Recombinant proteases for cell detachment

Abstract

The present invention relates to compositions suitable for cell detachment, comprising a polypeptide having protease activity. The invention further relates to polypeptides having protease activity, polynucleotides encoding said polypeptides, nucleic acid constructs and expression vectors comprising said polynucleotides, recombinant host cells comprising said nucleic acid constructs or expression vectors, methods for producing said polypeptides, granules comprising said polypeptides, fermentation broth formulations comprising said polypeptides, and methods and uses utilizing said polypeptides.

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 compositions suitable for cell detachment, comprising a polypeptide having protease activity. The invention further relates to polypeptides having protease activity, polynucleotides encoding said polypeptides, nucleic acid constructs and expression vectors comprising said polynucleotides, recombinant host cells comprising said nucleic acid constructs or expression vectors, methods for producing said polypeptides, granules comprising said polypeptides, fermentation broth formulations comprising said polypeptides, and methods and uses utilizing said polypeptides. [Background technology]

[0003] Cell detachment is an important step during the passaging of cells when grown as adherent cells as well as cell clusters. The detachment step preferably involves the use of proteolytic enzymes, as they are gentle yet effective in not only releasing cells from the surface to which they adhere, but also lysing cell clusters formed in suspension cultures.

[0004] Accutase® and Accumax® (both available, for example, from Innovative Cell Technologies, Inc.) are commercially available products for cell detachment that contain a mixture of enzymes with proteolytic and collagenolytic activity isolated from invertebrate sources. A problem associated with these products is that regulatory agencies generally do not allow the use of animal-derived products in drug development and production processes, preventing their application in the development and production of cell-based therapies. Another problem with these products is that, as a result of the products being isolated from animal sources, there is an inherent risk of batch-to-batch variability in terms of composition and activity.

[0005] TrypLE™ (available, for example, from ThermoFisher Scientific) is a commercially available trypsin product that can be used for cell detachment. TrypLE™ is recombinantly produced and is therefore not of animal origin. However, a problem associated with TrypLE™ is that not all cell types are adequately detached when treated with trypsin alone, which limits the broad applicability of this product.

[0006] It is an object of the present invention to provide an enzyme solution that addresses the shortcomings associated with current products for cell detachment. In particular, it is an object of the present invention to provide an enzyme solution that meets regulatory requirements, is useful for detaching many different cell types, and can be produced in a uniform manner without batch-to-batch variation. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a recombinantly expressed alternative to animal-derived enzyme products currently used in cell detachment processes. The inventors analyzed Accutase® products and identified a single protease (SEQ ID NO: 1) as a major contributor to cell detachment. SEQ ID NO: 1 has an increased P1 preference for the amino acid residues Leu, Tyr, and Phe. Without being bound by theory, it is speculated that the P1 preference profile exhibited by SEQ ID NO: 1 results in effective yet gentle cleavage of cell surface proteins involved in surface attachment and cell-cell adhesion.

[0008] In a first aspect, the present invention relates to a composition suitable for cell detachment, comprising a polypeptide having protease activity and having at least 70% sequence identity to SEQ ID NO: 1, wherein said polypeptide is present in an amount of at least 10% by weight of the total polypeptide material of the composition.

[0009] In a second aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:1.

[0010] In a third aspect, the present invention relates to a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:1 operably linked to a heterologous promoter.

[0011] In a fourth aspect, the present invention relates to a recombinant host cell comprising in its genome a nucleic acid construct or an expression vector of the third aspect.

[0012] In a fifth aspect, the present invention relates to a method for producing a polypeptide having at least 70% sequence identity to SEQ ID NO:1 and having protease activity, comprising: (a) culturing a recombinant host cell according to the fourth aspect under conditions conducive to expression of the polypeptide; and optionally (b) recovering the polypeptide.

[0013] In a sixth aspect, the present invention relates to a fermentation broth formulation comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 and having protease activity.

[0014] In a seventh aspect, the present invention relates to a method for cell detachment comprising contacting a cell with a composition according to the first aspect, wherein the cell is attached to a surface and / or another cell.

[0015] In an eighth aspect, the present invention relates to the use of the composition of the first aspect in a cell exfoliation process. [Brief explanation of the drawings]

[0016] [Figure 1]SDS-PAGE analysis of type IV collagen degradation. Lane 1: Protein ladder. Lane 2: 0.44 μg / mL desalted Accutase with type IV collagen substrate. Lane 3: 0.1 μg / mL desalted Accutase with type IV collagen substrate. Lane 4: Desalted Accutase without substrate. Lane 5: Type IV collagen substrate only.

[0017] [Figure 2] A schematic overview of the hPSC setup is shown, indicating the time points (circled) at which hPSC monolayer detachment and assessment of cluster formation were performed.

[0018] [Figure 3] A schematic overview of the hPSC setup is shown, indicating the time points (circled) at which hPSC cluster dissociation and repopulation assessment were performed. DETAILED DESCRIPTION OF THE INVENTION

[0019] Array Overview SEQ ID NO: 1 is the S1 protease isolated from the Accutase® product.

[0020] SEQ ID NO:2 is the codon-optimized DNA sequence gene encoding the S1 protease of SEQ ID NO:1.

[0021] SEQ ID NO: 3 is the β-glucosidase secretion signal from Aspergillus aculeatus.

[0022] definition cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as a spliced ​​mature mRNA.

[0023] Cell detachment: The term "cell detachment" refers to the process of detaching or releasing smaller groups of cells, or even single cells, from cell culture, particularly 2D and 3D cell culture. 2D cell culture includes adherent cell culture, in which cells are grown as a monolayer attached to the surface of a cell culture vessel (e.g., a culture flask or a Petri dish), and the cells adhere to each other and / or the surface of the cell culture vessel. 3D cell culture includes suspension culture, in which cells are grown as cell clusters suspended in agitated growth medium, and the cells adhere to each other. 3D cell culture also includes enriched medium culture (e.g., agarose culture or Matrigel culture), and scaffold culture, in which cells are grown on a structural scaffold. The terms "cell detachment" and "cell dissociation" are used interchangeably herein.

[0024] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which generally begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0025] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from different genes) to the polynucleotide encoding a polypeptide, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include a promoter, and transcription and translation stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of a polynucleotide encoding a polypeptide.

[0026] Expression: The term "expression" refers to any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0027] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, the coding sequence operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0028] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide, wherein the fragment has protease activity. In one aspect, the fragment has chymotrypsin activity. In one aspect, the fragment has type I collagenase activity. In one aspect, the fragment has type IV collagenase activity.

[0029] Heterologous: The term "heterologous" with respect to a host cell means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that the regulatory sequences of the polypeptide or nucleic acid, e.g., the promoter, are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0030] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of the invention has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from cells.

[0031] Isolated: The term "isolated" refers to a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including, but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell, or other material is therefore in a form that is not found in nature. Isolated polypeptides include, but are not limited to, culture broths containing the polypeptide expressed and secreted in a host cell.

[0032] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0033] Nucleic Acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode specific amino acid sequences. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0034] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that is otherwise modified to contain a segment of nucleic acid in a non-naturally occurring manner, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0035] Operably linked: The term "operably linked" means that the specified components are in a relationship (including, but not limited to, juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0036] Passaging: The term "passaging" refers to the process of removing some or all of the cells from a culture and transferring them to fresh growth medium. Passaging of cells may also be called subculturing. In some embodiments, passaging results in a single cell suspension.

[0037] Protease: The term "protease" refers to a polypeptide having protease activity (EC 3.4; also known as peptidase activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group contains several subgroups, including EC 3.4.21 (serine endopeptidases), which further contain several subgroups, including EC 3.4.21.62 (subtilisins). The terms "protease" and "polypeptide having protease activity" are used interchangeably herein.

[0038] For purposes of the present invention, protease activity (EC3.4) may be determined according to the protease activity assay described in the Examples herein.

[0039] For purposes of the present invention, trypsin activity (EC 3.4.21.4) may be determined according to the trypsin activity assay described in the Examples herein.

[0040] For purposes of the present invention, chymotrypsin activity (EC 3.4.21.1) may be determined according to the chymotrypsin activity assay described in the Examples herein.

[0041] For purposes of the present invention, type I collagenase activity may be determined according to the type I collagenase activity assay described in the Examples herein.

[0042] For purposes of the present invention, type IV collagenase activity may be determined according to the type IV collagenase activity assay described in the Examples herein.

[0043] Purified: The term "purified" refers to a nucleic acid, polypeptide (e.g., protease), or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more pure (e.g., on a weight percent or molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is substantially increased after applying a purification or concentration procedure. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0044] In one aspect, the term "purified," as used herein, refers to a polypeptide (e.g., a protease) or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a polypeptide that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which the polypeptide is obtained. In one aspect, a polypeptide has been separated from a portion of the soluble components of the organism and culture medium from which the polypeptide is recovered. A polypeptide can be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0045] Thus, a polypeptide (e.g., a protease) can be purified so that only trace amounts of other proteins (e.g., other polypeptides) are present. The term "purified," as used herein, can refer to the removal of other components, particularly other proteins, most particularly other enzymes, present in the cell from which the polypeptide originated. A polypeptide can also be "substantially pure," i.e., free from other components from the organism that produces the polypeptide (e.g., the host organism in the case of a recombinantly produced polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in a preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in a preparation (e.g., a composition suitable for cell detachment). As used herein, a "substantially pure polypeptide" can mean a polypeptide preparation that contains up to 10%, preferably up to 9%, preferably up to 8%, preferably up to 7%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, more preferably up to 2%, more preferably up to 1%, more preferably up to 0.5, more preferably up to 0.1%, more preferably up to 0.05%, more preferably up to 0.01%, even more preferably up to 0.005%, and most preferably up to 0.001%, by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0046] Thus, a substantially pure polypeptide (e.g., a protease) is preferably at least 90% pure, preferably at least 91%, more preferably at least 92% pure, more preferably at least 93% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, more preferably at least 99% pure, more preferably at least 99.5% pure, more preferably at least 99.9% pure, more preferably at least 99.95%, more preferably at least 99.99% pure, even more preferably at least 99.995% pure, and most preferably at least 99.999% pure, by weight of total polypeptide material present in a preparation (e.g., a composition suitable for cell detachment). Polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide using well-known recombinant or classical purification methods.

