EP1098964A1 - A polypeptide-polymer conjugate with improved wash performance - Google Patents

Abstract

The present invention relates to a polypeptide-polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymers is homo-polymers, graft, block, alternate, or ramdom co-polymers. The invention also relates to industrial compositions and products comprising a conjugate of the invention and to the use of said conjugate for improving the wash performance of industrial composition and products such as detergent compositions.

Description

TITLE: A polypeptide-polymer conjugate with improved wash performance

FIELD OF THE INVENTION 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 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 30 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. DURAZYM^® (Novo Nordisk A/S), RELASE^ε (Novo Nordisk A/S) , MAXAPEM⁰ (Gist-Brocades N.V.), PURAFECT^® (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 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 (i.e. pharmaceuticals) the typical potential immune response is an IgG and/or IgM response, while polypeptides which are inhaled through the respiratory system (i.e. 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, e.g., ethylene oxide (EO) , polyethylene glycol

(PEG) , or propylene oxide (PO) , 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 polymer-polypeptide conjugates have improved wash performance in comparison to the unmodified polypeptide.

SUMMARY OF THE INVENTION In it first aspect the present invention relates to 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:

EO_xPO_y (I)

wherein x=l-99%, y=l-99% and x+y=100% covalently to a parent polypeptide, used for industrial application, the wash performance is improved when compared to the parent polypeptide. In both cases the respiratory allergenicity may also be reduced when compared to the parent enzyme. I the latter case the respiratory allergenicity may even be reduced when compared to a corresponding conjugate coupled with PEG or other homopolymers .

In a second aspect the invention relates to compositions for use in industrial products comprising a conjugate of the invention. In a third aspect the invention relates to the use of conjugates for improving wash performance and a final aspect the invention relates to a method for improving wash performance of polypeptides.

Industrial polypeptides

Polypeptides used for industrial applications often have an enzymatic and/or anti-microbial activity. Industrial 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 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 (i.e. IgE response) as a conse- quence 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 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 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. 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 (S. Fδrster and . 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 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 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 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 polypeptide- polymer 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 polypeptide- polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymer is characterized by the general formula:

EO_xPO_y ( I ;

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

The polymer is preferably characterized by the general formula: (I) wherein x=10-90%, y=10-90%, 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, 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 (I) 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 IgGlA and IgGIB (see e.g. Prentø, 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 IgGlA and IgGIB is a measure of the allergenicity level.

A rat strain suitable for intratracheal exposure to polypep- 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 (i.e. poly-OH) , polyamines (i.e. poly-NH₂) and polycarboxyl acids (i.e. 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) , PEG- oxycarbonylimidazole (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), PEG50stearate (Myrj 53) , PEGlOOstearate (Myrj 59) , and polyoxyethylene 25 propylene glycol stearate, polyoxyethylene ethers, including 2 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, 5 Octyl Ether, 5 Decyl Ether, 5 Lauryl Ether, 5 Myristyl Ether, 5 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, 10 Cetyl Ether, 10 Stearyl Ether, 10 Oleyl Ether, 20 Cetyl Ether, 20 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 :

EO_xPO_y(I)

wherein x=l-99%, y=l-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. 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) -block- poly (ethylene glycol) -block-poly (propylene glycol)mono butyl ether; poly (ethylene glycol) -block-poly (propylene glycol) -block- poly (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 (-OCH₂CH₂-) _x [-OCH (CH₃) CH₂-] _y (-OCH₂CH₂-) _zOH, having the average molecule weight (M_n) of 1,100 and the ethylene glycol content of 10 wt%, M_n= 1,900 and 50 wt%, M_n= 2,000 and 10 wt%, M_n= 2,800 and 10 wt%, M_n=2,800 and 15 wt%,M_n= 2,900 and 40 wt%, M_n= 4,400 and 30 wt%, M_n= 5,800 and 30 wt%, M_n= 8,400 and 80 wt% .

