNA.

[0244] Embodiment 131. The method of any one of embodiments 1 to 130, wherein the microbial fermentation product contains less than 1 ng / g of recombinant DNA.

[0245] Embodiment 132. The method of any one of embodiments 1 to 131, wherein the microbial fermentation product contains less than 0.5 ng / g of recombinant DNA.

[0246] Embodiment 133. The method of any one of embodiments 1 to 132, wherein the microbial fermentation product contains less than 0.1 ng / g of recombinant DNA.

[0247] Embodiment 134. The method of any one of embodiments 1 to 133, wherein the microbial fermentation product contains less than 0.05 ng / g of recombinant DNA.

[0248] Embodiment 135. The method of any one of embodiments 1 to 134, wherein the microbial fermentation product contains less than 0.01 ng / g of recombinant DNA.

[0249] Embodiment 136. The method of any one of embodiments 1 to 135, wherein the microbial fermentation product comprises DNase.

[0250] Embodiment 137. A microbial fermentation product comprising less than 10 ng / g of recombinant DNA.

[0251] Embodiment 138. The microbial fermentation product of embodiment 137, comprising less than 5 ng / g of recombinant DNA.

[0252] Embodiment 139. The microbial fermentation product of any one of embodiments 1 to 138, comprising less than 1 ng / g of recombinant DNA.

[0253] Embodiment 14. The microbial fermentation product of any one of embodiments 1 to 139, comprising less than 0.5 ng / g of recombinant DNA.

[0254] Embodiment 141. The microbial fermentation product of any one of embodiments 1 to 140, comprising less than 0.1 ng / g of recombinant DNA.

[0255] Embodiment 142. The microbial fermentation product of any one of embodiments 1 to 141, comprising less than 0.05 ng / g of recombinant DNA.

[0256] Embodiment 143. A microbial fermentation product according to any one of embodiments 1 to 142, comprising less than 0.01 ng / g of recombinant DNA.

[0257] Embodiment 144. A microbial fermentation product according to any one of embodiments 1 to 143, further comprising a fungal DNase or a variant thereof.

[0258] Embodiment 145. The microbial fermentation product of embodiment 144, wherein the fungal DNase or variant thereof is NUC3 nuclease.

[0259] Embodiment 146. A microbial fermentation product according to any one of embodiments 1 to 145, comprising a protein of interest.

[0260] Embodiment 147. The microbial fermentation product of embodiment 146, wherein the protein of interest is a recombinant protein of interest.

[0261] Embodiment 148. A microbial fermentation product according to any one of embodiments 1 to 147, comprising at least 0.1% w / w of the protein of interest.

[0262] Embodiment 149. A microbial fermentation product according to any one of embodiments 1 to 148, comprising at least 0.5% w / w of the protein of interest.

[0263] Embodiment 150. A microbial fermentation product according to any one of embodiments 1 to 149, comprising at least 1% w / w of the protein of interest.

[0264] Embodiment 151. The microbial fermentation product of embodiment 150, wherein the protein of interest is an enzyme.

[0265] Embodiment 152. A microbial fermentation product according to any one of embodiments 1 to 151, produced by a method according to any one of embodiments 1 to 151.

[0266] Chemicals were of at least reagent grade.

[0267] DNase "Bacterial nuclease" is the Bacillus cibi DNase having the amino acid sequence set forth in SEQ ID NO:1.

[0268] "Fungal nuclease" is an Aspergillus oryzae DNase having the amino acid sequence set forth in SEQ ID NO:2.

[0269] Other DNases were also used for comparison, the donor strains being indicated in the corresponding examples.

[0270] Detection of gDNA and resDNA was performed by PCR and digital PCR (dPCR) assays as described in Example 1. The specific assays used will be described in the individual examples. EXAMPLES

[0271] Example 1 Detection of residual recombinant DNA The method used to demonstrate the absence of residual recombinant DNA in each sample can be divided into two steps: step i) extraction of DNA from the samples, including a lysis step, and step ii) detection of the recombinant DNA (target) by PCR amplification using either conventional PCR techniques followed by gel electrophoresis or digital PCR techniques.

[0272] DNA extraction including a lysis step The DNA extraction method was based on a commercial kit (Maxwell RSC PureFood GMO & Authentication kit, Promega). The standard proteinase K treatment was modified to allow efficient removal of proteins, thereby reducing the possibility of PCR inhibition by the sample matrix.

