WO1996040791A1 - Modification of polypeptides - Google Patents

Modification of polypeptides Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
kda
use according
polyalkylene oxide
polypeptides
Prior art date
Application number
PCT/DK1996/000249
Other languages
French (fr)
Inventor
Arne Agerlin Olsen
Thomas Christian Beck
Original Assignee
Novo Nordisk A/S
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 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/en

Links

Classifications

    • 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, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • 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

Abstract

The invention relates to a process for producing polyalkylene oxide-polypeptide conjugates with reduced allergenicity comprising the steps of: a) generating a polyalkylene oxide halogen formiate; and b) conjugating at least one polyalkylene oxide halogen formiate to attachment groups on the polypeptide. Further, the invention is also related to a polyalkylene oxide-polypeptide conjugate with reduced allergenicity, an activated polyalkylene oxide for conjugating polypeptides, and the use of said process for reducing the allergenicity of industrial polypeptides. Finally it is the object of the invention to provide the use of said polypeptide conjugate for a number of industrial purposes, such as the use in personal care products and in detergent compositions.

Description

Title: Modification of polypeptides
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
Due to the strongly extended use of polypeptides, such as e- specially proteins and enzymes, for industrial and pharmaceu¬ tical purposes an increasing number of people are daily exposed to polypeptides. 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.
For more than two decades scientists all over the world have made efforts to develop a technology making it possible to eliminate the immunogenicity, allergenicity, and/or antige¬ nicity of polypeptides for therapeutic use. Even though the "perfect" technology has not yet been developed some good results have been achieved. Most of the developed technologies involve chemical or genetic modification of polypeptides leading to larger and/or heavier polypeptide molecules. It seems that the molecular weight has an influence on the immune system's response towards polypep¬ tides. However, today no precise commonly accepted explanation of the advantageous effect of larger and/or heavier polypeptide molecules exists. Further, it is possible to find exceptions from the rule. Even though a polypeptide such as e .g. plant pollen is both large and heavy it is known to cause an allergic response for certain people.
One of the technologies, which has shown good results on po¬ lypeptides for therapeutic uses, involves modification of polypeptides by means of covalent attachment of strands of polyalkylene oxides (PAO) , such as polyalkylene glycol (PAG) , to polypeptide molecules.
In general such chemical attachment of polyalkylene oxides (PAO) to polypeptides is recognized to lead to polypeptides having increased stability, increased resistance to proteolytic inactivation, reduced immunogenicity, antigenicity and/or allergenicity, and a low toxicity.
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.
To effect covalent attachment of polyalkylene glycol (PAG) to a polypeptide the hydroxyl end-groups of the polymer must first be converted into reactive functional groups. This process is frequently referred to as "activation", and the product is called "activated", "derivatized" or "functionalized". To obtain a selective and specific attachment of the polymer strand to the polypeptide a linker molecule having a suitable coupling group is normally used. Various PAG activation methods are known. One of the first com¬ monly used methods involved activating the hydroxyl end-group of PAG using cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578). However this method is not suitable for modifying polypeptide for foods and pharmaceuticals as cyanuric chloride (2,4,6-trichloro-s-triazine) is toxic and the activated PAG is non-specific, as it may react with polypep¬ tides having functional groups other that amines, such as free essential cysteine or tyrosine residues.
Other methods commonly used for activation of insoluble po¬ lymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichloro- hydrin, divenylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R.F. Taylor, (1991) , "Protein immobilisation. Fundamental and applications". Marcel Dekker, N.Y.; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques". Academic Press, N.Y.). Some of the methods concern activation of insol¬ uble polymers but are also applicable to activation of soluble polymers e .g. periodate, trichlorotriazine, sulfonylhalides, divenylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemis¬ try.
Additional methods for activation of polymers can also be found in WO 94/17039, US patent no. 5,324,844, WO 94/18247, WO 94/04193, US patent no. 5,219,564, US patent no. 5,122,614, WO 90/13540 (Enzon) , and US patent no. 5,281,698 (Cetus) , and more WO 93/15189 (Veronese) and for conjugation between activated polymers and enzymes e .g. Coagulation Factor VIII (WO 94/15625) , haemoglobin (WO 94/09027) , oxygen carrying molecule (US patent no. 4,412,989), ribonuclease and superoxide dis u- tase (Veronese at al., (1985), App. Biochem. Biotech., 11, p. 141-45) .
Summarized the polymer activation research has been concen¬ trated on finding activated PAO-polymers: 1) being reactive under mild process conditions, 2) having a high degree of selectivity towards specific attachment groups on the poly¬ peptide, and 3) which, during the conjugation process release only non-toxic residues.
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. However the product has limited stabil¬ ity in aqueous media.
The above described polypeptide conjugation technology is de¬ signed to modify polypeptides for therapeutic use, for which selectiveness and controlled specific attachment are important. First of all because 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. Secondly because 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.
Prior art patents
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. The preparations of conjugated polypeptides have re¬ duced 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.
Furthermore, WO 90/13540 discloses a process for synthesis of SC-PEG via PEG-chloroformiate by treating PEG with phosgene (0=C-C12) . The resulting PEG-chloroformiate is then reacted with N-hydroxysuccinimide (HOSu) followed by triethylamine (TEA) to yield the desired activated SC-PEG. During protein modification 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. In the application it is asserted that (m)PEG chloroformiate readily reacts with amines, alcohols, phenols, and carboxylic acids.
Discussion of prior art
According to prior art it is known to conjugate polypeptides to a number of polyalkylene oxides (PAO) , including polyalkylene glycols (PAG) , such as polyethylene glycols (PEG) . 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. However, the demand for selectivity and specific attachment makes the polypeptide modification process cumbersome, slow, and reduces the process yield. Even though the selectiveness of the acti¬ vated PAO does not affect the advantageous properties of the reaction end product in a negative manner, it certainly will affect the cost of the process, as it is necessary to add a surplus amount of activated PAO-polymer to obtain a suitable absolute process yield.
It is to be understood that 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. Further, 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.
The urge to obtain selective attachment of the polymer to the polypeptide is less important in the context of polypeptides for non-therapeutic purposes, as the demand for selectivity is a consequence of the "in-the-body" use.
Further, 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.
Therefore, it would be desirable to be able to obtain 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. SUMMARY OF THE INVENTION
It is the object of the present invention to provide polyal¬ kylene oxide-polypeptide conjugates for industrial purposes having reduced allergenicity.
The present inventors have surprisingly found an advantageous process for producing industrial polypeptides having reduced allergenicity using less process steps than expected.
In the first aspect 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
a) generating a polyalkylene oxide halogen formiate, and
b) conjugating at least one polyalkylene oxide halogen formiate to attachment groups on the polypeptide.
Further 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 - (0-R2)a - (0-R3)b - (0-R4)c - O - C=0 - Y
wherein
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.
Also contemplated according to the invention is polyalkylene oxide-polypeptide conjugates having the general formula
(Rl - (0-R2)a - (0-R3)b - (0-R4)c - 0 - C=0 - X -)n polypeptide
wherein
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.
Finally 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.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows the results of the IT study in Brown Norway rats intratracheally exposed to immunized
1: Carezyme© core (CC)
2: Core Carezyme©-PEG 5,000 (CC/chlPEG5000)
3: Glycine-PEG 15,000 (Gly/SPEG15000)
4: 0.9% NaCl (NaCl)
The sera IgG and IgE antibodies were raised against Carezyme© core
Figure 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:
1: Subtilisin Novo without PMSF (SN)
2: Subtilisin Novo with PMSF (SN-PMSF)
3: Subtilisin Novo-mPEG 15,000 (SN/SPEG15000)
4: Glycine-PEG 15,000 (Gly/SPEG15000)
6: 0.9% NaCl (NaCl)
The serum IgG and IgE antibodies were raised against Subtilisin
Novo without PMSF.
