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WO1996040791A1 - Modification de polypeptides - Google Patents

Modification de polypeptides Download PDF

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Publication number
WO1996040791A1
WO1996040791A1 PCT/DK1996/000249 DK9600249W WO9640791A1 WO 1996040791 A1 WO1996040791 A1 WO 1996040791A1 DK 9600249 W DK9600249 W DK 9600249W WO 9640791 A1 WO9640791 A1 WO 9640791A1
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WO
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Prior art keywords
polypeptide
kda
use according
polyalkylene oxide
polypeptides
Prior art date
Application number
PCT/DK1996/000249
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English (en)
Inventor
Arne Agerlin Olsen
Thomas Christian Beck
Original Assignee
Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU58936/96A priority Critical patent/AU5893696A/en
Publication of WO1996040791A1 publication Critical patent/WO1996040791A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/24—Organic nitrogen compounds
    • A21D2/26—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/86—Polyethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/0005—Other compounding ingredients characterised by their effect
    • C11D3/0078—Compositions for cleaning contact lenses, spectacles or lenses
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/37—Polymers
    • C11D3/3703—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C11D3/3719—Polyamides or polyimides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/70—Biological properties of the composition as a whole
    • A61K2800/72—Hypo-allergenic

Definitions

  • the present invention relates to a process for producing poly ⁇ alkylene oxide-polypeptide conjugates with reduced allergeni ⁇ city, a polyalkylene oxide-polypeptide conjugate with reduced allergenicity, an activated polyalkylene oxide for conjugating polypeptides, the use of said process for reducing the aller ⁇ genicity of industrial polypeptides, the use of said polypep ⁇ tide conjugate for a number of industrial purposes, such as the use in personal care products and in detergent compositions.
  • polypeptides such as e- specially proteins and enzymes
  • This exposure may inflict problems for people having an enhanced inclination for eliciting an allergic re ⁇ sponse toward polypeptides or to people frequently in direct contact with relatively large amounts of polypeptides.
  • the latter group of people includes employees handling the manufacturing of products containing polypeptides, profes ⁇ sionals, such as hair dressers, using products containing polypeptides, and to some extent also end-users of cosmetics and toiletry products.
  • PAO polyalkylene oxides
  • PAG polyalkylene glycol
  • PAO polyalkylene oxides
  • PAO-polymer polyethylene glycol (PEG) and methoxypolyethylene glycol (mPEG) , capped on one end with a functional group and reactive with amines on the polypeptide molecule, are often used.
  • Poly(ethylene glycol) succinoyl-N-hydroxysuccinimide ester (SS- PEG) (Abuchowski et al., (1984), Cancer Biochem. Biophys., 7, 175-186) is an example of such an activated PAO releasing only non-toxic residues, which are readily separable from the PAO- polypeptide conjugate.
  • SS- PEG Poly(ethylene glycol) succinoyl-N-hydroxysuccinimide ester
  • polypeptide conjugation technology is de ⁇ signed to modify polypeptides for therapeutic use, for which selectiveness and controlled specific attachment are important.
  • pharmaceutical polypeptides are often to be introduced into the bloodstream of the human body, which ob ⁇ viously lines up very high demands to the quality of the product.
  • pharmaceutical products have to be approved by national health authorities, such as e .g. the Food and Drug Administration (FDA) in the United States of America, who demand highly controlled production processes and thorough testing of the products.
  • FDA Food and Drug Administration
  • US patent no. 4,179,337 discloses a process for conjugation of polyalkylene glycol (PEG) to polypeptides for therapeutic use, such as insulin and certain enzymes, all having a physiological activity.
  • PEG polyalkylene glycol
  • the preparations of conjugated polypeptides have re ⁇ cuted immunogenicity and have further a prolonged lifetime in the bloodstream as compared to the parent polypeptides.
  • the surplus amounts of polymer necessary to conjugate the polypep- tide makes the method expensive.
  • WO 90/13540 (Enzon Inc.) concerns activated PEG derivatives, namely, poly(ethylene glycol) succinimide carbonates (SC-PEG) and the bifunctional derivative of PEG, namely, poly(ethylene glycol-bis-succinimide carbonate (BSC-PEG) . Furthermore, the heterobifunctional derivatives of PEG, which in one end have the succinimide carbonate group and in the other end have a group which readily reacts with amino groups of proteins to afford PEG attachment through stable urethane linkages.
  • SC-PEG poly(ethylene glycol) succinimide carbonates
  • BSC-PEG poly(ethylene glycol-bis-succinimide carbonate
  • the resulting PEG-chloroformiate is then reacted with N-hydroxysuccinimide (HOSu) followed by triethylamine (TEA) to yield the desired activated SC-PEG.
  • HOSu N-hydroxysuccinimide
  • TAA triethylamine
  • SC-PEG only releases non-toxic material (N- hydroxysuccinimide) .
  • EP 632 082 (Heyleclina) describes the preparation of activated (methoxy)poly(alkylene glycol) carbamate (C-(m)PEG) from (m)PEG via (m)PEG-chloroformiate.
  • the described activated (m)PEG car ⁇ bamate has a high degree of selectivity, as it only reacts with amine groups.
  • (m)PEG chloroformiate readily reacts with amines, alcohols, phenols, and carboxylic acids.
  • PAO polyalkylene oxides
  • PAG polyalkylene glycols
  • PEG polyethylene glycols
  • the general method involves activating a PAO-polymer to obtain a functiona ⁇ lized group which selectively attaches to a specific chemical group on the polypeptide.
  • the selective attachment is advan ⁇ tageous in connection with pharmaceuticals, as pharmaceuticals often are to be introduced into the human bloodstream.
  • the demand for selectivity and specific attachment makes the polypeptide modification process cumbersome, slow, and reduces the process yield.
  • conjugation of polypeptides which are not intended to enter the circulatory system of the human body is a totally different matter, as such polypeptides are not intended to elicit a physiological response.
  • unin ⁇ tended exposure of the human body to such polypeptides will not involve direct contact with the bloodstream, as the polypeptide is not e .g. injected into the human circulatory system or intramuscular. The contact is much less pronounced, and when occurring, it will take place in the form of inhalation of polypeptides though the mouth, or as nasal or dermal contact.