[0273] First, 200 μl of the sample was treated with proteinase K (QIAgen catalog number 19133) in CTAB buffer at a final concentration of 12.5 mg proteinase K / ml sample. The mixture was then incubated at 40° C. for 1 hour to ensure efficient removal of protein. DNA was then extracted using a Maxwell RSC instrument according to the procedure described in the commercial kit, and DNA was eluted in 55 μl of DNase-free water.

[0274] Detection of residual recombinant DNA using PCR amplification PCR amplification is a common method used for the detection of extremely small amounts of target DNA. In these experiments, both conventional PCR and digital PCR technologies were used. The difference between the two PCR methods is as follows: conventional PCR is a qualitative method in which the amplified PCR target is detected by agarose gel electrophoresis. On the other hand, digital PCR is a quantitative method based on TaqMan technology, which can quantify the amount of amplified PCR target.

[0275] The primers used for both techniques are specific for the recombinant DNA in question (Table 1) and amplify target fragments with a size less than 150 bp. For the digital PCR technique, which is based on TaqMan technology, sequence-specific oligonucleotides (probes) conjugated with fluorophores and quencher moieties are also required (Table 1). The design of primers, probes and amplicons was based on the basic requirements, using a web tool, as described by Rodriguez et al. in Chapter 3 of Design of primers and Probes for Quantitative Real-time PCR methods in Methods in Molecular Biology 1275, Springer Protocols (editor C. Basu).

[0276] [Table 1]

[0277] [Table 2]

[0278] [Table 3]

[0279] [Table 4]

[0280] [Table 5]

[0281] [Table 6]

[0282] Conventional PCR method PCR amplification was performed on 10 μL of extracted DNA using the appropriate primer set for a given target at a concentration of 400 nM for each primer. One tube of Illustra PureFood PCR Bead System from GF Healthcare (Cat. No. 27-9557-02) was used per reaction. PCR reactions were performed under the following thermocycler conditions:

[0283] [Table 7]

[0284] After PCR amplification, the PCR reactions were visualized on a 2.2% agarose gel (Lonza FlashGel system, Cat. No. 57031). FlashGel DNA markers 100bp-4kb (Lonza Cat. No. 50473) were used for band size estimation.

[0285] Digital PCR Digital PCR (dPCR) uses the procedure of end-point PCR, but divides the PCR reaction into many single compartments, in which the template is randomly distributed to all available compartments. After PCR, amplification is detected by measuring the fluorescence in all positive compartments. Using Poisson statistics, the average amount of target DNA per sample can be calculated. The number of filled compartments is determined by a reference fluorescent dye present in the reaction mixture. Absolute quantification of the amount of target DNA in each sample can then be calculated.

[0286] For these experiments, the QIAGEN digital system QIAcuity ONE was used with the QIAcuity Probe PCR kit (cat. no. 250101). The kit contains a 4x concentrated ready-to-use Master mix optimized for use with the QIAcuity nanoplate (cat. no. 25001) which distributes each sample into 26,000 compartments. The procedure described by the supplier was optimized for primer and probe concentrations and for PCR cycle conditions. For these experiments, primer and probe concentrations of 800 nM, respectively, were used.

[0287] To ensure uniform distribution of the template across the QIAcuity Nanoplate, the extracted DNA should be fragmented by restriction digestion prior to sectioning. For these experiments, the restriction enzyme EcoRI was chosen at a concentration of 0.25 U / 40 μl reaction. This restriction enzyme was chosen because it does not cut within the target fragment. For each dPCR reaction, 5 μl of template DNA obtained using DNase-free water was used in a total reaction volume of 40 μl.

[0288] The mixture was then transferred to a nanoplate, sealed, and incubated at room temperature for a minimum of 10 minutes to allow the restriction enzymes to act.

[0289] Immediately afterwards, the nanoplate was run under the following thermal cycle conditions:

[0290] [Table 8]

[0291] The results of the dPCR analysis are given as copy number of target PCR fragment / μl of total reaction volume. From this value, the amount of ng recombinant DNA / g sample in starting material can be calculated based on the following assumptions: · 100% efficient DNA purification is obtained. The average genome weight of Bacillus is 4.2 × 10 6 It is predicted to be a base pair (see JT Revors, 1996, Genome size in bacteria. Antonie van Leeuwenhocek 69(4):293-303). · The molecular weight of a base pair is 650 daltons. 1 Dalton is 1.6605 x 10 -15 Equivalent to ng. The weight of the Bacillus genome is 4.5 × 10 -6 It is estimated to be ng.

