CZ2002211A3 - Protease conjugates with sterically protected epitope regions - Google Patents

Abstract

The present disclosure relates to subtilisin protease conjugate comprising a protease moiety and one or more addition moieties. Each addition moiety is covalently attached to an epitope protection position of the protease moiety. The protease conjugates have decreased immunogenicity relative to a parent protease. The present disclosure further relates to cleaning and personal care compositions comprising the protease conjugates.

Description

Protease conjugate with epitope protected position

Technical area

The present invention relates to a protease conjugate comprising a protease moiety and one or more addition moieties, wherein the protease moiety comprises a first epitope region, a second epitope region and a third epitope region, and each addition moiety is covalently linked to an epitope protected position of the protease moiety. The present invention also describes cleaning and personal care compositions comprising said protease conjugates.

State of the art

Enzymes represent the largest group of naturally occurring proteins. One group of enzymes are proteases, which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins is typically exploited by the inclusion of naturally occurring and genetically engineered proteases in cleaning compositions, particularly those suitable for laundry applications.

In the cleaning field, the most widely used proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms, with some being produced by other organisms such as fungi. See Siezen et al.: Homology Modelling and Protein Engineering Strategies of Subtilases, the Family of Subtilisin-Like Serine Proteases, Protein Engineering 1991, 4(7), pp. 719-737. Unfortunately, the effectiveness of natural proteases in their natural environment is often not optimal in the artificial environment of a cleaning agent. In particular, properties of proteases such as thermal stability, pH stability, oxidative stability, and substrate specificity are not necessarily optimized for use outside the protease's natural environment.

Several approaches have been used to alter the natural amino acid sequence of serine proteases. The goal was to increase the efficiency of the proteases in unnatural laundry environments. These approaches include genetic engineering of the proteases and/or chemical modification of the proteases to increase thermal stability and improve oxidative stability under completely different conditions.

However, since such modified proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause an immunological and/or allergenic response (collectively described herein as an immunogenic response) in mammals.

Furthermore, although genetic design and chemical modification of proteases have been instrumental in the ongoing search for more effective proteases for laundry applications, these proteases have not been commercially exploited in personal care products and low-impact detergents. The primary reason for the absence of these proteases in products such as soaps, gels, body lotions, shampoos, and low-impact dishwashing detergents is the problem of human sensitivity leading to unwanted immunogenic responses. It would therefore be highly advantageous to have a personal care product or low-impact detergent that provides the cleaning properties of proteases without eliciting an immunogenic response.

Currently, the immunogenic response to proteases can be minimized by immobilizing, granulating, coating, or dissolving chemically modified proteases, thereby avoiding airborne transmission. These methods, which expose the consumer to airborne proteases, still carry risks associated with prolonged tissue contact with the final product and worker exposure to dust or aerosol containing proteases during manufacturing.

It has also been proposed that reducing the immunogenicity of proteases can be achieved by attaching polymers to the protease. See, e.g., U.S. Patent No. 4,179,337, Davis et al., issued December 18, 1979, U.S. Patent No. 5,856,451, Olsen et al., Novo Nordisk, issued May 5, 1979.

• · · • ♦ · • · · · · · · · · · · · · January 1999, WO 99/00489, Olsen et al., Novo Nordisk, published January 7, 1999, WO 98/30682, Olsen et al., Novo Nordisk, published July 16, 1998, and WO 98/36026, Von der Osten et al., published August 13, 1998. However, these proposals did not suggest the importance of attaching polymers to amino acid regions of the protease that are responsible for the immune response (i.e., epitopes).

It has recently been discovered that subtilisin protease contains three epitope regions and that attachment of one or more polymers, polypeptides or other moieties to one or more of these regions would effectively significantly reduce the immunogenicity of the protease. See, e.g., U.S. Patent Application Serial No. 09/088,812, Weisgerber et al., The Procter & Gamble Co., filed June 2, 1998.

The present inventors have discovered that steric protection near one or more epitope regions of a protease is an alternative mechanism for preventing or inhibiting epitope presentation and for reducing the immunogenicity of the protease. Accordingly, the present inventors provide modified subtilisins wherein the modification exists in the region of steric proximity to one or more epitope regions. Accordingly, the present inventors have discovered subtilisin proteases that elicit a reduced immunogenic response while still retaining their activity as a potent and active protease. Accordingly, the present protease conjugates are suitable for use in several types of compositions including, but not limited to, laundry detergents, dishwashing detergents, hard surface cleaners, skin care, hair care, beauty care, oral care, and contact lens compositions.

The essence of the invention

The present invention relates to protease conjugates comprising a protease moiety and one or more addition moieties, each addition moiety being covalently linked to an epitope protected position of the protease moiety, wherein,

a) the epitope protected positions for the first epitope region are selected from the group consisting of positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215 and 216 corresponding to subtilisin BPN',

b) the epitope protected positions for the second epitope region are selected from the group consisting of positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145 corresponding to subtilisin BPN', and

c) the epitope protected positions for the third epitope region are selected from the group consisting of positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272 and 275 corresponding to subtilisin BPN', and each additional part independently has the structure

Ri

I x - ,

I r ₂ wherein X is selected from the group consisting of zero and a binding moiety, R ₁ is selected from the group consisting of zero, a first polypeptide, and a first polymer, and R ₂ is selected from the group consisting of zero, a second polypeptide, and a second polymer, wherein at least one of X, R 1 , and R ₂ is not zero.

The protease conjugates of the present invention have reduced immunogenicity relative to the parent protease. Such proteases are therefore suitable for use in several types of compositions including, but not limited to, laundry detergents, dishwashing detergents, hard surface detergents, skin care detergents, hair care detergents, beauty care detergents, oral care detergents, and contact lens detergents.

The following section describes the invention in detail.

The essential components of the present invention are described herein below. Also included herein are non-limiting descriptions of various optional and preferred components useful in embodiments of the present invention.

The present invention may include, consist of, or consist essentially of any required or optional components and/or limitations described herein.

All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated relative to the entire composition unless otherwise indicated.

The amounts of all ingredients or compositions are reported as the active amount of such ingredient or composition and are reported without impurities, for example residual solvents or by-products, which may be present in commercially available sources.

All documents referenced herein, including all patents, patent applications, and publications, are hereby incorporated by reference in their entirety.

Reference is made throughout the specification to trade names for materials including, but not limited to, enzymes. The present inventors do not intend to be limited to the materials under a particular trade name. Equivalent materials (e.g., those obtained from other sources under different names or catalog numbers) may be used for the protease conjugates and compositions of the present invention in addition to those referred to herein by trade name.

As used herein, abbreviations will be used to describe amino acids. Table I shows a list of abbreviations used herein.

• 4 4 4 4444

Table I

amino acid three-letter abbreviation one-letter abbreviation alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamine Gin Q glutamic acid Glu E glycine Gly G histidine His H isoleucine lle 1 leucine Leu L lysine Lys To methionine Meth M phenylalanine Phe F proline For P serine Shit With threonine Thr T tryptophan Suffering w tyrosine Tyr Y valine Val in

The following section of the description provides definitions of the terms used.

The term mutation, as used herein, refers to a change in a gene sequence and/or the amino acid sequence produced by those gene sequences. Among • · · ·

• · « • · · · · · · · · · · · · · · · · · · · · · · mutations include deletions, substitutions, and additions of amino acid residues to the sequence of the natural protein.

The term parent, as used herein, means a protein (native or variant) that is used for further modification to form a protease conjugate of the invention.

The term native, as used herein, refers to a protein, such as a protease or other enzyme, produced by unmutated organisms.

The term variant, as used herein, means a protein that has an amino acid sequence that differs from the corresponding native protein.

All molecular weights of polymers as used herein are expressed as weight average molecular weights.

Although the conjugates of the present invention are not limited to those comprising subtilisin BPN' and variants thereof, all amino acid numbering as used herein is referenced to the amino acid sequence of subtilisin BPN', which is represented by SEQ ID NO: 1.