Figure 4 shows as figure 3 except that the sera IgG and IgE antibodies were raised against Subtilisin Novo with PMSF.
Figure 5 as figure 3 except that the sera IgG and IgE antibodies were raised against Subtilisin Novo-mPEG 15,000.
DETAILED DESCRIPTION OF THE INVENTION
It is the object of the invention to provide a process for conjugating polyalkylene oxides and polypeptides to obtain an industrial product with reduced allergenicity.
In the context of the present invention "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. Examples of such 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 C02 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.
As described above it is known to produce polyalkylene oxide- polypeptide conjugates by first generating a polyalkylene glycol chloroformiate by using phosgene (Cl2~C=0) and thereafter reacting it with N-hydroxysuccinimide (HOSu) followed by triethylamine (TEA) to yield the desired activated PEG-derivative.
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. However the process is cumbersome and slow, and costly.
In the first aspect 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:
a) generating a polyalkylene oxide halogen formiate, and
b) conjugating at least one polyalkylene oxide halogen formiate to attachment groups on the polypeptide.
In comparison to the corresponding prior art process mentioned above, at least one process step has been eliminated, and still a product with reduced allergenicity is obtained.
Polymer
According to the invention the polyalkylene oxide (PAO) can be an polyalkylene glycol (PAG) , such as an methoxypolyalkylene glycol (mPAG) .
More specifically the PAG may advantageously be a polyethylene glycol (PEG) , such as a ethoxypolyethylene glycol (mPEG) .
PAO-polymers having a molecular weight (Mr) 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.
Note that all polymer molecular weights mentioned in this ap¬ plication are average molecular weights.
Advantages of the invention
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.
Summarized, the process cost per kilogram is reduced in com¬ parison with the corresponding prior art process for producing therapeutic polypeptides, which of course is especially im¬ portant when processing very large amounts of polypeptides.
According to the above described prior art document EP 0 632 082-A1 (see page 19, line 41) mPEG chloroformiate readily reacts with a number of chemical groups.
This is a simplification, as this is only correct when the reaction takes place in organic solvent. In an aqueous reaction solution chloroformiate will mainly react (and attach) with amine groups on the polypeptide and reaction with (attachment to) other groups will be much less pronounced. Therefore, when using the process of the invention a conjugation product consisting of PAO-polymers conjugated to a number of chemical groups on the polypeptide can be obtained, and if desired a conjugation product mainly consisting of PAO-polymers conju¬ gated to the amine groups on the polypeptide can be obtained.
Consequently 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.
Immunogenicitv. antigenicity and allergenicity
"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¬ duced into circulatory system of animals and humans, is capable of stimulating an immunologic response.
The term "antigen" refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.
Further, 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) .
It is, in the context of enzymes for industrial applications, important to distinguish between allergens mediating allergic responses e .g. intradermally, and respiratory allergens causing allergic responses by contact with cell-bound IgE in the respiratory tract.
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 number of in vitro animal models exist for assessment of the allergenicity of polypeptides. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a rat model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitized animals. According to these models the alleged allergens are introduced intratra¬ cheally into the animals. 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. Prentø, 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.
Other animals such as rabbits may also be used for comparable studies.
In 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.
Polypeptides
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.
In an embodiment of the invention the polypeptide is a protein having a biological activity, such as anti-microbial activity.
In a preferred embodiment of the invention the protein is an enzyme selected from the group of proteases, lipases, trans- ferases, carbohydrases, oxidoreductases, and phytases.
Especially contemplated is enzymes with a molecular weight in the range between about 10 kDa and 100 kDa.
In the cases of the polypeptide having a biological or enzyma¬ tic activity, said activity is substantially maintained.
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.
In Example 1 and Example 2 the preparation of a 15,000 and 5,000 methoxypolyethylene glycol chloroformiate, respectively, is described. However, according to the invention the halogen may also be any other halogen, such as Br or F. In step b) of the process of the invention the attachment groups on the polypeptide can be any group selected from the group including amines, hydroxyls, alcohols, phenols, or carboxylic acids.
More specifically the 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 - (0-R2)a - (0-R3) - (0-R4)c - 0 - C=0 - Y
wherein
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 - (0-R2)a - (0-R3)b - (0-R4)c - 0 - C=0 - X -)n polypeptide
wherein 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.
Preferably the polypeptide conjugate of the invention is produced by the process of the invention.
In a preferred embodiment of the invention R2, R3 and R4 are CH2-CH2, CH2-CH-CH3, or CH2-CH2-CH2-CH2.
Activated PAO-polymers having a molecular weight (Mr) between 1 and 60 kDa may be used as starting material for the process of the invention. Preferred are polymers having a molecular weight (Mr) of between 2 kDa and 35 kDa, especially between 2 kDa and 25 kDa.
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.
A conjugate of the invention can be stored by freezing e .g. at about 18°C.
Compositions
The invention also relates to compositions comprising at least one polypeptide conjugate of the invention.
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.
Examples of said enzymes/polypeptides include proteases, lipases, oxidoreductases, carbohydrases, transferases, such as transglutaminases, anti-microbial polypeptides, and phytases.
Detergent Compositions
According to the invention, 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. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in patent GB 1483591. 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. 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. It may also contain 0-40% of non-ionic surfactant such as alcohol ethoxylate (AEO or AE) , alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolygly- coside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154) .
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. One, more or all these enzymes/polypeptides may be modified according to the invention. Normally 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) .
The detergent builders may be subdivided into phosphorus- containing and non-phosphorous-containing types. Examples of phosphorus-containing inorganic alkaline detergent builders include the water-soluble salts, especially alkali metal pyrophosphates , orthophosphates, polyphosphates and phospho¬ nates. Examples of 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.
Examples of 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.
The detergent composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. The bleaching agents may be coated or incapsulated. Examples of 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 H20 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylene- diamine (TAED) or nonanoyloxybenzenesulfonate (NOBS) .
Examples of 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.
In dishwashing detergents the oxygen bleaches are preferred, for example in the form of an inorganic persalt, preferably with a bleach precursor or as a peroxy acid compound. Typical examples of 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.
The pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e .g. in the range of 7-11.
Particular forms of laundry detergent compositions within the scope of the invention include:
1) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
Linear alkylbenzenesulfonate (cal¬ 7 - 12% culated as acid)
Alcohol ethoxysulfate (e.g. Cι _ιs alcohol, 1-2 EO) or alkyl sulfate 1 - 4% (e.g. C16-i8)
Alcohol ethoxylate (e.g. Ci4_ι5 alco¬ hol, 7 EO) 5 - 9%
Sodium carbonate (as Na Cθ3) 14 - 20%
Soluble silicate (as Na20,2Si0 ) 2 - 6%
Zeolite (as NaAlSiOd 15 - 22%
Sodium sulfate (as Na Sθ4) 0 - 6%
Sodium citrate/citric acid (as C6H5Na307/C6H807) 0 - 15%
Sodium perborate (as NaB03.H20) 11 - 18%
TAED 2 - 6%
Carboxymethylcellulose 0 - 2%
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)
2) 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 ethoxysulfate (e.g. C12_!8 alcohol, 1-2 EO or alkyl sulfate 1 - 3% (e.g. C16_18)
Alcohol ethoxylate (e.g. C14..15 alco¬ hol, 7 EO) 5 - 9%
Sodium carbonate (as Na2C03) 15 - 21%
Soluble silicate (as Na20,2Si02) 1 - 4%
Zeolite (as NaAlSiθ4) 24 - 34%
Sodium sulfate (as Na2Sθ4) 4 - 10%
Sodium citrate/citric acid (as C6H5Na307/C6H8θ7) 0 - 15%
Carboxymethylcellulose 0 - 2%
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)
3) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
Linear alkylbenzenesulfonate (cal¬ 5 - 9% culated as acid)
Alcohol ethoxylate (e.g. Cι2_ι5 alco¬ hol, 7 EO) 7 - 14%
Soap as fatty acid (e .g. Cιg-22 fatty 1 - 3% acid)