[0292] The content of residual recombinant DNA in the original sample can then be calculated using the following formula: ngDNA / ml sample = 0.04 x Df x D where Df is the dilution factor of the DNA preparation before dPCR analysis and D is the result of the dPCR analysis expressed as copy number / μl in a total reaction volume of 40 μl.

[0293] Example 2 Digestion of genomic DNA in distilled water using bacterial nuclease from Bacillus cibi and fungal nuclease from Aspergillus oryzae Experimental procedure To understand nuclease degradation of purified genomic Bacillus licheniformis DNA (gDNA) in a simple, "clean" matrix, all samples in Example 2 were prepared in distilled water (DW). To assess gDNA degradation, two positive control samples were prepared in DW with a total volume of 300 μL / sample. The control samples were spiked with 1000 ng / g gDNA at pH 9. A total of 5 mM MgCl2 was added to one of the two control samples. Another positive control sample was prepared in DW and spiked with 10 ng / g gDNA. gDNA degradation using bacterial nucleases was investigated in a dose-response fashion, with final concentrations ranging from 0.0005 g / L to 0.5 g / L in a factor of 10 concentration increase. Additionally, MgCl2 (cofactor) was added to a final concentration of 5 mM to a spare sample with a final concentration of 0.005 g / L. A final concentration of 0.5 g / L was used for the fungal nuclease. The total reaction volume was 300 μL at pH 9 and the samples were incubated at 37° C. for 24 hours with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. The ability of the nuclease to degrade gDNA was assessed by conventional PCR analysis using the primers shown in Table 4.

[0294] result Both bacterial and fungal nucleases were able to degrade gDNA in DW down to <10 ng / g at all concentrations tested, regardless of whether additional MgCl2 was added (Table 7).

[0295] [Table 9]

[0296] Example 3 Degradation of DNA in enzyme concentrates using bacterial and fungal nucleases. Experimental procedure All samples in Example 3 were prepared using enzyme concentrates recovered from Bacillus licheniformis fermentation broth (FB). The enzyme concentrates had residual DNA (resDNA) concentrations of >>10ng / g. Residual DNA is the amount of recombinant DNA derived from B. licheniformis host cells. resDNA degradation using bacterial nucleases was investigated in a dose-response manner, with final concentrations ranging from 0.0005g / L to 0.5g / L in a factor of 10 concentration increase. Additionally, MgCl2 (cofactor) was added to a final concentration of 5mM to a reserve sample with a final concentration of 0.005g / L. A final concentration of 0.5g / L was used for the fungal nuclease. The total reaction volume was 400μL at pH 6.5 and samples were incubated at 37°C for 24 hours with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. Two positive control samples were used: enzyme concentrate and DW spiked with 10 ng / g gDNA. resDNA degradation was assessed by conventional PCR analysis using the primers shown in Table 3.

[0297] result The fungal nuclease was able to degrade bacterial host cell resDNA down to <<10 ng / g in the enzyme concentrate. The bacterial nuclease was unable to degrade bacterial host cell resDNA to significant levels at any of the concentrations tested, regardless of whether additional MgCl2 was added (Table 8).

[0298] [Table 10]

[0299] Example 4 Degradation of DNA in enzyme concentrates using bacterial and fungal nucleases at various pH values. Experimental procedure In Example 3, the fungal nuclease was the only nuclease effective in degrading resDNA, so the test was repeated at various pHs in enzyme concentrates recovered from Bacillus licheniformis FB. The enzyme concentrates had a resDNA concentration of >10 ng / g. A final concentration of 0.5 g / L was used for both nucleases. The total reaction volume was 400 μL at pH 4, 5, 6, 7, and 8 for the fungal nuclease and at pH 4, 6, and 8 for the bacterial nuclease, and the samples were incubated at 37°C for 24 hours with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. The enzyme concentrate without any added nuclease was used as a positive control sample. The resDNA degradation was evaluated by conventional PCR analysis using the primers shown in Table 1.

[0300] result The fungal nuclease was able to degrade bacterial host cell resDNA in the enzyme concentrate to <<10 ng / g at all pHs investigated, but was most efficient (<<<10 ng / g) at pH 4. The bacterial nuclease was unable to degrade significant levels of bacterial host cell resDNA in the enzyme concentrate at any of the pHs tested, although a slight effect was observed at pH 4 (Table 9).

