EP3703661A1 - Feste matrixzusammensetzungen mit gesenktem gefrierpunkt für die schmelzgranulation von enzymen - Google Patents

Feste matrixzusammensetzungen mit gesenktem gefrierpunkt für die schmelzgranulation von enzymen

Info

Publication number
EP3703661A1
EP3703661A1 EP18804821.9A EP18804821A EP3703661A1 EP 3703661 A1 EP3703661 A1 EP 3703661A1 EP 18804821 A EP18804821 A EP 18804821A EP 3703661 A1 EP3703661 A1 EP 3703661A1
Authority
EP
European Patent Office
Prior art keywords
fpdsm
enzyme
urea
solid
betaine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18804821.9A
Other languages
English (en)
French (fr)
Inventor
Nathaniel T. Becker
Joseph C. Mcauliffe
Peyman Moslemy
Jared Enwei TAN
Luther WHITE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Danisco US Inc
Original Assignee
Danisco US Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Danisco US Inc filed Critical Danisco US Inc
Publication of EP3703661A1 publication Critical patent/EP3703661A1/de
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1611—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the present disclosure is generally related to the fields of protein chemistry, formulation chemistry, enzymology, rheology, physical chemistry and the like.
  • the disclosure is related to stabilized protein compositions and formulations thereof.
  • the disclosure is related to storage stable protein compositions and formulations thereof.
  • the disclosure is related to one or more freezing point depressed solid matrix (FPDSM) systems, melt granulation methods thereof and formulations thereof, wherein the FPDSM system encapsulates and stabilizes the protein compositions thereof, during the melt granulation process and subsequent storage.
  • FPDSM freezing point depressed solid matrix
  • proteins such as enzymes, transport proteins, structural proteins, defense proteins, regulatory proteins, receptor proteins, and the like have many remarkable biological properties, they are all subject to denaturation and degradation at elevated temperatures, and loss of activity over time at ambient temperature.
  • proteins e.g., enzymes
  • proteins can lose activity as result of proteolysis, unfolding, misfolding, aggregation, thermal instability, thermal stress, mechanical stress, sub-optimal buffer conditions, pH and the like.
  • One particular factor that limits the lifetime (activity) of a protein is its susceptibility to enzymatic and non-enzymatic chemical reactions which degrade the protein.
  • proteins e.g., enzymes
  • proteins are subject to various forms of non-enzymatic denaturation and degradation including, but not limited to, unfolding, oxidative degradation of cysteine and methionine sulfur atoms, the deamidation of glutamine and asparagine residues, peptide-bond hydrolysis, cross-linking reactions between the protein amino groups and natural reducing sugars, and the like.
  • Other factors influencing protein degradation and/or protein conformational stability include, but are not limited to, conditions such as temperature, pH, salt concentration, water activity, oxygen concentration, AG of folded versus unfolded protein, solvent/buffer systems, pressure, electromagnetic radiation (e.g., uv light), molecular contaminants, microbial contaminants, and the like.
  • protein denaturation, degradation, protein stability and/or protein activity are significantly influenced by the temperatures at which the protein is expressed, produced, formulated, recovered, stored, used and the like.
  • the net free energy (AG) stabilizing a folded versus unfolded protein in an aqueous solution at 25°C is generally about 5-15 kcal/mol, which is approximately the amount energy needed to dissociate (break) about one to three non- covalent protein (intramolecular) interactions (e.g., electrostatic, Van der Walls, H-bonding).
  • the stability (and activity) of a typical industrial enzyme such as a protease or amylase (e.g., for use in a laundry or dish cleaning) relates to the entire life cycle of the enzyme, which includes production (e.g., fermentation, downstream processing and formulation), distribution (e.g., transport and storage) and the final application/end-use (e.g., see, Misset, 1992; Chotani et al, 2017; Garske etal, 2017).
  • the production phase of an enzyme can generally be subdivided into three processing steps, which include but are not limited to, (i) fermentation of the production organism (host cell) wherein the enzyme is either secreted into the culture medium or the host cells lysed to recover the enzyme, (ii) downstream processing of the fermentation broth (e.g., protein purification methods such as precipitation, ultrafiltration, chromatography, spray drying, etc.) to yield either a liquid or solid enzyme composition, and (iii) formulation of the enzyme composition to yield optimal performance and maximal stability of the final enzyme product.
  • the formulated enzyme product is (iv) distributed by transportation and often stored at temperatures around 20°-39°C for a period time before (v) the final end use/application. It therefore becomes evident that such industrial enzymes are subjected to numerous environmental insults throughout their chain of production, formulation and final storage thereafter.
  • WO2014/131906 provides Example 17, "Protein stabilization in animal tissue samples", wherein mouse liver was added to molten choline chloride/trifluoroacetamide (1 :2) or PBS buffer (control) and incubated at 24°C for up to 18 days prior to protein extraction.
  • the present disclosure is directed to stable solid protein compositions, formulations thereof and melt granulation methods and compositions thereof, wherein the solid protein of interest (POI) is first dispersed in a molten (FPDSM) solvent system of the disclosure at an elevated temperature (i.e., between 40°C to about 100°C) and then subsequently cooled and solidified (i.e., about 20°C to 40°C ), such that the POI is entrapped in a "freezing point depressed solid matrix" (FPDSM) in its solid state.
  • FPDSM molten
  • a FPDSM composition of the disclosure comprises at least two FPDSM components, wherein each single (FPDSM) component of the FPDSM composition does not melt below 100°C (e.g., see Table 1, FPDSM Component Melting Points), and optionally further comprising water, wherein the FPDSM composition is a solid at a temperature between about 20°C to 40°C.
  • molten solvent systems for solubilizing or stabilizing proteins
  • maintenance of the composition in the "liquid state” e.g., for purposes such as extraction of the POI, solubilization of the POI, biocatalysis using solubilized enzymes, and the like.
  • the present disclosure is directed to molten solvent systems (i.e., a FPDSM system of the disclosure) wherein a POI is embedded (e.g., entrapped) within the solvent system as it solidifies, such that the POI is immobilized and thereby not immediately available as a biocatalyst or reactant.
  • FPDSM molten solvent system
  • FPDSM molten solvent system
  • Applicant discovered herein that it is not necessary to maintain specific molar ratios of the FPDSM components in order to achieve the desired properties of the instant disclosure, namely providing a molten solvent system (FPDSM) formulation wherein the POI is stable prior to solidification of the molten (FPDSM) system, and upon solidification of the FPDSM system, forms a solid matrix composition thereof useful for encapsulating, delivering and stabilizing enzymes at a temperature between about 20°C to 40°C.
  • thermally labile proteins e.g., enzymes
  • can retain most of their activity i.e. at least about 70% of their activity
  • a surprising advantage of the present disclosure is that functional proteins (e.g., enzymes) suspended within the FPDSM can survive during suspension in the molten FPDSM, thereby maintaining high recovery of functional protein conformation and enzymatic activity, e.g., at least 70% recovery of initial enzyme activity, after being subjected to high temperatures (i.e., temperatures which usually denature most proteins; e.g., temperatures as high as 80 ° C, 90 ° C or 100 ° C) for periods of at least one minute to five minutes, prior to subsequent cooling and solidification from a melt (e.g., in a melt granulation process).
  • high temperatures i.e., temperatures which usually denature most proteins; e.g., temperatures as high as 80 ° C, 90 ° C or 100 ° C
  • the high recovery of active enzyme has been demonstrated for compositions that include water up to 10% w/w.
  • the FPDSM (entrapped) enzyme compositions of the disclosure demonstrate excellent storage stability at room temperature (20°C-25°C)
  • the instant disclosure addresses such long felt and unmet needs for methods, formulations, including granular formulations and compositions thereof which mitigate protein degradation, instability and/or inactivation, including such methods, formulations and compositions which mitigate the degradation and/or loss of activity of solid protein formulations which are stored for a period of time (i.e., hours, days, weeks, months) before their end use.
  • the present disclosure is directed to stable solid protein compositions, formulations thereof and methods thereof.
  • the disclosure is directed to methods for preparing/formulating one or more "freezing point depressed solid matrix" (FPDSM) systems which stabilize solid protein compositions.
  • FPDSM freezing point depressed solid matrix
  • the disclosure is directed to methods for inhibiting the degradation of solid protein compositions formulated with one or more FPDSM systems of the disclosure.
  • the disclosure is directed to a solid enzyme formulation comprising a freezing point depressed solid matrix (FPDSM) system and a solid enzyme composition entrapped therein, wherein the FPDSM system comprises at least two distinct FPDSM components which at least two distinct FPDSM components do not melt at a temperature below 100°C until combined, wherein the combined FPDSM system is a liquid at a temperature between 40°C to about 100°C and a solid at a temperature between about 20°C to 40°C, wherein recovery of the active enzyme from the solidified FPDSM system is at least 70% active enzyme relative to the activity of the solid enzyme composition.
  • the at least two component FPDSM further comprises up to 10% (w/w) water.
  • the water content is about 0.05% to 10% w/w. In certain embodiments, the water content is about 1% to about 10% w/w, about 2% to about 10% w/w, about 3% to about 10% w/w, about 4% to about 10% w/w, or about 5% to about 10% w/w.
  • the FPDSM system comprises a combination of at least two components selected from the group consisting of ammonium chloride, choline chloride, betaine, betaine hydrochloride, citric acid, malonic acid, tartaric acid, fructose, sucrose, glucose, lactose, maltose, urea and sorbitol.