Ala 1 Gin Shit Val For 5 Tyr Gly Val Shit Gin 10 Ile Lys Ala For Ala 15 Leo His Shit Gin Gly 20 Tyr Thr Gly Shit Asn 25 Val Lys Val Ala Val 30 Ile Asp Shit Gly Ile 35 Asp Shit Shit His For 40 Asp Leo Lys Val Ala 45 Gly Gly Ala Shit Met 50 Val For Shit Glu Thr 55 Asn For Phe Gin Asp 60 Asn Asn Shit His Gly 65 Thr His Val Ala Gly 70 Thr Val Ala Ala Leu 75 Asn Asn Shit Ile Gly 80 Val Leu Gly Val Ala 85 For Shit Ala Shit Leu 90 Tyr Ala Val Lys Val 95 Leo Gly Ala Asp Gly 100 Shit Gly Gin Tyr Shit 105 Suffering Ile Ile Asn Gly 110 Ile Glu

• · · · • ·

Suffering Ala Ile 115 Ala Asn Asn Meth Asp Val 120 Ile Asn Met Ser 125 Leo Gly Gly For Ser 130 Gly Shit Ala Ala Leu 135 Lys Ala Ala Val Asp 14 0 Lys Ala Val Ala Ser 145 Gly Val Val Val Val 150 Ala Ala Ala Gly Asn Glu 155 Gly Thr Shit Gly 160 Shit Shit Shit Thr Val 165 Gly Tyr For Gly Lys Tyr Pro 170 Shit Val Ile 175 Ala Val Gly Ala Val 1Θ0 Asp Shit Shit Asn Gin 185 Arg Ala Ser Phe Shit 190 Shit Val Gly For Glu 195 Leo Asp Val Meth Ala Pro 200 Gly Val Ser Ile 205 Gin Shit Thr Leo For 210 Gly Asn Lys Tyr Gly 215 Ala Tyr Asn Gly Thr 220 Shit Meth Ala Shit For 225 His Val Ala Gly Ala 230 Ala Ala Leu Ile Leu Ser 235 Lys His For Asn 240 Suffering Thr Asn Thr Gin 245 Val Arg Sir Sir Leu Glu Asn 250 Thr Thr Thr 255 Lys Leo Gly Asp Ser 260 Phe Tyr Tyr Gly Lys 265 Gly Leu Ile Asn Val 270 Gin Ala

Ala Ala Gin 275

The amino acid sequence of subtilisin BPN' is further described by Wells et al.: Nucleic Acids Research 1983, //, 7911-7925.

Protease conjugates of the present invention are compounds that comprise a protease moiety and one or more addition moieties, wherein the protease moiety and the addition moiety are linked by a covalent attachment (i.e., a covalent bond).

Protease moieties: Protease moieties according to the invention mean proteases similar to subtilisin, either natural type or variants thereof. The term subtilisin-like protease, as used herein, means such a protease which has • · 4 • · ♦ ·

at least 50%, preferably 80% amino acid sequence identity with the subtilisin BPN' sequences. Natural subtilisin-like proteases are produced, for example, by the microorganisms Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. A discussion of subtilisin-like serine proteases and their homologues can be found in Siezen et al.: Homology Modelling and Protein Engineering Strategies of Subtilases, the Family of Subtilisin-Like Serine Proteases, Protein Engineering 1991, 4(7), 719-737.

Preferred protease moieties for use in the present invention include, for example, those obtained from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis, subtilisin BPN, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K and thermitase including A/C Alcalase ^(R) (commercially available from Novo Industries, Copenhagen, Denmark), Esperase ^(R) (Novo Industries), Savinase ^(R) (Novo Industries), Maxatase ^(R) (commercially available from Genencor International Inc.), Maxacal ^(R) (Genencor International Inc.), Maxapem 15 ^(R) (Genencor International Inc.) and variants of the foregoing proteases. Particularly preferred protease moieties for use in the present invention are those obtained from Bacillus amyloliquefaciens and variants thereof. The most preferred protease moieties in the present invention are subtilisin BPN' and variants thereof.

Particularly preferred variants of subtilisin BPN', hereinafter referred to as Protease A, for use as the parent amino acid sequence of the invention are described in U.S. Patent No. 5,030,378, Venegas, issued July 9, 1991, which are characterized by the amino acid sequence of subtilisin BPN' with the following mutations:

a) Gly at position 166 is substituted with an amino acid residue selected from Asn, Ser, Lys, Arg, His, Gln, Ala and Glu; Gly at position 169 is substituted with Ser and Met at position 222 is substituted with an amino acid residue selected from Gln, Phe, His, Asn, Glu, Ala and Thr, or

b) Gly at position 160 is substituted by Ala and Met at position 222 is substituted by

Alas.

• ·

Further preferred variants of subtilisin BPN', hereinafter referred to as Protease B, for use as the parent amino acid sequence of the invention are described in European Patent No. 251,446, Genencor International, Inc., published January 7, 1988, and characterized by the amino acid sequence of native subtilis BPN' with mutations at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys213, Tyr214, Gly215 and Ser221, or at two or more of the above positions combined with one or more mutations at positions selected from Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.

Other preferred variants of subtilisin BPN' for use in the invention are hereinafter referred to as Protease C, are described in WO 95/10615, Genencor International, Inc., published April 20, 1995, and are characterized by the amino acid sequence of native subtilisin BPN' with a mutation at position Asn76 in combination with mutations at one or more of the other positions selected from Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274.

Other preferred variants of subtilisin BPN' for use in the invention are hereinafter referred to as Protease D, are described in U.S. Patent No. 4,760,025, Estell et al., issued July 26, 1988, and are characterized by the amino acid sequence of native subtilisin BPN' with mutations at one or more of the amino acid positions selected from the group consisting of Asp32, Ser33, His64, Tyr104, Asp155, Glu156, Gly166, Gly169, Phe189, Tyr217, and Met222.

More preferred protease moieties of the invention are selected from the group consisting of subtilisin BPN', Protease A, Protease B, Protease C and Protease D, with Protease D being most preferred.

• · ·

Without wishing to be bound by theory, the protease portions of the invention have three epitope regions: a first epitope region, a second epitope region, and a third epitope region. The first epitope region occurs at positions 70 to 84 corresponding to subtilisin BPN', the second epitope region corresponds to positions 103 to 126 corresponding to subtilisin BPN', and the third epitope region corresponds to positions 220 to 246 corresponding to subtilisin BPN'. See, e.g., U.S. Patent Application Serial No. 09/088,912, Weisgerber et al., The Procter & Gamble Co., filed June 2, 1998, accompanying U.S. Provisional Patent Application Serial No. 60/144,991, Rubingh et al.: Serine Protease Variants Having Amino Acid Substitutions and Deletions in Epitope Regions, filed July 22, 1999, and accompanying U.S. Provisional Patent Application No. 60/144,980, Sikorski et al.: Serine Protease Variants Having Amino Acid Substitutions in Epitope Regions, filed July 22, 1999.

The present inventors have surprisingly discovered epitope protected positions that are in steric proximity to at least one preceding epitope region. It has further been discovered that these epitopes are protected from hydrolysis and thus are exposed to covalent attachment of at least one additional moiety to an amino acid of the protease moiety at the epitope protected position.

The epitope-protected position that is relevant for covalent modification by the addition moieties depends on which epitope requires protection. Most preferably, at least one addition moiety is covalently linked to an epitope-protected position of the first epitope region.

It has been discovered that the epitope protected positions of the first epitope region are positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215 and 216 corresponding to subtilisin BPN'. Preferred epitope protected positions of the first epitope region are positions 1,2, 3, 4, 5, 6, 7,12,17, 40, 41, 43, 67, 86, 87, 89, 206, 209, 214 and 215 corresponding to subtilisin BPN'. The most preferred epitope protected positions of the first epitope region are positions 1, 2, 3, 4, 5, 17, 40, 41, 43, 67, 86, 87 and 214 corresponding to subtilisin BPN'.

• 4

4

4 ·

4 4 4

44444 · 4 ·« ·

It was further discovered that the epitope protected positions for the second epitope region are positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145 corresponding to subtilisin BPN'. Preferably, the epitope protected positions for the second epitope region are positions 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132, 134, 138 and 141 corresponding to subtilisin BPN'.

It has further been discovered that the epitope protected positions for the third epitope region are selected from the group consisting of positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272 and 275 corresponding to subtilisin BPN'. Preferably, the epitope protected positions for the third epitope region are positions 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265 and 275 corresponding to subtilisin BPN'.

In a preferred embodiment of the present invention, the protease portion comprises a modified sequence of the parent amino acid sequence. The parent amino acid sequence may be any of the proteases described above with the same preferred limitations as described above. In this embodiment, the parent amino acid sequence is substituted at one or more residues of the parent amino acid with a substituting amino acid, thereby preparing a protease portion suitable for coupling to one or more of the present addition moieties. According to the present invention, the substitution should be made at one or more epitope protected positions. The epitope protected positions and their preferred limitations are described above.

In order to best achieve selective attachment at one or more epitope protected positions of one or more addition moieties to the protease moiety, the substitution should be made with a substituting amino acid that does not occur in (is unique to) the parent amino acid sequence. In this regard, any substituting amino acid that is unique to the parent amino acid sequence may be used. For example, since the cysteine residue does not occur in the natural ·· · • · ··

amino acid sequence of subtilisin BPN', the substitution of subtilisin BPN' with one or more cysteine residues at one or more epitope protected positions is suitable for the present invention. If the cysteine residue occurs at a position other than the epitope protected position of the parent amino acid sequence, it is preferred to substitute a different amino acid residue at each of these positions to allow selective condensation with one or more addition moieties at the epitope protected position. Cysteine is the most preferred substituting amino acid for substitutions at one or more epitope protected positions.