[0047] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form a group different from that found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been altered from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions different from those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0048] Recover: The terms "recover" and "recovery" refer to removing a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, for example, by collecting polypeptide crystals, by chromatography, by filtration, such as depth filtration (using filter aids or packed filter media, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filters, candle filters, horizontal leaf filters, or similar, with sheet or pad filtration in a framework or modular setup) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross-flow, dynamic cross-flow, or dead-end operation), or by centrifugation (using decanter centrifuges, disc centrifuges, hydrocyclones, or similar), or by using particle size fractionation to precipitate proteins and collect the polypeptide from the broth medium, and using related solid-liquid separation methods. Recovery encompasses isolation and / or purification of the polypeptide.

[0049] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0050] For purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), 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), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The -nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output, labeled "longest identity," is calculated as follows: (Identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0051] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program in the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The Needle output, labeled "longest identity," is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0052] Detailed Description of the Invention The present invention provides a recombinantly expressed alternative to animal-derived enzyme products currently used in cell detachment processes. The inventors analyzed Accutase® products and identified a single protease (SEQ ID NO: 1) as the primary contributor to cell detachment. SEQ ID NO: 1 has an increased P1 preference for the amino acid residues Leu, Tyr, and Phe. Without being bound by theory, it is speculated that the P1 preference profile exhibited by SEQ ID NO: 1 results in effective yet gentle cleavage of cell surface proteins involved in surface attachment and cell-cell adhesion. As can be seen from the examples herein, the protease of SEQ ID NO: 1 exhibits improved cell detachment compared to Accutase®. The inventors then successfully constructed recombinant host cells capable of expressing SEQ ID NO: 1, thereby enabling the bioengineered production of the key active component of Accutase® that complies with the regulatory constraints imposed on clinical development and pharmaceutical production of cell therapies.

[0053] Polypeptides The present invention relates to polypeptides having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1.

[0054] In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0055] In one embodiment, the polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids. Preferably, the N-terminal and / or C-terminal extension is 1 to 25 amino acids, such as 1 to 20, 1 to 15, 1 to 10, or 1 to 5 amino acids.

[0056] In one embodiment, the polypeptide is a fragment containing at least 200 amino acid residues, at least 210 amino acid residues, or at least 220 amino acid residues of SEQ ID NO:1.

[0057] In one embodiment, the polypeptide has increased P1 selectivity for Leu, Tyr, and Phe. Preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position. Preferably, P1 preference is determined according to Example 2 herein.

[0058] In one embodiment, the polypeptide has trypsin activity. Trypsin activity (EC 3.4.21.4) can be determined according to the trypsin activity assay described in the Examples herein.

[0059] In one embodiment, the polypeptide has chymotrypsin activity. Chymotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0060] In one embodiment, the polypeptide has type I collagenase activity. Type I collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0061] In one embodiment, the polypeptide has type IV collagenase activity. Type IV collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0062] In another aspect, the polypeptide is derived from SEQ ID NO: 1 by substituting, deleting, or adding one or several amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 1 comprising substitutions, deletions, and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO: 1 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, e.g., an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding module.

[0063] Important amino acids in a polypeptide can be identified using art-known procedures 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 to identify amino acid residues critical to the molecule's activity and / or specificity, and the resulting molecules are tested for protease activity and / or P1 preference (see also Hilton et al., 1996, J. Biol. Chem. 271;4699-4708). The active site of a polypeptide can also be determined by physical analysis of its structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. 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. Additionally, the identification of important amino acids can be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or within a polypeptide or protein family, including polypeptides / proteins typically derived from a common ancestor with similar three-dimensional structure, function, and significant sequence similarity. Additionally, or alternatively, protein structure prediction tools can be used for protein structure modeling to identify important 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.

[0064] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods 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, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0065] To detect the activity of cloned, mutagenized polypeptides expressed by host cells, mutagenesis / shuffling methods can be combined with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and easily sequenced using standard methods in the art. These methods allow for the rapid determination of individual important amino acid residues within a polypeptide.

[0066] In one aspect, the polypeptide is isolated.

[0067] In another embodiment, the polypeptide is purified.

[0068] Compositions suitable for cell detachment The present invention also relates to a composition suitable for cell detachment comprising a polypeptide of the present invention, wherein the polypeptide is present in an amount of at least 10%, e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more by weight of the total polypeptide material of the composition.

[0069] In one embodiment, the polypeptide of the invention is present in an amount of at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material of the composition.

[0070] In one embodiment, the polypeptide is present in an amount of at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material present in the composition.

[0071] In preferred embodiments, the polypeptide is present in an amount of at least 99%, e.g., at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material present in the composition.

[0072] In more preferred embodiments, the polypeptide is present in an amount of at least 99.9%, such as at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material present in the composition.

[0073] In a most preferred embodiment, the polypeptide is present in an amount of at least 99.99%, such as at least 99.995%, at least 99.999%, or more, by weight of the total polypeptide material present in the composition.

[0074] In alternative embodiments, the polypeptide is present in an amount of at least 10%, e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more by weight of the total polypeptide material of the composition.

[0075] In one embodiment, the polypeptide has increased P1 selectivity for Leu, Tyr, and Phe. Preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position. Preferably, P1 preference is determined according to Example 2 herein.

[0076] In one embodiment, the polypeptide has trypsin activity. Trypsin activity (EC 3.4.21.4) can be determined according to the trypsin activity assay described in the Examples herein.

[0077] In one embodiment, the polypeptide has chymotrypsin activity. Chymotrypsin activity (EC 3.4.21.1) can be determined according to the chymotrypsin activity assay described in the Examples herein.

[0078] In one embodiment, the polypeptide has type I collagenase activity. Type I collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0079] In one embodiment, the polypeptide has type IV collagenase activity. Type IV collagenase activity can be determined according to the type I collagenase activity assay described in the Examples herein.

[0080] In one aspect, the composition suitable for cell detachment is a liquid composition. Preferably, the composition is an aqueous composition to ensure compatibility with media commonly used in cell culture. In some embodiments, the liquid composition is lyophilized. In another aspect, the composition is a solid composition, preferably a lyophilized composition.

[0081] To ensure that the liquid composition has a pH value compatible with cell culture conditions, the composition may contain an aqueous buffer solution. The composition may contain 1 to 99% by weight of the aqueous buffer solution, for example, 5 to 95%, 10 to 90%, 15 to 85%, 20 to 80%, or 25 to 75%. Alternatively, the composition may contain at least 5% by weight of the aqueous buffer solution, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% or more.

[0082] In some embodiments, the liquid composition has a pH value of about 5 to about 9, e.g., pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5, or pH 9. More preferably, the composition has a pH value of about 7 to about 8, e.g., pH 7, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, or pH 8. Even more preferably, the composition has a pH value of about 7 to about 7.5, e.g., pH 7.1, pH 7.2, pH 7.3, pH 7.4, or pH 7.5. Most preferably, the composition has a pH value of about 7.4.

[0083] In some embodiments, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate. Preferably, the aqueous buffer is a HEPES buffer, a TRIS buffer, or a phosphate (e.g., PBS) buffer.

[0084] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.1 μg / ml to about 100 μg / ml, e.g., about 0.5 μg / ml to about 50 μg / ml, about 1 μg / ml to about 20 μg / ml, or about 1 μg / ml to about 10 μg / ml.

[0085] In some embodiments, the liquid composition comprises a concentration of about 0.1 μg / ml to about 20 μg / ml, e.g., about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, The polypeptide of the present invention may be present in an amount of about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml, or about 20 μg / ml.

[0086] In some embodiments, the liquid composition comprises a polypeptide of the present invention in an amount of about 0.5 μg / ml to about 5 μg / ml, e.g., about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, or about 4 μg / ml, or about 5 μg / ml.

[0087] In some embodiments, the liquid composition comprises a polypeptide of the present invention in an amount of about 1 μg / ml to about 10 μg / ml, e.g., about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml.

[0088] In some embodiments, the liquid composition comprises a polypeptide of the present invention in an amount of about 1 μg / ml to about 20 μg / ml, e.g., about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml, or about 20 μg / ml.

[0089] In a preferred embodiment, the liquid composition comprises the polypeptide of the present invention in an amount of 1 μg / ml to 20 μg / ml, for example, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, or 20 μg / ml, more preferably 1 μg / ml to 10 μg / ml, and most preferably 1 μg / ml to 5 μg / ml.

[0090] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.1 mg / ml to about 100 mg / ml, e.g., about 0.5 mg / ml to about 50 mg / ml, about 1 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0091] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.1 mg / ml to about 20 mg / ml, e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, or about 20 mg / ml.

[0092] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 0.5 mg / ml to about 5 mg / ml, e.g., about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, or about 4 mg / ml, or about 5 mg / ml.

[0093] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 1 mg / ml to about 10 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml.

[0094] In some embodiments, the liquid composition comprises a polypeptide of the invention in an amount of about 1 mg / ml to about 20 mg / ml, e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, or about 20 mg / ml.

[0095] In a preferred embodiment, the liquid composition comprises the polypeptide of the invention in an amount of 1 mg / ml to 20 mg / ml, for example, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 μg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml, more preferably 1 mg / ml to 10 mg / ml, and most preferably 1 mg / ml to 5 mg / ml.