  • the at least two components of the FPDSM system are combined at a molar ratio ranging from about 1 : 10 to about 10: 1, wherein the combined molar ratio of the FPDSM system components are in a liquid state at a temperature between 40°C to about 100°C, and a solid at a temperature between about 20°C to 40°C.
  • the enzyme retains at least 85% enzymatic activity after twenty-four (24) hours of storage at 25°C. In other embodiments, the enzyme retains at least 95% enzymatic activity after twenty -four (24) hours of storage at 25 °C. In certain other embodiments, the enzyme retains at least 99% enzymatic activity after twenty -four (24) hours of storage at 25°C. In yet another embodiment, the enzyme retains at least 80% enzymatic activity after seven (7) days of storage at 25 °C. In certain other embodiments, the enzyme retains at least 75% enzymatic activity after twenty-eight (28) days of storage at 25°C.
  • the FPDSM formulated solid enzyme composition retains a higher enzymatic activity than an unformulated solid enzyme composition, wherein both the FPDSM formulated enzyme and the solid enzyme composition are held at the same temperature for the same amount of time before enzymatic activity is assayed.
  • the enzyme is selected from the group consisting of acetyl esterases, aryl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -galactosidases, a-glucanases, glucan lysases, endo- -glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isome
  • the FPDSM system comprises choline chloride and citric acid at a molar ratio between about 1:2 (choline chloride/citric acid) to about 2: 1 (choline chloride/citric acid).
  • the FPDSM system comprises choline chloride and sorbitol at a molar ratio between about 2: 1 (choline chloride/sorbitol) to about 3: 1 (choline chloride/sorbitol).
  • the FPDSM system comprises anhydrous betaine and urea at a molar ratio between about 1 : 1 (betaine/urea) to about 1 :4 (betaine/urea).
  • the FPDSM system comprises ammonium chloride and urea at a molar ratio between about 1 :2 (ammonium chloride/urea) to about 1 :3 (ammonium chloride/urea).
  • the FPDSM system comprises at least two different enzymes.
  • the solid enzyme composition is a spray dried enzyme, a lyophilized enzyme, a precipitated enzyme, a crystalized enzyme, a spray cooled enzyme, spray chilled enzyme or a spray granulated enzyme.
  • the FPDSM formulated solid enzyme comprises a solid form selected from a solid enzyme pellet, a solid enzyme granule, a solid enzyme powder, a solid enzyme film and a solid enzyme extrudate.
  • the disclosure is related to a solid enzyme composition homogenously distributed and entrapped in a FPDSM system formulation of the disclosure.
  • the disclosure is directed to a method for stabilizing a solid enzyme composition from loss of activity, the method comprising (a) selecting and preparing a FPDSM system comprising at least two distinct FPDSM system components and optionally up to 10% water, wherein the FPDSM system components are selected such that when combined and heated, form a liquid at a temperature between 40°C to about lOOC, and when subsequently cooled, form a solid at a temperature between about 20°C to 40°C, (b) adding and mixing a solid enzyme composition to the FPDSM system of step (a) at a temperature higher than 40°C and lower than about 100°C, and (c) lowering the temperature of the step (b) mixture to a temperature between about 20°C to 40°C, wherein the FPDSM system solidifies to form a solid FPDSM system that entraps and encapsulates the enzyme.
  • the recovery of the FPDSM formulated enzyme relative to the recovery of solid enzyme composition is at least 70%.
  • the enzyme retains at least 85% enzymatic activity after twenty -four (24) hours of storage at 25 °C.
  • the enzyme retains at least 95% enzymatic activity after twenty-four (24) hours of storage at 25 °C.
  • the enzyme retains at least 99% enzymatic activity after twenty-four (24) hours of storage at 25 °C.
  • the enzyme retains at least 80% enzymatic activity after seven (7) days of storage at 25°C.
  • the enzyme retains at least 75% enzymatic activity after twenty-eight (28) days of storage at 25°C.
  • the FPDSM formulated solid enzyme composition retains a higher enzymatic activity than an unformulated solid enzyme composition, wherein both the FPDSM formulated enzyme and the solid enzyme composition are stored at the same temperature for the same amount of time before enzymatic activity is assayed.
  • the FPDSM system comprises a combination of at least two components selected from the group consisting of ammonium chloride, choline chloride, betaine, betaine hydrochloride, citric acid, malonic acid, tartaric acid, fructose, sucrose, glucose, lactose, maltose, urea and sorbitol.
  • the FPDSM system comprises a combination selected from the group consisting of urea/lactose, urea/glucose, urea/maltose, urea/fructose, urea/ammonium chloride, urea/betaine, urea/betaine hydrochloride, urea/choline chloride, betaine/glucose, betaine/fructose, betaine/lactose, betaine/citric acid, betaine/sucrose, choline chloride/glucose, choline chloride/malonic acid, choline chloride/ tartaric acid, choline chloride/citric acid, choline chloride/ sorbitol, citric acid/glucose and citric acid/maltose.
  • urea/lactose urea/glucose
  • urea/maltose urea/fructose
  • urea/ammonium chloride ure
  • two components of the FPDSM system are combined at a molar ratio ranging from about 1 : 10 to about 10: 1, wherein the combined molar ratio of the FPDSM system components are in a liquid state at a temperature between 40°C to about 100°C, and a solid at a temperature between about 20°C to 40°C.
  • the FPDSM system comprises choline chloride and citric acid at a molar ratio between about 1 :2 (choline chloride/citric acid) to about 2: 1 (choline chloride/citric acid).
  • the FPDSM system comprises choline chloride and sorbitol at a molar ratio between about 2: 1 (choline chloride/sorbitol) to about 3: 1 (choline chloride/sorbitol).
  • the FPDSM system comprises anhydrous betaine and urea at a molar ratio between about 1 : 1 (betaine/urea) to about 1 :4 (betaine/urea).
  • the FPDSM system comprises ammonium chloride and urea at a molar ratio between about 1:2 (ammonium chloride/urea) to about 1 :3 (ammonium chloride/urea).
  • the FPDSM system comprises betaine and glucose at a molar ratio of about 5:2 (betaine/glucose) and about 0.05% to 10% weight water. In another embodiment, the FPDSM system comprises betaine and urea at a molar ratio of about 1 : 1 (betaine/glucose) and about 0.05% to 10% weight water. In other embodiments of the method, the FPDSM system comprises urea and glucose at a molar ratio of about 3: 1 (urea/glucose) and about 0.05% to 10% weight water.
  • the FPDSM system comprises urea and maltose at a molar ratio of about 6: 1 (urea/maltose) and about 0.05% to 10% weight water. In yet other embodiments, the FPDSM system comprises urea and lactose at a molar ratio of about 6: 1 (urea/lactose) and about 0.05% to 10% weight water. In another embodiment, the FPDSM system comprises urea and lactose at a molar ratio of about 9: 1 (urea/lactose) to about 1.5: 1 (urea/lactose) and about 4.0% to 6.0% weight water. In other embodiments of the method, the water content is about 0.05% to 10% w/w. In certain other embodiments, the water content is about 5% to 10% w/w.
  • the enzyme is selected from the group consisting of acetyl esterases, aryl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -galactosidases, a-glucanases, glucan lysases, endo- -glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerase
  • the FPDSM system comprises at least two different enzymes.
  • the solid enzyme composition is a spray dried enzyme, a lyophilized enzyme, a precipitated enzyme, a crystalized enzyme, a spray cooled enzyme, spray chilled enzyme or a spray granulated enzyme.
  • the solidified FPDSM system of step (c) comprises a solid form selected from a solid pellet, a solid granule, a solid powder, a solid film and a solid extrudate.
  • the disclosure is related to a solid enzyme composition homogenously distributed and entrapped in a FPDSM system prepared by a method of the disclosure.
  • the disclosure is related to a method for inhibiting the degradation of a solid enzyme composition stored for at least twenty-four hours at 25 °C, the method comprising (a) selecting and preparing a FPDSM system comprising at least two distinct FPDSM system components and optionally up to 10% water, wherein the FPDSM system components are selected such that when combined and heated, form a liquid at a temperature between 40°C to about 100°C, and when subsequently cooled, form a solid at a temperature between about 20°C to 40°C, (b) adding and mixing a solid enzyme composition to the FPDSM system of step (a) at a temperature higher than 40°C and lower than about 100°C, and (c) lowering the temperature of the step (b) mixture to a temperature between about 20°C to 40°C, wherein the FPDSM system solidifies to form a solid FPDSM system that entraps and encapsulates the enzyme.
  • the recovery of the FPDSM formulated enzyme relative to the recovery of solid enzyme composition is at least 70%.
  • the enzyme retains at least 85% enzymatic activity after twenty-four (24) hours of storage at 25°C.
  • the enzyme retains at least 95% enzymatic activity after twenty-four (24) hours of storage at 25 °C.
  • the enzyme retains at least 99% enzymatic activity after twenty-four (24) hours of storage at 25°C.
  • the enzyme retains at least 80% enzymatic activity after seven (7) days of storage at 25°C.
  • the enzyme retains at least 75% enzymatic activity after twenty-eight (28) days of storage at 25°C.
  • the FPDSM formulated solid enzyme composition retains a higher enzymatic activity than an unformulated solid enzyme composition, wherein both the FPDSM formulated enzyme and the solid enzyme composition are stored at the same temperature for the same amount of time before enzymatic activity is assayed.
  • the FPDSM system comprises a combination of at least two components selected from the group consisting of ammonium chloride, choline chloride, betaine, betaine hydrochloride, citric acid, malonic acid, tartaric acid, fructose, sucrose, glucose, lactose, maltose, urea and sorbitol.
  • the FPDSM system comprises a combination selected from the group consisting of urea/lactose, urea/glucose, urea/maltose, urea/fructose, urea/ammonium chloride, urea/betaine, urea/betaine hydrochloride, urea/choline chloride, betaine/glucose, betaine/fructose, betaine/lactose, betaine/citric acid, betaine/sucrose, choline chloride/glucose, choline chloride/malonic acid, choline chloride/ tartaric acid, choline chloride/citric acid, choline chloride/ sorbitol, citric acid/glucose and citric acid/maltose.