Other preferred block polymers are block polymers having the general formula: H [-OCH (CH₃) CH₂-] _x (-OCH,CH₂-) _y [-OCH (CH₃) CH₂-] _zOH, having the average molecule weight (M_n) of 2,000 and the ethylene glycol content of 50 wt%, M_n= 2,700 and 40 wt%, and M_n= 3,300 and 10 wt%. 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-co- propylene glycol), especially poly (ethylene glycol-co-propylene glycol) having an an average molecule weight M_n of 2,500 and 75 wt% ethylene glycol and an average molecule weight M_n of 12,000 and 75 wt% ethylene glycol; poly (ethylene glycol-co-propylene glycol) mono butyl ether, especially poly (ehtylene glycol-co- propylene glycol)monobutyl ether having an M_n of 970 and 50 wt% ethylene glycol, an M_n of 1,700 and 50 wt% ethylene glycol and an M_n 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. 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, trichlorotri- azine etc. (see R.F. Taylor, (1991), "Protein immobilisation. Fundamental and applications", Marcel Dekker, N.Y.; S.S. Wong, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized Affinity Ligand Techniques", Academic Press, N.Y.). Some of the methods concern activation of 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 e- xample 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 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 ortho- pyridyl 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. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K. , Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K. , 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 (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

5 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

10 (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 Al, (1993), Looze, Y.) 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.

15 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 al . ) . The reactivity of these

20 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

25 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,

30 375-378. 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

35 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. B.) thus introducing an 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 al.). Lysine residues may also be used as the backbone.

General overviews of polymer activation and PEG fuctionalization for the preparation of relevant conjugates is given in Zaplisky, S., Bioconjugate Chem., 1995, 6, 150-165, Hermanson, G.T., 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 (i.e. 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

15 to 60 kDa, using any suitable technique known in the art.

The term "parent" polypeptide is intended to indicate any uncoupled polypeptide (i.e. 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. The parent polypeptide may be a naturally-occurring (or wild- type) 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 co- polymers are coupled in the area close to the active site.

In the present context "spread broadly" means positioned so 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 A² (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 polymers (i.e. 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 (E.C. 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, (E.C. 2), such as transglutaminases (TGases) ; Isomerases (E.C. 5) , such as Protein disulfide Isomerases (PDI) .

Parent Proteases

Parent proteases (i.e. 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 (E.C.) numbers:

3.4.11 (i.e. 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 (i.e. so-called serine endopeptidases) , including

3.4.21.1 (Chy otrypsin) , 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 (i.e. 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 (i.e. so-called aspartic endopeptidases), including 3.4.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin) ; and

3.4.24 (i.e. so-called metalloendopeptidases) , including 3.4.24.28 (Bacillolysin) .

Examples of relevant subtilisins comprise subtilisin BPN¹, 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 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 A/S), WO 91/00345 (Nove Nordisk A/S), EP 525 610

(Solvay) and WO 94/02618 (Gist-Brocades N.V.).

The C-component disclosed in EP 482,879 Bl (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. 5.

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 (i.e. number of attachment groups, position of attachment groups etc.). Specific examples of suitable parent proteases having a suitable number of attachment groups are indicated in Table 1 below:

Table 1:

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 (i.e. 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 (i.e. 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 (i.e. Lysine residues) on the surface plus one N-terminal amino group. (See SEQ ID NO: 5)

Parent Lipases

Parent lipases (i.e. 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 (E.C.) numbers: 3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (3.1.1.3) Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase

A₂.

Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference: 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- 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 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 { e . g. described in WO 90/09446) .

Parent Oxidoreductases Parent oxidoreductases (i.e. 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 (E.C.) numbers: Glycerol-3-phosphate dehydrogenase NAD+ (1.1.1.8), Glycerol-3- phosphate dehydrogenase NAD(P)" (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase NADP (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamate dehydrogenase NAD(P)⁺ (1.4.1.3), Glutamate dehydrogenase NADP^* (1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine dehydrogenase NADP⁺ (1.4.1.8), Leucine dehydrogenase (1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acid oxidase 5 (1.4.3.2.), D-Amino-acid oxidase (1.4.3.3) , L-Glutamate oxidase (1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase (1.4.3.14), L-Aspartate oxidase (1.4.3.16), D-amino-acid dehydrogenase (1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase (1.6.4.5), Protein disulfide reductase

10 (glutathione) (1.8.4.2), Laccase (1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), 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,

15 Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.

