AU770911B2 - A polypeptide-polymer conjugate with improved wash performance - Google Patents

Description

WO 00/04138 PCT/DK99/00406 1 TITLE: A polypeptide-polymer conjugate with improved wash performance FIELD OF THE INVENTION s The present invention relates to a polypeptide-polymer conjugate wherein the polymer is a homopolymer graft, block, alternate, or random co-polymer coupled to the surface of the polypeptide. The invention also relates to industrial compositions and products comprising a conjugate of the o0 invention, the use of a polypeptide-polymer conjugate of the invention for improving the wash performance of industrial compositions and products, and finally a method for improving the wash performance of polypeptides.

BACKGROUND OF THE INVENTION In the detergent industry enzymes have for more than years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof.

Commercially most important enzymes are proteases.

An increasing number of commercially used enzymes e.g.

proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYMO (Novo Nordisk RELASE (Novo Nordisk MAXAPEMO (Gist-Brocades PURAFECTO (Genencor International, Inc.).

However, even though a number of useful enzyme variants have been described in the literature, there is still a need for new improved enzyme or enzyme variants for a number of industrial uses.

As polypeptides may potentially cause an undesired immune response dependent on the way of challenge typically an IgG and/or IgE response, techniques for reducing it have been developed during the last three decades.

WO 97/24421 and WO 97/24427 discloses the immobilization of enzymes by covalent binding on an activated polymer. The immobilization of one or more enzymes using an activated polymer has inter alia shown improved antigenicity profile of the enzyme protein. The inventors state that the advantages can be achieved SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 2 by structurally modifying the enzyme without affecting the enzyme performance profile in the detergent solution.

Although, immobilization should avoid the formation of airborne material, this method still represent a risk of dust or aerosol formation during handling and processing of the immobilisation step.

Another technique is the coupling technique where a number of polymeric molecules are coupled to the polypeptide in question. When using this technique the immune system have difficulties recognizing the epitopes (on the polypeptide's surface) responsible for the formation of antibodies, thereby reducing the immune response.

For polypeptides introduced directly into the circulatory system of the human body to give a particular physiological effect pharmaceuticals) the typical potential immune response is an IgG and/or IgM response, while polypeptides which are inhaled through the respiratory system industrial polypeptide) potentially may cause an IgE response (i.e.

allergic response).

One of the theories explaining the reduced immune response is that the polymeric molecule(s) shield(s) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation. Another theory or at least a partial factor is that the heavier the conjugate is the more reduced immune response is obtained.

Typically the polymers used for coupling to polypeptide to form conjugates are homopolymers, i.e. consisting of one repeating unit, ethylene oxide polyethylene glycol (PEG), or propylene oxide polypropylene glycol (PPG).

Saccharides, such as dextran have also been used.

US patent no. 4,179,337 concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG).

WO 96/17929 (Novo Nordisk A/S) concerns modified polypeptide conjugates coupled to polymeric molecules, in particular polyethylene glycol (PEG).

The present inventors have now surprisingly found that the SUBSTITUTE SHEET (RULE 26) 8, JAN. 2004 15:38 SPRUSON AND FERGUSON 61292615486 NO. 4927 P. 7 3 polymer-polypeptide conjugates have improved wash performance in comparison to the unmodified polypeptide, Summary Of The Invention Herein disclosed is a polypeptide-polymer conjugate with improved wash performance.

The present inventors have found that when coupling homo-polymers with a molecular weight in the range of 0.1 kDa to 60 kDa to a parent polypeptide with a molecular weight of between 4 kDa and 100 kDa the wash performance of the polypeptide is improved compared to the wash performance of the parent polypeptide.

The present inventors have further found that when coupling graft, block, alternate, or random co-polymers with the general formula: EOxPO,

(I)

wherein x=1-99%, y=1-99% and x+y=100% covalently to a parent polypeptide, used for industrial application, the wash performance is improved when compared to the parent 15 polypeptide.

In both cases the respiratory allergenicity may also be reduced when compared to S* the parent enzyme. In the latter case the respiratory allergenicity may even be reduced when compared to a corresponding conjugate coupled with PEG or other homopolymers.

Also herein disclosed are compositions for use in industrial products comprising a 20 conjugate of the invention.

Also herein disclosed is the use of conjugates for improving wash performance and a final aspect the invention relates to a method for improving wash performance of polypeptides.

."Thus, according to an embodiment of the invention, there is provided the use of a 25 polypeptide-polymer conjugate with improved wash performance for improving the wash performance of industrial compositions, wherein the polymer has a molecular weight in the range of from 100 Da to 350 Da, and wherein the polypeptide-polymer conjugate has reduced respiratory allergenicity relative to the unconjugated polypeptide.

Industrial polypeptides Polypeptides used for industrial applications often have an enzymatic and/or antimicrobial activity. Industrial ASlM7Sap COMS ID No: SMBI-00562040 Received by IP Australia: Time 15:40 Date 2004-01-08 WO 00/04138 PCT/DK99/00406 4 polypeptides are (in contrast to pharmaceutical polypeptides) not intended to be introduced into the circulatory system of the body.

Therefore, it is not very likely that industrial polypeptides, such as enzymes, used as active ingredients in industrial compositions and/or products (defined below), such as detergents, such as laundry and dish washing detergens, composition for treating textiles, and personal care products, including cosmetics, come into direct contact with the io circulatory system of the body of humans or animals, as such polypeptides (or products comprising such polypeptides) are not injected (or the like) into the bloodstream.

Thus, in the case of the industrial polypeptide the potential risk is respiratory allergy IgE response) as a consequence of inhalation of polypeptides through the respiratory passage.

In the context of the present invention "industrial polypeptides" are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the circulatory system of the body of humans and/or animals.

Examples of such polypeptide include polypeptides with enzymatic activity as defined below.

However, when coupling one or more polymers to an enzyme the performance of said enzyme will remain approximately the same or decrease as compared to the parent enzyme.

The catalytic performance of an enzymes depends among many things on the contact between the enzyme and the substrate at the active site(s). When coupling polymers to an enzyme the polymers will normally be distributed in a random manner on the surface of the enzyme. Also polymers will be coupled to the enzyme near the active site of said enzyme which leads to steric or spatial hindrance. One will therefore expect that the enzyme performance will be adversely affected.

The present inventors have surprisingly found that enzyme performance may be increased by conjugation to polymers.

DETAILED DESCRIPTION OF THE INVENTION SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 The present inventiors have now surprisingly suceeded in providing polypeptide-polymer conjugates, wherein the catalytic performance of the polypeptide is improved.

The present invention relates to a polypeptide-polymer conjugate suitable for industrial applications and incorporation as active ingredients in industrial products. Conjugates of the invention may also have reduced respiratory allergenicity.

The term "polypeptide-polymer conjugate" means in the context of the present invention that one or more polymers have o0 been covalent bound to the polypeptide.

The term "reduced allergenicity" means in the context of the present invention that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals), which can lead to an allergic state, is decreased when inhaling a modified polypeptide of the invention in comparison to the corresponding parent polypeptide. The term "respiratory allergenicity" may be used instead. The term "improved wash performance" means in the context of the present invention that the delta reflectance value of test material washed with the conjugate has increased compared to the delta reflectance value of test material washed with the parent enzyme (non-conjugate).

When the term "improved wash performance" is used in connection with e.g. skin care products, where no delta reflectance values are available, the term means that the cleansing effect has improved compared to the cleansing effect when using the parent enzyme (non-conjugate).

The present inventors have found that when a parent unmodified polypeptide is coupled to homo-polymers, graft, block, alternate, or random co-polymers the wash performance is improved. The potential allergenic response caused by inhalation of the polypeptide may also be reduced in comparison to a corresponding parent unmodified polypeptide.

In one aspect in the invention relates to conjugates, wherein the parent polypeptide may be coupled to polymeric molecules with a molecular weight in the range from 100 Da up to 10,000 Da, preferably 100 Da to 5,000 Da, more preferably 100 to 2,000 Da, especially 100 to 1,000 Da.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 6 It is advantageous to couple short/light polymeric molecules to the polypeptide in question as short/light polymeric molecules are known to have less tendency to inhibit a functional activity of the polypeptide. For instance, the active site of an enzyme coupled to polymeric molecules having a molecular weight as defined according embodiments of the present invention is easier accessible for the substrate in comparison to the corresponding enzyme coupled to larger/heavier polymeric molecules as the spatial hindrance by the polymeric molecules is less pronounced. Further, a polypeptide-polymer conjugate with smaller/lighter polymeric molecules has improved stability in comparison to a corresponding conjugate with larger/heavier polymeric molecules coupled to the polypeptide, as deformation of the polypeptide structure is minimal due to the fact that less weight is pulling the polypeptide structure in diffrent directions.

Another advantage of using small polymeric molecules is that they are cheaper to purchase as polymers are sold per kilo. This reduces the cost of producing a conjugate of the invention.

Furthermore, in comparison to immobilized enzymes, e.g.

multiple covalent attachment of enzymes to an activated polymer with multiple reactive groups,conjugates of the invention display individual polymer molecules covalenty attached to the protein surface. Thus avoiding cross-linking of enzymes, leads to more equal and increased distribution of the catalyst in the application medium. This may lead to a better performance of the conjugates of the invention per unit protein in comparison to immobilized enzymes.

It is well known, that a polymer can adopt different conformation/morphologies depending mainly, but not only on its molecular architecture, the solvent (here water), the temperature, and the concentration Forster and M.

Antonietti, Adv. Mater, 1998, 10, No.3, pp 195-217). Those conformation/morphologies include micelles of various shapes, lamellae, ordered cylinders, or bicontinous structures. The molecular conformation of co-polymers in aqueaous media like a solvated random coil, an extended coil, a rod-like polymer, a SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 7 hypercoil, and a vesicle are well known (Water soluble polymers, M. J. Comstock Ed., ACS Symposium Series, 1991).

Thus, without being limited to any theory it is believed that a graft, block, alternate, or random co-polymer linked to the polypeptide surface adopts a conformation in water which yields to a better shielding of the surface as does a more hydrophilic homopolymer. Also synergistic effects due to the formation of supramolecular structures may reduce the accessibility of the polypeptide surface. Furthermore, an o0 increased repulsion of the more lipophilic copolymer (in comparison to a PEG homopolymer) with the antibody might play a role.

Further, the more rigid structure (compared to homopolmers) of graft, block, alternate, or random co-polymer may make it is more difficult for the antibody to "find its way" (through the more ridgid polymers and the adopted conformation) to the epitope on the polypeptide surface responsible for the IgE formation which results in an allergic response.

The hydrophobicity of the polymer is also believed to have an influence on the potential allergenicity of a polypeptidepolymer conjugate.

By a proper choice of polymer and molecular architecture optimal coverage with respect shielding epitopes on the surface of the polypeptide can be obtained. Furthermore, by adjusting the properties of the attached polymers, optimized properties for different formulations, e.g. detergents, can be obtained.

In another aspect the invention relates to a polypeptidepolymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymer is characterized by the general formula: EO.POy

(I)

wherein x=1-99%, y=1-99%, and x+y=100%.

The polymer is preferably characterized by the general formula: wherein x=10-90%, y=10-90%, and x+y=100%.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 8 In a preferred embodiment of the invention the polymer consists of ethylene oxide units and propylene oxide units in a ration (EO unit: PO unit) of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10.

In a preferred embodiment said polymer has a molecular weight from 100 to 100,000 Da, in particular 100 to 50,000 Da, especially 100 to 10,000 Da.

In a more preferred embodiment said polymer has a molecular weight from 100 to 12,000 Da, more preferred from 300 to 3,000 Da.

In an embodiment of the invention the polymer is a diblock, triblock, multiblock polymer. The general formula should be interpreted as comprising polymers, wherein the EO units and PO units are placed independently.

Assessment of allergenicity Allergenicity may be assessed on the basis of inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent polypeptide with the corresponding modified polypeptide according to the invention.

A number of in vivo animal models exist for assessment of the allergenicity of polypeptide. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitised animals. According to these models the alleged allergens are introduced intratracheally into the animals.

A suitable strain of guinea pigs, the Dunkin Hartley strain, do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgGIB (see e.g. Prento, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level.

A rat strain suitable for intratracheal exposure to polypep- SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 9 tides, such as enzymes, is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.

