Title:  Granule containing enzyme, corn starch and sugar layered on an inert particle

United States Patent:  6,790,643

Issued:  September 14, 2004

Inventors:  Becker; Nathaniel T. (Hillsborough, CA); Green; Thomas S. (Montara, CA)

Assignee:  Genencor International, Inc. (Palo Alto, CA)

Appl. No.:  180785

Filed:  June 25, 2002

Abstract

Granules that include a protein core are described. The protein core includes a protein matrix which includes a protein mixed together with a starch and optionally sugar such as sucrose. The protein matrix can be layered over a seed particle or the protein core can be homogeneous. The protein can be an enzyme or a therapeutic protein. A barrier layer may surround the protein core, and a coating can be applied to the seed particle, the protein matrix and/or the barrier layer.

SUMMARY OF THE INVENTION

The present invention provides a granule that includes a protein core that includes a protein matrix. The protein matrix includes at least one protein (e.g., one or more enzymes) mixed together with a starch. Optionally, a barrier layer can be layered over the protein core or a barrier material can be included in the protein core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

The present invention further provides a granule that includes a protein core that includes a protein matrix layered over a seed particle. The protein matrix includes at least one protein (e.g., one or more enzymes) mixed together with a starch. Optionally, a barrier layer can be layered over the enzyme core or a barrier material can be included in the enzyme core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

The present invention also provides a granule that includes an enzyme core that includes an enzyme matrix. The enzyme matrix includes one or more enzymes mixed together with a starch. Optionally, a barrier layer can be layered over the enzyme core or a barrier material can be included in the enzyme core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

The present invention additionally provides a granule that includes an enzyme core that includes an enzyme matrix layered over a seed particle. The enzyme matrix includes one or more enzymes mixed together with a starch. Optionally, a barrier layer can be layered over the enzyme core or a barrier material can be included in the enzyme core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

The other features, aspects and advantages of the present invention will become apparent from the following detailed description, in conjunction with the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is a granule that includes a protein core that includes a protein matrix. The protein matrix includes one or more proteins mixed together with a starch. Optionally, a barrier layer can be layered over the protein core or a barrier material can be included in the protein core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

A further embodiment of the invention is a granule that includes a protein core that includes a protein matrix layered over a seed particle. The protein matrix includes one or more proteins mixed together with a starch. Optionally, a barrier layer can be layered over the protein core or a barrier material can be included in the protein core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

Another embodiment of the invention is a granule that includes an enzyme core that includes an enzyme matrix. The enzyme matrix includes one or more enzymes mixed together with a starch. Optionally, a barrier layer can be layered over the enzyme core or a barrier material can be included in the enzyme core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

A further embodiment of the invention is a granule that includes an enzyme core that includes an enzyme matrix layered over a seed particle. The enzyme matrix includes one or more enzymes mixed together with a starch. Optionally, a barrier layer can be layered over the enzyme core or a barrier material can be included in the enzyme core. Also, optionally, a coating can be applied over the seed particle, the enzyme matrix and/or the barrier layer.

A "protein core", an "enzyme core" or a "core" includes a protein matrix, for example, an enzyme matrix in the case of an enzyme core. The matrix can be homogenous throughout the core or can be layered over a seed particle. There can be one or more layers between the seed particle and the matrix or the matrix and the barrier layer, for example, a coating such as polyvinyl alcohol (PVA).

Seed particles are inert particles upon which the enzyme matrix can be layered which can be composed, for example, of inorganic salts, sugars, sugar alcohols, small organic molecules such as organic acids or salts, minerals such as clays or silicates or a combination of two or more of these. Suitable soluble ingredients for incorporation into seed particles include: sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, sodium sesquicarbonate, urea, citric acid, citrate, sorbitol, mannitol, oleate, sucrose, lactose and the like. Soluble ingredients can be combined with dispersible ingredients such as talc, kaolin or bentonite. Seed particles can be fabricated by a variety of granulation techniques including: crystallization, precipitation, pan-coating, fluid-bed coating, fluid-bed agglomeration, rotary atomization, extrusion, prilling, spheronization, drum granulation and high shear agglomeration. In the granules of the present invention, if a seed particle is used then the ratio of seed particles to granules is 1:1.