Bilirubin oxidases may be derived from Myrothechecium verrucaria. 0 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 A/S), which are herby incorporated as reference,

25 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

30 A/S) . 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,

35 1984, Verlag Chemie, Weinheim, vol. 3.

Contemplated laccases include the laccases disclosed in WO 96/00290 and WO 95/33836 from Novo Nordisk. 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 (e.g. starches) of especially five and six member ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) 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 (E.C.) numbers: α-amylase (3.2.1.1) β-amylase (3.2.1.2), glucan 1,4-α- glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1, 3 (4) -β- glucanase (3.2.1.6), endo-1, 4-β-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), β-glucosidase (3.2.1.21), α-galactosidase (3.2.1.22), β-galactosidase (3.2.1.23), amylo-1, 6-glucosidase (3.2.1.33), xylan 1, 4-β-xylosidase (3.2.1.37), glucan endo-1, 3- β-D-glucosidase (3.2.1.39), α-dextrin endo-1, 6-glucosidase

(3.2.1.41), sucrose α-glucosidase (3.2.1.48), glucan endo-1, 3- α-glucosidase (3.2.1.59), glucan 1, 4-β-glucosidase (3.2.1.74), glucan endo-1, 6-β-glucosidase (3.2.1.75), arabinan endo-1, 5-α- arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase (3.2.1.132) and xylose isomerase (5.3.1.5).

Examples of relevant carbohydrases include α-1, 3-glucanases derived from Trichoderma harzianum; α-1, 6-glucanases derived from a strain of Paecilomyces; β-glucanases derived from Bacillus subtilis; β-glucanases derived from Humicola insolens; β-glucanases derived from Aspergillus niger; β-glucanases derived from a strain of Trichoderma; β-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1, 4-α-D-glucosidases (glucoamylases) derived from Aspergillus niger; α-amylases derived from Bacillus subtilis; α-amylases derived from Bacillus amyloliquefaciens; α-amylases derived from Bacillus stearothermophilus; α-amylases derived from Aspergillus oryzae; α-amylases derived from non-pathogenic microorganisms; α- galactosidases derived from Aspergillus niger; Pentosanases, 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; endo- glucanase derived from non-pathogenic mold; pullulanases derived from Bacillus acidopullyticus; β-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 A/S) . 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. Parent Transferases

Parent transferases (i.e. enzymes classified under the Enzyme

Classification number E.C. 2 in accordance with the Recommen- dations (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 groups (E.C. 2.1); transferases transferring aldehyde or residues (E.C 2.2); acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4); transferases transferring alkyl or aryl groups, other that methyl groups (E.C. 2.5); transferases transferring nitrogeneous groups (2.6). In a preferred embodiment the parent transferease is a transglutaminase E.C 2.3.2.13 (Protein-glutamine m- glutamyltransferase) .

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 gamma- glutamyl-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 polycephalu (Klein et al., Journal of Bacteriology, Vol. 174, p. 2599-2605) ; transglutaminases derived from Streptomyces sp., including Strepto yces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Strep- toverticillium sp., including Streptoverticillium mobaraense, 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 Syte atic 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. 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 ory- zae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species) .

Examples of parent phytases include phytases selected from those classified under the Enzyme Classification (E.C.) numbers: 3-phytase (3.1.3.8) 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 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 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, 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 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. 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 H₂0₂, which then will initiate the oxidation of for instance SCN^" or I^" into anti- microbial reagents (SCNO^" or I₂) 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 (R. 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 (a) an amino-functional active ingredient, (b) transglutaminase to catalyse cross-linking of the active ingredient to the skin, hair or nails, and (c) a carrier is known from US patent no. 5,490,980.

A cosmetic composition suitable for application to mammalian skin, hair or nails comprising: (a) at least one corneocyte envelope protein in an amount sufficient to provide a protective layer on said skin, hair or nails; (b) 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; (c) calcium ions in an amount sufficient to activate the transglutaminase; and (d) 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, e.g., one or more of polyethylene gly- col, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, alginil acid, carboxymethyl cellulose, starch, and hydroxypropyl cellulose. The modification is done, e.g., 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 ef- fective 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, 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 Examples %

Surfactants Soap (sodium salt) 83 -87

Sequestering agents Ethylenediamine tetraacetate 0.1-0.3 Consistency regulators Sodium chloride approx.