More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al.,(1996), Fundamental and Applied Toxicology, 33, p. 1-10.

Other animals such as e.g. rabbits may also be used for comparable studies.

The polymeric molecule The polymeric molecules coupled to the polypeptide may be any suitable polymeric molecule with a molecular weight as defined according to the invention, including natural and synthetic homo-polymers, such as polyols poly-OH), polyamines poly-NH,) and polycarboxyl acids poly- COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.

Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylen glycols, PEG-glycidyl ethers (Epox-PEG), PEGoxycarbonylimidazole (CDI-PEG), Branched PEGs, star-shaped PEGs, poly-vinyl alcohol (PVA), poly-carboxylates, poly- (vinylpyrolidone), poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydrid, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-straches and hydroxy propyl-starches, glycogen, agaroses and derivates thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, bio-polymers, polyoxyethylene esters, including stearate, e.g.

PEG8stearate (Myrj 45), PEG40stearate (Myrj 52), (Myrj 53), PEGl00stearate (Myrj 59), and polyoxyethylene propylene glycol stearate, polyoxyethylene ethers, including 2 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Ethyl Ether, 2 Pentyl Ether, 2 Cetyl Ether, 2 Stearyl Ether, 2 Oleyl Ether, 3 Hexyl Ether, 3 Octyl Ether, 3 Decyl Ether, 3 Lauryl Ether, 3 Myristyl Ether, 3 Cetyl Ether, 3 Stearyl Ether, 4 Heptyl Ether, 4 Octyl Ether, 4 Decyl Ether, 4 Lauryl Ether, 4 Myristyl Ether, 4 Cetyl Ether, 4 Stearyl Ether 5 Hexyl Ether, Octyl Ether, 5 Decyl Ether, 5 Lauryl Ether, 5 Myristyl Ether, Cetyl Ether, 5 Stearyl Ether, 6 Decyl Ether, 6 Lauryl Ether, 6 Myristyl Ether, 6 Cetyl Ether, 6 Stearyl Ether, 7 Decyl Ether, 7 Lauryl Ether, 7 Myristyl Ether, 7 Stearyl Ether, 8 Decyl Ether, 8 Lauryl Ether, 8 Myristyl Ether, 8 Cetyl Ether, 8 Stearyl Ether, 9 Lauryl Ether, 10 Lauryl Ether, 10 Tridecyl Ether, Cetyl Ether, 10 Stearyl Ether, 10 Oleyl Ether, 20 Cetyl Ether, Isohexadecyl Ether, 20 Stearyl Ether, 20 Oleyl Ether, 21 Stearyl Ether, 23 Lauryl Ether, 100 Stearyl Ether, and polyoxyethylenesorbitans, Including Monolaurate, Monooleate, Monopalmitate, Monostearate, Trioleate, Tristearate.

Preferred polymeric molecules are non-toxic polymeric molecules such as polyethylene glycol (PEG) which further requires a relatively simple chemistry for its covalent coupling to attachment groups on the enzyme's surface.

Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially PEG, are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking, which is undesirable.

The polymer coupled to the polypeptide may also be a graft, block, alternate, or random co-polymer having the general formula: EOPOy(I) wherein x=1-99%, y=1-99%, and x+y=100%.

In a preferred embodiment of the invention the polymer consists of ethylene oxide units and propylene oxide units in a ration (EO unit: PO unit) of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 or 90:10.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 11 In a preferred embodiment said polymer has a molecule weight from 100 to 100,000 Da, in particular 100 to 50,000 Da, especially 100 to 10,000 Da.

In an embodiment of the invention invention the polymer is a diblock, triblock, multiblock polymer.

Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: poly(ethylene glycol-co-propylene glycol); poly(ethylene glycol-co-propylene glycol) mono butyl ether; poly(ethylene glycol-co-propylene glycol) mono methyl ether.

Preferred polymers are non-toxic polymers composed of e.g.

PEG and PPG co-polymers. Polymers requiring a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface are preferred.

Examples of specific block polymers which may be used to couple to the surface of the polypeptide are: poly(propylene glycol)-block-poly(ethyleneglycol)-block-poly(propylene glycol);poly(ethylene glycol)-block-poly(propylene glycol)block-poly(ethylene glycol); poly(propylene glycol)-blockpoly(ethylene glycol)-block-poly(propylene glycol)mono butyl ether; poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol)mono butyl ether; poly(propylene glycol)block-poly(ethylene glycol)-block-poly(propylene glycol)mono methyl ether; poly(ethylene glycol)-block-poly(propylene glycol) -block-poly(ethylene glycol)mono methyl ether.

Preferred block polymers are block polymers having the general formula: H(-OCH 2 CH2-) [-OCH (CH 3 CH-] y(-OCH 2

CH

2

OH,

having the average molecule weight of 1,100 and the ethylene glycol content of 10 wt%, 1,900 and 50 wt%, Mn= 2,000 and 10 wt%, Ms= 2,800 and 10 wt%, M,=2,800 and 15 wt%,Mn= 2,900 and 40 wt%, 4,400 and 30 wt%, 5,800 and 30 wt%, M,= 8,400 and 80 wt%.

Other preferred block polymers are block polymers having the general formula: H [-OCH CH 2 [-OCH (CH 3

CH

2

OH,

having the average molecule weight (Mn) of 2,000 and the ethylene glycol content of 50 wt%, 2,700 and 40 wt%, and Mn= 3,300 and 10 wt%.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 12 Examples of specific block polymers are p7120: Pluronics, commercial available from BASF (Germany), Tergitol commercial available from Union Carbide (USA), Synperonic commercial available from Fluka (Switzerland).

Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: poly(ethylene glycol-copropylene glycol), especially poly(ethylene glycol-co-propylene glycol) having an an average molecule weight M, of 2,500 and wt% ethylene glycol and an average molecule weight M, of 12,000 and 75 wt% ethylene glycol; poly(ethylene glycol-co-propylene glycol) mono butyl ether, especially poly(ehtylene glycol-copropylene glycol)monobutyl ether having an Mn of 970 and 50 wt% ethylene glycol, an M, of 1,700 and 50 wt% ethylene glycol and an Mn of 3,900 and 50 wt% ehtylene glycol; poly(ethylene glycol-co-propylene glycol) mono methyl ether.

Preferred polymers are non-toxic polymers composed of e.g.

PEG and PPG co-polymers. Polymers requiring a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface are preferred.

Examples of specific EO-oligomers are: diethylene glycol, diethylene glycol monomethylether, triethylene glycol, triethylene glycol monomethylether, tetraethylene glycol, tetraethylene glycol monomethylether, pentaeethylene glycol, pentaethylene glycol monomethylether, hexaethylene glycol, hexaethylene glycol monomethylether, heptaethylene glycol, heptaethylene glycol monomethylether, or linear unbranched C2- C14 monoalkylethers of ethylene glycol and ethylene glycol oligomers with 2-7 ethyleneoxide units.

The graft, block, alternate or radom co-polymers may be star-shaped or branched.

Preparation of suitable polymers Polymers to be attached to the surface of the parent polypeptide may be prepared using standard techniques known in the art. Further, various polymers is commercially available from companies such as BASF (Germany), Union Carbide (USA), Aldrich, Shearwater, Sigma (USA) etc.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 13 Activation of polymers If the polymer to be conjugated with the polypeptide in question is not active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the block or co- polymer to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.

Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature.

Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R.F. Taylor, (1991), "Protein immobilisation.

Fundamental and applications", Marcel Dekker, 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, Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, 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 chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Am.

Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 14 Press).

Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.

Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the orthopyridyl disulfide. Also vinylsulfone (US patent no. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.

Techniques involving coupling electrophilically activated PEGs to the amino groups of Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem.

Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.

Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.

Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (US patent no. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 Al, (1993), Looze, etc.

The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.

Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.

Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (US patent no.

5,349,001, (1994), Greenwald et The reactivity of these compounds are very high but may make the hydrolysis to fast.

PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (US patent no.

5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.

Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., (1977), J. Biol.

Chem., 252, 3578-3581; US patent no. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378. Also the polymer can be coupled to the polypeptide through a pyrimidine ring (see US 4,144,128, US 4,195,128 and US 4,298,395).

Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (US patent no.

5,321,095, (1994), Greenwald, R. thus introducing an SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 16 additional amide linkage.

As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine.

This can be done e.g. by the use of 1,3-diamino-2-propanol.

PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et Lysine residues may also be used as the backbone.

General overviews of polymer activation and PEG fuctionalization for the preparation of relevant conjugates is io given in Zaplisky, Bioconjugate Chem., 1995, 6, 150-165, Hermanson, Academic Press, San Diego, 1996, and S. Herman, G. Hooftman, E. Schacht, Journal of Bioactive and Compatible polymers, Vol.10, 1995, 145-187.

Position of the.coupled block or co-polymer(s) Virtually all ionized groups, such as the amino group of Lysine residues, are on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, (1993), "Proteins", W.H.

Freeman and Company, New York). Therefore, the number of readily accessible attachment groups amino groups) on the polypeptide's surface equals the number of Lysine residues in the primary structure of the polypeptide plus the N-terminus amino group.

According to the invention from 1 to 100 polymers, preferably 4 to 50 polymeric molecules, 5 to 35 polymers are coupled to the parent polypeptide in question.

The parent polypeptide The modified polypeptides of the invention may be prepared on the basis of parent polypeptides, typically having a molecular weight in the range from 4 to 100 kDa, preferably from to 60 kDa, using any suitable technique known in the art.

The term "parent" polypeptide is intended to indicate any uncoupled polypeptide a polypeptide to be modified). The polypeptide may preferably be of microbial origin, such as bacterial, filamentous fungus or yeast origin, or it may be of plant origin.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 17 The parent polypeptide may be a naturally-occurring (or wildtype) polypeptide or may be a variant thereof.

When choosing a parent polypeptide it is advantageous to use a polypeptide with the a high number of attachment groups.

Further, in a preferred embodiment of the invention the polymers are spread broadly over the surface of the parent polypeptide. For enzymes it is preferred that no block or copolymers are coupled in the area close to the active site.

In the present context "spread broadly" means positioned so o0 that the polymeric molecules coupled to the attachment groups of the polypeptide shields different parts of the polypeptide surface, preferable the whole or close to the whole surface area to make sure that the relevant epitope(s) being recognisable are shielded and hereby not recognised by the immune system's antibodies when a low allergenic enzyme should be obtained. It is believed that the surface area of interaction between the polypeptide and an antibody lies in the range about 500 A 2 (26 x 19A) (see Sheriff et al. (1987), Proc. Natl. Acad. Sci. USA, Vol. 84, p. 8075).

For enzymes it is preferred, to ensure a minimal loss of enzymatic activity, not to couple polymers in a close distance of the active site. Generally seen it is preferred that no polymers are attached within 5 A, preferred 10 A from the active site.

Further, polypeptides having coupled polymers at known epitope recognisable by the immune system or close to said epitope are also considered advantageous according to the invention. If the position of the epitope(s) is(are) unknown it is advantageous to couple as many polymers to the attachment groups available on the surface of the polypeptide. It is preferred that said attachment groups are spread broadly over the surface of the polypeptide in a suitable distance from the active site.

Parent polypeptides fulfilling the above claims to the distribution of coupled polymers on the surface of the polypeptide are preferred according to the invention.

For enzymes especially enzymes having no or only very few SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 18 polymers 0 to 2) coupled within a distance of 0 to 5 A, preferably 0 to 10 A from the active site are preferred.

The enzyme activity The parent enzyme may have any activity known to be used in industrial composition and products as defined below.

Contemplated enzyme activities include Oxidoreductases 1, "Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Hydrolases E.C. 3, including proteases, especially Serin proteases such as subtilisins, and lipolytic enzymes; Transferases, such as transglutaminases (TGases); Isomerases such as Protein disulfide Isomerases (PDI).

Parent Proteases Parent proteases enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include proteases within this group.

Examples include proteases selected from those classified under the Enzyme Classification numbers: 3.4.11 so-called aminopeptidases), including 3.4.11.5 (Prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (Bacterial leucyl aminopeptidase), 3.4.11.12 (Thermophilic aminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase).

