KR20020021396A - Protease conjugates having sterically protected epitope regions - Google Patents

Abstract

The present disclosure relates to a subtilisin protease conjugate comprising a protease moiety and one or more addition moieties. Each addition portion is covalently attached to the epitope protecting position of the protease portion. Protease conjugates have reduced immunogenicity compared to parental proteases. Furthermore, the present disclosure relates to cleaning and personal care compositions containing protease conjugates.

Description

PROTEASE CONJUGATES HAVING STERICALLY PROTECTED EPITOPE REGIONS}

Enzymes make up the largest group of naturally occurring proteins. One group of enzymes includes proteases that catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has typically been utilized by introducing naturally occurring proteases and genetically engineered proteases into cleaning compositions, particularly those suitable for laundry applications.

In the field of washing, the most widely used protease is serine protease. Most of the serine proteases are produced by bacterial individuals, while some are produced by other individuals, such as fungi. Siezen et al., "Homology Modeling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases", Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the effectiveness of wild type proteases in the natural environment is not often optimized in the artificial environment of the cleaning composition. In particular, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability and substrate specificity, are not necessarily optimized for use outside the natural environment of the protease.

For the purpose of increasing the protease efficacy in non-natural cleaning environments, some approaches have been adopted to change the wild type amino acid sequence of the serine protease. Such approaches include genetic redesign and / or chemical modification of proteases to enhance thermal stability and improve oxidative stability under a wide variety of conditions.

However, since the modified protease is foreign to mammals, it is a potential antigen. As an antigen, the protease causes an immunogenic and / or allergic reaction in the mammal (herein collectively referred to as an immunogenic response).

In addition, although genetic redesign and chemical modification of proteases excel in the ongoing search for more effective proteases for laundry applications, these proteases are not commercially used in personal care compositions and housekeeping detergents. For example, the primary reason for the lack of such proteases in products such as soaps, gels, bash washes, shampoos and housekeeping dishwashing detergents is the problem of human sensitization causing undesirable immunogenic reactions. Thus, it would be very beneficial to provide a personal care composition or housekeeping detergent that provides the cleansing properties of the protease without causing an immunogenic response.

Recently, immunity to proteases can be minimized by immobilizing, granulating, coating or dissolving chemically modified proteases to prevent them from airborne. The method solves the consumer's exposure to protonated proteases, but still provides the risks associated with prolonged tissue contact with the final composition and worker exposure to protease containing dust or aerosol during manufacture.

It has also been suggested that immunogenicity reduction of the protease can be achieved by attaching the polymer to the protease. See, eg, US Pat. No. 4,179,337, Davis et al., Dec. 18, 1979; US Patent No. 5,856,451, Olsen et al., Novo Nordisk application, Jan. 5, 1999; WO 99/00489, Olsen et al., Novo Nordisk application, published Jan. 7, 1999; WO 98/30682, Olsen et al., Novo Nordisk application, published July 16, 1998; And WO 98/35026, Von Der Osten et al., August 13, 1998. However, the above proposals did not suggest the importance of polymer attachment of the protease to the amino acid region (ie epitope) involved in the immunogenic response.

Recently, it has been discovered that subtilisin proteases contain three epitope regions, and that in order to significantly reduce the immunogenicity of the protease, a conjugate of one or more polymers, polypeptides or other groups must be attached to one or more such regions. There is a bar. See, eg, US Patent Application No. 09 / 088,912, Weisgerber et al., The Procter & Gamble Co. Applicant filed June 2, 1998.

The inventors have discovered that steric protection near one or more epitope regions of a protease is an alternative mechanism to inhibit or delay epitope presentation and reduce the immunogenicity of the protease. Thus, we provide herein a modified subtilisin, where the modification occurs in a region stericly close to one or more epitope regions. Therefore, the inventors have discovered subtilisin proteases that cause a reduced immunogenic response but retain their activity as an efficient and active protease. Thus, the protease conjugates of the present invention are suitable for use in various types of compositions including, but not limited to, laundry, tableware, hard surfaces, skin care, hair care, beauty care, oral care, and contact lens compositions.

Summary of the Invention

The present invention relates to a protease conjugate comprising a protease moiety and one or more addition moieties, each addition moiety covalently attached to the epitope protecting position of the protease moiety,

(a) The epitope protection position for the first epitope region is at positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44 corresponding to subtilisin BPN '. , 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215 and 216;

(b) The epitope protection position for the second epitope region is at positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99 corresponding to subtilisin BPN '. , 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145;

(c) The epitope protecting position for the third epitope region is at positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, corresponding to subtilisin BPN '. 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271,272 and 275;

Each additional portion independently has the following structure:

Wherein X is selected from nothing and the linking moiety; R ₁ is selected from nothing, the first polypeptide and the first polymer; R ₂ is nothing, the second polypeptide and the second One or more of X, R ₁ and R ₂ are none).

Protease conjugates of the invention have reduced immunogenicity compared to parental proteases. Thus, the protease conjugates are suitable for use in many types of compositions including, but not limited to, laundry, tableware, hard surfaces, skin care, hair care, cosmetic care, oral care, and contact lens compositions.

The present invention relates to chemically modified subtilisin proteases useful in compositions such as, for example, personal care compositions, laundry compositions, hard surface cleansing compositions, and housekeeping relief compositions.

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

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

All percentages and ratios are calculated by weight unless otherwise specified. All percentages are calculated based on the total composition unless otherwise indicated.

All component or composition levels are referenced to the activity level of the component or composition, and impurities such as residual solvents or by-products that may be present in commercial sources are excluded.

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

Reference is made herein to, but is not limited to, brand names for substances including enzymes. The inventors do not intend to limit the substance to a particular brand name herein. Substances equivalent to the referenced brand name (eg those sold by different companies with different names or catalog (reference) numbers) may be used in place of the protease conjugates and compositions herein.

In this application, abbreviations will be used to describe amino acids. Table I provides a list of abbreviations used herein.

Justice

As used herein, the term “variation” means a change in the gene sequence and / or amino acid sequence produced by the gene sequence. Variants include deletions, substitutions and additions of amino acid residues to wild type protein sequences.

As used herein, the term “parent” refers to a protein (wild type or variant) that is used for further modification to form the protease conjugate herein.

As used herein, the term “wild type” means a protein, eg a protease or other enzyme, produced by an unmutated individual.

As used herein, the term “variant” means a protein having an amino acid sequence that is different from the corresponding wild type protein.

All polymer molecular weights as used herein are expressed in weight average molecular weight.

As used herein, the conjugates of the present invention are not limited to those comprising subtilisin BPN 'and variants thereof, but all amino acid number assignments are those of subtilisin BPN' Reference was made to the amino acid sequence. The amino acid sequence of subtilisin BPN 'is described in Wells et al. Nucleic Acids Research, Vol. II, pp. 7911-7925 (1983).

Protease Conjugates of the Invention

Protease conjugates of the invention are compounds comprising a protease moiety and one or more addition moieties, wherein the protease moiety and the addition moiety are linked via covalent attachment (ie, covalent bonds).

Protease moiety

The protease portion herein is a subtilisin-like protease that is wild type or variant thereof. As used herein, the term “subtilisin-like protease” means a protease having at least 50%, preferably at least 80%, amino acid sequence homology with the sequence of subtilisin BPN ′. Wild-type subtilisin Shin like protease, for example, Bacillus al knife pillar's (Bacillus alcalophilus), Bacillus amyl Lowry kepa when Enschede (Bacillus amyloliquefaciens), Bacillus amyl Rosa Kariya kusu (Bacillus amylosaccharicus), Bacillus Lee Kenny formate miss (Bacillus licheniformis ), Bacillus lentus and Bacillus subtilis microorganisms. A discussion of subtilisin-like serine proteases and their homologues is described in Siezen et al., "Homology Modeling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases", Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991).