0.5 Dyestuffs < 0 . 1

Optical brighteners < 0 . 1

Antioxidants 2, 6-bis (1, 1-Dimethylethyl) - 0 . . 1-0 . , 3

4-methyl phenol (BHT) Whitening agents Titanium dioxide 0 . . 1-0 . . 3 Fragrances 1 . . 0-2 . , 0

Enzymes Protease/Lipase 0- -5

Water Balance

Syndet (Synthetic Detergents) Ingredients Examples %

Surfactants Lauryl sulfate 30-50 Lauryl sulfo succinate 1-12

Refatting agents Fatty alcohols 10-20 Plasticizers Stearyl mono/diglycerides 0-10 Fillers Starches 0-10 Active agents Salicylic acid 0-1 Dyestuffs < 0.2 Fragrances 0-2 Enzymes Protease/Lipase 0-5 Water Balance

Foam bath and shower bath

Ingredients Examples % % Foam bath Shower bath Surfactants Lauryl ether sulfate 10-20 10-12

Coco amidopropyl dimethyl betaine 2-4 2-4

Ethoxylated fatty acids 0.5-2

Refatting agents Fatty alcohols 0.5-3

Ethoxylated fattyalcoho. 0.5-5 0-4 Enzymes Protease/Lipase 0-5 0-5

Ingredients Examples % Foam bath Shower bath Foam stabilizers Fatty acid alkanol amide 0.2-2 0-4 Conditioners Quaternized hydroxypropyl cellulose 0-0.5

Thickeners Sodium chloride 0-3 0-3

Pearlescent agents Ethyleneglycol stearate 0-2

Active agents Vegetable extracts 0-1 0-1 Preservatives 5-Bromo-5-nitro-l, 3- dioxane 0.1 0.1

Dyestuffs 0.1-0.2 0.1 Fragrances 0.3-3 0.3-2 Enzymes Protease/Lipase 0-5 0-5 Water Balance Balance Hair shampoo

Ingredients Examples % Surfactants Lauryl ether sulfate 12-16

Coco fatty acid amidopropyl 2-5 dimethyl betaine Fatty acid polyglycol esters 0-2

Foam boosters Fatty acid ethanol amides 0. 5-2 . Conditioners Quaternized hydroxyethyl 0.4-1 cellulose Protein hydrolysates 0.2-1

Refatting agents Ethoxylated lanolin alcohols 0.2-1 Additives Anti-dandruff agents 0-1

Preservatives 5-Bromo-5-nitro-l, 3-dioxane 0 . 1-0 , Pearlescent agents Ethyleneglycol stearate 0-2 Dyestuffs < 0. 1 pH-Regulators Acids/Bases 0 . 1-1 Fragrances 0 . 3-0 , Enzymes Protease/Lipase 0-5 Water Balance

Hair rinse and hair conditioner

Ingredients Examples % % Hair rinse Hair conditiner Surfactants Fatty alcohol polyglycol ethers 0.1-0.2 1.5-2.5 Cetyl trimethyl ammonium chloride 0.5-1 Dimethyl benzyl stearyl ammonium 0.5-1 chloride

Refatting agents Cetyl/Stearyl mono/ diglyceride 0.5-1.5 1.5-2.5

Consistency regulators Fatty alcohols 1-2.5 2.5-3.5 Thickeners Methyl hydroxypropyl 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 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 than conventional granular detergents, i.e. from 550 to 950 g/1. 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 60% 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 25 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 CIO 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 R0(A)mS03M 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 (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or sub- stituted-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 ROS03M 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, e.g., an alkali metal cation (e.g. sodium, potassium, lithium) , or ammonium or substituted ammonium. 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 olefinsul- fonates, 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 ammopolyphosphonates. 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 30%.

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 (e.g. laccases). 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 (e.g. 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 25%.