3.4.21 so-called serine endopeptidases), including 3.4.21.1 (Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.19 (Glutamyl endopeptidase), 3.4.21.25 (Cucumisin), 3.4.21.32 (Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin); 3.4.22 so-called cysteine endopeptidases), including 3.4.22.2 (Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7 (Asclepain), 3.4.22.14 (Actinidain), 3.4.22.30 (Caricain) and 3.4.22.31 (Ananain); 3.4.23 so-called aspartic endopeptidases), including 3.4.23.1 (Pepsin 3.4.23.18 (Aspergillopepsin 3.4.23.20 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 19 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin); and 3.4.24 so-called metalloendopeptidases), including 3.4.24.28 (Bacillolysin).

Examples of relevant subtilisins comprise subtilisin BPN', subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin, Bacillus PB92 protease, proteinase K, Protease TW7, and Protease TW3.

Specific examples of such readily available commercial o0 proteases include Esperase®, Alcalase®, Neutrase®, Durazym®, Savinase®, Pyrase®, Pancreatic Trypsin NOVO (PTN), Bio-Feed Pro, Clear-Lens Pro, Everlase®, Kanase®, Relase®, V8Proteinase® (all enzymes available from Novo Nordisk A/S).

Examples of other commercial proteases include Maxatase®, Maxacal®, Maxapem®, Opticlean®, Properase® and Purafect® marketed by Genencor International.

It is to be understood that also protease variants are contemplates as the parent protease. Examples of such protease variants are disclosed in EP 130.756 (Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496- 500, WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Nove Nordisk WO 91/00345 (Nove Nordisk EP 525 610 (Solvay) and WO 94/02618 (Gist-Brocades The C-component disclosed in EP 482,879 B1 (Shionogi) should also be mentioned.

The activity of proteases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. Contemplated proteolytic enzymes include proteases selected from the group of acidic aspartic proteases, cysteine proteases, serine proteases, such as subtilisins, or metallo proteases, with the above indicated properties number of attachment groups, position of attachment groups etc.).

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Specific examples of suitable parent proteases having a suitable number of attachment groups are indicated in Table 1 below: Table 1: Enzyme Number of Molecular Reference attachment weight groups kDa PD498 13 29 Seq. ID No. 2 WO 93/24623 Savinase® 6 27 von der Osten et al., (1993), Journal of Biotechnology, 28, p. Proteinase K 9 29 Gunkel et al., (1989), Eur. J. Biochem, 179, p. 185-194 Proteinase R 5 29 Samal et al, (1990), Mol. Microbiol, 4, p. 1789-1792 Proteinase T 14 29 Samal et al., (1989), Gene, 85, p. 329-333 Subtilisin DY 13 27 Betzel et al. (1993), Arch. Biophys, 302, no.

2, p. 499-502 Lion Y 15 46 SEQ ID NO. 4 JP 04197182-A Jal6 5 28 WO 92/17576 Thermolysin 12 34 Titani et al., (1972) Nature New Biol. 238, p. 35-37, and SEQ ID NO Alcalase® 10 27 von der Osten et al., (a natural (1993), Journal of subtilisin Biotechnology, 28, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Carlsberg p. variant) The subtilisin PD498 has a molecular weight of 29 kDa, and as can be seen from SEQ ID NO: 2, 12 Lysine groups for polymer attachment on the surface of the enzyme plus one N-terminal amino group. As mentioned above preferred enzymes have Lysines spread broadly over the surface. PD498 has no Lysine residues in a distance of 0-10 A from the active site which makes it especially suitable in modified form. Further, the Lysine residues are spread broadly on the surface of the enzyme (i.e.

away from the active site).

The enzyme Subtilisin DY has a molecular weight of 27 kDa and has 12 amino groups Lysine residues) on the surface of the enzyme and one N-terminal amino group (see SEQ ID NO: 3).

The parent protease Lion Y has a molecular weight of 46 kDa and has 14 amino groups Lysine residues) on the surface of the enzyme plus one N-terminal amino group (see SEQ ID NO: 4).

The neutral metallo protease Thermolysin has a molecular weight of about 34 kDa and has 11 amino groups Lysine residues) on the surface plus one N-terminal amino group. (See SEQ ID NO: Parent Lipases Parent lipases enzymes classified under the Enzyme Classification number E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include lipases within this group.

Examples include lipases selected from those classified under the Enzyme Classification numbers: 3.1.1 so-called Carboxylic Ester Hydrolases), including Triacylglycerol lipases, Phosphorlipase

A

2 Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference: SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 22 Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var.

thermoidea and H. insolens (US 4,810,414) Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/04361), or Ps. plantarii or Ps.

gladioli (US patent no. 4,950,417 (Solvay enzymes)) or Ps.

alcaligenes and Ps. pseudoalcaligenes (EP 218 272) or Ps.

mendocina (WO 88/09367; US 5,389,536).

Fusarium, e.g. F. oxysporum (EP 130,064) or F. solani pisi (WO 90/09446).

Mucor (also called Rhizomucor), e.g. M. miehei (EP 238 023).

Chromobacterium (especially C. viscosum) Aspergillus (especially A. niger).

Candida, e.g. C. cylindracea (also called C. rugosa) or C.

antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 and WO 89/02916).

Geotricum, e.g. G. candidum (Schimada et al., (1989), J.

Biochem., 106, 383-388) Penicillium, e.g. P. camembertii (Yamaguchi et al., (1991), Gene 103, 61-67).

Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci.

Biotech. Biochem 56, 716-719) or R. oryzae.

Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO 91/16422).

Specific examples of readily available commercial lipases include Lipolase®, Lipolase® Ultra, Lipozyme®, Palatase®, Novozym® 435, Lecitase® (all available from Novo Nordisk A/S).

Examples of other lipases are Lumafast®, Ps. mendocian lipase from Genencor Int. Inc.; Lipomax®, Ps. pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes. Other lipases are available from other companies.

It is to be understood that also lipase variants are contem- SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 23 plated as the parent enzyme. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.

The activity of the lipase can be determined as described in "Methods of Enzymatic Analysis", Third Edition, 1984, Verlag Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (available on request from Novo Nordisk A/S).

Contemplated lipolytic enzymes include Humicola lanuginosa lipases, e.g. the one described in EP 258 068 and EP 305 216, Humicola insolens, a Rhizomucor miehei lipase, e.g. as described io in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. Antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as described in EP 331 376, a Pseudomonas sp. lipase as disclosed in WO 95/14783, a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253- 260), a B. stearothermophilus lipase (JP 64/744992) and a B.

Pumilus lipase (WO 91/16422). Other types of lipolytic include cutinases, e.g. derived from Humicola insolens, Pseudomonas mendocina (WO 88/09367), or Fusarium solani pisi described in WO 90/09446).

Parent Oxidoreductases Parent oxidoreductases enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include oxidoreductases within this group.

Examples include oxidoreductases selected from those classified under the Enzyme Classification numbers: Glycerol-3-phosphate dehydrogenase NAD+ Glycerol-3phosphate dehydrogenase NAD(P)' Glycerol-3-phosphate 1-dehydrogenase NADP Glucose oxidase Hexose oxidase Catechol oxidase Bilirubin oxidase Alanine dehydrogenase Glutamate dehydrogenase Glutamate dehydrogenase NAD(P)' SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 24 Glutamate dehydrogenase NADP* L-Amino acid dehydrogenase Serine dehydrogenase Valine dehydrogenase NADP* Leucine dehydrogenase Glycine dehydrogenase L-Amino-acid oxidase D-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase Protein-lysine 6-oxidase L-lysine oxidase L-Aspartate oxidase D-amino-acid dehydrogenase Protein disulfide reductase Thioredoxin reductase Protein disulfide reductase (glutathione) Laccase Catalase Peroxidase Lipoxygenase (1.13.11.12), Superoxide dismutase (1.15.1.1) Said Glucose oxidases may be derived from Aspergillus niger.

Said Laccases may be derived from Polyporus pinsitus, Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.

Bilirubin oxidases may be derived from Myrothechecium verrucaria.

The Peroxidase may be derived from e.g. Soy bean, Horseradish or Coprinus cinereus.

The Protein Disulfide reductase may be any mentioned in any of the DK patent applications no. 768/93, 265/94 and 264/94 (Novo Nordisk which are herby incorporated as reference, including Protein Disukfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli.

Specific examples of readily available commercial oxidoreductases include Gluzyme® (enzyme available from Novo Nordisk However, other oxidoreductases are available from others.

It is to be understood that also variants of oxidoreductases are contemplated as the parent enzyme.

The activity of oxidoreductases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 3.

Contemplated laccases include the laccases disclosed in WO 96/00290 and WO 95/33836 from Novo Nordisk.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Other oxidoreductases include catalase, glucose oxidase, peroxidase, haloperoxidase, superoxide dismutase, and lipoxygenase.

Parent Carbohydrases Parent carboydrases may be defined as all enzymes capable of breaking down carbohydrate chains starches) of especially five and six member ring structures enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) io in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)).

Also included in the group of carbohydrases according to the invention are enzymes capable of isomerizing carbohydrates e.g.

six member ring structures, such as D-glucose to e.g. five member ring structures like D-fructose.

Examples include carbohydrases selected from those classified under the Enzyme Classification numbers: a-amylase 0-amylase glucan 1,4-aglucosidase cellulase endo-1,3(4)-0glucanase endo-1,4-p-xylanase dextranase chitinase polygalacturonase (3.2.1.15), lysozyme -glucosidase a-galactosidase P-galactosidase amylo-1,6-glucosidase xylan 1,4-p-xylosidase glucan endo-1,3- P-D-glucosidase a-dextrin endo-1,6-glucosidase sucrose a-glucosidase glucan endo-1,3a-glucosidase glucan 1,4-p-glucosidase (3.2.1.74), glucan endo-1,6-p-glucosidase arabinan arabinosidase lactase (3.2.1.108), chitonanase (3.2.1.132) and xylose isomerase Examples of relevant carbohydrases include a-1,3-glucanases derived from Trichoderma harzianum; a-1,6-glucanases derived from a strain of Paecilomyces; P-glucanases derived from Bacillus subtilis; P-glucanases derived from Humicola insolens; SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 26 P-glucanases derived from Aspergillus niger; P-glucanases derived from a strain of Trichoderma; P-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-a-D-glucosidases (glucoamylases) derived from Aspergillus niger; a-amylases derived from Bacillus subtilis; a-amylases derived from Bacillus amyloliquefaciens; a-amylases derived from Bacillus stearothermophilus; a-amylases derived from Aspergillus oryzae; a-amylases derived from non-pathogenic microorganisms; agalactosidases derived from Aspergillus niger; Pentosanases, 1o xylanases, cellobiases, cellulases, hemi-cellulases deriver from Humicola insolens; cellulases derived from Trichoderma reesei; cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum; endoglucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; P-galactosidases derived from Kluyveromyces fragilis; xylanases derived from Trichoderma reesei; Specific examples of readily available commercial carbohydrases include Alpha-Gal@, Bio-Feed@ Alpha, Bio-Feed@ Beta, Bio-Feed® Plus, Bio-Feed@ Plus, Novozyme@ 188, Carezyme@, Celluclast@, Cellusoft@, Ceremyl@, Citrozym@, Denimax®, Dezyme@, Dextrozyme@, Finizym@, Fungamyl@, Gamanase@, Glucanex@, Lactozym@, Maltogenase®, Pentopan®, Pectinex@, Promozyme@, Pulpzyme@, Novamyl®, Termamyl@, AMG (Amyloglucosidase Novo), Maltogenase@, Sweetzyme@, Aquazym@, Natalase@ (all enzymes available from Novo Nordisk Other carbohydrases are available from other companies.

It is to be understood that also carbohydrase variants are contemplated as the parent enzyme.

The activity of carbohydrases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 4.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 27 Parent Transferases Parent transferases enzymes classified under the Enzyme Classification number E.C. 2 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include transferases within this group.

The parent transferases may be any transferase in the subgroups of transferases: transferases transferring one-carbon io groups transferases transferring aldehyde or residues (E.C acyltransferases 2.3); glucosyltransferases transferases transferring alkyl or aryl groups, other that methyl groups transferases transferring nitrogeneous groups In a preferred embodiment the parent transferease is a transglutaminase E.C 2.3.2.13 (Protein-glutamine mglutamyltransferase).

Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the epsilon-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transferases form intramolecular or intermolecular gammaglutamyl-epsilon-lysyl crosslinks.

Examples of transglutaminases are described in the pending DK patent application no. 990/94 (Novo Nordisk A/S).