  • urea/lactose urea/glucose
  • urea/maltose urea/fructose
  • urea/ammonium chloride ure
  • two components of the FPDSM system are combined at a molar ratio ranging from about 1 : 10 to about 10: 1, wherein the combined molar ratio of the FPDSM system components are in a liquid state at a temperature between 40°C to about 100°C, and a solid at a temperature between about 20°C to 40°C.
  • the FPDSM system comprises choline chloride and citric acid at a molar ratio between about 1:2 (choline chloride/citric acid) to about 2: 1 (choline chloride/citric acid).
  • the FPDSM system comprises choline chloride and sorbitol at a molar ratio between about 2: 1 (choline chloride/sorbitol) to about 3: 1 (choline chloride/sorbitol).
  • the FPDSM system comprises anhydrous betaine and urea at a molar ratio between about 1 : 1 (betaine/urea) to about 1 :4 (betaine/urea).
  • the FPDSM system comprises ammonium chloride and urea at a molar ratio between about 1 :2 (ammonium chloride/urea) to about 1 :3 (ammonium chloride/urea).
  • the FPDSM system comprises betaine and glucose at a molar ratio of about 5:2 (betaine/glucose) and about 0.05% to 10% weight water.
  • the FPDSM system comprises betaine and urea at a molar ratio of about 1 : 1 (betaine/glucose) and about 0.05% to 10% weight water.
  • the FPDSM system comprises urea and glucose at a molar ratio of about 3: 1 (urea/glucose) and about 0.05% to 10% weight water. In certain other embodiments, the FPDSM system comprises urea and maltose at a molar ratio of about 6: 1 (urea/maltose) and about 0.05% to 10% weight water. In another embodiment, the FPDSM system comprises urea and lactose at a molar ratio of about 6: 1 (urea/lactose) and about 0.05% to 10% weight water.
  • the FPDSM system comprises urea and lactose at a molar ratio of about 9: 1 (urea/lactose) to about 1.5: 1 (urea/lactose) and about 4.0% to 6.0% weight water.
  • the FPDSM system water content is about 0.05% to 10% w/w. In another embodiment, the water content is about 5% to 10% w/w.
  • the enzyme is selected from the group consisting of acetyl esterases, aryl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -galactosidases, a-glucanases, glucan lysases, endo- -glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, is
  • the FPDSM system comprises at least two different enzymes.
  • the solid enzyme composition is a spray dried enzyme, a lyophilized enzyme, a precipitated enzyme, a crystalized enzyme, a spray cooled enzyme, spray chilled enzyme or a spray granulated enzyme.
  • the solidified FPDSM system of step (c) comprises a solid form selected from a solid pellet, a solid granule, a solid powder, a solid film and a solid extrudate.
  • the disclosure is related to a solid enzyme composition homogenously distributed and entrapped in a FPDSM system prepared by a method of the disclosure.
  • Figure 1 shows the viscosity (cP) of a two-component FPDSM system comprising betaine and urea at 95°C, plotted as a function of the betaine mole fraction.
  • Figure 2 shows the percent (%) recovery of a subtilisin protease, as the percentage of residual enzyme activity remaining after addition to a molten FPDSM composition, with subsequent solidification at room temperature (21° C in this particular case).
  • the percent residual activity is plotted as a function of water added to a FPDSM system set forth in Table 3.
  • Figure 3 shows a plot of percent (%) recovery of a subtilisin protease, as a function of the mixing temperature (°C) used to entrap the enzyme in a FPDSM system set forth in Table 2 of the specification.
  • the compositions of Figure 3 are the same as those of Figure 2; accordingly, the composition with higher percentages of water had lower melting points.
  • the present disclosure is generally related to solid protein compositions and specific formulations thereof which stabilize the solid protein compositions. More specifically, in certain embodiments, the disclosure is directed to formulations which stabilize solid protein compositions and concomitantly mitigate or inhibit certain environmental factors associated with enzymatic and non-enzymatic protein degradation. More particularly, Applicant of the instant disclosure has discovered that certain combinations (i.e., mixtures) of "solvent components" in specified ranges of molar ratios are particularly suitable for mitigating protein degradation, protein instability and/or protein inactivation, most particularly, thermally-induced protein inactivation.
  • Thermally-induced protein inactivation can arise by well-known biophysical and biochemical processes such as protein unfolding, protein aggregation, oxidation, glycation, deamidation, and the like.
  • Storage-stable protein formulations can be produced using a variety of protein formulation, particle formation, and drying processes.
  • certain embodiments of the disclosure are directed to storage stable solid protein compositions (e.g., a lyophilized solid protein composition, a spray dried solid protein composition, a spray cooled solid protein composition, a spray chilled solid protein composition, a spray granulated solid protein composition and the like), formulations thereof and methods thereof, wherein the solid protein of interest (POI) is entrapped in a "freezing point depressed solid matrix" (FPDSM) system of the disclosure.
  • POI solid protein of interest
  • FPDSM freezing point depressed solid matrix
  • the disclosure is directed to methods for preparing/formulating one or more "freezing point depressed solid matrix" (hereinafter "FPDSM”) systems which encapsulate solid protein compositions.
  • FPDSM freezing point depressed solid matrix
  • the disclosure is related to one or more solid protein compositions formulated in a FPDSM system described herein.
  • the disclosure is related to methods for inhibiting the degradation of solid protein compositions formulated with one or more FPDSM systems described herein.
  • the disclosure is related to stable solid protein compositions, formulations thereof, granulation processes thereof and methods thereof, wherein the solid protein of interest is distributed in a (molten liquid) FPDSM system of the disclosure, wherein each individual (FPDSM system) component is a solid that does not melt below 100°C, wherein the combined (molten liquid) FPDSM system solidifies and entraps the protein of interest at a temperature of about 20°C to 40°C.
  • certain embodiments are directed to one or more proteins of interest (e.g., an enzyme) formulated in a tunable FPDSM system of the disclosure, wherein the FPDSM system is tuned to be a liquid at a temperature between 40°C to about 100°C, and a solid at a temperature between about 20°C to 40°C.
  • proteins of interest e.g., an enzyme
  • the disclosure is related to solid protein compositions of interest (e.g., a protease, an amylase, a glucoamylase, a lipase, etc.) formulated in a tunable FPDSM system of the disclosure, wherein such FPDSM formulations mitigate the degradation and/or loss of activity of the solid protein composition, particularly solid protein compositions which are stored for a period of time (i.e., hours, days, weeks, months) before their end use.
  • solid protein compositions of interest e.g., a protease, an amylase, a glucoamylase, a lipase, etc.
  • a “freezing point depressed solid matrix” (abbreviated herein as, "FPDSM”), a freezing point depressed solid matrix “system” or a “FPDSM system” may be used interchangeably.
  • a “FPDSM system” comprises at least two (2) distinct FPDSM components, wherein each distinct (FPDSM system) component individually does not melt below 100°C, yet in combination the at least two (2) distinct FPDSM components melt at a temperature between 40°C to about 100°C.
  • a “FPDSM system” of the disclosure, comprising at least two (2) distinct FPDSM components further comprises up to about 10% (w/w) water.
  • a "distinct” FPDSM system component or simply a “FPDSM component”, as used in phrases such as a "distinct FPDSM system component", a “distinct FPDSM solvent component”, “individual FPDSM component” and the like includes, but is not limited to, FPDSM components selected from the group consisting of ammonium chloride (NH4CI), choline chloride, betaine ( ⁇ , ⁇ , ⁇ ,- trimethylglycine), betaine hydrochloride, citric acid, malonic acid, tartaric acid, sorbitol, sucrose, fructose, glucose, lactose, maltose, urea, and the like.
  • NH4CI ammonium chloride
  • choline chloride betaine ( ⁇ , ⁇ , ⁇ ,- trimethylglycine)
  • betaine hydrochloride citric acid, malonic acid, tartaric acid
  • sorbitol sucrose, fructose, glucose, lactose, maltose, urea, and
  • a "solid protein”, a “solid protein composition”, a “solid enzyme composition”, and the like specifically refer to a protein composition in its solid dried state (e.g., by means of lyophilization, spray drying, crystallization, spray cooling, spray chilling, spray granulated, and the like), in contrast to a "liquid protein composition", which liquid protein compositions are substantially hydrated (dissolved) in water or an aqueous buffer thereof.
  • solid enzyme a "solid” protein, a “solid” enzyme composition and the like
  • dry protein a “dry” enzyme, a “dried” enzyme, a “dried” protein, a “dried” enzyme composition and the like.
  • protein will be used hereinafter when referring to a “solid protein” composition of the disclosure.
  • solid protein composition of the disclosure.
  • molten solvent system particularly refers to a FPDSM system of the disclosure, wherein the at least two (2) FPDSM components have been combined and mixed at a temperature between 40°C to about 100°C, wherein the FPDSM system has been tuned to be in a liquid state at a temperature between 40°C to about 100°C (i. e., melt below about 100°C).
  • a “solid” or “solidified” FPDSM system of the disclosure particularly refers to the same FPDSM system, wherein the FPDSM system has been cooled to a temperature between about 20°C to 40°C, such that the FPDSM system solidifies, thereby entrapping and encapsulating the solid protein composition therein.
  • room temperature means a temperature between about 20°C to about 28°C.
  • ambient temperature means a temperature between about 20°C to about 40°C.
  • enzyme recovery is calculated as the weight percentage of active enzyme in the solidified FPDSM material relative to the enzyme activity in the initial solid spray dried enzyme powder.