  • polypeptides for pharmaceutical purposes is produced in amounts of a few kilograms, while industrial polypeptides is produces in amounts of many 1000 kilograms. Techniques used for producing polypeptides for therapeutic purposes cannot always advantageously be adapted for producing polypeptides for industrial purposes.
  • PAO- polypeptide conjugates for purposes where the polypeptide is not intended to enter the body, the conjugate of which has reduced allergenicity using a minimum of process steps.
  • the present inventors have surprisingly found an advantageous process for producing industrial polypeptides having reduced allergenicity using less process steps than expected.
  • the invention relates to a process for pro ⁇ ducing polyalkylene oxide-polypeptide conjugates with reduced allergenicity, using a polyalkylene oxide (PAO) as the starting material, comprising the steps of
  • the invention is directed towards an activated polyalkylene oxide capable of linking to attachment groups on a polypeptide, whose activated PAO has the generic structure
  • Rl is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated,
  • R3 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated,
  • R4 is an alkyl group, which may be straight, branched, disubstituted, or unsaturated, a is an integer between 1 and 1000, b is an integer between 0 and 1000, c is an integer between 0 and 1000, and Y is a halogen or a nitrile.
  • polyalkylene oxide-polypeptide conjugates having the general formula
  • Rl is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated,
  • R3 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated,
  • R4 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated, a is an integer between 1 and 1000, b is an integer between 0 and 1000, c is an integer between 0 and 1000, n is an integer between 1 and 100, characterized in that X is a coupling group between the polymer and an polypeptide, which has been formed by reaction with a polyalkylene oxide halogen formiate.
  • the invention relates to the use of the above process for reducing the allergenicity of polypeptides for industrial purposes, which include the use in personal care products and detergents.
  • Figure 1 shows the results of the IT study in Brown Norway rats intratracheally exposed to immunized
  • FIG. 2 as figure 1 except that the sera IgG and IgE antibodies were raised against Carezyme ⁇ core-mPEG 5,000
  • Figure 3 shows the results of the IT study in Brown Norway rats intratracheally exposed to immunizsed:
  • the serum IgG and IgE antibodies were raised against Subtilisin
  • Figure 4 shows as figure 3 except that the sera IgG and IgE antibodies were raised against Subtilisin Novo with PMSF.
  • FIG. 5 as figure 3 except that the sera IgG and IgE antibodies were raised against Subtilisin Novo-mPEG 15,000.
  • “industrial polypep ⁇ tides” are defined as polypeptides, such as proteins and en ⁇ zymes, which are not to be introduced into the circulatory system of the body of humans and animals.
  • polypeptides which are specifically contemplated, are polypeptides used in products such as detergents, household article products, agrochemicals, personal care products, such as cosmetics, toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for cleaning hard surfaces, compositions used for manufacturing food and feed etc.
  • the "circulatory system" of the body of humans and animals means, in the context of the present invention, the system which mainly consists of the heart and blood vessels.
  • the heart delivers the necessary energy for maintaining blood circulation in the vascular system.
  • the circulation system functions as the organisms transportation system, when the blood transports O2, nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood re ⁇ moves C0 2 from the tissue to the lungs and residual substances to e .g. the kidneys. Furthermore the blood is of importance for the temperature regulation and the defence mechanisms of the body, which include the immune system.
  • HOSu N-hydroxysuccinimide
  • TAA triethylamine
  • the above conjugation process is advantageous for producing polypeptides for therapeutic purposes, as a selective attachment to amine groups on the polypeptides will be obtained.
  • the process is cumbersome and slow, and costly.
  • the present invention relates to a process for producing polyalkylene oxide-polypeptide conjugates using a polyalkylene oxide (PAO) as the starting material.
  • the conjuga ⁇ tion process comprises the steps of:
  • the polyalkylene oxide (PAO) can be an polyalkylene glycol (PAG) , such as an methoxypolyalkylene glycol (mPAG) .
  • PAG polyalkylene glycol
  • mPAG methoxypolyalkylene glycol
  • the PAG may advantageously be a polyethylene glycol (PEG) , such as a ethoxypolyethylene glycol (mPEG) .
  • PEG polyethylene glycol
  • mPEG ethoxypolyethylene glycol
  • PAO-polymers having a molecular weight (M r ) between 1 kDa and 60 kDa, such as between 2 kDa and 35 kDa, especially between 2 kDa and 25 kDa, may be used according to the invention.
  • M r molecular weight
  • Polypeptides for industrial purposes need not attach to speci ⁇ fic attachment groups on the polypeptide. Actually it may in certain cases even be an advantage to obtain a less specific attachment, as the attachment of PAO will be more homogeneous, and the addition of a large surplus amount of PAO is not needed. Further, the process runs much faster, and is less cumbersome to control.
  • the process of the invention gives the possibility of obtaining a specifically designed conjugated PAO-polypeptide product.
  • the conjugation product is dependant on the reaction medium.
  • the present inventors have developed the process of the in ⁇ vention especially for large scale processing of industrial polypeptides.
  • Another advantage of the invention is that the prepared poly- peptide-polymer conjugate has an improved stability in compari ⁇ son to the parent polypeptide.
  • the process has all of the above mentioned advantages, and only a minimum of process steps are used to obtain polypeptides with reduced allergenicity, in comparison to the corresponding prior art processes.
  • Immunogenicity is a wider term than "antigenicity” and "al ⁇ lergenicity”, and expresses the immune systems response to the presence of foreign substances. Said foreign substances are called immunogens, antigens and allergens depending of the type of immune response the elicit.
  • an “immunogen” may be defined as a substance which, when intro ⁇ quizted into circulatory system of animals and humans, is capable of stimulating an immunologic response.
  • antigen refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.
  • an "allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals) .
  • Assessment of the allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administered parent polypeptides with the correspon ⁇ ding polypeptides with reduced allergenicity modified according to the invention.
  • a suitable strain of guinea pigs, the Dunkin Hartley strain does not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgGIA and IgGIB (see e .g. Prent ⁇ , ATLA, 19, p. 8- 14, 1991), which are responsible for their allergenic response to inhaled polypeptides including proteases. Therefore when using the Dunkin Hartley animal model, the relative amount of IgGIA and IgGIB is a measure of the allergenicity level.
  • a rat strain suitable for intratracheal exposure to polypep ⁇ tides and enzymes is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.
  • Example 6 it is shown that by using the process of the invention the allergenicity of polypeptides, such as the enzymes Carezyme ⁇ core and Subtilisin Novo, is reduced.