[0096] In some embodiments, the liquid composition includes ethylenediaminetetraacetic acid (EDTA). Preferably, the liquid composition includes EDTA in an amount of about 0.01 mM to about 100 mM, e.g., about 0.05 mM to about 50 mM, about 0.1 mM to about 10 mM, or about 0.5 mM to about 5 mM. Preferably, the liquid composition includes EDTA in an amount of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 0.95 mM, about 1 mM, about 1.5 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. More preferably, the liquid composition comprises EDTA in an amount of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 0.95 mM, about 1 mM, about 1.5 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. Most preferably, the liquid composition comprises EDTA in an amount of about 1 mM.

[0097] In some embodiments, the liquid composition comprises magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ In some embodiments, the liquid composition is substantially free of magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ In some embodiments, the liquid composition does not contain magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) is not included.

[0098] In some embodiments, the liquid composition comprises a phosphate buffer (e.g., PBS), EDTA, and magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free.

[0099] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 8, preferably about 7 to about 7.5, and most preferably about pH 7.4; the liquid composition further comprises EDTA in an amount of about 0.1 mM to about 10 mM, preferably about 0.5 mM to about 5 mM, and most preferably about 1 mM; and the liquid composition contains magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free.

[0100] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 7.5, most preferably about pH 7.4; the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; and the liquid composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) is not included.

[0101] In a preferred embodiment, the liquid composition comprises a phosphate buffer solution (e.g., PBS) having a pH value of about 7 to about 7.5, most preferably about pH 7.4; the liquid composition further comprises EDTA in an amount of about 0.5 mM to about 5 mM, most preferably about 1 mM; and the liquid composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) does not contain: The composition contains SEQ ID NO: 1 in an amount of 0.1 μg / ml to 20 μg / ml.

[0102] In a preferred embodiment, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH value of about pH 7.4; the composition further comprises EDTA in an amount of about 1 mM; and the composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca2+ ) is not included.

[0103] In a preferred embodiment, the liquid composition comprises a phosphate buffer (e.g., PBS) having a pH value of about pH 7.4; the composition further comprises EDTA in an amount of about 1 mM; and the composition contains magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ ) does not contain: The composition contains SEQ ID NO: 1 in an amount of 1 μg / ml to 20 μg / ml.

[0104] The liquid composition may further comprise an enzyme stabilizer (examples include a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid).

[0105] In some embodiments, a filler or carrier material is included to increase the volume of the liquid composition. Suitable fillers and carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the composition contains from about 5% to about 90% of such materials.

[0106] In one aspect, the liquid composition comprises 20-80% w / w of a polyol. In one embodiment, the liquid composition comprises 0.001-2% w / w of a preservative.

[0107] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of a polypeptide of the present invention (e.g., SEQ ID NO: 1); (b) 20-80% w / w polyol; (c) optionally 0.001 to 2% w / w of a preservative; and (d) water The present invention relates to a liquid composition comprising:

[0108] In another embodiment, the present invention provides (a) 0.001 to 25% w / w of a polypeptide of the present invention (e.g., SEQ ID NO: 1); (b) 0.001-2% w / w of a preservative; (c) optionally 20 to 80% w / w of a polyol; and (d) water The present invention relates to a liquid composition comprising:

[0109] In another embodiment, the liquid composition comprises one or more additives selected from the group consisting of polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetates, and phosphates, preferably sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0110] In one embodiment, the liquid composition comprises glucose in an amount of about 0.1 g / L to about 10 g / L, e.g., about 0.1 g / L, about 0.2 g / L, about 0.3 g / L, about 0.4 g / L, about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L. In a preferred embodiment, the liquid composition comprises glucose in an amount of about 0.5 g / L to about 5 g / L, most preferably about 1 g / L.

[0111] In another embodiment, the liquid composition comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol. In one embodiment, the liquid formulation comprises 20% to 80% polyol, e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0112] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid composition comprises 0.02-1.5% w / w of the preservative, e.g., 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% w / w of the preservative, 0.05-1% w / w of the preservative, or 0.1-0.5% w / w of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0113] In one aspect, the composition further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, DNase, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0114] In a preferred embodiment, the composition further comprises DNase.

[0115] Polynucleotides The present invention also relates to polynucleotides encoding the polypeptides of the present invention. The polynucleotides may be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0116] The polynucleotide may also be mutated by introducing nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but that correspond to the codon usage of the host organism in which the enzyme is intended to be produced, or that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, e.g., Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0117] In one aspect, the polynucleotide is isolated.

[0118] In another embodiment, the polynucleotide is purified.

[0119] Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a polynucleotide of the invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0120] Polynucleotides may be manipulated in a variety of ways to bring about expression of a polypeptide. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0121] promoter The control sequence may be a promoter, which is a polynucleotide recognized by a host cell to direct expression of a polynucleotide encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in a host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0122] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY; Davis et al., 2012, supra; and Song et al., 2016, PLOS One 11(7):e0158447.

[0123] Examples of promoters suitable for directing transcription of the polynucleotides of the invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0124] Examples of promoters useful for expression in yeast hosts are described by Smolke et al., 2018, "Synthetic Biology: Parts, Devices and Applications" (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications", Fungal Biology.

[0125] Terminator The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator functional in the host cell may be used in the present invention.

[0126] Preferred terminators for bacterial host cells may be obtained from the genes for alkaline protease of Bacillus clausii (aprH), alpha-amylase of Bacillus licheniformis (amyL), and ribosomal RNA of Escherichia coli (rrnB).

[0127] Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as the terminators described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology, e.g., Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I.

[0128] Preferred terminators for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other terminators useful for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0129] mRNA stabilizers A regulatory sequence may also be an mRNA stabilizing region downstream of the promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0130] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the SP82 gene of Bacillus subtilis (Hue et al., 1995, J. Bacteriol. 177;3465-3471).

[0131] Examples of mRNA stabilization regions in fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824, and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0132] Leader sequence The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5' terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0133] Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12):3051-3059, and Kaberdin and Blaesi, 2006, FEMS Microbiol. Rev. 30(6):967-979.

[0134] Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0135] Suitable leaders for yeast host cells may be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0136] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a polynucleotide and, upon transcription, is recognized as a signal by a host cell and adds polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0137] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0138] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0139] signal peptide The control sequence may also be a signal peptide coding region linked to the N-terminus of a polypeptide, encoding a signal peptide that directs the polypeptide into the secretory pathway of the cell. The 5' end of the coding sequence of a polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence heterologous to the coding sequence. A heterologous signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a heterologous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of the host cell may be used.

[0140] Effective signal peptide coding sequences for bacterial host cells include those obtained from the genes for maltogenic amylase from Bacillus NCIB 11837, subtilisin from Bacillus licheniformis, β-lactamase from Bacillus licheniformis, α-amylase from Bacillus stearothermophilus, neutral protease from Bacillus stearothermophilus (nprT, nprS, nprM), and prsA from Bacillus subtilis. Additional signal peptides have been described by Freudl, 2018, Microbial Cell Factories 17:52.

[0141] Effective signal peptide coding sequences for filamentous fungal host cells include those obtained from genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as those described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0142] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0143] Propeptide The regulatory sequence may also be a propeptide coding sequence encoding a propeptide located at the N-terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or, in some cases, a zymogen). Propolypeptides are generally inactive and can be converted to active polypeptides by catalytic or autocatalytic cleavage of the propeptide. Propeptide coding sequences may be obtained from the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, laccase of Myceliophthora thermophila (WO 95 / 33836), aspartic proteinase of Rhizomucor miehei, and α-factor of Saccharomyces cerevisiae.

[0144] When both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence. Additionally, or alternatively, when both a signal peptide sequence and a propeptide sequence are present, the polypeptide may include only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may include a mixture of mature polypeptides and polypeptides that include either partial or full-length propeptide and / or signal peptide sequences.

[0145] Regulatory sequences It may also be desirable to add regulatory sequences that regulate polypeptide expression relative to the growth of the host cell. Examples of regulatory sequences include those that activate or inactivate gene expression in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory sequences for prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences include those that allow for gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase, which is amplified in the presence of methotrexate, and metallothionein genes, which are amplified with heavy metals.

[0146] transcription factors A regulatory sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor that regulates the transcription of the gene encoding the protein of interest, for example, by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23, and also in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0147] Expression vector The present invention also relates to recombinant expression vectors comprising the polynucleotides of the present invention, promoters, and transcriptional and translational stop signals. Various nucleotide and control sequences may be ligated together to create recombinant expression vectors that may contain one or more convenient restriction sites, allowing for the insertion or substitution of a polynucleotide encoding a polypeptide at such sites. Alternatively, a polynucleotide may be expressed by inserting a nucleic acid construct containing the polynucleotide into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0148] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0149] The vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal element independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be such that, upon introduction into a host cell, it is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA or transposon to be introduced into the genome of the host cell, may be used.

[0150] Vectors preferably contain one or more selectable markers which permit easy selection of transformed, transfected, transduced cells, etc. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0151] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0152] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0153] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to autonomously replicate in the intended host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication and functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0154] Two or more copies of a polynucleotide of the present invention may be inserted into a host cell to increase production of the polypeptide. For example, two, three, four, five, or more copies are inserted into the host cell. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide; cells containing an amplified copy of the selectable marker gene, and thus cells containing additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selection agent.

[0155] Recombinant host cells The present invention also relates to recombinant host cells containing a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0156] Thus, in one aspect, the recombinant host cell comprises a polynucleotide that encodes a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the recombinant host cell comprises a polynucleotide that encodes a polynucleotide that comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0157] A construct or vector containing a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The choice of host cell will largely depend on the gene encoding the polypeptide and its source. The polypeptide may be native or heterologous to the recombinant host cell. Also, at least one of the one or more control sequences may be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may contain a single copy of a polynucleotide of the invention, or at least two copies, e.g., three, four, five or more copies.