  • a “stable” enzyme composition” e.g., as used in phrases such as “a stable enzyme composition”, “a storage stable enzyme composition” or “a FPDSM stabilized enzyme" particularly refers to a solid enzyme composition formulated in a FPDSM system the disclosure.
  • the enzymatic activity of the "stable" (FPDSM formulated) enzyme composition is relative to (vis-a-vis) the activity of the same (unformulated) enzyme composition (e.g., a solid enzyme, spray-dried control) when the solid enzyme (spray -dried control) is assayed (measured) under the same conditions.
  • a solid enzyme, spray-dried control e.g., a solid enzyme, spray-dried control
  • weight percent or “weight %” are abbreviated hereinafter “wt%” or “% w/w”.
  • the term protein or enzyme "melting temperature” (assuming two-state protein folding (F U)), is the denaturation midpoint “T m ", which is defined as the temperature at which both the folded (F) and unfolded (U) states are equally populated at equilibrium.
  • a "protein of interest” may be abbreviated as "POI", wherein a POI of the disclosure includes, but is not limited to, enzymes, transport proteins, structural proteins, defense or immune related proteins (cytokines; e.g., IL-12), regulatory proteins, receptor proteins, immunoglobulins, cytokines (e.g., IL- 12), and the like.
  • cytokines e.g., IL-12
  • cytokines e.g., IL-12
  • cytokines e.g., IL-12
  • immunoglobulins cytokines
  • cytokines e.g., IL- 12
  • a POI according to the instant disclosure is any solid protein composition contemplated for formulation and stabilization thereof in a FPDSM system of the disclosure.
  • polypeptide and “protein” are used interchangeably and include reference to polymers comprising amino acid residues linked by peptide bonds.
  • the conventional one (1) letter or three (3) letter codes for amino acid residues are used
  • polypeptide amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the terms also encompass amino acid polymers that have been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification).
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • a "variant" protein refers to a protein that is derived from a parent (or reference) protein by the substitution, addition, or deletion of one or more amino acids, typically by recombinant DNA techniques. Variant proteins may differ from a parent protein by a small number of amino acid residues and may be defined by their level of primary amino acid sequence homology/identity with a parent (reference) protein.
  • the disclosure are directed to one or more storage stable protein compositions, wherein the protein composition is distributed and encapsulated (entrapped) within an FPDSM system, wherein the FPDSM system comprises at least two FPDSM components (each of which individual FPDSM components does not melt below 100°C), wherein the FPDSM system is fine tuned to be a molten liquid at a temperature between 40°C to about 100°C, and a solid at a temperature between about 20°C to 40°C.
  • the processes for producing the FPDSM compositions of the disclosure can be generically described as melt solidification or melt granulation processes. More particularly, in all of these processes, a dry form of the protein (e.g., produced by processes such as lyophilization, spray drying, crystallization, precipitation, spray chilling and the like) is suspended in the molten (liquid) FPDSM solvent system and subsequently solidified at ambient temperatures (e.g., about 20°C to 40°C).
  • the resulting solidified formulations can be of any size or shape (i.e., monoliths, tablets, granules or powders).
  • melt solidification is melt granulation.
  • the molten FPDSM system in which the POI is suspended
  • the molten FPDSM system is atomized or formed into particles or granules, which are flowable and provide protection to the POI.
  • Granules are typically particles with diameters between about 50 microns and 1000 microns, or as large as 5000 microns.
  • melt granulation processes included spray cooling, spray chilling, spinning disk atomization, melt extrusion, and the like.
  • Examples of individual components suitable for combining in a FPDSM system of the disclosure are listed below in Table 1. Note that the melting point (in some cases, a decomposition point) of each individual component is greater than 100°C (i.e., none of these individual (distinct) components melts below 100°C). However, as stated above and further discussed below, when the components are combined in mixtures in certain ratios (and/or with the addition of up to 10% w/w water), the resulting (combined) FPDSM systems will melt at a temperature between 40°C to about 100°C, and yet will solidify at a temperature between about 20°C to 40°C.
  • a protein of interest is formulated in a tunable FPDSM system of the disclosure, wherein the POI is combined (added) with a FPDSM system at a temperature between 40°C to about 100°C.
  • the combined FPDSM/POI formulation is subsequently cooled to a temperature between about 20°C to 40°C (/ ' . e. , a temperature at which the FPDSM system is a solid), wherein the FPDSM encapsulates (entraps) the protein composition within a solid matrix as the FPDSM system transitions from a liquid state to a solid state during the cooling process.
  • a protein composition is formulated in a FPDSM system of the disclosure and encapsulated by cooling the FPDSM system as generally described above, wherein the encapsulated POI is substantially stabilized against various forms of environmental protein degradation. More particularly, certain embodiments are related to protein compositions formulated in a FPDSM system of the disclosure, wherein the FPDSM encapsulated composition may be stored at temperatures up to about 40°C or less for a period of time (e.g., an hour, a day, a week, a month, etc.) prior the end use of the particular protein (POI) composition.
  • a period of time e.g., an hour, a day, a week, a month, etc.
  • an elegant benefit of the tunable FPDSM systems of the instant disclosure, and more particularly the protein compositions formulated therein (and granules thereof), is that the protein composition (or granules thereof) is readily reconstituted for end use by dilution of the solid FPDSM composition in water (or an aqueous buffered solvent).
  • a protein composition is formulated in a FPDSM system of the disclosure, wherein the FPDSM encapsulated composition (or granules thereof) may be held at temperatures up to about 40°C or less for a period of time prior the end use of the particular protein composition, with minimal loss of protein activity thereof.
  • a FPDSM system of the disclosure comprises at least two (2) distinct FPDSM components.
  • a FPDSM system of the disclosure may additionally comprise up to about 10% (w/w) water.
  • such distinct FPDSM components are selected from the group consisting of ammonium chloride (NH4CI), choline chloride, betaine ( ⁇ , ⁇ , ⁇ ,-trimethylglycine), betaine hydrochloride, citric acid, malonic acid, tartaric acid, sorbitol, sucrose, fructose, glucose, lactose, maltose and urea.
  • an at least two component FPDSM system comprises betaine and monosaccharide, wherein the monosaccharide is selected from the group consisting of glucose, galactose, fructose, mannose, erythrose, ribose, glyceraldehyde, dihydroxyacetone, glycerol, sorbitol, mannitol and dulcitol.
  • any POI e.g., enzyme
  • T m thermal denaturation profile and melting temperature
  • optimal FPDSM systems are provided, wherein the protein composition is combined with the FPDSM system at temperatures between 40°C to about 100°C.
  • T m of a protein is generally defined as the temperature at which both the folded (F) and unfolded (U) states are equally populated at equilibrium.
  • a novel protein e.g., a novel enzyme or novel variant thereof
  • a combination of more than one protein e.g., a combination of two different enzymes
  • a denaturation profile and/or a protein melting temperature (T m ) profile may readily determine a denaturation profile and/or a protein melting temperature (T m ) profile using routine laboratory techniques and methods available in the art including, but not limited to, differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence anisotropy (polarized light), Fourier transform infrared spectroscopy (FTIR), hydrophobic fluorophores, intrinsic (tryptophan) fluorescence lifetime changes, light scattering, ultracentrifugation sedimentation velocity analysis and the like.
  • DSC differential scanning calorimetry
  • CD circular dichroism
  • fluorescence anisotropy polarized light
  • FTIR Fourier transform infrared spectroscopy
  • hydrophobic fluorophores intrinsic (tryptophan) fluorescence lifetime changes, light scattering, ultracentrifugation sedimentation velocity analysis and the like.
  • subtilisin protease i.e., Properase ® enzyme
  • FPDSM systems of the disclosure at elevated temperatures (e.g. 80-100°C), wherein it is generally known in the art that subtilisin proteases (e.g., including, but not limited to Properase ® ) are relatively unstable at temperatures of about 55°C and above.
  • the water (e.g., up to about 10% w/w) in such FPDSM systems is sequestered, and hence less available than "free" water to interact with the POI in a manner that would lead to inactivation or other manifestations of irreversible structural destabilization.
  • FIG. 2 of the disclosure demonstrates excellent enzyme recovery in the FDPSM at up to 10% w/w water, wherein it is possible that even more than 10% w/w water can be incorporated into the FDPSM without adverse impact on enzyme recovery.
  • the melting point of a solid matrix suitable for the formulation of enzymes can be depressed, and thereby "tuned” to more enzyme or protein friendly temperatures, by means of the two levers of the instant disclosure comprising: (1) blending two or more solid components to form a FPDSM blend; and (2) incorporating water, to further depress the melting point.
  • the compositions of the instant disclosure comprise FPDSM systems suitable for entrapping enzymes and thereby improving their recovery and stability.
  • Certain embodiments of the disclosure are directed to one or more proteins of interest (e.g., an enzyme) formulated in a tunable FPDSM system of the disclosure, wherein the FPDSM system is tuned to be a liquid at temperatures of 40°C to about 100°C, and a solid at temperatures between about 20°C to 40°C. More particularly, in certain embodiments the disclosure is directed to a protein composition of interest formulated in a tunable FPDSM system of disclosure, wherein such FPDSM systems significantly mitigate the degradation and/or loss of activity of the protein composition, particularly such formulated protein compositions which are stored for a period of time (i.e., days, weeks, months) before their end use.
