AU2933302A - Modified polypeptides with high activity and reduced allergenicity - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority a. a a. a.

a.

Related Art: Name of Applicant: The Procter Gamble Company Actual Inventor(s): David Weisgerger, Donn Nelton Rubingh Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: MODIFIED POLYPEPTIDES WITH HIGH ACTIVITY AND REDUCED ALLERGENICITY Our Ref: 666213 POF Code: 44135/44135 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6006q 1A MODIFIED POLYPEPTIDES WITH HIGH ACTIVITY AND REDUCED ALLERGENICITY This application is a divisional application of Australian Patent Application 85834/98 the entire contents of which are herein incorporated by reference.

TECHNICAL FIELD The present invention relates to modified polypeptides, specifically lipase and protease enzymes, with high activity and reduced allergenicity.

BACKGROUND OF THE INVENTION An increasing number of commercial products containing active polypeptides are becoming available. The majority of these products utilize an enzyme as the polypeptide. Enzymes are polypeptides which react with a compound, or substrate, to break down that compound. Enzymes are divided into numerous classes based on the class of substrate they react upon. Each class of enzyme generally catalyzes the severing of different of chemical bonds resulting in the specific selection of activity.

The lipase class of enzymes are known for their ability to hydrolyze ester bonds created between, but not limited to, hydrocarbons and polyalcohol backbone substrates. Examples of these substrates are mono-, di-, and triglyceride 20 Spolyglycerol esters. The protease class of enzymes are known for their ability to hydrolyze proteins. Naturally occurring and bio-engineered protease enzymes are incorporated into household cleaning detergents to hydrolyze proteinaceous dirt and stains, into personal care products to remove dirt and dead skin, into oral cleansing products to facilitate plaque removal in the mouth, and medicines to affect undesired 25 proteins in the body.

It is known that current commercial cleansing products are made more effective by the incorporation of protease polypeptides. U.S. Patent Number 4,261,868 (Hora et U.S. Patent Number 4,404,115 (Tai), U.S. Patent Number 4,318,818 (Letton et European Patent Application 130,756 (published Jan. 9, 1985) and U.S. Patent 5,030,378 (Venegas) all disclose the use of protease polypeptides in cleansing or detergent products.

It is also realized, however, that polypeptides are potential antigens, and may cause allergic reactions in humans, under certain conditions. The human immune system can produce specific antibodies upon exposure to polypeptides. This process of producing specific antibodies is referred to as "immunization" when a clinically beneficial response is obtained. When the response leads to hypersensitivity, however, it is referred to as "sensitization". Allergenic sensitization to polypeptides has been observed in environments where humans are regularly exposed to the polypeptide. Such environments include manufacturing facilities, where workers can be exposed to uncontrolled dust or aerosol containing a polypeptide, or the marketplace, where consumers' repeated use of products containing polypeptides has, on occasion, caused an allergic reaction.

Presently, allergic responses to polypeptides can be minimized by immobilizing, granulating, coating or dissolving the polypeptides to avoid their becoming airborne. These methods, while addressing consumer exposure to airborne polypeptides, still leave the risks associated with extended tissue contact with the finished product and exposure to enzyme-containing dust or aerosol during manufacturing.

o Another way of diminishing allergic response has been to select polypeptides of human origin. While this approach minimizes allergenicity problems, it is not a 15 complete solution since it is often not possible to find such a polypeptide which also S* has the activity properties desired.

A third proposition for decreasing allergenicity has been to reduce .the size of the polypeptide molecules (see JP Patent Publication Number 4,112,753). However, .size reduction can cause a significant reduction in enzyme activity.

A fourth approach to reduce the allergenicity of polypeptides is through epitope mapping and alteration of the polypeptide amino acid sequence to deliver a polypeptide with reduced allergenicity. This approach usually requires a large investment of development time and money.

In the medical field, suggestions have been made to diminish the 25 immunogenicity of polypeptides through yet another method. This method involves attaching unreactive polymers to the polypeptide. U.S. Patent No. 4,179,337 (Davis, et al.) relates to polypeptides coupled to substantially straight chain polyethylene glycol (PEG) or polypropylene glycol (PPG) polymer moieties. While PEG/PPG coupling was found to mitigate the allergenicity of the polypeptide, only 15% of the physiological activity was maintained. PCT Application WO 96/17929 (Olsen, et al., published June 13, 1996) relates to the modification of polypeptides by conjugating them with suitable polymers. The Olsen application describes modified polypeptides which demonstrate a reduction in allergenicity of from 25% to 66% compared to the parent polypeptide, while maintaining from 39% to 100% of the activity of the parent.

Monfardini, et al, "A Branched Monomethoxypoly(ethylene glycol) for Protein Modification", American Chemical Society, 1995) describes efforts to increase the activity of native polypeptides by conjugating branched monomethoxypolyethylene glycol (mPEG) polymers to the reactive enzyme group. Monfardini, et al. teaches conjugation of enzymes with linear mPEG polymers having molecular weight 5000 KD and branched mPEG polymers having a molecular weight of 5000 KD per branch. Conjugation to ribonuclease, catalase, trypsin and asparaginase is shown.

Enzymatic activity levels of conjugated enzyme are shown to range from 86% to 133% of the activity of the respective parent enzyme. No allergenicity data is presented.

It would be highly desirable to develop an enzyme-based compound which would virtually eliminate allergenic responses while maintaining the desired high levels of enzymatic activity. If this were accomplished it would provide manufacturers and consumers with safer ways to utilize the benefits of enzyme technology.

It is an object of the present invention to provide a modified enzyme compound which delivers this high activity and yet shows reduced stimulation of and resulting activation of the immune system. It is also an object to provide compositions of use of this modified enzyme compound.

SUMMARY OF THE INVENTION The present invention relates to a modified polypeptide which has an enzymatic activity level of greater than about 70% of the parent polypeptide and an allergenic response level of less than about 33% of the parent polypeptide.