  • a polypeptide having "reduced allergenicity" according to the invention indicates that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals) , which can lead to an allergic state, is significantly decreased when inhaling a modified polypeptide of the invention in comparison to the corresponding parent polypeptide.
  • the polypeptide to be conjugated according to the invention may be of plant, animal or microbial origin, although the polypep ⁇ tides preferably is of microbial origin, such as of bacterial or fungal origin.
  • polypeptide is a protein having a biological activity, such as anti-microbial activity.
  • the protein is an enzyme selected from the group of proteases, lipases, trans- ferases, carbohydrases, oxidoreductases, and phytases.
  • enzymes with a molecular weight in the range between about 10 kDa and 100 kDa.
  • a “substantially” maintained activity is in the context of the present invention defined as an activity which is at least be ⁇ tween 20% and 30%, preferably between 30% and 40%, more pre ⁇ ferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the parent polypeptide.
  • the maximal number of polymer molecules which can be conjugated to the polypeptides depend to the number of attachment groups on the polypeptide. In general between 1 and 25 polymer molecules, preferably from 1 to 10 polymer molecules, are conjugated to each polypeptide molecule. This is significantly less than corresponding prior art techniques. Consequently the expense to polymers is reduced. To some extent it entails that the activity of the enzyme is substantially retained, as it is to be anticipated that the activity to a certain extent varies inversely with the number and the size of polymers conjugated to the polypeptide.
  • the polyalkylene oxide halogen formiate generated in step a) is preferably a polyalkylene oxide chloroformiate, such as metho- xypolyalkylene glycol chloroformiate, and may be generated by using phosgene.
  • Example 1 and Example 2 the preparation of a 15,000 and 5,000 methoxypolyethylene glycol chloroformiate, respectively, is described.
  • the halogen may also be any other halogen, such as Br or F.
  • the attachment groups on the polypeptide can be any group selected from the group including amines, hydroxyls, alcohols, phenols, or carboxylic acids.
  • polyalkylene oxide halogen formiate may if desired attach to the side chain of e .g. serine, threonine, tyrosine, lysine, arginine, aspartate, glutamate in the poly ⁇ peptide chain.
  • Another aspect of the invention is to provide an activated po ⁇ lyalkylene oxide (PAO) capable of linking to attachment groups on a polypeptide, which activated PAO has the generic structure
  • Rl is hydrogen, methyl, hydroxyl or methoxy
  • R2 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated,
  • R3 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated,
  • R4 is an alkyl group, which may be straight, branched, disub ⁇ stituted, or unsaturated, a is an integer between 1 and 1000, b is an integer between 0 and 1000, c is an integer between 0 and 1000, and Y is a halogen or nitrile
  • the halogen Y may be either of Cl, Br, or F, although Cl is preferred.
  • the activated PAO of the invention may preferably have a molecular weight in the range of from about 1 kDa to 60 kDa, preferably from about 2 kDa to 35 kDa, especially from about 2 kDa to 25 kDa. It is also the object of the invention to provide a polyalkylene oxide-polypeptide conjugate having the general formula
  • Rl, R2, R3, R4, a, b and c are defined as above, n is an integer between 1 and 100 and X is a coupling group between the polymer and an polypeptide.
  • the coupling group has been formed by reacting the activated PAO (polyalkylene oxide halogen formiate) of the invention with an attachment group on the polypeptide.
  • Said attachment group may be e .g. an amine, hydroxyl, alcohol, phenol, and/or carboxylic acid on the polypeptide.
  • the attachment group may constitute the side chain of serine, threonine, tyrosine, lysine, arginine, aspartate, glutamate in the polypeptide chain.
  • R2 and R4 are CH 2 -CH 2 , CH 2 -CH-CH 3 , or CH 2 -CH 2 -CH 2 -CH 2 .
  • Activated PAO-polymers having a molecular weight (M r ) between 1 and 60 kDa may be used as starting material for the process of the invention.
  • the polypeptide to be conjugated is preferably a protein having a biological activity or an enzyme as mentioned above.
  • a PAO-polypeptide conjugate of the invention has a total mo- lecular weight in the range of 50 kDa to 250 kDa, preferably between 80 and 200 kDa.
  • the composition may further comprise other enzymes/polypeptides and/or ingredients normally used in e .g. detergents, including soap bars, household articles, agrochemicals, personal care products, such as cleaning preparations e .g. for contact lenses, cosmetics, toiletries, oral and dermal pharmaceuticals, compositions used for treating textiles, compositions for cleaning hard surfaces, compositions used for manufacturing food, e.g. baking, and feed etc.
  • detergents including soap bars, household articles, agrochemicals, personal care products, such as cleaning preparations e .g. for contact lenses, cosmetics, toiletries, oral and dermal pharmaceuticals, compositions used for treating textiles, compositions for cleaning hard surfaces, compositions used for manufacturing food, e.g. baking, and feed etc.
  • enzymes/polypeptides include proteases, lipases, oxidoreductases, carbohydrases, transferases, such as transglutaminases, anti-microbial polypeptides, and phytases.
  • the enzymes/polypeptides may typically be a component of a detergent composition, e.g., a laundry detergent composition or a dishwashing detergent composition. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabili ⁇ zed liquid, or a protected enzyme.
  • Non-dusting granulates may be produced, e .g. , as disclosed in US 4',106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Other enzyme stabilizers are well known in the art.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e .g. as powder, granules, paste or liquid.
  • a liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
  • the detergent composition comprises one or more surfactants, each of which may be anionic, non-ionic, cationic, or amphoteric (zwitterionic) .
  • the detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzene- sulfonate (LAS) , alpha-olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxysulfate (AEOS or AES) , secondary alkanesulfonates (SAS) , alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap.
  • anionic surfactant such as linear alkylbenzene- sulfonate (LAS) , alpha-olefinsulfonate (AOS) , alkyl sulfate (fatty alcohol sulfate) (AS) , alcohol ethoxy
  • the detergent composition may additionally comprise one or more enzymes/polypeptides, such as amylases, pullulanase, esterase, lipase, cutinase, protease, cellulase, peroxidase, or oxidase, e.g., laccase, and anti-microbial polypeptides.
  • enzymes/polypeptides such as amylases, pullulanase, esterase, lipase, cutinase, protease, cellulase, peroxidase, or oxidase, e.g., laccase, and anti-microbial polypeptides.