  • a period of time i.e., days, weeks, months
  • FPDSM system (solvent) components were screened at the indicated (component 1): (component 2) molar ratios specified in the Tables 2 and 3: ammonium chloride, choline chloride, betaine ( ⁇ , ⁇ , ⁇ ,-trimethylglycine), betaine hydrochloride, citric acid, malonic acid, tartaric acid, sorbitol, fructose, sucrose, glucose, lactose, maltose, urea and water.
  • the FPDSM system components (Table 2 or Table 3) were screened in the Example 1 with the specific parameters that: (a) the combined FPDSM system components melt (form a liquid) at a temperature between 40°C to about 100°C (i.e., to mitigate thermal denaturation/inactivation of the protein) and (b) that the FPDSM system should quickly solidify at a temperature between about 20°C to 40 ° C, thereby quickly forming a robust solid matrix, suitable for encapsulation of proteins. As described in Example 2, the FPDSM systems meeting the aforementioned screening parameters were subsequently prepared/formulated with an active POI (i.e., a subtilisin protease).
  • an active POI i.e., a subtilisin protease
  • the POI formulations presented in Example 2 are FPDSM systems comprising added water (e.g. 5% w/w), wherein the following solvent component combinations were assessed: (i) anhydrous betaine/urea (1 : 1 mole ratio) and 5% (weight) water, and (ii) urea/lactose (6: 1 mole ratio) and 5% (weight) water.
  • Example 2 FPDSM systems were assayed as described in Example 3, wherein the percent (%) recovered protease activity of the anhydrous betaine/urea (1 : 1 mole ratio) + 5% water formulation was 71.8% and the percent (%) recovered protease activity of the urea/lactose (6: 1 mole ratio) + 5% water was 87.3%. (e.g., see, Table 5)
  • Example 4 the ability to fine tune such FPDSM systems are demonstrated in Example 4.
  • certain FPDSM systems which did not melt (i.e., form a liquid) at a temperature feasible for formulating the subtilisin protease i.e., at a temperature of 40°C to about 100°C were re-formulated (i.e., fine-tuned) with the addition of water (e.g., up to about 10% w/w).
  • Example 6 the ability to fine tune such FPDSM systems by the addition of a specified weight percent of water, thereby lowering the FPDSM system mixing temperature, significantly enhances the recovered protease activity of these formulations (e.g., see, Table 6, urea/ammonium chloride (1:4) + 10% water and betaine/urea (1 : 1) + 5% water).
  • a comparison of the urea/glucose (1 : 1) + 5% water (FPDSM system) relative to the urea/glucose (1 : 1) + 10% water (FPDSM system) indicates that the water addition does not significantly reduce the mixing temperature going from 5% water (i.e., 80°C) to 10% water (i.e., 80°C), whereas the recovered protease activity from the same formulations indicate that the water in such FPDSM systems improves the recovery of active protease (e.g., the Properase ® relative residual activity of the urea/glucose (1 : 1) + 5% water (FPDSM system) was 79.7%, whereas the Properase ® relative residual activity of the urea/glucose (1 : 1) + 10% water (FPDSM system) increased to 93%).
  • active protease e.g., the Properase ® relative residual activity of the urea/glucose (1 : 1) + 5% water (FPDSM
  • Example 5 provides an exemplary analysis of the protease activity (i.e., Properase ® ) as a function of different mixing temperatures, which analysis is readily extended to other proteins of interest contemplated herein. For example, as shown in FIG. 3., there was an evident decrease in protease activity as the mixing temperature generally exceeded 80°C.
  • protease activity i.e., Properase ®
  • Example 6 and Example 7 of the disclosure further describe methods for granulating such protein encapsulated FPDSM systems, which protein/FPDSM system granules are particularly suitable for short- term or long-term storage thereof.
  • Example 8 of the disclosure demonstrates the storage stability of such protein/FPDSM system formulations.
  • a subtilisin protease was formulated in a FPDSM system of the disclosure, wherein the storage stability of the protease/FPDSM system formulation was evaluated at 25°C for a period of 28 days. More specifically, as presented in Table 7, the protease activity at days 7, 14, 21 and 28, remained well above 98% recovered activity. Furthermore, when compared to the activity of the control protease (i.e., spray dried protease) at days 7, 14, 21 and 28, the recovered protease activity of all FPDSM systems demonstrate that greater protease activity was maintained relative to the spray dried protease.
  • the control protease i.e., spray dried protease
  • Example 9 of the disclosure further describes a second POI (i.e., an amylase) formulated and stabilized in a FPDSM system of the disclosure. More particularly, Example 9 describes the maximum solubility of an a-amylase protein of interest in a choline chloride/urea (1 :2 ratio) FPDSM system (e.g., see, FIG. 4)
  • the disclosure is directed to such FPDSM systems.
  • the disclosure is related to one or more solid protein compositions formulated and stabilized in such FPDSM systems of the disclosure.
  • the disclosure is related to storage stable solid enzyme compositions comprising a FPDSM system formulation disclosed herein, wherein such (FPDSM stabilized) solid enzyme compositions retain at least about 50% to 100% enzymatic activity after at least 24-hours of storage at 25°C. III. FPDSM Stabilized Proteins of Interest and Recovered Activity Thereof
  • a protein of interest can be any endogenous or heterologous protein or a variant thereof.
  • the protein can contain one or more disulfide bridges or is a protein whose functional form is a monomer or a multimer, i.e. , the protein has a quaternary structure and is composed of a plurality of identical (homologous) or non-identical (heterologous) subunits.
  • certain embodiments of the disclosure are directed to one or more proteins of interest formulated in a protein stabilizing FPSDM system of the disclosure.
  • a protein of interest is an enzyme, a hormone, a growth factor, a cytokine, an antibody, a receptor, and the like.
  • a POI is an enzyme selected from acetyl esterases, aryl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinases, deoxyribonucleases, epimerases, esterases, a-galactosidases, ⁇ -galactosidases, a-glucanases, glucan lysases, endo- -glucanases, glucoamylases, glucose oxidases, a-glucosidases, ⁇ -glucosidases, glucuronidases, hemi
  • a POI or a variant POI thereof is an enzyme selected from Enzyme Commission (EC) Number EC 1, EC 2, EC 3, EC 4, EC 5 or EC 6.
  • a POI is an oxidoreductase enzyme, including, but not limited to, an EC 1 (oxidoreductase) enzyme selected from EC 1.10.3.2 (e.g., a laccase), EC 1.10.3.3 (e.g., L- ascorbate oxidase), EC 1.1.1.1 (e.g., alcohol dehydrogenase), EC 1.1 1.1.10 (e.g., chloride peroxidase), EC 1.11.1.17 (e.g., peroxidase), EC 1.1.1.27 (e.g., L-lactate dehydrogenase), EC 1.1.1.47 (e.g., glucose 1- dehydrogenase), EC 1.1.3.X (e.g., glucose oxidase), EC 1.1.3.10 (e.g., pyranose oxidase), EC 1.13.1 l .X (e.g., dioxygena
  • l .X e.g., fatty acid reductase
  • EC 1.2.1.10 e.g., acetaldehyde dehydrogenase
  • EC 1.5.3.X e.g., fructosyl amine reductase
  • EC 1.8.1.X e.g., disulfide reductase
  • EC 1.8.3.2 e.g., thiol oxidase
  • a POI is a transferase enzyme, including, but not limited to, an EC 2 (transferase) enzyme selected from EC 2.3.2.13 (e.g., transglutaminase), EC 2.4.
  • an EC 2 (transferase) enzyme selected from EC 2.3.2.13 (e.g., transglutaminase), EC 2.4.
  • l .X e.g., hexosyltransferase
  • EC 2.4.1.40 e.g., altemasucrase
  • EC 2.4.1.18 e.g., 1,4 alpha-glucan branching enzyme
  • EC 2.4.1.19 e.g., cyclomaltodextrin glucanotransferase
  • EC 2.4.1.2 e.g., dextrin dextranase
  • EC 2.4.1.20 e.g., cellobiose phosphorylase
  • EC 2.4.1.25 e.g., 4-alpha-glucanotransferase
  • EC 2.4.1.333 e.g., 1,2-beta-oligoglucan phosphor transferase
  • EC 2.4.1.4 e.g., amylosucrase
  • EC 2.4.1.5 e.g., dextransucrase
  • EC 2.4.1.69
  • a POI is a hydrolase enzyme, including, but not limited to, an EC 3 (hydrolase) enzyme selected from EC 3.1.X.X (e.g., an esterase), EC 3.1.1.1 (e.g., pectinase), EC 3.1.1.14 (e.g., chlorophyllase), EC 3.1.1.20 (e.g., tannase), EC 3.1.1.23 (e.g., glycerol-ester acylhydrolase), EC 3.1.1.26 (e.g., galactolipase), EC 3.1.1.32 (e.g., phospholipase Al), EC 3.1.1.4 (e.g., phospholipase A2), EC 3.1.1.6 (e.g., acetylesterase), EC 3.1.1.72 (e.g., acetylxylan esterase), EC 3.1.1.73 (e.g., ferase).
  • X e.g., serine-type carboxypeptidase
  • EC 3.4.16.5 e.g., carboxypeptidase C
  • EC 3.4.19.3 e.g., pyroglutamyl-peptidase I
  • EC 3.4.21.X e.g., serine endopeptidase
  • EC 3.4.21.1 e.g., chymotrypsin
  • EC 3.4.21.19 e.g., glutamyl endopeptidase
  • EC 3.4.21.26 e.g., prolyl oligopeptidase
  • EC 3.4.21.4 e.g., trypsin
  • EC 3.4.21.5 e.g., thrombin
  • EC 3.4.21.63 e.g., oryzin
  • EC 3.4.21.65 e.g., thermomycolin
  • EC 3.4.21.80 e.g., strept
  • X e.g., metalloendopeptidase
  • EC 3.4.24.39 e.g., deuterolysin
  • EC 3.4.24.40 v., serralysin
  • EC 3.5.1.1 e.g., asparaginase
  • EC 3.5.1.1 1 e.g., penicillin amidase
  • EC 3.5.1.14 e.g., N-acyl-aliphatic-L-amino acid amidohydrolase
  • EC 3.5.1.2 e.g., L-glutamine amidohydrolase
  • EC 3.5.1.28 e.g., N-acetylmuramoyl-L- alanine amidase
  • EC 3.5.1.4 e.g., amidase
  • EC 3.5.1.44 e.g., protein-L-glutamine amidohydrolase
  • EC 3.5.1.5 e.g., urease
  • a POI is a lyase enzyme, including, but not limited to, an EC 4 (lyase) enzyme selected from EC 4.1.2.10 (e.g., mandelonitrile lyase), EC 4.1.3.3 (e.g., N-acetylneuraminate lyase), EC 4.2.1.1 (e.g., carbonate dehydratase), EC 4.2.2.- (e.g., rhamnogalacturonan lyase), EC 4.2.2.10 (e.g., pectin lyase), EC 4.2.2.22 (e.g., pectate trisaccharide-lyase), EC 4.2.2.23 (e.g. , rhamnogalacturonan endolyase) and EC 4.2.2.3 (e.g. , mannuronate-specific alginate lyase).
  • an EC 4 (lyase) enzyme selected from
  • a POI is an isomerase enzyme, including, but not limited to, an EC 5 (isomerase) enzyme selected from EC 5.1.3.3 (e.g. , aldose 1-epimerase), EC 5.1.3.30 (e.g., D-psicose 3- epimerase), EC 5.4.99.1 1 (e.g., isomaltulose synthase) and EC 5.4.99.15 (e.g., (l ⁇ 4)-a-D-glucan 1-a-D- glucosylmutase).
  • an EC 5 (isomerase) enzyme selected from EC 5.1.3.3 (e.g. , aldose 1-epimerase), EC 5.1.3.30 (e.g., D-psicose 3- epimerase), EC 5.4.99.1 1 (e.g., isomaltulose synthase) and EC 5.4.99.15 (e.g., (l ⁇ 4)-a-D-glucan 1-a-D-
  • a POI is a ligase enzyme, including, but not limited to, an EC 6 (ligase) enzyme selected from EC 6.2.1.12 (e.g., 4-coumarate: coenzyme A ligase) and EC 6.3.2.28 (e.g., L-amino- acid alpha-ligase).