Embodiments of the present invention relate to modified polypeptides with reduced allergenicity and high enzymatic activity comprising the formula: SA-Bn wherein A is an enzyme selected from the group consisting of lipase enzymes and protease enzymes, and mixtures thereof; B is a twin polymer moiety, having a total molecular weight of from about 0.5 kilodaltons (KD) to about 40 KD, having the formula R

X

R

2 conjugated to the enzyme; wherein R 1 and R 2 are essentially straight chain polymers, having a molecular weight ranging from about 0.25 KD to about 20 KD; wherein the ratio of the molecular weights ofR 1 and R 2 is from about 1:10 to about 10:1; wherein'X is a linking moiety which links the twin moiety to a single site on the enzyme; and n is the number of twin polymer moieties conjugated to the enzyme, and represents an integer from about 1 to about DETAILED DESCRIPTION OF THE INVENTION The modified polypeptide of the present invention is represented by the formula: A-Bn containing, as essential components, a enzyme, A, and a plurality, n, of twin polymer moieties, B. While not intending to be limited by theory, it is believed that the conjugation of the twin polymer moieties to the enzyme provides a balanced stearic hindrance of the activated surface of the enzyme as to allow for high activity but simultaneously prevent stimulation of the immune system and subsequent antibody formation responsible for allergic reaction.

As used herein, the phrase "amino acid sequence" refers to a specific configuration of the amino acids comprising a polypeptide. The following is a list of S* abbreviations used herein to describe amino acids: Amino Acid Three-letter Abbreviation One-letter Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D S 20 Cysteine Cys C Glutamine Gin Q Glutamic Acid Glu Q .Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V No amino acid at position Xaa As used herein, the term "mutation" refers to the genetic alteration of an organism, which in turn alters the amino acid sequence of the enzyme produced by that organism. The mutation of an enzyme has been often found to alter the properties of the enzyme.

As used herein, the term "wild-type" refers to an enzyme produced by unmutated hosts.

As used herein, the term "variant", means an enzyme having an amino acid sequence which differs from that of the wild-type enzyme due to the genetic mutation of the host producing that enzyme.

As used herein, the term "parent polypeptide" is defined as the enzyme, wildtype or variant, with no additional conjugation of polymer moieties. The activity and allergenicity of the parent polypeptide are usually well known from their development and use in medical and/or consumer products.

The essential components of the present invention, as well as a non-exclusive list of preferred and optional ingredients, are described in detail below.

ENZYME

S An essential component of the present invention is an active enzyme. Any enzyme can be used in the modified polypeptide herein. Preferred enzymes are 15 selected from the group consisting of protease enzymes and lipase enzymes.

Mixtures of proteases and lipases are also included.

Lipase enzymes are 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).

Examples of lipases include lipases derived from the following microorganisms.

The indicated patent publications are incorporated herein by reference: Humicola, 4,810,414) Pseudonomas (WO 89/04361, U.S. 4,950,417, EP 218 272, WO 88/09367, U.S. 5,389,536) 25 Fusarium (EP 130 064, WO 90/09446) Mucor (EP 238 023) Chromobacterium Aspergillus Candida (WO 88/02775, WO 94/01541, WO 89/02916) Geotricum Penicillium Rhizopus Bacillus (WO 91/16422) Specific examples of commercial lipases include Lipolase®, Lipolase T M Ultra, Lipozyme®, Palatase®, Novozym435, Lecitase® (all available from Novo Nordisk LumafastTM and Lipomax (available from Genencor Int., Inc.).

Protease enzymes are classified under the Enzyme Classification number E.G.

3.4 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (TUBMB).

Useful proteases are also described in PCT publications: WO 95/300 10 published November 9, 1995 by The Procter Gamble Company; WO 95/30011 published November 9, 1995 by The Procter Gamble Company; WO 95/29979 published November 9, 1995 by The Procter Gamble Company. Preferred protease enzymes for use in the modified polypeptides herein are subtilisin, chymotrypsin and elastasetype protease enzymes.

Especially preferred for use herein are subtilisin-type protease enzymes.

Subtilisin enzymes are naturally produced by Bacillus alcalophilus, Bacillus ainyloliquefaciens, Bacillus :rnylosaccharicus, Bacillus licheniformis, Bacillus .A particularly preferred substilisin-type enzyme is bacterial serine protease enzyme, and variants thereof, obtained from Bacillus amyloliquefaciens, Bacillus lichen ifonnis and/or Bacillus subtilis, including Novo Industries A/S AlcalaseS, Esperase®& Savinase® (Copenhagen, Denmark), Gist-brocades' Maxatase®, 20 Maxacal® and Maxapemn 1 50 (protein engineered Maxacal®) (Delft, Netherlands), and subtilisin BPN and BPN', which are comnmercially available.

Especially preferred are protease enzymes, and variants thereof, obtained from Bacillus amyloliquefaciens. One known enzyme is BPN. The wild-type BPN' from Bacillus amyloliquefaciens is characterized by the amino acid sequence: 1 10 AlaGin Ser Val Pro TyrGly Val Ser Gln le LysAlaPro AlaLeuHis SerGlnGly :25 30 TyrThrGlySerAsnValLysValAlaVaI Ile AspSerGly le AspSer SerHis Pro AspLeuLysValAlaGlyGlyAla SerMetValPro SerGluThrAsnProPheGlnAsp AsnAsnSerHisGlyThrHis ValAlaGlyThrValAlaAlaLeuAsnAsnSer Ile Gly 100 ValLeuGly Val Ala Pro Ser Ala SerLeuTyrAlaValLysValLeuGlyAlaAspGly 110 120 SerGlyGlnTyrSerTrp Ile le AsnGly le GluTrp Ala le AlaAsnAsnMetAsp, 130 140 Val le AsnMetSerLeuGlyGlyPro SerGlySerAlaAlaLeuLysAlaAlaValAsp 150 160 LysAlaVal Ala SerGlyValValValValAlaAlaAlaGlyAsnGluGlyThrSerGly 170 180 Ser Ser SerThrValGlyTyrProGlyLysTyrPro SerVal Ile Ala VaIGlyAlaVal 190 200 AspSer SerAsnGinArgAlaSerPheSer Ser VaiGlyPro GluLeuAspVaiMetAla 210 220 Pro Gly Val Ser le GlnSerThrLeuProGyAsnLysTyrGlyAlaTyrAsflyThr 230 240 SerMetAla Ser Pro His ValAlaGlyAlaAlaAlaLeu le LeuSerLysHis ProAsn 4 250 260 TrpThrAsnThrGlnValArgSer SerLeuGluAsnThrTbrThrLysLeuGlyAspSer 270 275 PheTyrTyrGlyLysLysGlyLeu le AsnAsnValGlnAlaAlaAlaGil Variants of BPN', hereafter referred to as "Protease are disclosed in U.S.