The parent transglutaminase may the of human, aminal (e.g.

bovine) or microbially origin.

Examples of such parent transglutaminases are animal derived Transglutaminase, FXIIIa; microbial transglutaminases derived from Physarum polycephalum (Klein et al., Journal of Bacteriology, Vol. 174, p. 2599-2605); transglutaminases derived from Streptomyces sp., including Streptomyces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Streptoverticillium sp., including Streptoverticillium mobaraense, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 28 Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., US 5,156,956; Andou et al., US 5,252,469; Kaempfer et al., Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al., International Journal of Sytematic Bacteriology, Vol. 44, p. 285-292; Andou et al., US 5,252,469; Williams et al., Journal of General Microbiology, Vol. 129, p. 1743-1813).

It is to be understood that also transferase variants are contemplated as the parent enzyme.

io The activity of transglutaminases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10.

Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S).

Parent Phytases Parent phytases are included in the group of enzymes classified under the Enzyme Classification number E.C. 3.1.3 (Phosphoric Monoester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)).

Phytases are enzymes produced by microorganisms which catalyse the conversion of phytate to inositol and inorganic phosphorus Phytase producing microorganisms comprise bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomyces cerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species).

Examples of parent phytases include phytases selected from those classified under the Enzyme Classification numbers: 3-phytase and 6-phytase (3.1.3.26).

The activity of phytases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 29 Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2 A.

Isomerases Parent isomerases are included in the group of enzymes classified under the Enzyme Classification number E.C. 5 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB).

An example of a parent isomerase is Protein Disulfide Isomerase. Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).

Industrial composition In a further aspect of the invention relates to an "industrial composition" comprising a modified polypeptide with improved wash performance.

In the context of the present invention an "industrial composition" means a composition which is not intended to be introduced into the circulatory system. In other words it means a composition which is not intended for intradermally, intravenously or subcutaneously administration.

As mentioned above the main problem for polypeptides, such as enzymes, for industrial application is the potential risk of respiratory allergy caused by inhalation through the respiratory system i.e. intratracheally or intranasal exposure.

Examples of "industrial composition" are polypeptides, especially enzymes and anti-microbial polypeptides, used in compositions or products such as detergents, including laundry and dish washing detergents, household article products, agro-chemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, compositions used for treating/processing textiles, compositions for hard surface cleaning etc. Especially contemplated according to the invention are skin care products and detergents.

Skin Care Products SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 In the context of the present invention "skin care products" cover all personal care products used for cleansing, care and/or beautification of the skin of the body and further other products, such as hair care products, which during use may come in contact with the skin or respiratory system. Also corresponding products for animals are contemplated according to the present invention.

Specific examples of skin care products contemplated according to the present invention are soap, cosmetics, i cleansing cream, cleansing lotion, cleansing milk, cream soap, whitening powder, powder soap, cake soap, transparent soap, nail polish remover, shampoo, balsam, hair rinse, etc.

Enzyme activities suitable for Skin Care is Skin care compositions of the invention comprise conjugates with improved wash or cleansing effect and e.g. reduced allergenicity of the invention and further ingredients known to be used in skin care compositions A number of enzyme activities are known to be used skin care compositions.

Proteases Proteases are effective ingredients in skin cleaning products. Proteases remove the upper layer of dead keratinous skin cells and thereby makes the skin look brighter and more fresh. Further, proteases also improves the smoothness of the skin.

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.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCTIDK99/00406 31 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.

Oxidoreductases The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e.g.

glucose) that ensures production of H 2 0 2 which then will initiate the oxidation of for instance SCN- or I- into antimicrobial reagents (SCNO' or 12) by a peroxidase (usually lactoperoxidase). This enzymatic complex is known in nature from e.g. milk and saliva.

It is being utilised commercially as anti-microbial system 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.

Another application of oxidoreductases are oxidative hair dyeing using oxidases, peroxidases and laccases (See e.g. WO 96/00290 or WO 95/33836 from Novo Nordisk).

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 leads to destruction of fatty membranes, collagen, and cells.

The application of free radical scavengers such as Superoxide dismutase into cosmetics is well-known L.

Goldemberg, DCI, Nov. 93, p. 48-52).

Protein disulfide isomerase (PDI) is also an oxidoreductase.

It may be utilised 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).

Transglutaminase Skin care compositions for application to human skin, hair or nails comprise an amino-functional active ingredient, transglutaminase to catalyse cross-linking of the active SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 32 ingredient to the skin, hair or nails, and a carrier is known from US patent no. 5,490,980.

A cosmetic composition suitable for application to mammalian skin, hair or nails comprising: at least one corneocyte envelope protein in an amount sufficient to provide a protective layer on said skin, hair or nails; a transglutaminase in an amount sufficient to form covalent bonds between the corneocyte envelope protein and externally exposed corneocyte proteins present in the stratum corneum of said skin, hair or nails; calcium ions in an amount sufficient to activate the transglutaminase; and a cosmetically acceptable vehicle, wherein the composition comprises an emulsion having two phases and wherein the corneocyte envelope protein is contained in one of the phases and the transglutaminase is contained within the other phase (see US patent no. 5,525,336).

JP 3083908 describes a skin cosmetic material contains a transglutaminase modified with a water-soluble substance. The modifying substance is, one or more of polyethylene glycol, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, alginil acid, carboxymethyl cellulose, starch, and hydroxypropyl cellulose. The modification is done, by introducing reactive groups and bonding to the enzyme. For providing a material mild to the skin, causing less time-lapse discolouring and odorising, and having good effects of curing rough skin, retaining moisture, and conditioning the skin beautifully.

The Skin Care Products of the invention In the third aspect the invention relates to a skin care product comprising a skin care composition of the invention. The term "skin care products" are defined above.

A skin care product of the invention may comprise from an effective amount of modified enzymes of the invention. Such effective amounts known to the skilled person may will often lie in the range from above 0 to 5% of the final skin care product.

Contemplated skin care products of the invention include, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 33 without being limited thereto, the following products: soap, cosmetics, cleansing cream, cleansing lotion, cleansing milk, cream soap, powder soap, cake soap, transparent soap, nail polish remover, shampoo, balsam, hair rinse, etc.

General skin care product formulations The term "ingredients used in skin care products" is meant to cover all ingredients which are known to be used in skin care product formulations. Examples of such ingredients ingredients can be found in "Cosmetics and Toiletries" edited by Wilfried Umbach and published by Ellis Horwood, Limited, England, (1991), and "Surfactants in Consumer Products", edited by J. Falbe and published by Spring-Verlag, (1987).

In the following a non exhausting list of guide formulations are listed. These provide an overwiev of formulations of important skin care products contemplated according to the invention.

Toilet soap Ingredients Surfactants Sequestering agents Consistency regulators Dyestuffs Optical brighteners Antioxidants Whitening agents Fragrances Enzymes Water Examples Soap (sodium salt) Ethylenediamine tetraacetate Sodium chloride 2,6-bis(1,1-Dimethylethyl)- 4-methyl phenol(BHT) Titanium dioxide Protease/Lipase Balance 83 -87 0.1-0.3 approx.

0.1 0.1 0.1-0.3 0.1-0.3 1.0-2.0 Syndet (Synthetic Detergents) Ingredients Surfactants Examples Lauryl sulfate 30-50 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Refatting agents Plasticizers Fillers Active agents Dyestuffs Fragrances Enzymes Water Lauryl sulfo succinate Fatty alcohols Stearyl mono/diglycerides Starches Salicylic acid Protease/Lipase Balance 1-12 10-20 0-10 0-10 0-1 0.2 0-2 Foam bath and shower bath Ingredients Foam bath Surfactants Refatting agents Enzymes Ingredients Foam bath Foam stabilizers Conditioners Thickeners Pearlescent agents Active agents Preservatives Examples Shower bath Lauryl ether sulfate Coco amidopropyl dimethyl betaine Ethoxylated fatty acids Fatty alcohols Ethoxylated fattyalcoho.

Protease/Lipase 10-20 2-4 0.5-2 0.5-3 0.5-5 0-5 10-12 2-4 0-4 Examples Shower bath Fatty acid alkanol amide 0.2-2 Quaternized hydroxypropyl cellulose Sodium chloride 0-3 Ethyleneglycol stearate 0-2 Vegetable extracts 0-1 5-Bromo-5-nitro-1,3- 0-4 0-0.5 0-3 0-1 0.1 0.1 0.3-2 dioxane Dyestuffs Fragrances Enzymes Water 0.1 0.1-0.2 0.3-3 0-5 Balance Protease/Lipase Balance SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Hair shampoo Ingredients Surfactants Examples Lauryl ether sulfate Coco fatty acid amidopropyl dimethyl betaine Fatty acid polyglycol esters Fatty acid ethanol amides Quaternized hydroxyethyl cellulose Foam boosters Conditioners io Protein hydrolysates Refatting agents Ethoxylated lanolin alcohols Additives Anti-dandruff agents Preservatives 5-Bromo-5-nitro-1,3-dioxane Pearlescent agents Ethyleneglycol stearate Dyestuffs pH-Regulators Acids/Bases Fragrances Enzymes Protease/Lipase Water Balance 12-16 0-2 0.5-2.5 0.4-1 0.2-1 0.2-1 0-1 0.1-0.3 0-2 0.1 0.1-1 0.3-0.5 Hair rinse and hair conditioner Ingredients Hair rinse Surfactants Examples Hair conditiner Fatty alcohol polyglycol ethers Cetyl trimethyl ammonium chloride Dimethyl benzyl stearyl ammonium chloride Cetyl/Stearyl mono/ diglyceride Fatty alcohols Methyl hydroxypropyl 0.1-0.2 1.5-2.5 0.5-1 0.5-1 0.5-1.5 1.5-2.5 Refatting agents Consistency regulators Thickeners 1-2.5 2.5-3.5 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 36 cellulose 0.3-0.6 0.4-0.8 Conditioners Quaternized hydroxyethyl cellulose 0.1-0.3 0.3-0.4 Preservatives p-Hydroxy benzoic acid ester 0.1-0.3 0.1-0.3 Dyestuffs <0.1 <0.1 pH-Regulators Acids/Bases 0,1-1 0.1-1 Fragrances 0.2-0.5 0.2-0.5 Enzymes Protease/Lipase 0-5 Water Balance Balance Detergent disclosure The detergent compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations, including biofilm removal and dishwashing operations.

In biofilm removal alginic acid lyase should be mentioned as a preferred enzyme (see JP10127281 A K.K. GUNZE and TANABE SEIYAKU CO hereby incorporated by reference).

The detergent composition of the invention comprises the conjugate of the invention and a surfactant. Additionally, it may optionally comprise a builder, another enzyme, a suds suppresser, a softening agent, a dye-transfer inhibiting agent and other components conventionally used in detergents such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes.

The detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms. The pH (measured in aqueous solution at use con-centration) will usually be neutral or alkaline, e.g. in the range of 7-11. Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 37 than conventional granular detergents, i.e. from 550 to 950 g/l.

The enzyme conjugate of the invention, or optionally another enzyme incorporated in the detergent composition, is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition. However, the enzyme dosage depends on the allergenicity and improved wash performance of the enzymes, i.e. by a low allergenicity a higher dosage can be used and by improved wash performance a lower dosage can be used.

Surfactant system: The surfactant system may comprise nonionic, anionic, cationic, ampholytic, and/or zwitterionic surfactants. The surfactant system preferably consists of anionic surfactant or a combination of anionic and nonionic surfactant, e.g. 50-100 of anionic surfactant and 0-50 nonionic. The laundry detergent compositions may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein.

The surfactant is typically present at a level from 0.1% to by weight. Some examples of surfactants are described below.

Nonionic surfactant: The surfactant may comprise polyalkylene oxide (e.g.

polyethylene oxide) condensates of alkyl phenols. The alkyl group may contain from about 6 to about 14 carbon atoms, in a straight chain or branched-chain. The ethylene oxide may be present in an amount equal to from about 2 to about 25 moles per mole of alkyl phenol.

The surfactant may also comprise condensation products of primary and secondary aliphatic alcohols with about 1 to about SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 38 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, and generally contains from about 8 to about 22 carbon atoms.

Further, the nonionic surfactant may comprise polyethylene oxide conden-sates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.