Other preferred amino acid substitutions include lysine. When the substitution amino acid is lysine, it is preferred to mutate lysine residues that occur at positions other than the epitope protected position of the parent amino acid sequence to another amino acid residue such that the functionalization of one or more lysine residues at the epitope protected position is selective. For example, a lysine residue occurs at position 43 of subtilisin BPN', which is an epitope protected position as defined herein. Site-selective mutation of all other lysine residues occurring in the subtilisin BPN' sequence may be performed, followed by selective functionalization of the lysine residue at position 43 of the addition moiety. Also, amino acid residues at any epitope protected position may be mutated to lysine (for example), followed by selective functionalization at those positions of the addition moiety.

Addition Moieties: The protease conjugates of the present invention comprise one or more addition moieties, each addition moiety being covalently linked to one amino acid residue of an epitope protected site as described herein. An addition moiety may be any chemical structure. Preferably, the addition moiety sterically hinders the epitope protected site to which it is linked or any other epitope protected site as defined herein. Non-limiting examples of addition moieties include organic molecules including, but not limited to, molecules having a molecular weight of less than 1600, preferably less than 800, more preferably less than 400, and most preferably less than 300, polypeptides, and polymers. The term polypeptide, as used herein, means a molecule comprising two or more • · • ···

·· ·· amino acid residues. The term polymer' ¹ as used herein means any molecule that contains two or more identical (preferably five or more identical) monomeric units.

The addition part has the structure

Ri

X-,

I r ₂ wherein X is selected from the group consisting of zero and a binding moiety, R 1 is selected from the group consisting of zero, a first polypeptide, and a first polymer, and R ₂ is selected from the group consisting of zero, a second polypeptide, and a second polymer, wherein at least one of X, R 1 , and R ₂ is not zero.

The protease conjugate preferably contains from 1 to 15, more preferably from 2 to 10, and most preferably from 1 to 5 addition moieties.

When R1 and _R2 independently represent polypeptide moieties or polymer moieties, R1 and _R2 may be identical or different. Preferably, when R1 represents a polypeptide moiety, _R2 is selected from zero and a polypeptide moiety, most preferably it is zero. Most preferably, when R1 represents a polypeptide moiety, _R2 is selected from zero and an identical polypeptide moiety and most preferably it is zero. Preferably, when R1 represents a polymer moiety, _R2 is selected from zero and an identical polymer moiety. Most preferably, when R1 represents a polymer moiety, _R2 is selected from zero and an identical polymer moiety. If at least one of R1 and _R2 represents a first polymer and a second polymer, then X preferably does not represent zero.

Polypeptide moieties: Polypeptide moieties described herein include those comprising two or more amino acid residues. Preferred polypeptide moieties are selected from proteins, including enzymes. Preferred enzymes include proteases, cellulases, • 9

9· 9999 lipases, amylases, peroxidases, microperoxidases, hemicellulases, xylanases, phospholipases _; esterases, cutinases, pectinases, keratinases, reductases (including, for example, NADH reductase), oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccases, transferases, isomerases (including, for example, glucose isomerase and xylose isomerase), lyases, ligases, synthetases and fruit-derived enzymes (including, for example, papain). More preferred enzymes for use as polypeptide moieties include proteases, cellulases, amylases, lipases and fruit-derived enzymes, with proteases being more preferred.

Examples of lipases for use as a polypeptide moiety include those derived from the following microorganisms: Humicola, Pseudomonas, Fusarium, Mucor, Chymobacterium, Aspergillus, Candida, Geotricum, Penicillium, Rhizopus, and Bacillus.

Examples of commercial lipases include Lipolase ^(R) , Lipolase Ultra ^(R) , Lipozyme ^(R) , Palatase ^(R) , Novozym435 ^(R) , and Lecitase ^(R) (all commercially available from Novo Nordisk, Copenhagen, Denmark), Lumafast ^(R) (commercially available from Genencor, Int., Rochester, NY), and lipomax ^(R) (Genencor, Int.).

Examples of proteases for use as the polypeptide moiety include serine proteases, chymotrypsin, and elastase-type enzymes. Most preferred proteases for use as the polypeptide moiety include serine proteases as defined hereinabove in the discussion of protease moieties.

Most preferably, when the polypeptide portion is a serine protease, the polypeptide portion independently has the definition of a protease portion as described hereinabove. Preferably, as described hereinabove, the polypeptide portion has a modified amino acid sequence of a parent amino acid sequence in which there is a modification at one or more epitope protected positions as described hereinabove (wherein the parent amino acid sequence may be referred to as the second parent amino acid sequence). In this case, one of the binding portions (wherein the binding portion is zero) or the protease portions (wherein the binding portion is ...

does not mean zero) is covalently attached to the polypeptide portion by a single substituting amino acid present at one of the epitope protected positions of the polypeptide portion. When the polypeptide portion is a serine protease, the same preferred groupings of epitope protected positions as described above for protease portions and their corresponding parent amino acid sequences apply.

Most preferably, when the polypeptide portion is a serine protease, the polypeptide portion and the protease portion are the same. In this case, the polypeptide portion and the protease portion are most preferably linked by a disulfide bridge, wherein X is zero and most preferably R ₂ is zero.

Polymeric moieties; The addition moieties of the invention may include a polymeric moiety. The term polymeric moiety, as used herein, means any molecule that contains two or more identical (preferably five or more identical) monomeric units. Examples of suitable polymeric moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvinyl pyrrolidones, celluloses, dextrans, starches, glycogen, agaroses, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and chitosan hydrolysates. Preferred polyalkylene oxides include polyethylene glycols, methoxypolyethylene glycols, and polypropylene glycols. Preferred dextrans include carboxymethyldextrans. Preferred celluloses include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose, and hydroxypropylcellulose. Preferred starches include hydroxyethyl starches and hydroxypropyl starches. More preferred polymers are polyalkylene oxides. The most preferred polymer moiety is polyethylene glycol.

When R1 and _R2 independently represent polymer moieties, ^R1 and _R2 preferably have a combined molecular weight (i.e., the molecular weight of R1 plus the molecular weight of _R2 ) of from 200 to 40,000, more preferably from 500 to 40,000, even more preferably from 500 to 20,000, and most preferably from 1,000 to 10,000.

· • * >4 44«· • · « • ♦ ·« •4 444·

When R1 and _R2 represent polymeric moieties, ^R1 and _R2 independently preferably have a molecular weight of from 100 to 20,000, more preferably from 250 to 20,000, even more preferably from 500 to 10,000, and most preferably from 500 to 5,000.

When both R1 and _R2 represent polymeric moieties, the molecular weight ratio of R1 to _R2 is preferably in the range of from 1:10 to 10:1, more preferably from 1:5 to 5:1 and most preferably from 1:3 to 3:1.

When R1 represents a polymer moiety and _R2 represents zero, _R1 preferably has a molecular weight of from 100 to 40,000, more preferably from 500 to 40,000, even more preferably from 500 to 20,000, and most preferably from 1,000 to 10,000.

Binding Moieties: X, as used herein, can be zero or a binding moiety that is optionally covalently linked to one or more polypeptide moieties or to one or more polymeric moieties or to both and is also covalently linked to an amino acid residue at an epitope protected position of the protease moiety. A binding moiety can generally be any small molecule, i.e., a molecule that has a molecular weight of less than 1600, preferably less than 800, more preferably less than 400, and most preferably less than 300. Most preferred binding moieties include those that are capable of covalently linking to a cysteine residue or a lysine residue, most preferably a cysteine residue.

Examples of binding moieties and related chemistry are described in U.S. Patent No. 5,446,090, Harris, issued August 29, 1995; U.S. Patent No. 5,171,264, Merrill, issued December 15, 1992; U.S. Patent No. 5,162,430, Rhee et al., issued November 10, 1992; U.S. Patent No. 5,153,265, Shadle et al., issued October 6, 1992; U.S. Patent No. 5,122,614, Zalipsky, issued June 16, 1992, in Goodson et al.: Site-Directed Pegylation of Recombinant lnterleukin-2 at its Glycosylation Site, Biotechnology 1990, 8(4), 343-346, Kogan: The Synthesis of Substituted Methoxy-Poly(ethylene glycol) Derivatives Suitable for Selective Protein Modification, Synthetic Communications 1992, 22, 2417-2424; and Ishii et al.: Effects of the • Φ** • · • · · « ···

State of the Succinimido-Ring on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled αα-Tropomyosin, Biophysical Journal 1986, 50, 75-80. The most preferred linking moiety is a substituted (e.g., alkyl) or unsubstituted succinimide.