Patent 5,030,378 (issued to Venegas, July 9, 1991) as characterized by the BPN' amino acid sequence with the following mutations: the Gly at position Glyl166 is replaced with Asn, Ser, Lys, Arg, His, Gin, Ala or Giu; the Gly at position Giy1 69 is replaced with Ser; the Met at position Met222 is replaced with Gin, Phe, Cys, His, Asn, Glu, Ala or Thr; or the Gly at position Glyl66 is replaced with Lys and the Met at position Met222 is replaced with Cys; or the Gly at position Glyl6O is replaced with Ala and the Met at position Met222 is replaced with Ala.

*.20 Additional variants of BPN, heretoforth referred to as "Protease are disclosed by Genencor International, Inc. (San Francisco, California) European Patent EP-B-25 1,446 (granted December 28, 1994 and published January 7, 1988) as **.*characterized by the wild-type BPN' amino acid with the mutations in one or more of the following amino acids: Tyr2l, Tbr22, Ser24, Asp36, Ala 45, Ala48, Ser49, **25 Met5O, His67, Ser87, Lys94, Val95, Gly97, Serl~l, GlylO2, GlylO3, I1el07, GlyllO0, Met 124, Glyl27, Glyl28, Prol29, Leu135, Lys17O, Tyrl'7l, Prol72, Aspl97, Met 199, Ser 204, Lys2l3, Tyr214, Gly215, and Ser22l; or two or more of the amino acids listed above and Asp32, Ser33, TyrlO4, Ala152, Asn155, Glu156, Gly166, Gl169, Phel89, Tyr217, and Met222 wherein both mutations cannot be made on the Asp32, Ser33, TyrlO4, Ala152, Asn155, Glu1.56, Gly166, Gly169, Phel.89, Tyr2 17, and Met222 amino acids.

Another preferred BPN' variant protease, hereafter referred to as "Protease D", is described in WO 95/1 0615 published April 20, 1995 by Genencor International as characterized by the wild-type BPN' amino acid with mutation to position Asn76, in combination with mutations in one or more other amino acid positions selected from the group consisting of Asp99, Serl~l, GlnlO3, TyrlO4, SerlO5, 11e107, AsnlO9, Asn123, Leu126, Gly127, Gly128, Leul135, Glul56, Gly166, Glu195, Aspl97, Ser2O4, Gln206, Pro2lO, Ala2l6, Tyr2l7, Asn2lS, Met222, Ser26O, Lys265, and/or Ala274.

Another preferred BPN' variant protease, hereafter referred to as "Protease F", is described in U.S. Patent Number 4,760,025, issued to Estell, et al. on July 26, 1988 as characterized by the wild-type BPN' amino acid with mutation to one or more amino acid positions selected from the group consisting of Asp32, Ser33, His64, Tyrl04, Asn155, Glu156, Gly166, Glyl69, Phel89, Tyr217, and Met222.

Preferred proteolytic enzymes, then, are selected from the group consisting of Alcalase®, BPN', Protease A, Protease B, Protease D, and Protease F, and mixtures thereof. Protease F is most preferred.

TWIN POLYMER MOIETIES The enzyme employed in the present invention is modified by conjugation of a plurality, n, of twin polymer moieties to the enzyme, wherein n is the average number of moieties conjugated to a polypeptide. The average number of moieties per polypeptide can range from about 1 to about 15, preferably from about 2 to about and more preferably from about 3 to about The twin polymer moiety has a total molecular weight of from about 0.5 KD to *about 40 KD, preferably from about 0.5 KD to about 20 KD, and more preferably from about 1.0 KD to about 10 KD.

The twin polymer moiety of the present invention has the following structure

R

X-

R

2 20 wherein R 1 and R 2 are essentially straight chain polymers having a molecular weight of from about 0.5 kilodaltons (KD) to about 20 KD, preferably from about 1.0 KD to about 10 KD and more preferably from about 2 KD to about 5 KD, and X is a linking moiety which connects the twin polymer moiety to a single site on the enzyme. The ratio of the molecular weights of R 1 and R 2 can range from 1:10 to about 10:1, preferably from 1:5 to about 5:1 and more preferably from 1:3 to about 3:1.

Examples of the suitable polymers which comprise the twin polymer moiety include polyethylene glycols, methoxypolyethylene glycols, polypropylene glycols, polyvinyl alcohols, poly-carboxylates, poly-vinylpyrolidones, poly-D,L-amino acids, dextrans including carboxymethyldextrans, celluloses including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethyl cellulose, carboxyethyl cellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches including hydroxyethyl-starches and hydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers. Mixtures of polymers can also be used to form the twin polymer moiety. The preferred polymer is polyethylene glycol.

Suitable linking moieties can be taken from the class of materials capable of being functionalized appropriately to link two polymer chains, while maintaining functionality to 'reactive groups on desired peptide groups within the enzyme.

Examples of linking moieties and related chemistry are disclosed in U.S. Patent 5,446,090, Harris, issued August 29, 1995; U.S. Patent Number 5,171,264, Merrill, issued December 15, 1992; U.S. Patent 5,162,430, Rhee et al., issued November 1992; U.S. Patent Number 5,153,265, Shadle et al., issued October 6, 1992; and U.S.

Patent 5,122,614, Zalipsky, issued June 16, 1992, all herein incorporated by reference.