Sodium carbonate (as Na C0 ) 10 - 17%
Soluble silicate (as Na20,2Si0 ) 3 - 9%
Zeolite (as NaAlSiθ4) 23 - 33%
Sodium sulfate (as Na2S04) 0 - 4%
Sodium perborate (as NaB0 .H 0) 8 - 16%
TAED 2 - 8% τ,hosphonate (e.g. EDTMPA) 0 - 1%
Carboxymethylcellulose 0 - 2%
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)
4) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
Linear alkylbenzenesulfonate (cal¬ 8 - 12% culated as acid)
Alcohol ethoxylate (e.g. Cι2_15 alco¬ hol, 7 EO) 10 - 25%
Sodium carbonate (as Na2C03) 14 - 22%
Soluble silicate (as Na20,2Si02) 1 - 5%
Zeolite (as NaAlSi0 ) 25 - 35%
Sodium sulfate (as Na2S0 ) 0 - 10%
Carboxymethylcellulose 0 - 2%
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)
5) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate (cal¬ 15 - 21% culated as acid)
Alcohol ethoxylate (e .g. Cι _ιs alco¬ hol, 7 EO or C12_i5 alcohol, 5 EO) 12 - 18%
Soap as fatty acid (e.g. oleic acid) 3 - 13%
Alkenylsuccinic acid (C12_i ) 0 - 13%
Aminoethanol 8 - 18%
Citric acid 2 - 8%
Phosphonate 0 - 3%
Polymers (e.g. PVP, PEG) 0 - 3%
Borate (as B407) 0 - 2%
Ethanol 0 - 3%
Propylene glycol 8 - 14%
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)
6) An aqueous structured liquid detergent composition compris¬ ing
Linear alkylbenzenesulfonate (calculated as acid) 15 - 21%
Alcohol ethoxylate (e.g. Cι2_i5 alcohol, 7 EO, 3 - 9% or Cι2_i5 alcohol, 5 EO)
Soap as fatty acid (e.g. oleic 3 - 10% acid)
Zeolite (as NaAlSi04) 14 - 22%
Potassium citrate 9 - 18%
Borate (as B4O7) 0 - 2%
Carboxymethylcellulose 0 - 2%
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
Glycerol 0 - 5%
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)
7) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
Fatty alcohol sulfate 5 - 10%
Ethoxylated fatty acid monoethanol¬ 3 - 9% amide
Soap as fatty acid 0 - 3%
Sodium carbonate (as Na C03) 5 - 10%
Soluble silicate (as Na20,2Si02) 1 - 4%
Zeolite (as NaAlSi0 ) 20 - 40%
Sodium sulfate (as Na2S04) 2 - 8%
Sodium perborate (as NaB03.H 0) 12 - 18%
TAED 2 - 7%
Polymers (e.g. maleic/acrylic acid 1 - 5% copolymer, PEG)
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)
8) A detergent composition formulated as a granulate comprising
Linear alkylbenzenesulfonate (calculated as acid) 8 - 14%
Ethoxylated fatty acid monoethanol¬ 5 - 11% amide
Soap as fatty acid 0 - 3%
Sodium carbonate (as Na2C0 ) 4 - 10%
Soluble silicate (as Na20,2Si02) 1 - 4%
Zeolite (as NaAlSi04) 30 - 50%
Sodium sulfate (as Na2Sθ4) 3 - 11%
Sodium citrate (as C6H5Na307) 5 - 12%
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)
9) A detergent composition formulated as a granulate comprising
Linear alkylbenzenesulfonate (calculated as acid) 6 - 12%
Nonionic surfactant 1 - 4%
Soap as fatty acid 2 - 6%
Sodium carbonate (as Na2C03) 14 - 22%
Zeolite (as NaAlSiθ4) 18 - 32%
Sodium sulfate (as Na S04) 5 - 20%
Sodium citrate (as C6H5Na307) 3 - 8%
Sodium perborate (as NaB03.H20) 4 - 9%
Bleach activator (e.g. NOBS or TAED) 1 - 5%
Carboxymethylcellulose 0 - 2%
Polymers (e.g. polycarboxylate or 1 - 5% PEG)
Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
Minor ingredients (e.g. optical 0 - 5% brightener, perfume)
10) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate (calculated as acid) 15 - 23%
Alcohol ethoxysulfate (e.g. C12-i5 alcohol, 2-3 EO) 8 - 15%
Alcohol ethoxylate (e.g. C12-15 al¬ cohol, 7 EO, 3 - 9% or C12_15 alcohol, 5 EO)
Soap as fatty acid (e.g. lauric 0 - 3% acid)
Aminoethanol 1 - 5%
Sodium citrate 5 - 10%
Hydrotrope (e.g. sodium 2 - 6% toluensu1fonate)
Borate (as B4O7) 0 - 2%
Carboxymethylcellulose 0 - 1%
Ethanol 1 - 3%
Propylene glycol 2 - 5%
Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
Minor ingredients (e.g. polymers, dispersants, perfume, optical 0 - 5% brighteners)
11) An aqueous liquid detergent composition comprising
Linear alkylbenzenesulfonate (calculated as acid) 20 - 32%
Alcohol ethoxylate (e.g. Cι2_ι5 alco¬ hol, 7 EO, 6 - 12% or Cι _i5 alcohol, 5 EO)
Aminoethanol 2 - 6%
Citric acid 8 - 14%
Borate (as B4O7) 1 - 3%
Polymer (e.g. maleic/acrylic acid copolymer, anchoring polymer such as, e.g., lauryl 0 - 3% methacrylate/acrylic acid copolymer)
Glycerol 3 - 8%
Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
Minor ingredients (e.g. hydrotropes, dispersants, perfume, optical 0 - 5% brighteners)
12) 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)
Sodium carbonate (as Na2C03) 8 - 25%
Soluble silicates (as Na20, 2Si0 ) 5 - 15%
Sodium sulfate (as Na2Sθ4) 0 - 5%
Zeolite (as NaAlSiθ4) 15 - 28%
Sodium perborate (as NaB03.4H20) 0 - 20%
Bleach activator (TAED or NOBS) 0 - 5%
Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
Minor ingredients (e.g. perfume, 0 - 3% optical brighteners)
13) Detergent formulations as described in 1) - 12) wherein all or part of the linear alkylbenzenesulfonate is replaced by (C 12 _C 18) alkyl sulfate.
14) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
(Cι2-Cιs) alkyl sulfate 9 - 15%
Alcohol ethoxylate 3 - 6%
Polyhydroxy alkyl fatty acid amide 1 - 5%
Zeolite (as NaAlSiθ4) 10 - 20%
Layered disilicate (e.g. SK56 from Hoechst) 10 - 20%
Sodium carbonate (as Na2C03) 3 - 12%
Soluble silicate (as Na20,2Si02) 0 - 6%
Sodium citrate 4 - 8%
Sodium percarbonate 13 - 22%
TAED 3 - 8%
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)
15) A detergent composition formulated as a granulate having a bulk density of at least 600 g/1 comprising
(Cι2-Cιs) alkyl sulfate 4 - 8%
Alcohol ethoxylate 11 - 15%
Soap 1 - 4%
Zeolite MAP or zeolite A 35 - 45%
Sodium carbonate (as Na2C03) 2 - 8%
Soluble silicate (as Na20,2Si0 ) 0 - 4%
Sodium percarbonate 13 - 22%
TAED 1 - 8%
Carboxymethyl cellulose 0 - 3%
Polymers (e.g. polycarboxylates and 0 - 3% PVP)
Enzymes including modified enzymes 0.0001 - 0.5% (calculated as pure enzyme protein)
Minor ingredients (e.g. optical 0 - 3% brightener, phosphonate, perfume)
16) Detergent formulations as described in 1) - 15) which contain a stabilized or encapsulated peracid, either as an additional component or as a substitute for already specified bleach systems.
17) Detergent compositions as described in 1) , 3) , 7) , 9) and 12) wherein perborate is replaced by percarbonate.
18) Detergent compositions as described in 1), 3), 7), 9), 12), 14) and 15) which additionally contain a manganese catalyst. 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.
19) 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. The detergent may also comprise anionic surfactant and/or a bleach system.
Particular forms of dishwashing detergent compositions within the scope of the invention include:
1) POWDER AUTOMATIC DISHWASHING COMPOSITION
Nonionic surfactant 0.4 - 2.5%
Sodium metasilicate 0 - 20%
Sodium disilicate 3 - 20%
Sodium triphosphate 20 - 40%
Sodium carbonate 0 - 20%
Sodium perborate 2 - 9%
Tetraacetylethylenediamine (TAED) 1 - 4%
Sodium sulphate 5 - 33%
Enzymes including modified enzymes 0.0001 - 0.5%
2) POWDER AUTOMATIC DISHWASHING COMPOSITION
Nonionic surfactant - 2% (e.g. alcohol ethoxylate)
Sodium disilicate - 30%
Sodium carbonate 10 - 50%
Sodium phosphonate - 5%
Trisodium citrate dihydrate - 30%
Nitrilotrisodium acetate (NTA) - 20%
Sodium perborate monohydrate - 10%
Tetraacetylethylenediamine (TAED) - 2%
Polyacrylate polymer (e.g. maleic acid/acrylic acid co¬ - 25% polymer)
Enzymes including modified enzymes 0.0001 - 0.5%
Perfume 0.1 - 0.5%
Water - 10
3) POWDER AUTOMATIC DISHWASHING COMPOSITION
Nonionic surfactant 0.5 - 2.0%
Sodium disilicate 25 - 40%
Sodium citrate 30 - 55%
Sodium carbonate 0 - 29%
Sodium bicarbonate 0 - 20%
Sodium perborate monohydrate 0 - 15%
Tetraacetylethylenediamine (TAED) 0 - 6%
Maleic acid/acrylic 0 - 5% acid copolymer
Clay 1 - 3%
Poly(amino acids) 0 - 20%
Sodium polyacrylate 0 - 8%
Enzymes including modified enzymes 0.0001 - 0.5% 4) POWDER AUTOMATIC DISHWASHING COMPOSITION
Nonionic surfactant 1 2%
Zeolite MAP 15 42%
Sodium disilicate 30 34%
Sodium citrate 0 12%
Sodium carbonate 0 20%
Sodium perborate monohydrate 7 15%
Tetraacetylethylenediamine (TAED)
0 3%
Polymer 0 4%
Maleic acid/acrylic acid copolymer 0 5%
Organic phosphonate 0 4%
Clay 1 2%
Enzymes including modified enzymes 0.0001 - 0.5%
Sodium sulphate Balance
5) POWDER AUTOMATIC DISHWASHING COMPOSITION
Nonionic surfactant 1 7%
Sodium disilicate 18 30%
Trisodium citrate 10 24%
Sodium carbonate 12 20%
Monopersulphate (2 KHS05.KHS04.K2S0 ) 15 21%
Bleach stabilizer 0.1 2%
Maleic acid/acrylic acid copolymer 0 6%
Diethylenetriaminepentaacetate, pentasodium salt 0 2.5%
Enzymes including modified enzymes 0.0001 - 0.5%
Sodium sulphate, water Balance 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM
Nonionic surfactant 0 - 1.5%
Octadecyl dimethylamine N-oxide dihydrate 0 - 5%
80:20 wt.C18/C16 blend of octadecyl dimethylamine N-oxide dihydrate and hexadecyldimethyl amine N-oxide 0 - 4% dihydrate
70:30 wt.C18/C16 blend of octadecyl bis (hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis 0 - 5% (hydroxyethyl)amine N-oxide anhydrous
Cι3-Ci5 alkyl ethoxysulfate with an average degree of ethoxylation of 3 0 - 10%
Cι2-C15 alkyl ethoxysulfate with an average degree of ethoxylation of 3 0 - 5%
C 13~"C 15 ethoxylated alcohol with an average degree of ethoxylation of 12 0 - 5%
A blend of Cι2-Cιs ethoxylated alco¬ hols with an average degree of 0 - 6.5% ethoxylation of 9
A blend of Cι3-Cιs ethoxylated alco¬ hols with an average degree of 0 - 4% ethoxylation of 30
Sodium disilicate 0 - 33%
Sodium tripolyphosphate 0 - 46%
Sodium citrate 0 - 28%
Citric acid 0 - 29%
Sodium carbonate 0 - 20%
Sodium perborate monohydrate 0 - 11.5%
Tetraacetylethylenediamine (TAED) 0 - 4%
Maleic acid/acrylic acid copolymer 0 - 7.5%
Sodium sulphate 0 - 12.5%
Enzymes including modified enzymes 0.0001 - 0.5% 7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION
Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%
Alkali metal silicate 3.0 - 15.0%
Alkali metal phosphate 20.0 - 40.0%
Liquid carrier selected from higher glycols, polyglycols, polyoxides, 25.0 - 45.0% glycolethers