  • enzymes/polypeptides such as amylases, pullulanase, esterase, lipase, cutinase, protease, cellulase, peroxidase, or oxidase, e.g., laccase, and anti-microbial polypeptides.
  • the detergent contains 1-65% of a detergent builder, but some dishwashing detergents may contain even up to 90% of a detergent builder, or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA) , ethylenedia inetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) .
  • zeolite diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA) , ethylenedia inetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTMPA) , alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (
  • the detergent builders may be subdivided into phosphorus- containing and non-phosphorous-containing types.
  • phosphorus-containing inorganic alkaline detergent builders include the water-soluble salts, especially alkali metal pyrophosphates , orthophosphates, polyphosphates and phospho ⁇ nates.
  • non-phosphorus-containing inorganic builders include water-soluble alkali metal carbonates, borates and silicates as well as layered disilicates and the various types of water-insoluble crystalline or amorphous alumino silicates of which zeolites is the best known representative.
  • suitable organic builders include alkali metal, am ⁇ monium or substituted ammonium salts of succinates, malonates, fatty acid malonates, fatty acid sulphonates, carboxymethoxy succinates, polyacetates, carboxylates, polycarboxylates, ami- nopolycarboxylates and polyacetyl carboxylates.
  • the detergent may also be unbuilt, i.e. essentially free of detergent builder.
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP) , po- lyethyleneglycol (PEG) , poly(vinyl alcohol) (PVA) , polycarboxy ⁇ lates such as polyacrylates, polymaleates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • CMC carboxymethylcellulose
  • PVP poly(vinylpyrrolidone)
  • PEG po- lyethyleneglycol
  • PVA poly(vinyl alcohol)
  • polycarboxy ⁇ lates such as polyacrylates, polymaleates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type.
  • the bleaching agents may be coated or incapsulated.
  • inorganic chlor ⁇ ine/bromine-type bleaches are lithium, sodium or calcium hypochlorite or hypobromite as well as chlorinated trisodium phosphate.
  • the bleaching system may also comprise a H 2 0 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylene- diamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) .
  • TAED tetraacetylethylene- diamine
  • NOBS nonanoyloxybenzenesulfonate
  • organic chlorine/bromine-type bleaches are he ⁇ terocyclic N-bromo and N-chloro i ides such as trichloro- isocyanuric, tribromoisocyanuric, dibro oisocyanuric and dichloroisocyanuric acids, and salts thereof with water solubilizing cations such as potassium and sodium.
  • Hydantoin compounds are also suitable.
  • the bleaching system may also comprise peroxyacids of, e .g. , the amide, imide, or sulfone type.
  • oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound.
  • suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates and perphosphates.
  • Preferred activator materials are TAED or NOBS.
  • the enzymes of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative such as, e .g. , an aromatic borate ester, and the composition may be formulated as described in, e . g. , WO 92/19709 and WO 92/19708.
  • the enzymes of the invention may also be stabilized by adding reversible enzyme inhibitors, e.g., of the protein type as described in EP 0 544 777 BI.
  • the detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors) , suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil- redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.
  • fabric conditioners including clays, deflocculant material, foam boosters/foam depressors (in dishwashing detergents foam depressors) , suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil- redeposition agents, dyes, dehydrating agents, bactericides, optical brighteners, or perfume.
  • the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e .g. in the range of 7-11.
  • laundry detergent compositions within the scope of the invention include:
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Alcohol ethoxysulfate e.g. C ⁇ _ ⁇ s alcohol, 1-2 EO
  • alkyl sulfate 1 - 4% e.g. C 16 -i 8
  • Alcohol ethoxylate e.g. Ci 4 _ ⁇ 5 alco ⁇ hol, 7 EO 5 - 9%
  • Soluble silicate (as Na 2 0,2Si0 ) 2 - 6%
  • Zeolite (as NaAlSiOd 15 - 22%
  • Polymers e.g. maleic/acrylic acid copolymer, PVP, PEG 0 - 3%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. suds suppressors, perfume , optical 0 - 5% brightener, photobleach
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Linear alkylbenzenesulfonate (cal ⁇ culated as acid) 6 - 11%
  • Alcohol ethoxylate e.g. C 14 .. 15 alco ⁇ hol, 7 EO 5 - 9%
  • Soluble silicate (as Na 2 0,2Si0 2 ) 1 - 4%
  • Polymers e.g. maleic/acrylic acid copolymer, PVP, PEG 1 - 6%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. suds 0 - 5% suppressors, perfume
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Alcohol ethoxylate e.g. C ⁇ 2 _ ⁇ 5 alco ⁇ hol, 7 EO 7 - 14%
  • Soap as fatty acid e .g. C ⁇ g- 22 fatty 1 - 3% acid
  • Soluble silicate (as Na 2 0,2Si0 ) 3 - 9%
  • Polymers e.g. maleic/acrylic acid copolymer, PVP, PEG 0 - 3%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. suds suppressors, perfume, optical 0 - 5% brightener
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Alcohol ethoxylate e.g. C ⁇ 2 _ 15 alco ⁇ hol, 7 EO 10 - 25%
  • Soluble silicate (as Na 2 0,2Si0 2 ) 1 - 5%
  • Polymers e.g. maleic/acrylic acid copolymer, PVP, PEG 1 - 3%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. suds 0 - 5% suppressors, perfume
  • An aqueous liquid detergent composition comprising
  • Linear alkylbenzenesulfonate (cal ⁇ 15 - 21% culated as acid)
  • Alcohol ethoxylate e .g. C ⁇ _ ⁇ s alco ⁇ hol, 7 EO or C 12 _i 5 alcohol, 5 EO 12 - 18%
  • Polymers e.g. PVP, PEG 0 - 3%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. dispersants, suds suppressors, perfume, optical 0 - 5% brightener
  • An aqueous structured liquid detergent composition compris ⁇ ing
  • Alcohol ethoxylate e.g. C ⁇ 2 _i 5 alcohol, 7 EO, 3 - 9% or C ⁇ 2 _i 5 alcohol, 5 EO
  • Soap as fatty acid e.g. oleic 3 - 10% acid
  • Polymers e .g. PEG, PVP 0 - 3%
  • Anchoring polymers such as, e .g. , lauryl methacrylate/acrylic acid 0 - 3% copolymer; molar ratio 25:1; MW 3800