Preferable examples of these linking moieties are: a) Twin-polymer-succinimide to couple to Lysine, Tyrosine, Histidine, etc., where an amide or ester linkage is formed: o HN, HN-pEGM b) Twin-polymer-carbodiimidecoupling to Lysine, Tyrosine, Histidine. etc., where an amide or ester linkage is formed 0 N 0 *NH

NH

MPEG' O 2 8 MPEG^ NH 0 2

N

RWa NH HN PEGM HNPEGM c) Twin-polymer-CH20H coupling to Glutamic or Aspartic acid forming an ester linkage 0 MPEG NH H MPEGNH OH O

MPEGNH

IH+ Catalyst d) Twin-polymer-aldehyde coupling to Lysine forming an imine or amine linkage depending on whether reducing agent NaCNBH 3 is used.

The preferred linking moiety, X, is an activated lysine succinimidyl ester of the form C- NH

(CH),

CH

C-NH C-NH-

II

This activated lysine succinidyl ester reacts with the amino acid group of lysine, arginine and histidine peptides of the enzyme. Therefore, the most preferred S structure of the twin polymer moiety of the present invention is o R 1

NH

(CH

2 4 d p CH R C--NH C-NH- The polypeptide of the present invention can also comprise combinations of twin polymer moieties to achieve the activity and reduced allergenicity required.

ENZYMATIC ACTIVITY AND ALLERGENICITY The modified polypeptides of the present invention provides both high enzymatic activity and significantly reduced allergenicity when compared to their respective parent polypeptides. The particular modified polypeptides of the present inventions have an enzymatic activity level of greater than about 70%, preferably greater than about 80%, and more preferably greater than about 90%, of the parent polypeptide as measure by the Enzymatic Activity Method set forth hereinafter in the Analytical Methods Section. Moreover, the particular modified polypeptides of the present inventions have an allergenic response level of less than about 33%, preferably less than about 20%, more preferably less than about 10% and most preferably less than about of the parent polypeptide as measure by the Allergenic Response Method set forth hereinafter in the Analytical Methods Section.

METHOD OF MANUFACTURE In reaction vessel, add polypeptide, in solution of 0.2M Borate buffer at pH. Add one-fourth of the activated twin-polymer, maintaining the reaction temperature at approximately 25 C and let react 30 minutes. Repeat the addition of activated twin-polymer every 30 minutes over a 2 hour period. Buffer exchange through YM30 Amicon setup at 4 0 C with 0.01M KH 2 PO4, 5.5 pH buffer. Remove excess reactants via filtration.

COMPOSITIONS OF USE The modified polypeptides herein can be used in any application which is suitable for the respective parent polypeptide. The modified polypeptides are used at levels of greater than about 0.001%, preferably greater than about 0.01%, and most preferably greater than about 0.1% and at levels less than about 20%, preferably less than about 10%, and most preferably less than For example the modified polypeptides herein can be incorporated into laundry compositions, hard surface cleansing products, light duty cleansing compositions, 15 automatic dishwasher detergent compositions, leave-on and rinse-off hair conditioners, hair shampoos, leave-on and rinse-off facial acne preparations, facial milks and conditioners, shower gels, foaming and non-foaming facial cleansers, cosmetics, hand and body lotions, leave-on facial moisturizers, cosmetic and cleansing wipes, oral cleansing compositions and enzymatic contact lens cleansing 20 solutions. These products are all manufactured using standard procedures using S standard materials known in the respective arts.

Examples of each type of composition are shown in the references below, all herein incorporated by reference.

Personal cleansing compositions 25 Skin cleansers U.S. Patent 5,641,479, Linares et al, issued June 24, 1997; U.S.

Patent Number 5,599,549, Wivell et al., issued February 4, 1997; U.S. Patent Number 5,585,104, Ha et al., issued December 17, 1996; U.S. Patent 5,540,852, Kefauver et al., issued July 30, 1996; and U.S. Patent 5,510,050, Dunbar et al., issued April 23, 1996.

Facial acne preparations U.S. Patent 5,612,324, Guang Lin et al., issued March 18, 1997; U.S. Patent 5,587,176, Warren et al., issued December 24, 1996; U.S.

Patent 5,549,888, Venkateswaran, issued August 27, 1996; and U.S. Patent 5,470,884, Corless et al., issued November 28, 1995.

Shower gels U.S. Patent Number 5,650,384, Gordon et al., issued July 22, 1997; and U.S. Patent 5,607,678, Moore et al., issued March 4, 1997.

Hair conditioners and shampoos U.S. Patent 5,624,666, Coffindaffer et al., issued April 29, 1997; U.S. Patent 5,618,524, Bolich, Jr. et al., issued April 8, 1997; U.S.

Patent 5,612,301, Inman, issued March 18, 1997; U.S. Patent 5,573,709, Wells, issued November 12, 1996; U.S. Patent 5,482,703, Pings, issued January 9, 1996; and U.S. Patent Number Re. 34,584, Grote et al., Reissued April 12, 1994.

Topical skin care compositions Cosmetics U.S. Patent 5,641,493, Date et al., issued June 24, 1997; U.S. Patent 5,605,894, Blank et al., issued February 25,1997; U.S. Patent 5,585,090, Yoshioka et al., issued December 17, 1996.

Hand, face, and body lotions U.S. Patent 4,939,179, Cheney et al., issued July 3, 1990; and U.S. Patent 5,607,980, McAtee et al., issued March 4, 1997.

Cosmetic and cleansing wipes U.S. Patent Number 4,045,364, Richter et al., issued August 30, 1977; European Patent Application, EP 0 619 074, Touchet et al., published October 12, 1994; and U.S. Patent Number 4,975,217, Brown-Skrobot et al., issued December 4, 1990 Laundry cleansing compositions Liquid fabric detergents U.S. Patent 4,261,868, Hora et al., issued April 14, 1981; U.S. Patent 4,404,115, Tai, issued September 13, 1983, U.S. Patent 4,318,818, Letton et al., issued March 9, 1982.