Preferred protease moieties for use herein 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 A / S Alcalase (Commercially available from Denmark, Copenhagen, Novo Industries), Esperase Novo Industries, Savinase Novo Industries, Maxatase (Commercially available from Genencor International Inc.), Maxacal (Genencor International Inc.), Maxapem 15 Termitase including Genencor International Inc., and variants of the foregoing. Particularly preferred protease moieties for use herein include those obtained from Bacillus amyloliquefaciens and variants thereof. Most preferred protease moieties herein are subtilisin BPN 'and variants thereof.

Particularly preferred variants of subtilisin BPN 'for use as parental amino acid sequences herein, referred to as "protease A", are disclosed in US Pat. No. 5,030,378 (Venegas, issued Sep. 9, 1991), Characterized by the subtilisin BPN 'amino acid sequence 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; Met at position 222 is substituted with an amino acid residue selected from Gln, Phe, His, Asn, Glu, Ala and Thr;

(b) Gly at position 160 is substituted with Ala, and Met at position 222 is substituted with Ala.

Further preferred variants of subtilisin BPN 'for use as parental amino acid sequences herein, referred to as "protease B", are disclosed in EP 251,446 (Genencor International, Inc., published on January 7, 1988). And 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, Has mutations in Gly128, Pro129, Leu135, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys213, Tyr214, Gly215 and Ser221; Wild type subtilisin BPN 'amino acid sequence combining two or more positions described above and one or more mutations at a position selected from Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222 It is characterized by.

Variants of other subtilisin BPN's preferred for use herein, referred to hereinafter as "protease C", are described in WO 95/10615 (Genencor International, Inc. application, published on April 20, 1995), Variation at position Asn76 and Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218 The mutation at one or more other positions selected from Met222, Ser260, Lys265 and Ala274 is characterized by a combined wild type subtilisin BPN 'amino acid sequence.

Variants of other subtilisin BPN's preferred for use herein, referred to hereinafter as "protease D", are described in US Pat. No. 4,760,025 (Estell et al., July 26, 1988), Asp32, Ser33 , His64, Tyr104, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222, characterized by a wild type subtilisin BPN 'amino acid sequence having a mutation at one or more amino acid positions.

More preferred protease moieties herein 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 moiety herein has three epitope regions: a first epitope region, a second epitope region and a third epitope region. The first epitope region is at positions 70-84 corresponding to subtilisin BPN '; The second epitope region is at positions 103-126 corresponding to subtilisin BPN '; The third epitope region is at positions 220-246 corresponding to subtilisin BPN '. See, eg, US Patent Application No. 09 / 088,912, Weisgerber et al., The Procter & Gamble Co. Applicant filed June 2, 1998; Pending US Provisional Patent Application No. 60 / 144,991, Rubingh et al., “Serine Protease Variants Having Amino Acid Substitutions and Deletions in Epitope Regions,” filed on July 22, 1999; And pending U.S. Provisional Patent Application 60 / 144,980, Sikorski et al., "Serine Protease Variants Having Amino Acid Substitutions in Epitope Regions", July 22, 1999.

Surprisingly, we have found epitope protection sites that are stericly close to one or more of the epitope regions. Furthermore, it has been found that by covalently attaching one or more addition moieties to amino acids of the protease moiety at an epitope protecting position, the epitope is protected from hydrolysis, thereby protecting the exposure of the epitope.

Suitable epitope protection sites for covalent modification to the addition portion are based on which epitope is intended to be protected. Most preferably, at least one addition portion is covalently attached to the epitope protected position in the first epitope region.

Epitope protective positions for the first epitope region are positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44, 45, 67 corresponding to subtilisin BPN '. , 86, 87, 89, 206, 209, 210, 212, 213, 214, 215 and 216. Preferably, the epitope protecting position for the first epitope region is at positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86 corresponding to subtilisin BPN '. , 87, 89, 206, 209, 214 and 215. Most preferably, the epitope protective positions for the first epitope region are positions 1, 2, 3, 4, 5, 17, 40, 41, 43, 67,86, 87, and 214 corresponding to subtilisin BPN '. .

Furthermore, the epitope protection position for the second epitope region is at positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99, 100 corresponding to subtilisin BPN '. , 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145. Preferably, the epitope protective 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 '. to be.

Furthermore, the epitope protecting position for the third epitope region is at positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, 173, corresponding to the subtilisin BPN '. It was found selected from the group consisting of 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272 and 275. Preferably, the epitope protecting position for the second epitope region is at positions 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265 corresponding to the subtilisin BPN '. And 275.

In a preferred embodiment of the invention, the protease moiety includes a modified sequence of the parent amino acid sequence. The parent amino acid sequence may be any protease described above, with the same preferred limitations as described above. In such embodiments, the parent amino acid sequence replaces one or more parent amino acid residues with a replacement amino acid to produce a protease moiety suitable for attaching one or more additional moieties of the invention. In accordance with the present invention, substitutions must be made at one or more epitope protecting positions. Epitope protective sites and their preferred limitations are described above.

At one or more epitope protecting positions, one or more additional moieties must be substituted with a replacement amino acid that is not in the parent amino acid sequence (unique to that sequence) in order to best attach it to the protease moiety. In view of the above, any substituted amino acid unique to the parent amino acid sequence can be used. For example, since there is no cysteine residue in the wild type amino acid sequence of subtilisin BPN ', it is suitable for the present invention to substitute subtilisin BPN' with one or more cysteine residues in one or more epitope protecting positions. If there are cysteine residues at positions other than the epitope protecting position of the parent amino acid sequence, it is preferred to substitute each of these positions with another amino acid residue so that one or more additional moieties can be selectively coupled to the epitope protecting position. . Cysteine is the most preferred substituted amino acid for substitution at one or more epitope protecting positions.

Other preferred substituted amino acids include lysine. If the substituted amino acid is lysine, it is preferred to lysine the lysine residue at a position other than the epitope protecting position of the parent amino acid sequence to another amino acid residue to selectively make functionalization of at least one lysine residue at the epitope protecting position. . For example, there is a lysine residue at position 43 of the subtilisin BPN 'which is an epitope protective position as defined herein. Site-selective variation of all other lysine residues in the subtilisin BPN 'sequence may be followed by selective functionalization of the lysine residue at position 43 with the addition portion. Alternatively, amino acid residues (for example) may be mutated (eg) to lysine at any epitope protecting position, followed by selective functionalization with the addition moiety.

Added part

The protease conjugates of the invention comprise one or more addition moieties, each addition covalently attached to one amino acid residue at the epitope protecting position described herein. The addition portion can be of any chemical structure. Preferably, the addition portion stericly hides the attached epitope protecting position or any other epitope protecting position as defined above. Non-limiting examples of addition moieties include, but are not limited to, molecules having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300; Polypeptides; And polymers. The term "polypeptide" as used herein, means a molecule comprising two or more amino acid residues. As used herein, the term "polymer" means any molecule comprising two or more identical (preferably five or more identical) monomeric units.

Preferably, the addition portion has the following structure:

Wherein X is selected from nothing and the linking moiety; R ₁ is selected from the group consisting of nothing, the first polypeptide and the first polymer; R ₂ is nothing, the second polypeptide And a second polymer, at least one of X, R ₁ and R ₂ is none).

Preferably, the protease conjugate comprises 1 to about 15, more preferably about 2 to about 10 and most preferably about 1 to about 5 addition portions.

When R ₁ and R ₂ are each independently a polypeptide moiety or a polymer moiety, R ₁ and R ₂ may be the same or different. Preferably, when R ₁ is a polypeptide moiety, R ₂ is selected from nothing and polypeptide moiety, most preferably none. Most preferably, when R ₁ is a polypeptide moiety, R ₂ is selected from nothing and the same polypeptide moiety, most preferably none. Preferably, when R ₁ is a polymer moiety, R ₂ is selected from nothing and the polymer moiety. Most preferably, when R ₁ is a polymer moiety, R ₂ is selected from nothing and the same polymer moiety. When at least one of R ₁ and R ₂ is the first polymer and the second polymer, respectively, X is preferably nothing.