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. dispersants, suds suppressors, per ⁇ 0 - 5% fume, optical brighteners
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Soluble silicate (as Na 2 0,2Si0 2 ) 1 - 4%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. optical brightener, suds suppressors, per ⁇ 0 - 5% fume
  • a detergent composition formulated as a granulate comprising
  • Soluble silicate (as Na 2 0,2Si0 2 ) 1 - 4%
  • Zeolite (as NaAlSi0 4 ) 30 - 50%
  • Polymers e.g. PVP, maleic/acrylic 1 - 5% acid copolymer, PEG
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. suds 0 - 5% suppressors, perfume
  • a detergent composition formulated as a granulate comprising
  • Bleach activator e.g. NOBS or TAED 1 - 5%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • An aqueous liquid detergent composition comprising
  • Alcohol ethoxysulfate e.g. C 12 -i 5 alcohol, 2-3 EO 8 - 15%
  • Alcohol ethoxylate e.g. C 12 - 15 al ⁇ cohol, 7 EO, 3 - 9% or C 12 _ 15 alcohol, 5 EO
  • Soap as fatty acid e.g. lauric 0 - 3% acid
  • Hydrotrope e.g. sodium 2 - 6% toluensu1fonate
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • An aqueous liquid detergent composition comprising
  • Alcohol ethoxylate e.g. C ⁇ 2 _ ⁇ 5 alco ⁇ hol, 7 EO, 6 - 12% or C ⁇ _i 5 alcohol, 5 EO
  • Polymer e.g. maleic/acrylic acid copolymer, anchoring polymer such as, e.g., lauryl 0 - 3% methacrylate/acrylic acid copolymer
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Anionic surfactant linear alkylbenzenesulfonate, alkyl sulfa ⁇ te, alpha-olefinsulfonate, alpha- 25 - 40% sulfo fatty acid methyl esters, alkanesulfonates, soap
  • Nonionic surfactant e.g. alcohol 1 - 10% ethoxylate
  • Soluble silicates (as Na 2 0, 2Si0 ) 5 - 15%
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Layered disilicate e.g. SK56 from Hoechst 10 - 20%
  • Soluble silicate (as Na 2 0,2Si0 2 ) 0 - 6%
  • Polymers e.g. polycarboxylates and 0 - 5% PVP
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • Minor ingredients e.g. optical brightener, photo bleach, perfume, 0 - 5% suds suppressors
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
  • Soluble silicate (as Na 2 0,2Si0 ) 0 - 4%
  • Polymers e.g. polycarboxylates and 0 - 3% PVP
  • Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
  • the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching". Nature, 369, (1994), p. 637-639.
  • Detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
  • a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
  • the detergent may also comprise anionic surfactant and/or a bleach system.
  • dishwashing detergent compositions within the scope of the invention include:
  • Nonionic surfactant 0.4 - 2.5%
  • TED Tetraacetylethylenediamine
  • Nonionic surfactant - 2% e.g. alcohol ethoxylate
  • NTA Nitrilotrisodium acetate
  • Polyacrylate polymer e.g. maleic acid/acrylic acid co ⁇ - 25% polymer
  • Nonionic surfactant 0.5 - 2.0%
  • TED Tetraacetylethylenediamine
  • TED Tetraacetylethylenediamine
  • Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
  • Liquid carrier selected from higher glycols, polyglycols, polyoxides, 25.0 - 45.0% glycolethers
  • Stabilizer e.g. a partial ester of phosphoric acid and a 0.5 - 7.0% alkanol
  • Foam suppressor e.g. silicone 0 - 1.5%
  • Liquid nonionic surfactant e.g. alcohol ethoxylates 2.0 - 10.0%
  • Stabilizing system e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol 0.5 - 7.0% ethers
  • Clay gel thickener e.g. bentonite 0.0 - 10.0%
  • Liquid carrier selected from higher lycols, polyglycols, polyoxides and Balance glycol ethers 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION
  • Oleic acid 0 - 10%
  • TED Tetraacetylethylenediamine
  • the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching". Nature, 369, (1994), p. 637-639.
  • conjugated enzymes with reduced allergenicity of the invention are of interest.
  • Proteases are well-known active ingredients for cleaning of contact lenses. They hydrolyze the proteinaceous soil on the lens and thereby makes it soluble. Removal of the protein soil is essential for the wearing comfort.
  • Proteases are also effective ingredients in skin cleaning products, where they remove the upper layer of dead keratinaseous skin cells and thereby make the skin look brighter and more fresh.
  • Proteases are also used in oral care products, especially for cleaning of dentures, but also in dentifrices.
  • proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.
  • Lipases can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in bath and shower products such as creams and lotions as active ingredients for skin care.
  • Lipases can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.
  • hair cleaning products e.g. shampoos
  • Lipases are also effective ingredients in products for cleaning of contact lenses, where they remove lipid deposits from the lens surface.
  • oxidase usually glucose oxidase
  • substrate e.g. glucose
  • peroxidase usually lactoperoxidase
  • Anti-microbial systems comprising the combination of an oxidase and a peroxidase are known in the cleaning of contact lenses.
  • Oxidoreductases Another application of oxidoreductases is oxidative hair dyeing using oxidases, peroxidases and laccases .
  • the free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells.
  • free radical scavengers such as Superoxide dismutase into cosmetics is well-known (R. L. Goldemberg, DCI, NOV. 93, p. 48-52) .
  • Protein disulfide isomerase is also an oxidoreductase. It can be utilized for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds) .
  • Carbohvdrases are also an oxidoreductase. It can be utilized for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds) .
  • Plaque formed on the surface of teeth is composed mainly of polysaccharides. They stick to the surface of the teeth and the microorganisms.
  • the polysaccharides are mainly ⁇ -1,6 bound glucose (dextran) and ⁇ -1,3 bound glucose (mutan) .
  • the applica ⁇ tion of different types of glucanases such as mutanase and dex- tranase helps hydrolysing the sticky matrix of plaque, making it easier to remove by mechanical action.
  • biofilm for instance the biofilm formed in lens cases can be removed by the action of glucanases.
  • Anti-microbial polypeptides have widespread applications such as for preservation of cosmetic products, anti-acne products, deodorants and shampoos. Further such polypeptides may be use in contact lens products.