25 Granular fabric detergents U.S. Patent 5,569,645, Dinnewell et al., issued October :29, 1996; U.S. Patent 5,554,587, Scott, issued September 10, 1996; U.S. Patent 5,458,810, Fredj et al., issued October 17, 1995; U.S. Patent 4,379,080, Murphy, issued April 5, 1983; U.S. Patent 4,412,934, Chung et al., issued November 1, 1983.

Other cleansing compositions Oral cleaning compositions (including dentifrice compositions, mouthwashes, lozenges, chewing gum, and denture cleansing tablets) U.S. Patent 5,096,700, Seibel, issued March 17, 1992; U.S. Patent 5,028,414, Sampathkumar, issued July 2, 1991 and U.S. Patent 5,028,415, Benedict, et al., issued July 2, 1991.

13 Enzymatice contact lens cleaning solution U.S. Patent 4,863,627, Davies, et al., September 5, 1989; U.S. Patent Re. 32,672, Huth, et al., reissued May 24, 1988; and U.S. Patent 4,609,493, Schafer, issued September 2, 1986.

Hard surface cleansing products U.S. Patent 4,943,392, Hastedt et al., issued July 24, 1990.

Light duty dish cleansing compositions U.S. Patent Number 5,599,400, Mao et al., issued February 4, 1997; U.S. Patent Number 5,545,354, Ofosu-Asante, issued August 13, 1996; and U.S. Patent Number 5,635,466, Burdon et al., issued June 3, 1997.

Automatic dishwasher detergent compositions U.S. Patent Number 5,616,277, Raleigh et al., issued April 1, 1997; U.S. Patent Number 5,614,485, Painter, issued 15 March 25, 1997; U.S. Patent Number 5,578,136, Taylor et al., issued November 26, 1996; and U.S. Patent Number 5,559,089, Hartman et al., issued September 24, 1996.

*e *o o* ANALYTICAL METHODS ENZYMATIC ACTIVITY METHOD The enzymatic activity of a polypeptide or a modified polypeptide is assayed by measuring the rate of reaction of the polypeptide or modified polypeptide with a substrate.

Substrates For proteases: Enzymatic activity of proteases is measured using the substrate succiny-Ala-Ala-Pro-Phep-Nitroaniline (PNA). Proteases cleave the bond between the peptide and p-nitroaniline to give a visible yellow color absorbing at 410 nm.

For lipases: Enzymatic activity of lipases is measured using the substrate pnitrophenyl carbrilate. Lipases cleave the bond between the caprilate and the pnitrophenyl to give a visible yellow color absorbing at 410 nm.

Equipment: 15 Any calibrated spectrophotometer with the capability to measure the rate of change of absorbance at 410 nm. can be used.

Materials: Buffer Solution: 0.1M Tris (Tris Hydroxy Methyl Amino Methane), 0.01M CaC1 2 pH 8.6.

20 (For example mix 21.7 g.Tris (Tris Hydroxy Methyl Amino Methane), 2.6 g. CaC1 2 2H 2 0 and 1.8 L distilled deionized filtered H 2 0).

Substrate Solution: 20 mg of the appropriate substrate is dissolved into S. ml dimethyl sulfoxide (DMSO).

Polypeptide Solutions: A solution of modified polypeptide and a solution of parent polypeptide having equal polypeptide concentrations as measured by spectrophotometric absorbance at 280 nm.

Working solution: 252.5 pl of substrate solution is diluted up to 25 ml with buffer solution.

Procedure: 1. Mix 10 pil of test polypeptide solution and 990 pl. buffer solution in flask.

2. In separate vessel add 50 pl of solution from step 1. to 950 pl buffer solution.

3. In spectrophotometer flask, add 990 pl of working solution.

4. Add 10 pl of solution from step 2 to spectrophotometer flask. Record the absorbance at 410 nm as a function of the time and ABS/min. The temperature should be controlled (20 25C depending on the protease).

Data and Results The Enzymatice Activity Level is the ratio of the slope of absorbance versus time (Abs/min) of the modified polypeptide to the slope of absorbance versus time of the parent polypeptide and multiplied by 100 to present the activity as a percent of the parent.

ALLERGENIC RESPONSE METHOD The allergenic response of polypeptides is measured utilizing ELISA (Enzyme Linked Immunosorbant Assay) technique. Antibody binding is quantitated for both parent and modified polypeptide with the amount bound for the modified polypeptide, at equal polypeptide concentrations, expressed as a percentage of the amount bound to the parent. Reductions in the percentage of antibody bound to the modified polypeptide is predictive of reduced in-vivo immune response.

Procedure: 15 1. A microtiter plate is coated with 100 LL/well rabbit anti-Enzyme-base antibody (2 jpg/mL in 15mM sodium carbonate, 35mM sodium bicarbonate buffer, pH 9.6) overnight. Unbound coating antibody is washed out with wash buffer NaCI, 13mM Trizma-base, 0.2% BSA, 0.5% Tween 20, pH then 20 blocked one hour with 100 tL/well 2% BSA in water.

2. A series of Enzyme standards ranging from 0.2 20 ng/mL are prepared in sample prep buffer (6.6mM Trizma-base, 0.5M NaC1, 1mM CaC12*2H20, 30mM Na 2 S203, 0.1% BSA, 0.1% Tween 20, pH 3. For each modified Enzyme sample, the parent material (unmodified 25 Enzyme) is required at the same concentration (by protein level) as a reference, as measured by spectrophotometry at 280 nm. The sample and its reference are then diluted equally into sample prep buffer to bring them into the range of the standard curve.

4. Standards, samples and references are added to the coated, blocked and washed plate at 50 tL/well. Sample prep buffer is used for the blank. Then 50 pL/well of a dilute solution of rabbit anti-Enzyme antibody, alkaline phosphatase conjugate in assay buffer NaCI, 50mM Trizma-base, 1.5% BSA, 0.15% Tween 20, pH 8.4) is added. The plate is incubated for 2 hrs. at 37 0 C, then emptied and washed.