The "protein matrix", "enzyme matrix" or "matrix" is an admixture of one or more proteins such as an enzyme and a starch. Optionally, the matrix can include a sugar, such as sucrose. The selected components can be mixed, for example, in solution or as a slurry. The protein can be applied from a solution or applied in slurry form as a suspension of crystals or precipitated protein. The matrix of the present invention comprises between about 20-80% of the weight of the granule.

By burying a protein within a matrix, the protein can be better protected from the twin dangers of attrition and activity loss. Also, to achieve a low dusting granular protein product, it is necessary to control the shape and size distribution of the granules. Uniform and reproducible size and shape also contribute to granule stability, since particle breakup and re-agglomeration would bring some protein near the granule surface.

Surprisingly, it has been found that by combining a starch with a protein, the protein can be applied uniformly to individual seed particles at rapid rates without agglomeration or attrition. The resulting particle size distribution can be precisely controlled, based on knowledge of the starting seed size distribution and the amount of solids to be added. The resulting particles are approximately spherical in shape, have high cohesive strength, and are resistant to attrition and penetration by moisture and inactivating substances.

Starches have high water solubility or dispersibility. A matrix formula can be easily prepared which includes starches and enzymes as a solution or slurry with high total solids concentration. Total solution or slurry solids concentrations of 20-50% w/w or more can be formulated. These concentrated mixtures are highly desirable in that they can be formed into granules with a minimal need for evaporating water, an advantage in any granulation and drying process.

Proteins that are within the scope of the present invention include pharmaceutically important proteins such as hormones or other therapeutic proteins and industrially important proteins such as enzymes.

Any enzyme or combination of enzymes may be used in the present invention. Preferred enzymes include those enzymes capable of hydrolyzing substrates, e.g. stains. These enzymes are known as hydrolases which include, but are not limited to, proteases (bacterial, fungal, acid, neutral or alkaline), amylases (alpha or beta), lipases, cellulases and mixtures thereof. Particularly preferred enzymes are subtilisins and cellulases. Most preferred are subtilisins such as described in U.S. Pat. No. 4,760,025, EP Patent 130 756 B1 and EP Patent Application WO 91/06637, which are incorporated herein by reference, and cellulases such as Multifect L250.TM. and Puradax.TM., commercially available from Genencor International. Other enzymes that can be used in the present invention include oxidases, transferases, dehydratases, reductases, hemicellulases and isomerases.

The matrix of the granules of the present invention may further comprise one or more synthetic polymers or other excipients as known to those skilled in the art. Suitable synthetic polymers include polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and polyethylene oxide/polypropylene oxide.

The matrix may also further comprise plasticizers and anti-agglomeration agents. Suitable plasticizers useful in the present invention include polyols such as glycerol, propylene glycol, polyethylene glycol (PEG), urea, or other known plasticizers such as triethyl citrate, dibutyl or dimethyl phthalate or water. Suitable anti-agglomeration agents include fine insoluble or sparingly soluble materials such as talc, TiO₂, clays, amorphous silica, magnesium stearate, stearic acid and calcium carbonate.

The granules of the present invention can further comprise a barrier layer. A barrier layer is used to slow or prevent the diffusion of substances that can adversely affect the protein or enzyme into the matrix. The barrier layer is made up of a barrier material and can be coated over the protein core or the barrier material can be included in the protein core. Suitable barrier materials include, for example, inorganic salts or organic acids or salts.

The granules of the present invention can also comprise one or more coating layers. For example, such coating layers may be one or more intermediate coating layers or such coating layers may be one or more outside coating layers or a combination thereof. Coating layers may serve any of a number of functions in a granule composition, depending on the end use of the enzyme granule. For example, coatings may render the enzyme resistant to oxidation by bleach, bring about the desirable rates of dissolution upon introduction of the granule into an aqueous medium, or provide a barrier against ambient moisture in order to enhance the storage stability of the enzyme and reduce the possibility of microbial growth within the granule.

Suitable coatings include water soluble or water dispersible film-forming polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), cellulose derivatives such as methylcellulose, hydroxypropyl methylcellulose, hydroxycellulose, ethylcellulose, carboxymethyl cellulose, hydroxypropyl cellulose, polyethylene glycol, polyethylene oxide, gum arabic, xanthan, carrageenan, chitosan, latex polymers, and enteric coatings. Furthermore, coating agents may be used in conjunction with other active agents of the same or different categories.