[0158] The host cell can be any microbial cell, such as a prokaryotic or fungal cell, useful for the recombinant production of the polypeptides of the invention.

[0159] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, 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.

[0160] Prokaryotic host cells include 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, but not limited to, a Bacillus pumilus, a Bacillus stearothermophilus, a Bacillus subtilis, and a Bacillus thuringiensis cell. In one embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, a Bacillus licheniformis, or a Bacillus subtilis cell.

[0161] In a preferred embodiment, the recombinant host cell is a Bacillus licheniformis cell.

[0162] In a preferred embodiment, the recombinant host cell is a Bacillus subtilis cell.

[0163] For purposes of the present invention, the class / genus / species of Bacillus shall be defined as set forth in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0164] Bacterial host cells may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0165] A bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0166] Methods for introducing DNA into prokaryotic host cells 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, and transduction, where the DNA is introduced as a linear or circular polynucleotide. Those skilled in the art will be able to easily 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.

[0167] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0168] Fungal cells may be transformed by processes including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic methods, and shock wave-mediated transformation, as reviewed in Li et al., 2017, Microbial Cell Factories 16:168, and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, Christensen et al., 1988, Bio / Technology 6:1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27:53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and the DNA may be introduced as a linear or circular polynucleotide.

[0169] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). For purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0170] Yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, and the like. The yeast host cell may be a Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0171] In a preferred embodiment, the recombinant host cell is a Pichia pastoris (Komagataella phaffii) cell.

[0172] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivisions Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0173] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neomyces, and others. The filamentous fungal host cell may be a Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell. In a more preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0174] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestrisThe cell may be a Trichoderma terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0175] In a preferred embodiment, the recombinant host cell is an Aspergillus niger cell.

[0176] In a preferred embodiment, the recombinant host cell is an Aspergillus oryzae cell.

[0177] In a preferred embodiment, the recombinant host cell is a Trichoderma reesei cell.

[0178] In one aspect, the recombinant host cell is isolated.

[0179] In another embodiment, the recombinant host cell is purified.

[0180] Production method The present invention also relates to a method for producing a polypeptide of the invention, comprising: (a) culturing a host cell, the wild-type of which produces a polypeptide having protease activity and at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, under conditions conducive to production of the polypeptide; and, optionally, (b) recovering the polypeptide. In a preferred embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0181] The present invention also relates to a method for producing a polypeptide of the invention, comprising: (a) culturing a recombinant host cell of the invention under conditions conducive to the production of a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; and, optionally, (b) recovering the polypeptide. In a preferred embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0182] In one aspect, the recombinant host cell is a prokaryotic host cell, preferably a bacterial cell. In one embodiment, the recombinant host cell is a Bacillus cell, preferably a 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 In a preferred embodiment, the recombinant host cell is a Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cell. In a preferred embodiment, the recombinant host cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, or Bacillus subtilis cell.

[0183] In a preferred embodiment, the recombinant host cell is a Bacillus licheniformis cell.

[0184] In a preferred embodiment, the recombinant host cell is a Bacillus subtilis cell.

[0185] In one aspect, the recombinant host cell is a filamentous fungal host cell. In one embodiment, the recombinant host cell is selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis girvescens, and the like. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutushirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambusinum sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersoniiemersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride cells. In a preferred embodiment, the recombinant host cell is an Aspergillus niger, Aspergillus oryzae, or Trichoderma reesei cell. In a preferred embodiment, the recombinant host cell is an Aspergillus niger cell.

[0186] In a preferred embodiment, the recombinant host cell is an Aspergillus niger cell.

[0187] In a preferred embodiment, the recombinant host cell is an Aspergillus oryzae cell.

[0188] In a preferred embodiment, the recombinant host cell is a Trichoderma reesei cell.

[0189] In one aspect, the recombinant host cell is a yeast host cell. In one embodiment, the recombinant host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, or a Saccharomyces lactis or Saccharomyces carlsbergensis cell. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0190] In a preferred embodiment, the recombinant host cell is a Pichia pastoris (Komagataella phaffii) cell.

[0191] The host cells or recombinant host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells can be cultured in shake flask cultures or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation) in laboratory or industrial fermentors in a suitable medium and under conditions that allow for expression and / or isolation of the polypeptide. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., compositions published in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0192] Polypeptides can be detected using methods known in the art that are specific for the polypeptides, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or assays that determine the relative or specific activity of the polypeptide.

[0193] The polypeptide may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, lyophilization, evaporation, or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0194] Polypeptides may be purified by various procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0195] In an alternative embodiment, the polypeptide is not recovered.

[0196] Protease Granules The present invention also relates to enzyme granules / particles comprising a polypeptide of the invention, i.e., a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0197] In one embodiment, the granules comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0198] The core may have a diameter, measured as an equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm. The core diameter, measured as an equivalent spherical diameter, can be determined using laser diffraction, for example, using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0199] In one embodiment, the core comprises a polypeptide of the invention.

[0200] The core may contain additional materials such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0201] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0202] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components, typically as a homogeneous blend.

[0203] The core may comprise inert particles into which the polypeptide has been absorbed or to which the polypeptide has been applied, for example, by fluidized bed coating.

[0204] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm.

[0205] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during handling, or to color the granules. Optional coatings include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA).

[0206] The coating may be applied in an amount of at least 0.1%, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15% by weight of the core, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0207] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm, or at least 5 μm thick. In some embodiments, the coating thickness is less than 100 μm, for example less than 60 μm, or less than 40 μm.

[0208] The coating must encapsulate the core unit by forming a substantially continuous layer. By substantially continuous layer, it is to be understood that the coating is substantially free of holes, so that there are few uncoated areas of the core unit. The layer or coating must, in particular, be uniform in thickness.

[0209] The coating may further contain other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0210] The salt coating can comprise at least 60% by weight salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0211] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm, or at least 8 μm, hi certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm.

[0212] The salt may be added from a salt solution in which the salt is completely dissolved, or from a salt suspension in which the particles are less than 50 μm, for example less than 10 μm or less than 5 μm.

[0213] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water-soluble, in particular having a solubility of at least 0.1 g in 100 g of water at 20° C., preferably at least 0.5 g per 100 g of water, for example at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0214] The salts may be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (having less than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal salts of sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates, or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0215] The salt in the coating may have a constant humidity of more than 60%, in particular more than 70%, 80% or more than 85% at 20° C., or may be another hydrated form of such a salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0216] Specific examples of suitable salts are NaCl (CH20°C = 76%), Na2CO3 (CH20°C = 92%), NaNO3 (CH20°C = 73%), Na2HPO4 (CH20°C = 95%), Na3PO4 (CH20°C = 92%), NH4Cl (CH20°C = 79.5%), (NH4)2HPO4 (CH20°C = 93.0%), NH4H2PO4 (CH20°C = 93.1%), (NH4)2SO4 (CH20°C = 81.1%). %), KCl (CH20°C = 85%), KHPO4 (CH20°C = 92%), KHPO4 (CH20°C = 96.5%), KNO3 (CH20°C = 93.5%), NaSO4 (CH20°C = 93%), KSO4 (CH20°C = 98%), KHSO4 (CH20°C = 86%), MgSO4 (CH20°C = 90%), ZnSO4 (CH20°C = 90%), and sodium citrate (CH25°C = 86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0217] The salts may be in anhydrous form or may be hydrated salts, i.e., crystalline salt hydrates containing bound water of crystallization, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO·7H2O), zinc sulfate heptahydrate (ZnSO·7H2O), sodium phosphate dibasic heptahydrate (NaHPO·7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0218] Preferably, the salt is added as a solution of the salt, for example using a fluidized bed.

[0219] The coating material can be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0220] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0221] The cores can be prepared by granulating a blend of ingredients by methods including granulation techniques such as, for example, crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0222] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include following known feed and granule formulation techniques. (a) Spray-dried products, in which a liquid polypeptide-containing solution is atomized in a spray-drying tower to form droplets that are dried as they pass through the tower to form a polypeptide-containing particulate material. Microparticles can be produced in this manner (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (b) Layered products, in which a polypeptide is coated as a layer around preformed inert core particles, typically by atomizing a polypeptide-containing solution in a fluidized bed apparatus in which the preformed core particles are fluidized, allowing the polypeptide-containing solution to adhere to the core particles and dry completely, leaving a layer of polypeptide on the surface of the core particles. If useful core particles of the desired particle size can be found, particles of the desired particle size can be obtained in this manner. Products of this type are described, for example, in WO 97 / 23606. (c) Absorbed core particles, in which the polypeptide is absorbed onto and / or into the core rather than coated as a layer around the core. Such a process is described in WO 97 / 39116. (d) Extruded or pelletized products, in which a polypeptide-containing paste is pressed into pellets or extruded under pressure through small orifices and cut into particles, which are then dried. Such particles are usually quite large because the material from which the extrusion orifices are made (usually a perforated plate) limits the allowable pressure drop across the extrusion orifices. Also, when small orifices are used, very high extrusion pressures increase the generation of heat within the polypeptide paste, which is detrimental to the polypeptide (Michael S. Showell (editor); Powdered Detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). (e) Prilled products, in which a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed into a cooling chamber, for example by a rotating disk atomizer, causing the droplets to rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product has the polypeptide uniformly distributed throughout the inert material, rather than concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technique. (f) Mixer-granulated products, in which a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. When the liquid and powder are mixed in the appropriate ratio and the moisture from the liquid is absorbed by the dry powder, the dry powder components adhere and begin to aggregate, building particles to form granules containing the polypeptide. Such processes are described in U.S. Pat. No. 4,106,991, European Patent Nos. 170360, 304332, and 304331, and International Publication Nos. 90 / 09440 and 90 / 09428. In certain embodiments of this process, various high-shear mixers can be used as granulators. Granules consisting of polypeptide, fillers, and binders are mixed with cellulose fibers to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust. (g) Size reduction, in which cores are produced by grinding or crushing larger particles, pellets, tablets, briquettes, etc., containing the polypeptide. The desired core particle fraction is obtained by sieving the ground or crushed product. The large and small particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons. (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere to each other, forming granules. (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other methods known in the feed or enzyme industry for drying granules may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25 to 90°C. For some polypeptides, it is important that the polypeptide-containing cores contain a small amount of water before coating with salt. If water-sensitive polypeptides are coated with salt before removing excess water, the excess water may become trapped within the core, which may adversely affect the activity of the polypeptide. After drying, the cores preferably contain 0.1 to 10% w / w water.