P-nitrophenylphosphate substrate solution (1 mg/mL in diethanolamine buffer) is added to the wells at 100 4tL/well. The plate is incubated at 37 0 C until sufficient color has developed, about minutes. Absorbances are read in a microtiter plate reader in the dual wavelength mode at 405 nm with reference wavelength of 620 nm.

6. The net absorbances of the standards are plotted against their concentrations to generate a standard curve. The concentrations of the samples and their references are calculated from the curve. The "percent antibody binding retained" is calculated by dividing the concentration of the sample by the concentration of its reference and multiplying by 100.

EXAMPLES

The following are nonlimiting examples of the modified polypeptides of the present :'invention.

15 EXAMPLE 1 Protease B is conjugated with an average of three (n 3) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 5000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease B and 15 ml of 0.2M Borate, pH 8.5, buffer 20 solution to a reaction vessel. The reaction temperature is maintained at approximately 25 Approximately 240 mg Twin PEG 10K Succinimide (Shearwater Polymers, Inc.) is added to the reaction vessel and reacted for minutes. Three more additions 240 mg Twin PEG Succinimide is made every minutes for a total added of 960 mg. of Twin PEG 10K Succinimide added over a 2 25 hour period. The solution buffers are exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 2 Protease F is conjugated with an average of eight (n 8) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 2000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease F and 15 ml of 0.2M Borate,.pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 OC. Approximately 240 mg Twin PEG 4K Succinimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 240 mg Twin PEG 4K Succinimide is made every 30 minutes for a total added of 960 mg. of Twin PEG 4K Succinimide added over a 2 hour period. The solution buffers are exchanged with 0.01M KH 2 P0 4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 3 Protease F is conjugated with an average of five (n 5) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 2000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease F and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 OC. Approximately 150 mg Twin PEG 4K Succinimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 150 mg Twin PEG 4K Succinimide is made every 30 minutes for a total added of 600 mg. of Twin PEG 4K Succinimide added over a 2 hour period. The solution buffers are exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

.:EXAMPLE 4 Protease A is conjugated with an average of five (n 5) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 20 4000 KD and an activated carbodiimide. The modified polypeptide is prepared by adding of 20 mg Protease A and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 °C.

Approximately 300 mg Twin PEG 8K Carbodiimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 300 mg Twin PEG Succinimide is S. 25 made every 30 minutes for a total added of 1200 mg. of Twin PEG 8K Carbodiimide added over a 2 hour period. The solution buffers are exchanged with 0.01M

KH

2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE Protease F is conjugated with an average of eight (n 8) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 5000 KD and an activated carbodiimide. The modified polypeptide is prepared by adding of 20 mg Protease F and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 OC.

Approximately 640 mg Twin PEG 10K Carbodiimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 640 mg Twin PEG Succinimide is made every 30 minutes for a total added of 2560 mg. of Twin PEG 8K Carbodiimide 18 added over a 2 hour period. The solution buffers are exchanged with 0.01M

KH

2 P0 4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 6 Protease F is conjugated with an average of eight (n 8) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 5000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease F and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 oC. Approximately 640 mg Twin PEG 10K Succinimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 640 mg Twin PEG Succinimide is made every 30 minutes for a total added of 2560 mg. of Twin PEG 10K Succinimide added over a 2 hour period. The solution buffers are S1 exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 7 Protease B is conjugated with an average of three (n 3) twin polymer moieties consisting of two polyethylene glycol moieties, each with a molecular weight of 20 10,000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease B and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 OC. Approximately 480 mg Twin PEG 20K Succinimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 480 mg Twin 25 PEG 20K Succinimide is made every 30 minutes for a total added of 19200 mg. of Twin PEG 10K Succinimide added over a 2 hour period. The solution buffers are exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 8 19 Protease A is conjugated with an average of three (n 3) twin polymer moieties consisting of two polyvinyl alcohol moieties, each with a molecular weight of 20,000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by adding of 20 mg Protease A and 15 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 Approximately 960 mg Twin PVA 40K Succinimide is added to the reaction vessel and reacted for 30 minutes. Three more additions 960 mg Twin PVA Succinimide is made every 30 minutes for a total added of 3840 mg. of Twin PVA 40K Succinimide added over a 2 hour period. The solution buffers are exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

EXAMPLE 9 X Protease B is conjugated with an average of four (n 4) twin polymer moieties, where the moieties are a equal molar mixture of two polyethylene glycol moieties.

One moiety has twin polyethylene glycol moieties, each with a molecular weight of 1000 KD and the other has twin polyethylene glycol moieties, each with a molecular weight of 5000 KD. Both contain an activated lysine succinimidyl ester linking agent. The modified polypeptide is prepared by adding of 20 mg Protease B and 20 ml of 0.2M Borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 Approximately 320 mg of an equal molar mixture of Twin PEG 2K Succinimide and Twin PEG 10K Succinimide (both from Shearwater Polymers, Inc.) is added to the reaction vessel and reacted for minutes. Three more additions of 320 mg of the Twin PEG mixture is made every 25 30 minutes for a total added of 1280 mg. of Twin PEG mixture added over a 2 hour period. The solution buffers are exchanged with 0.01M KH 2

PO

4 5.5 pH buffer and filtered to remove excess reactants.

The following examples further describe and demonstrate embodiments within the scope of the present invention. In the following examples, all ingredients are listed at an active level. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention.

Ingredients are identified by chemical or CTFA name.

EXAMPLES 10-13 Bodvwash Products Ex. 10 Ex. 11 Ex. 12 Ex. 13 55.00 Water Disodium EDTA 0.20 Glycerine 3.00 Polyquatemium 0O 0.40 Sodium/Magnesium Laureth-3-3.6 Sulphate 12.00 Cocamide MEA 2.80 Sodium Lauraphoacetate 6.00 Myristic Acid 1.60 Magnesium Sulphate Hepta Hydrate 0.30 Trihydroxystearin 0.50 PEG-6 Caprylic/Capric Triglycerides 3.00 Sucrose Polyesters of Cottonate Fatty Acid 3.00 Sucrose Polyesters of Behenate Fatty Acid 3.00 15 Petrolatum 0.00 Mineral Oil 0.00 DMDM Hydantoin 0.08 Modified Polypeptides of Example 1 9 0.10 Citric Acid 1.40 (Wgt 55.00 55.00 55.00 0.20 0.20 0.20 3.00 3.00 3.00 0.40 0.40 0.40 12.00 12.00 12.00 2.80 2.80 2.80 6.00 6.00 6.00 1.60 1.60 1.60 0.30 0.30 0.30 0.50 0.50 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.00 0.00 4.00 8.00 0.00 0.00 0.00 6.00 0.08 0.08 0.08 2.00 2.00 5.00 1.40 1.40 1.40 0 10 10 .s.