Polypeptide part

Polypeptide portions described herein include those comprising two or more amino acid residues. Preferred polypeptide moieties are selected from proteins comprising enzymes. Preferred enzymes include proteases, cellulases, lipases, amylases, peroxidases, microperoxidases, hemicelluloses, xylanases, phospholipases, esterases, cutinases, pectinase, keratinase, reductase (Eg, including NADH reductase), oxidase, phenol oxidase, lipoxygenase, riginase, pullulanase, tanase, pentosanase, malanase, b-glucanase, arabino Cidase, hyaluronidase, chondroitinase, laccase, transferase, isomerase (including, for example, glucose isomerase and xylose isomerase), lyase, ligase, shin Terases and fruit based enzymes (including, for example, papain). Preferred enzymes for use as polypeptide moieties include proteases, cellulases, amylases, lipases and fruit based enzymes, with proteases even more preferred.

Examples of lipases for use as a polypeptide portion include those derived from the following microorganisms: the trailing coke (Humicola), pseudomonas switch (Pseudonomas), Fusarium (Fusarium), non-cor (Mucor), chromotherapy tumefaciens (Chromobacterium ), Aspergillus peogil Russ (Aspergillus), Candida (Candida), Keio tree glutamicum (Geotricum), Penny room Solarium (Penicillium), Rhizopus crispus (Rhizopus) and Bacillus (Bacillus).

Examples of commercially available lipases include Lipolase , Lipolase Ultra , Lipozyme , Palatase , Novozym435 And Lecitase (All commercially available from Novo Nordisk, Copenhagen, Denmark), Lumafast (Commercially available from Rochester Genencor, Int., NY) and Lipomax (Genencor, Int.).

Examples of proteases for use as polypeptide moieties include enzymes of the serine protease, chymotrypsin and elastase types. Most preferred proteases for use as polypeptide moieties, as defined herein in the discussion of “protease moieties”, include serine proteases.

Most preferably, when the polypeptide portion is a serine protease, the polypeptide portion is independently accompanied by the definition of the protease portion described herein above. Preferably, as described above, the polypeptide portion has a modified amino acid sequence of the parent amino acid sequence, wherein the modification is at one or more epitope protecting positions as described herein above (the second amino acid sequence is referred to as "second "Parent amino acid sequence). In this case, one of the linking moiety (the linking moiety is nothing) or the protease moiety (the connecting moiety is nothing) is one of the polypeptides via one substitutional amino acid present at one epitope protecting position of the polypeptide moiety. Covalently attached to the part. Where the polypeptide portion is a serine protease, for the protease portion and its corresponding parent amino acid sequence, the same preferred epitope protection sites as described herein above apply.

Most preferably, when the polypeptide portion is a serine protease, the polypeptide portion and the protease portion are equivalent portions. In this case, the polypeptide moiety and the protease moiety are most preferably attached via disulfide bonds, where X is nothing and most preferably R ₂ is nothing.

Polymer part

Addition moieties herein may include polymer moieties. As used herein, the term “polymer moiety” means any molecule comprising two or more identical (preferably five or more identical) monomeric units. Examples of suitable polymer moieties include polyalkylene oxides, polyalcohols, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidone, cellulose, dextran, starch, glycogen, agarose, guar gum, pullulan, inulin And hydrosylate of xanthan gum, carrageenan, pectin, alginic acid hydrosylate and chitosan. Preferred polyalkylene oxides include polyethylene glycol, methoxypolyethylene glycol and polypropylene glycol. Preferred dextrans include carboxymethyldextran. Preferred celluloses include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose and hydroxypropyl cellulose. Preferred starches include hydroxyethyl starch and hydroxypropyl starch. More preferred polymers are polyalkylene oxides. The most preferred polymer part is polyethylene glycol.

When R ₁ and R ₂ are each independently a polymer moiety, R ₁ and R ₂ are preferably from about 0.2 kD (kilodaltons) to about 40 kD, more preferably from about 0.5 kD to about 40 kD, even more preferred Preferably a combined molecular weight (ie, a molecular weight of R ₁ plus a molecular weight of R ₂ ) of about 0.5 kD to about 20 kD, most preferably about 1 kD to about 10 kD.

When R ₁ and R ₂ are each polymer moieties, R ₁ and R ₂ are each independently, preferably from about 0.1 kD to about 20 kD, more preferably from about 0.25 kD to about 20 kD, even more preferably It has a molecular weight of about 0.5 kD to about 10 kD, most preferably about 0.5 kD to about 5 kD.

When R ₁ and R ₂ are each polymer moieties, the molecular weight ratio of R ₁ to R ₂ is preferably from about 1:10 to about 10: 1, more preferably from about 1: 5 to about 5: 1, most preferably Preferably in the range of about 1: 3 to about 3: 1.

When R ₁ is a polymer moiety and R ₂ is nothing, R ₁ is preferably from about 0.1 kD to about 40 kD, more preferably from about 0.5 kD to about 40 kD, even more preferably from about 0.5 kD to It has a molecular weight of about 20 kD, most preferably about 1 kD to about 10 kD.

Connection part

As used herein, X may be nothing, a linking moiety that is optionally covalently attached to one or more polypeptide moieties or one or more polymer moieties, or both, and may also be an amino acid at one epitope protecting position of the protease moiety. Covalently attached to the residue. The linking moiety may generally be any small molecule, ie, a molecule having a molecular weight of less than about 1600, preferably less than about 800, more preferably less than about 400, and most preferably less than about 300. Most preferred linking moieties include those that are capable of covalently binding to cysteine residues or lysine residues, most preferably cysteine residues.

Examples of linking moieties and related chemistries are disclosed below: US Pat. No. 5,446,090, Harris, Aug. 29, 1995; US Patent No. 5,171,264, Merrill, Dec. 15, 1992; US Patent No. 5,162,430, Rhee et al., November 10, 1992 US Patent No. 5,153,265, Shadle et al., October 6, 1992; U.S. Patent No. 5,122,614, Zalipsky, June 16, 1992. Goodson et al., "Site-Directed Pegylation of Recombinant Interleukin-2 at its Glycosylation Site", Biotechnology, Vol. 8, No. 4, pp. 343-346 (1990); Kogan, "The Synthesis of Substituted Methoxy-Poly (ethylene glycol) Derivatives Suitable for Selective Protein Modification", Synthetic Communications, Vol. 22, pp. 2417-2424 (1992); And Ishii et al., "Effects of the State of the Succinimido-Ring on the Fluorescence and Structural Properties of Pyrene Maleimide-Labeled αα-Tropomyosin", Biophysical Journal, Vol. 50, pp. 75-80 (1986)]. Most preferred linking moieties are substituted (eg alkyl substituted) or unsubstituted succinimides.

As an additional example, 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; Bis [sulfone succinimidyl] suvrate; 1,5-difluoro-2,4-dinitrobenzene; Dimethyl adipimate. 2 HCl; Dimethyl pimelimide-2 HCl; Dimethyl suverimidate. 2 HCl; Disuccinimidyl glutarate; Disuccinimidyl suverrate; m-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- (p-maleimidophenyl) -butyrate; Succinimidyl-6-[(beta-maleimidopropionamido) hexanoate]; Bis [2- (sulfosuccinimidyloxycarbonyloxy) -ethyl] sulfone; N- [gamma-maleimidobutyryloxy] sulfonsuccinimide ester; N-hydroxysulfosuccinimidyl-4-azidobenzoate; N- [kappa-maleimidodecanoyloxy] sulfosuccinimide ester; m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; Sulfosuccinimidyl [4-azidosalicylamido] hexanoate; Sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate; Sulfosuccinimidyl 6- [4'-azido-2'-nitrophenylamino] hexanoate; Sulfosuccinimidyl 4- [p-azidophenyl] butyrate; Sulfosuccinimidyl [4-iodoacetyl] aminobenzoate; Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate; And sulfosuccinimidyl 4- (p-maleimidophenyl) -butyrate. Each of these reagents was prepared by Pierce Chemical Co., Rockford, Illinois. Is commercially available.