[0223] Non-dusting granules may be produced, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art.

[0224] The granules may further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. The more granules in which each enzyme is present, the more uniform the enzyme distribution will be, and the less physical separation of different enzymes due to different particle sizes will be. A method for purifying multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0225] Another example of the formulation of polypeptides through the use of co-granules is disclosed in WO 2013 / 188331.

[0226] The present invention also relates to protected polypeptides prepared according to the methods disclosed in EP 238216.

[0227] Fermentation Broth Composition or Cell Composition The present invention also relates to a fermentation broth formulation or cell composition comprising a polypeptide of the invention, i.e., a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0228] The fermentation broth formulation or cell composition further comprises additional components used in the fermentation process, such as, for example, cells (including host cells containing genes encoding a polypeptide of the invention used to produce a polypeptide of interest), cell debris, biomass, fermentation medium, and / or fermentation products. In some embodiments, the composition is a killed whole broth containing organic acids, killed cells and / or cell debris, and culture medium.

[0229] The term "fermentation broth," as used herein, refers to a preparation produced by cell fermentation with no or minimal recovery and / or purification. For example, a fermentation broth is produced when a microbial culture is grown to saturation and incubated under carbon-limited conditions that allow protein synthesis (e.g., expression of enzymes by the host cells) and secretion into the cell culture medium. A fermentation broth can contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, a fermentation broth is unfractionated and includes spent culture medium and cellular debris present after microbial cells (e.g., filamentous fungal cells) have been removed, for example, by centrifugation. In some embodiments, a fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0230] In some embodiments, the fermentation broth formulation or cell composition comprises a first organic acid component comprising an organic acid of at least 1-5 carbons and / or a salt thereof, and a second organic acid component comprising an organic acid of at least 1-6 or more carbons and / or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing, and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.

[0231] In one aspect, the composition contains an organic acid and optionally further contains dead cells and / or cell debris, hi some embodiments, the dead cells and / or cell debris are removed from the cell-killed whole broth to obtain a composition free of these components.

[0232] The fermentation broth formulation or cell composition may further comprise preservatives and / or antimicrobial (e.g., bacteriostatic) agents, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

[0233] 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., filamentous fungal cells) have been grown to saturation and incubated under carbon-limited conditions that allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains spent cell culture medium, extracellular enzymes, and killed filamentous fungal 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.

[0234] Whole broth or cell compositions as described herein are typically liquid but may contain insoluble components such as dead cells, cell debris, culture medium components, and / or insoluble enzymes, etc. In some embodiments, the insoluble components may be removed to obtain a clarified liquid composition.

[0235] The whole broth formulations and cell compositions of the present invention may be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0236] Methods and Uses The present invention also relates to methods for detaching cells, comprising contacting the cells with a polypeptide of the invention or a composition comprising a polypeptide of the invention, wherein the cells are attached to a surface and / or another cell. In one embodiment, the detached cells are attached to a surface. In one embodiment, the detached cells are part of a cell monolayer. In one embodiment, the detached cells are part of a cell cluster.

[0237] In one embodiment, a polypeptide of the invention has protease activity and has at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0238] In one embodiment, the polypeptide has increased P1 selectivity for Leu, Tyr, and Phe. Preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position. Preferably, P1 preference is determined according to Example 2 herein.

[0239] The methods of the present invention may be used to exfoliate any type of cell, including, but not limited to, A375 metastatic melanoma cells, beta cells, BHK cells, bone marrow stem cells, cardiomyocytes, CHO cells, COS cells, D54 glioma cells, dopaminergic progenitor cells, fibroblasts, HEK293 cells, HeLa cells, hepatocytes, hepatocyte progenitor cells, human stem cells, HT1080 fibrosarcoma cells, immortalized mouse testicular germ cells, keratinocytes, L929 cells, M24 metastatic melanoma cells, macrophages, Madin-Darby canine kidney cells, mesenchymal stem cells, MG63 cells, NIH / 3T3 cells, NT2 cells, primary chick embryonic neurons, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells, and Vero cells.

[0240] In a preferred embodiment, the cells are mammalian cells, most preferably human cells.

[0241] The method of the present invention can be used to explant any type of stem cell or stem cell derivative. Thus, the stem cells can be totipotent stem cells (e.g., fertilized egg cells), pluripotent stem cells (e.g., embryonic stem cells), multipotent stem cells (e.g., mesenchymal stem cells), oligopotent stem cells (e.g., hematopoietic stem cells), or unipotent stem cells (e.g., muscle stem cells). In one embodiment, the stem cells are human stem cells. In one embodiment, the stem cells are human pluripotent stem cells, human multipotent stem cells, human oligopotent stem cells, or human unipotent stem cells. In a preferred embodiment, the stem cells are human pluripotent stem cells. In another preferred embodiment, the stem cells are human induced pluripotent stem cells.

[0242] In some embodiments, the detached cells are attached to a surface, such as a plastic or glass surface. In some embodiments, the detached cells are attached to another cell. In some embodiments, the attached cell is part of a cell cluster.

[0243] In some embodiments, the detached cells are attached to biomaterials, extracellular matrix (ECM)-coated surfaces, and / or other scaffolds made from natural polymers (e.g., collagen, hyaluronic acid, fibrin, alginate, gelatin, etc.) or synthetic polymers (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL), etc.).

[0244] In one embodiment, the detached cells are pluripotent stem cells, preferably human pluripotent stem cells. Preferably, the pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0245] In one embodiment, the detached cells are induced pluripotent stem cells, preferably human induced pluripotent stem cells. Preferably, the induced pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0246] In one embodiment, the detached cells are cardiomyocytes, preferably human cardiomyocytes. Preferably, the cardiomyocytes are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0247] In one embodiment, the detached cells are stem cell-derived cardiomyocytes, preferably human stem cell-derived cardiomyocytes. Preferably, the stem cell-derived cardiomyocytes are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0248] The present invention also relates to the use of the polypeptides of the present invention in cell detachment processes. In one embodiment, the polypeptides of the present invention have protease activity and have at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0249] In one embodiment, the polypeptide has increased P1 selectivity for Leu, Tyr, and Phe. Preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position. Preferably, P1 preference is determined according to Example 2 herein.

[0250] The polypeptides of the present invention may be used in any type of cell exfoliation process, including, but not limited to, A375 metastatic melanoma cells, beta cells, BHK cells, bone marrow stem cells, cardiomyocytes, CHO cells, COS cells, D54 glioma cells, dopaminergic progenitor cells, fibroblasts, HEK293 cells, HeLa cells, hepatocytes, hepatocyte progenitor cells, human stem cells, HT1080 fibrosarcoma cells, immortalized mouse testicular germ cells, keratinocytes, L929 cells, M24 metastatic melanoma cells, macrophages, Madin-Darby canine kidney cells, mesenchymal stem cells, MG63 cells, NIH / 3T3 cells, NT2 cells, primary chick embryonic neurons, Sf9 insect cells, U251 glioma cells, vascular endothelial cells, vascular smooth muscle cells, and Vero cells.

[0251] In a preferred embodiment, the exfoliated cells are mammalian cells, most preferably human cells.

[0252] The polypeptides of the present invention can be used in any type of stem cell detachment process. Thus, the cells to be detached can be totipotent stem cells (e.g., fertilized egg cells), pluripotent stem cells (e.g., embryonic stem cells), multipotent stem cells (e.g., mesenchymal stem cells), oligopotent stem cells (e.g., hematopoietic stem cells), or unipotent stem cells (e.g., muscle stem cells). In one embodiment, the stem cells are human stem cells. In one embodiment, the stem cells are human pluripotent stem cells, human multipotent stem cells, human oligopotent stem cells, or human unipotent stem cells. In a preferred embodiment, the stem cells are human pluripotent stem cells. In another preferred embodiment, the stem cells are human induced pluripotent stem cells.

[0253] In one embodiment, the cells to be exfoliated are stem cell derivatives, preferably pluripotent stem cell derivatives, most preferably human pluripotent stem cell derivatives.

[0254] In some embodiments, the detached cells are attached to a surface, such as a plastic or glass surface. In some embodiments, the detached cells are attached to another cell. In some embodiments, the attached cell is part of a cell cluster.

[0255] In some embodiments, the detached cells are attached to biomaterials, extracellular matrix (ECM)-coated surfaces, and / or other scaffolds made from natural polymers (e.g., collagen, hyaluronic acid, fibrin, alginate, gelatin, etc.) or synthetic polymers (e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL), etc.).