The hydrogen peroxide releasing agent can be used in combination with bleach activators such as tetra- acetylethylenediamine (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 hydro- phobic esters (e.g. 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. 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 BPNXumbering) .

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 anti- rat IgE (Serotec MCA193; dilution 1:200). 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

K2HP04 1.04 g

KH2P04 0.32 g

- Washing buffer PBS, 0.05% (v/v) Tween 20 - Blocking buffer PBS, 2% (wt/v) Skim Milk powder

- Dilution buffer PBS, 0.05% (v/v) 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)

- CaCl₂ (Sigma) - Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184)

- W-Hydroxy succinimide (Fluka art. 56480))

- Phosgene (Fluka art. 79380)

- Lactose (Merck 7656) - PMSF (phenyl methyl sulfonyl flouride) from Sigma -Succinyl-Alanine-Alanine-Proline-Phenylalanine-para- nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.

- mPEG (Fluka)

Protease model detergent 95 i s an in-house detergent formulation : 25 % STP (Na₅P₃O₁₀ ) 25 Na₂S0₄

10 % Na₂C0₃ 20 % LAS (Nansa 80S) 5 % NI (Dobanol 25-7) 5 % Na₂Si₂0₅

0.5 % Carboxymethylcellulose (CMC) 9.5 % 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)

Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.

SLT: Fotometer from SLT Lablnstruments 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 2 " long metal probe is used. This probe is instilled in the trachea of the rats approximately 1 cm below the epiglottis, and 0.1 ml of a solution of the molecules is deposited.

The test animals are Brown Norway rats (BN) in groups of 10. 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 (50microL/well) . Incubate over night at 4°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.

5) Dilute specific polyclonal anti-enzyme antiserum serum (pig) for detecting bound antibody in Dilution buffer. Incubate

50 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 H₂0,/ml in substrate Buffer. Make the solution just before use. Incubate for 10 minutes. 50 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. 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 p- nitroaniline to give a visible yellow colour absorbing at 405 nm.

Buffer: e.g. Britton and Robinson buffer pH 8.3 Substrate: 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO) . 100 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_{405 nm}/min. The temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.

EXAMPLES

Example 1

Activation of poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) 1.900 (_50 wt% ehtyleneqlycol) with N-succinimidyl carbonate

Poly (ethylene glycol) -block-poly (propylene glycol) -block- poly (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 azeotropically. The solution was cooled to 20 ^CC 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 5 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

10 precipitation of triethylamine hydrochloride (Et3N.HCl) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et3N.HCl. The filtrate was evaporated to dryness under reduced pressure to yield 97 % (mole/mole) of an oil. NMR Indicating >

15 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)

(50 wt% ehtyleneglycol) (CDC13) δ: 1.15 bs (1=330 -CH3 in PPG), 2.69 s (1=1.7 unreacted NHS), 2.83 s (1= 41, succinimide), 3.41 20 m (1=110, CH-CH2 in PPG), 3.55 m (1=220, CH-CH2 in PPG), 3.61 m (1=440 main peak), 4.46 t (1=19, CH2-0-CO- in PEG).

25

Example 2

Activation of poly (ethylene glycol) -co- (propylene glycol) 30 monobutyl ether 970 (ca. 50 wt% ethyleneglycol) with N- succinimidyl 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 35 normal pressure to dry the reactants azeotropically. The solution was cooled to 0°C and phosgene in toluene (1.93 M, 5 mole/mole polymer) was added. The mixture was then stirred at 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 5 oil. i\J-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 (Et₃N.HCl) could be observed. The mixture was stirred overnight at room temperature. The mixture

10 was then filtered using a glass frit (G5) to remove insoluble Et₃N.HCl. 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. ^-NMR (400MHz), CDC1₃) δ: 0.91 t (1=1000 -CH₃ butyl), 1.15 bs (1=8744 -

15 CH₃ in propylene glycol), 1.39 m (1=1320 CH₃-CH₂-CH₂- butyl), 1.55 m (1=656 -CH₂-0- butyl), 2.68 s (1=60.8 unreacted NHS), 2.83 s (1= 963.2, succinimide), 3.40 (1=3059, CH-CH₂ in propylene glycol), 3.55 m (1=2678, CH-CH₂ in propylene glycol), 3.61 m (1=1764 main peak, -CH₂-CH₂- in ethylene glycol), 4.46 m

20 (CH₂-0-CO-) .