Optional part

Protease conjugates may additionally be attached to an epitope protecting position, eg, one or more small molecules, polypeptides, and free of other residues or addition portions (referred to herein as “additional portions”) of proteases not illustrated herein. And / or one or more chemical structures, including polymers. Additional moieties may include polypeptide moieties, polymer moieties, and linking moieties as described herein above. In addition, for example, from about 100 Da to about 5000 Da, preferably from about 100 Da to about 2000 Da, more preferably from about 100 Da to about 1000 Da, even more preferably from about 100 Da to about 750 Da, most At least one polymer (most preferably polyethylene glycol), preferably having a molecular weight of about 300 Da, may be covalently attached to the protease moiety at residues other than those exemplified herein. The polymer moiety may be attached directly to the protease moiety herein at any position of the protease moiety using techniques described herein and well known in the art, including via the linking moiety as described herein. Non-limiting examples of such selective types of polymer bonding are shown in WO 99/00849 (Olsen et al., Novo Nordisk A / S, published Jan. 7, 1999).

Manufacturing method

Protease moieties that have substitutions in one or more epitope protecting positions (or any other position of the protease moiety) are made by mutating the nucleotide sequence encoding the parent amino acid sequence. Such methods are well known in the art; One non-limiting example of the method is shown below:

Phagemids (pSS) including wild type subtilisin BPN 'gene (Mitchison, C. and JA Wells, "Protein Engineering of Disulfide Bonds in Subtilisin BPN'", Biochemistry, Vol. 28, pp. 4807-4815 (1989)) -5) E. coli ( Escherichiacoli ) dut-ung- strain CJ236 and transformed into VCSM13 helper phage (Kunkel et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzymology, Vol 154, pp. 367- 382 by Yuckenberg et al. (1987) using "Site-Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors", Directed Mutagenesis-A Practical Approach, McPherson, MJ, pp. 27-48 (1991). Thus, a single chain uracil containing DNA template is prepared. Zoller, MJ and M. Smith, "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, Vol. 10, pp. 6487-6500 (1982), using primer site directed mutagenesis, modified in the methods disclosed in essence, to prepare all variants (essentially as Yuckenberg et al., Supra).

Oligonucleotides are prepared using a 380B DNA synthesizer (Applied Biosystems Inc.). The mutation reaction product is transformed with E. coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations were identified by DNA sequencing, and the isolated DNA was identified as Bacillus subtilis expressing strain PG632 ("Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-Amino Acid Fragment of Human Parathyroid Hormone" by Saunders et al. ", Gene, Vol. 102, pp. 277-282 (1991) and Yang et al." Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the Cloned Gene to Createan in vitro-Derived Deletion Mutation ", Journal of Bacteriology , Vol. 160, pp. 15-21 (1984)).

Fermentation is carried out as follows. Bacillus subtilis cells (PG632) containing the subject protease were cultured in 1 liter of LB broth containing 10 g / L glucose to the log phase medium and a total of 9 liters of Biostat C fermenter (Braun Biotech, Inc., Allentown, Inoculated in PA). Fermentation medium contains yeast extract, casein hydrosulfate, soluble-partially hydrolyzed starch (Maltrin M-250), antifoam, buffer and trace minerals ("Biology of Bacilli: Applications to Industry", Doi, RH and M. McGloughlin (1992)). The broth is kept constant at pH 7.5 during the fermentation run. For antibiotic selection of the mutated plasmid, kanamycin (50 μg / mL) is added. Cells are incubated at 37 ° C. for 18 hours until A ₆₀₀ is about 60, to recover the product.

The fermentation broth is followed by steps to obtain pure protease. The broth is run vertically through a 0.16 μm membrane to remove Bacillus subtilis cells. Cell-free broth is then ultrafiltered and concentrated with a 8,000 molecular weight cutoff membrane. The pH is adjusted to 5.5 with concentrated MES buffer (2- (N-morpholino) ethanesulfonic acid). The protease is subsequently purified by cation exchange chromatography with S-Sepharose and eluted with a NaCl gradient. See Scopes, R. K., "Protein Purification Principles and Practice", Springer-Verlag, New York (1984).

The active protease concentrations of the fractions collected during gradient gradient elution are measured using the pNa assay (Analytical Biochemistry, Vol. 99, pp. 316-320 (1979), DelMar et al.). The assay measures the rate at which p-nitroaniline is released as the protease hydrolyzes succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (sAAPF-pNA), a soluble synthetic substrate. The rate of yellow formation from the hydrolysis reaction is measured at 410 nm on the spectrometer and is proportional to the concentration of active protease moiety. In addition, the absorbance measurement at 280 nm is used to measure the total protein concentration. Protease purity is obtained with the active protease / total protein ratio and used to identify the fractions for pooling stock solutions.

To prevent autolysis of the protease during storage, the same amount of propylene glycol is obtained from the chromatography column and added to the pooled fractions. Upon completion of the purification process, the purity of the stock protease solution was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and turkey egg whites with trypsin inhibitor type II-T (Sigma Chemical Company, St. Louis, MO) Absolute enzyme concentration is determined by active site titration using

In preparation for use, the protease stock solution is eluted through a Sephadex-G25 (Pharmacia, NJ, Pharmacia) size exclusion column to remove propylene glycol and exchange buffer. MES buffer in enzyme stock solution is exchanged with 0.01 M KH ₂ PO ₄ solution, pH 5.5.

Protease conjugates can be prepared using the prepared protease to be functionalized with one or more addition moieties. Precursor of the addition portion (the precursor of the addition portion reacts with the precursor of the protease portion to form a protease conjugate consisting of the addition portion and the protease portion), thereby activating the reactivity to the precursor of the protease portion. . Such activation is well known in the art. Non-limiting examples of methods for preparing protease conjugates are provided below.

Example 1

A protease comprising one cysteine residue at one epitope protecting position is coupled with the polymer moiety according to the above scheme using the following method (wherein “P” is the protease moiety minus the thiol group produced by cysteine substitution) N is the number of repeating monomer units of polyethylene glycol (eg n = 77)).

A variant of subtilisin BPN 'was prepared by replacing tyrosine at position 217 with leucine and replacing serine at position 3 with cysteine. The variant is made to a concentration of about 2 mg / mL in phosphate buffer (pH 5.5). The pH is then raised to 7.5 with dilute sodium hydroxide. The variant is mixed with monomethyl polyethylene glycol maleimide at 25: 1 so that the variant is in excess in the activated polymer. After mixing at room temperature for one hour, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and filtered through a molecular weight cutoff ultrafilter to remove excess polymer. The concentrate contains purified protease conjugates.

Example 2

The protease moiety comprising one cysteine residue at one epitope protecting position is coupled with the polymer moiety according to the scheme above using the following method (wherein "P" subtracts the thiol group resulting from cysteine substitution at the protease moiety). N is the number of repeating monomer units of each polyethylene glycol (eg n = 77)).

A variant of subtilisin BPN 'was prepared by replacing tyrosine at position 217 with leucine and replacing phenylalanine at position 17 with cysteine. Variants are made to a concentration of about 2 mg / mL in phosphate buffer (pH 5.5). The pH is then raised to 7.5 with dilute sodium hydroxide. The variant is mixed with dimethyl polyethylene glycol maleimide at 25: 1 so that the variant is in excess in the activated polymer. After mixing at room temperature for one hour, the pH of the mixture is adjusted to 5.5 with dilute phosphoric acid and filtered through a molecular weight cutoff ultrafilter to remove excess polymer. The concentrate contains purified protease conjugates.

Example 3

Selective coupling of the succinimide-protecting polymer to lysine at one or more epitope protecting positions, where "MPEG" and "PEGM" are equivalent and represent monomethyl polyethylene glycol and "P" is indicated in the protease moiety. Minus lysine amine groups):

Example 4

The carbodiimide-protecting polymer is selectively coupled to lysine at one or more epitope protecting positions, where "MPEG" and "PEGM" are equivalent and represent monomethyl polyethylene glycol, and "P" is indicated in the protease moiety. Minus lysine amine groups, X and X 'are side chains comprising a carbodiimide moiety, for example alkyl):

Example 5

A protease moiety comprising one cysteine residue at one epitope protecting position is coupled with an alkyl maleimide using the following method (wherein "P" represents the protease moiety minus a thiol group resulting from cysteine substitution, "R" is an alkyl group). In this example, R ₁ and R ₂ are each none, and the linking moiety is derived from alkyl maleimide.