Stabilizer (e.g. a partial ester of phosphoric acid and a
Figure imgf000043_0001
0.5 - 7.0% alkanol)
Foam suppressor (e.g. silicone) 0 - 1.5%
Enzymes including modified enzymes 0.0001 - 0.5%
8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION
Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%
Sodium silicate 3.0 - 15.0%
Alkali metal carbonate 7.0 - 20.0%
Sodium citrate 0.0 - 1.5%
Stabilizing system (e.g. mixtures of finely divided silicone and low molecular weight dialkyl polyglycol 0.5 - 7.0% ethers)
Low molecule weight polyacrylate polymer 5.0 - 15.0%
Clay gel thickener (e.g. bentonite) 0.0 - 10.0%
Hydroxypropyl cellulose polymer 0.0 - 0.6%
Enzymes including modified enzymes 0.0001 - 0.5%
Liquid carrier selected from higher lycols, polyglycols, polyoxides and Balance glycol ethers 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION
12~C14 fatty acid 0 - 0.5%
Block co-polymer surfactant 1.5 - 15.0%
Sodium citrate 0 - 12%
Sodium tripolyphosphate 0 - 15%
Sodium carbonate 0 - 8%
Aluminium tristearate 0 - 0.1%
Sodium cumene sulphonate 0 - 1.7%
Polyacrylate thickener 1.32 - 2.5%
Sodium polyacrylate 2.4 - 6.0%
Boric acid 0 - 4.0%
Sodium formate 0 - 0.45%
Calcium formate 0 - 0.2%
Sodium n-decydiphenyl oxide disulphonate 0 - 4.0%
Monoethanol amine (MEA) 0 - 1.86%
Sodium hydroxide (50%) 1.9 - 9.3%
1,2-Propanediol 0 - 9.4%
Enzymes including modified enzymes 0.0001 - 0.5%
Suds suppressor, dye, perfumes, water
Balance
10) LIQUID AUTOMATIC DISHWASHING COMPOSITION
Alcohol ethoxylate 0 - 20%
Fatty acid ester sulphonate 0 - 30%
Sodium dodecyl sulphate 0 - 20%
Alkyl polyglycoside 0 - 21%
Oleic acid 0 - 10%
Sodium disilicate monohydrate 18 - 33%
Sodium citrate dihydrate 18 - 33%
Sodium stearate 0 - 2.5%
Sodium perborate monohydrate 0 - 13%
Tetraacetylethylenediamine (TAED) 0 - 8%
Maleic acid/acrylic acid copolymer 4 - 8%
Enzymes including modified enzymes 0.0001 - 0.5%
11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH PARTICLES
Sodium silicate 5 - 10%
Tetrapotassium pyrophosphate 15 - 25%
Sodium triphosphate 0 - 2%
Potassium carbonate 4 - 8%
Protected bleach particles, e.g. chlorine 5 - 10%
Polymeric thickener 0.7 - 1.5%
Potassium hydroxide 0 - 2%
Enzymes including modified enzymes 0.0001 - 0.5%
Water Balance
11) Automatic dishwashing compositions as described in 1) , 2) , 3), 4), 6) and 10), wherein perborate is replaced by per¬ carbonate.
12) Automatic dishwashing compositions as described in 1) - 6) which additionally contain a manganese catalyst. 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.
Personal care applications
Also for personal care products the conjugated enzymes with reduced allergenicity of the invention are of interest.
Proteases
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.
Further, proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.
Lipases
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.
Lipases are also effective ingredients in products for cleaning of contact lenses, where they remove lipid deposits from the lens surface.
Oxidoreductases
The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g. glucose) that ensures production of H202, which then will initiate the oxidation of for instance SCN" or I~ into antimicrobial reagents (SCNO~ or I2) by a peroxidase (usually lactoperoxidase) . This enzymatic complex is known in nature from e.g. milk and saliva.
It is utilized commercially as anti-microbial systems in oral care products (mouth rinse, dentifrice, chewing gum) , where it also can be combined with an amyloglucosidase to produce the glucose. These systems are also known in cosmetic products for preservation.
Anti-microbial systems comprising the combination of an oxidase and a peroxidase are known in the cleaning of contact lenses.
Another application of oxidoreductases is oxidative hair dyeing using oxidases, peroxidases and laccases .
Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair) .
The free radicals activate chain reactions that lead to destruction of fatty membranes, collagen, and cells. The application of free radical scavengers such as Superoxide dismutase into cosmetics is well-known (R. L. Goldemberg, DCI, NOV. 93, p. 48-52) .
Protein disulfide isomerase (PDI) 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
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 α-1,6 bound glucose (dextran) and α-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.
Also other kinds of biofilm for instance the biofilm formed in lens cases can be removed by the action of glucanases.
Anti-microbial polypeptides
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.
Food and Feed
Further conjugated enzymes or polypeptides with reduced aller¬ genicity according to the invention may advantageously be used in the manufacturing of food and feed.
Proteases
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.
To coagulate casein in milk when producing cheese proteases such as rennet or chymosin may be used.
In the brewery industry proteases are used for brewing with unmalted cereals and for controlling the nitrogen content.
In animal feed products proteases are used so to speak to expand the animals digestion system.
Lipases
The application of 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.
The flavour development of blue roan cheeses (e.g. Danablue) , certain Italian cheese types and other dairy products contain¬ ing butter fat are dependent on the degradation of milk fat into free fatty acids. Lipases may be used for developing flavour in such products.
In the oil- and fat producing industry lipases are used e.g. to minimize the amount of undesirable side-products, to modify fats by interesterification, and to synthesis of esters.
Oxidoreductases
Further oxidoreductases with reduced allergenicity according to the invention may advantageously be used in the manufacturing of food and feed.
Several oxidoreductases are used for baking, glucose oxidase, lipoxygenase, peroxidase, catalase and combinations hereof. Traditionally, 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.
Gluzyme™ (Novo Nordisk 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.
Carbohydrases
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. Selectively modifies the gelatinized starch by cleaving from the non-reducing end of the starch molecules, low molecular wight sugars and dextrins. The starch is modified in such a way that retrogradation is less likely to occur. The produced low-molecular-weight sugars improve the baked goods1 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 α-amylases which further provide good and uniform structure of the bread crumb. Said α-amylases are endoenzymes that produce maltose, dextrins and glucose. Cereal and some bacterial α-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.
By hydrolyzing the pentosans1 fraction in flour, it will lose a great deal of its water-binding capacity, and the water will then be available for starch and gluten. The gluten becomes more pliable and extensible, and the starch gelatinize more easily. Pentosanases can be used in combination with or as an alternative to emulsifiers.
Further 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 α-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. If high maltose syrups are desired, β-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.
In the sugar industry, it is common practice to speed up the break-down of present starch in cane juices. Thereby the starch content in the raw sugar is reduced, and filtration at the refinery facilitated.
Furthermore dextranases are used to break down dextran in raw sugar juices and syrups.
In the alcohol industry α-amylases are advantageously used for thinning of starch in distilling mashes.
In the brewing industry α-amylases are used for adjunct liquefaction.
In the dairy industry β-galactosidases (lactase) are used when producing low lactose milk for persons suffering from lactose malabsorption.
When flavoured milk drinks are produced from lactase-treated milk, the addition of sugar can be reduced without reducing the sweetness of the product.
In the production of condensed milk, lactose crystallization can be avoided by lactase treatment, and the risk of thickening caused by casein coagulation in lactose crystals is thus reduced.
When producing ice cream made from lactase-treated milk (or whey) no lactose crystals will be formed and the defect, sandiness, will not occur. Further, xylanases are known to be used within a number of food/feed industrial applications as described in WO 94/21785 (Novo Nordisk A/S) .
α-amylases are used in the animal feed industry to be added to cereal-containing feed to improve the digestibility of starch.
Anti-microbial polypeptides
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)
Transferases
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) .
Further producing 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) .
Furthermore for preparation of gels for yoghurt, mousses, chee¬ se, puddings, orange juice, from milk and milk-like products, and binding of chopped meat product, improvement of taste and texture of food proteins (see WO 94/21120 and WO 94/21129 from Novo Nordisk A/S) .
Phvtases
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)
Further phytases may be used in soy processing. 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) .
Also for baking purposes 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)
A high phytase activity koji mold is known to be used for producing refined sake (see JP-0-6070749-A) . Textile applications
Proteases
Proteases are used for degumming and sand-washing of silk.
Lipases
Lipases are used for removing fatty matter containing hydro- phobic esters (e.g. triglycerides during, the
Figure imgf000055_0001
of textiles (see e.g. WO 93/13256 from Novo Nordisk A/S) .
Oxidoreductases
In bleach clean-up of textiles catalases may serve to remove excess hydrogen peroxide.