  • a POI of the disclosure is an industrial protease or amylase enzyme, which protease or amylase is stabilized and formulated in an FPDSM system of the disclosure.
  • a POI is neutral protease (or "metalloproteases") or an alkaline (or "serine”) protease.
  • Bacillus subtilisin proteins are exemplary serine proteases for use and stabilization in a FPDSM composition of the disclosure. More particularly, a wide variety of Bacillus subtilisins have been identified and sequenced, for example, subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 147 and subtilisin 309 (e.g. , WO 1989/06279 and Stahl et al., 1984).
  • variant proteases suitable for formulation in a FPDSM system of the disclosure such as PCT Publication Nos. WO1999/20770; WO1999/20726; WO1999/20769; WO1989/06279; U.S. RE34,606; U.S. Patent Nos. 4,914,031; 4,980,288; 5,208,158; 5,310,675; 5,336,611; 5,399,283; 5,441,882; 5,482,849; 5,631,217; 5,665,587; 5,700,676; 5,741 ,694; 5,858,757; 5,880,080; 6, 197,567 and 6,218,165.
  • a POI is an amylase formulated and stabilized in an FPDSM system of the disclosure.
  • amylase enzymes and variants thereof are known to one skilled in the art.
  • International PCT Publication NO. WO2006/037484 and WO 2006/037483 describe variant a-amylases having improved solvent stability
  • Publication No. WO 1994/18314 discloses oxidatively stable a-amylase variants
  • Publication No. WO 1999/ 19467, WO2000/29560 and WO2000/60059 disclose Termamyl-like ⁇ -amylase variants, Publication No.
  • WO2008/112459 discloses a- amylase variants derived from Bacillus sp. number 707
  • Publication No. W01999/43794 discloses maltogenic a-amylase variants
  • Publication No. WO1990/11352 discloses hyper-thermostable a-amylase variants
  • Publication No. WO2006/089107 discloses ⁇ -amylase variants having granular starch hydrolyzing activity.
  • a POI formulated and stabilized in a FPDSM system of the disclosure is a peptide, a peptide hormone, a growth factor, a clotting factor, a chemokine, a cytokine, a lymphokine, an antibody, a receptor, an adhesion molecule, a microbial antigen (e.g. , HBV surface antigen, HPV E7, etc.), variants thereof, fragments thereof and the like .
  • Other types of proteins (or variants thereof) of interest may be those that are capable of providing nutritional value to a food or to a crop.
  • Non-limiting examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g., a higher lysine content than a non-transgenic plant).
  • certain embodiments are directed to FPDSM stabilized protein compositions. More particularly, certain embodiments are related to a solid enzyme composition stabilized in a FPDSM system of the disclosure.
  • the activity of an enzyme of interest e.g., a protease
  • the activity of an FPDSM formulated enzyme composition is measured after two (2) hours, after twelve (12) hours, after one (1) day, after seven (7) days, after twenty -one (21) days, etc. of storage at room temperature (RT; about 20-25°C).
  • RT room temperature
  • certain embodiments are related to recovered enzymatic activity of a FPDSM formulated enzyme composition which has been stored for a period of time prior to its end-use.
  • assays There are various assays known to those of ordinary skilled in the art for detecting and measuring activity of proteins.
  • proteases there are assays based on the release of acid-soluble peptides from casein or hemoglobin measured as absorbance at 280 nm or colorimetrically, using the Folin method (e.g., Bergmeyer et al, 1984).
  • Other assays involve the solubilization of chromogenic substrates (see e.g., Ward, 1983).
  • Other exemplary assays include succinyl-Ala-Ala-Pro-Phe-para-nitroanilide assay (SAAPFpNA) and the 2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay).
  • SAAPFpNA succinyl-Ala-Ala-Pro-Phe-para-nitroanilide assay
  • TNBS assay 2,4,6-trinitrobenzene sulfonate sodium salt assay
  • Applicant screened various solvent components (see, Table 1) to identify solvent component mixtures (see, Tables 2 and 3) meeting specific pre-determined parameters which are relevant to the stability and formulation requirements of one or more protein compositions of the disclosure.
  • the solvent component mixtures were screened for solvent component mixtures having melting points lower than 100°C, and an ability to solidify at a temperature above 20°C.
  • Other considerations for solvent component mixtures may include suitable viscosity and non- hygroscopic behavior.
  • Viscosity A Brookfield viscometer equipped with Spindle 18 was used to determine the viscosity of the betaine/urea compositions at different molar ratios. About Ten (10) grams of each composition was placed in the sample container and heated to about 10°C above the apparent melting temperature (i.e., the apparent T m ; identified from the initial screen with a continuous running water bath attached to a thermocouple). The viscosity, measured in cP (centiPoise), was recorded after one (1) minute. Table 4 and FIG. 1 show the viscosity of betaine/urea compositions at different molar ratios of mixture components at 95°C. TABLE 2
  • the solvent component mixtures contemplated in the present disclosure need to (A) melt at a temperature between 40°C to about 100°C; and (B) quickly solidify at a temperature between about 20°C to 40°C, thereby quickly forming a robust protein (encapsulated) solid mixture.
  • samples of solvent component mixtures were prepared as follows: water at a specific weight percent (wt %) was added to a container, followed by the addition of thirty (30) grams of the solvent mixture components in the specified molar ratio. The solvent components and water were then mixed at 10 ° C above the predicted melting temperature on a hot plate.
  • subtilisin protease preparation was subsequently poured onto a sheet of aluminum foil and left at room temperature (i.e., 21°C in this particular case) to solidify.
  • the solvent components tested in the present example were: (1) anhydrous betaine (i.e., ⁇ , ⁇ , ⁇ -trimethylglycine), (2) urea, (3) lactose and (4) water. More particularly, the following solvent component mixtures were formulated for encapsulating and stabilizing the solid enzyme (Properase®): (A) anhydrous betaine/urea (1 : 1 mole ratio) and 5% (weight) water at 85°C, and (B) urea/lactose (6: 1 mole ratio) and 10% (weight) water at 80°C (e.g., see Table 5).
  • Etrase® anhydrous betaine/urea (1 : 1 mole ratio) and 5% (weight) water at 85°C
  • B urea/lactose (6: 1 mole ratio) and 10% (weight) water at 80°C (e.g., see Table 5).
  • FPDSM formulated (protease) enzyme i. e., Properase ® ; formulated as described above in Example 2
  • the solidified enzyme cast from encapsulation was transferred to a tared 250 mL Nalgene bottle and weighed.
  • Tris buffer pH 8.6
  • the resulting dissolved liquid (subtilisin protease) preparation was tested for proteolytic activity against the substrate N-succinyl-L-Ala- L-Ala-L-Pro-L-Phe-/J>-nitroanilide (hereinafter, "Suc-AAPF-pNA"; Sigma, St. Louis, MO, Product No. S- 7388), wherein Suc-AAPF-pNA was used as to make standard curve.
  • the Suc-AAPF-pNA control and UFC purified protease (Properase ® ) were used as standards, wherein Suc-AAPF-pNA was used as substrate (as generally described in Example 4 of U.S. Patent Publication No. US2015/0147768).
  • Suc-AAPF-pNA assay was generally performed as follows: In order to determine protease activity, the hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-p-nitroanilide (suc- AAPF-pNA) was measured.
  • the reagent solutions used were: 100 mM Tris/HCl, pH 8.6, containing 0.005% TWEEN ® -80 (Tris dilution buffer); 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl.sub.2 and 0.005% TWEEN ® -80 (Tris/Ca buffer); and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388).
  • suc-AAPF-pNA working solution 1 ml suc-AAPF-pNA stock solution was added to 50 ml Tris/Ca buffer and mixed repeatedly.
  • the assay was performed by adding 5 ⁇ of diluted culture supernatant to each well containing 150 ⁇ Tris dilution buffer, immediately followed by the addition of 100 ⁇ of 2 mg/ml suc-AAPF-pNA working solution. The solutions were mixed for 5 seconds, and the absorbance change in kinetic mode (1 1 readings in 2 minutes) was read at 405 nm in an MTP reader, at 25°C The protease activity was expressed as AU absorbance at 405 nm.
  • Anhydrous Betaine Urea (1: 1) + 5% weight water; heated to 85°C, enzyme added and hand-mixed for 60 seconds.
  • Properase® protease enzyme was added to FPDSM solvent component mixtures at different temperatures (e.g., see, FIG. 3). For example, specific temperatures were chosen wherein a specific solvent component mixture would remain a molten liquid system. Thus, as set forth in FIG. 3, the Properase ® enzymatic activity (as measured by the "Suc-AAPF-pNA assay" described above) was recorded and plotted as a function of temperature. As presented in FIG. 3, there is an evident decrease in protease activity at mixing temperatures generally exceeding 80-85 ° C. EXAMPLE 6
  • a protein of interest is formulated in an approximately 420 grams of a solvent component mixture comprising urea/betaine (1 : 1) + water (4.5%) formulation, according to the following protocol.
  • a protein of interest was formulated in an approximately 1 OOg solvent component mixture comprising urea/lactose (6: 1) and water (4.4%), according to a similar protocol as described above in Example 6.
  • Procedure Set up / Get ready a 95 ° C and an 85 ° C hot water bath.
  • proteins e.g., enzymes
  • protein degradation e.g., heat, oxidation, UV light degradation, contaminants, and the like
  • a protease enzyme e.g., Properase ®
  • FPDSM system i.e., comprising urea and lactose
  • the solvent component mixtures were prepared using lactose and urea with a bulk congealing method as described in the following exemplary procedure for a 30 g batch, consisting of 90 mol% urea and 10 mol% lactose (i. e., urea/lactose; 9: 1) and approximately 4.5 wt% water and approximately 5.4 wt% (Properase ® ) enzyme.
  • the ground materials were subsequently screened using a Retsch Vibratory Sieve Shaker and size- classified.
  • the residual protease activity was greater than 88% during the first month (i. e., 28-days) of storage for all urea/lactose solvent composition ratios tested (i. e., 9: 1, 8.5 : 1.5, 3 : 1 and 6:4).
EP18804821.9A 2017-11-02 2018-11-01 Feste matrixzusammensetzungen mit gesenktem gefrierpunkt für die schmelzgranulation von enzymen Pending EP3703661A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762580581P 2017-11-02 2017-11-02
PCT/US2018/058665 WO2019089898A1 (en) 2017-11-02 2018-11-01 Freezing point depressed solid matrix compositions for melt granulation of enzymes