A variant of subtilisin BPN 'was prepared by replacing tyrosine at position 217 with leucine and replacing serine at position 86 with cysteine. 20 mL of a solution of the variant is prepared at a concentration of about 1 mg / mL in 0.01 M KH ₂ PO ₄ buffer (pH 7). To the solution, 1.5 equivalents of alkyl maleimide (eg methyl maleimide) is added to the solution. The solution is gently mixed at room temperature for about 1 hour. The resulting protease conjugates are obtained from solution by standard methods.

Example 6

Protease moieties comprising one cysteine residue at one epitope protecting position are formed into duplexes using the following method (wherein "P" represents the protease moiety minus the thiol group produced by cysteine substitution). In this example, the protease moiety and the polypeptide moiety are equivalent (X is nothing).

A variant of subtilisin BPN 'was prepared by replacing tyrosine at position 217 with leucine and replacing serine at position 214 with cysteine. 20 mL of a solution of the variant is prepared at a concentration of about 1 mg / mL in 0.01 M KH ₂ PO ₄ buffer (pH 8.6). Oxygen is gently bubbled through the solution at room temperature for about 1 hour to form the desired protease conjugate duplex. The resulting protease conjugates are obtained from solution by standard methods.

Analytical Method

The protease conjugates of the invention can be tested for enzyme activity and immunogenic reactions using the following methods, both of which are known to those skilled in the art. Other methods well known in the art may alternatively be used.

Protease Conjugate Activity

The protease activity of the protease conjugates of the invention can be analyzed by methods well known in the art. Two such methods are shown herein below:

How skin flakes are active

Scotch Using # 375OG tape, the human skin flakes are repeatedly peeled from the subject's legs until the tape is substantially opaque by the flakes. The tape is then cut and prepared into 1 inch by 1 inch pieces. To a 10 mm × 35 mm Petri dish, add 2 mL of control enzyme (0.75 mg / mL) (eg subtilisin BPN ′) or the protease conjugate to be tested to 0.01 M KH ₂ PO ₄ pH 5.5 buffer. . To this solution, 1 mL of a 2.5% sodium laurate pH 8.6 solution is added. Mix the solution gently on a platform shaker. While continuing to mix gently, the pre-prepared piece of tape is immersed in the solution (with flakes up) for 10 minutes. The piece of tape is then rinsed gently in tap water for 15 seconds. Pipette Stevenel Blue Stain (3 mL, available from Sigma Chemical Co., St. Louis, MO) into a clean Petri dish. Leave the rinsed piece of tape (flap up) for 3 minutes while gently mixing the rinsed tape pieces. A piece of tape is removed from the dye and subsequently rinsed in two 300 mL distilled water beakers for 15 seconds. Allow the tape pieces to air dry. The intensity of the color between the tape pieces obtained from the control enzyme and the tape pieces obtained from the protease conjugate is compared using the naked eye or colorimeter. Protease conjugate tape pieces that exhibit less color intensity compared to control enzyme tape pieces indicate that the protease conjugates have higher activity.

Stained Collagen Activity Method

0.1 M Tris buffer (tris-hydroxymethyl-aminomethane) (pH 8.6) containing 0.01 M CaCl ₂ , 50 mL and azocol (collagen impregnated with azo dye, Missouri, St. Louis, Sigma Chemical Co.) ) Add 0.5 g. The mixture is incubated at 25 ° C. with gentle mixing using a platform shaker. Filter 2 mL of the mixture through a 0.2 micron syringe filter, then read the absorbance of the mixture at 520 nm as 0 using a spectrometer. Control enzyme (eg subtilisin BPN ') or 1 ppm of the protease conjugate to be tested is added to 48 mL of the remaining Tris / Azocol mixture. Every 2 minutes for a total of 10 minutes, 2 mL of the control / protease conjugate containing solution is filtered through a 0.2 micron syringe filter. For each filtered sample, the absorbance at 520 mn is read immediately. Graph the results over time. The slope of the control and test conjugates indicates the relative activity of the samples. The higher the slope, the higher the activity. Test protease conjugate activity (tilt) can be expressed as a percentage of control activity (tilt).

Mouse Intranasal Tests for Immunogenicity

The potential immunogenicity of the protease conjugates of the present invention can be measured by methods known in the art or by mouse intranasal tests for immunogenicity as shown herein below. Such tests are described in Robinson et al., "Specific Antibody Responses to Subtilisin Carlsberg (Alcalase) in Mice: Development of an Intranasal Exposure Model", Fundamental and Applied Toxicology, Vol. 34, pp. 15-24 (1996) and Robinson et al., "Use of the Mouse Intranasal Test (MINT) to Determine the Allergenic Potency of Detergent Enzymes: Comparison to the Guinea Pig Intratracheal (GPIT) Test", Toxicological Science, Vol. 43, pp. 39-46 (1998), which are similar to the assays described in the text, which can be used in place of the tests shown below herein.

Female BDF1 mice (Charles River Laboratories, Portage, MI) weighing about 18 to about 20 g are used for the test. Mice are isolated one week before administration. Mice are housed in cages bedded with wood chips in rooms controlled by humidity (30-70%), temperature (67-77 ° F) and a 12-hour light cycle. Purina on Mouse Mouse feed (Purina Mills, Richmond, IN) and water are supplied freely.

The potential antigen to be tested (subtilisin BPN 'as a positive control or protease conjugate of the invention) is administered to a group of five mice. Prior to administration, each mouse is anesthetized by intraperitoneal injection of a mixture of Ketaset (88.8 mg / kg) and Rompun (6.67 mg / kg). The anesthetized animal is placed on the palm of the back with the back down and intranasally administered 5 μl of protease in buffer solution (0.01 M KH ₂ PO ₄ , pH 5.5). The same dose is administered to each group, but various doses may be tested. The dosing solution is gently placed outside of each nasal cavity for inhalation by the mouse. Administration is repeated at 3, 10, 17 and 24 days.

Serum samples are taken on day 29. Enzyme-specific IgG1 antibodies in mouse serum are measured by antigen capture ELISA method. Standard ED ₅₀ values can be used to compare the immunogenicity of protease conjugates to the immunogenicity of subtilisin BPN '.

Compositions of the Invention

The protease conjugates herein can be used for any use suitable for each parent protease. One example of the above includes a cleaning composition. Because of the preferably reduced immunogenic nature of the protease conjugates of the present invention, the protease conjugates can be further used for applications that have historically not benefited from the use of proteases. Examples of such uses include those in which protease conjugates inevitably come into intimate contact with mammalian skin (especially human skin), such as in the use of a personal care composition.

Cleaning composition

Protease conjugates may be used in cleaning compositions including, but not limited to, laundry compositions, hard surface cleaning compositions, household alleviation cleaning compositions including dish cleaning compositions, and automatic dishwasher detergent compositions.

The cleaning compositions herein contain an effective amount of at least one protease conjugate of the invention and a cleaning composition carrier.

As used herein, "effective amount of protease" and the like means the amount of protease conjugate necessary to obtain the proteolytic activity required for a particular cleaning composition. Such effective amounts are readily identified by those skilled in the art and are based on a number of factors, such as the particular protease conjugate used, the cleaning application, the specific composition of the cleaning composition and the need for a liquid or dry (eg granule, bar) composition. Preferably, the cleaning composition contains about 0.0001% to about 10%, more preferably about 0.001% to about 1%, most preferably about 0.01% to about 0.1% of one or more protease conjugates of the present invention. do. Several examples of various cleaning compositions in which protease conjugates can be employed are discussed in more detail below.