Anionic surfactants: Suitable anionic surfactants include alkyl alkoxyla-ted sulfates which are water soluble salts or acids of the formula RO(A)mSO3M wherein R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24 alkyl com-ponent, preferably a C12-C20 alkyl or hydroxyalkyl, more pre-ferably C12-C18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxy-lated sulfates as well as alkyl propoxylated sulfates are contemplated herein. Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethyla-mine, mixtures thereof, and the like.

Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO3M wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl, and M is H or a cation, an alkali metal cation sodium, potassium, lithium), or ammonium or substituted ammonium.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 39 Other anionic surfactants include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C8- C22 primary or secondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates.

Alkylbenzene sulfonates are suitable, especially linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms.

The laundry detergent compositions typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.

Builder system: The compositions according to the present invention may further comprise a builder system. Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate (EDTA), metal ion sequestrants such as aminopolyphosphonates. Phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Detergency builder salts are normally included in amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to Other detergent enzyme activities: The detergent composition may, in addition to the conjugate of the invention with a specific activity, further comprise other enzyme activities e.g. also in the form of an enzyme conjugate as described according to the present invention, providing cleaning performance and/or fabric care benefits, e.g.

proteases, lipases, cutinases, amylases, cellulases, peroxidases, haloperoxidases, oxidases laccases).

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Specific examples of contemplated enzymes are listed above in the section "The enzyme activity".

Bleaching agents: The detergent composition (especially in the case of a granular detergent) may also comprise a bleaching agents, e.g.

an oxygen bleach or a halogen bleach. The oxygen bleach may be a hydrogen peroxide releasing agent such as a perborate PB1 or PB4) or a percarbonate, or it may e.g. be a percarboxylic acid. The parti-cle size may be 400-800 microns. When present, oxygen bleching compounds will typically be present at levels of from about 1% to about The hydrogen peroxide releasing agent can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzene-sulfonate (NOBS), 3,5-trimethyl-hexsanoloxybenzene-sulfonate (ISONOBS) or pentaacetylglucose (PAG).

The halogen bleach may be, e.g. a hypohalite bleaching agent, for example, trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are nor-mally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.

Textile applications Proteases Proteases are used for degumming and sand-washing of silk.

Lipases Lipases are used for removing fatty matter containing hydrophobic esters triglycerides) during the finishing 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.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 41 Carbohydrases Cellulolytic enzymes are widely used in the finishing of denim garments in order to provide a localized variation in the colour density of the fabric (Enzyme facilitated "stone wash").

Also cellulolytic enzymes find use in the bio-polishing process. Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance 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 fabric, the so called desizing. Desizing of fabrics sized with a size containing starch or modified starch is preferably facilitated by use of amylolytic enzymes.

MATERIAL AND METHODS Materials Enzymes: PD498: Protease of subtilisin type shown in WO 93/24623. The sequence of PD498 is shown in SEQ ID NO: 1 and 2.

Subtilisin DY: Protease of the subtilisin type shown in SEQ ID NO: 3 isolated from Bacillus sp. variant (Betzel et al. (1993), Archives of Biophysics, Vol. 302, No. 2, p. 499-502).

Savinase®.

Savinase variant R247K (Arginine in position 247 has been replaced with Lysine using the BPN'numbering).

ELISA reagents: Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, P217, dilution 1:1000).

Rat anti-mouse IgE (Serotec MCA419; dilution 1:100). Mouse antirat IgE (Serotec MCA193; dilution 1:200).

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 42 Biotin-labelled mouse anti-rat IgGl monoclonal antibody (Zymed 03-9140; dilution 1:1000) Biotin-labelled rat anti-mouse IgGl monoclonal antibody (Serotec MCA336B; dilution 1:2000) Streptavidin-horse radish peroxidase (Kirkegard Perry 14-30- 00; dilution 1:1000).

Buffers and Solutions: PBS (pH 7.2 (1 liter)) NaCl 8.00 g KC1 0.20 g K2HPO4 1.04 g KH2PO4 0.32 g Washing buffer PBS, 0.05% Tween Blocking buffer PBS, 2% (wt/v) Skim Milk powder Dilution buffer PBS, 0.05% Tween 20, 0.5% (wt/v) Skim Milk powder Citrate buffer (0.1M, pH 5.0-5.2 (1 liter)) NaCitrate 20.60 g Citric acid 6.30 g Stop-solution (DMG-buffer) Sodium Borate, borax (Sigma) 3,3-Dimethyl glutaric acid (Sigma) CaC12 (Sigma) Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184) N-Hydroxy succinimide (Fluka art. 56480)) Phosgene (Fluka art. 79380) Lactose (Merck 7656) PMSF (phenyl methyl sulfonyl flouride) from Sigma -Succinyl-Alanine-Alanine-Proline-Phenylalanine-paranitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.

mPEG (Fluka) Protease model detergent '95 is an in-house detergent formulation: STP (NasP0O 1 0 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 43 NaSO, Na 2

CO

3 LAS (Nansa NI (Dobanol 25-7) 5 Na 2 Si 2 zO Carboxymethylcellulose (CMC) water EMPA 116: Blood, milk, Indian ink on cotton EMPA 117: Blood, milk, Indian ink on PE/BO Colouring substrate: OPD: o-phenylene-diamine, (Kementec cat no. 4260) Test Animals: Brown Norway rats (from Charles River, DE) Equipment: XCEL II (Novex) ELISA reader (UVmax, Molecular Devices) HPLC (Waters) PFLC (Pharmacia) column, Mono-Q, Mono S from Pharmacia, SW.

SLT: Fotometer from SLT LabInstruments Size-exclusion chromatograph (Spherogel TSK-G2000 SW).

Size-exclusion chromatograph (Superdex 200, Pharmacia, SW) Amicon Cell Filtron Ultrasette with an Omega 10K membrane Miniwash Robot J&M Tidas MMS/16 photometer equipped with a CLX 75W Xenon lamp and fibre optics Methods: Intratracheal (IT) stimulation of Brown Norway rats For IT administration of molecules disposable syringes with a 2k" long metal probe is used. This probe is instilled in the trachea of the rats approximately 1 cm below the epiglottis, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 44 and 0.1 ml of a solution of the molecules is deposited.

The test animals are Brown Norway rats (BN) in groups of Weight at time of start is more than 200 grams and at termination approximately 450 grams.

ELISA procedure to determine relative concentrations of IgE antibodies in Brown Norway rats.

A three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum anti-bodies.

1) Coat the ELISA-plate with 10 mg mouse anti-rat IgE Buffer 1 Incubate over night at 4 0

C.

2) Empty the plates and block with Blocking buffer for at least hour at room temperature (200 microL/well). Shake gently.

Wash the plates 3 times with Washing Buffer.

3) Incubate with rat sera (50 microL/well), starting from undiluted and continue with 2-fold dilutions. Keep some wells free for buffer 4 only (blanks). Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer.

4) Dilute the enzyme in Dilution buffer to the appropriate protein concentration.

Incubate 50 microL/well for 30 minutes at room temperature.

Shake gently. Wash the plates 3 times in Washing Buffer.

Dilute specific polyclonal anti-enzyme antiserum serum (pig) for detecting bound antibody in Dilution buffer. Incubate microl/well for 30 minutes at room temperature. Shake gently.

Wash the plates 3 times in Washing Buffer.

6) Dilute Horseradish Peroxidase-conjugated anti-pig-antibody in Dilution buffer. Incubate 50 microL/well at room temperature for 30 minutes. Shake gently. Wash the plates 3 times in Washing Buffer.

7) Mix 0.6 mg ODP/ml 0.4 microL HO,/ml in substrate Buffer.

Make the solution just before use. Incubate for 10 minutes. microL/well.

8) To stop the reaction, add 50 microL Stop Solution/well.

9 Read the plates at 492 nm with 620 nm as reference.

Data is calculated and presented in Lotus.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Determination of the molecular weight Electrophoretic separation of proteins was performed by stan-dard methods using 4-20% gradient SDS polyacrylamide gels (Novex). Proteins were detected by silver staining. The molecular weight was measured relatively to the mobility of Mark-12® wide range molecular weight standards from Novex.

Protease activity Analysis with Suc-Ala-Ala-Pro-Phe-pNa: Proteases cleave the bond between the peptide and pnitroaniline 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 ml 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

ABS

40 5 m/min. The temperature should be controlled (20-50°C depending on protease). This is a measure of the protease activity in the sample.

EXAMPLES

Example 1 Activation of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) 1.900 (50 wt% ehtyleneglycol) with N-succinimidyl carbonate Poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol) 1.900 (50 wt% ehtyleneglycol)from ALDRICH was dissolved in toluene (5 ml/g of polymer). About 20% was distilled off at normal pressure to dry the reactants SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 46 azeotropically. The solution was cooled to 20°C and phosgene in toluene (1.93 M, 7 mole/mole polymer) was added. The mixture was then stirred at room temperature overnight. The solvent and excess phosgene was removed in vacuo and the intermediate bis(chloroformate) was obtained as an oil.

Toluene (dry 4 ml/g polymer) was added to redissolve the oil. N-Hydroxy succinimide (NHS) (2.4 mole/mole polymer) was added and the mixture was cooled with an ice-bath. Triethylamine (2.2 mole/mole polymer) was added dropwise at 0 C. Immediate precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature.

The mixture was filtered using a glass frit (G5) to remove the Et3N.HC1. The filtrate was.evaporated to dryness under reduced pressure to yield 97 (mole/mole) of an oil. NMR Indicating 90% activation and <8 o/o (mole/mole) of unbound NHS. 1H-NMR (400MHz) for poly(ethylene glycol)-block-poly(propylene glycol)block-poly(ethylene glycol) 1.900 bis(succinimidyl carbonate) wt% ehtyleneglycol)(CDCl3) 6: 1.15 bs (1=330 -CH3 in PPG), 2.69 s (1=1.7 unreacted NHS), 2.83 s 41, succinimide), 3.41 m (I=110, CH-CH2 in PPG), 3.55 m (I=220, CH-CH2 in PPG), 3.61 m (1=440 main peak), 4.46 t (1=19, CH2-O-CO- in PEG).

Example 2 Activation of poly(ethylene glycol)-co-(propylene glycol) monobutyl ether 970 (ca. 50 wt% ethyleneglycol) with Nsuccinimidyl carbonate Poly(ethylene glycol)-co-(propylene glycol) monobutyl ether 970 (ca. 50 wt% ethyleneglycol) from ALDRICH was dissolved in toluene (4 ml/g of polymer). About 25% was distilled off at normal pressure to dry the reactants azeotropically. The solution was cooled to 0°C and phosgene in toluene (1.93 M, mole/mole polymer) was added. The mixture was then stirred at SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 47 room temperature for 21 hours. The solvent and excess phosgene were removed in vacuo and the intermediate chloroformate was obtained as an oil.

Toluene (dry 2 ml/g polymer) was added to redissolve the oil. N-Hydroxy succinimide (NHS) (1.2 mole/mole polymer) was added at room temperature. Triethylamine (1.1 mole/mole polymer) was added dropwise at 0°C. Immediate precipitation of triethylamine hydrochloride (Et 3 N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture was then filtered using a glass frit (G5) to remove insoluble Et 3 N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 89 (mole/mole) of an oil. NMR Indicating 72% activation and <5 o/o (mole/mole) of unbound NHS. 1

H-NMR

(400MHz), CDC13) 6: 0.91 t (I=1000 -CH3 butyl), 1.15 bs (1=8744 CH3 in propylene glycol), 1.39 m (1=1320 CH3-CH2-CH 2 butyl), 1.55 m (1=656 -CH 2 butyl), 2.68 s (1=60.8 unreacted NHS), 2.83 s 963.2, succinimide), 3.40 m (1=3059, CH-CH 2 in propylene glycol), 3.55 m (1=2678, CH-CH, in propylene glycol), 3.61 m (1=1764 main peak, -CH2-CH 2 in ethylene glycol), 4.46 m

(CH

2 Example 3 Activation of mPEG 350 with N-succinimidyl carbonate mPEG 350 was dissolved in toluene (4 ml/g of mPEG). About 20% was distilled off at normal pressure to dry the reactants azeotropically. The solution was cooled to 20°C and phosgene in toluene (1.93 M 1.5 mole/mole mPEG) was added. The mixture was then stirred at room temperature over night. The mixture was evaporated under reduced pressure and the intermediate chloroformate was obtained as an oil.