As further examples, the following non-limiting reagents can be used to form the linking moiety: N-[alpha-maleimidoacetoxy]succinimide ester, N-5-azido-2-nitrobenzoyloxysuccinimide, bismaleimidohexane, N-[beta-maleimidopropyloxy]succinimide ester, bis[2-(succinimidyloxycarbonyloxy)-ethyl]sulfone, bissulfosuccinimidyl]suberate, 1,5-difluoro-2,4-dinitrobenzene, dimethyl adipimate.2 HCl, dimethyl pimelimidate.2 HCl, dimethyl suberimidate.2 HCl, disuccinimidyl glutarate, disuccinimidyl suberate, 2-maleimidobenzoyl-N-hydroxysuccinimide ester, N-hydroxysuccinimidyl-4-azidosalicylic acid, N-succinimidyl-6-[4'-azido-2'-nitrophenylamino]hexanoate, N-hydroxysuccinimidyl-2,3-dibromopropionate, succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate, succinimidyl-4-(4-maleimidophenyl)-butyrate, succinimidyl-6-[(beta-maleimidopropionamido)hexanoate, bis[2-(sulfosuccinimidyloxycarbonyloxy)-ethyl]sulfone, N-[gamma-maleimidobutyryloxy]sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-[kappa-maleimidonedecanoyloxy]sulfosuccinimide ester, 3-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl[4-azidosalicylamidohexanoate, sulfosuccinimidyl-7-azido-4-methylcoumarin-3-acetate, sulfosuccinimidyl-6″[4′″azido-2′-nitrophenylamino]hexanoate, sulfosuccinimidyl 4-[4-azidophenyl]butyrate, sulfosuccinimidyl [4-iodoacetyl]aminobenzoate, sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, and sulfosuccinimidyl 4-(4-maleimidophenyl)butyrate. Each of these reagents is commercially available from Pierce Chemical Co., Rockford, IL.

Optional Particles: The protease conjugate may further comprise one or more other chemical structures including (for example) one or more small molecules, polypeptides and/or polymers linked to another protease residue not listed herein, or even to an epitope-protected site not bearing an addition moiety (hereinafter referred to as accessory particles). Accessory particles may include polypeptide particles, polymer particles and binding particles as hereinbefore described.

» · • · · * • ····» • · · ·· · ···· • · • * • · « · · ·· ♦·

«4·· described. Further, for example, one or more polymers (most preferably polyethylene glycol) having a molecular weight of from 100 to 5000, preferably from 100 to 2000, more preferably from 100 to 1000, even more preferably from 100 to 750, and most preferably about 300 may be covalently linked to the protease moiety at residues other than those listed herein. Such polymer particles may be linked directly to the protease moiety at any location on the protease moiety, using methods described herein and well known in the art (including the linker moiety as described herein). Non-limiting examples of polymer conjugation of this optional type are disclosed in WO 99/00849, Olsen et al., Novo Nordisk A/S, published January 7, 1999.

Method of Preparation: Protease particles having substitutions at one or more epitope protected positions (or any other location on the particle) are prepared by mutating the nucleotide sequences that encode the parent amino acid sequence. Such methods are well known in the art; a non-limiting example of one such method is provided below:

Phagemid (pSS-5) containing the native subtilisin BPN' gene (Mitchinson C. and Wells J. A.: Protein Engineering of Disulfide Bonds in Subtilisin BPN', Biochemistry, 1989, 28, 4807-4815.) is transformed into Escherichia coli dut- ung strain CJ236 and a single-stranded uracil-containing DNA template is produced using the helper phage VCSM13 (Kunkel et al.: Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection, Methods in Enzymology 1987, 154, 2B7-382, as modified by Yuckenberg et al.: Site-directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors, Directed Mutagenesis - A Practical Approach, McPherson M. J., ed., pp. 27-48 (1991). Primer-directed mutagenesis modified by the method described in Zoller M. J. and Smith M.: Oligonucleotide - Directed Mutagenesis Using M13 - Derived Vectors: An Efficient and General Procedure for the Production of Point Mutations in any Fragment of DNA, Nucleic Acids Research 1982, 10, 6487-6500, is used to produce all mutants (essentially as reported by Yuckenberg et al., supra).

Oligonucleotides are produced using a 380B DNA synthesizer (Applied Biosystems Inc.). The mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing. The isolated DNA is transformed into Bacillus subtilis expression strain PG632 (Saunders et al.: Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-Amino Acid Fragment of Human Parathyroid Hormone, Gene 1991, 102. 277-282, and Yang et al.: Cloning of the Neutral Protease Gene of Bacillus Subtilis and the Use of the Cloned Gene to Create an in vitro - Derived Deletion Mutation, Journal of Bacteriology 1984, 160, 15-21.

The fermentation is as follows. Bacillus subtilis (PG632) cells containing the protease of interest were grown to mid-log phase in one liter of LB medium containing 10 g/L glucose and inoculated into a Biostat C fermenter (Braun Biotech, Inc., Allentown, Pa.) with a total volume of 9 liters. The fermentation medium contained yeast extract, casein hydrolysate, soluble, partially hydrolyzed starch (Maltrin M-250), antifoaming agent, buffers, and trace materials (see Biology of Bacilli: Applications to Industry, Doi R. H. and M. McGloughlin, eds. (1992)). The medium was maintained at a constant pH of 7.5 throughout the fermentation. Kanamycin (50 μg/ml) was added for antibiotic selection of the mutagenized plasmid. The cells were grown for 18 hours at 37°C to Aeoo 60 and the product was isolated.

The fermentation broth is removed during the following steps to obtain the pure protease. The broth is purified from Bacillus subtilis cells by tangential flow against a 0.16µm membrane. The cell-free broth is then concentrated by ultrafiltration through a 8000 molecular weight cut-off membrane. The pH is adjusted to 5.5 with concentrated MES buffer (2-(N-morpholino)ethanesulfonic acid). The protease is further purified by cation exchange chromatography with S-Sepharose, eluting with a NaCl gradient. See Scopes R. K: Protein Purification Principles and Practice, Springer-Verlag, New York (1984).

• ·

The pNA assay (DelMar et al.: Analytical Biochemistry 1979, 99, 316-320) is used to determine the concentration of active protease in the fractions collected during gradient elution. This assay measures the rate at which 4-nitroanaline is released as the protease hydrolyzes a suitable synthetic substrate, succinyl-alanine-alanine-proline-phenylalanine-4-nitroaniline (sAAPF-pNA). The rate of yellow color production from the hydrolysis reaction is measured spectrophotometrically at 410 nm and is directly proportional to the concentration of the active protease moiety. The absorbance measurement at 280 nm is further used to determine the total protein concentrations. The ratio of active protease/total protein indicates the purity of the protease and is used to identify the fractions to be pooled for the stock solution.

To avoid protease autolysis during storage, an equal weight of propylene glycol is added to the pooled fractions obtained from the chromatography column. After the purification procedure is complete, the purity of the protease stock solution is checked by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and the absolute enzyme concentration is determined by active site titration using the trypsin inhibitor type ll-T: turkey egg white (Sigma Chemical Company, St. Louis, Missouri).

When prepared for use, the protease stock solution is eluted through a Sephadex G25 column (Pharmacia, Piscataway, New Jersey) to remove propylene glycol and exchange the buffer. The MES buffer in the enzyme stock solution is exchanged for 0.01 M sodium dihydrogen phosphate solution, pH 5.5.

The prepared protease can be used for functionalization with one or more addition moieties to form a protease conjugate. The addition moiety precursor (the addition moiety precursor reacts with the protease moiety precursor to form a protease conjugate consisting of the addition moiety and the protease moiety) is preferably activated to increase reactivity with the protease moiety precursor. This activation is well known in the art. Non-limiting examples of methods for preparing a protease conjugate are provided herein below.

Examples of embodiments of the invention

Example 1

O

P h—SH

A protease containing a cysteine residue at one epitope protected position is fused to a polymer moiety according to the above scheme using the following method (where P represents the protease moiety minus the thiol group resulting from the cysteine substitution and n represents the number of repeating monomeric units of polyethylene glycol (e.g. n = 77)).

A variant of subtilisin BPN' is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine for serine at position 3. A concentration of 2 mg of this variant/ml is obtained in phosphate buffer (pH 5.5). The pH is then raised to 7.5 with dilute sodium hydroxide. This variant is mixed with monomethyl polyethylene glycol maleimide in a 25:1 excess of activated polymer to variant. After stirring for one hour at room temperature, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and the mixture is filtered through an ultrafilter with a molecular weight cut-off to remove excess polymer. The concentrate contains the purified protease conjugate.

Example 2

_H3C

OH,ď L ,OΌn (CH ₂ )7 o

y

O^NH /

NH

O

NH

O

n —SH

H,C h ₃ co

oΌY ^H o

O NH (CH ₂ )/

NH

O

NH

A protease moiety containing a cysteine residue at one epitope protected position is fused to a polymer moiety according to the above scheme using the following method (where P represents the protease moiety minus the thiol group resulting from the substitution with cysteine and n represents the number of repeating monomeric units of polyethylene glycol (e.g. n = 77)).