In addition to the protease conjugates of the present invention, the cleaning compositions of the present invention additionally contain a cleaning composition carrier comprising one or more cleaning composition materials compatible with the protease conjugates. As used herein, the term “cleaning composition material” means any material selected for the particular type of cleaning composition desired and the form of the product (eg, liquid, granules, bars, sprays, sticks, pastes, gels), These materials are also compatible with the protease conjugates used in the composition. The particular selection of the cleaning composition material is readily carried out taking into account the surface material to be cleaned, the desired form of the composition for the cleaning conditions in use (eg, through the use of a wash detergent). As used herein, the term “compatibility” means that the cleaning composition material does not reduce the proteolytic activity of the protease conjugate to the extent that the protease is not as effective as desired, under normal use conditions. Specific cleaning composition materials are illustrated in detail below.

The protease conjugates of the invention can be used in a variety of detergent compositions where high sussing and good cleansing are desired. Thus, protease conjugates can be used with various conventional ingredients to provide fully formulated hard surface cleaners, dish cleaning compositions, fabric laundry compositions, and the like. The composition may be in the form of a liquid, granules, bars or the like. The composition may be formulated with a "concentrated" detergent containing from about 30% to about 60% by weight surfactant.

The cleaning compositions herein may optionally and preferably contain various surfactants (eg, anionic, nonionic or zwitterionic surfactants). The surfactant is typically present at a level of about 5% to about 35% of the composition.

Non-limiting examples of useful surfactants herein include the conventional C ₁₁ -C ₁₈ alkyl benzene sulfonates and primary and random alkyl sulfates, the formula _{CH 3 (CH 2) x (} CHOS0 3 - M +) CH 3 and CH _{3 (CH 2) y (CHOSO 3} - M +) CH 2 CH 3 ( and the formula, x and (y + l) is about 7 or more, and preferably an integer greater than or equal to about 9, M is a water-soluble cation, especially sodium) the C ₁₀ -C ₁₈ secondary (2,3) alkyl sulfates, C _1O -C _l8 alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C _1O -C ₁₈ alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylate), C ₁₀ -C _l8 alkyl polyglycosides and their corresponding sulfated polyglycosides, C ₁₂ -C ₁₈ α-sulfonated fatty acid esters, C ₁₂ -C ₁₈ alkyl and Alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy / propoxy), C ₁₂ -C ₁₈ betaines and sulfobetaines (“sultaines”), C ₁₀ -C ₁₈ amine oxides and the like. Alkyl alkoxy sulfates (AES) and alkyl alkoxy carboxylates (AEC) are preferred herein. It is also preferred, depending on the purpose of the formulator, to use the surfactant in combination with an amine oxide and / or a betaine or sultaine surfactant. Other common useful surfactants are listed in the standard text. In particular, U.S. Patent No. 5,194,639 Useful surfactants (, 1993. Connor, etc. 3. 16. issued) C _1O -C _l8 N- include methylglutaryl erucamide disclosed.

Various other ingredients useful in detergent cleaning compositions can be contained in the compositions herein, including, for example, other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, and solvents for liquid formulations. If an additional increase in foaming is needed, a foam booster, such as C ₁₀ -C ₁₆ alcoholamide, may be incorporated into the composition, typically at levels of about 1% to about 10%. C ₁₀ -C ₁₄ monoethanol and diethanol amides represent a typical group of such foam boosters. The use of high foaming auxiliary surfactants such as the amine oxides, betaines and sultaines mentioned above with the foam boosters is also advantageous. If desired, soluble magnesium salts such as MgCl ₂ , MgSO ₄ , and the like may be added at levels typically of about 0.1% to about 2% to provide additional foaming.

The liquid detergent compositions herein may contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol and isopropanol are suitable. Monohydric alcohols are preferred for dissolving the surfactant, but include polyols, such as from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (eg, 1,3-propanediol, ethylene glycol, Glycerin and 1,2-propanediol) may also be used. The composition may contain about 5% to about 90%, typically about 10% to about 50% of said carrier.

Detergent compositions herein will preferably be formulated such that the wash water has a pH of about 6.8 to about 11, during use in aqueous cleaning operations. Thus, the final product is typically formulated in this range. Techniques for adjusting pH to recommended use levels include, for example, the use of buffers, alkalis and acids. Such techniques are well known to those skilled in the art.

When formulating the hard surface cleaning composition and the fabric cleaning composition of the present invention, it can be expected that the formulator adopts various builders in the level of about 5% to about 50% by weight. Typical builders include 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates and the like. Other conventional builders are listed as standard formulations.

As such, formulators can be expected to adopt various additional enzymes, such as cellulases, lipases, amylases and proteases, into the composition, typically at levels of about 0.001% to about 1% by weight. Various detergents and fabric care enzymes are well known in the laundry detergent art.

Various bleach compounds such as percarbonate, perborate and the like can be used in the composition, typically at levels of about 1% to about 15% by weight. If desired, the composition may also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate and the like, which are also known in the art. Use levels typically range from about 1% to about 10% by weight.

Pollution release agents, in particular contamination release agents of the anionic oligoester type, chelating agents, in particular aminophosphonates and ethylenediaminedisuccinates, clay soil removers, in particular ethoxylated tetraethylene pentamine, dispersants, in particular polyacrylates and polyas Paratates, varnishes, in particular anionic varnishes, antifoams, in particular silicones and secondary alcohols, fabric softeners, in particular smectite clays, and the like, can all be used in the composition at levels ranging from about 1% to about 35% by weight. Standard formulations and published patents include complex and detailed descriptions of these conventional materials.

Enzyme stabilizers can also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably about 1% to about 10%), sodium formate (preferably about 0.1% to about 1%) and calcium formate (preferably about 0.1% to about 1%) ) Is included.

Variants of the invention are useful in hard surface cleaning compositions. As used herein, "hard surface cleaning composition" means a liquid and granular detergent composition for cleaning hard surfaces such as floors, walls, bathroom tiles and the like. The hard surface cleaning compositions of the present invention comprise an effective amount of at least one protease conjugate of the present invention, preferably from about 0.001% to about 10% by weight, more preferably from about 0.01% to about 5% of the protease conjugate of the composition. Wt%, even more preferably about 0.05 wt% to about 1 wt%. In addition to containing one or more protease conjugates, the hard surface cleaning composition typically contains a surfactant and a water soluble sequestration builder. However, in certain embodied products, such as sprayed window cleaners, surfactants are sometimes not used because they can produce residues of films and / or stripes on the glass surface.

If present, the surfactant component may contain as much as 0.1% of the composition herein, but typically the composition will contain from about 0.25% to about 10%, more preferably from about 1% to about 5% of the surfactant. will be.

Typically the composition will contain from about 0.5% to about 50%, preferably from about 1% to about 10% of detergent builders.

Preferably, the pH should be in the range of about 7-12. If adjustment is required, conventional pH adjusters may be used, for example sodium hydroxide, sodium carbonate or hydrochloric acid.

Solvents may be contained in the compositions. Useful solvents include glycol ethers such as diethylene glycol monohexyl ether, diethylene glycol monobutyl ether, ethylene glycol monobutyl ether, ethylene glycol monohexyl ether, propylene glycol monobutyl ether, dipropylene glycol monobutyl ether, and diols Such as, but not limited to, 2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, the solvent is typically present at a level of about 0.5% to about 15%, more preferably about 3% to about 11%.

Additionally, if the composition is applied to the surface at "full strength" and then the surface is not rinsed, a high volatility solvent such as isopropanol or ethanol is added to the composition of the present invention to promote faster evaporation of the composition from the surface. Can be used. When used, volatile solvents are typically present at levels of about 2% to about 12% in the composition.

Examples 7-12

Liquid hard surface cleaning composition

Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Protease Conjugate of Example 3 0.05% 0.50% 0.02% 0.03% 0.30% 0.05% EDTA - - 2.90% 2.90% - - Sodium citrate - - - - 2.90% 2.90% NaC ₁₂ alkyl-benzene sulfonate 1.95% - 1.95% - 1.95% - NaC ₁₂ alkylsulfate - 2.20% - 2.20% - 2.20% NaC ₁₂ (ethoxy) sulfate - 2.20% - 2.20% - 2.20% C ₁₂ Dimethylamine Oxide - 0.50% - 0.50% - 0.50% Sodium cumene sulfonate 1.30% - 1.30% - 1.30% - Hexyl carbitol 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 formulations are adjusted to pH 7.