After evaporation dichloromethane and toluene dry 4 ml/g mPEG) was added to re-dissolve the colorless oil. N-Hydroxy succinimide (NHS) (1.5 mole/mole mPEG.) was added as a solid and then triethylamine (1.1 mole/mole mPEG) at 0°C. Immediate precipitation of triethylamine hydrochloride (Et 3 N.HC1) could be observed. The mixture was stirred overnight at room temperature.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 48 The mixture was filtered using a glass frit (G5) to remove the Et 3 N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 98 (mole/mole) of an oil. NMR Indicating 95% activation and <10 o/o (mole/mole) HNEt 3 C1. 'H-NMR (400 MHz) for mPEG 350 succinimidylcarbonate (CDCl 3 6: 1.42 t (1=1.4

CH

3 in HNEt3Cl), 2.68 s (1=3.4 unreacted NHS), 2.84 s 6.2 succinimide), 3.10 dq 1.0 CH, i HNEt 3 Cl), 3.38 s (1=5.8 CH 3 i OMe), 3.64 bs (1=50 main peak), 4.47 t CH, in PEG).

Example 4 Activation of PEG 300 with N-succinimidvl carbonate PEG 300 was dissolved in toluene (13 ml/g of mPEG). About was distilled off at normal pressure to dry the reactants azeotropically. The solution was cooled to 20 0 C and phosgene in toluene (1.93 M 3.8 mole/mole PEG) was added. The mixture was then stirred at room temperature for 20 hours. The mixture was evaporated under reduced pressure and the intermediate bis(chloroformate) was obtained as an oil.

After evaporation dry toluene (10 ml/g PEG) was added to redissolve the colorless oil. N-Hydroxy succinimide (NHS) mole/mole mPEG.) was added as a solid and then triethylamine (2.4 mole/mole mPEG) at 0 0 C. Immediate precipitation of triethylamine hydrochloride (Et 3 N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et 3 N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 90 (mole/mole) of an oil. NMR Indicating >55 activation and <5 o/o (mole/mole) HNEt 3 Cl. 'H-NMR (400 MHz,CDC1 3 8: 1.11 t (1=1.8 CH3 in NEt 3 2.69 s (1=1.39 unreacted NHS), 2.84 s 20.0 succinimide), 3.64 bs (1=113 main peak), 4.44 m (I=10.0, CH, in PEG).

Example Activation of mPEG 550 with N-succinimidyl carbonate mPEG 550 was dissolved in toluene (9 ml/g of mPEG). About was distilled off at normal pressure to dry the reactants SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCTIDK99/00406 49 azeotropically. The solution was cooled to 20 0 C and phosgene in toluene (1.93 M 1.5 mole/mole mPEG) was added. The mixture was then stirred at room temperature for overnight. The mixture was evaporated under reduced pressure and the intermediate chloroformate was obtained as an oil.

After evaporation dry toluene (4 ml/g mPEG)and dry dichloromethane (3 ml/g mPEG) was added to re-dissolve the colorless oil. N-Hydroxy succinimide (NHS) (1.2 mole/mole mPEG) was added as a solid and then triethylamine (1.2 mole/mole mPEG) at 0°C. Immediate precipitation of triethylamine hydrochloride (Et 3 N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit to remove the Et 3 N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 89 (mole/mole) of a is viscous oil. NMR Indicating >77 activation and <2 o/o (mole/mole) HNEt 3 C1. 'H-NMR (400MHz) for mPEG 550 succinimidylcarbonate (CDC13) 6: 1.41 t (1=4.2 CH 3 in HNEt 3 Cl), 2.69 s (1=24.4 unreacted NHS), 2.84 s 81 succinimide), 3.10 dq 3.7 CH 2 i HNEt 3 C1), 3.38 s (1=97 CH 3 i OMe),3.64 bs (I=1250 main peak), 4.44 m (1=41, CH, in PEG).

Example 6 Conjugation of PD498 protease with activated mPEG 350 62 mg of PD498 was incubated in 50 mM Sodium Borate, pH 9.7, with 20 mg (=200Il) of activated mPEG 350 with Nsuccinimidyl carbonate (prepared according to Example in a final volume of 6 ml. The reaction was carried out at ambient temperature using magnetic stirring. Reaction time was 2 hour.

The reaction was stopped by adding 0.5 M succinic acid to a final pH of The molecular weight of the obtained derivative was approximately 33 kDa, corresponding to about 11 moles of mPEG attached per mole PD498.

Compared to the parent enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- Nitroanilide).

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCTIDK99/00406 Example 7 Conjugation of Subtilisin DY protease with activated mPEG 350 Subtilisin DY was conjugated to mPEG 350 with N-succinimidyl carbonate using the same procedure as described in Example 2.

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 departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.

Example 8 Conjugation of Savinase variant R247K with activated mPEG-350 21 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 16 mg of N-succinimidyl carbonate activated mPEG 350 in a reaction volume of approximately 2 ml.

The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.

The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with mM Sodium Borate, 5mM succinic acid, ImM CaClI, pH Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

3o Example 9 Conjugation of Savinase variant R247K with activated mPEG-550 21 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 25 mg of N-succinimidyl carbonate activated mPEG 550 in a reaction volume of approximately 2 ml.

The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval by addition of 0.5 M NaOH. The reaction time was 2 hours.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 51 The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with mM Sodium Borate, 5mM succinic acid, ImM CaCI 2 pH Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

Example Conjugation of Savinase variant R247K with activated bis-PEG- 300 21 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9.5 with 14 mg of N-succinimidyl carbonate activated bis-PEG 300 in a reaction volume of approximately 2 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval by addition of 0.5 M NaOH. The reaction time was 2 hours.

The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with mM Sodium Borate, 5mM succinic acid, 1mM CaC 2 pH Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

Example 11 Conjugation of Savinase with activated bis-PEG-200 827 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9 with 420 mg of N-succinimidyl carbonate activated bis-PEG 200 in a reaction volume of approximately ml. The reaction wascarried out at ambient temperature using magnetic stirring while keeping the pH within the interval by addition of 0.5 M NaOH. The reaction time was 2 hours.

The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by untra-filtration using a Filtron- Ultrasette with 10kD cut-off.

Compared to the parent enzyme, residual activity was close to SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 52 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

Example 12 Conjugation of Savinase with activated bis-PEG-300 827 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9 with 610 mg of N-succinimidyl carbonate activated bis-PEG 300 in a reaction volume of approximately ml. The reaction was carried out at ambient temperature using io magnetic stirring while keeping the pH within the interval by addition of 0.5 M NaOH. The reaction time was 2 hours.

The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by untra-filtration using a Filtron- Ultrasette with 10kD cut-off.

Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

Example 13 Conjugation of Savinase with activated bis-PEG-600 827 mg of the Savinase variant was incubated in 50 mM Sodium Borate pH 9 with 1000 mg of N-succinimidyl carbonate activated bis-PEG 600 in a reaction volume of approximately 100 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval by addition of 0.5 M NaOH. The reaction time was 2 hours.

The reaction was stopped by adding 1M HC1 to a final pH of Reagent excess was removed by untra-filtration using a Filtron- Ultrasette with 10kD cut-off.

Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-pnitro-anilide).

Example 14 Conjugation of Savinase with activated PEG 1000 2 g of Savinase was incubated in 50 mM Sodium Borate, pH 9 with 2.8 g of N-succinimidyl carbonate activated PEG 1000 in SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 53 a final volume of approximately 200 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping pH within the interval of pH 8.5-9.0 by addition of 0.5 M NaOH. Reaction time was 2 hour. The reaction was stopped by adding 1 M HC1 to a final pH of Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette.

Compared to the parent Savinase enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala- Pro-Phe-p-Nitro-anilide).

Example Conjugation of Savinase with activated PEG 2000 2 g of Savinase was incubated in 50 mM Sodium Borate, pH 9 with 7.8 g of N-succinimidyl carbonate activated PEG 2000 in a final volume of approximately 200 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping pH within the interval of pH 8.5-9.0 by addition of 0.5 M NaOH. Reaction time was 2 hour. The reaction was stopped by adding 1 M HC1 to a final pH of Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette.

Compared to the parent Savinase enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala- Pro-Phe-p-Nitro-anilide).

Example 16 Conjugation of Savinase with poly(ethylene glycol)-blockpoly(propylene glycol)-block-poly(ethylene glycol) 1900 bis(succinimidyl carbonate) (50 wt ethylene glycol).

148 mg Savinase in 3 ml buffer was adjusted to pH 9.0 with 0.5 N NaOH. 450 mg of the activated block polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 54 with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M succinic acid. The reaction mixture was purified by gelfiltering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 130% compared to the parent enzyme.

Example 17 Conjugation of Savinase with poly(ethylene glycol)-blockpoly(propylene glycol)-block-poly(ethylene glycol) 2900 bis(succinimidyl carbonate) (40 wt ethylene glycol).

148 mg Savinase in 3 ml buffer was adjusted to pH 9.0 with N NaOH. 700 mg of the activated block polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at with 0.5 N NaOH. After 2 h pH was adjusted to 6.5 with 0.5 M succinic acid. The reaction mixture was purified by gelfiltering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 84% compared to the parent enzyme.

Example 18 Conjugation of Savinase with poly(ethylene glycol)-blockpoly(propylene glycol)-block-poly(ethylene glycol) 8400 bis(succinimidyl carbonate) (80 wt ethylene glycol).

148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with N NaOH. 2000 mg of the activated block polymer was dissolved in 6 ml 1 mM HC1, and was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at 9.0 with 0.5 N NaOH.

After 2 h pH was adjusted to 6.5 with 0.5 M succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 103% compared to the parent enzyme.

Example 19 Conjugation of Savinase with poly(ethylene glvcol)-blockpoly(propylene glycol)-co-poly(ethvlene glycol) 12000 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCTIDK99/00406 bis(succinimidyl carbonate) (75 wt ethylene glycol).

148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with N NaOH. 2800 mg of the activated co polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M succinic acid. The reaction mixture was purified by gelfiltering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 140% compared to the parent o0 enzyme.

Example Conjugation of Savinase with poly(ethylene glycol)-blockpoly(propylene glycol)-co-poly(ethylene glycol) 970 bis(succinimidyl carbonate) (50 wt ethylene glycol).

148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with N NaOH. 340 mg of the activated co polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M succinic acid. The reaction mixture was purified by gelfiltering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 124% compared to the parent enzyme.

Example 21 Brown Norway Rat intratrachaeal (IT) trials of PD498 conjugates of small mPEG polymers PD498 samples with known protein concentration (measured by optical density and amino acid sequence analysis for derivatives) were diluted to 0.75 microG protein/ml.

The diluted samples were aliquoted in 1.5 ml fractions for individual immunizations. These fractions were stored under stable conditions at -20 0 C until use. The analyses were performed at the beginning and at the end of the study. For each immunization and each analysis a new fraction was taken.

SUBSTITUTE SHEET (RULE 26) WO00/04138 PCT/DK99/00406 56 Enzyme conjugates were conjugated with N-succinimidyl carbonate activated mPEG 350, 550, 750 as described in the examples above. The corresponding parent enzymes were used as controls.

The following samples were tested: Group 1: PD498 (parent uncopled enzyme control) Group 2: PD498-PEG 750 Group 3: PD498-PEG 550 Group 4: PD498-PEG 350 Rats were immunized weekly 15 times with 100 microL of a 0.9% (wt./vol.) NaCl solution (control group), or 100 microL of the PD498 protein dilutions mentioned above.

Each group comprised 10 Brown Norway rats. Blood samples (2 ml) were colllected from the eyes one week after every second immunization, but before the following immunization. Serum was obtained by blood cloothing, and centrifugation.

Specific IgE levels were determined using the ELIAS assay specific for rat IgE described above. The sera were titrated at dilution, starting from undiluted. Optical density was measured at 492/620 nm.

The result of the IT trials are shown in the following table illustrating the total optical density per 100 microL of serum at the end of the study, as observed in Brown Norway rats with the respective PD498 derivatives.

The result of the PD498 conjugate trials is shown in Table 2 below: Table 2: Number of un- PEG PEG PEG NaCI immuni- modified 350 550 750 zations 0 0.3 0.3 0.3 0.3 0.3 (0.6) 5.3 2.7 1.6 1.5 0.3 (0.6) SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 57 Value in parenthesis: Standard error of the mean value determined.