[0256] In one embodiment, the detached cells are pluripotent stem cells, preferably human pluripotent stem cells. Preferably, the pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0257] In one embodiment, the detached cells are induced pluripotent stem cells, preferably human induced pluripotent stem cells. Preferably, the induced pluripotent stem cells are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0258] In one embodiment, the detached cells are cardiomyocytes, preferably human cardiomyocytes. Preferably, the cardiomyocytes are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0259] In one embodiment, the detached cells are stem cell-derived cardiomyocytes, preferably human stem cell-derived cardiomyocytes. Preferably, the stem cell-derived cardiomyocytes are detached from a surface, preferably a plastic or glass surface, or from a cell cluster.

[0260] Preferred Embodiments 1) The composition comprises a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, wherein the polypeptide comprises at least 50%, e.g., at least 55%, at least A composition suitable for cell detachment, wherein the composition is present in an amount of 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more.

[0261] 2) The composition of embodiment 1, wherein the polypeptide has an increased P1 preference for Leu, Tyr, and Phe; preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position.

[0262] 3) A composition according to any of embodiments 1 to 2, wherein the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0263] 4) A composition according to any one of embodiments 1 to 3, wherein the polypeptide is a variant of a fragment of SEQ ID NO: 1.

[0264] 5) The composition of any of embodiments 1 to 4, wherein the polypeptide is present in an amount of at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material of the composition.

[0265] 6) The composition according to any one of embodiments 1 to 5, which is a liquid composition; preferably an aqueous composition.

[0266] 7) The composition of embodiment 6, wherein the liquid composition comprises an aqueous buffer; preferably, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate; most preferably, the aqueous buffer comprises phosphate or bicarbonate.

[0267] 8) The composition of any of embodiments 6 to 7, having a pH value of about 5 to about 9; more preferably about 7 to about 8; most preferably about 7 to about 7.5.

[0268] 9) The composition according to any one of embodiments 6 to 8, comprising EDTA; preferably, the composition comprises EDTA in an amount of about 0.1 mM to about 10 mM.

[0269] 10) The composition contains magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ 10. The composition of any one of embodiments 6 to 9, wherein the composition does not comprise:

[0270] 11) The composition according to any of embodiments 6 to 10, wherein the polypeptide is present in an amount of about 0.1 μg / ml to about 20 μg / ml; preferably, in an amount of about 1 μg / ml to about 20 μg / ml.

[0271] 12) An isolated polynucleotide encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:1; preferably, the polynucleotide encodes a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO:1.

[0272] 13) A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:1 operably linked to a heterologous promoter; preferably, the polynucleotide encodes a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO:1.

[0273] 14) A recombinant host cell comprising in its genome a nucleic acid construct or an expression vector according to embodiment 13.

[0274] 15) The recombinant host cell of embodiment 14, which is a B. subtilis cell, a B. licheniformis cell, an A. niger cell, an A. oryzae cell, a T. reesei cell, or a P. pastoris (K. phaffii) cell.

[0275] 16) A method for producing a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 and having protease activity, comprising: (a) culturing a recombinant host cell of any of embodiments 14-15 under conditions conducive to expression of the polypeptide; and, optionally, (b) recovering the polypeptide.

[0276] 17) The method of embodiment 16, wherein the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0277] 18) Granules or particles comprising a polypeptide having at least 70% sequence identity with SEQ ID NO:1 and having protease activity; preferably, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0278] 19) A fermentation broth formulation comprising a polypeptide having at least 70% sequence identity to SEQ ID NO:1 and having protease activity; preferably, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:1.

[0279] 20) A method for cell detachment, comprising contacting a cell with a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, or a composition comprising said polypeptide, wherein the cell is attached to a surface and / or another cell.

[0280] 21) The method of embodiment 20, wherein the polypeptide has an increased P1 preference for Leu, Tyr, and Phe; preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position.

[0281] 22) A method according to any one of embodiments 20 to 21, wherein the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0282] 23) A method according to any one of embodiments 20 to 22, wherein the polypeptide is a variant of a fragment of SEQ ID NO: 1.

[0283] 24) The method of any one of embodiments 20 to 23, wherein the polypeptide has a purity of at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material present in the composition.

[0284] 25) The method of any one of embodiments 20 to 24, wherein the composition is a liquid composition; preferably an aqueous composition.

[0285] 26) The method of embodiment 25, wherein the liquid composition comprises an aqueous buffer; preferably, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate; most preferably, the aqueous buffer comprises phosphate or bicarbonate.

[0286] 27) The method of any one of embodiments 25-26, wherein the liquid composition has a pH value of about 5 to about 9; more preferably about 7 to about 8; most preferably about 7 to about 7.5.

[0287] 28) The method of any one of embodiments 25 to 27, wherein the liquid composition comprises EDTA; preferably, the composition comprises EDTA in an amount of about 0.1 mM to about 10 mM.

[0288] 29) The liquid composition contains magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ 29. The method of any one of embodiments 25 to 28, wherein the method does not include

[0289] 30) The method of any one of embodiments 25 to 29, wherein the liquid composition comprises the polypeptide in an amount of about 0.1 μg / ml to about 20 μg / ml; preferably, the liquid composition comprises the polypeptide in an amount of about 1 μg / ml to about 20 μg / ml.

[0290] 31) The method of any one of embodiments 20 to 30, wherein the cells are human cells.

[0291] 32) The method of any one of embodiments 20 to 31, wherein the cells are stem cells or stem cell derivatives; preferably, the cells are pluripotent stem cells, induced pluripotent stem cells, or (stem cell-derived) cardiomyocytes.

[0292] 33) The method of any one of embodiments 20 to 32, wherein the surface is a plastic surface or a glass surface.

[0293] 34) Use of a polypeptide having protease activity and having at least 70%, for example at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, or a composition comprising said polypeptide, in a cell detachment process.

[0294] 35) The use according to embodiment 34, wherein the polypeptide has an increased P1 preference for Leu, Tyr, and Phe; preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position.

[0295] 36) Use according to any of embodiments 34 to 35, wherein the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1.

[0296] 37) The use according to any one of embodiments 34 to 36, wherein the polypeptide is a variant of a fragment of SEQ ID NO: 1.

[0297] 38) Use according to any of embodiments 34 to 37, wherein the polypeptide has a purity of at least 90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material present in the composition.

[0298] 39) Use according to any of embodiments 34 to 38, wherein the composition is a liquid composition; preferably an aqueous composition.

[0299] 40) The use of embodiment 39, wherein the liquid composition comprises an aqueous buffer; preferably, the aqueous buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), phosphate, or bicarbonate; most preferably, the aqueous buffer comprises phosphate or bicarbonate.

[0300] 41) The use according to any one of embodiments 39 to 40, wherein the liquid composition has a pH value of about 5 to about 9; more preferably about 7 to about 8; most preferably about 7 to about 7.5.

[0301] 42) The use according to any one of embodiments 39 to 41, wherein the liquid composition comprises EDTA; preferably, the composition comprises EDTA in an amount of about 0.1 mM to about 10 mM.

[0302] 43) The liquid composition contains magnesium ions (Mg 2+ ) and / or calcium ions (Ca 2+ ) is substantially free of magnesium ions (Mg 2+ ) or calcium ions (Ca 2+ 43. The use according to any one of embodiments 39 to 42, wherein the use does not comprise:

[0303] 44) Use according to any of embodiments 39 to 43, wherein the liquid composition contains the polypeptide in an amount of about 0.1 μg / ml to about 20 μg / ml; preferably, the liquid composition contains the polypeptide in an amount of about 1 μg / ml to about 20 μg / ml.

[0304] 45) Use according to any of embodiments 34 to 44, in which the cells are human cells.

[0305] 46) Use according to any of embodiments 34 to 45, in which the cells are stem cells or stem cell derivatives; preferably, the cells are pluripotent stem cells, induced pluripotent stem cells, or (stem cell-derived) cardiomyocytes.

[0306] 47) Use according to any one of embodiments 34 to 46, in which the cells are detached from the surface or from cell clusters.

[0307] 48) The use according to embodiment 47, wherein the surface is a plastic surface or a glass surface. [Example]

[0308] Materials and Methods Accutase desalting Lyophilized Accutase® XL (Sigma Aldrich, total volume) was dissolved in 25 mL of MilliQ water, and 10 mL was loaded onto a HiPrep 26 / 10 desalting chromatography column (Sigma Aldrich) equilibrated with 50 mM Tricine, 10 mM CaCl2, pH 7.5 at a flow rate of 10 mL / min using an AEKTA explorer 100. Elution continued in the same buffer. The first peak fraction was collected and analyzed by absorbance at 280 nm (A 280 ) and stored at −20° C. Desalted Accutase was used for proteolytic activity assays (trypsin, chymotrypsin, collagenase type I, and collagenase type IV).

[0309] Trypsin activity assay The assay was performed in a 96-well format using a total well volume of 200 μL. The enzyme concentration ranged from 0.5 to 200 μg / mL. The substrate (Nα-benzoyl-L-arginine ethyl ester, BAEE, Merck) was diluted in 67 mM phosphate buffer, pH 7.5. The final BAEE substrate solution was 0.28 mg / mL. Samples were diluted in 67 mM phosphate buffer, pH 7.5 (13.544 g sodium phosphate dibasic heptahydrate, 2.274 g sodium phosphate monobasic monohydrate, add milliQ water to 1 L). Samples were tested over a concentration range of 5 to 100 μg / mL. 15 μL of diluted sample was added to 185 μL of BAEE substrate solution. The reaction was monitored over 10 minutes by measuring the absorbance at 253 nm (A253) at 27 °C.