A variant of subtilisin BPN' is prepared with a substitution of leucine for tyrosine at position 217 and a substitution of cysteine for phenylalanine at position 17. A concentration of 2 mg of this variant/ml is obtained in phosphate buffer (pH 5.5). The pH is then raised to 7.5 with dilute sodium hydroxide. This variant is mixed with dimethylpolyethylene glycol maleimide in a 25:1 excess of activated polymer to variant. After stirring for one hour at room temperature, the pH of the mixture is adjusted to • · 4 · (· · · · • 4 · · · · • · · 4 · ·

44444»· · • 4 4 4 * « · · 4 • · · • 44 · · •4 · 44 ·· 44 ····

5.5 and the mixture is filtered through an ultrafilter with a molecular weight cutoff to remove excess polymer. The concentrate contains the purified protease conjugate.

Example 3

The succinimide-protected polymer is selectively fused to lysine at one or more epitope protected positions (wherein MPEG and PEGM are equivalent and represent monomethyl polyethylene glycols and P represents the protease moiety minus the lysine amine group indicated):

pH 8.5

PEGM

Example 4

The carbodiimide-protected polymer is selectively condensed with lysine at one or more epitope-protected positions (wherein MPEG and PEGM are equivalent and represent monomethyl polyethylene glycols, P represents the protease moiety minus said lysine amine group, and X and X' represent side chains that contain the carbodiimide moiety, e.g., alkyls):

• · • · · · • ·

Example 5

A protease moiety containing a cysteine residue at one epitope protected position is fused with an alkylmaleimide using the following method (where P represents the protease moiety minus the thiol group derived from the cysteine substitution and R represents an alkyl group). In this example, R and _R are zero and the linking moiety is derived from the alkylmaleimide.

• · • · * ·* ♦ · ······

A variant of subtilisin BPN' is prepared with a leucine to tyrosine substitution at position 217 and a cysteine to serine substitution at position 86. A 20 ml solution of this variant at a concentration of 1 mg/ml in 0.01 M KH ₂ PO ₄ buffer (pH 7) is prepared. 1.5 equivalents of an alkylmaleimide (e.g. methylmaleimide) is added to this solution. The solution is gently stirred at room temperature for one hour. The resulting protease conjugate is obtained from this solution by standard methods.

Example 6

equivalent

A protease moiety containing a cysteine residue at one epitope protected position will form a dimer using the following method (where P represents the protease moiety minus the thiol group resulting from the cysteine substitution). In this example, the protease moiety and polypeptide moiety are equivalent (and X represents zero).

Analytical methods

The present protease conjugates can be tested for enzymatic activity and immunogenic response using the following two methods known to one skilled in the art. Alternatively, other methods well known in the art can be used.

• · · · «φ φφ • ♦ * » · • φ · ·

Protease Conjugate Activity

The protease activity of the protease conjugate of the present invention can be assayed by methods well known in the art. Two such methods are set forth hereinbelow.

Method for determining skin flake activity

Using Scotch ^(R) #3750G tape, flakes are repeatedly peeled from the skin of a human leg until the tape is essentially matted with flakes. The tape is then cut to a size of 2.5 x 2.5 cm and allowed to lie. To a Petri dish (10 mm by 35 mm) is added 2 ml of 0.75 mg/ml of the control enzyme (e.g., subtilisin BPN') or protease conjugate to be tested in 0.01 M potassium dihydrogen phosphate buffer, pH 5.5. To this solution is added 1 ml of 2.5% sodium laurate solution, pH 8.6. This solution is gently agitated on a horizontal shaker. Pre-prepared squares of tape are allowed to soak in the solution (flaky side up) for 10 minutes with continued gentle agitation. The tape square is then gently rinsed with tap water for 15 seconds. Stevenel Blue Stain (3 ml, commercially available from Sigma Chemical Corp., St. Louis, Mo., as Stevenel Blue Stain) is pipetted into a clean Petri dish. The rinsed tape square is placed in the stain for three minutes (flake side up) with gentle agitation. The tape square is removed from the stain and washed successively in two beakers of 300 ml distilled water for 15 seconds each (one rinse). The tape square is allowed to air dry. The intensity of the color between the tape square obtained from the control enzyme and the tape square obtained from the protease conjugate is compared visually or using a chromometer. When compared to the control enzyme tape square, the protease conjugate tape square showing lower color intensity indicates higher protease conjugate activity.

9 9 • 991

Method for determining activity by collagen staining

Combine 50 ml of 0.1 M Tris buffer (tris-hydroxymethylaminomethane) containing 0.01 M CaCl ₂ to give a pH of 8.5 and 0.5 g of azocoll (collagen impregnated with an azo dye, commercially available from Sigma Chemical Co., St. Louis, Mo.). Incubate this mixture at 25°C with gentle agitation on a horizontal shaker. Filter the mixture through a 0.2 μm needle filter and read the absorbance of this mixture at 520 nm as zero on a spectrophotometer. To the remaining 48 ml of the tris/azocoll mixture is added 1.10 ^-4 % of the control enzyme (i.e., 1 ppm; e.g., subtilisin BPN') or protease conjugate to be tested. Every two minutes for a total of ten minutes, filter 2 ml of the solution containing the control/protease conjugate through a 0.2 μm syringe filter. For each filtered sample, the absorbance is read immediately at 520 nm. The results are plotted against time. The slopes of the control and test conjugate indicate the relative activities of these samples. A higher slope indicates a higher activity. The assay activity of the protease conjugate (slope) can be expressed as a percentage of the activity of the control (slope).

Mouse intranasal immunogenicity test

The immunogenic potential of the protease conjugates of the present invention can be determined using methods known in the art or by the mouse intranasal immunogenicity assay described herein. This assay is similar to the assays described in Robinson et al.: Specific Antibody Responses to Subtilisin Carlsberg (Alcalase) in Mice: Development of an Intranazal Exposure Model, Fundamental and Applied Toxicology 1996, 34, 15-24, and Robinson et al.: Use of the Mouše Intranasal Test (MINT) to Determine the Allergenic Potency of Detergent Enzymes: Comparison to the Guinea Pig Intratracheal (GPIT) Test, Toxicological Science 1998, 43, 39-46, both of which can be used in place of the assay described herein.

Female BDF1 mice (Charles River Laboratories, Portage, Mi.) weighing 18 to 20 grams are used in this test. These mice are quarantined for one week prior to dosing. The mice are placed in cages with wood shavings bedding• · 4 ·

44 • 4 4 «4 · · ·· 4444 us in rooms with controlled humidity (30 to 70% w/w), temperature (19.5-25°C), and a 12-hour light/dark cycle. Mice are fed Purina ^(R) mouse chow (Purina Mills, Richmond, In.) with water ad libitum.

The potential antigen to be tested (either subtilisin BPN' as a positive control or the protease conjugate of the present invention) is administered to a group of five mice. Prior to administration, each mouse is anesthetized by an intraperitoneal (ip) injection of a mixture of Ketaset (88.8 mg/kg) and Rompun (6.67 mg/kg). The anesthetized animal is held in the palm of the hand, back down, and 5 μΙ of protease in buffer (0.01 M KH ₂ PO ₄ , pH 5.5) is administered intranasally. Although each group receives the same dosage, different doses can be tested. The administered solutions are carefully placed on the outside of each nostril and allowed to be inhaled by the mouse. The administration is repeated on days 3, 10, 17, and 24.

Sera samples are collected on day 29. Enzyme-specific IgG1 antibody in mouse serum is measured by antigen capture ELISA. The immunogenicity of the protease conjugate can be compared to subtilisin BPN' using standard ED50 values.

Compositions of the present invention

The protease conjugates of the present invention can be used in any application that is suitable for the respective parent protease. One such example is cleaning compositions. Due to the desirable properties of reduced immunogenicity of the present protease conjugates, the protease conjugates can further be used in applications for which the use of proteases has historically been minimally advantageous. Examples of such applications include those in which the protease conjugate necessarily comes into close contact with mammalian skin (particularly human skin), such as in the use of personal care products.

* · · · · · ·

Cleaning products

The present compositions may be used in cleaning compositions including, but not limited to, laundry detergents, hard surface cleaners, low-impact cleaning compositions including dishwashing compositions, and automatic dishwashing detergent compositions.

The cleaning compositions of the present invention comprise an effective amount of one or more protease conjugates of the present invention and a cleaning composition carrier.

The term effective amount of protease conjugate or the like, as used herein, means that amount of protease conjugate that is necessary to achieve the proteolytic activity required in a specific cleaning composition. Such effective amounts can be readily determined by one skilled in the art and are determined by many factors, such as the particular protease conjugate being used, the cleaning application, the specific composition of the cleaning composition, whether a liquid or dry (e.g., granular, cube) composition is desired, and the like. The cleaning compositions preferably contain from 0.0001% to 10%, more preferably from 0.001% to 1%, and most preferably from 0.01% to 0.1% of one or more protease conjugates of the present invention. Several examples of various cleaning compositions in which proteases may be used are discussed in detail below.