As can be seen from the Table 2 the specific IgE response level of the rats exposed intratracheally with the PD498 conjugate with small polymers coupled thereto is reduced in comparison to rats having been exposed intratracheally with the parent unmodified enzymes. Thus, the allergenicity is reduced.

Example 22 Brown Norway Rat intratrachaeal (IT) trials of a Subtilisin DY conjugate The Brown Norway rat IT study described in Example 5 was repeated comparing a Subtilisin DY-PEG750 conjugate with the corresponding parent Subtilisin DY enzyme (see SEQ ID NO: 3) The result of the Subtilisin DY-PEG750 trial is shown in table 3: Table 3: Number of un- PEG NaCI immuni- modified 750 zations 0 0.3 0.3 0.3 (0.6) 7.2 1.9 0.3 (0.6) Value in paranthesis: standard error of the mean value determined As can be seen from the Table 3 the specific IgE response level of the rats exposed intratracheally with the Subtilisin DY conjugate with a 750 Da polymer coupled thereto is reduced in comparison to rats having been exposed intratracheally with the parent unmodified enzyme.

Thus, the allergenicity is reduced.

Example 23 Skin care formulations comprising a PD498-PEG conjugate SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 58 The following skin care formulations comprising conjugates of the invention were prepared: Lotion (to make 100 g) Oil phase: Liquid Paraffin 35 g Cetyl Alcohol 5 g Tween 80 7 g Water phase: Mono Propylene Glycol (MPG) 10 g 0.4% citric acid buffer* pH 5.8 42.9 g Methyl Paraben 0.1 g PD498-SPEG550 10 mg (as enzyme protein) The Oil phase and the water phase were mixed separately and heated to 800C. The oil phase was poured slowly into the water phase while stirring. The mixture was cooled to apprx. 35 0 C and the PD498-SPEG550 conjugate was added. The lotion was cooled rapidly.

0.4% citric acid monohydrate, pH adjusted to 5.9 **Will usually be supplied as a formulation with MPG. MPG in the water phase should be adjusted according to the amount of MPG in the enzyme formulation.

Gel (to make 100g) MPG 20 g* ad.100g Citric Acid 0.4g** Carbapol 940 1 g PD498-SPEG350 10 mg (as enzyme protein) The ingredients were mixed in the above order. The pH was adjusted to 5.6 before addition of carbapol. After addition of carbapol the pH was adjusted again.

Adjust according to amount in enzyme formulation.

**pH 5.6 SUBSTITUTE SHEET (RULE 26) WO 00/04138 WO 0/0138PCT/DK99/00406 59 Example 24 Wash performance of PEG-Savinase and EOPO-Savinase Table 4: Experimental setup No. 1 Detergent Model detergent 95, 3.0 g/l Water hardness 6 0 dH (2:1 Ca/Mg) Enzymes Protease Concentration (concentrations Savinase 8. 1 X 10 M are based on Savinase-PEG10O0bis 1.1 X iO-1 M absorbance Svns-E20bs 12x1measurements at -4iaePE20bs1. O 280 nm) Savinase-PEG4000bis 1.4 x 10- M Savinase-PEG6000bis 1.1 X 10-4 M Savinase-PEGlO000bis 1.1 X 10-4 M Wash time 15 min.

Temperature 15 0

C

Enzyme conc. 10 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA117 Table 5: Exper imental setup No. 2 Detergent Tide powder detergent, 1.0 g/l Water hardness 6 0 dH (2:1.Ca/Mg) Enzymes Protease Concentration (concentrations Savinase 8. 1 X i0- M are based on Savinase-PEGlOO0bis 1.1 X 10-1 M abs orbance Svns-E20bs 12x1measurements at SaiaePG0bs1.xi0 M 280.nm) Savinase-PEG4000bis 1.4 x 10-' M 1.1 X 10-1 M 1.1 X i0-1 M Wash time 10 min.

Temperature 25 0

C

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Enzyme conc. 0; 3; 6; 9; 15; 25 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA117 Table 6: Experimental setup No.3 Detergent Tide powder detergent, 1.0 g/l Water hardness 6 0 dH (2:1 Ca/Mg) Enzymes Protease Concentration (concentrations Savinase 8.1 x 10- 4

M

are based on Savinase-PEG1000bis 1.1 x 10- 4

M

absorbance asurensa Savinase-PEG2000bis 1.2 x 10- M measurements at 280 nm) Wash time 10 min.

Temperature 25 0

C

Enzyme conc. 0; 3; 6; 9; 15; 25 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA117 Table 7: Experimental setup No.4 Detergent Model detergent 95, 3.0 g/1 Water hardness 6 0 dH (EMPA117), 18 0 dH (grass) (2:1 Ca/Mg) Enzymes Protease Concentration (concentrations Savinase 6.8 x 10- 4

M

are based on Savinase-PEG200bis 1.7 mg/ml 6.4 x absorbance 10-5 M measurements at Savinase-PEG300bis 1.6 mg/ml 5.8 x 280 nm) I0-5 M Savinase-PEG600bis 6.5 mg/ml 2.4 x SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406

M

Savinase-PEGl000bis 10-1 M Savinase-PEGl000bis 4

M

PS174-PEG1000bis 5

M

9.6 mg/ml 3.6 x 3.4 mg/ml 1.3 x 1.9 mg/ml 6.9 x Wash time 15 min.

Temperature 15 0

C

Enzyme conc. 10 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA117 Table 8: Experimental setup Detergent Omo Color, 4 g/l Water hardness 18 0 dH (EMPA116) Enzymes Protease Remission Delta Remission Savinase 25.8 4.3 Savinase- PEG300bis 26.2 4.8 Savinase- EOsPOo 0 27.2 5.8 Wash time 20 min.

Temperature 30 0

C

Enzyme conc. 2.5 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA116 Table 9: Experimental setup 6: Detergent Omo Color, 4 g/l SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Water hardness 18 0 dH (EMPA116) Enzymes Protease Remission Delta Remission Savinase 28.2 6.7 Savinase- EOsPOo 0 28.4 Wash time 20 min.

Temperature 30 0

C

Enzyme conc. 5.0 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA116, Table 10: Experimental setup 7: Detergent Wisk HDP, 1 g/l Water hardness 6 0 dH (EMPA117) Enzymes Protease Remission Delta Remission Savinase 13.7 2.7 Savinase- PEG300bis 14.6 3.7 SavinasemPEG350 14.4 3.4 Savinase- EOsPO 0 14.1 3.1 Wash time 10 min.

Temperature 25 0

C

Enzyme conc. 5.0 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA116, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 63 Table 11: Experimental setup 8: Detergent Wisk HDP, 1 g/l Water hardness 60dH (EMPA117) Enzymes Protease Remission Delta Remission Savinase 15.0 Savinase- PEG300bis 16.1 5.6 SavinasemPEG350 15.8 5.3 Savinase- EOsoPOso 16.0 Wash time 10 min.

Temperature 25 0

C

Enzyme conc. 10.0 nM Test method Miniwash robot 3 repetitions Swatch/volume 3 x 6 cm test material in 50 ml detergent solution Test material EMPA117 pH of the detergent solution was adjusted to 10.5 with HCl/NaOH. Water hardness was adjusted by adding CaCl 2 and MgC 2 1 to deionized water (see also Surfactants in Consumer Products Theory, Technology and Application, Springer Verlag 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HC1.

Proteases present in the commercial powder detergents were inactivated by heating a detergent solution to 85 0 C for minutes in a microwave oven.

Reflectance measurements of the test material were done at 460 nm using a J&M Tidas MMS/16 photometer equipped with a CLX 75W Xenon lamp and fibre optics. Each textile piece was measured individually with other textile pieces (same settings) as background.

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 64 SAS 6.12 software was used to make an analysis of variance and a t-test comparison (Student-Newman-Keuls) at significance on the experimental data.

The wash performance of the different Savinase® variants was evaluated by comparing delta reflectance (DR) values: DR Rprotease RBlank DR: Delta reflectance Rprotease: RBlank: Reflectance of test material washed with conjugated protease Reflectance of test material washed with nonconjugated protease Results The capital letters designate statistical groupings within each column based on a t-test (SNK, a=0.05). If two are in the same group (same letter), they cannot be separated statistically.

Table 12:Mean reflectance value and statistics Exp. No. 1 Reflectance Blind 9.5 F Savinase 14.3 B Savinase-PEGl000bis 14.7 A Savinase-PEG2000bis 14.5 B Root MSE 0.2 R-square 0.99 Table 13: Mean reflectance values SUBSTITUTE SHEET (RULE 26) WO 00/04138 WO 00/ 138PCT/DK99/00406 6 nM 14.2 15.8 15.0 9 nM 15.4 16.9 15.3 nM 16.3 17.4 16.3 nM 16.6 17.6 17.6 Table 14: Mean reflectance values Exp. No 3 EMPA117 Savinase PEG1000 PEG2000 Blind 11.5 12.5 12.0 3 nM 13.8 15.1 15.5 6 nM 14.7 16.3 15.8 9 nM 15.1 17.1 16.6 nM 16.0 18.3 17.2 nM 16.4 18.3 18.3 Table 15: Mean reflectance value Exp. No. 4 EMPA117 Blind 9.8 D Savinase 14.9 AB Savinase-PEG200bis 15.2 AB Savinase-PEG300bis 15.3 AB Savinase-PEG10G0bis 15.5 A Savinase variant R247K- 15.2 AB PEGiQO0bis Root MSE 0.4 R-square 0.96 As shown in the above tables the wash performance of PEG- Savinase and EOPO-Savinase have improved compared to the wash performance of non-conjugated Savinase SUBSTITUTE SHEET (RULE 26) EDITORIAL NOTE APPLICATION NUMBER 48983/99 The following Sequence Listing pages 1-6 are part of the description.

The claims pages follow on pages 66-67 WO 00/04138 SEQUENCE LISTING <110> NOVO NORDIS1( A/S e120>' A polypeptide -polymer conjugate with improved wash performance <130> 5625,HkBk <140> <141> <160> <170> Patentln Ver. 2.1 PCTIDK99/00406- <210> 2.

<211> 840 <212> DNA 4213> Bacillus sp. PD498, NCIME No. 40484 <400> 1 tggtcaccga cctgctgcct ggagtggatt gacagggaca gctgctgata gccgtacggg cgctatgctg tccacaactc gctgcaggga gcagtaggtg gtggatgtca tacatgtctg agtcaaggta tctggcactg atgaccctta gggatgtaac ataaccaccc ataacccaat cgaacaatgg tccttgatgc ctgatcaagg ttaagagtgc atgacaatgt ccattgactc ctgctccagg gtacgtccat agaataacgt gaacaaactt ctattctgCt ccgtggaagc tgatcttgca ggatcttaac aattggcgta caatggaagt ggcaaaggta cgtcgactat atcccgtaca caatgatcga tgtgaacata ggcatcc Oct acaaatccgc caagtatggt taccagtatg agcactcaaa agaaaagtaa, ggacatggta gccggtatgg ggctcacttg ctcaacctct gcatggaaca.