Suitable PVAs for incorporation in the coating layer(s) of the granule include partially hydrolyzed, fully hydrolyzed and intermediately hydrolyzed PVAs having low to high degrees of viscosity. Preferably, the outer coating layer comprises partially hydrolyzed PVA having low viscosity. Other vinyl polymers which may be useful include polyvinyl acetate and polyvinyl pyrrolidone. Useful copolymers include, for example, PVA-methylmethacrylate copolymer and PVP-PVA copolymer.

The coating layers of the present invention may further comprise one or more of the following: plasticizers, extenders, lubricants, pigments, and optionally additional enzymes. Suitable plasticizers useful in the coating layers of the present invention are plasticizers including, for example, polyols such as sugars, sugar alcohols, or polyethylene glycols (PEGs), urea, glycol, propylene glycol or other known plasticizers such as triethyl citrate, dibutyl or dimethyl phthalate or water. Suitable pigments useful in the coating layers of the present invention include, but are not limited to, finely divided whiteners such as titanium dioxide or calcium carbonate or colored pigments and dyes or a combination thereof. Preferably such pigments are low residue pigments upon dissolution. Suitable extenders include sugars such as sucrose or starch hydrolysates such as maltodextrin and corn syrup solids, clays such as kaolin and bentonite and talc. Suitable lubricants include nonionic surfactants such as Neodol, tallow alcohols, fatty acids, fatty acid salts such as magnesium stearate and fatty acid esters.

Adjunct ingredients may be added to the enzyme granules of the present invention. Adjunct ingredients may include: metallic salts; solubilizers; activators; antioxidants; dyes; inhibitors; binders; fragrances; enzyme protecting agents/scavengers such as ammonium sulfate, ammonium citrate, urea, guanidine hydrochloride, guanidine carbonate, guanidine sulfamate, thiourea dioxide, monoethanolamine, diethanolamine, triethanolamine, amino acids such as glycine, sodium glutamate and the like, proteins such as bovine serum albumin, casein and the like etc.; surfactants including anionic surfactants, ampholytic surfactants, nonionic surfactants, cationic surfactants and long-chain fatty acid salts; builders; alkalis or inorganic electrolytes; bleaching agents; bluing agents and fluorescent dyes and whiteners; and caking inhibitors.

For granules having a matrix that includes, in addition to starch, a sugar (such as sucrose), it may be desirable to keep the sugar content of the matrix very low (e.g., substantially less than the starch content). For example, in a particular spray coating process, similar to that described in the examples below, amylase granules having a 1:1 corn starch:sucrose matrix were found to be very tacky and had a tendency to agglomerate during the spray cycle. A drastic reduction in the sucrose content alleviated the tackiness of the matrix. Accordingly, in certain circumstances, it is preferred to employ a matrix having a high starch content relative to the sucrose content. In one preferred embodiment, the ratio (w/w) of starch to sucrose is much greater than 1:1, e.g., in the range of about 5:1 to 15:1. For example, the ratio can be about 10:1.

In an exemplary formulation, sucrose is present in the matrix in an amount of from about 0.5% (w/w) to about 8% (w/w), relative to the total weight of the granules; and preferably in an amount of about 2% (w/w). In one particularly preferred embodiment, corn starch is present in the matrix in an amount of about 23% and sucrose is present in the matrix in an amount of about 2% (again, w/w, relative to the total weight of the granules). The sucrose content of this embodiment can be increased above 2%, but preferably should not exceed equal parts of corn starch. For example, for every 1% increase of sucrose added to the matrix, an equal amount of corn starch is subtracted from the matrix. Thus, if the sucrose content is increased from 2% up to 5%, the corn starch content would be adjusted down from 23% to 20%. In this example, the maximum sucrose content would be 12.5%, equaling the calculated corn starch content.

While little or no sugar may be useful in certain circumstances (e.g., as just discussed), it should be appreciated that other circumstances (e.g., where agglomeration does not present a significant problem) may call for a higher sugar content in the matrix.

The granules described herein may be made by methods known to those skilled in the art of enzyme granulation, including pan-coating, fluid-bed coating, prilling, disc granulation, spray drying, extrusion, centrifugal extrusion, spheronization, drum granulation, high shear agglomeration, or combinations of these techniques.

Claim 1 of 19 Claims

It is claimed:

1. A granule comprising:

an admixture layered over an inert particle, the admixture consisting essentially of; (I) an enzyme, (ii) a corn starch, and (iii) a sugar, wherein the ratio (w/w) of corn starch to sugar is greater than 1:1; and

a barrier layer surrounding the admixture.

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