[0301] [Table 11]

[0302] Example 5 Degradation of DNA in fermentation broth and flocculated fermentation broth using fungal nucleases at various pH levels. Experimental section All samples in Example 5 were prepared using FB or flocculated fermentation broth (fFB) derived from Bacillus licheniformis.

[0303] Preparation of fermentation broth samples Fungal nuclease was added directly to the FB at pH 7.5 + / - 0.5 to achieve a final nuclease concentration of 0.5 g / L. FB without any added fungal nuclease was used as a positive control sample. Samples were incubated at 37°C for 24 hours with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 2.

[0304] Preparation of fFB samples To generate fFB, FB was treated as follows. 1. Dilute with water 2. Add divalent salt 3. Add polyaluminium chloride (PAC) 4. Adjust the pH to 4, 6 or 8. 5. Add fungal nuclease to achieve a final concentration of 0.5 g / L 6. Incubate at 37°C for 24 hours with gentle agitation.

[0305] A positive control sample was generated similarly, except for step 5. After the incubation period, the nuclease reaction was stopped by freezing the samples. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 2.

[0306] result The fungal nuclease significantly degraded bacterial host cell resDNA compared to the control samples, regardless of whether it was added to the FB or the fFB. Furthermore, the efficiency of the nuclease was similar at all pHs tested (Table 10).

[0307] [Table 12]

[0308] Example 6 DNA degradation during recovery of enzymes from fermentation broth. Experimental section All samples in Example 6 are derived from different streams during the recovery of enzyme from Bacillus licheniformis FB.

[0309] Preparation of FBs Before starting the fermentation, fungal nuclease was added to the tank to achieve a final nuclease concentration of 0.5 g / L + / - 0.1. The pH during fermentation and harvest was 7.5 + / - 0.5. FB without any fungal nuclease added was used as a positive control batch. FB was collected and incubated at 5°C for 22-24 hours before harvesting was started. After the incubation period, the nuclease reaction was stopped by freezing the samples. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 2.

[0310] Collection Process To generate fFB, FB was treated as follows. 1. Dilute with water 2. Add divalent salt 3. Add polyaluminium chloride (PAC) 4. Adjust pH to 7.5+ / -0.5 5. Add cationic polymer 6. Add anionic polymer 7. The fFB was centrifuged to separate the liquid phase (supernatant) from the biomass. 8. Filter the supernatant through a filter with a cutoff size of 5.0 to 0.3 μm to generate a filtrate. 9. The filtrate was concentrated using ultrafiltration (UF) with a membrane with a cut-off size of 10 kDa to produce a UF-concentrate.

[0311] result The fungal nuclease significantly degraded bacterial host cell resDNA in the FB, in the supernatant and in the UF-concentrate of the fFB compared to the control FB (Table 11).

[0312] [Table 13]

[0313] Example 7 Degradation of DNA in enzyme concentrates using DNases from various organisms Experimental procedure All samples in Example 7 were prepared using enzyme concentrate with a resDNA concentration of >>10ng / g, recovered from B. licheniformis FB. resDNA degradation using nucleases from different organisms was investigated at a final nuclease concentration of 0.5g / L. The enzyme concentrate had a pH of 5.9 and the samples were incubated at 25°C for 1 hour with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. The enzyme concentrate was used as a positive control sample. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 5.

[0314] result The fungal nuclease (from A. oryzae) degraded bacterial host cell resDNA to <<1 ng / g in the enzyme concentrate, but none of the other nucleases from various organisms tested were able to degrade resDNA to <10 ng / g (Table 12).

[0315] [Table 14]

[0316] Example 8 Degradation of DNA in enzyme concentrates using DNases from various organisms Experimental procedure All samples in Example 8 were prepared using enzyme concentrates with resDNA concentrations >10ng / g, recovered from Bacillus licheniformis FB. resDNA degradation using nucleases from different organisms was investigated at a final nuclease concentration of 0.5g / L. The enzyme concentrates had a pH of 5.0 and the samples were incubated at 25°C for 1 hour with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. The enzyme concentrates were used as positive control samples. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 2.