100.00 100.00 100.00 a Water a1s.

100.00 r EXAMPLES 14-17 Facewash Products Water Disodium EDTA Citric Acid Sodium Laureth-3 Sulfate Sodium Laureth-4 Carboxylate Laureth-12 Polyquaternium 10 Polyquaternium 25 Glycerine Sodium Lauroamphoacetate Lauric Acid Myristic Acid Ex. 14 Ex. 15 Ex. 16 Ex. 17 (Wgt 50.00 50.00 50.00 50.00 0.10 0.10 0.20 0.20 0.00 0.00 1.40 1.40 3.00 3.50 0.00 0.00 3.00 3.50 0.00 0.00 1.00 1.20 0.00 0.00 0.00 0.00 0.40 0.40 0.30 0.30 0.00 0.00 3.00 3.00 3.00 3.00 0.00 0.00 6.00 6.00 6.00 6.00 3.00 3.00 0.00 0.00 3.00 3.00 Magnesium Sulphate Hepta Hydrate 2.30 2.00 2.00 2.00 Triethanol Amine 4.00 4.00 4.00 4.00 Trihydroxystearin 0.50 0.50 0.50 0.50 Sucrose Polyesters of Behenate Fatty Acid 2.00 2.00 0.00 0.00 Sucrose Polyesters of Cottonate Fatty Acid 3.00 2.00 0.00 0.00 PEG-6 Caprylic/Capric Triglycerides 0.00 0.00 0.00 2.00 Petrolatum 0.00 0.00 4.00 0.00 Mineral Oil 0.00 0.00 0.00 2.00 Cocamidopropyl Betaine 2.00 3.00 1.80 1.80 Lauryl Dimethyl Amine Oxide 1.00 1.20 1.20 1.20 Dex Panthenol 1.00 0.25 0.25 0.00 DMDM Hydantoin 0.08 0.08 0.08 0.08 Modified Polypeptide of Examples 1 9 1.00 2.00 0.50 0.50 Fragrance 0.20 0.20 0.20 0.20 15 Water q.s. q.s. qs.

100.00 100.00 100.00 100.00 EXAMPLE 18-19 Leave-on Skin Moisturizing Composition Ex. 18 Ex. 19 20 (Wgt Glycerine 5.00 0.00 Stearic Acid 3.00 0.00 Cl1-13 Isoparaffin 2.00 0.00 Glycol Stearate 1.50 0.00 S 25 Propylene Glycol 0.00 3.00 Mineral Oil 1.00 10.00 Sesame Oil 0.00 7.00 Petrolatum 0.00 1.80 Triethanolamine 0.70 0.00 Cetyl Acetate 0.65 0.00 Glyceryl Stearate 0.48 2.00 TEA Stearate 0.00 2.50 Cetyl Alcohol 0.47 0.00 Lanolin Alcohol 0.00 1.80 DEA-Cetyl Phosphate 0.25 0.00 Methylparaben 0.20 0.20 Propylparaben 0.12 0.10 Carbomer 934 0.11 0.00 Disodium EDTA 0.10 0.00 Modified Polypeptide of Examples 1 9 0.10 Water q.s. q.s EXAMPLE 20 Cleansing Wipe Composition Cleansing composition (Wgt Propylene Glycol 1.00% Ammonium Lauryl Sulfate 0.60% Succinic Acid 4.00% Sodium Succinate 3.20% Triclosan® 0.15% Modified Polypeptide of Examples 1 9 0.05% 15 Water q.s. to 100% The cleansing composition above is impregnated onto a woven absorbent sheet comprised cellulose and/or polyester at about 250% by weight of the absorbent sheet EXAMPLES 21-24 Shampoo 20 Ex.21 Ex.22 Ex.23 Ex. 24 (Wgt Water 50.00 50.00 50.00 50.00 Ammonium Lauryl Sulfate 10.00 10.00 8.00 6.00 Ammonium Laureth Sulfate 4.00 3.00 2.00 2.00 Cocamide MEA 2.00 2.00 2.00 2.00 Ethylene Glycol Distearate 2.00 2.00 2.00 2.00 Cetyl Alcohol 2.00 2.00 2.00 2.00 Stearyl Alcohol 1.20 1.20 1.20 1.20 Glycerin 1.00 1.00 1.00 1.00 Polyquaternium 10 0.50 0.25 0.00 0.00 Polyquatemium 24 0.00 0.00 0.50 0.25 Ammonium Lauryl Sulfate 1.50 1.50 1.50 1.50 Sodium Chloride 0.10 0.10 0.10 0.10 Sucrose Polyesters of Cottonate Fatty Acid 3.00 3.00 0.00 0.00 Sucrose Polyesters of Behenate Fatty Acid 2.00 3.00 0.00 0.00 Polydimethyl Siloxane 0.00 0.00 3.00 2.00 Cocaminopropyl Betaine 0.00 1.00 3.00 3.00 Lauryl Dimethyl Amine Oxide Decyl Polyglucose DMDM Hydantoin Modofied Polypeptides of Examples 1 9 Phenoxyethanol Fragrance Water EXAMPLE 25 Liquid Dish Detergent 1.50 0.00 0.15 2.00 0.50 0.50 100.00 1.50 0.00 0.15 5.00 0.50 0.50 100.00 1.50 1.00 0.15 0.10 0.50 0.50 100.00 1.50 1.00 0.15 5.00 0.50 0.50 100.00 (Wgt. C12 Ethoxy Sulfate 12.00 2-methoxy Undecanoic Acid 4.50 C12 Ethoxy Carboxylate 4.50 Cl 12 Alcohol Ethoxylate 3.00 15 C12 Amine Oxide 3.00 Sodium Cumene Sulfonate 2.00 Ethanol 4.00 Mg++ (as MgC12) 0.20 Ca++ (as CaCl2) 0.40 20 Modified Polypeptide of Example I 9 1.00 Water q.- 100.00 EXAMPLE 26-2 7 Laundry Detergent Powders Ex. 26 C 13 Linear Alkylbenzene Sulfonate 22.0 Phosphate (as sodium tripolyphosphates) 23.0 Sodium Carbonate 23.0 Sodium Silicate 14.0 Zeolite 8.2 2-butyl Octanoic Acid 0.0 Sodium C12-14 Secondary Alkyl Sulfate 0.0 Sodium Citrate 0.0 Optical Brighter 0.0 Diethyaenetriaminepentaacetic acid 0.4 Sodium Sulfate 5.5 Ex. 27 (Wgt 12.0 0.0 0.0 0.0 26.0 0.1 0.0 17.0 Modified Polypeptide of Examples 1 9 3.0 0.2 Water qs q.