  • conjugated enzymes or polypeptides with reduced aller ⁇ genicity may advantageously be used in the manufacturing of food and feed.
  • the gluten in wheat flour is the essential ingredient respon ⁇ sible for the ability of flour to be used in baked foodstuffs.
  • Proteolytic enzymes are sometimes needed to modify the gluten phase of the dough, e.g. a hard wheat flour can be softened with a protease.
  • Neutrase® is a commercially available neutral etallo protease that can be used to ensure a uniform dough quality and bread texture, and to improve flavour.
  • the gluten proteins is degraded either moderately or more extensively to peptides, whereby close control is necessary in order to avoid excessive softening of the dough.
  • Proteases are also used for modifying milk protein.
  • proteases are used for brewing with unmalted cereals and for controlling the nitrogen content.
  • proteases are used so to speak to expand the animals digestion system.
  • lipase in the baking industry is rather new. Addition of lipase results in improved dough properties and an improved bread aking quality in terms of larger volume, impro ⁇ ved crumb structure and whiter crumb colour. The observed ef ⁇ fect can be explained by a mechanism where the lipase changes the interaction between gluten and some lipids fragment during dough mixing. This results in an improved gluten network.
  • lipases are used e.g. to minimize the amount of undesirable side-products, to modify fats by interesterification, and to synthesis of esters.
  • oxidoreductases with reduced allergenicity may advantageously be used in the manufacturing of food and feed.
  • oxidoreductases are used for baking, glucose oxidase, lipoxygenase, peroxidase, catalase and combinations hereof.
  • bakers strengthen gluten by adding ascorbic acid and potassium bromate.
  • Some oxidoreductases can be used to replace bromate in dough systems by oxidation of free sulfydryl units in gluten proteins. Hereby disulphide linkages are formed resulting in stronger, more elastic doughs with greater resistance.
  • GluzymeTM Novartis A/S is a glucose oxidase preparation with catalase activity that can be used to replace bromate.
  • the dough strengthen is measured as greater resistance to mechan ⁇ ical shock, better oven spring and larger loaf volume.
  • Flour has varying contents of amylases leading to differences in the baking quality. Addition of amylases can be necessary in order to standardize the flour.
  • Amylases and pentosanases generally provide sugar for the yeast fermentation, improve the bread volume, retard retrogradation, and decrease the staling rate and stickiness that results from pentosan gums. Examples of carbohydrases are given below.
  • Certain maltogenic amylases can be used for prolonging the shelf-life of bread for two or more days without causing gumminess in the product.
  • the starch is modified in such a way that retrogradation is less likely to occur.
  • the produced low-molecular-weight sugars improve the baked goods 1 water retention capacity without creating the intermediate-length dextrins that result in gumminess in the finished product.
  • the enzyme is inactivated during bread baking, so it can be considered a processing aid which does not have to be declared on the label. Overdosing of Novamyl can almost be excluded.
  • the bread volume can be improved by fungal ⁇ -amylases which further provide good and uniform structure of the bread crumb.
  • Said ⁇ -amylases are endoenzymes that produce maltose, dextrins and glucose.
  • Cereal and some bacterial ⁇ -amylases are inacti ⁇ vated at temperatures above the gelatinization temperature of starch, therefore when added to a wheat dough it results in a low bread volume and a sticky bread interior.
  • Fungamyl has the advantage of being thermolabile and is inactivated just below the gelatinization temperature.
  • Enzyme preparations containing a number of pentosanase and hemi-cellulase activities can improve the handling and stabil ⁇ ity of the dough, the freshness, the crumb structure and the volume of the bread.
  • Pentosanases can be used in combination with or as an alternative to emulsifiers.
  • carbohydrases are utilized for producing syrups from starch, which are widely used in soft drinks, sweets, meat products, dairy products, bread products, ice cream, baby food, jam etc.
  • the conversion of starch is normally carried out three steps. First the starch is liquefied, by the use of ⁇ -amylases. Maltodextrins, primarily consisting of oligosaccharides and dextrins, are obtained.
  • the mixture is then treated with an amyloglucosidase for hydrolyzing the oligosaccharides and dextrins into glucose. In this way a sweeter product is obtained.
  • an amyloglucosidase for hydrolyzing the oligosaccharides and dextrins into glucose.
  • ⁇ -amylases alone or in combination with a pullulanase (de-branching enzyme) may be used.
  • the glucose mixture can be made even sweeter by isomerization to fructose. For this an immobilized glucose isomerase can be used.
  • dextranases are used to break down dextran in raw sugar juices and syrups.
  • ⁇ -amylases are advantageously used for thinning of starch in distilling mashes.
  • ⁇ -galactosidases (lactase) are used when producing low lactose milk for persons suffering from lactose malabsorption.
  • flavoured milk drinks are produced from lactase-treated milk, the addition of sugar can be reduced without reducing the sweetness of the product.
  • lactose crystallization can be avoided by lactase treatment, and the risk of thickening caused by casein coagulation in lactose crystals is thus reduced.
  • ⁇ -amylases are used in the animal feed industry to be added to cereal-containing feed to improve the digestibility of starch.
  • Certain bacteriolytic enzymes may be used e.g. to wash car ⁇ casses in the meat packing industry (see US patent no. 5,354,681 from Novo Industri A/S)
  • Transgluta inases with reduced allergenicity according to the invention may advantageously be used in the manufacturing of food and feed.
  • Transglutaminases has the ability to crosslinking protein.
  • This property can be used for gelling of aqueous phases containing proteins. This may be used for when producing of spreads (DK patent application no. 1071/84 from Novo Nordisk A/S).
  • Transglutaminases are used for improvement of baking quality of flour e.g. by modifying wheat flour to be used in the pre ⁇ paration of cakes with improved properties, such as improved taste, dent, mouth-feel and a higher volume (see JP 1-110147) .
  • paste type food material e.g. used as fat substitution in foods as ice cream, toppings, frozen desserts, mayonnaises and low fat spreads (see WO 93/22930 from Novo Nordisk A/S) .
  • Phytases of the invention may advantageously be used in the manufacturing of food, such as breakfast cereal, cake, sweets, drink, bread or soup etc., and animal feed.
  • Phytases may be used either for exploiting the phosphorus bound in the phytate/phytic acid present in vegetable protein sources or for exploiting the nutritionally important minerals bound in phytic acid complexes.
  • Microbial phytase may be added to feedstuff of monogastric animals in order to avoid supplementing the feed with inorganic phosphorus (see US patent no. 3,297,548)
  • Soyabean meal may contain high levels of the anti-nutritional factor phytate which renders this protein source unsuitable for application in baby food and feed for fish, calves and other non-ruminants, since the phytate chelates essential minerals present therein (see EP 0 420 358) .
  • phytases may be used. Bread with better quality can be prepared by baking divided pieces of a dough containing wheat flour etc. and phytase (see JP-0- 3076529-A)
  • Proteases are used for degumming and sand-washing of silk.
  • Lipases are used for removing fatty matter containing hydro- phobic esters (e.g. triglycerides during, the of textiles (see e.g. WO 93/13256 from Novo Nordisk A/S) .
  • catalases may serve to remove excess hydrogen peroxide.
  • Cellulolytic enzymes are widely used in the finishing of denim garments in order to provide a localized variation in the co ⁇ lour density of the fabric (Enzyme facilitated "stone wash") .
  • Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and ap ⁇ pearance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278.
  • the threads are exposed to con ⁇ siderable mechanical strain.
  • they are usually reinforced by coating (sizing) with a gelatinous substance (size) .
  • the most common sizing agent is starch in native or modified form. A uniform and durable finishing can thus be obtained only after removal of the size from the fa ⁇ bric, the so called desizing.
  • Desizing of fabrics sized with a size containing starch or modified starch is preferably facili ⁇ tated by use of amylolytic enzymes.
  • Such hard surface cleaning compositions comprising enzymes may also advantageously be used in the transportation sector, for instance for washing cars and for general vessel wash.
  • the invention relates to the use of the conjugate of the invention or a composition of the invention in products comprising polypeptides.
  • conjugate or compositions of the invention can advantageously be used for personal care products, such as hair care and hair treatment products.
  • skin care products and cosmetics are contemplated, such as skin cream, skin milk, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, nourishing essence, skin lotion, milky lotion, calamine lotion, hand cream, powder soap, transparent soap, sun oil, sun screen, shaving foam, shaving cream, baby oil lipstick, lip cream, creamy foundation, face powder, powder eye-shadow, powder, foundation, make-up base, essence powder, whitening powder.
  • conjugate of the invention can be used advantageously.
  • Such products include contact lenses cleaning and disinfection products.
  • detergents such as washing powder, soap, soap bars, liquid soap are also contemplated.
  • Tween 20 Poly oxyethylene sorbitan mono laurate (Merck cat no.
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • OPD o-phenylene-diamine
  • Proteases cleave the bond between the peptide and p- nitroaniline to give a visible yellow colour absorbing at 405 nm.
  • Buffer e.g. Britton and Robinson buffer pH 8.3
  • Substrate 100 mg suc-AAPF-pNA is dissolved into 1 ml dimethyl sulfoxide (DMSO) . 100 ⁇ l of this is diluted into 10 ml with Britton and Robinson buffer.
  • DMSO dimethyl sulfoxide
  • the substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS 405 nm/min.
  • the temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.
  • Enzymatic activity was measured as release of blue dye from azurine-crosslinked HE-cellulose (Cellazyme-C®) .
  • the reaction was carried out at 40°C in 20 mM Na-phosphate pH 7 for 10 minutes. Release of dye was monitored by reading the absorbance at 595 nm in a UVmax® Elisa-reader.
  • cellulytic activity was measured as described in "EAL-SM-0373.01/01" (available from Novo Nordisk on request) .
  • a three layer sandwich ELISA is used to determine relative con ⁇ centrations of total Ig or IgE antibodies.
  • the immunizing molecule is used as coating antigen with 10 ⁇ g per ml and 50 ⁇ l per well, in neutral phosphate buffer, incu ⁇ bated overnight at 4°C. All remaining binding spots on the well surface are blocked in 2 % skim milk, 200 ⁇ l per well in phos ⁇ phate buffer for at least 30 minutes at room temperature (RT) . All sera to be tested with this antigen are added at 50 ⁇ l per well to this plate using a 8-channel pipette in dilution series from 10 x diluted followed by 3-fold dilutions. Dilutions are made in phosphate buffer with 0.5% skim milk and 0.05% Tween20, incubated 2 hours on agitation platform at RT.
  • the "tracer" molecule(s) is(are) streptavidin-horse-radish peroxidase (when testing for total Ig) and biotin labelled mouse-anti-rat IgE coupled with Streptavidin horse-radish peroxidase (when testing for IgE).
  • 50 ⁇ l tracer molecules(s) per well diluted 2000 times in phosphate buffer with 0.5 % skim milk and 0.05% Tween20 are incubated for 2 hours on an agitation platform at room temperature (RT) .
  • Controls (blanks) are identical sequence but without rat sera.
  • 50 ⁇ l per well Streptavidin horse radish peroxidase, diluted 2000 times is incubated 1 hour on an agitation platform.
  • the colouring substrate (50 ⁇ l per well) is OPD (6 mg) and H 2 0 2
  • test animals are Brown Norway rats (BN) in groups of 10. Weight at time of start is more than 250 grams and at termination approximately 450 grams.
  • Electrophoretic separation of proteins was performed by stan ⁇ dard methods using 4-20% gradient SDS poly acrylamide gels (Novex) . Proteins were detected by silver staining. The mole ⁇ cular weight was measured relative to the mobility of Mark-12® wide range molecular weight standards from Novex.
  • the resulting Subtilisin Novo-mPEG-15,000 conjugate was purified by size-exclusion chromatography using a Superdex-75 column in an HPLC-system.
  • the residual activity of the conjugate was assessed by using suc-AAPF-pNP and casein as substrates: ⁇ peptide substrate : 95% CM-casein : 60%
  • reaction was performed at ambient temperature using magnetic stirring. At time 120 minutes the reaction was terminated by addition of 0.5 ml of 2 M Glycine.
  • the resulting Subtilisin Novo-mPEG-5,000 conjugate was purified by size-exclusion chromatography using a Superdex-75 column in an HPLC-system.
  • the residual activity of the conjugate is assessed by using suc-AAPF-pNP as substrate.
  • the conjugated retained activity towards the peptide substrate.
  • the catalytic core-domain of Carezyme ⁇ was prepared according to Boisset, C. et al. (1995), FEBS Lett. 376, p. 49-52.
  • the reaction was performed at ambient temperature using magnetic stirring. At time 30 minutes the reaction was terminated by addition of 1 ml of 2 M Glycine.
  • the resulting Cellulase-core-domain-mPEG-5,000 conjugate was purified by desalting in 0.05 M NaHC0 3 and concentrated to the original volume using an Amicon Cell.
  • the obtained conjugate was PEGylated a second time using 1.0 g of methoxypolyethylene glycol 5,000 chloroformiate at otherwise identical conditions.
  • PEGylated Carezyme ⁇ core-mPEG-5,000 conjugate was purified by desalting in water and concentrated to approximately 1 mg/ml of protein using an Amicon Cell. The conjugate was stored at 4°C.
  • the amount of total Ig (assessed as IgG) and IgE (allergic response) of BN rats exposed intratracheally with the modified polypeptides i.e. PEGylated Carezyme ⁇ core and PEGylated Subtilisin Novo
  • the modified polypeptides i.e. PEGylated Carezyme ⁇ core and PEGylated Subtilisin Novo

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Abstract

Cette invention concerne un procédé de production de conjugués d'oxyde de polyalkylène-polypeptide présentant une allergénicité réduite; ce procédé consistant à: a) générer un formiate d'halogène d'oxyde de polyalkylène; et b) conjuguer au moins un formiate d'halogène d'oxyde de polyalkylène avec des groupes de fixation situés sur le polypeptide. Cette invention concerne également un conjugué d'oxyde de polyalkylène-polypeptide présentant une allergénicité réduite, un oxyde de polyalkylène activé utile pour conjuguer les polypeptides ainsi que l'utilisation dudit procédé pour réduire l'allergénicité des polypeptides industriels. L'objet de cette invention est d'utiliser ledit conjugué polypeptidique dans un grand nombre d'application industrielles, par exemple dans des produits d'hygiène et de beauté ou des compositions détergentes.
PCT/DK1996/000249 1995-06-07 1996-06-07 Modification de polypeptides WO1996040791A1 (fr)

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US6284246B1 (en) 1997-07-30 2001-09-04 The Procter & Gamble Co. Modified polypeptides with high activity and reduced allergenicity
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US6030933A (en) * 1995-12-29 2000-02-29 The Procter & Gamble Company Detergent compositions comprising immobilized enzymes
US6106828A (en) * 1996-02-15 2000-08-22 Novo Nordisk A/S Conjugation of polypeptides
WO1997030148A1 (fr) * 1996-02-15 1997-08-21 Novo Nordisk A/S Conjugaison de polypeptides
US6284246B1 (en) 1997-07-30 2001-09-04 The Procter & Gamble Co. Modified polypeptides with high activity and reduced allergenicity
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6642011B2 (en) 1998-04-15 2003-11-04 Genencor International, Inc. Human protease and use of such protease for pharmaceutical applications and for reducing the allergenicity of non-human proteins
EP1997897A1 (fr) 1998-04-15 2008-12-03 Genencor International, Inc. Protéines mutantes disposant d'une réponse allergénique inférieure chez les humains et procédés de construction, d'identification et de production de telles protéines
US6936249B1 (en) 1998-04-15 2005-08-30 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6586224B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
EP2133098A1 (fr) 2000-01-10 2009-12-16 Maxygen Holdings Ltd Conjugués G-CSF
EP1982732A2 (fr) 2000-02-11 2008-10-22 Maxygen Holdings Ltd. Molécules de type facteur VII ou VIIA
EP2319541A1 (fr) 2000-02-11 2011-05-11 Bayer HealthCare LLC Conjugués de type facteur VII ou VIIA
EP2080771A2 (fr) 2001-02-27 2009-07-22 Maxygen Aps Nouvelles molécules de type interféron beta
US6645512B2 (en) 2001-03-07 2003-11-11 The Procter & Gamble Company Topical composition comprising a functionally acylating cosmetic bonding agent
US6713075B2 (en) 2001-03-07 2004-03-30 The Procter & Gamble Company Topical composition comprising an aldehyde or ketone-based cosmetic bonding agent
US6565865B2 (en) 2001-03-07 2003-05-20 The Procter & Gamble Company Topical composition comprising a three membered cyclic compound-based cosmetic bonding agent
WO2002072057A3 (fr) * 2001-03-07 2003-02-13 Procter & Gamble Composition topique comportant un agent de fixation cosmetique acylisant
US6589542B2 (en) 2001-03-07 2003-07-08 The Procter & Gamble Company Topical composition comprising a functionally alkylating cosmetic bonding agent
US6929939B2 (en) 2001-03-23 2005-08-16 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
WO2002077187A2 (fr) 2001-03-23 2002-10-03 Genencor International, Inc. Proteines provoquant une reaction immunogene modifiee, et methodes de production et d'utilisation desdites proteines
US7476528B2 (en) 2001-03-23 2009-01-13 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6607737B2 (en) 2001-05-30 2003-08-19 The Procter & Gamble Company Topical composition comprising an activated, trans-structured cosmetic bonding agent
US6613341B2 (en) 2001-05-30 2003-09-02 The Procter & Gamble Company Topical composition comprising a substituted cosmetic bonding agent
US7332320B2 (en) 2001-12-31 2008-02-19 Genencor International, Inc. Protease producing an altered immunogenic response and methods of making and using the same
WO2004000366A1 (fr) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Glycoformes du facteur vii pegylees
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EP2641611A2 (fr) 2003-10-17 2013-09-25 Novo Nordisk A/S Thérapie combinée
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US9260472B2 (en) 2004-02-02 2016-02-16 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
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US7638299B2 (en) 2004-07-21 2009-12-29 Ambrx, Inc. Biosynthetic polypeptides utilizing non-naturally encoded amino acids
US7838265B2 (en) 2004-12-22 2010-11-23 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
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US7736872B2 (en) 2004-12-22 2010-06-15 Ambrx, Inc. Compositions of aminoacyl-TRNA synthetase and uses thereof
US7939496B2 (en) 2004-12-22 2011-05-10 Ambrx, Inc. Modified human growth horomone polypeptides and their uses
US7846689B2 (en) 2004-12-22 2010-12-07 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
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US7959926B2 (en) 2004-12-22 2011-06-14 Ambrx, Inc. Methods for expression and purification of recombinant human growth hormone mutants
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