Publications (1)

Publication Number Publication Date
EP3703661A1 true EP3703661A1 (de) 2020-09-09

Family

ID=64362697

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18804821.9A Pending EP3703661A1 (de) 2017-11-02 2018-11-01 Feste matrixzusammensetzungen mit gesenktem gefrierpunkt für die schmelzgranulation von enzymen

Country Status (2)

Country Link
EP (1) EP3703661A1 (de)
WO (1) WO2019089898A1 (de)

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4760025A (en) 1984-05-29 1988-07-26 Genencor, Inc. Modified enzymes and methods for making same
US5310675A (en) 1983-06-24 1994-05-10 Genencor, Inc. Procaryotic carbonyl hydrolases
US5763257A (en) 1984-05-29 1998-06-09 Genencor International, Inc. Modified subtilisins having amino acid alterations
US5182204A (en) 1984-05-29 1993-01-26 Genencor International, Inc. Non-human carbonyl hydrolase mutants, vectors encoding same and hosts transformed with said vectors
JP2599946B2 (ja) 1986-01-15 1997-04-16 アムジェン，インコーポレイテツド ズブチリシン類似体
US4980288A (en) 1986-02-12 1990-12-25 Genex Corporation Subtilisin with increased thermal stability
US4914031A (en) 1987-04-10 1990-04-03 Amgen, Inc. Subtilisin analogs
DK6488D0 (da) 1988-01-07 1988-01-07 Novo Industri As Enzymer
CN1056187C (zh) 1988-02-11 2000-09-06 金克克国际有限公司 新的蛋白水解酶及其在洗涤剂中的应用
DE3909096A1 (de) 1989-03-20 1990-09-27 Garabed Antranikian Alpha-amylase
US5665587A (en) 1989-06-26 1997-09-09 Novo Nordisk A/S Modified subtilisins and detergent compositions containing same
DK97190D0 (da) 1990-04-19 1990-04-19 Novo Nordisk As Oxidationsstabile detergentenzymer
US5482849A (en) 1990-12-21 1996-01-09 Novo Nordisk A/S Subtilisin mutants
EP0583339B1 (de) 1991-05-01 1998-07-08 Novo Nordisk A/S Stabilisierte enzyme und waschmittelzusammensetzungen
DE69415659T3 (de) 1993-02-11 2010-05-12 Genencor International, Inc., Palo Alto Oxidativ stabile alpha-amylase
US6509183B1 (en) 1993-02-25 2003-01-21 Kao Corporation DNA fragment containing gene for alkaline pullulanase
DE4411223A1 (de) 1994-03-31 1995-10-05 Solvay Enzymes Gmbh & Co Kg Verwendung alkalischer Proteasen in gewerblichen Textilwaschverfahren
ATE423192T1 (de) 1997-10-13 2009-03-15 Novozymes As Mutanten der alpha-amylase
AR015977A1 (es) 1997-10-23 2001-05-30 Genencor Int Variantes de proteasa multiplemente substituida con carga neta alterada para su empleo en detergentes
US6287839B1 (en) 1997-11-19 2001-09-11 Genencor International, Inc. Cellulase producing actinomycetes, cellulase produced therefrom and method of producing same
US6562612B2 (en) 1997-11-19 2003-05-13 Genencor International, Inc. Cellulase producing actinomycetes, cellulase produced therefrom and method of producing same
NZ505820A (en) 1998-02-27 2002-10-25 Novozymes As Enzyme variants based on the 3D structure of maltogenic alpha-amylase that have an altered pH optimum, thermostability, specific activity, cleavage pattern and ability to reduce the staling of bread
US6835550B1 (en) 1998-04-15 2004-12-28 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
US6197565B1 (en) 1998-11-16 2001-03-06 Novo-Nordisk A/S α-Amylase variants
US6268328B1 (en) 1998-12-18 2001-07-31 Genencor International, Inc. Variant EGIII-like cellulase compositions
ES2496568T3 (es) 1999-03-30 2014-09-19 Novozymes A/S Variantes de alfa-amilasa
AU7405200A (en) * 1999-10-01 2001-05-10 Novozymes A/S Spray dried enzyme product
FI108728B (fi) 1999-10-12 2002-03-15 Carbozyme Oy Menetelmä perheen G/11 ksylanaasien stabiilisuuden parantamiseksi ja optimaalisen pH-alueen muuttamiseksi
US7718411B1 (en) 2004-08-05 2010-05-18 Danisco Us Inc. Trichoderma reesei G/11 xylanases with improved stability
EP1250423B1 (de) 2000-01-12 2008-09-03 Novozymes A/S Pullulanase variante mit vorbestimmten eigenschaften und verfahren zur deren herstellung
PT1263941E (pt) 2000-03-08 2011-01-20 Danisco Variantes de xilanase tendo sensibilidade alterada a inibidores de xilanase
KR100390565B1 (ko) 2000-06-20 2003-07-07 주식회사 씨티씨바이오 만난아제를 생산하는 신규한 바실러스 속 ｗｌ-1 균주
EP1444246B1 (de) 2001-10-26 2015-12-30 Danisco US Inc. Phytaseenzyme von trichoderma reesei, nukleinsäuren, die für solche phytaseenzyme codieren, vektoren und wirtszellen, in die diese eingebaut sind, und verfahren zu deren herstellung und verwendung
AU2002356855A1 (en) 2001-10-26 2003-05-12 Genencor International, Inc. Phytase enzymes, nucleic acid sequences encoding phytase enzymes and vectors and host cells incorporating same
CA2472723C (en) 2002-01-16 2013-12-17 Genencor International, Inc. Multiply-substituted protease variants
AU2003258157A1 (en) 2002-08-12 2004-02-25 Genencor International, Inc. Mutant e. coli appa phytase enzymes
CN101410520B (zh) 2003-04-29 2012-05-30 金克克国际有限公司 新颖的杆菌029cel纤维素酶
JP4757191B2 (ja) 2003-04-30 2011-08-24 ジェネンコー・インターナショナル・インク 新規なバチルスｍＨＫｃｅｌセルラーゼ
WO2004099369A2 (en) 2003-04-30 2004-11-18 Genencor International, Inc. Novel bacillus bagcel cellulase
RU2006131052A (ru) * 2004-01-30 2008-03-10 БАСФ Акциенгезельшафт (DE) Стабилизированные композиции фосфатаз
WO2005093050A2 (en) 2004-03-25 2005-10-06 Genencor International, Inc. Cellulase fusion protein and heterologous cellulase fusion construct encoding the same
DE102004047776B4 (de) 2004-10-01 2018-05-09 Basf Se Gegen Di- und/oder Multimerisierung stabilisierte Alpha-Amylase-Varianten, Verfahren zu deren Herstellung sowie deren Verwendung
DE102004047777B4 (de) 2004-10-01 2018-05-09 Basf Se Alpha-Amylase-Varianten mit erhöhter Lösungsmittelstabilität, Verfahren zu deren Herstellung sowie deren Verwendung
AU2005290934C1 (en) 2004-10-04 2013-05-09 Dupont Nutrition Biosciences Aps Citrobacter freundii phytase and homologues
GB0422052D0 (en) 2004-10-04 2004-11-03 Dansico As Enzymes
GB0423139D0 (en) 2004-10-18 2004-11-17 Danisco Enzymes
WO2006071598A1 (en) 2004-12-23 2006-07-06 Genencor International, Inc. Neutral cellulase catalytic core and method of producing same
WO2006089107A1 (en) 2005-02-18 2006-08-24 Genencor International, Inc. Polypeptides having alpha-amylase and granular starch hydrolyzing activity
KR20080059198A (ko) * 2005-10-12 2008-06-26 제넨코 인터내셔날 인코포레이티드 안정적이고 내구적인 활성제-함유 과립
US20080090747A1 (en) 2006-07-18 2008-04-17 Pieter Augustinus Protease variants active over a broad temperature range