In another embodiment of the invention, the dishwashing composition contains one or more variants of the invention. As used herein, “dishwashing composition” means any form of dishwashing composition, including but not limited to granules and liquid forms.

Examples 13-16

Liquid tableware detergent

Example 13 Example 14 Example 15 Example 16 Protease Conjugate of Example 1 0.05% 0.50% 0.02% 0.40% C ₁₂ -C ₁₄ N-methyl glucamide 0.90% 0.90% 0.90% 0.90% C ₁₂ ethoxy (1) sulphate 12.0% 12.0% 12.0% 12.0% 2-methyl undecanoic acid 4.50% 4.50% 4.50% 4.50% C ₁₂ ethoxy (2) carboxylate 4.50% 4.50% 4.50% 4.50% C ₁₂ Alcohol Ethoxylate (4) 3.00% 3.00% 3.00% 3.00% C ₁₂ amine oxide 3.00% 3.00% 3.00% 3.00% Sodium cumene sulfonate 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 formulations are adjusted to pH 7.

Examples 17-19

Liquid fabric cleaning composition

Example 17 Example 18 Example 19 Protease Conjugate of Example 4 0.05% 0.03% 0.30% Sodium C ₁₂ -C ₁₄ Alkyl Sulfate 20.0% 20.0% 20.0% 2-butyl octanoic acid 5.0% 5.0% 5.0% Sodium citrate 1.0% 1.0% 1.0% C ₁₀ Alcohol Ethoxylate (3) 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 composition

The protease conjugates of the present invention are particularly suitable for personal care compositions such as leave-on and rinse-off hair conditioners, shampoos, leave-on and rinse-off acne compositions, facial milks and Conditioners, shower gels, soaps, foamy and non-bubble facial cleansers, cosmetics, hands, face, and body lotions, moisturizers, patches and masks, rib-on facial moisturizers, cosmetic and cleansing wipes, oral care compositions, sanitary napkins And suitable for use in contact lens care compositions. The personal care composition of the present invention contains one or more protease conjugates of the present invention and a personal care carrier.

For illustrative purposes, the protease conjugates of the present invention are suitable for inclusion in the compositions described in the following references: US Pat. No. 5,641,479, Linares et al., June 24, 1997 (skin cleanser); US Patent No. 5,599,549 Wivell et al., Feb. 4, 1997 (Her skin cleanser); US Patent No. 5,585,104, Ha et al., Dec. 17, 1996, Hur (skin cleanser); US Patent No. 5,540,852, Kefauver et al., July 30, 1996, Hur (skin cleanser); U.S. Patent No. 5,510,050, Dunbar et al., April 23, 1996 (Her skin cleanser); U.S. Patent No. 5,612,324, Guang Lin et al., March 18, 1997 Grant (anti-acne preparations); U.S. Patent No. 5,587,176, Warren et al., Dec. 24, 1996, issued (anti-acne preparations); U.S. Patent No. 5,549,888, Venkateswaran, Aug. 27, 1996, Grant (anti-acne preparations); US Patent No. 5,470,884, Corless et al., Nov. 28, 1995, Grant (anti-acne preparations); U.S. Patent No. 5,650,384, Gordon et al., July 22, 1997, Hurer (Shower Gel); U.S. Patent No. 5,607,678, Moore et al., March 4, 1997, Heryer (Shower Gel); U.S. Patent No. 5,624,666, Coffindaffer et al., April 29, 1997 (Hair conditioner and / or shampoo); U.S. Patent No. 5,618,524, Bolich et al., April 8, 1997 (Hair conditioner and / or shampoo); U.S. Patent No. 5,612,301, Inman, March 18, 1997 Grant (hair conditioner and / or shampoo); U.S. Patent No. 5,573,709, Wells, November 12, 1996 (Hair Conditioner and / or Shampoo); US Patent No. 5,482,703, Pings, Jan. 9, 1996 (Hair Conditioner and / or Shampoo); U.S. Patent No. Re. 34,584, Grote et al., April 12, 1994 reauthorization (hair conditioner and / or sampu); U.S. Patent No. 5,641,493, Date et al., June 24, 1997, Huer (Cosmetic); US Patent No. 5,605,894, Blank et al., Feb. 25, 1997, Huer (Cosmetic); U.S. Patent No. 5,585,090, Yoshioka et al., Dec. 17, 1996, Hur (cosmetics); U.S. Patent No. 4,939,179, Cheney et al., July 3, 1990; hand (hand, face and / or body lotion); U.S. Patent No. 5,607,980, McAtee et al., March 4, 1997, grant (hand, face and / or body lotion); U.S. Patent No. 4,045,364, Richter et al., Aug. 30, 1977 (Her cosmetic and cleansing wipes); European Patent Application EP 0 619 074, Touchet et al., Published October 12, 1994 (cosmetic and cleansing wipes); U.S. Patent No. 4,975,217, Brown-Skrobot et al., Dec. 4, 1990 (permanent cosmetic and cleansing wipes); U.S. Patent No. 5,096,700, Seibel, March 17, 1992, Feder (oral cleaning composition); US Patent No. 5,028,414, Sampathkumar, July 2, 1991; U.S. Patent 5,028,415, Benedict et al., July 2, 1991; U.S. Patent 5,028,415, Benedict et al., July 2, 1991; US Patent No. 4,863,627, Davies et al., September 5, 1989 (contact lens cleaning solution); U.S. Patent No. Re. 32,672, Huth et al., May 24, 1988 Reauthorization (contact lens cleaning solution); And US Pat. No. 4,609,493, Schafer, Sept. 2, 1986 (contact lens cleaning solution).

To further illustrate the oral cleaning composition of the present invention, pharmaceutically acceptable amounts of one or more of the protease conjugates of the present invention are contained in a composition useful for removing proteinaceous stains from teeth or dentures. As used herein, “oral cleansing compositions” include dentoxes, toothpastes, cream toothpastes, powdered toothpastes, mouthwashes, oral sprays, oral gels, chewing gums, medicinal mint drops, lozenges, oral sachets, Tablets, biogels, prophylactic pastes, dental therapeutic solutions, and the like. The oral cleansing composition is preferably about 0.0001% to about 20%, more preferably about 0.001% to about 10%, even more preferably about 0.01% to about 5% by weight of the composition. At least two protease conjugates of the invention, and a pharmaceutically acceptable carrier. As used herein, “pharmacologically acceptable” is suitable for use in contact with tissues of humans and lower animals without inappropriate toxicity, incompatibility, instability, hypersensitivity, allergic reactions, etc. Means a drug, medicament, or inactive ingredient that is taken

Typically, the pharmaceutically acceptable oral cleansing carrier component of the oral cleansing components of the oral cleansing composition is generally from about 50% to about 99.99% by weight of the composition, preferably from about 65% to about 99.99% by weight, More preferably from about 65% to about 99% by weight.

Pharmaceutically acceptable carrier components and optional components that may be included in the oral cleaning composition of the present invention are well known to those skilled in the art. A wide variety of composition types, carrier components, and optional components useful for oral cleaning compositions are disclosed in the references cited above.

In another embodiment of the invention, the denture cleaning composition for denture cleaning outside the oral cavity contains one or more protease conjugates of the invention. The denture cleaning composition is an effective amount, preferably about 0.0001% to about 50%, more preferably about 0.001% to about 35%, even more preferably about 0.01% to about 20% by weight of the composition At least one protease conjugate, and a denture cleaning carrier. Various denture cleaning composition formats such as foam tablets are well known in the art (see US Pat. No. 5,055,305, Young) and generally suitable for incorporation of one or more protease conjugates for removal of proteinaceous stains from dentures. Do.