[0310] Chymotrypsin activity assay Assays were performed in a 96-well format using a total well volume of 200 μL. Enzyme concentrations ranged from 20 to 500 μg / mL. The substrate (Nα-benzoyl-L-tyrosine ethyl ester, BTEE, Sigma) was dissolved in 1 mL of 96% ethanol and diluted in 4 mL of 80 mM Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol) containing 17 mM β-cyclodextrin, pH 7.5. The final BTEE substrate solution was 1.72 mg / mL. Samples were diluted in 80 mM Tris (assay buffer), pH 7.5. Samples were tested over a concentration range of 50 to 500 μg / mL. 20 μL of diluted sample was added to 120 μL of assay buffer and 60 μL of BTEE substrate solution. The reaction was monitored over a 10 minute period by measuring the absorbance at 256 nm (A256) at 27°C.

[0311] Type I collagenase activity assay 30 μL of 1 mg / mL collagen FITC (Merck) suspended in 1 mM acetic acid, DPBS (Ca 2+ and Mg 2+ Twenty microliters of enzyme at 0.25–7.5 μg / mL in Dulbecco's phosphate-buffered saline (DMSO) (Merck) was mixed with 100 μL of 0.5 M Tricine (Sigma), pH 7.4. Reactions were carried out at 37°C and stopped after 30, 60, or 90 minutes by adding 150 μL of ice-cold DPBS, followed by centrifugation (13,000 g for 5 minutes). Reactions were quantified by measuring 100 μL of supernatant in duplicate at 485 nm excitation and 535 nm emission (detection).

[0312] Type IV collagenase activity assay The enzyme was diluted to 1, 0.25, and 0.1 mg / mL in 100 mM Tricine pH 7.4 and mixed with 40 μL of 5 mg / mL type IV collagen (human placenta, Sigma-Aldrich), and the pH was adjusted with 10 μL of 0.5 M Tricine pH 7.4. After 15 min, the reaction was stopped by adding 105 μL of 10% trichloroacetic acid precipitation, incubated at 5°C for 10 min, and then centrifuged (13,000 g; 3 min). The supernatant was removed, and the precipitate was solubilized in 50 μL of sample buffer (200 μL of 4x Laemmli Sample buffer (Bio-Rad item no. 161-0747), 40 μL of reducing agent (Bio-Rad), 30 μL of 2 M Tris, and 130 μL of milliQ water). The samples were subjected to SDS-PAGE analysis using gels, Tris / glycine / SDS buffer, and Bio-Rad Precision Plus protein ladder. Enzyme activity was scored as no activity (0), less activity than Accutase (desalted sample) (-), or equal (+) or greater activity (++). For an example, see Figure 1, where degradation of a first band at less than 200 kDa, a second band at 150 kDa, and a third band at 100 kDa was observed.

[0313] Protease activity assay The proteolytic activity of a polypeptide can be determined by a method utilizing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, a blocked peptide that can be cleaved by endoproteases. After proteolytic cleavage, yellow free pNA molecules are released and can be measured by visible spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is manufactured, for example, by Bachem (catalog number L1400, dissolved in DMSO).

[0314] The sample containing the polypeptide to be analyzed is diluted in the remaining activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / ml in 100 mM Tris, pH 8.6). The solution is mixed at room temperature and the absorbance is measured at 405 nm every 20 seconds for 5 minutes. The sample should be diluted to a level where the slope is linear. The slope of the time-dependent absorbance curve (absorbance per minute) is directly proportional to the proteolytic activity of the polypeptide under a given set of conditions.

[0315] Evaluation of stem cell detachment and cluster dissociation Evaluation of human pluripotent stem cell detachment and cluster dissociation Human pluripotent stem cells (hPSCs) were grown as attached monolayers in the early stages and as clusters in later stages of production. After detachment of the hPSC monolayer, the formation of uniform clusters continued for suspension growth and expansion. See Figures 2 and 3 for a schematic overview of the process and the metrics at which assessments were performed.

[0316] Assessment of hPSC surface detachment and cluster formation: Remove spent medium from the T-flask and rinse with wash buffer (Ca 2+ and Mg 2+ PBS without HCl; 0.04-0.3 mL / cm 2 ) was added. The cell monolayer was washed to remove the buffer. 2+ and Mg 2+ The enzyme solution was prepared by dissolving SEQ ID NO: 1 to a concentration of 8.3 μg / mL in Dulbecco's PBS buffer without HCl, followed by pre-warming to room temperature and adding 0.5 mM ethylenediaminetetraacetic acid (EDTA).

[0317] Add the enzyme solution to the cell culture vessel (0.04–0.3 mL / cm 2) and the vessel was incubated at 37°C for 3–20 minutes. Culture medium was added to the vessel, and the cell suspension was mixed by pipetting to obtain a single cell suspension. After surface detachment, the cells were centrifuged, resuspended in an appropriate volume, and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). The total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [1]. To estimate cluster formation efficiency, the fold change was determined on day 1 after passaging. The number of cells within the clusters was determined using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [2]. To estimate cell proliferation, the daily fold change was determined on day 3 after seeding according to [2]. The size and size distribution of the clusters (coefficient of variation of cluster diameter) were determined on day 3 after passaging using a Biorep Islet Cell Counter according to [3] and [4]. The evaluated parameters were scored according to Table 1 below and the mean score was calculated.

[0318] [Table 1]

[0319] Dissociation of hPSC clusters and assessment of cluster re-formation: hPSC clusters were centrifuged and washed (Ca 2+ and Mg 2+ Ca-free PBS; 0.1–1 mL per mL of original working volume, typically 0.25 mL / mL. 2+ and Mg 2+ The enzyme solution was prepared by dissolving SEQ ID NO: 1 to a concentration of 4 μg / mL in Dulbecco's PBS buffer without EDTA, followed by pre-warming to room temperature and adding 0.5 mM EDTA.

[0320] The enzyme solution was then added to the cells (0.025–0.5 mL per mL of working volume; typically 0.2 mL / mL), and the cells were incubated at 37°C for 3–15 minutes. Culture medium was added to the vessel, and the cell suspension was mixed by pipetting to obtain a single cell suspension. After dissociation of the clusters, the cells were centrifuged, resuspended in an appropriate volume, and counted using a NucleoCounter NC202 (ChemoMetec, Denmark). The total cell number, cell viability, and percentage of aggregation (% aggregation) were recorded according to [1]. To estimate the cluster formation efficiency, the fold change on day 1 after passaging was determined. The number of cells within the clusters was determined using a NucleoCounter NC200 (ChemoMetec, Denmark) according to [2]. To estimate cell proliferation, the daily fold change was determined on day 3 after seeding according to [2]. Cluster size and size distribution (coefficient of variation of cluster diameter) were determined on day 3 after passage using a Biorep Islet Cell Counter according to [3] and [4]. The evaluated parameters were scored according to Table 2 below, and the average score was calculated.

[0321] After the second passage at the cluster stage, the pluripotent phenotype was confirmed by staining with antibodies specific for the surface markers Oct4 (BD) and Nanog (Nordic BioSite AP) using fluorescence-activated single-cell sorting (FACS) analysis.

[0322] [Table 2]

[0323] Assessment of cardiomyocyte cluster dissociation Dissociation of CM08: Stem cell-derived cardiomyocyte (CM) clusters were cultured in a shake flask format in RPMI medium supplemented with 1% ascorbic acid and 2% B-27 supplement (Gibco) in a shaking incubator (70 rpm; 37°C). At the CM08 stage (8 days after differentiation), 3 mL of clusters were harvested by centrifugation (300 g; 1 min), and the supernatant medium was removed. Ca 2+ and Mg 2+ The cells were washed with 3 mL of PBS without Ca and centrifuged (300 g; 1 min) to remove the supernatant. 2+ and Mg 2+ The enzyme solution was prepared by dissolving SEQ ID NO:1 to a concentration of 8 μg / mL in Dulbecco's PBS buffer without HCl, preheating it to room temperature, and adding 0.5 mM EDTA. 1 mL of the enzyme solution was added to the CM clusters, and the clusters were incubated (70 rpm; 37°C) until the medium became turbid with single cells. After confirming that the clusters had completely disintegrated, 2 mL of culture medium (preheated to 37°C) was added to stop the enzyme reaction.

[0324] Analysis of CM08: After adding culture medium, images were taken and cells were counted using an ICC4 automated cell counter (BioRep) to determine the total number of live and dead cells, cell viability, and remaining clusters (not fully dissociated into a single-cell suspension).

[0325] Cluster re-formation culture: Incubate in an incubator at 70 rpm and 37°C for 10 6 The recovered cells were resuspended in culture medium to re-form clusters at a density of 10 viable cells / mL. The medium was completely replaced every 2–3 days, and cluster re-formation was observed for 7 days. After that, clusters were harvested at the CM15 stage.

[0326] CM15 Collection and Analysis: The number of clusters, their size distribution, and morphology were examined using an ICC4 automated cell counter (BioRep). Clusters were then harvested and analyzed for cluster reconstitution yield ([total viable cells / total seeded viable cells] * 100) and mean cluster size estimate (pIEQ divided by total cluster number) [1, 2].

[0327] Rating and Scoring: Parameters important for dissociation and subsequent cluster formation were evaluated and scored (see Table 3). The average score was calculated for the enzyme solutions.

[0328] [Table 3]

[0329] Example 1a: Purification of SEQ ID NO: 1 from Accutase Accutase™ XL (lyophilized, e.g., available from Merck) was dissolved in milliQ water at room temperature, the pH adjusted to 8.0, filtered, and applied to a 6.4 mL volume of Butyl Sepharose FF (Cytiva) column packed in an XK16 / 20 column (Cytiva) (flow rate 3.5 mL / min) equilibrated with 50 mM sodium phosphate, pH 7.0 (39 mL of 0.5 M NaH2PO4·2H2O and 61 mL of 0.5 M Na2HPO4, plus 1 L of MilliQ water) and 1.5 M ammonium sulfate, pH 7.0. After a wash step with the equilibration buffer, the protein was eluted with 50 mM sodium phosphate, pH 7.0, using a 5 mL / min gradient over 40 column volumes. Liquid chromatography-mass spectrometry (LC-MS) confirmed the molecular weight of SEQ ID NO:1.