Example 3

Activation of mPEG 350 with N-succinimidyl carbonate mPEG 350 was dissolved in toluene (4 ml/g of mPEG) . About

25 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

30 chloroformate was obtained as an oil.

After evaporation dichloromethane and toluene (1:2, dry 4 ml/g mPEG) was added to re-dissolve the colorless oil. I\-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

35 precipitation of triethylamine hydrochloride (Et₃N.HCl) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et₃N.HCl. The filtrate was evaporated to dryness under reduced pressure to yield 98 % (mole/mole) of an oil. NMR Indicating 85 - 95% activation and <10 o/o (mole/mole) HNEt₃Cl . ^JH-NMR (400 MHz) for mPEG 350 succinimidylcarbonate (CDC1₃) δ: 1.42 t (1=1.4 CH₃ in HNEt,Cl), 2.68 s (1=3.4 unreacted NHS), 2.84 s (1= 6.2 succinimide), 3.10 dq (1= 1.0 CH, i HNEt₃Cl) , 3.38 s (1=5.8 CH₃ i OMe), 3.64 bs (1=50 main peak), 4.47 t (1=3.0, CH, in PEG).

Example 4

Activation of PEG 300 with N-succinimidyl carbonate

PEG 300 was dissolved in toluene (13 ml/g of mPEG) . About 25% 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 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 re- dissolve the colorless oil. W-Hydroxy succinimide (NHS) (3.0 mole/mole mPEG.) was added as a solid and then triethylamine

(2.4 mole/mole mPEG) at 0°C. Immediate precipitation of triethylamine hydrochloride (Et₃N.HCl) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et₃N.HCl. 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) HNEt₃Cl. H-NMR (400 MHz,CDCl₃) δ: 1.11 t (1=1.8 CH₃ in NEt₃) , 2.69 s (1=1.39 unreacted NHS), 2.84 s (1= 20.0 succinimide), 3.64 bs (1=113 main peak), 4.44 m (1=10.0, CH, in PEG) .

Example 5

Activation of mPEG 550 with N-succinimidyl carbonate mPEG 550 was dissolved in toluene (9 ml/g of mPEG) . About 10% 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 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 (ΝHS) (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 (Et₃Ν.HCl) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et₃N.HCl. The filtrate was evaporated to dryness under reduced pressure to yield 89 % (mole/mole) of a viscous oil. NMR Indicating >77 % activation and <2 o/o (mole/mole) HNEt₃Cl. 'H-NMR (400MHz) for mPEG 550 succinimidylcarbonate (CDC1₃) δ: 1.41 t (1=4.2 CH₃ in HNEt₃Cl) , 2.69 s (1=24.4 unreacted NHS), 2.84 s (1= 81 succinimide), 3.10 dq (1= 3.7 CH₂ i HNEt₃Cl) , 3.38 s (1=97 CH₃ i OMe),3.64 bs (1=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 («200μl) of activated mPEG 350 with N- succinimidyl carbonate (prepared according to Example 1) , 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 6.0.

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) . 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 9.0- 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 6.0. Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with 50 mM Sodium Borate, 5mM succinic acid, ImM CaCl₂, pH 6.0. Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-anilide) .

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 9.0- 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 6.0. Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with 50 mM Sodium Borate, 5mM succinic acid, ImM CaCl₂, pH 6.0. Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-anilide) .

Example 10 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 9.0-

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 6.0.

Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with

50 mM Sodium Borate, 5mM succinic acid, ImM CaCl₂, pH 6.0.

Compared to the parent enzyme, residual activity was close to

100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-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 30 ml. The reaction was, carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 8.5-

9.0 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 6.0. Reagent excess was removed by untra-filtration using a Filtron-

Ultrasette with lOkD cut-off.

Compared to the parent enzyme, residual activity was close to 100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-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 30 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 8.5-

9.0 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 6.0.