In another embodiment of the invention, the contact lens cleaning composition contains one or more protease conjugates of the invention. The contact lens cleaning composition is an effective amount, preferably from about 0.01% to about 50% by weight, more preferably from about 0.01% to about 20% by weight, even more preferably from about 1% to about 5% by weight of the composition. % Of one or more protease conjugates, and a contact lens cleaning carrier. Various contact lens cleaning composition formats, such as tablets, liquids and the like, are well known in the art and are generally suitable for incorporation of one or more protease conjugates of the invention for removal of proteinaceous stains from contact lenses.

Examples 20-23

Contact Lens Cleaning Solution

Example 20 Example 21 Example 22 Example 23 Protease Conjugate of Example 5 0.01% 0.5% 0.1% 2.0% Glucose 50.0% 50.0% 50.0% 50.0% Nonionic Surfactant (Polyoxyethylene-Polyoxypropylene Copolymer) 2.0% 2.0% 2.0% 2.0% Anionic Surfactants (Polyoxyethylene-Alkylphenylether Sodium 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 24-27

Body wash products

Example 24 Example 25 Example 26 Example 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% Polyquaternium 10 0.4% 0.4% 0.4% 0.4% Sodium Lauret Sulfate 12.0% 12.0% 12.0% 12.0% Cocamide MEA 2.8% 2.8% 2.8% 2.8% Sodium Laurapoacetate 6.0% 6.0% 6.0% 6.0% Myristic acid 1.6% 1.6% 1.6% 1.6% Magnesium Sulfate 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 Polyester of Cotonate Fatty Acid 3.0% - - - Sucrose Polyester of Behenate Fatty Acid 3.0% - 4.0% - Petrolatum - 4.0% 8.0% - Mineral oil - - - 6.0% DMDM Hydantoin 0.08% 0.08% 0.08% 0.08% Protease Conjugate of Example 6 0.1% 2.0% 2.0% 5.0% Citric acid 1.40% 1.40% 1.40% 1.40%

Examples 28-31

Face wash products

Example 28 Example 29 Example 30 Example 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 Lauret-3 Sulfate 3.0% 3.5% - - Sodium laureth-4 carboxylate 3.0% 3.5% - - Lauret-12 1.0% 1.2% - - Polyquaternium 10 - - 0.4% 0.4% Polyquaternium 25 0.3% 0.3% - - glycerin 3.0% 3.0% 3.0% 3.0% Sodium Lauro Ampo Acetate - - 6.0% 6.0% Lauric acid 6.0% 6.0% 3.0% 3.0% Myristic acid - - 3.0% 3.0% Magnesium Sulfate Heptahydrate 2.3% 2.0% 2.0% 2.0% Triethanol amine 4.0% 4.0% 4.0% 4.0% Trihydroxystearin 0.5% 0.5% 0.5% 0.5% Sucrose Polyester of Behenate Fatty Acid 2.0% 2.0% - - Sucrose Polyester of Cotonate Fatty Acid 3.0% 2.0% - - PEG-6 Caprylic / Capric Triglycerides - - - 2.0% Petrolatum - - 4.0% - Mineral oil - - - 2.0% Cocamidopropyl 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 of Example 2 1.0% 2.0% 0.5% 0.5% Spices 0.2% 0.2% 0.2% 0.2%

Examples 32-33

Rib-on Skin Moisturizing Composition

Example 32 Example 33 glycerin 5.0% - Stearic acid 3.0% - C _11-13 isoparaffin 2.0% - Glycol stearate 1.5% - Propylene glycol - 3.0% Mineral oil 1.0% 10.0% Sesame oil - 7.0% Petrolatum - 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 of Example 4 0.1% 0.5% water 84.32% 71.1%

Example 34

Cleansing wipe composition

Propylene glycol 1.0% Ammonium Lauryl Sulfate 0.6% Succinic acid 4.0% Sodium succinate 3.2% Triclosan 0.15% Protease Conjugate of Example 1 0.05% water 91.0%

The composition is injected onto an absorbent woven sheet composed of cellulose and / or polyester, which is about 250% by weight of the absorbent sheet.

Claims (14)

A protease conjugate comprising a protease portion comprising a first epitope region, a second epitope region and a third epitope region and at least one addition portion covalently attached to the epitope protecting position of the protease portion, respectively. (a) The epitope protection position for the first epitope region is at positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 36, 40, 41, 43, 44 corresponding to subtilisin BPN '. , 45, 67, 86, 87, 89, 206, 209, 210, 212, 213, 214, 215 and 216; (b) The epitope protection position for the second epitope region is at positions 25, 26, 27, 46, 47, 48, 49, 50, 51, 52, 53, 54, 91, 99 corresponding to subtilisin BPN '. , 100, 101, 102, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140, 141, 144 and 145; (c) The epitope protecting position for the third epitope region is at positions 9, 10, 22, 23, 24, 62, 63, 143, 146, 154, 155, 156, 157, 172, corresponding to subtilisin BPN '. Protease conjugates selected from the group consisting of 173, 187, 189, 195, 197, 203, 204, 253, 254, 256, 265, 267, 269, 271, 272 and 275. The protease conjugate according to claim 1, wherein each addition portion independently has the following structure: Wherein X is selected from the group consisting of nothing and linking moieties; R ₁ is selected from the group consisting of nothing, the first polypeptide and the first polymer; R ₂ is nothing, A second polypeptide and a second polymer; at least one of X, R ₁ and R ₂ is nothing. 3. The protease conjugate of claim 2, wherein the protease moiety has a modified amino acid sequence of the parent amino acid sequence, wherein the modified amino acid sequence includes a substitution of an amino acid at one or more epitope protecting positions, wherein each addition portion is A protease conjugate covalently attached to one substituted amino acid. 4. The protease conjugate according to claim 3, wherein the replacing amino acid is cysteine. The method according to claim 4, wherein the parent amino acid sequence is subtilisin BPN ', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, thermitase, protease A, protease B, Protease conjugates selected from the group consisting of Protease C and Protease D, and variants thereof. The method of claim 5, (a) The epitope protection position for the first epitope region is at positions 1, 2, 3, 4, 5, 6, 7, 12, 17, 40, 41, 43, 67, 86 corresponding to subtilisin BPN '. , 87, 89, 206, 209, 214 and 215; (b) Epitope protective 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 '. Is selected from the group consisting of; (c) the epitope protecting position for the third epitope region is at positions 22, 23, 24, 143, 146, 155, 173, 189, 197, 203, 204, 253, 254, 265, and corresponding to subtilisin BPN '; A protease conjugate selected from the group consisting of 275. 7. The protease conjugate of claim 6, wherein R ₁ and R ₂ are each none. The method of claim 6 wherein, R ₁ is subtilisin new BPN ', subtilisin new Carlsbad Berg, subtilisin new DY, subtilisin Seen 309, proteinase Kinase K, Thermal Mita kinase, protease A, Protease B, Protease C, and Protease D, and a protease conjugate, which is the first polypeptide selected from the group consisting of variants thereof. The method of claim 8, wherein the first polypeptide corresponds to positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 17, 22, 23, 24, 25, 26 corresponding to subtilisin BPN '. , 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 From the group consisting of 187, 189, 195, 197, 203, 204, 206, 209, 210, 212, 213, 214, 215, 216, 253, 254, 256, 265, 267, 269, 271, 272 and 275 At a selected position, a protease conjugate covalently attached to the linking portion or protease portion. 10. The protease conjugate of claim 9, wherein X is absent and the protease moiety and the first polypeptide are covalently attached via disulfide bonds. The protease conjugate of claim 6, wherein R ₂ is nothing and the first polymer is polyethylene glycol. 12. The protease conjugate of claim 11, wherein at least one addition portion is covalently attached to an epitope protecting position for an epitope region selected from the group consisting of a first epitope region, a second region and a third region. A cleaning composition comprising the protease conjugate according to claim 1 and a cleaning composition carrier. A personal care composition comprising the protease conjugate according to claim 1 and a personal care carrier.