[0317] result The fungal nuclease (from A. oryzae) degraded bacterial host cell resDNA to <<<1ng / g in the enzyme concentrate. The bacterial nuclease and nucleases from Neosartorya massa, Arthrographis sp. 07MA20, Rhizoctonia solani and Morchella costata degraded resDNA to <10ng / g. Nucleases from Pyrenochaetopsis sp., Arthrinium arundinis, Cladosporium cladosporioides, Penicillium quercetorum, Phialophora geniculate, and Acremonium chrysogenum were unable to degrade resDNA to <10 ng / g (Table 13).

[0318] [Table 15]

[0319] Example 9 Degradation of DNA in enzyme concentrates using DNases from various organisms Experimental procedure All samples in Example 9 were prepared using enzyme concentrate with a resDNA concentration of >>>10ng / g, recovered from Bacillus subtilis FB. resDNA degradation using nucleases from different organisms was investigated at a final nuclease concentration of 0.5g / L. The enzyme concentrate had a pH of 6.2 and the samples were incubated at 25°C for 1 hour with gentle agitation. After the incubation period, the nuclease reaction was stopped by freezing the samples. The enzyme concentrate was used as a positive control sample. resDNA degradation was assessed by dPCR analysis using the primers / probes shown in Table 6.

[0320] result The fungal nuclease (from A. oryzae) degraded bacterial host cell resDNA to <<1 ng / g in the enzyme concentrate. The bacterial nuclease and the nucleases from Neosartorya massa, Pyrenochaetopsis sp., Arthrinium arundinis and Phialophora geniculata were unable to degrade resDNA to <10 ng / g (Table 14).

[0321] [Table 16]

Claims

1. (a) providing a fermentation broth comprising microbial host cells, recombinant DNA derived from the microbial host cells, and a protein of interest produced by the microbial host cells; (b) subjecting the fermentation broth to a flocculation or precipitation step to provide a fermentation broth supernatant; (c) subjecting the fermentation broth supernatant to a membrane filtration step, the membrane having a size exclusion limit of less than 100 kDa or less than 1 μm, to provide a fermentation product; 1. A method for reducing the amount of DNA in a microbial fermentation product, comprising: The method, wherein a fungal DNase or a variant thereof is added to the fermentation medium before or during fermentation, to the fermentation broth in or after step (a), to the fermentation broth supernatant after step (b), or to the fermentation product after step (c).

2. The method of claim 1 , wherein the protein of interest is heterologous to the microbial host cell.

3. 10. The method of claim 1, wherein the fermentation broth of step (a) comprises the protein of interest in an amount of at least 0.1% w / w.

4. 2. The method of claim 1, wherein the protein of interest is an enzyme, a heme-containing protein, a cell signaling protein, or a ligand-binding protein, preferably an enzyme.

5. 10. The method of claim 1, wherein the membrane filtration of step (c) comprises a microfiltration step.

6. 10. The method of claim 1, wherein the membrane filtration of step (c) comprises an ultrafiltration step.

7. 10. The method of claim 1, wherein some or all of the microbial host cells of step (a) are mechanically or enzymatically disrupted / homogenized.

8. 2. The method of claim 1, wherein the fungal DNase or variant thereof has at least 90% amino acid sequence identity to SEQ ID NO: 2, and the variant exhibits DNase activity.

9. 2. The method of claim 1, wherein the fungal DNase or variant thereof comprises a DNase_NucA_NucB domain and further comprises the motif [LV][PTA][FY][DE][VAGPH][CFY][WY][AT][IM][CYQ] and / or any of the motifs GPYCK (SEQ ID NO: 3) or WF[QE]IT (SEQ ID NO: 4).

10. The microbial host cells are selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Fusarium, and the like.

2. The method of claim 1, wherein the fungus is a strain selected from the group consisting of the genera Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma.

11. 2. The method of claim 1, wherein the microbial host cell is a strain selected from the group consisting of bacterial host cells.

12. 10. The method of claim 1, wherein the microbial fermentation product contains less than 10 ng / g of recombinant DNA.

13. A microbial fermentation product containing less than 10 ng / g of recombinant DNA.

14. 14. The microbial fermentation product of claim 13, further comprising a fungal DNase or a variant thereof.

15. 15. The microbial fermentation product of claim 13 or 14, further comprising a protein of interest.

16. 16. The microbial fermentation product of claim 15, wherein the protein of interest is a recombinant protein of interest.

17. 14. The microbial fermentation product according to claim 13, produced by the method according to any one of claims 1 to 12.