100.0 100.0 EXAMPLE 28 bLiguid Laundry Detergent C 13 -C 17 Sodium Paraffin Sulfonate 10.00 Laureth-8 5.00 Sodium Lauroamphodipropionate 5.00 Enzyme 1.00 Ethanol 4.00 Propylene Glycol 6.00 Polyquaternium- 10 0.50 SCitric Acid 2.00 Triethanolamine to pH Perfume 1.00 9*99Modified Polypeptide of Examples 1 9 2.00 *WaterI

S

100.00 EXAMPLE 29-30 Hard Surface Cleaners Ex. 29 Sodium C12 Alkylbenzene Sulfonate 1.95 Sodium C12 Alkyl Sulfate 0.00 Sodium C12 Disthyleneglycol Monohexyl Ether Sulfate C12 Dimethylamine Oxide 0.00 Sodium Cumene Sulfonate 1.30 Hexyl Carbitol 6.30 Modified Polypeptide of Examples 1 9 0.10 Water a.s 100.00 Ex. 0.00 2.20 0.00 2.20 0.50 0.00 6.30 5.00 100.00 4* EXAMPLE 31 Dentifrice Composition 15 Sorbitol (70% aqueous solution) Polyethylene Glycol (MW=600) Silica dental abrasive Sodium Flouride Titanium Dioxide Sodium Saccharin Sodium Alkyl Sulfate (27.9% aqueous sol.) Flavor Carboxyvinyl Polymer 25 Carrageenan Modified Polypeptide of Examples 1 9 Water a.s.

(Wgt 35.0 20.0 0.243 0.286 0.3 0 100.0 EXAMPLE 32 Mouthwash Composition (Wgt SDA 40 Alcohol 8.00 Flavor 0.08 Sodium Fluoride 0.05 Glycerine 10.00 Sweetener 0.02 Benzoic acid 0.05 Sodium hydroxide 0.20 Modified Polypeptide of Examples 1 910.00 Wateras 100.00 EXAMPLE 33 Lozenpe Composition (Wgt Sorbitol 17.50 Mannitol 17.50 Starch 13.60 Sweetener 1.20 Flavor 11.70 Color 0.10 Modified Polypeptide of Examples I 9 0.05 15 Corn syrup Is 100.00 p. a.

a a ,a a. at a. a a a tt a.

a a a a 'a a a a. a at a a a.

ta a a a ta a a aaaa *a a.

at a at a a a.

EXAMPLE 34 Enzymatic Contact Lens Cleaning Solution (Wgt 20 Glucoes 50.00 Nonionic Surfactant 2.00 (Polyoxyethylene-polyoxypropylefle copolymer Anionic Surfactant 1.00 (Polyoxyethlene-alkylphenylether sodium suifricester) 25 Sodium Chloride 1.00 Borax 0.30 Modified Polypeptide of Examples 1 9 1.00 Water -q 100.00

Claims (7)

1. A modified polypeptide characterized in that it has an enzymatic activity level of greater than 70% of the parent polypeptide and an allergenic response level of less than 33% of the parent polypeptide.

2. A modified polypeptide with reduced allergenicity and high activity characterized in that it comprises the formula: A-Bn wherein: A is an enzyme; B is a twin polymer moiety having a total molecular weight of from KD to 40 KD, conjugated to the proteolytic enzyme, having the formula: R/ X *R/ wherein RI and R 2 are essentially straight chain polymers, having a molecular weight ranging from 0.25 KD to 20 KD; wherein the ratio of the molecular weights ofR 1 and R 2 is from 1:10 to 10:1; Sand wherein X is a linking moiety which links the twin polymer moiety to a single site on the enzyme; and c) n is from 1 to

3. A modified polypeptide according to Claim 2 wherein: the enzyme, A. is selected from the group consisting of lipase enzymes and protease enzymes, and mixtures thereof; and wherein the modified polypeptide has an enzymatic activity level of greater than of the parent polypeptide and an allergenic response level of less than 33% of the parent polypeptide.

4. A modified polypeptide according to either of Claim 2 or Claim 3, wherein the enzyme, A, is selected from the group consisting of lipase enzymes and protease enzymes selected from the group consisting of subtilisin, chymotrypsin, and elastase-type enzymes, and mixtures thereof. 29~ were not lodged with this application A q 29 A topical skin care composition comprising greater than 0.001% and less than of the modified polypeptide according to any of the preceding claims.

16. A shower, gel comprising greater than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims.

17. A leave-on skin moisturiser composition comprising greater than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims.

18. A cosmetic composition comprising greater than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims. S S' 19. A cleansing wipe composition comprising a cleansing composition comprising greater than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims. DATED: 28 March, 2002 o PHILLIPS ORMONDE FITZPATRIC Attorneys for: THE PROCTER GAMBLE COMPANY OS