Carbohydrases
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") .
Also cellulolytic enzymes find use in the bio-polishing pro¬ cess. 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.
During the weaving of textiles, the threads are exposed to con¬ siderable mechanical strain. In order to prevent breaking, 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. Oral and dermal pharmaceuticals
Proteases
Different combinations of highly purified proteases (e.g. Trypsin and Chymotrypsin) are used in pharmaceuticals to be taken orally, and dermal pharmaceuticals for combating e.g inflammations, edemata and injuries.
Leather production
Transferase
Transglutaminase is known to be used for casein finishing of leather by acting as a hardening agent (see WO 94/13839 from Novo Nordisk) .
Hard surface cleaning
Cleaning of hard surfaces e.g. in the food industry is often difficult, as equipment used for producing dairies, meat, sea food products, beverages etc. often have a complicated shape. The use of surfactant compositions in the form gels and foams comprising enzymes have proven to facilitate and improve hard surface cleaning. Enzymes, which advantageously may be added in such surfactant compositions, are in particular proteases, lipases, amylases and cellulases.
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.
Finally the invention relates to the use of the conjugate of the invention or a composition of the invention in products comprising polypeptides.
First of all the conjugate or compositions of the invention can advantageously be used for personal care products, such as hair care and hair treatment products. This includes products such as shampoo, balsam, hair conditioners, hair waving compositions, hair dyeing compositions, hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.
Further oral care products are contemplated such as dentifrice, mouth washes, chewing gum.
Also 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.
Also for contact lenses hygiene products the conjugate of the invention can be used advantageously. Such products include contact lenses cleaning and disinfection products.
The use for detergents such as washing powder, soap, soap bars, liquid soap are also contemplated.
METHODS AND MATERIALS
Materials:
Methoxypolyethylene glycol 15.000 (mPEG from Shearwater)
Methoxypolyethylene glycol 5.000 (mPEG from Fluka)
Subtilisin Novo (from Novo Nordisk A/S)
Carezyme© core (from Novo Nordisk A/S)
Succinyl-Alanine-Alanine-Proline-Phenylalanine-para- nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.
ELISA reagents:
Horse Radish Peroxidase labeled anti-rat-Ig ( Dako, DK, P162, 031; dilution 1:1000).
Biotin-labelled mouse-anti-rat-IgE (Zymed 03-9740; dilution
1:1000) .
Streptavidin-horse radish peroxidase (Kirkegard & Perry 14-30-
00; dilution 1:1000).
Solutions:
Stop-solution (DMG-buffer)
Sodium Borate, borax (Sigma)
3,3-Dimethyl glutaric acid (Sigma)
CaCl2 (Sigma)
Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka)
Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no.
822184) l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka)
N-Hydroxy succinimide (Fluka art. 56480))
Phosgene (Fluka art. 79380)
Lactose (Merck 7656)
PMSF (phenyl methyl sulfonyl flouride) from Sigma
Colouring substrate:
OPD: o-phenylene-diamine, (Kementec cat no. 4260)
Test Animals:
Brown Norway rats (from Charles River, DE)
Ecfuipment:
XCEL II (Novex)
ELISA reader (UVmax, Molecular Devices)
HPLC (Waters)
PFLC (Pharmacia)
Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.
SLT: Fotometer from SLT Lablnstruments
Size-exclusion chromatograph (Spherogel TSK-G2000 SWG) .
Size-exclusion chromatograph (Superdex 200, Pharmacia, SW)
Amicon Cell Protease activity
Analysis with Suc-Ala-Ala-Pro-Phe-pNA:
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 μl of this is diluted into 10 ml with Britton and Robinson buffer.
Analysis
The substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS405 nm/min.
The temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.
Analysis with Casein
The analysis is performed according to standard Novo Nordisk procedures described in AF 220 which are hereby included as reference (and available of request) .
Carezyme® activity
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. In addition, cellulytic activity was measured as described in "EAL-SM-0373.01/01" (available from Novo Nordisk on request) . ELISA Ig/IgE test system
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 μg per ml and 50 μl 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 μl 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 μl 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 μl 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 μl per well Streptavidin horse radish peroxidase, diluted 2000 times is incubated 1 hour on an agitation platform.
The colouring substrate (50 μl per well) is OPD (6 mg) and H202
(4 μl of a 30% solution) per 10 ml citrate buffer pH 5.2. The reaction is stopped using 100 μl per well 2 N H2S0 . All readings on SLT are performed at 486 nm and 620 nm as refer¬ ence. Data is calculated and presented in Lotus.
Intratracheal (IT) stimulation of rats
For IT administration of molecules disposable syringes with a 2 " long metal probe are used. This probe is instilled in the trachea of the rats approximately 1 cm below the epiglottis, and 0.1 ml of a solution of the molecules is deposited. The animals are stimulated 4 times, with 5 days between the last stimulation and exsanguination.
The 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.
Determination of the molecular weight
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.
EXAMPLES
EXAMPLE 1
Preparation of mPEG 15.000 chloroformiate
10 gram mPEG 15,000 was suspended in 60 ml toluene of which 15 ml was distilled off to remove any trace of water. After cooling to ambient temperature some precipitate formed, which re-dissolved upon addition of 10 ml anhydrous dichloromethane. 1.7 ml phosgene (1.93 M in toluene 5 eqv.) was added without any detectable reaction. After 14 hours at ambient temperature the mixture was evaporated to dryness to remove phosgene. To improve the crystalline structure it is preferable to re- crystallise from toluene (dry 5 ml/ g mPEG) . Yield after filtration and drying generally exceeds 98% and activation degree is better than 90% by NMR. ^-NMR for mPEG 15,000 (CDC13) δ 3.38 s (1= 2.6 CH3 i OMe), 3.40* dd (I = 4.5 0/00, 13C satellite), 3.64 bs (I = 1364 main peak), 3.89* dd (I = 4.8 0/00,, 13C satellite), 4.46 q* (I = 1.8, CH α to chloroformiat) . When stored in an desiccator at 22°C a decrease in activation degree of 40% was detected after 3 months and a new peak was detected at δ 4.37. When stored for 5 months a decrease of 70% was detected and the same peak at δ 4.37 was seen. When stored at -18°C no change was detected after 3 months.
EXAMPLE 2
Preparation of mPEG 5.000 chloroformiate
10 gram mPEG 5,000 was suspended in 60 ml toluene of which 15 ml was distilled off to remove any trace of water. After cooling to ambient temperature some precipitate formed, which redissolved upon addition of 10 ml anhydrous dichloromethane. 15.2 ml phosgene (1.93 M in toluene 5 eqv.) was added without any detectable reaction. After 14 hours at ambient temperature the mixture was evaporated to dryness to remove phosgene. To improve the crystalline structure the mixture was re- crystallised.
EXAMPLE 3
Conjugation of protease with mPEG 15.000-chloroformiate To a solution (10 ml) of 100 mg of highly purified Subtilisin Novo in 0.1 M Borate (pH 9.5, 0.5 M NaCl) 3 x 550 mg of methoxypolyethylene glycol-15,000-chloroformiate was added sequential at 0, 30 & 75 minutes.
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%
EXAMPLE 4
Conjugation of a protease with mPEG 5,000 chloroformiate To a solution (10 ml) of 100 mg of highly purified Subtilisin Novo in 0.1 M Borate (pH 9.5) and 50% dimethylformamide (DMF) 3 x 367 mg of methoxypolyethylene glycol 5,000 chloroformiate was added sequential at 0, 30 & 75 minutes.
The 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.
EXAMPLE 5
Conjugation of a cellulase with mPEG 5.000 chloroformiate
The catalytic core-domain of Carezyme© was prepared according to Boisset, C. et al. (1995), FEBS Lett. 376, p. 49-52.
To a solution (12 ml) of 100 mg of the purified Carezyme© core in 0.1 M NaHC03f 0.2 M Na-Borate (pH 8.5) 1.5 g of methoxypolyethylene glycol 5,000 chloroformiate was added.
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 NaHC03 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.
The final two times 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.
EXAMPLE 6
IT-Studies in Brown Norway Rats.
To assess the reduction in allergenicity of enzymes resulting from modification according to the invention Carezyme© core
(unmodified), Carezyme© core-PEG 5,000 (modified. Example 5), Subtilisin Novo (unmodified), and Subtilisin Novo-PEG 15,000
(modified. Example 3), Glycine-PEG 15,000, 0.9% NaCl (control) was introduced intratracheally (IT) into Brown Norway rats
(BN).
Sera from immunized BN rats were tested in the ELISA assay (described above) to elucidate whether the molecules had pene¬ trated the lung epithelias and activated the immune response system giving rise to a total Ig (assessed as IgG) and IgE response.
The results of the IT tests are shown in Figure 1 to 5.
As can be seen from the Figures 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) has been reduced in comparison to the BN rats having been exposed intratracheally with the parent unmodified polypeptides.
As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without depart¬ ing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the sub¬ stance defined by the following claims.
As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without depart¬ ing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the sub¬ stance defined by the following claims.