US7968691B2 (en) 2006-08-23 2011-06-28 Danisco Us Inc. Pullulanase variants with increased productivity
BRPI0718226B1 (pt) 2006-10-06 2020-10-27 Danisco Us Inc., Genencor Division célula hospedeira de bacillus sp. recombinante, cultura da mesma e métodos de produção da referida célula e de proteína aditivo para detergentes
BRPI0806886A2 (pt) 2007-01-30 2014-04-29 Novozymes As Polipeptídeo, fitase, variante de um polipeptídeo, polinucleotídeo, construto de ácido nucleico, célula hospedeira recombinante, método para produzir o polipeptídeo, e, uso da fitase ou de uma composição
EP2115143B1 (de) 2007-02-07 2014-07-16 Danisco US, Inc., Genencor Division Varianten von buttiauxella sp phytasen mit veränderten eigenschaften
WO2008112459A2 (en) 2007-03-09 2008-09-18 Danisco Us Inc., Genencor Division Alkaliphilic bacillus species a-amylase variants, compositions comprising a-amylase variants, and methods of use
DK2132316T3 (da) 2007-03-23 2015-06-22 Danisco Us Inc Forøget amylaseproduktion ved n-terminal addition til modent amylaseprotein
CN101815783A (zh) 2007-05-30 2010-08-25 丹尼斯科美国公司 地衣芽孢杆菌α-淀粉酶的改良变体
RU2009149406A (ru) 2007-05-30 2011-07-10 ДАНИСКО ЮЭс, ИНК., ДЖЕНЕНКОР ДИВИЖН (US) Варианты альфа-амилазы с повышенными уровнями продукции в процессах ферментации
CA2693084A1 (en) 2007-06-08 2008-12-18 Benjamin S. Bower Heterologous and homologous cellulase expression system
CA2698765A1 (en) 2007-09-07 2009-03-19 Danisco Us Inc. Beta-glucosidase enhanced filamentous fungal whole cellulase compositions and methods of use
US8247198B2 (en) 2007-09-21 2012-08-21 Friedrich Srienc Enzymatic processing in deep eutectic solvents
EP2222842B1 (de) 2007-11-20 2014-10-15 Danisco US Inc. Glucoamylase-varianten mit veränderten eigenschaften
DK2283124T3 (en) 2008-04-18 2016-09-05 Danisco Us Inc BUTTIAUXELLA SP. PHYTASE VARIANTS
US9090887B2 (en) 2008-06-06 2015-07-28 Danisco Us Inc. Variant alpha-amylases from Bacillus subtilis and methods of use, thereof
BRPI0913402B1 (pt) 2008-06-06 2019-07-02 Danisco Us Inc. Alfa amilases (amys) variantes de geobacillus stearothermophilus com propriedades melhoradas
EP2626422A1 (de) 2008-06-06 2013-08-14 Danisco US Inc. Zusammensetzungen und Verfahren mit Cellulasevarianten mit verminderter Affinität gegenüber cellulosefreien Materialien
JP5508431B2 (ja) 2008-11-11 2014-05-28 ダニスコ・ユーエス・インク スブチリシン変異体を含む組成物及び方法
MX339402B (es) 2008-11-11 2016-05-25 Danisco Us Inc Composiciones y metodos que comprenden variantes de serina proteasa.
EA201170879A1 (ru) 2008-12-23 2012-02-28 Даниско А/С Полипептиды с ксиланазной активностью
GB0922467D0 (en) 2009-04-24 2010-02-03 Danisco Feed supplement
DK3591046T3 (da) 2009-05-19 2021-07-26 Dupont Nutrition Biosci Aps Amylasepolypeptide
KR20120062643A (ko) 2009-06-03 2012-06-14 다니스코 유에스 인크. 향상된 발현, 활성 및/또는 안정성을 갖는 셀룰라아제 변이체, 및 이의 용도
US8728790B2 (en) 2009-12-09 2014-05-20 Danisco Us Inc. Compositions and methods comprising protease variants
NL2004835C2 (en) 2010-06-07 2011-12-08 Univ Leiden Process for extracting materials from biological material.
EP2712363A1 (de) 2011-04-29 2014-04-02 Danisco US Inc. Waschmittelzusammensetzungen mit geobacillus tepidamans mannanase und verfahren zu ihrer verwendung
US20140073548A1 (en) 2011-04-29 2014-03-13 Danisco Us Inc. Detergent compositions containing bacillus sp. mannanase and methods of use thereof
JP6084617B2 (ja) 2011-09-14 2017-02-22 デュポン ニュートリション バイオサイエンシーズ エーピーエス 酵素
WO2013127069A1 (en) 2012-02-29 2013-09-06 Danisco Us Inc. Thermostable xylanase from thermobifida fusca and methods of use thereof
US20150320081A1 (en) * 2012-07-18 2015-11-12 Danisco Us Inc. Melt-delayed granule
WO2014047520A1 (en) 2012-09-20 2014-03-27 Danisco Us Inc. Microtiter plates for controlled release of culture components to cell cultures
BR112015012904A2 (pt) 2012-12-07 2017-09-12 Danisco Us Inc composições e métodos de uso
WO2014099525A1 (en) 2012-12-21 2014-06-26 Danisco Us Inc. Paenibacillus curdlanolyticus amylase, and methods of use, thereof
GB201303666D0 (en) 2013-03-01 2013-04-17 Goldsborough Andrew S Sample fixation and stabilisation
ES2676895T5 (es) 2013-03-11 2022-04-27 Danisco Us Inc Variantes combinatorias de alfa-amilasa
GB201308843D0 (en) * 2013-03-14 2013-07-03 Verenium Corp Phytase formulation
GB201401680D0 (en) 2014-01-31 2014-03-19 Dupont Nutrition Biosci Aps Compositions and methods comprising a xylanase enzyme variant
US20180092379A1 (en) * 2015-03-19 2018-04-05 Danisco Us Inc. Stable granules with low internal water activity

Also Published As

Publication number Publication date
WO2019089898A1 (en) 2019-05-09

Similar Documents

Publication Publication Date Title
JP4790957B2 (ja) 酵素粒質物
EP3180429B1 (de) Waschmittel und zusammensetzungen mit enzymatischen polymerteilchen
EP2732018B1 (de) Lagerstabile enzymgranulate
EP2992076B1 (de) Mikroverkapselung von wasch und reinigungsmittelenzymen
EP2840134B1 (de) Enzymkörnchen
JP3312364B2 (ja) 被覆した酵素含有顆粒
EP3122859B1 (de) Mikroverkapselung unter verwendung von kleinen aminen
US9222060B2 (en) Triggered release
US7273736B2 (en) Method for preparing an enzyme containing granule
JP2005531308A (ja) 顆粒の安定化
US20210187466A1 (en) Process for preparation of microcapsules
WO2010105820A1 (en) Improved blends containing proteases
JP2004508040A (ja) 潤滑化粒体
EP3703661A1 (de) Feste matrixzusammensetzungen mit gesenktem gefrierpunkt für die schmelzgranulation von enzymen
Simi et al. Encapsulation of crosslinked subtilisin microcrystals in hydrogel beads for controlled release applications
US11753605B2 (en) Multi-core granules
CN111770788A (zh) 使用氨基糖低聚物进行微囊化
Easa et al. Optimizing the preparation of cross-linked enzyme aggregates (CLEA)-amylase from supermeal worm (Zophobas Morio)
EP4206309A1 (de) Proteinpartikel mit verbesserter weisse
JP2003289862A (ja) タンパク質組成物およびタンパク質の保存方法、搬送方法
WO2019125683A1 (en) Enzyme-containing, hot-melt granules comprising a thermotolerant desiccant
US20230193162A1 (en) Low dusting granules
US20200318037A1 (en) Low dusting granules

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20200429

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230530

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20240227