Reagent excess was removed by untra-filtration using a Filtron-

Ultrasette with lOkD cut-off. Compared to the parent enzyme, residual activity was close to

100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-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 8.5-

9.0 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 6.0.

Reagent excess was removed by untra-filtration using a Filtron-

Ultrasette with lOkD cut-off. Compared to the parent enzyme, residual activity was close to

100% towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p- nitro-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 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 6.0.

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 15

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 6.0.

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) -block- poly (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 9.0 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 gel- filtering 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) -block- poly (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 0.5 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 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 84% compared to the parent enzyme.

Example 18

Conjugation of Savinase with poly (ethylene glycol) -block- poly (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 0.5 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 glycol) -block- poly (propylene glycol) -co-poly (ethylene glycol) 12000 bis (succinimidyl carbonate) (75 wt % ethylene glycol).

148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with 0.5 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 9.0 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 gel- filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 140% compared to the parent enzyme.

Example 20

Conjugation of Savinase with poly (ethylene glycol) -block- poly (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 0.5 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 9.0 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 gel- filtering 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°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. 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:

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:

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 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 80°C. The oil phase was poured slowly into the water phase while stirring. The mixture was cooled to apprx. 35°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 lOOg)

MPG 20 g*

H20 ad.lOOg

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 Example 24

Wash performance of PEG-Savinase and EOPO-Savinase

Table 4 : Experimental setup No . 1

Table 5 : Experimental setup No . 2

Detergent Tide powder detergent, 1.0 g/1

Water hardness 6°dH (2:1 Ca/Mg)

Enzymes Protease Concentration

(concentrations Savinase 8.1 x 10^"" M are based on Savinase-PEGlOOObis 1.1 x 10^"" M absorbance measurements at Savinase-PEG2000bis 1.2 x 10^"" M 280 nm) Savinase-PEG4000bis 1.4 x 10^"" M

Savinase-PEG600Obis 1.1 x 10^"" M

Savinase-PEGlOOOObis 1.1 x 10^"" M

Wash time 10 min.

Temperature 25°C

Table 6: Experimental setup No.3

Table 7: Experimental setup No.4

Detergent Model detergent 95, 3.0 g/1

Water hardness 6°dH (EMPA117), 18°dH (grass) (2:1 Ca/Mg)

Enzymes Protease Concentration

(concentrations Savinase 6.8 x 10^"" M are based on Savinase-PEG20Obis 1.7 mg/ml ~ 6.4 x absorbance 10^"5 M measurements at 280 nm) Savinase-PEG300bis 1.6 mg/ml ~ 5.8 x 10^"5 M

Savinase-PEG60Obis 6.5 mg/ml - 2.4 x

Table 8: Experimental setup 5:

Table 9: Experimental setup 6:

Detergent Omo Color, 4 g/1

Table 11: Experimental setup 8:

pH of the detergent solution was adjusted to 10.5 with HCl/NaOH. Water hardness was adjusted by adding CaCl₂ and MgCl₂ 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°C for 5 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. SAS 6.12 software was used to make an analysis of variance and a t-test comparison (Student-Newman-Keuls) at 95% significance on the experimental data.

The wash performance of the different Savinase® variants was evaluated by comparing delta reflectance (DR) values:

DR = Rprotease ^{~ R}Blank

DR: Delta reflectance

R Protease ^; Reflectance of test material washed with conjugated protease

^RBlank^: Reflectance of test material washed with non- conjugated protease

Results

The capital letters designate statistical groupings within each column based on a t-test (SNK, α=0.05). If two are in the same group (same letter) , they cannot be separated statistically.

Table 12:Mean reflectance value and statistics

Table 13 : Mean reflectance values

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

Claims

Patent Claims

1. A polypeptide-polymer conjugate with improved wash performance.

2. The conjugate according to claim 1, wherein the conjugate has reduced allergenicity.

3. The conjugate according to claim 1 or 2, wherein the polypeptide is an enzyme.

4. The conjugate according to any of the proceeding claims, wherein the parent polypeptide has a molecular weight of between

4 kDa and 100 kDa conjugated to a homopolymer with a molecular weight in the range of 0.1 kDa to 60 kDa, preferably in the range of 100 Da to 10,000 Da, especially in the range of 100 Da to 2,000 Da.