In addition to the present protease conjugates, the present cleaning compositions further comprise a cleaning composition carrier comprising one or more cleaning composition materials compatible with the protease conjugate. The term cleaning composition material, as used herein, means any material selected for the type of cleaning composition desired and the product form (e.g., liquid, granule, bar, spray, stick, paste, gel), wherein such materials are also compatible with the protease conjugate used in the composition. The specific selection of cleaning composition materials is readily made based on consideration of the surface of the material to be • « · · · ·

·· ♦ · ♦

purified, in the desired composition form for the cleaning conditions during use (e.g., when used as a laundry detergent). The term compatible, as used herein, means a cleaning composition material that does not reduce the proteolytic activity of the protease conjugate to such an extent that the protease is not as effective as desired during normal use situations. Examples of specific cleaning composition materials are detailed herein below.

The protease conjugates of the present invention can be used in a variety of detergent compositions where high sudsing and good cleaning are desired. The protease conjugates can therefore be used with a variety of conventional additives to provide fully formulated hard surface cleaners, dishwashing detergents, fabric washing detergents and the like. These compositions can be in the form of liquids, granules, bars and the like. These compositions can be formulated as concentrated detergents containing from up to 30% to 60% by weight of surfactants.

The cleaning compositions of the invention may optionally, and preferably, contain various surfactants (e.g., anionic, nonionic, or zwitterionic surfactants). These surfactants are typically present in an amount of from 5% to 35% by weight of the compositions.

Non-limiting examples of surfactants useful in the present invention include conventional C11-C18 alkylbenzene sulfonates and primary and random alkyl sulfates, C10-C18 secondary (2,3) alkyl sulfates of the general formulae _CH3 ( _CH2 ) _X ( _CHOSO3 )'M ⁺ )CH3 and CH3(CH2)y(CHOSO3)'M ⁺ ) _CH2CH3 , in which x and (y+1) are at least 7, preferably at least 9, and M is a water-solubilizing cation, especially sodium, C10-C18 alkyl alkoxy sulfates (especially EO1-5 ethoxy sulfates), C10-C18 alkyl alkoxy carboxylates (especially EO1-5 ethoxy carboxylates), C10-C18 alkyl polyglycosides, and their corresponding sulfated polyglycosides, α-sulfonated fatty acid esters of 12 to 18 carbon atoms, alkyl of 12 to 18 carbon atoms and alkylphenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy), betaines of 12 to 18 carbon atoms and sulfobetaines (sultaines), amine oxides of 10 to 18 carbon atoms and the like. Preferred according to the invention are alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC). The use of such surfactants in combination with amine oxide and/or betaine or sultaine surfactants is also advantageous, at the discretion of the formulator. Other conventionally useful surfactants are listed in conventional texts. Particularly useful surfactants include the N-methyl glucamides of 10 to 18 carbon atoms described in U.S. Pat. No. 5,111,111. 5,194,639, Connor et al., issued March 16, 1993.

Various other ingredients useful in detergent cleaning compositions may be included in the compositions of the invention, including, for example, other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, and solvents for liquid formulations. If additional foaming is desired, foaming agents such as C10-16 alkanolamides may be included in the compositions, typically in amounts of 1% to 10% by weight. Monoethanol and C10-14 diethanolamides illustrate a typical group of such foaming enhancers. It is also preferred to use such foaming enhancers with high foaming co-surfactants such as the aforementioned amine oxides, betaines, and sultaines. If desired, soluble magnesium salts, such as magnesium chloride, magnesium sulfate, and the like, may be added in amounts typically from 0.1% to 2% by weight to provide additional foaming.

The liquid detergent compositions of the invention may contain water and other solvents as carriers. Primary or secondary alcohols of low molecular weight are suitable, examples of which are methanol, ethanol, propanol and isopropanol. Monohydric alcohols are preferred for solubilizing the surfactants, but polyols such as those containing from 2 to 6 carbon atoms and from 2 to 6 hydroxyl groups (e.g. 1,3-propanediol, ethylene glycol, glycerin and 1,2-propanediol) may also be used. These compositions may contain from 5% to 90%, typically from 10% to 50% by weight of such carriers.

♦ 4 ··♦·

The detergent compositions of the invention will preferably be formulated so that during use in aqueous cleaning operations the wash water has a pH between 6.8 and 11. The final products are therefore formulated to typically be within this range. Methods of adjusting the pH for recommended use values include, for example, the use of buffers, alkalis and acids. These methods are well known to those skilled in the art.

When formulating hard surface cleaning and fabric cleaning compositions of the present invention, the formulator may wish to use a variety of building blocks in amounts from 5% to 50% by weight. Typical building blocks include 1 to 10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. A list of other conventional building blocks is provided in the usual formulations.

Similarly, the formulator may wish to use various other enzymes in the compositions, such as cellulases, lipases, amylases, and proteases, typically in amounts from 0.001% to 1% by weight. Various cleaning and fabric care enzymes are well known in the laundry detergent art.

Various bleaching compounds such as percarbonates, perborates and the like may be used in such compositions, typically in amounts from 1% to 15% by weight. If desired, these compositions may also contain bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate and the like, which are also known in the art. The amounts used are typically in the range of from 1% to 10% by weight.

Soil release agents, especially of the anionic oligoester type, chelating agents, especially aminophosphonates and ethylenediamide succinates, clay soil release agents, especially ethoxylated tetraethylenepentamine, dispersing agents, especially polyacrylates and polyaspartates, brighteners, especially anionic brighteners, suds suppressors, especially silicones and secondary alcohols, fabric softeners, especially smectite clays and the like, may be used in these compositions in amounts ranging from 1% to 35% by weight. Standard codes and published patents contain many detailed descriptions of such conventional materials.

Enzyme stabilizers may also be used in cleaning compositions. These enzyme stabilizers include propylene glycol (preferably from 1% to 10% by weight), sodium formate (preferably from 0.1% to 1% by weight), and calcium formate (preferably from 0.1% to 1%).

The variants herein are useful in hard surface cleaning compositions. The term hard surface cleaning composition as used herein refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tiles and the like. Hard surface cleaning compositions of the present invention comprise an effective amount of one or more protease conjugates of the present invention, preferably from 0.001% to 10%, more preferably from 0.01% to 5%, even more preferably from 0.05% to 1% by weight of the protease conjugate by weight of the composition. In addition to comprising one or more protease conjugates, these hard surface cleaning compositions typically comprise a surfactant and a water-soluble scavenger builder. However, in some specialty products such as window cleaning sprays, surfactants are sometimes not used because they can produce a filmy and/or streaky residue on the glass surface.

The surfactant component, if present, may constitute as little as 0.1% by weight of the compositions of the invention, but typically such compositions will contain from 0.25% to 10%, more preferably from 1% to 5% by weight of surfactant.

The compositions will typically contain from 0.5% to 50% detergent builder, preferably from 1% to 10% by weight.

··· ·

99

9 9 9 ·9· ·· 9 9 9 9 9 9

9999 · · 9 999 9 9

The pH should be in the range of 7 to 12. If pH adjustment is necessary, conventional pH adjusting agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used.

Solvents may be included in the compositions. Useful solvents include, but are not limited to, glycol ethers such as diethylene glycol monohexyl ether, diethylene glycol monobutyl ether, ethylene glycol monobutyl ether, ethylene glycol monohexyl ether, propylene glycol monobutyl ether, and dipropylene glycol monobutyl ether, and diols such as 2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, such solvents are typically present in amounts from 0.5% to 15%, more preferably from 3% to 11% by weight.

Highly volatile solvents such as isopropanol or ethanol may also be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after full strength application of the composition to the surface. When used, volatile solvents are typically present in amounts of from 2% to 12% by weight of the compositions.

Examples 7 to 12

Liquid hard surface cleaners (amounts of ingredients are given in % by weight) ·· ·*»·

ex.7 ex.8 ex.9 ex.1O ex.11 ex.12 protease conjugate from ex. 3 0.05 0.50 0.02 0.03 0.30 0.05 EDTA - - 2.90 2.90 - - sodium citrate - - - - 2.90 2.90 alkyl(with 12 carbon atoms)benzene- sodium sulfonate 1.95 - 1.95 - 1.95 - C12 alkyl sulfate sodium - 2.20 - 2.20 - 2.20 12-carbon ethoxysulfate sodium - 2.20 - 2.20 - 2.20 12-membered dimethylamine oxide carbon - 0.50 - 0.50 - 0.50 sodium cumenesulfonate 1.30 - 1.30 - 1.30 - hexylcarbitol 6.30 6.30 6.30 6.30 6.30 6.30 water 90.4 88.3 87.53 85.87 87.25 85.85

All preparations are adjusted to a pH of 7.