Claims

PATENT CLAIMS
1. A process of producing polyalkylene oxide-polypeptide conjugates with reduced allergenicity using a polyalkylene oxide (PAO) as the starting material, comprising the steps of
a) generating a polyalkylene oxide halogen formiate, and
b) conjugating at least one polyalkylene oxide halogen formiate to attachment groups on the polypeptide.
2. The process according to claim 1, wherein the polyalkylene oxide (PAO) is an polyalkylene glycol (PAG) or and methoxypolyalkylene glycol ( PAG) .
3. The process according to claim 2, wherein the PAG or mPAG is an polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG) .
4. The process according to any of claims 1 to 3, wherein the polypeptide is a protein.
5. The process according to any of the claims 1 to 4, wherein the polypeptide has anti-microbial activity.
6. The process according to claims 4 and 5, wherein the protein has biological activity.
7. The process according to any of claims 4 to 6, wherein the protein has enzymatic activity.
8. The process according to claim 7, wherein the enzyme is a protease, a lipase, a transferase, a carbohydrase, an oxidoreductase, or a phytase.
9. The process according to any of claims 1 to 8, wherein the polyalkylene oxide halogen formiate is a polyalkylene oxide chloroformiate.
10. The process according to claim 9, wherein the polyalkylene oxide chloroformiate is a polyalkylene glycol chloroformiate.
11. The process according to claim 10, wherein the polyalkylene glycol chloroformiate is a polyethylene glycol chloroformiate.
12. The process according to claim 10, wherein the polyethylene glycol chloroformiate is a methoxypolyethylene glycol chloroformiate.
13. The process according to any of claims 1 to 12, wherein step a) is mediated by phosgene (C12-C=0) .
14. The process according to any of claims 1 to 13, wherein the attachment groups are at least one selected from the groups including amines, alcohols, phenols, or carboxylic acids.
15. The process according to claim 14, wherein the polyalkylene oxide halogen formiate attach to the side chain serine, threonine, tyrosine, lysine, arginine, aspartate, glutamate in the polypeptide chain.
16. An activated polyalkylene oxide capable of linking to attachment groups on a polypeptide, whose activated PAO has the generic structure
Rl - (0-R2)a - (0-R3)b - (0-R4)c - O - C=0 - Y
wherein
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 nitrile
17. The activated PAO according to claim 16, wherein the halogen is Cl, Br, or F.
18. The activated PAO according to claims 16 and 17, wherein R2, R3 and R4 is CH2-CH2, CH2-CH-CH3, or CH2-CH2-CH2-CH2.
19. The activated PAO according to any of claims 16 to 18, wherein the molecular weight lies between 1 kDa and 60 kDa, such as between 2 kDa and 35 kDa, especially between 2 kDa and 25 kDa.
20. A polyalkylene oxide-polypeptide conjugate having the general formula
(Rl - (0-R2)a - (0-R3)b - (0-R4)c - O - C=0 - X -)n polypeptide
wherein
Rl, R2, R3, R4, a,b and c, are defined in claim 16, n is an integer between 1 and 100, and X is a coupling group between the polymer and an polypeptide.
21. The conjugate according to claim 20 being produced by the process according to claims 1 to 15.
22. The conjugate according to claims 20 and 21, wherein X is an attachment group selected from the group including amines, alcohols, phenols, and/or carboxylic acid group on the polypeptide.
23. The conjugate according to any of claims 20 to 22, wherein R2 , R3 and R4 are2-CH2 , CH2-CH-CH3 , or CH2-CH2-CH2-CH2 .
24. The conjugate according to any of claims 20 to 23, wherein the polypeptide is a protein.
25. The conjugate according to any of claims 20 to 24, wherein the polypeptide has anti-microbial activity.
26. The conjugate according to claims 24 and 25, wherein the protein has biological activity.
27. The conjugate according to any of claims 24 to 26, wherein the protein has enzymatic activity.
28. The conjugate according to claim 27, wherein the enzyme is a protease, a lipase, a transferase, a carbohydrase, an oxido- eductase, or a phytase.
29. The conjugate according to any of claims 20 to 28, wherein the total molecular weight lies 50 kDa and 250 kDa, preferably between 80 kDa and 200 kDa.
30. The conjugate according to any of claims 20 to 29, wherein the molecular weight of the PAO lies between 1 kDa and 60 kDa, such as between 2 kDa and 35 kDa, especially between 2 kDa and 25 kDa.
31. A composition comprising a polyalkylene oxide-polypeptide conjugate according to any of claims 20 to 30, which further comprises other enzymes/polypeptides and/or ingredients normally used in detergents, including soap bars, household articles, agrochemicals, personal care products, including cleaning preparations for contact lenses and skin and hair cleaning preparations, cosmetics, toiletries, oral and dermal pharmaceuticals, composition for treating textiles, composi¬ tions for cleaning hard surfaces, and compositions used for manufacturing food and feed.
32. The composition according to claim 31, wherein one, more or all of said other enzymes/polypeptides are modified according to any of claims 1 to 15 or are conjugates according to any of claims 16 to 30.
33. The composition according to claims 31, comprising at least one enzyme/polypeptide from the group comprising proteases, li¬ pases, oxidoreductases, carbohydrases, transferases, such as transglutaminases, anti-microbial polypeptides, and phytases.
34. Use of a conjugate according to any of claims 20 to 30 or compositions according to claims 31 and 33, in industrial products comprising a polypeptide.
35. The use according to claim 34 in personal care products.
36. The use according to claim 35 for hair care or hair treatment products.
37. The use according to claim 36 for shampoo, balsam, hair conditioners, hair waving compositions, hair dyeing composi¬ tions, hair tonic, hair liquid, hair cream, shampoo, hair rinse, hair spray.
38. The use according to claim 35 in oral care products.
39. The use according to claim 38 for dentifrice, mouth washes, chewing gum.
40. The use according to claim 35, in skin care products.
41. The use according to claim 40, in skin cream, skin milk, cleansing cream, cleansing lotion, cleansing milk, cold cream, cream soap, nourishing essence, skin lotion, milky lotion, ca- lamine lotion, hand cream, powder soap, transparent soap, sun oil, sun screen, shaving foam, shaving cream, and baby oil.
42. The use according to claim 35 in cosmetics.
43. The use according to claim 42 for lipstick, lip cream, creamy foundation, face powder, powder eye-shadow, powder, foundation, make-up base, essence powder, whitening powder.
44. The use according to claim 34 for contact lenses hygiene products.
45. The use according to claim 44 for contact lenses cleaning and disinfection products.
46. The use according to claim 34 in detergents.
47. The use according to claim 46 in washing powder.
48. The use according to claim 46 in liquid detergents.
49. The use according to claim 46 in dishwash detergents.
50. The use according to claim 46 for soap, soap bars, liquid soap.
51. The use according to claim 34, in oral and dermal pharmaceuticals.
52. The use according to claim 34, in agrochemicals.
53. The use according to claim 34, in food or feed.
54. The use according to claim 53, in baking products.
55. The use according to claim 34, in products for processing textiles.
56. The use according to claim 34, in compositions for cleaning hard surfaces.
57. A method for reducing the allergenicity of industrial polypeptides by using the process according to claims 1 to 15.
PCT/DK1996/000249 1995-06-07 1996-06-07 Modification of polypeptides WO1996040791A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU58936/96A AU5893696A (en) 1995-06-07 1996-06-07 Modification of polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK0639/95 1995-06-07
DK63995 1995-06-07

Publications (1)

Publication Number Publication Date
WO1996040791A1 true WO1996040791A1 (en) 1996-12-19

Family

ID=8095867

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1996/000249 WO1996040791A1 (en) 1995-06-07 1996-06-07 Modification of polypeptides

Country Status (2)

Country Link
AU (1) AU5893696A (en)
WO (1) WO1996040791A1 (en)

Cited By (73)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030148A1 (en) * 1996-02-15 1997-08-21 Novo Nordisk A/S Conjugation of polypeptides
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
US6284246B1 (en) 1997-07-30 2001-09-04 The Procter & Gamble Co. Modified polypeptides with high activity and reduced allergenicity
WO2002072057A2 (en) * 2001-03-07 2002-09-19 The Procter & Gamble Company Topical composition comprising a functionally acylating cosmetic bonding agent
WO2002077187A2 (en) 2001-03-23 2002-10-03 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6565865B2 (en) 2001-03-07 2003-05-20 The Procter & Gamble Company Topical composition comprising a three membered cyclic compound-based cosmetic bonding agent
US6569663B1 (en) 1998-03-26 2003-05-27 The Procter & Gamble Company Serine protease variants having amino acid substitutions
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
US6586223B1 (en) 1999-07-22 2003-07-01 The Procter & Gamble Company Subtilisin protease variants having amino acid substitutions in defined epitope regions
US6589542B2 (en) 2001-03-07 2003-07-08 The Procter & Gamble Company Topical composition comprising a functionally alkylating cosmetic bonding agent
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
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
WO2004000366A1 (en) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Pegylated factor vii glycoforms
US6713075B2 (en) 2001-03-07 2004-03-30 The Procter & Gamble Company Topical composition comprising an aldehyde or ketone-based cosmetic bonding agent
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
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
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
WO2006009901A2 (en) 2004-06-18 2006-01-26 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
WO2006134173A2 (en) 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
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
WO2008030558A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Modified human plasma polypeptide or fc scaffolds and their uses
EP1982732A2 (en) 2000-02-11 2008-10-22 Maxygen Holdings Ltd. Factor VII or VIIA-like molecules
EP1997897A1 (en) 1998-04-15 2008-12-03 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
WO2009067636A2 (en) 2007-11-20 2009-05-28 Ambrx, Inc. Modified insulin polypeptides and their uses
EP2080771A2 (en) 2001-02-27 2009-07-22 Maxygen Aps New interferon beta-like molecules
EP2133098A1 (en) 2000-01-10 2009-12-16 Maxygen Holdings Ltd G-CSF conjugates
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
US7736872B2 (en) 2004-12-22 2010-06-15 Ambrx, Inc. Compositions of aminoacyl-TRNA synthetase and uses thereof
EP2213733A2 (en) 2006-05-24 2010-08-04 Novo Nordisk Health Care AG Factor IX analogues having prolonged in vivo half life
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
EP2263684A1 (en) 2003-10-10 2010-12-22 Novo Nordisk A/S IL-21 derivatives
EP2284191A2 (en) 2004-12-22 2011-02-16 Ambrx, Inc. Process for the preparation of hGH
US7947473B2 (en) 2004-12-22 2011-05-24 Ambrx, Inc. Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid
EP2327724A2 (en) 2004-02-02 2011-06-01 Ambrx, Inc. Modified human growth hormone polypeptides and their uses
US8012931B2 (en) 2007-03-30 2011-09-06 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
WO2011107591A1 (en) 2010-03-05 2011-09-09 Rigshospitalet Chimeric inhibitor molecules of complement activation
WO2011143274A1 (en) 2010-05-10 2011-11-17 Perseid Therapeutics Polypeptide inhibitors of vla4
US8093356B2 (en) 2005-06-03 2012-01-10 Ambrx, Inc. Pegylated human interferon polypeptides
US8114630B2 (en) 2007-05-02 2012-02-14 Ambrx, Inc. Modified interferon beta polypeptides and their uses
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
US8278418B2 (en) 2008-09-26 2012-10-02 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
WO2013004607A1 (en) 2011-07-01 2013-01-10 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
US8420792B2 (en) 2006-09-08 2013-04-16 Ambrx, Inc. Suppressor tRNA transcription in vertebrate cells
EP2633866A2 (en) 2003-10-17 2013-09-04 Novo Nordisk A/S Combination therapy
WO2013185115A1 (en) 2012-06-08 2013-12-12 Sutro Biopharma, Inc. Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use
WO2014036492A1 (en) 2012-08-31 2014-03-06 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
WO2015006555A2 (en) 2013-07-10 2015-01-15 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
US8961544B2 (en) 2010-08-05 2015-02-24 Lifebond Ltd. Dry composition wound dressings and adhesives comprising gelatin and transglutaminase in a cross-linked matrix
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
US9017664B2 (en) 2006-12-15 2015-04-28 Lifebond Ltd. Gelatin-transglutaminase hemostatic dressings and sealants
US9044456B2 (en) 2008-06-18 2015-06-02 Lifebond Ltd. Cross-linked compositions
US9066991B2 (en) 2009-12-22 2015-06-30 Lifebond Ltd. Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices
US9121024B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
WO2020056066A1 (en) 2018-09-11 2020-03-19 Ambrx, Inc. Interleukin-2 polypeptide conjugates and their uses
WO2020082057A1 (en) 2018-10-19 2020-04-23 Ambrx, Inc. Interleukin-10 polypeptide conjugates, dimers thereof, and their uses
WO2020168017A1 (en) 2019-02-12 2020-08-20 Ambrx, Inc. Compositions containing, methods and uses of antibody-tlr agonist conjugates
WO2021183832A1 (en) 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
WO2022040596A1 (en) 2020-08-20 2022-02-24 Ambrx, Inc. Antibody-tlr agonist conjugates, methods and uses thereof
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
WO2022212899A1 (en) 2021-04-03 2022-10-06 Ambrx, Inc. Anti-her2 antibody-drug conjugates and uses thereof