5. The conjugate according to claim 4, wherein the parent polypeptide, in particular an enzyme, has a molecular weight from 15 to 60 kDa.

6. The conjugate according to any of claims 1-5, wherein from 1 to 100 polymeric molecules, preferably 4 to 50, especially 5 to 35 polymeric molecules, are coupled covalently to the parent enzyme.

7. The conjugate according to claims 1 to 6, wherein the polymeric molecule is selected from the group comprising a natural or synthetic homo- and heteropolymers .

8. The conjugate according to claim 7, wherein the polymeric molecule is selected from the group comprising synthetic polymeric molecules including branched PEGs, star-shaped PEGs, PEG ethers and PEG esters.

9. The conjugate according to any of the preceeding claims, wherein the polymeric material is selected from a group comprising PEGs with a molecular weight of from 100 to 6,000 Da, preferable 100 to 2,000 Da.

5

10. The conjugate according to claim 7, wherein the polymeric molecule is selected from the group comprising naturally occurring polymeric molecules including dextrans, including car- boxymethyl-dextrans, and celluloses such as methylcellulose,

10 carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenin, pectin and alginic acid.

15

11. The comjugate according to claim 1-3, wherein the polypeptide-polymer conjugate is a graft, block, alternate, or random co-polymer coupled to the surface of the polypeptide.

20 12. The conjugate according to claim 11, wherein the polypeptide-polymer conjugate has coupled one or more polymers covalently to the parent polypeptide, wherein the polymer is characterized by the general formula:

25 EO_xPO_y (I)

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

13. The conjugate according to claims 12, wherein the polymer 30 has a molecular weight from 100 to 100,000 Da, in particular 100 to 50,000 Da, especially 100 to 10,000 Da.

14. The conjugate according to claim 12 to 13, wherein the parent polypeptide has a molecular weight from 1 to 100 kDa, in

35 particular an enzyme, has a molecular weight from 15 to 60 kDa.

15. The conjugate according to any of claims 12 to 14, wherein the block or co-polymer (s) comprise ethylene oxide units (EO) and propylene oxide units (PO) in a ratio in the range from

10:90 or 20:80 or 30:70 or 40:60 or 50:50 or 60:40 or 70:30 or 80:20 or 90:10.

16. The conjugate according to any of claims 12 to 15, wherein from 1 to 100 polymeric molecules, preferably 4 to 50, especially 5 to 35 polymeric molecules, are coupled covalently to the parent enzyme.

17. The conjugate according to any of claims 4 to 10 or 11 to 16, wherein the polypeptide is of microbial origin, such as bacterial, filamentous fungus or yeast origin or of plant origin.

18. The conjugate according to claim 17, wherein the polypeptide is an enzyme from the group of hydrolase, including proteases, including serine proteases, such as subtilisins and metallo proteases, or carbohydrases, or lipases, or oxidoreductases, such as a laccase and haloperoxidases, or superoxide dismutase.

19. The conjugate according to claim 18, wherein the parent protease is selected from the group including PD498, Savinase®, Savinase variant R247K, ProteinaseK, ProteinaseR Thermitase, Subtilisin DY, Lion Y, Alcalase®, ProteinaseT, and JA16.

20. The conjugate according to claim 18, wherein the parent carbohydrase is selected from the group including Novamyl®, Fungamyl®, Natalase®, and Termamyl®.

21. An industrial composition comprising a conjugate according to claim 1-20.

22. The industrial composition according to claims 21, being a detergent, such as a laundry detergent composition, dishwahs composition, or hard surface cleaning composition.

23. The use of a conjugate of any of claims 1-20 for improving the wash performance of industrial compositions according to any of claims 21-22.

24. The use according to claim 23 for reducing the respiratory allergenicity.

25. A method for improving the wash performance of a polypeptide comprising coupling homo-polymers as defined in any of claims 4,

6-10 to a parent polypeptide of any claims 4, 5, 17-20 and/or coupling block or co-polymers as defined in any of claims 11, 12, 13, 15, 16 to a parent polypeptide of any claims 14, 17-20.