In another embodiment of the present invention, dishwashing compositions comprise one or more variants of the present invention. Dishwashing composition, as used herein, refers to all forms of dishwashing compositions including, but not limited to, granular and liquid forms.

Examples 13 to 16

Liquid dishwashing detergent (amounts of ingredients are given in % by weight) ·· · • · · · · · · · · 9 * • ······· 9

9 9 9 9

9 9 9 ·· ·«**

99·9

ex.13 ex.14 ex.15 ex. 16 protease conjugate from example 1 0.05 0.50 0.02 0.40 N-methylglucamide with 12 to 14 carbon atoms 90 0.90 0.90 0.90 12-carbon ethoxy(1)sulfate 12.0 12.0 12.0 12.0 2-methylundecanoic acid 4.50 4.50 4.50 4.50 ethoxy(2)carboxylate with 12 carbon atoms 4.50 4.50 4.50 4.50 alcohol ethoxylate(4) with 12 carbon atoms 3.00 3.00 3.00 3.00 amine oxide with 12 carbon atoms 3.00 3.00 3.00 3.00 sodium cumenesulfonate 2.00 2.00 2.00 2.00 ethanol 4.00 4.00 4.00 4.00 Mg ²⁺ (as MgCl ₂ ) 0.20 0.20 0.20 0.20 Ca ²⁺ (as CaCl ₂ ) 0.40 0.40 0.40 0.40 water 65.45 65 65.48 65.1

All preparations are adjusted to pH 7.

Examples 17 to 19

Liquid detergents for cleaning fabrics (amounts of ingredients are given in % by weight) *· * • 9 9

9 9 9

9 ··♦*

9 9 9

9 99 ex.17 ex.18 ex. 19

protease conjugate from example 4 0.05 0.03 0.30 sodium alkyl sulfate with 12 to 14 carbon atoms 20.0 20.0 20.0 2-Butyloctanoic acid 5.0 5.0 5.0 sodium citrate 1.0 1.0 1.0 alcohol ethoxylate(3) with 10 carbon atoms 13.0 13.0 13.0 monoethanolamine 2.50 2.50 2.50 water/propylene glycol/ethanol (100:1:1) 58.45 58.47 58.20

Personal care products

The present protease conjugates are particularly suitable for use in personal care products, such as leave-in or rinse-out hair conditioners, shampoos, leave-in or rinse-out acne products, facial lotions and conditioners, shower gels, soaps, foaming and non-foaming facial cleansers, cosmetics, hand, face and body washes, moisturizers, patches and masks, leave-in facial moisturizers, cosmetic and cleansing pads, oral care products, menstrual products and contact lens care products. The present personal care products comprise one or more protease conjugates of the present invention and a personal care carrier.

By way of illustration, the present protease conjugates are suitable for inclusion in the compositions described in the following references: U.S. Patent No. 5,641,479, Linares et al., issued June 24, 1997 (skin cleansing compositions), U.S. Patent No. 5,599,549, Wivell et al., issued February 4, 1997 (skin cleansing compositions), U.S. Patent No.

• * · • ♦ · · · 9 • · ···· • · · ·· · t* ··«· • · « • « · • · 9 • · · · · 99

99 • 9 9 9

9 9

9 9

9·9

9999

585,104, Ha et al., issued December 17, 1996) (skin cleansing compositions), U.S. Patent No. 5,540,852, Kefauver et al., issued July 30, 1996 (skin cleansing compositions), U.S. Patent No. 5,510,050, Dunbar et al., issued April 23, 1996 (skin cleansing compositions), U.S. Patent No. 5,612,324, Guang Lin et al., issued March 18, 1997 (anti-acne compositions), U.S. Patent No. 5,587,176, Warren et al., issued December 24, 1996 (anti-acne compositions), U.S. Patent No. 5,549,888, Venkateswaran, issued August 27, 1996 (anti-acne compositions), U.S. Patent No. 5,470,884, Corless et al., issued November 28, 1995 (anti-acne preparations), U.S. Patent No. 5,650,384, Gordon et al., issued July 22, 1997 (shower gels), U.S. Patent No. 5,607,678, Moore et al., issued March 4, 1997 (shower gels), U.S. Patent No. 5,624,666, Coffindaffer et al., issued April 29, 1997 (hair conditioners and/or shampoos), U.S. Patent No. 5,618,524, Bolich et al., issued April 8, 1997 (hair conditioners and/or shampoos), U.S. Patent No. 5,612,301, Imman, issued March 18, 1997 (hair conditioners and/or shampoos), U.S. Patent No. 5,573,709, Wells, issued November 12

1996 (hair conditioners and/or shampoos), U.S. Patent No. 5,482,703, Pings, issued January 9, 1996 (hair conditioners and/or shampoos), reissued U.S. Patent No. 34,584, Grote et al., reissued April 12, 1994 (hair conditioners and/or shampoos), U.S. Patent No. 5,641,493, Dáte et al., issued June 24, 1997 (cosmetics), U.S. Patent No. 5,605,894, Blank et al., issued February 25, 1997

1997 (cosmetics), U.S. Patent No. 5,585,090, Yoshioka et al., issued December 17, 1996 (cosmetics), U.S. Patent No. 4,939,179, Cheney et al., issued July 3, 1990 (washes for hands, face and/or body), U.S. Patent No. 5,607,980, McAtee et al., issued March 4, 1997 (washes for hands, face and/or body), U.S. Patent No. 4,045,364, Richter et al., issued August 30, 1997 (cosmetic and cleansing pads), European Patent Application 0 619,074, Touchet et al., published October 12, 1994 (cosmetic and cleansing pads), U.S. Patent No. 4,975,217, Brown-Skrobot et al., issued December 4, 1990 (cosmetic and cleansing pads), U.S. Patent No. 5,096,700, Seibel, issued March 17, 1992 (oral cleansers), U.S. Patent No. 5,028,414, Sampathkumar, issued July 2, 1991 (oral cleansers), U.S. Patent No. 5,028,415, Benedict et al., issued July 2, 1991 (oral cleansers), U.S. Patent No. 5,028,415, Benedict et al., issued July 2, 1991 • 9 • 9 • 9 · • 9 4 • · · 9 • · 9 ···

9 9 • · · • 9 9

9 9 9 • 9 9 9

9* 99 »·> *···

1991 (oral cleansers), U.S. Patent No. 4,863,627, Davies et al., September 5, 1989 (contact lens cleaning solution), Reissue U.S. Patent No. 32,672, Huth et al., May 24, 1988 (contact lens cleaning solutions), and U.S. Patent No.

609,493, Schafer, issued September 2, 1986 (contact lens cleaning solutions).

To further illustrate the oral cleaning compositions of the present invention, compositions useful for removing protein stains from teeth and dentures include a pharmaceutically acceptable amount of one or more protease conjugates of the present invention. The term oral cleaning compositions, as used herein, includes dentifrices, toothpastes, dental gels, tooth powders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylactic pastes, dental medicated solutions, and the like. The oral cleaning compositions preferably comprise from 0.0001% to 20% of one or more protease conjugates of the present invention, more preferably from 0.001% to 10%, even more preferably from 0.01% to %wt. of the composition, and a pharmaceutically acceptable carrier. The term pharmaceutically acceptable, as used herein, means that the drugs, medicinal preparations or inert ingredients described by the term are suitable for use in contact with human and lower animal tissues without undue toxicity, incompatibility, instability, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio.

The pharmaceutically acceptable carrier components of the oral cleansing composition will typically generally constitute from 50% to 99.99%, preferably from 65% to 99.99%, more preferably from 65% to 99% by weight of the composition.

Pharmaceutically acceptable carrier ingredients and optional ingredients that may be included in the oral cleansing compositions of the present invention are well known to those skilled in the art. Various types of compositions, carriers and optional ingredients useful in oral cleansing compositions are described in the references cited hereinabove.

*· · • · 4 • · · <

• · ···· • · · ·· · «··· • » · ♦ · · • · · · • · · · · · • · ·

Φ Φ · · • · · • Φ Φ • Φ · ····

In another embodiment of the present invention, compositions for cleaning dentures outside the oral cavity comprise one or more protease conjugates of the present invention. Such denture cleaning compositions comprise an effective amount of one or more protease conjugates, preferably in an amount of from 0.0001% to 50%, more preferably from 0.001% to 35%, even more preferably from 0.01% to 20% by weight of the composition and the denture cleaning carrier. Various types of denture cleaning compositions, such as effervescent tablets and the like, are well known in the art (see, e.g., U.S. Patent No. 5,055,305, Young) and are generally suitable for including one or more protease conjugates for removing protein stains from dentures.