[0330] Example 1b: Recombinant expression of SEQ ID NO: 1 Cloning and expression of SEQ ID NO:1 was performed using the strategy described in U.S. Patent Application Publication No. 2019 / 0225988, which uses three overlapping fragments for integration at the niiA / niiD (nitrite reductase / nitrate reductase) locus of the Aspergillus oryzae host strain ColS1300. The central fragment corresponding to the gene encoding the polypeptide of SEQ ID NO:1 was codon-optimized for Aspergillus oryzae (provided as SEQ ID NO:2) and ordered as a synthetic gene containing overlapping sequences for the two flanking fragments. SEQ ID NO:1 was expressed by replacing the gene's native secretion signal with the β-glucosidase secretion signal from Aspergillus aculeatus (amino acid sequence: MKLSWLVAAALTAASVVSA; provided as SEQ ID NO:3).

[0331] The three amplified overlapping fragments were transformed into COLS1300. Transformants were plated onto plates containing minimal medium supplemented with NaNO3, and the plates were incubated at 30°C for 3 days. During this process, only spores that successfully recombined the integration cassette into the host cell chromosome and reconstituted the niiA and niaD sites were able to germinate and survive. Recombinant Aspergillus oryzae clones containing sequences confirmed to have integrated the expression construct were grown in liquid culture in 24-well deep-well plates (Corning) containing 2 ml of YPG medium (WO 05 / 066338) at 30°C for 5 days without shaking.

[0332] The supernatant containing the enzyme was collected and MS analysis was used to confirm the expression of SEQ ID NO:1.

[0333] Example 2: Determining the specificity of proteases The specificity of a protease can be defined according to its P1 preference, where the P1 position is defined as the amino acid residue located N-terminal to the cleavage site of a polypeptide (Biochemical and Biophysical Research Communications, volume 27, issue 2, 20 April 1967, pages 157-162).

[0334] Preference is defined as the observed frequency being higher than that expected by random cleavage when counting the number of cleavage sites from protease digestion performed on a complex protein substrate containing high sequence diversity.

[0335] Specifically, purified protease samples are incubated on a 10 kDa cutoff spin filter with yeast protein extract (Promega V7341) for 16 hours at 37°C in 100 mM HEPES, 1 mM CaCl2, pH 7. Prior to incubation, the substrate is denatured by trichloroacetic acid (TCA) precipitation, reduced by the addition of dithiothreitol (DTT), and alkylated by the addition of iodoacetamide (IAA).

[0336] Three reactions were performed with protease:substrate ratios of 1:1250, 1:6250, and 1:30000. The resulting protease digests were collected after centrifugation by collecting the flow-through. Additional washes may be required to improve peptide recovery. The protease digests were acidified with TFA and directly analyzed by LC-MS / MS, e.g., Evosep One (Evosep) / timsTOF Pro (Bruker Daltonik).

[0337] To identify peptides, the data are searched against the UniProt yeast reference proteome using the Mascot search engine (Matrix science) with the following search parameters: Enzymes: None Peptide mass tolerance: ±25 ppm Fragment mass tolerance: ±0.05 Da Maximum number of missed cuts: 0

[0338] The N- and C-termini of the identified peptides are used to deduce protease cleavage sites. The extent of proteolysis must be sufficiently low to reflect the initial preferred cleavage site. Knowledge of the amino acid sequence of the protein from which the peptide is derived identifies the amino acids present at the subsite (e.g., P1) at the time of proteolytic cleavage.

[0339] Preference for an amino acid in a subsite (e.g., P1) is calculated by comparing the sum of the intensities of identified peptides containing this amino acid in the subsite (e.g., P1) to the prevalence expected in random cleavage of the protein.

[0340] Based on this method, the P1 preference of SEQ ID NO: 1 was evaluated. SEQ ID NO: 1 has an increased P1 preference for the amino acid residues Leu, Tyr, and Phe. When the P1 preference of SEQ ID NO: 1 was determined with all 20 standard amino acids and then ranked, Leu, Tyr, and Phe were among the five most preferred amino acid residues at the P1 position.

[0341] Example 3: Proteolytic activity of SEQ ID NO: 1 and Accutase The total protein concentration of Accutase was determined to be 20 μg / mL. The trypsin, chymotrypsin, collagen type I, and collagen type IV activities of Accutase were assessed and normalized to 100%, and the enzymatic activity of SEQ ID NO: 1 is reported relative to Accutase (see Table 4).

[0342] [Table 4]

[0343] Example 4: hPSC monolayer detachment and cluster formation of SEQ ID NO: 1 SEQ ID NO:1 and Accutase were evaluated and scored for hPSC monolayer detachment and cluster formation (Table 5).

[0344] [Table 5]

[0345] The overall performance of SEQ ID NO: 1 was improved compared to Accutase, in particular the rate of aggregation was reduced while the cluster formation efficiency was improved.

[0346] Example 5: Dissociation and re-formation of hPSC clusters with SEQ ID NO: 1 SEQ ID NO:1 and Accutase were evaluated and scored for dissociation and reformation of hPSC clusters (Table 6).

[0347] [Table 6]

[0348] The overall performance of SEQ ID NO: 1 was improved compared to Accutase, particularly in terms of cluster formation efficiency, cell proliferation, and cluster uniformity.

[0349] Example 6: Dissociation and re-formation of cardiomyocyte clusters with SEQ ID NO: 1 SEQ ID NO:1 and Accutase were evaluated and scored for dissociation and reformation of CM clusters (Table 7).

[0350] [Table 7]

[0351] The overall performance of SEQ ID NO: 1 was improved compared to Accutase, especially in terms of the cluster regeneration yield and average cluster size.

[0352] References 1) ChemoMetec, Application Note No. 2026. Rev. 1.4. Count&Viability - Via2-Cassette(TM) 2) ChemoMetec, Application Note No. 2028. Rev. 1.3. Aggregated Cells - Via2-Cassette(TM) 3) BioRep User manual for AUTOMATIC ISLET CELL COUNTER 4, Ref. ICC-04: 4) Fully Automated Islet Cell Counter(ICC) for the Assessment of Islet Mass, Purity, and Size Distribution by Digital Image Analysis. Peter Buchwald, Andres Bernal, Felipe Echeverri, Alejandro Tamayo-Garcia, Elina Linetsky and Camillo Ricordi. Cell Transplantation. Vol. 25, pp. 1747-1761, 2016.

Claims

1. 1. A composition suitable for cell detachment, comprising a polypeptide having protease activity and having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, wherein the polypeptide is present in an amount of at least 10% by weight of the total polypeptide material of the composition.

2. 2. The composition of claim 1, wherein the polypeptide has an N-terminal and / or C-terminal extension of one or more amino acids compared to SEQ ID NO: 1; preferably an N-terminal and / or C-terminal extension of 1 to 25 amino acids.

3. 2. The composition of claim 1, wherein the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:

1.

4. 4. A composition according to any one of claims 1 to 3, wherein the polypeptide is present in an amount of at least 15%, such as at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more by weight of the total polypeptide material of the composition.

5. 5. The composition of any one of claims 1 to 4, wherein the polypeptide is present in an amount of at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material of the composition.

6. 6. The composition of any one of claims 1 to 5, wherein the polypeptide is present in an amount of at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, at least 99.99%, at least 99.995%, at least 99.999% or more by weight of the total polypeptide material of the composition.

7. 7. The composition of claim 1, wherein the polypeptide has an increased P1 preference for Leu, Tyr, and Phe; preferably, Leu, Tyr, and Phe are among the five most preferred amino acid residues at the P1 position.

8. The composition according to any one of claims 1 to 7, which is a liquid composition; preferably an aqueous composition.

9. 9. The composition of claim 8, comprising an aqueous buffer; preferably a phosphate buffer.

10. The composition of any one of claims 8 to 9, having a pH value of from about 5 to about 9; more preferably from about 7 to about 8; most preferably from about 7 to about 7.

5.

11. The composition of any one of claims 8 to 10, comprising EDTA; preferably, the composition comprises EDTA in an amount of about 0.1 mM to about 10 mM.

12. Magnesium ions (Mg ２＋ ) and / or calcium ions (Ca ２＋ The composition according to any one of claims 8 to 11, which is substantially free of

13. 13. The composition of any one of claims 8 to 12, wherein the polypeptide is present in an amount of from about 0.1 μg / ml to about 100 μg / ml, such as from about 0.5 μg / ml to about 50 μg / ml, from about 1 μg / ml to about 20 μg / ml, or from about 1 μg / ml to about 10 μg / ml.

14. 1. An isolated polynucleotide encoding a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; preferably, the polynucleotide encodes a polypeptide that comprises, consists of, or consists essentially of SEQ ID NO:

1.

15. 1. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, operably linked to a heterologous promoter; preferably, the polynucleotide encodes a polypeptide comprising, consisting of, or consisting essentially of SEQ ID NO:

1.

16. 16. A recombinant host cell comprising in its genome the nucleic acid construct or expression vector of claim 15, preferably a B. subtilis cell, a B. licheniformis cell, an A. niger cell, an A. oryzae cell, a T. reesei cell, or a P. pastoris (K. phaffii) cell.

17. 17. A method for producing a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1, the method comprising: (a) culturing a recombinant host cell of claim 16 under conditions conducive to expression of the polypeptide; and optionally (b) recovering the polypeptide.

18. A fermentation broth formulation comprising a polypeptide having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; preferably, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO:

1.

19. 14. A method for cell detachment comprising contacting a cell with the composition of any one of claims 1 to 13, wherein the cell is attached to a surface and / or another cell.

20. Use of a composition according to any one of claims 1 to 13 in a cell detachment process.

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