ttccaaccag aaagcatcat gcatcaaccg cacgtggccg caggccattg aaaatcaact gaccacaaaa cggtggcggt taaaagggta cccatgttgc caccagatac acagcattgc cccttggttg aaggagctgt cttcttaccc tctccaatta ttccgaataa gtttggctgc agcaaaccgc caaacaaagc cacctcaacc ccttgattcc cgactttatc cggtactgtt gaagatcctt ctcaggtatc cgaatgcaac agtcgttgct taatgccatt cggaacgtgg tggctactcc tttgttggca cgataagat c tgtaagatac 120 180 240 300 360 420 480 540 600 660 720 780 840 <210> 2 c211> 280 <212> PRT <213> Bacillus sp. PD498, NCIMB No. 40484 <400> 2 Trp Ser Pro 1 Asn Thr Ser Gin Thr Val Leu Ala Arg s0 Asn Asp Pro 5 Tltr Pro Mla Tyr Tyr Ser Tyr Gin Tyr Gly Pro Gin Ser Ser Th~r Ala Trp Asp Val Thr Arg Gly 25 Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp 40 Gly Tyr Asp Phe Ile Asp Arg Asp .Asn Lys Val Ile Asn Pro Met Asp, LeU Asn Gly His Gly Thr His Val Mla Gly Thr Val SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406- Ala Thr Leu Lys Lys 145 Ala Pro Ser Asn Thr 225 Ser Ala Asn Ala Lys Asp Val 130 Ser Ala Asn Phe Ile 210 Ser Gin Asp Ser Asp Ile Ser 115 Leu Ala Giy Ala Ser 195 Ala Met Gly Lys Asn 275 Thr Asn Leu Ala 100 Ile Ala Asn Leu Vai Asp Asn Asp 165 Ile Ala 180 Asn Tyr Ser Thr Ala Ser Lys Asn 245 Ile Ser 260 Asn Gly Ile Gly Val Ala Val Ser Ser Tyr 150 Asn Val Gly Val Pro 230 Asn Gly Arg Gly Leu 135 Ala Val Gly Thr Pro 215 His Val Thr Val Ile 120 Gly Trp Ser Ala Trp 200 Asn Val Gin Gly Leu 105 Arg Cys Asn Arg Ile 185 Val Asn Ala Ile Thr 265 90 Asp Ala Tyr Ala Glu Cys Lys Gly Thr Phe 170 Asp Ser Asp Val Gly Tyr Gly Leu 235 Arg Gln 250 Asn Phe Gly Asn Ala Asn 140 Ala Gin Asn Thr Ser 220 Ala Ala Lys Met Ala Gly Ser 110 Asp Gin 125 Ser Thr Val Val Pro Ala Asp Arg 190 Ala Pro 205 Tyr Met Ala Leu Ile Glu Tyr Gly 270 Pro Asp Gly Ser Gly Ala Thr Leu Val Ala 160 Ser Tyr 175 Lys Ala Gly Val Ser Gly Leu Ala 240 Gin Thr 255 Lys Ile Lys Ala Val Arg Tyr .280 <210> 3 <211> 274 <212> PRT' <213> Bacillus sp. variant <400> 3 Ala Gin Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val 1 5 .10 Gin Ala Gin Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp 25 Thr Gly Ile Ala (Ala/Ser) Ser His Thr Asp Leu Lys Val Val Gly Gly Ala 40 Ser Phe Val Ser Gly Giu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly 55 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 3 Thr His Vai Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val 70 75 s0 Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn 90 Ser Ser Giy Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Giu Trp 100 105 110 Ala Thr Gin Asn Gly Leu Asp Vai Ile Asn Met Ser Leu Gly Gly Pro 115 120 125 Ser Gly Ser Thr Ala Leu Lys Gin Ala Val Asp Lys Ala Tyr Ala Ser 130 135 140 Gly Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly.Ser 145 150 155 160 Gin Asn Thr le Gly Tyr Pro Ala Lys Tyr Asp Ser Vai le Ala Val 165 170 175 Giy Ala Vai Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 CAla/Ser) Giu Leu Giu Val Met Ala Pro Gly Val Ser Val Tyr Ser Th~r Tyr 195 200 205 Pro Ser Asn Th~r Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Thr Leu 225 230 235 240 Ser Ala Ser Gin Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu 245 250 255* Giy Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Vai Giu Ala Ala 260 265 270 Ala Gin <210> 4 <~211> 433 <212> PRT <213> Bacillus sp. Y <400> 4 Asn Asp Val Ala Arg Gly Ile Val Lys Ala Asp Val Ala Gin Asn Asn 1 5 10 Tyr Giy Leu Tyr Gly Gin Gly Gin Leu Val Ala Val. Ala Asp Thr'Giy 25 Leu Asp Thr Giy Arg Asn Asp Ser Ser Met His Glu Ala Phe Arg Gly 40 SUBSTITUTE SHEET (RULE 26) PCT/DK99/00406 WO 00/04138 Lys Ile Thr Ala Leu Tyr Ala Leu Gly Arg 55

II

Pro 1 Leu 2 Met Lell] Trp( Asp I 145 Asn Asfl.

Ser Ala Phe 225 Ala Thr Wys lie Gly 305 Asn LSfl ~sn !sp Phe Gly Glu Glu Ala Ile Thr 210 lie Asn Pro AsE Ala 29C Trj Git Gly I Lys C Ser I Ser 115 Ala Tyr' Gly Ile Ala 195 Arg Leu yr Ile Arg 275 Gly Gly x Ala [is ;ly er LO0 31n Pro Val Pro Thr 180 Asp Asp Ser Asn Val 260 Gl Ala Ar5 Thi Gly IJ Met 2 Gly Ala Val Arg.

As 165 Val As Gly Ala Ser 245 Ala Ile Thr Val Ala 325 hr 70 Uia fly rrp Asn Asn 150 Ser Gly Pro Arg Arg 230 Lys G13 Th AsI Thn 31( Le His I Pro Leu Asn Gly 135 Asn.

Gly Ala Asn Ile 215 Ser STyr Asfl Pro Val 295 c Leu a Ala lal .In fly kla 120 Ala Asp Thr Thr His 200 Lys Ser Ala Val Lys 280 Gl Asp Th2 Ala G Ala Gly I 105 Gly I Tyr Met Ile Glu.

185 lie Pro Leu Tyr Ala 265 Pro Leu Lys Gly ly5 ns 90 .eu Ua hr rhr Ser 170 Asn Ala Asp Ala Met 250 Gin Ser Gly Ser Gin 330 'hr I jer N 75 .eu Pro krg Ala Val 155 Ala Tyr Gin Val Pro 235 Gly Leu Leu Tyr Leu 315 Lys Lsn lal Ial Ser lie Asn 140 Leu Pro Arg Phe Thr 220 Asp Gly Arc lie Prc Asi Al.

Asn Ala Leu Gly 2 Phe Gin Asn Leu 110 His Thr 125 Ser Arg Phe Ala Gly Thr Pro Ser 190 Ser Ser 205 Ala Pro Ser Ser Thr Ser Glu His 270 Lys Ala 285 Ser Gly 1 Val Ala i Thr Tyr er .sn Ser ksn Asn Gln Ala Ala 175 Phe Arg Gly Phe Met 255 Phe Ala Asp Tyr Ser 335 Asp Ala Ile Thr Ser Val Gly 160 Lys Gly Gly Thr Trp 240 Ala Ile Leu Gln Val 320 Phe Gin Ala Gin Gly Lys Pro Leu Lys 345 lie Ser Leu Val Trp Thr Asp 350 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 Ala Pro Gly Ser Thr Thr Ala Ser Tyr Thr Leu Val Asn Asp Leu Asp 355 360 365 Leu Val Ile Thr Ala Pro Asn Gly Gin Lys Tyr Val Gly Asn Asp Phe 370 375 380 Ser Tyr Pro Tyr Asp Asn Asn Trp Asp Gly Arg Asn Asn Val Glu Asn 385 390 395 400 Val Phe Ile Asn Ala Pro Gin Ser Gly Thr Tyr Ile Ile Glu Val Gin 405 410 415 Ala Tyr Asn Val Pro Ser Gly Pro Gin Arg Phe Ser Leu Ala Ile Val 420 425 430 His <210> <211> 316 <212> PRT <213> Bacillus thermoproteolyticus <400> Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly Asp 1 5 10 Gin Lys Asn Ile Asn Thr Thr Tyr Ser Thr Tyr Tyr Tyr Leu Gin Asp 25 Asn Thr Arg Gly Asp Gly Ile Phe Thr Tyr Asp Ala Lys Tyr Arg Thr 40 Thr Leu Pro Gly Ser Leu Trp Ala Asp Ala Asp Asn Gin Phe Phe Ala 55 Ser Tyr Asp Ala Pro Ala Val Asp Ala His Tyr Tyr Ala Gly Val Thr 70 75 Tyr Asp Tyr Tyr Lys Asn Val His Asn Arg Leu Ser Tyr Asp Gly Asn 90 Asn Ala Ala Ile Arg Ser Ser Val His Tyr Ser Gin Gly Tyr Asn Asn 100 105 110 Ala Phe Trp Asn Gly Ser Glu Met Val Tyr Gly Asp Gly Asp Gly Gln 115 120 125 Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Val Val Ala His Glu Leu 130 135 140 Thr His Ala Val Thr Asp Tyr Thr Ala Gly Leu Ile Tyr Gin Asn Glu 145 150 155 160 Ser Gly Ala Ile Asn Glu Ala Ile Ser Asp Ile Phe Gly Thr Leu Val 165 170 175 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 6 Glu Phe Tyr Ala Asn Lys Asn Pro Asp Trp Glu Ile Gly Glu Asp Val 180 165 190 Tyr Thr Pro Gly Ile Ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro 195 200 205 Ala Lys Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr 210 215 220 Gin Asp Asri Gly Gly Val His Ile Asn Ser Gly Ile Ile Asn Lys Ala 225 230 235 240 Ala Tyr Leu Ile Ser Gin Gly Gly Thr His Tyr Gly Val Ser Val Val 245 250 255 Gly Ile Gly Arg Asp Lys Leu Gly Lys Ile Phe Tyr Arg Ala Leu Thr 260 265 270 Gin Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gin Leu Arg Ala Ala Ala 275 280 285 Val Gin Ser Ala Thr Asp Leu Tyr Giy Ser Thr Ser Gin Giu Val Ala 290 295 300 Ser Val Lys Gin Ala Phe Asp Ala Vai Gly Val Lys 305 310 315 SUBSTITUTE SHEET (RULE 26)

Claims (21)

1. Use of a polypeptide-polymer conjugate with improved wash performance for improving the wash performance of industrial compositions, wherein the polymer has a molecular weight in the range of from 100 Da to 350 Da, and wherein the polypeptide- polymer conjugate has reduced respiratory allergenicity relative to the unconjugated polypeptide. S2. The use according to claim 1, wherein the polypeptide is an enzyme.

3. The use according to any one of the proceeding claims, wherein the parent polypeptide is conjugated to a homopolymer, a graft, block, alternate, or random co- polymer with a molecular weight in the range of 100 Da to 350 Da.

4. The use according to any one of the proceeding claims, wherein the polymeric molecule is selected from the group comprising natural or synthetic homo- and heteropolymers. The use according to claim 4, wherein the polymeric molecule is selected 15 from the group comprising synthetic polymeric molecules including branched PEGs, star- S* shaped PEGs, PEG ethers and PEG esters.

06. The use according to any of claims 1 to 3, wherein the block or co- ::polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 10:90. 0 20 7. The use according to any of claims I to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 20:80. 8 The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of30:70.

9. The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of40:60.

10. The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 50:50.

11. The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 60:40.

12. The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 70:30,

13. The use according to any of claims I to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 80:20. A51778lpoe COMS ID No: SMBI-00562040 Received by IP Australia: Time 15:40 Date 2004-01-08 8.-JAN. 2004 15:39 SPRUSON AND FERGUSON 61292615486 NO. 4927 P. 9 67

14. The use according to any of claims 1 to 3, wherein the block or co- polymer(s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio of 90:10. The use according to any of claims 1 to 3, wherein the polypeptide is of microbial origin.

16. The use of claim 15, wherein the peptide is of bacterial origin.

17. The use of claim 15, wherein the peptide is of filamentous fungal origin.

18. The use of claim 15, wherein the peptide is of yeast origin.

19. The use of claim 15, wherein the peptide is of plant origin.

20. The use according to any one of claims 15 to 19, wherein the polypeptide is an enzyme from the group of hydrolases, oxidoreductases, or superoxide dismutases.

21. The use according to claim 20, wherein said polypeptide is a serine protease.

22. The use according to claim 20, wherein said polypeptide is a subtilisin. 15 23. The use according to claim 20, wherein said polypeptide is a metallo- protease.

24. The use according to claim 20, wherein said polypeptide is a laccase.

25. The use according to claim 20, wherein said polypeptide is a haloperoxidase.

26. The use according to any one of the proceeding claims, wherein the industrial composition is a detergent or a personal care product.

27. Use of a polypeptide-polymer conjugate with improved wash performance for improving the wash performance of industrial compositions, wherein the polymer has a molecular weight in the range of from 100 Da to 350 Da, and wherein the polypeptide- 25 polymer conjugate has reduced respiratory allergenicity relative to the unconjugated polypeptide, substantially as hereinbefore described with reference to any one of the Examples. Dated 8 January, 2004 Novozymes A/S r S S 5 C Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON A537781pC COMS ID No: SMBI-00562040 Received by IP Australia: Time 15:40 Date 2004-01-08