In another embodiment of the present invention, contact lens cleaning compositions comprise one or more protease conjugates of the present invention. These contact lens cleaning compositions comprise an effective amount of one or more protease conjugates, preferably from 0.01% to 50% of one or more protease conjugates, more preferably from 0.01% to 20%, even more preferably from 1% to 5% by weight of the composition, and a contact lens cleaning carrier. Various types of contact lens cleaning compositions, such as tablets, liquids, and the like, are well known in the art and are generally suitable for incorporating one or more protease conjugates of the present invention for removing protein stains from contact lenses.

Examples 20 to 23

Contact lens cleaning products (amounts of ingredients are given in % by weight)

ex.2O ex.21 ex.22 ex.23 protease conjugate from example 5 0.01 0.5 0.1 2.0 glucose 50.0 50.0 50.0 50.0 nonionic surfactant (copolymer polyoxyethylene/polyoxypropylene) 2.0 2.0 2.0 2.0 anionic surfactant (sodium salt polyoxyethylene-alkylphenyl ether- -sulfric ester) 1.0 1.0 1.0 1.0 sodium chloride 1.0 1.0 1.0 1.0 borax 0.30 0.30 0.30 0.30 water 45.69 45.20 45.60 43.70

• · ···<

Examples 24 to 27

Body wash products (ingredient amounts are given in % by weight)

ex.24 ex.25 ex.26 ex.27 water 62.62 65.72 57.72 60.72 disodium EDTA 0.2 0.2 0.2 0.2 glycerin 3.0 3.0 3.0 3.0 Polyquatemium 10 0.4 0.4 0.4 0.4 sodium laureth sulfate 12.0 12.0 12.0 12.0 coco amide MEA 2.8 2.8 2.8 2.8 sodium laurofoacetate 6.0 6.0 6.0 6.0 myristic acid 1.6 1.6 1.6 1.6 MgSO ₄ heptahydrate 0.3 0.3 0.3 0.3 trihydroxystearin 0.5 0.5 0.5 0.5 PEG-6 caprylic/capric triglycerides 3.0 - - - sucrose polyesters cotton fatty acids 3.0 - - - sucrose polyesters behen fatty acids 3.0 - 4.0 - vaseline - 4.0 8.0 - mineral oil - - - 6.0 DMDM hydantoin 0.08 0.08 0.08 0.08 protease conjugate from example 6 0.1 2.0 2.0 5.0 citric acid 1.40 1.40 1.40 1.40

Examples 28 to 31

Facial wash products (amounts of ingredients are given in % by weight) ·· ·· • ♦ · · • · · • · · • · * • · · · «

ex.28 ex.29 ex.30 ex.31 water 66.52 65.17 68.47 68.72 disodium EDTA 0.1 0.1 0.2 0.2 citric acid - - 1.4 1.4 sodium laureth-3-sulfate 3.0 3.5 - - sodium laureth-4-carboxylate 3.0 3.5 - - laureth-12 1.0 1.2 - - Polyquatemium 10 - - 0.4 0.4 Polyquatemium 25 0.3 0.3 - - glycerin 3.0 3.0 3.0 3.0 sodium lauroamphoacetate - - 6.0 6.0 lauric acid 6.0 6.0 3.0 3.0 myristic acid - - 3.0 3.0 MgSO ₄ heptahydrate 2.3 2.0 2.0 2.0 triethanolamine 4.0 4.0 4.0 4.0 trihydroxystearin 0.5 0.5 0.5 0.5 sucrose polyesters behen fatty acids 2.0 2.0 - - sucrose polyesters cotton fatty acids 3.0 2.0 - - PEG-6 caprylic/capric triglycerides - - - 2.0 vaseline - - 4.0 - mineral oil - - - 2.0 coco amidopropyl betaine 2.0 3.0 1.8 1.8 lauryl dimethylamine oxide 1.0 1.2 1.2 1.2 Dex Panthenol 1.0 0.25 0.25 -

• · · · • ·

DMDM hydantoin 0.08 0.08 0.08 0.08 protease conjugate from example 2 1.0 2.0 0.5 0.5 scent 0.2 0.2 0.2 0.2

• ·

Examples 32 to 33

Leave-in moisturizer (ingredient amounts are given in % by weight)

ex.32 ex.33 glycerin 5.0 - stearic acid 3.0 - isoparaffin with 11 to 13 carbon atoms 2.0 - glycol stearate 1.5 - propylene glycol - 3.0 mineral oil 1.0 10.0 sesame oil - 7.0 vaseline - 1.8 triethanolamine 0.7 - cetyl acetate 0.65 - glyceryl stearate 0.48 2.0 TEA stearate - 2.5 cetyl alcohol 0.47 - lanolin alcohol - 1.8 DEA-cetyl phosphate 0.25 - methylparaben 0.2 0.2 propylparaben 0.12 0.1 Carbomer 934 0.11 - disodium EDTA 0.1 - protease conjugate from example 4 0.1 0.5 water 84.32 71.1

Example 34

Product in the form of a cleansing pad (amounts of ingredients are given in % by weight)

ex.32 propylene glycol 1.0 ammonium lauryl sulfate 0.6 succinic acid 4.0 sodium succinate 3.2 Triclosan ^(R) 0.15 protease conjugate from example 1 0.05 water 91.0

The above composition is impregnated onto a woven absorbent sheet consisting of cellulose and/or polyester in an amount of 250% by weight of the absorbent sheet.

Claims (14)

PATENT CLAIMS A protease conjugate comprising a protease portion and one or more addition portions, wherein the protease portion comprises a first epitope region, a second epitope region, and a third epitope region, and each addition portion is covalently linked to an epitope protected position of the protease portion, wherein: (a) the epitope protected positions for the region of the first epitope are selected from the group consisting of positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67, 86; 87, 89, 206, 209, 210, 212, 213, 214, 215, and 216 corresponding to subtilisin BPN ', (b) the epitope protected positions for the second epitope region are selected from the group consisting of positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145 corresponding to the subtilisin BPN ', and (c) the epitope protected positions for the third epitope region are selected from the group consisting of positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272 and 275 corresponding to subtilisin BPN '. The protease conjugate of claim 1, wherein each addition moiety independently has a structure Ri I x -, r ₂ wherein X is selected from the group consisting of zero and the binding moiety, R 1 is selected from the group consisting of zero, the first polypeptide and the first polymer, and R ₂ is selected from the group consisting of zero, the second polypeptide and the second polymer wherein at least one of X, R 1 and R _{2 is} not zero. ·· ·· · · The protease conjugate of claim 1, wherein the protease moiety has a modified amino acid sequence of the parent amino acid sequence, wherein the modified amino acid sequence comprises amino acid substitution at one or more epitope protected positions and each addition moiety is covalently linked to a single amino acid substitution. The protease conjugate of claim 3, wherein the substituting amino acid is cysteine. The protease conjugate of claim 4, wherein the parent amino acid sequence is selected from the group consisting of subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, termitase, Protease A, Protease B, Protease C and Protease D and their variants. The protease conjugate of claim 4, wherein: a) the epitope protected positions of the first epitope region are selected from the group consisting of positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86, 87, 89, 206, 209 , 214 and 215 corresponding to subtilisin BPN ', (b) the epitope protected positions of the second epitope region are selected from the group consisting of positions 27, 47, 48, 50, 52, 102, 127, 128, 130, 131, 132,134,138 and 141 corresponding to subtilisin BPN '; and c) the epitope protected positions of the third epitope region are selected from the group consisting of positions 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254,265, and 275 corresponding to subtilisin BPN '. The protease conjugate of claim 6, wherein R 1 and R ₂ are both zero. The protease conjugate of claim 6, wherein R 1 is a first polypeptide selected from the group consisting of subtilisin BPN ', subtilisin Carlsberg, • subtilisin DY, subtilisin 309, proteinase K, termitases, Protease A, Protease B, Protease C and Protease D and variants thereof. The protease conjugate of claim 8, wherein the first polypeptide is covalently bound to a binding portion or a protease portion at a position of the first polypeptide selected from the group consisting of positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 12 17, 22, 23, 24, 25, 26, 27, 36, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 62, 63, 67 86, 87, 89, 91, 99, 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 143, 144, 145, 146 154, 155, 156, 157, 172, 173, 187, 189, 195, 197, 203, 204, 206, 209, 210, 212, 213, 214, 215, 216, 253, 254, 256, 265, 267 , 269, 271, 272, and 275 corresponding to subtilisin BPN '. The protease conjugate of claim 9, wherein X is zero and the protease moiety and the first polypeptide are covalently linked by a disulfide bridge. The protease conjugate of claim 6, wherein R ₂ is zero and the first polymer is polyethylene glycol. The protease conjugate of claim 11, wherein at least one addition portion is covalently linked to an epitope protected position for an epitope region selected from the group consisting of a first epitope region, a second region, and a third region. 13. A cleaning composition comprising the protease conjugate of claim 1 and a cleaning composition carrier. 14. A personal care composition comprising the protease conjugate of claim 1 and a personal care carrier.