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
DE3440141A1 (en) * 1984-11-02 1986-05-07 Heiner Dipl.-Chem. Dr. 8000 München Eckert Use of bistrichloromethyl carbonate as a proreagent for phosgene
WO1990013540A1 (en) * 1989-04-19 1990-11-15 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
WO1991001758A1 (en) * 1989-08-07 1991-02-21 Debiopharm S.A. Biologically active drug polymer derivatives
DD287951A5 (en) * 1989-09-15 1991-03-14 Adw Zi F. Molekularbiologie,De PROCESS FOR IMMOBILIZING BIOLOGICALLY ACTIVE COMPOUNDS ON POLYOXYALKYLENE GLYCOL AND ITS MONOALKOXY DERIVATIVES
EP0557199A1 (en) * 1992-02-20 1993-08-25 Transgene S.A. Polyethyleneglycol hirudin conjugate, process of preparation and use in the cure of thrombosis
WO1994004193A1 (en) * 1992-08-21 1994-03-03 Enzon, Inc. Novel attachment of polyalkylene oxides to bio-effecting substances
WO1994017039A1 (en) * 1993-01-19 1994-08-04 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
EP0632082A1 (en) * 1993-06-29 1995-01-04 "HEYLECINA", Société Anonyme Preparation of activated carbamates of poly(alkylene glycol) and their use
WO1996007670A1 (en) * 1994-09-09 1996-03-14 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
DE3440141A1 (en) * 1984-11-02 1986-05-07 Heiner Dipl.-Chem. Dr. 8000 München Eckert Use of bistrichloromethyl carbonate as a proreagent for phosgene
WO1990013540A1 (en) * 1989-04-19 1990-11-15 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
WO1991001758A1 (en) * 1989-08-07 1991-02-21 Debiopharm S.A. Biologically active drug polymer derivatives
DD287951A5 (en) * 1989-09-15 1991-03-14 Adw Zi F. Molekularbiologie,De PROCESS FOR IMMOBILIZING BIOLOGICALLY ACTIVE COMPOUNDS ON POLYOXYALKYLENE GLYCOL AND ITS MONOALKOXY DERIVATIVES
EP0557199A1 (en) * 1992-02-20 1993-08-25 Transgene S.A. Polyethyleneglycol hirudin conjugate, process of preparation and use in the cure of thrombosis
WO1994004193A1 (en) * 1992-08-21 1994-03-03 Enzon, Inc. Novel attachment of polyalkylene oxides to bio-effecting substances
WO1994017039A1 (en) * 1993-01-19 1994-08-04 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
EP0632082A1 (en) * 1993-06-29 1995-01-04 "HEYLECINA", Société Anonyme Preparation of activated carbamates of poly(alkylene glycol) and their use
WO1996007670A1 (en) * 1994-09-09 1996-03-14 Surface Engineering Technologies, Division Of Innerdyne, Inc. Electrophilic polyethylene oxides for the modification of polysaccharides, polypeptides (proteins) and surfaces

Cited By (143)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (en) * 1996-02-15 1997-08-21 Novo Nordisk A/S Conjugation of 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
US6495136B1 (en) 1998-03-26 2002-12-17 The Procter & Gamble Company Proteases having modified amino acid sequences conjugated to addition moieties
US6908757B1 (en) 1998-03-26 2005-06-21 The Procter & Gamble Company Serine protease variants having amino acid deletions and substitutions
EP1997897A1 (en) 1998-04-15 2008-12-03 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
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
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
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
US6566115B1 (en) 1999-07-22 2003-05-20 The Procter & Gamble Company Protease conjugates having sterically protected clip sites
US6946128B1 (en) 1999-07-22 2005-09-20 The Procter & Gamble Company Protease conjugates having sterically protected epitope regions
EP2133098A1 (en) 2000-01-10 2009-12-16 Maxygen Holdings Ltd G-CSF conjugates
EP1982732A2 (en) 2000-02-11 2008-10-22 Maxygen Holdings Ltd. Factor VII or VIIA-like molecules
EP2319541A1 (en) 2000-02-11 2011-05-11 Bayer HealthCare LLC Factor VII or VIIA-like conjugates
EP2080771A2 (en) 2001-02-27 2009-07-22 Maxygen Aps New interferon beta-like molecules
US6565865B2 (en) 2001-03-07 2003-05-20 The Procter & Gamble Company Topical composition comprising a three membered cyclic compound-based 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
WO2002072057A3 (en) * 2001-03-07 2003-02-13 Procter & Gamble Topical composition comprising a functionally acylating cosmetic bonding agent
US6645512B2 (en) 2001-03-07 2003-11-11 The Procter & Gamble Company Topical composition comprising a functionally acylating cosmetic bonding agent
US6589542B2 (en) 2001-03-07 2003-07-08 The Procter & Gamble Company Topical composition comprising a functionally alkylating cosmetic bonding agent
WO2002072057A2 (en) * 2001-03-07 2002-09-19 The Procter & Gamble Company Topical composition comprising a functionally acylating 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 (en) 2001-03-23 2002-10-03 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
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 (en) 2002-06-21 2003-12-31 Novo Nordisk Health Care Ag Pegylated factor vii glycoforms
EP2263684A1 (en) 2003-10-10 2010-12-22 Novo Nordisk A/S IL-21 derivatives
EP2641611A2 (en) 2003-10-17 2013-09-25 Novo Nordisk A/S Combination therapy
EP2633866A2 (en) 2003-10-17 2013-09-04 Novo Nordisk A/S Combination therapy
US8906676B2 (en) 2004-02-02 2014-12-09 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
US8097702B2 (en) 2004-02-02 2012-01-17 Ambrx, Inc. Modified human interferon polypeptides with at least one non-naturally encoded amino acid and their uses
US9260472B2 (en) 2004-02-02 2016-02-16 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
US8232371B2 (en) 2004-02-02 2012-07-31 Ambrx, Inc. Modified human interferon polypeptides and their uses
US8907064B2 (en) 2004-02-02 2014-12-09 Ambrx, Inc. Modified human four helical bundle polypeptides and their uses
EP2327724A2 (en) 2004-02-02 2011-06-01 Ambrx, Inc. Modified human growth hormone polypeptides and their uses
US9175083B2 (en) 2004-06-18 2015-11-03 Ambrx, Inc. Antigen-binding polypeptides and their uses
WO2006009901A2 (en) 2004-06-18 2006-01-26 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
US7846689B2 (en) 2004-12-22 2010-12-07 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7838265B2 (en) 2004-12-22 2010-11-23 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7883866B2 (en) 2004-12-22 2011-02-08 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
US8143216B2 (en) 2004-12-22 2012-03-27 Ambrx, Inc. Modified human growth hormone
US7947473B2 (en) 2004-12-22 2011-05-24 Ambrx, Inc. Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid
US8163695B2 (en) 2004-12-22 2012-04-24 Ambrx Formulations of human growth hormone comprising a non-naturally encoded amino acid
US7959926B2 (en) 2004-12-22 2011-06-14 Ambrx, Inc. Methods for expression and purification of recombinant human growth hormone mutants
US8178108B2 (en) 2004-12-22 2012-05-15 Ambrx, Inc. Methods for expression and purification of recombinant human growth hormone
EP2284191A2 (en) 2004-12-22 2011-02-16 Ambrx, Inc. Process for the preparation of hGH
US7736872B2 (en) 2004-12-22 2010-06-15 Ambrx, Inc. Compositions of aminoacyl-TRNA synthetase and uses thereof
US7829310B2 (en) 2004-12-22 2010-11-09 Ambrx, Inc. Compositions of aminoacyl-tRNA synthetase and uses thereof
US7816320B2 (en) 2004-12-22 2010-10-19 Ambrx, Inc. Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35
US8178494B2 (en) 2004-12-22 2012-05-15 Ambrx, Inc. Modified human growth hormone formulations with an increased serum half-life
US8080391B2 (en) 2004-12-22 2011-12-20 Ambrx, Inc. Process of producing non-naturally encoded amino acid containing high conjugated to a water soluble polymer
US8093356B2 (en) 2005-06-03 2012-01-10 Ambrx, Inc. Pegylated human interferon polypeptides
EP2360170A2 (en) 2005-06-17 2011-08-24 Novo Nordisk Health Care AG Selective reduction and derivatization of engineered proteins comprinsing at least one non-native cysteine
WO2006134173A2 (en) 2005-06-17 2006-12-21 Novo Nordisk Health Care Ag Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine
US9488660B2 (en) 2005-11-16 2016-11-08 Ambrx, Inc. Methods and compositions comprising non-natural amino acids
EP2213733A2 (en) 2006-05-24 2010-08-04 Novo Nordisk Health Care AG Factor IX analogues having prolonged in vivo half life
US7919591B2 (en) 2006-09-08 2011-04-05 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8618257B2 (en) 2006-09-08 2013-12-31 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US8053560B2 (en) 2006-09-08 2011-11-08 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
US9133495B2 (en) 2006-09-08 2015-09-15 Ambrx, Inc. Hybrid suppressor tRNA for vertebrate cells
US8420792B2 (en) 2006-09-08 2013-04-16 Ambrx, Inc. Suppressor tRNA transcription in vertebrate cells
US8022186B2 (en) 2006-09-08 2011-09-20 Ambrx, Inc. Modified human plasma polypeptide or Fc scaffolds and their uses
WO2008030558A2 (en) 2006-09-08 2008-03-13 Ambrx, Inc. Modified human plasma polypeptide or fc scaffolds and their uses
US9636433B2 (en) 2006-12-15 2017-05-02 Lifebond Ltd Gelatin-transglutaminase hemostatic dressings and sealants
US9017664B2 (en) 2006-12-15 2015-04-28 Lifebond Ltd. Gelatin-transglutaminase hemostatic dressings and sealants
US9655988B2 (en) 2006-12-15 2017-05-23 Lifebond Ltd Gelatin-transglutaminase hemostatic dressings and sealants
US9079971B2 (en) 2007-03-30 2015-07-14 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US10377805B2 (en) 2007-03-30 2019-08-13 Ambrx, Inc. Modified FGF-21 polypeptides comprising non-naturally encoding amino acids and their uses
US8383365B2 (en) 2007-03-30 2013-02-26 Ambrx, Inc. Methods of making FGF-21 mutants comprising non-naturally encoded phenylalanine derivatives
US10961291B2 (en) 2007-03-30 2021-03-30 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
US9975936B2 (en) 2007-03-30 2018-05-22 Ambrx, Inc. Nucleic acids encoding modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US8012931B2 (en) 2007-03-30 2011-09-06 Ambrx, Inc. Modified FGF-21 polypeptides and their uses
US9517273B2 (en) 2007-03-30 2016-12-13 Ambrx, Inc. Methods of treatment using modified FGF-21 polypeptides comprising non-naturally occurring amino acids
US8114630B2 (en) 2007-05-02 2012-02-14 Ambrx, Inc. Modified interferon beta polypeptides and their uses
EP2930182A1 (en) 2007-11-20 2015-10-14 Ambrx, Inc. Modified insulin polypeptides and their uses
WO2009067636A2 (en) 2007-11-20 2009-05-28 Ambrx, Inc. Modified insulin polypeptides and their uses
US8946148B2 (en) 2007-11-20 2015-02-03 Ambrx, Inc. Modified insulin polypeptides and their uses
US9938333B2 (en) 2008-02-08 2018-04-10 Ambrx, Inc. Modified leptin polypeptides and their uses
EP3103880A1 (en) 2008-02-08 2016-12-14 Ambrx, Inc. Modified leptin polypeptides and their uses
US9044456B2 (en) 2008-06-18 2015-06-02 Lifebond Ltd. Cross-linked compositions
US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
WO2010011735A2 (en) 2008-07-23 2010-01-28 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
EP3225248A1 (en) 2008-07-23 2017-10-04 Ambrx, Inc. Modified bovine g-csf polypeptides and their uses
US8569233B2 (en) 2008-09-26 2013-10-29 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
US8278418B2 (en) 2008-09-26 2012-10-02 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
US9121024B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9156899B2 (en) 2008-09-26 2015-10-13 Eli Lilly And Company Modified animal erythropoietin polypeptides and their uses
US9121025B2 (en) 2008-09-26 2015-09-01 Ambrx, Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
US9644014B2 (en) 2008-09-26 2017-05-09 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
EP3216800A1 (en) 2008-09-26 2017-09-13 Ambrx, Inc. Modified animal erythropoietin polypeptides and their uses
US10428333B2 (en) 2008-09-26 2019-10-01 Ambrx Inc. Non-natural amino acid replication-dependent microorganisms and vaccines
EP2805965A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified porcine somatotropin polypeptides and their uses
EP2805964A1 (en) 2009-12-21 2014-11-26 Ambrx, Inc. Modified bovine somatotropin polypeptides and their uses
US9066991B2 (en) 2009-12-22 2015-06-30 Lifebond Ltd. Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices
US10202585B2 (en) 2009-12-22 2019-02-12 Lifebond Ltd Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices
EP3815708A1 (en) 2010-03-05 2021-05-05 Omeros Corporation Chimeric inhibitor molecules of complement activation
WO2011107591A1 (en) 2010-03-05 2011-09-09 Rigshospitalet Chimeric inhibitor molecules of complement activation
WO2011143274A1 (en) 2010-05-10 2011-11-17 Perseid Therapeutics Polypeptide inhibitors of vla4
US8961544B2 (en) 2010-08-05 2015-02-24 Lifebond Ltd. Dry composition wound dressings and adhesives comprising gelatin and transglutaminase in a cross-linked matrix
US9567386B2 (en) 2010-08-17 2017-02-14 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US11439710B2 (en) 2010-08-17 2022-09-13 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
US10751391B2 (en) 2010-08-17 2020-08-25 Ambrx, Inc. Methods of treatment using modified relaxin polypeptides comprising a non-naturally encoded amino acid
WO2012024452A2 (en) 2010-08-17 2012-02-23 Ambrx, Inc. Modified relaxin polypeptides and their uses
US9452222B2 (en) 2010-08-17 2016-09-27 Ambrx, Inc. Nucleic acids encoding modified relaxin polypeptides
EP4302783A2 (en) 2010-08-17 2024-01-10 Ambrx, Inc. Modified relaxin polypeptides and their uses
US9962450B2 (en) 2010-08-17 2018-05-08 Ambrx, Inc. Method of treating heart failure with modified relaxin polypeptides
US10702588B2 (en) 2010-08-17 2020-07-07 Ambrx, Inc. Modified relaxin polypeptides comprising a non-naturally encoded amino acid in the A chain
US11311605B2 (en) 2010-08-17 2022-04-26 Ambrx, Inc. Methods of treating heart failure and fibrotic disorders using modified relaxin polypeptides
US8735539B2 (en) 2010-08-17 2014-05-27 Ambrx, Inc. Relaxin polypeptides comprising non-naturally encoded amino acids
US11786578B2 (en) 2010-08-17 2023-10-17 Ambrx, Inc. Modified relaxin polypeptides and their uses
US10253083B2 (en) 2010-08-17 2019-04-09 Ambrx, Inc. Therapeutic uses of modified relaxin polypeptides
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
US9382305B2 (en) 2011-07-01 2016-07-05 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
WO2013004607A1 (en) 2011-07-01 2013-01-10 Bayer Intellectual Property Gmbh Relaxin fusion polypeptides and uses thereof
EP3505534A1 (en) 2012-06-08 2019-07-03 Sutro Biopharma, Inc. Antibodies comprising sitespecific nonnatural amino acid residues, methods of their preparation and methods of their use
WO2013185115A1 (en) 2012-06-08 2013-12-12 Sutro Biopharma, Inc. Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use
EP3135690A1 (en) 2012-06-26 2017-03-01 Sutro Biopharma, Inc. Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use
EP4074728A1 (en) 2012-08-31 2022-10-19 Sutro Biopharma, Inc. Modified peptides comprising an azido group
WO2014036492A1 (en) 2012-08-31 2014-03-06 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
EP3584255A1 (en) 2012-08-31 2019-12-25 Sutro Biopharma, Inc. Modified amino acids comprising an azido group
WO2015006555A2 (en) 2013-07-10 2015-01-15 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
EP3336103A1 (en) 2013-07-10 2018-06-20 Sutro Biopharma, Inc. Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use
WO2015054658A1 (en) 2013-10-11 2015-04-16 Sutro Biopharma, Inc. Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use
US11248031B2 (en) 2014-10-24 2022-02-15 Bristol-Myers Squibb Company Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides
US9434778B2 (en) 2014-10-24 2016-09-06 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US9631004B2 (en) 2014-10-24 2017-04-25 Bristol-Myers Squibb Company Modified FGF-21 polypeptides comprising an internal deletion and uses thereof
US10189883B2 (en) 2014-10-24 2019-01-29 Bristol-Myers Squibb Company Therapeutic uses of modified FGF-21 polypeptides
US10377806B2 (en) 2014-10-24 2019-08-13 Bristol-Myers Squibb Company Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides and uses thereof
US11364281B2 (en) 2017-02-08 2022-06-21 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and pharmaceutical compositions thereof
US11185570B2 (en) 2017-02-08 2021-11-30 Bristol-Myers Squibb Company Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides
US10266578B2 (en) 2017-02-08 2019-04-23 Bristol-Myers Squibb Company Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof
WO2020056066A1 (en) 2018-09-11 2020-03-19 Ambrx, Inc. Interleukin-2 polypeptide conjugates and their uses
WO2020082057A1 (en) 2018-10-19 2020-04-23 Ambrx, Inc. Interleukin-10 polypeptide conjugates, dimers thereof, and their uses
WO2020168017A1 (en) 2019-02-12 2020-08-20 Ambrx, Inc. Compositions containing, methods and uses of antibody-tlr agonist conjugates
WO2021183832A1 (en) 2020-03-11 2021-09-16 Ambrx, Inc. Interleukin-2 polypeptide conjugates and methods of use thereof
WO2022040596A1 (en) 2020-08-20 2022-02-24 Ambrx, Inc. Antibody-tlr agonist conjugates, methods and uses thereof
WO2022212899A1 (en) 2021-04-03 2022-10-06 Ambrx, Inc. Anti-her2 antibody-drug conjugates and uses thereof

Also Published As

Publication number Publication date
AU5893696A (en) 1996-12-30

Similar Documents

Publication Publication Date Title
WO1996040791A1 (en) Modification of polypeptides
AU725287B2 (en) Conjugation of polypeptides
AU697440B2 (en) Polypeptide with reduced allergenicity
EP1002064B1 (en) A modified polypeptide
US6106828A (en) Conjugation of polypeptides
ES2220114T3 (en) VARIANTS OF LITTLE ALLERGEN PROTEINS.
ES2289824T3 (en) GLYCOSILATED PROTEINS WITH REDUCED ALLERGENICITY.
AU736806B2 (en) A modified enzyme for skin care
EP1088059B1 (en) A polypeptide-polymer conjugate
US6416756B1 (en) Modified protease having 5 to 13 covalently coupled polymeric molecules for skin care
US6638526B1 (en) Polypeptides conjugated to copolymers of ethylene oxide and propylene oxide to reduce allergenicity
AU731076B2 (en) Composition comprising polypeptide with reduced allergenicity

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA