JP2004518140A - Method for analyzing a granular composition by fluorescence analysis - Google Patents

Abstract

The present invention illuminates a granular composition comprising a purified bioactive compound with light capable of fluorescently exciting a fluorescent marker contained in the granular composition, and detects light emitted from the fluorescent marker And predicting the amount of a fluorescent marker in the particulate composition accessible to fluorescence excitation.

Description

[0001]
Field of the invention
The present invention relates to a method of analyzing the properties of a particulate composition comprising a biologically active compound by subjecting the particulate composition to a fluorescence analysis. The present invention also relates to a method for producing a specific product by subjecting the product to fluorescence analysis. Furthermore, the invention relates to a granulation and / or coating device suitable for preparing a granular composition comprising a biologically active compound comprising means for fluorescence analysis.
[0002]
Background of the Invention
The analysis of a chemical compound in a sample (fluorescence analysis) utilizing a compound (fluorescent substance) that emits fluorescence when excited by light is well known in the art. While fluorescence analysis has the advantage of being sensitive and accurate in well-defined samples, it also has significant disadvantages. For example, in complex samples, the fluorescence of the phosphor is often altered by other compounds present in the environment surrounding the phosphor (known as quenching), and is complex and / or poorly defined. It becomes difficult to use the sample quantitatively for fluorescence analysis. This applies especially to heterogeneous or solid-state samples, where light scattering must also be dealt with.
[0003]
During the last few years of electronics development, the emergence of more complex fluorescence imaging methods using camera detectors, making it possible to take pictures of emitted light on fluorescent samples, so that two-dimensional images are 3 shows the spatial distribution of the phosphor in the sample.
One example of fluorescent imaging of aleurone tissue for powder purification using ultraviolet excitation is known from the following reference: Dexter et al., Cereal Chemistry, Vol. 70 (1), 90-95, 1993.
See Kaufman et al., Powder Technology, Vol. 78 (3), 239-246, 1994 performed in situ visualization of coal particle distribution in liquid fluidized beds using fluorescence microscopy.
[0004]
More common examples of imaging in analysis are described in Buydens et al., Analytical Chimica Acta, Vol. 361 (1-2), 161-176, 1998, where online classification and multivariate image analysis for plastics in waste based on imaging using light near the infrared range. It has been reported.
[0005]
Watano et al., Pharmaceutical Bulletin, Vol. 48 (8), 1154-1159, 2000 reported on-line monitoring of grain growth in high shear granulation with an imaging processing system.
Watano et al., In U.S. Pat. No. 5,497,232, use a photographic camera, for example, a CCD camera of the type used in video cameras, to image grain growth in a fluidized bed or pan granulator. reported.
[0006]
Summary of the Invention
The present invention relates to a method for analyzing the properties of a granular composition comprising a purified biologically active compound by subjecting the granular composition to a fluorescence analysis. In order to improve the properties of the product and thus make it more commercially attractive, it usually requires incorporating the compound into the finished product, especially granulation. However, for bioactive compounds, granulation is often compulsory for the manufacturer because the product must be separated from the environment surrounding the bioactive compound to ensure safe handling of the product until applied in the intended use.
[0007]
The amount of bioactive compound that may escape from the granulated product, for example in the form of dust, must be kept to a minimum so that the person handling the product is not adversely affected by contact with the bioactive compound. No. Conversely, the active compound must be protected from the environment outside the granules and, when used, must remain stable and active. Once the active compound has been granulated, the granules comprising the biologically active compound are further coated to further inhibit release of the active compound from the granules and further enhance the stability of the active compound in the granules. It is known to improve. It is usually preferred to further improve the properties of the granules by increasing the thickness of the coating layer.
[0008]
One object of the present invention is to provide a method for measuring quality parameters of a granular composition comprising a purified bioactive compound, especially in the form of an imaging system. Such parameters include, for example, the amount of active compound released from the granules in the form of active dust during or after the process of making the granule composition. Another parameter is the thickness and / or the integrity of the coating applied to the granules to reduce dust formation and increase the stability of the active compound. Such thickness and / or integrity can be measured according to the invention during or after the coating process of the particulate composition comprising the biologically active compound. Another object of the present invention is to design a granulation apparatus and use this method online or inline in the manufacture of such a pharmaceutical composition, and to use this method in real time during the processing of the granular composition for the bioactive compound. Is to select the configuration of this method so that it can provide information about the level of dust comprising.
[0009]
For a particulate composition comprising a purified bioactive compound, by illuminating the composition with a fluorescent marker, e.g., light capable of fluorescence excitation of the bioactive compound itself, by detecting light emitted from the fluorescent marker Assess both the active compound restricted to the surface area of the granules and the active compound present in the composition in the dust particles, for example resulting from release of the active compound from the granules or insufficient granulation We have found what we can do. In a first aspect, the present invention provides a method of illuminating a particulate composition comprising a purified bioactive compound with light capable of fluorescently exciting a fluorescent marker contained in the particulate composition, comprising: Provided is a fluorescence analysis method comprising detecting emitted light and predicting the amount of a fluorescent marker in a particulate composition accessible to fluorescence excitation. The amount of accessible fluorescent marker can be related to the nature of the particulate composition, for example, the level of dust and / or the thickness or integrity of the coating.
[0010]
Further, in a second aspect, the present invention provides a bioactive compound purified in a granulator, a fluorescent marker, comprising a step of performing a fluorescence analysis according to the first aspect of the invention (see above). Provided is a method for producing a granule comprising an auxiliary granulating agent as required.
[0011]
Still further, in a third aspect, the present invention provides a granulation or coating apparatus comprising: (a) at least one chamber for granulating or coating the granulated material; At least one detector capable of detecting light; (c) means for projecting an illumination source onto a portion of the granules to be processed; (d) firing light emitted from the illuminated granules to the detector. A granulating or coating device comprising: (e) at least one device for selecting the wavelength of the illumination light or the emitted light.
Finally, in a fourth aspect, the invention provides the use of fluorescence analysis on a granule comprising a purified bioactive compound.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Fluorescence analysis
The present invention relates to a method for performing a fluorescence analysis on a granular composition comprising an active compound, as described above.
[0013]
The first step of the method comprises illuminating the particulate composition with a light beam capable of fluorescently exciting a fluorescent marker contained in the particulate composition. In this process, photons from the illuminating light are absorbed by the fluorescent marker, so that the electrons in the fluorescent compound gain energy and are brought to a particular increased energy level. An excited fluorescent marker is one in which electrons return to their original ground state energy levels and subsequently emit photons of light at wavelengths characteristic of the energy difference between the increased and original energy levels. This releases at least some of the acquired energy.
[0014]
The second step of the method comprises detecting light emitted from the particulate composition with a detector capable of converting emitted light into an electronic signal.
The third step of the method involves processing the electronic signal to predict the amount of fluorescent marker in the particulate composition accessible to fluorescence excitation, thereby reducing the amount of emitted light to one or two of the particulate composition. It is related to the above properties.
The interpretation and meaning of all terms relating to the basic principles of fluorescence analysis are known to those skilled in the art.
[0015]
Lighting of granular composition
Illumination of the granule composition can be performed using any suitable light source that emits lubricated granules capable of exciting fluorescent markers in the granule composition. The light source can be, for example, a conventional glow lamp, a more specialized xenon lamp or a strobe light bulb.
[0016]
The optical properties of the fluorescent marker compound are known, and the excitation of the known fluorescent marker can be optimized, and it is preferred to select a substantial portion of light at a wavelength appropriate for exciting the fluorescent marker. If it is desirable to avoid excitation of and emission from other compounds besides the fluorescent marker, which can interfere with the analysis (this can occur when using known fluorescent markers), light of a selected wavelength It may be desirable to filter the light beam so that only the particulate composition illuminates.
[0017]
This may be performed using one or more beam splitters and one or more bandpass filters, eg, high and / or low bandpass filters, or a grating monochromator that only passes light of a particular wavelength. Can be. These features are usually integrated in commercially available fluorometers, for example, available from Perkin Elmer, USA. It is known in the art that bandpass filters and monochromators pass light having a wavelength in a narrow range, typically within a few nm, for example, 0.5 to 10 nm. Thus, monochromatic light is to be understood as light of a wavelength within a narrow range determined by a bandpass filter or a grating monochromator.
[0018]
In certain embodiments, the light illuminating the particulate composition consists of 1 to 10 discrete monochromatic wavelengths, especially 1 to 4 discrete monochromatic wavelengths. In particular, the light illuminating the granular composition consists of one discrete monochromatic wavelength. In the step of illuminating the particulate composition, the illumination on the particulate composition is suitably performed using, for example, an optical arrangement comprising a mirror, a beam splitter (eg, a dichroic mirror) and / or fiber optics. Can emit light.
[0019]
Synchrotron radiation direction
The emitted light can be characteristic of the fluorescent marker, or of a chemical group or component contained within the fluorescent marker. Since fluorescent markers usually emit only one or two or more narrow ranges of wavelengths, it is preferable to filter the emitted light so that only the emitted light within these ranges reaches the detector. This can be achieved by filtering the emitted light with one or more bandpass filters or monochromators as described above. Thus, in a particular embodiment, only radiation of 1 to 10 discrete monochromatic wavelengths, in particular 1 to 4 discrete monochromatic wavelengths, is detected. In particular, the radiation reaching the detection consists of one discrete monochromatic wavelength.
[0020]
In addition, with respect to illumination, the step of detecting the emitted light suitably employs, for example, a mirror, a beam splitter (eg, a dichroic mirror), fiber optics and / or an optical arrangement comprising means for focusing the emitted light. Thus, the illumination light can be projected into the detector.
[0021]
Various detectors can be used to detect the emitted light. The detector can be a photomultiplier detector, a photodiode or photodiode array, a line scan camera, a CCD camera, an ICCD camera or any other type suitable for detecting emitted light. The particular camera is a camera-type detector, for example, a detector selected from the group consisting of a grayscale camera, a line scan camera, a photodiode array, a CCD (charge coupled device) camera and an ICCD (integrating CCD) camera. Can be. CCD and ICCD cameras can be the cameras of choice because they are more sensitive and can form two-dimensional images that show the spatial distribution of light-emitting particles or dust particles.
[0022]
This is called fluorescence imaging by trained analysts. In certain embodiments, two or more detectors are used to simultaneously record two or more selected wavelengths or two or more ranges of wavelengths. This is desirable if more than one fluorescent marker is to be measured, or if a particular fluorescent marker emits light of different wavelengths. In particular, this is achieved using an optical arrangement including one or more beam splitters and two or more bandpass filters or monochromators. In the most particular embodiment, an optical arrangement including two CCD cameras, a dichroic mirror, a bandpass filter and a lens is used, as shown schematically in FIG.
[0023]
Processing detected signals
Converting the emitted light into an electronic signal at the detector and converting this signal into a measurement, for example, a number, from which to predict the amount of emitted light and the amount of fluorescent markers accessible to excitation, perform the fluorescence analysis Such analyzers are abundantly available and are known to those skilled in the art. A fluorescent marker that compares data on light emitted from a granule composition with known properties with the amount of light emitted from an unknown granule composition, and thus is accessible to excitation in an unknown granule composition By predicting the amount of, this prediction can be performed appropriately.
[0024]
The output of most detectors, for example of the type based on photomultiplier or of the type based on some photodiodes, is an analog signal. Most detectors, including multiple single photodiode detectors, for example, multiple cameras, can have a built-in analog-to-digital converter that can convert analog signals to digital signals. Analog-to-digital converters are more suitable for computerized data processing. Depending on the fluorescent marker and nature of the particulate composition that is to be related to the amount of emitted light, the digital data resulting from the emitted light can be processed.
[0025]
This process is suitably performed in a computer system using software designed for such a process. Such software is LabView software as used in the examples herein, or provides the ability to perform the desired data processing relating the amount of emitted light to the properties of the particulate composition. You could be any other software. Data processing is an operation that is a data processing function included in commercially available software, such as particle counting, gauging, pattern matching (grayscale and color), statistics, limits, multivariate Image analysis, AMT, speckle analysis, area calculation, edge detection, morphological analysis, convolution, folding and unfolding, FFT, various filtration techniques, such as median filtration-all techniques are known to trained analysts Yes-can be included.
[0026]
In the process of transferring data to a computational unit, the computational unit must be equipped with hardware that typically obtains data to be stored in the computational unit from a detector. When using a CCD camera or other type of camera that produces a two-dimensional image of the phosphor particles, it is also advantageous to use software in the computing unit that can record the two-dimensional image in the form of a discrete digital still image. is there. Such software is known as a frame grabber program. The speed at which such software can record images usually depends on the speed of the computational unit, and for most fluorescence analysis purposes, a speed of about 15 frames per second is sufficient. This means recording a two-dimensional image at 15 / sec.
[0027]
Granular composition
Physical properties
The granule compositions of the present invention comprise a bioactive compound, a fluorescent marker, which can be the bioactive compound itself, and optionally auxiliary granulating and coating agents, which are processed into particles or granules. A composition comprising: Thus, the finished granules are the result of the processes and methods of the present invention. The term “granules” refers to predominantly spherical or spherical particles having an average size of 20-2000 μm, more particularly 100-1000 μm, most especially 200-800 μm, measured at the size of the macromolecule, in particular the longest diameter It should be understood as a structure close to. Preferably, the spherical structure has a ratio between the smallest dimension diameter (a) and the largest dimension diameter (b) of 1: 1 to 1: 5, in particular 1: 1 to 1: 3, (a) :( b)
[0028]
The "size distribution" (PSD) of a granule can be described by the mass average diameter of the individual particles. The average mass diameter of D50 is the diameter at which 50% by weight of the granules have a smaller diameter, but 50% by weight has a larger diameter. The values D10 and D90 are the diameters at which 10% and 90% respectively by weight of the granules have a diameter smaller than the value in question. “SPAN” indicates the width of the PSD and is expressed as:
(D90-D10) / D50
[0029]
For the purposes of the present invention, the PSD of the granules after granulation is usually as narrow as possible. According to the present invention, the use of fluorescence analysis to control the granulation process promotes a reduction in PSD, and thus the SPAN of the granulated granule composition is especially less than about 2.5, especially less than about 2.5. Less than 2.0, more particularly less than about 1.5, and most especially less than about 1.0.
[0030]
The granules are in particular coated with a coating agent. The coating forms a particularly homogeneous, cohesive continuous layer around the granules. The term coating agent, as used herein, should be understood as a single coating compound or a mixture of coating compounds. Thus, the coated granules consist of a granule core and a granule coating. In particular, the coating layer is relatively thick and further reduces dusting of the bioactive compound and improves stability. The thickness of the coating is the ratio between the average diameter of the coated granule core and the uncoated granule core (hereinafter D_G/ D_C), That is, a value obtained by dividing the average diameter of the coated granules by the average diameter of the granule core alone.
[0031]
For example, if a granule core of 100 μm diameter is coated with a 200 μm thick coating layer, the granules have a diameter (200 + 100 + 200) = 500 μm and D_G/ D_CIs 500 μm / 100 μm = 5. The coated granules according to the invention preferably have a D of at least 1.1._G/ D_CWhich means that the thickness of the coating is at least 5% of the average granular core diameter. More particularly D_G/ D_CIs at least 1.5, more particularly at least 2, more particularly at least 2.5, more particularly at least 3, most especially at least 4. However, D_G/ D_CIs especially about 100 or less, especially about 50 or less, more particularly 25 or less, most especially 10 or less. D_G/ D_CIs the most specific range from about 4 to about 6.
[0032]
Further, in the present invention, the coating is substantially free of enzymes. The term "substantially free of enzymes" as used herein means that there is no more than 5 mg of enzyme per gram of coating agent for the coating.
In view of the materials that make up the granules of the present invention (see below), these materials generally do not transmit visible light, as opposed to transparent materials such as glass, for example, usually the granules are not transparent. . However, when exposing particles comprising a biologically active compound to excitation light, such excitation light penetrates the particle surface and excites a suitable fluorescent compound present at and / or near the particle surface. We have found that light emitted from such fluorescent compounds escapes from the granules and is received by the detector.
[0033]
Furthermore, the moisture in the granules is typically less than 20 w / w%, in particular less than 15 w / w%, more particularly less than 10 w / w% and in the range of 4-8 w / w%. is there.
[0034]
Construction
Granules can be constructed by any of the granulation methods known in the art.
The construction of the granules of the present invention can be divided into the following non-exhaustive categories:
a) Spray-dried granules, wherein a liquid solution containing the biologically active compound is atomized in a spray dryer to form small liquid particles, which drop the biologically active compound while falling through the dryer. Form a granular material comprising. In this way, very small granules can be produced (Michael S. Showwell (editor); Powered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). For these granules, the biologically active compound is homogeneously mixed with any other auxiliary granulating agents present in the liquid solution.
[0035]
b) layered granules, wherein the bioactive compound is coated as a layer around the preformed core particles, wherein the solution containing the bioactive compound, and especially the auxiliary granulating agent, is typically preformed. In a fluidized bed apparatus in which the separated core particles are fluidized, the solution of the bioactive compound adheres to the core particles and dries to leave a dry layer of the bioactive compound on the surface of the core particles. If useful core particles of the required size can be found, the required size granules can be obtained in this way. Granules of this type are described, for example, in WO 97/23606.
[0036]
c) Rather than coating the bioactive compound as an absorbed core granule, here as a layer around the core, the bioactive compound is absorbed onto and / or into the core surface. Such a method is described in WO 97/39116.
[0037]
d) Extruded or pelletized granules, wherein the paste containing the biologically active compound is pressed in a mold or extruded under pressure through a small opening, cut into granules and subsequently dried. I do. Such particles are of considerable size since the material making up the extrusion opening (usually a plate with perforations) limits the allowable pressure applied to the extrusion opening. Also, when using small openings, very high extrusion pressures increase the exotherm in the paste, which can be detrimental to bioactive compounds. (Michael S. Showwell (editor); Powered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).
[0038]
e) Spray-cooled granules, wherein the powder of the biologically active compound is suspended in molten wax, and this suspension is sprayed into a cooling chamber, for example through a rotating disc-type atomizer. Liquid solidifies rapidly (Michael S. Showell (editor); Powered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). For these granules, the bioactive compound is homogeneously mixed with the wax instead of concentrating on the granule surface. References related to this technique are U.S. Pat. No. 4,016,040 and U.S. Pat. No. 4,713,245.
[0039]
f) Add the granules of the high shear mixer, here a liquid containing the biologically active compound, to the dry powder composition of the auxiliary granulating agent. The appropriate ratio of liquid and powder is mixed, and when the moisture of the liquid is absorbed into the dry powder, the dry powder adheres, agglomerates, builds up granules, and comprises particles comprising the biologically active compound. The body forms. For these granules, the active compound is homogeneously mixed with the auxiliary granulating agent. Such a method is disclosed in U.S. Pat. No. 4,106,991 (NOVO NORDISK) and related documents EP 170360 B1 (NOVO NORDISK), EP 304332 B1 (NOVO NORDISK), EP 304331 (NOVO NORDISK), WO 90/09440 (NOVO NORDISK). NOVO NORDISK) and WO 90/09428 (NOVO NORDISK).
[0040]
Dust particles in granular composition
Dust particles, which may be present in the granule composition, are fragments of whole granules, usually much smaller than granules, and are characterized by having no sphere characteristic of granules. Typically, the dust particles have an irregular, non-spherical, truncated structure, for example, rod or flake shape. Dust particles are typically much smaller than the average size of the granules, and most dust particles have a diameter of less than 20 μm, depending on the granule composition.
Dust particles, which are fragments of whole grains, have the same opacity and usually have the same moisture as the aforementioned whole grains.
Compound in granular composition
[0041]
Biologically active compounds
The granular compositions of the present invention comprise a biologically active compound, especially a purified biologically active compound. The term bioactive compound, as used herein, is understood as any compound that is active in a biological system, for example, a compound that interferes with and / or modifies a biological pathway or reaction. Should. The term “purified” should be understood as a biologically active compound that has been subjected to one or more purification steps prior to granulation, for example, to remove excess material and / or concentrate the active compound. is there. If the active compound is produced by a microbiological fermentation process, the purification step involves filtering, especially to remove biomass and other undesirable substances, including water, which result in a mixture that is enriched in the biologically active compound. Including a step selected from ultrafiltration, flocculation, sedimentation, evaporation, extraction and others.
[0042]
Bioactive compounds include, inter alia, organic compounds such as biocatalysts, therapeutics, herbicides, pesticides and fungicides. In particular, a biologically active compound can be produced by fermenting a microorganism that produces the active compound.
[0043]
In particular, the compounds are proteins and peptides, more particularly catalytic proteins, ie enzymes. Because proteins such as enzymes are used industrially in very large volumes and are known to cause adverse allergic reactions in humans and animals when exposed to such proteins. In addition, enzymes are widely used in household products, such as detergents for removing biological soils, and many industrial processes involve the handling of enzymes by humans. The enzyme can be any enzyme for which it is desirable to separate the enzyme from the surrounding environment through granulation of the enzyme.
[0044]
The classes of enzymes used herein are as follows: Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Pharmacy, Pharmacy. , 1992.
Accordingly, the types of enzymes that can be suitably incorporated into the granules of the present invention include: oxidoreductases (EC 1 .-.-.-), transferases (EC 2 .-.-.-), Hydrolases (EC 3 .-.-.-), lyases (EC 4 .-.-.-), isomerases (EC 5 .-.-.-) and ligases (EC 6 .-.-.-).
[0045]
In particular, the oxidoreductases in the context of the present invention are peroxidase (EC 1.11.1), laccase (EC 1.10.3.2) and glucose oxidase (EC 1.1.3.4) Is a transferase in any of the following subclasses:
[0046]
a) transferase for transferring one carbon group (EC 2.1);
b) transferases for transferring aldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3);
c) glycosyltransferase (EC 2.4);
d) a transferase (EC 2.5) that transfers an alkyl or aryl group other than methyl; and
e) Transferase to transfer the nitro generating group (EC 2.6).
A particular type of transferase in the context of the present invention is transglutaminase (protein-glutamine γ-glutamyltransferase; EC 2.3.2.13).
[0047]
Further examples of suitable transglutaminases are described in WO 96/06931 (Novo Nordisk A / S).
Particular hydrolases in the context of the present invention are as follows: carboxylate hydrolases (EC 3.1.1.1.-), for example lipases (EC 3.1.1.3); phytases (EC 3.1). 3.3-), for example, 3-phytase (EC 3.1.3.8) and 6-phytase (EC 3.1.3.26); glucosidase (EC 3.2, which is referred to herein as " Carbohydrases), such as α-amylase (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.
[0048]
In the context of the present invention, the term "carbohydrase" only refers to enzymes (ie glucosidases, EC 3.2) which are capable of breaking carbohydrate chains, in particular of 5- and 6-membered ring structures (eg starch or cellulose). Not only, but also used to mean an enzyme capable of isomerizing a carbohydrate, eg, a six-membered ring structure, eg, D-glucose, to a five-membered ring structure, eg, D-fructose.
[0049]
Relevant carbohydrases include the following (EC numbers in parentheses): α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), glucan 1,4-α-glucosidase (EC 3.2.1.3), cellulase (EC 3.2.1.4), endo-1,3 (4) -β-glucanase (EC 3.2.2.1. 6), endo-1,4-β-xylanase (EC 3.2.1.8), dextranase (EC 3.2.1.11), chitinase (EC 3.2.1.114), polygalacturo Nase (EC 3.2.1.15), lysozyme (EC 3.2.1.17), β-glucosidase (EC 3.2.1.21), α-galactosidase (EC 3.2.1.22) ), Β-galactosidase (EC 3.2.1. 3), amylo-1,6-glucosidase (EC 3.2.1.33), xylan 1,4-β-xylosidase (EC 3.2.1.37), glucan end-1,3-β-D -Glucosidase (EC 3.2.1.39), α-dextrin endo-1,6-α-glucosidase (EC 3.2.1.41), sucrose α-glucosidase (EC 3.2.1.48) , Glucan end-1,3-α-glucosidase (EC 3.2.1.59), glucan 1,4-β-glucosidase (EC 3.2.1.74), glucan end-1,6-β- Glycosidase (EC 3.2.1.75), arabinan endo-1,5-α-L-arabinosidase (EC 3.2.1.99), lactase (EC 3.2.1.108), Chitosanase (EC 3. 2.1.132) and xylose isomerase (EC 5.3.1.5).
[0050]
Examples of commercially available oxidoreductases (EC 1 .-.-.) Are GLUZYME^TM (Enzymes available from Novo Nordisk A / S). Examples of commercially available proteases (peptidases) are as follows: KANNASE^TM, EVERLASE^TM, ESPERASE^TM, ALCALASE^TM, NEUTRASE^TM, DURAZYM^TM, SAVINASE^TM, PYRASE^TM, PANCREATIC TRYPSIN NOVO (PTN), BIO-FEED^TM, PRO and CLEAR-LENS^TM PRO (all available from Novo Nordisk A / S, Bagsvaerd, Denmark).
[0051]
Other commercially available proteases include: MAXATASE^TM, MAXACAL^TM, MAXAPEM^TM, OPTICLAEAN^TMAnd PURAFECT^TM (Available from Genencor International Inc. or Gist-Brocades).
Examples of commercially available lipases are as follows: LIPPRIME^TM, LIPOLASE^TM, LIPOLASE^TM ULTRA, LIPOZYME^TM, PALATASE^TM, NOVOZYME^TM 435 and LECITASE^TM (All are available from Novo Nordisk A / S).
[0052]
Examples of other commercially available lipases are as follows: LUMAFAST^TM (Lipase of Pseudomonas mendocina from Genencor International Inc.); LIPOMAX^TM (Ps. Pseudoalcaligenes lipase from Gist-Brocades / Genencor International Inc .; and Bacillus species lipase from Solvay enzyme).
[0053]
Examples of commercially available carbohydrases are as follows: ALPHA-GAL^TM, BIO-FEED^TM ALPHA, BIO-FEED^TM BETA, BIO-FEED^TM PLUS, BIO-FEED^TM PLUS, NOVOZYME^TM 188, CELLUCLAST^TM, CELLUSOFT^TM, CEREMYL^TM, CITROZYME^TM, DENIMAX^TM, DEZYME^TM, DEXTROZYME^TM, FINIZYM^TM, FUNGAMYL^TM, GAMANASE^TM, GLUCANEX^TM, LACTOZYM^TM, MALTOGENASE^TM, PENTOPAN^TM, PECTINEX^TM, PROMOZYME^TM, PULPZYME^TM, NOVAMYL^TM, TERMAMYL^TM, AMG^TM (Amyloglucosidase Novo), MALTOGENASE^TM, SWEETZYME^TMAnd AQUAZYM^TM (All are available from Novo Nordisk A / S). Further carbohydrases are available from other suppliers.
[0054]
The amount of enzyme to be incorporated into the granules of the present invention will depend on the intended use of the granules. For many applications, the enzyme content will be as high as possible or practicable.
The enzyme content (calculated as pure enzyme protein) in the granules of the invention will typically range from about 0.5 to 50% by weight of the enzyme-containing granules.
[0055]
Auxiliary granulating agent
In particular, the granules of the present invention may be supplemented for purposes such as promoting granulation, controlling the density and volume of the granules, controlling the amount of active compound in the granules, stabilizing the active compound, and the like. Contains granules.
[0056]
Supplementary granulating agents include, but are not limited to:
a) Fillers, for example fillers customary in the field of granulation, water-soluble and / or insoluble inorganic salts, for example finely divided alkali sulphates, alkali carbonates and / or alkali chlorides), clays, for example , Kaolin (for example, Speshitte^TM, English China Clay), bentonite, talc, zeolites, and / or silicates.
[0057]
b) Binders, for example binders customary in the field of granulation, for example binders with or without a high melting point in all or part of the non-wax properties, for example polyvinylpyrrolidone, dextrin, Polyvinyl alcohol, cellulose derivatives such as hydroxypropylcellulose, methylcellulose or CMC. Suitable binders are carbohydrate binders, such as Glucidex 21D (available from Roquette Freres, France).
[0058]
c) Fiber materials, for example fibers commonly used in the field of granulation. Pure or impure cellulose in fiber form can be sawdust, pure fibrous cellulose, cotton, or pure or impure fibrous cellulose. Also, filter aids based on fibrous cellulose can be used. Several brands of cellulose in the form of fibers are sold, for example, CEPO and ARBOCELL. In the publication "Cepo Cellulose Powder" from Svenska Traejolsfavrikerna AB, for Cepo S / 20 cellulose, the approximate maximum fiber length is 500 μm, the approximate average fiber length is 160 μm, and the approximate maximum fiber width is Is 50 μm and the estimated average fiber width is 30 μm.
[0059]
It is also described that Cepo SS / 200 cellulose has an estimated maximum fiber length of 150 μm, an estimated average fiber length of 50 μm, a maximum fiber width of 45 μm, and an estimated average fiber width of 25 μm. . Cellulose fibers having these dimensions are very well suited for the purposes of the present invention. The languages "Cepo" and "Arbocel" are trademarks. Preferred fibrous cellulose is Arbocel^TM BFC200. Also, as described in EP 304331 B1, synthetic fibers can be used, and typical fibers can be made from polyethylene, polypropylene, polyester, especially nylon, polyvinyl format, poly (meth) acrylic compounds. it can.
[0060]
d) Liquid agents, for example, liquid agents commonly used in the field of granulation. Liquid agents are used in conventional mixer granulation processes to granulate the accumulation or agglomeration of conventional granulated component particles. Liquid agents are water and / or waxy substances. Liquid agents are always used in the liquid phase in the granular process, but can later solidify; thus, if a wax is present, dissolve or disperse it in water or melt.
[0061]
The term “waxy material” as used herein means a material having all of the following properties: 1) a melting point of 30-100 ° C., preferably 40-60 ° C .; It is tough and non-brittle in nature, and 3) the material is somewhat plastic in the greenhouse. Both water and wax are liquid agents, i.e., they are both active during the formation of granules; the wax remains as a component in the finished granules, but the water Portions are removed during the drying process. Examples of waxy substances are polyglycols, fatty alcohols, ethoxylated fatty alcohols, mono-, di- and triglycerol esters of higher fatty acids, such as glycerol monostearate, alkylaryl ethoxylates, and coconut ethanolamide. is there.
[0062]
If a large amount of wax is used, a relatively small amount of water is added, and vice versa. Thus, the liquid agent can be water alone, a waxy substance alone or a mixture of water and a waxy substance. When using a mixture of water and a waxy substance, the water and the waxy substance can be added in any order, for example, adding water first and then the waxy substance, or first adding the waxy substance Then, a solution or suspension of water or a waxy substance in water can be added. Also, when using a mixture of water and a wax, the wax can be soluble or insoluble (but dispersible) in the water. When water is used as the liquid agent, the water cannot be part of the finished mixer granules, since usually the majority of the water is dried off during subsequent drying of the mixer granules.
[0063]
e) Enzyme stabilizers or active ingredient protectants, for example enzyme stabilizers or active ingredient protectors commonly used in the field of granulation. Stabilizers or protectants fall into several categories: alkaline or neutral substances, reducing agents, antioxidants and / or metal ion salts of the first transition series. Each of these can be the same or used in combination with different categories of other protective agents. Examples of alkaline protectants are silicates, carbonates or bicarbonates of alkali metals, which provide a chemical scavenging action, for example, by actively neutralizing oxidants.
[0064]
Examples of reductive protecting agents are sulfites, thiosulfites or thiosulfates, while examples of antioxidants are methionine, butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA). The most preferred stabilizer or protecting agent is a thiosulfate, for example, sodium thiosulfate. Enzyme stabilizers can also be borates, borax, formate, dicarboxylic acids, tricarboxylic acids and reversible enzyme inhibitors, such as organic compounds with sulfhydryl groups or alkylated or arylated borates.
[0065]
f) Crosslinking agents, for example, those commonly used in the field of granulation. Crosslinking agents are enzyme-compatible surfactants, for example ethoxylated alcohols, especially alcohols having 10 to 80 ethoxy groups.
In addition, suspending agents, mediators (e.g. mediators or enzyme mediators that promote the bleaching action when dissolving the granules in the washing) and / or solvents can be incorporated as auxiliary granulating agents.
[0066]
Coating agent
The coating comprises one or more of the coating agents described in the following patent applications: WO 89/08694, WO 89/08695, EP 270 608 B1 and / or WO 00/01793. Other examples of coating agents can be found in the following patent applications: US Patent No. 4,106,991, EP 170360, EP 304332, EP 304331, EP 458849, EP 458845, WO 97/39116, WO. 92 / 12645A, WO 89/08695, WO 89/08694, WO 87/07292, WO 91/06638, WO 92/13030, WO 93/07260, WO 93/07263, WO 96/38527, WO 96/16151, WO 97/23606, U.S. Pat. No. 5,324,649, U.S. Pat. No. 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229 A, DD 263790, JP 61162185 A and / or J. P 58179492. The salt coatings described in WO 00/01793 are useful for coatings in the present invention.
[0067]
The coating agent can be selected from the list of auxiliary granulating agents mentioned above. In addition, coatings can be selected from the following list of polymers, chlorine scavengers, plasticizers, pigments, lubricants (eg, surfactants or antistatics) and fragrances.
Polymers useful in the coating layer include vinyl polymers or vinyl copolymers, such as polyvinyl alcohol (PVA) and / or polyvinyl pyrrolidone or derivatives thereof. Also included are isophthalic acid polymers.
[0068]
Useful plasticizers in the coating layer in the context of the present invention include, for example: polyols, such as sugars, sugar alcohols or polyethylene glycols (PEG) having a molecular weight of less than 1000; ureas, phthalate esters, For example, dibutyl or dimethyl phthalate; and water.
Suitable pigments include, but are not limited to, finely divided white extenders, such as titanium dioxide or kaolin, colored pigments, water-soluble colorants, and one or more pigments and water-soluble colorants. Combination with.
[0069]
As used in the context of the present invention, the term “lubricant” reduces surface friction, lubricates granule surfaces, reduces the tendency to accumulate static electricity, and / or reduces the friability of granules, Any substance. Also, by reducing the tackiness of the binder in the coating, a relevant role can be played in improving the coating process. Thus, the lubricant can act as an anti-agglomerating and wetting agent. Examples of suitable lubricants are lower polyethylene glycol (PEG) and ethoxylated fatty alcohols.
[0070]
In embodiments mainly intended for granules of detergent formulations, different "functional" components such as TAED, CMC, bleach, OBE, surfactants, fragrances and detergent formulations known to those skilled in the art. Other functional ingredients used in can be added to the coating. In addition, coatings may be used for specific applications in the pharmaceutical, agriculture, food, baking, additive, feed, cleaning or other industries where granules comprising bioactive compounds can be used as required. It can comprise selected functional components.
[0071]
In a particular embodiment of the present invention, the granules of the present invention have a high constant of at least 81%, as disclosed in WO 89/08694, which is incorporated herein by reference. Coated with a protective coating having a humidity of. Thus, in certain embodiments, the coating should act as a moisture and / or bleach barrier to stabilize the bioactive compound in the core. Further, in other embodiments, the coating unit acts as a mechanical barrier during a mechanical process, such as dosing or tableting. In some embodiments, the coating unit is sufficiently compressible and flexible that the core is resistant to the tableting process in structural terms and with respect to the biological activity of the active compound. This is most applicable for detergent formulations.
[0072]
In certain embodiments, the coating absorbs light from the excitation source and light emitted from fluorescent markers in the granules, such that when an area of the granule surface is coated with the coating, it is emitted from this area. Light detection is reduced.
[0073]
Fluorescent marker
The fluorescent marker contained in the granular composition of the present invention can be a compound that shows fluorescence when illuminated. The fluorescent marker is organic and, when illuminated with light in the X-ray, ultraviolet and / or visible region of the electromagnetic spectrum, for example light in the wavelength 10-700 nm, more particularly in the ultraviolet region, i.e. 10-380 nm, Shows fluorescence.
Furthermore, the fluorescent marker contained in the granule composition of the present invention, when excited, emits light in the ultraviolet, visible and / or near-infrared region of the electromagnetic spectrum, i.e., suitably at 185-2600 nm. Radiate.
[0074]
The fluorescent marker may belong to the group of bioactive compounds, auxiliary granulating agents and coatings, or may be a compound added to the granule composition solely for performing the fluorescence analysis according to the invention. it can. However, from a cost-saving standpoint, it is preferred that the fluorescent marker be the bioactive compound itself or an auxiliary granulating agent.
[0075]
Depending on the nature of the particulate composition to be evaluated by fluorescence analysis, for example the nature of the dust or the thickness of the coating, different fluorescent markers can be selected. In order to assess the nature of the dust, it is necessary to assess the amount of potentially damaging active compound in the dust particles present in the granular composition, for example, a bioactive compound as a fluorescent marker Is more appropriate, but a suitable auxiliary granulating agent can be selected as a fluorescent marker to assess the thickness of the coating.
[0076]
If the biologically active compound is a fluorescent marker having an aromatic amino acid residue responsible for the emission of fluorescent light, for example tyrosine or tryptophan, in particular belonging to a specific group of proteins and / or peptides, then in the fluorescence analysis, It is preferred to illuminate the particulate composition with a light source that emits a substantial portion of ultraviolet light, particularly at wavelengths of 10-380 nm, more particularly 200-400 nm or 200-300 nm, most particularly 260-280 nm. In this case, it is more preferable to detect only the emitted light having a wavelength of 200 to 700 nm, for example, 400 to 700 nm or 300 to 400 nm, particularly 280 to 360 nm or 325 to 375 nm. Further, in this case, it is necessary to select a detector capable of detecting emitted light of these wavelengths, for example, a CCD camera.
[0077]
When the fluorescent marker is one of the auxiliary granulating agents, it is preferable to illuminate the granular composition with a light source that emits light having a wavelength of 350 to 550 nm, more particularly 375 to 425 nm in the fluorescence analysis. Further, in this case, it is necessary to select a detector capable of detecting emitted light of these wavelengths, for example, a CCD camera. In the process of producing the granules, a fluorescent marker can be added to the process which serves only as a fluorescent marker for fluorescence analysis. A wide range of suitable fluorescent markers are available, for example, from Molecular Probes (Molecular Probes, USA).
[0078]
Fluorescence analysis in granulation and coating processes
The present invention also provides a method for predicting the properties of a granular composition, controlling and improving the method of manufacture, using the above-described fluorometric method, to produce a bioactive compound and optionally auxiliary granulation in a granulator. The present invention relates to a method for producing a granular composition comprising an agent.
Therefore, the present invention comprises a step of performing a fluorescence analysis of a fluorescent marker on granules formed in a granulator, comprising a purified bioactive compound, a fluorescent marker and, if necessary, an auxiliary granulating agent. A method for producing granules comprising the same in a granulator.
[0079]
In certain embodiments, the fluorescence analysis is performed during granulation in the granulation process, particularly online. This means that the fluorescence analysis is performed at an appropriate repetition rate two or more times online and in real time during the granulation process. The repetition rate depends, inter alia, on data processing from one or more detectors. In certain embodiments using a CCD or ICCD camera, measurements of about 15 flashes / second of emitted light are recorded in the granulation process in the form of a two-dimensional image. The term "formation of the granules" also includes coating the granules with a coating layer.
[0080]
In this embodiment, also the process further comprises changing at least one process parameter as a result of the fluorescence analysis. The process parameters to be varied can be parameters that affect the granulation process and / or the properties of the formed granules. These parameters include the supply of the granulating material to the granulator, ie, the bioactive compound and / or auxiliary granulating and / or coating agent, the gas supply to the granulator, the temperature in the granulator, It can be the pressure in the granulator, the pH in the granulator and the mechanical force applied to the granulated material. Process parameters can be manually changed through the automation system of the granulator.
[0081]
In a further embodiment, the fluorescence analysis according to the invention can also be suitably used to control the dusting properties of the finished granule composition after granulation. Accordingly, the present invention further provides a method for fluorescence analysis of active dust in a granular composition comprising a biologically active compound. This method can be used to discard or reprocess particulate compositions that do not meet the required quality for dust.
[0082]
In a further additional embodiment, the fluorescence analysis according to the invention is also suitable for controlling the coating thickness and / or homogeneity of the finished granule composition after granulation. Accordingly, the present invention further provides a method for fluorescence analysis of the coating thickness in a composition of coated granules comprising a biologically active compound. This method can be used to discard or reprocess particulate compositions that do not meet the required quality with respect to coating thickness.
[0083]
The imaging system can be used to evaluate quality parameters such as active dust values or coating thickness. In one embodiment, the evaluation is performed online, for example, during the coating process. Evaluation involves recording an image of the light emitted from the granules exposed to the excitation light source, especially by a camera, and comparing the recorded image with the recorded image of a sample with known quality parameter values (reference sample). It is implemented by doing. The fluorescence image of the reference sample and the corresponding quality parameters are used to provide a calibrating model, such as a partial least squares model or other modeling system suitable for producing a calibrating model of image data. The model is then used to predict quality parameter estimates from the fluorescent image of the unknown sample.
[0084]
Thus, the present invention also provides a method for estimating a quality parameter of a granular composition comprising a purified biologically active compound, comprising the following steps:
(A) illuminating a granule composition comprising a purified bioactive compound having known quality parameters with light capable of fluorescently exciting a fluorescent marker contained in the granule composition; Recording one or more images of light emitted from the body composition and subjecting the recorded images to data processing, particularly in the form of partial least squares data processing, providing a calibrated model;
[0085]
(B) illuminating the unknown particulate composition comprising the purified bioactive compound with light capable of fluorescence-exciting a fluorescent marker contained in the particulate composition, and radiating from the unknown particulate composition Recording at least one image of the applied light;
(C) comparing at least one image of the unknown granular composition with a calibrated model;
(D) Estimate the quality parameters of the unknown granular composition.
[0086]
Granulator
Granulation and / or coating devices comprising means for performing a fluorescence analysis on the granular composition according to the invention are also included in the scope of the invention. Therefore, the present invention comprises the following components:
(A) a granulation or coating apparatus comprising at least one chamber for processing the material into granules or coated granules, and
(B) a light source for illuminating the particles to be processed, at least one detector capable of detecting light emitted from the material to be processed, means for emitting illumination light onto a portion of the material to be processed, illumination An optical assembly comprising means for projecting light emitted from the exposed material to a detector, and at least one filtering device for filtering the light;
A granulation or coating apparatus comprising:
[0087]
The granulation or coating device can be any conventional granulation device, especially it can be selected from fluid bed granulator or coater, high shear mixer granulator, spray dryer, spray cooler, extruder Can be.
In an optical arrangement, the light source may be a conventional glow lamp, a more specialized xenon lamp or a strobe, which can emit in particular light of wavelength 10-700 nm, more particularly in the ultraviolet range, i.e. 10-380 nm. It is a light bulb.
In an optical arrangement, the detector is in particular a camera-type detector, more particularly a line scan camera, a CCD camera or an ICCD camera.
[0088]
Optionally, the optical arrangement comprises means for focusing the emitted light, for example a lens.
The means for emitting illumination light onto the material to be processed and for emitting light emitted from said material to a detector comprises one or more of fiber optics, mirrors, lenses, beam splitters and others.
[0089]
The optical arrangement includes at least one filtering device for filtering the illumination light and / or the emitted light. In certain embodiments, the device is a bandpass filter or a grating monochromator. In one aspect, one or more filtering devices are positioned such that only the emitted light is filtered and after the emitted light passes through one or more filters, it reaches one or more detectors. ing. In another aspect, at least two filtering devices are positioned to filter both illumination light and emitted light. The most preferred filters are the wavelength-selective filters described above for particular fluorescent markers. If two detectors are used, the optical arrangement also includes at least one beam splitter, for example, a dichroic mirror.
[0090]
In the most particular embodiment, the optical arrangement comprises a stroboscope light source, two CCD camera detectors, one bandpass filter for filtering the illumination light, and two, as depicted in FIG. Includes a bandpass filter, lens and two dichroic mirror beam splitters.
In one particular embodiment, the firing means emits illuminating light through an opening in the chamber onto a portion of the material being processed in the chamber, and projects light emitted from the material in the chamber to a detector. including.
[0091]
In a more particular embodiment, the granulator further comprises means for providing a purge flow of granules from the chamber. In this case, the optical assembly is positioned to allow fluorescence analysis of the material in the purge stream rather than the material present in the chamber. One reason this embodiment is preferred is that the granulation process usually involves considerable wear and tear of the granulator. When granulating bioactive compounds, such as enzymes, some auxiliary granulating agents can be clays or other inorganic substances.
[0092]
These substances can have a significant abrasive effect on the granulator. Therefore, it is common to observe that the inner surface of the granulator grinds several millimeters of steel per year. Wear and tear of this magnitude can be very detrimental to the sensitive devices of the optical assembly. The granules in the purge stream can be suitably recycled, and in this way, the fluorescence analysis does not interfere with the granulation process. The purge stream is made transparent to light by allowing the particles to properly pass through a portion of the purge stream, where the light can be projected. As an example, the purge stream can be transported from the granulation chamber through a transport system to an optical arrangement.
[0093]
The transport system can be, for example, one or more elements selected from chutes, pipes, conveyor belts, cyclones and others. Fluorescence analysis can be performed at some point in the transport system, where the granulated product can be accessed by illuminating light, from which emitted light can reach one or more detectors. . For example, such a point may be, for example, a point where a portion of the pipe material has been replaced with a transport material, such as glass, quartz or a polymeric material.
[0094]
In particular, in performing the fluorescence analysis, the means for providing a purge stream include the means for forming a single layer of granules, so that upon detection from the granules, there is little or no overlap with each other at the moment of detection. Try not to happen. This can be achieved by placing the granulated product on an inclined (non-horizontal) oscillating surface (eg, an oscillating chute), where the surface area, loading speed of the granules, and transport on the oscillating surface Adjust the speed (tilt angle) so that the shaking surface area is always greater than the area of the granules present on the surface.
[0095]
In this way, the granules will form a substantially monolayer of the granules transported over the surface. Fluorescence analysis can be performed anywhere in this single grain layer. In particular, also when the particles fall on one or more rims of the vibrating surface (comparable to a waterfall), the granules can leave the vibrating surface as a single layer and to avoid reflections from the vibrating surface Fluorescence analysis is performed at some point, particularly after the particles have left the vibrating surface, but a single layer of particles has been maintained.
[0096]
By measuring on the granules forming a single-grain layer, overlap is avoided and the light emitted from the granules can be more precisely focused, since the granules are distributed mainly only in two dimensions. . In this way, a sharply focused image of almost every individual grain passing through the fluorescence analysis point can be obtained with a two-dimensional detector, for example a CCD camera.
[0097]
As indicated above, the optical assembly is suitably connected to a granulation or coating device to allow for on-line or at-line fluorescence analysis of the particulate composition. An online analysis is to be understood as an analysis performed on granules when they are actually being granulated, for example by analyzing the granules in a granulator or a recycle purge stream. An at-line analysis is to be understood as an analysis performed on a non-recycled sample taken from the granulator downstream (eg at the outlet) after the granulation process or during granulation.
[0098]
The granulator can comprise other components, a computational unit that processes the data from the detector, and is equipped with specialized data handling hardware and software as needed. Also, the granulation device can comprise a control unit coupled to a computational unit that controls and regulates the granulation process based on the results of the fluorescence analysis. The control unit receives data from the computing unit and outputs these to one or more hardware units that control the granulation process, for example, feed flow, speed, temperature, airflow, and the like. , A PLC or other device that converts the data.
The manner of practicing the invention is demonstrated in the following examples. The experiments are merely examples of one aspect of the invention and should not be construed as limiting the invention.
[0099]
Example
Example 1.Fluorescence analysis of raw materials for producing enzyme granules
The fluorescence of eight different sources of enzyme granules and enzyme concentrates was measured on a LS50B (Perkin Elmer). The raw material was excited with internal light sources of different wavelengths of 230-500 nm in 10 nm steps and the emission from the material was recorded in 270-700 nm in 1 nm steps. The slit gap of the Perkin Elmer instrument for both excitation and emission was 4 nm. The fluorescence was measured by placing the material in a quartz container (cuvet) and measuring the fluorescence emission from the raw material at an angle displaced 22.5 degrees from the direction of the illumination light as required by the instrument design.
While some materials emitted significant fluorescence, the enzyme concentrate uniquely had a very strong emission at about 350 nm.
[0100]
Embodiment 2. FIG.Fluorescence analysis of enzyme granules
Fluorescence of uncoated enzyme granules and coated granules was measured on a LS50B (Perkin Elmer). The granules were excited with internal light sources of different wavelengths between 230 and 500 nm in 10 nm steps and the emission from the material was recorded in 270-700 nm in 1 nm steps. The slit gap of the Perkin Elmer instrument for both excitation and emission was 4 nm. The fluorescence was measured by placing the material in a quartz container (cuvet) and measuring the fluorescence emission from the raw material at an angle displaced 22.5 degrees from the direction of the illumination light as required by the instrument design.
The result showed a peak at about 350 nm corresponding to the enzyme concentrate. Another strong peak occurred at 450-500 nm due to the fluorescent compound in the granulation aid. The results show the possibility of detecting the enzyme in the granules.
[0101]
Embodiment 3 FIG.Online fluorescence analysis of granules
The fluorescence of the uncoated enzyme granules and the coated granules was measured using the optical assembly shown in FIG. Two camera detectors of Donpisha type "Progressive camera module" XC-8500CE1 / 2 "ITCCD 782 (H) x 582 (V) equipped with a 35 mm lens of CCIR addition at the sample introduction point. The light sources were an Oriel Xenon flash lamp 60000 / w Oriel attachment 60008 and an Oriel power supply 68826. The illumination light was filtered using a bandpass filter to a wavelength of 450 nm. Before arriving at the camera, the emitted light was split into two beams by a dichroic mirror beam splitter, and each beam was filtered by a band-pass filter, one of which was a wavelength 530 (green filter). nm light and pass through the other filter (red filter Filter) is passed through a light of a wavelength of 620 nm.
[0102]
Illumination and detection of emitted light was performed by loading the enzyme granules into a funnel having an outlet at a rate to allow the granules to pass, and performing the analysis on the granules leaving the funnel immediately after the funnel outlet.
A detector in a computational unit (normal personal computer) equipped with data acquisition hardware DAQ and SCR-68 breadboard and image processing software, Labview 4.1.1 IMAQ Vision and Universal Labview (all from National Instruments) To generate an instantaneous digital image of the phosphor particles passing the illumination light beam.
[0103]
As a result of these measurements, a series of images was obtained, which could be recorded and reproduced as a moving image. The uncoated granules glow and fluoresce, where the morphology and shape of the granules are clearly delineated, thus demonstrating the possibility of using fluorescence analysis in the production of enzyme granules. For coated granules, fluorescence was significantly reduced, indicating that the coating layer reduced access to fluorescent markers. Thus, the darker areas of the record indicate granules with a thicker coating, thus demonstrating the possibility of using fluorescence analysis in the production of the coated granules.
[0104]
Embodiment 4. FIG.Fluorescence analysis of enzyme granules with varying enzyme concentrations
Experiments were performed to evaluate whether a bioactive compound could also be a fluorescent marker. Four batches with varying contents of purified protease concentrate were made with a Lodige mixer. Protease contents were 0 (reference), 1, 4 and 12 KPNU, respectively. Where KPNU is the kilo NOVO protease unit, which is determined relative to the protease standard and is determined under standard conditions, ie, 50 ° C., pH 8.3, 9 minutes reaction time, 3 minutes At measurement time, it is based on the digestion of dimethyl casein (DMC) solution by proteolytic enzymes.
[0105]
The sample was excited with an Oriel xenon flash lamp 60000 / w Oriel attachment 60008 and Oriel power supply 68826. The illumination light was filtered using a bandpass filter to produce a light beam with a wavelength of 300-400 nm. The emitted light was detected with a 3-CCD camera (JAI) (M-90). The camera was fitted with a Computer 55 Telecentric lens. The signal from the detector was transferred to a computing unit (normal personal computer) equipped with an IFC51 frame grabber and Image-Pro Plus version 4.5.
[0106]
The recorded fluorescence images of the granules showed that the average intensity of emitted light increased significantly as a function of increasing concentrations of purified protease.
The four batches of granules were subsequently coated with a standard PEG-coating containing kaolin and titanium dioxide (PEG 4000). The recorded fluorescence images of the coated granules showed, in fact, that the average intensity of the emitted light increased significantly as a function of increasing concentrations of purified protease.
As indicated by this, the biologically active compound that is a protease in this experiment can also be a fluorescent marker.
[0107]
Embodiment 5 FIG.Fluorescence analysis of enzyme granules with varying coating thickness
Experiments were performed to study whether there was a correlation between coating thickness and emitted light intensity. A standard batch of granules containing the enzyme was coated with a standard PEG-coating containing kaolin and titanium dioxide (PEG 4000). The coating was sprayed onto the granules (Huetlin) and samples of the granules were withdrawn from the process when 10%, 25% and 50% of the coating were applied, respectively.
[0108]
The sample was excited with an Oriel xenon flash lamp 60000 / w Oriel attachment 60008 and Oriel power supply 68826. The illumination light was filtered using a bandpass filter to produce a light beam with a wavelength of 300-400 nm. The emitted light was detected with a 3-CCD camera (JAI) (M-90). The camera was fitted with a Computer 55 Telecentric lens. The signal from the detector was transferred to a computing unit (normal personal computer) equipped with an IFC51 frame grabber and Image-Pro Plus version 4.5.
[0109]
The recorded fluorescent images of the coated granules, in fact, show that the average intensity of the emitted light decreases as the concentration of the coating increases (ie the coating dose goes to 10%, 25% and 50%). That was shown.
As shown by this, the intensity of the fluorescent emission light can be used to predict the thickness of the coating.
[0110]
Embodiment 6 FIG.Fluorescence analysis of enzyme granules with varying amounts of enzyme-active dust
Experiments were performed to evaluate whether the amount of dust could be predicted using fluorescence analysis. Presumably, coated enzyme granules with flaws in the coating are known to lead to the problem of enzymatically active dust.
Eight samples with protease activity dust values of 29 ng / g to 2420 ng / g protease / g dust were subjected to online fluorescence analysis.
[0111]
Samples were excited with an Oriel xenon flash lamp 60000 / w Oriel attachment 60008 and Oriel power supply 68826. The illumination light was filtered using a bandpass filter to produce a light beam with a wavelength of 300-400 nm. The emitted light was detected with a 3-CCD camera (JAI) (M-90). The camera was fitted with a Computer 55 Telecentric lens. The signal from the detector was transferred to a computing unit (normal personal computer) equipped with an IFC51 frame grabber and Image-Pro Plus version 4.5.
[0112]
The sample was transported to the imaging system at a distance of approximately 10 cm.
The recorded fluorescence image of the granular composition showed, in fact, that for compositions with increasing amounts of protease active dust values, the average intensity of the emitted light increased.
As indicated by this, the intensity of the fluorescent emission light can be used to estimate the amount of bioactive dust in the particulate composition.
[Brief description of the drawings]
FIG.
FIG. 1: One example of an optical arrangement, where 1 = detector (CCD camera), 2 = light source, 3 = bandpass filter, 4 = dichroic mirror beam splitter, 5 = lens, 6 = funnel, 7 = Fluorescent particles passing through the illumination light beam.
FIG. 2
FIG. 2: One example of an on-line optical assembler, 1 = depiction showing the light emitted from the granules recorded by a CCD camera, 2 = CCD camera, 3 = light source, 4 = grains on a conveyor belt.

Claims (44)

Illuminating the particulate composition comprising the purified bioactive compound with light capable of fluorescently exciting a fluorescent marker contained in the particulate composition, detecting light emitted from the fluorescent marker, A fluorometric method comprising predicting the amount of a fluorescent marker in a particulate composition accessible to excitation. The method according to claim 1, wherein the granular composition is illuminated with a light source that emits ultraviolet light having a wavelength of 10 to 380 nm. 3. The method of claim 2, wherein the ultraviolet light comprises 1 to 10 discrete monochromatic wavelengths. 4. The method according to claim 3, wherein the UV radiation consists of one discrete monochromatic wavelength, in particular 260-280 nm. 2. The method according to claim 1, wherein detecting light emitted from the fluorescent marker comprises detecting emitted light at 1 to 10 discrete monochromatic wavelengths, in particular 185 to 2600 nm. 6. The method according to claim 5, wherein the fluorescent marker is a biologically active compound and the detection of the light emitted from the fluorescent marker comprises detecting the emitted light at one discrete monochromatic wavelength, in particular 300-400 nm. The method according to claim 1, wherein the detection is performed using at least one detector capable of converting the emitted light into an electronic signal. The method according to claim 7, wherein the detection is performed using a CCD camera or an ICCD camera capable of converting the emitted light into a digital two-dimensional image. 9. The method according to claim 8, wherein the detection is performed using at least two CCD cameras or ICCD cameras capable of converting the emitted light into a digital two-dimensional image. The method of claim 1, wherein the prediction is performed by comparing light emitted from the granular composition to light emitted from the granular composition containing a known amount of a fluorescent marker. The method of claim 10, wherein the prediction is real time. 2. The method according to claim 1, wherein the bioactive compound is a biocatalyst, a therapeutic agent, a herbicide, a pesticide and a fungicide. 13. The method according to claim 12, wherein the biologically active compound is selected from a protein and a peptide. 14. The method according to claim 13, wherein the biologically active compound is an enzyme, in particular selected from hydrolases and oxidoreductases. The method of claim 1, wherein the granular composition further comprises an auxiliary granulating agent. 16. The method according to claim 15, wherein the auxiliary granulating agent is selected from fibrous materials, binders, fillers, liquids, enzyme stabilizers, suspending agents, crosslinkers, mediators and / or solvents. 17. The method according to claim 16, wherein the fluorescent marker is an auxiliary granulating agent and the detection of the light emitted from the fluorescent marker comprises detecting one discrete monochromatic wavelength, in particular 350-500 nm emitted light. . The method of claim 1, wherein the granules comprise a core in which the bioactive compound is intimately mixed with the auxiliary granulating agent. The method of claim 1, wherein the granules comprise core particles coated with a layer comprising a bioactive compound and, in particular, an auxiliary granulating agent. 2. The method according to claim 1, wherein the granules have an average size of from 20 to 2000 [mu] m, in particular from 100 to 1000 [mu] m, more particularly from 200 to 800 [mu] m. The method according to claim 1, wherein the granules are coated with a coating agent. 22. A purified bioactive compound, a fluorescent marker and, if necessary, an aid, comprising a step of performing a fluorescent analysis of the fluorescent marker according to any one of claims 1 to 21 on granules formed in a granulator. A method for producing a granule comprising a static granulating agent in a granulating apparatus. 23. The method of claim 22, wherein the fluorescence analysis is performed online and in real time during the granulation process, and is repeated two or more times during the granulation process. 23. The method of claim 22, further comprising changing at least one process parameter as a result of the fluorescence analysis. 25. The process parameter of claim 24, wherein the process parameters are selected from a supply of a bioactive compound, a supply of an auxiliary granulating agent, a coating agent, a gas, a temperature, a pressure, a pH and a mechanical force applied to the granulated material. the method of. The granules comprising the bioactive compound and, optionally, an auxiliary granulating agent, are coated with a coating agent, and the parameter is further characterized by a coating process, which is the supply of the coating agent to the granulator. The method according to any one of 22 to 25. 23. The method of claim 22, wherein the fluorescence analysis is performed in-line in real time after the granulation process and repeated two or more times. The following components:
(A) a granulation or coating apparatus comprising at least one chamber for processing material into granules or coated granules; and (b) a light source illuminating the granules to be processed, from the granules to be processed. At least one detector capable of detecting emitted light, means for emitting illuminating light on a portion of the granules to be processed, means for emitting light emitted from the illuminated granules to a detector, And an optical assembly comprising at least one filtering device for filtering light.
A granulation or coating device comprising. 29. The device according to claim 28, wherein the granulation or coating device is selected from a fluid bed granulator or coater, a high shear mixer granulator, a spray dryer, a spray cooler, an extruder. 29. The device according to claim 28, wherein the light source is selected from a glow lamp, a xenon lamp or a strobe light bulb, which is capable of emitting light, especially at a wavelength of 10-700 nm. 29. The device according to claim 28, wherein the detector is a camera, in particular selected from a line scan camera, a CCD camera or an ICCD camera. 29. The device of claim 28, wherein the optical arrangement comprises means for focusing the emitted light. The means for emitting illumination light onto the particles to be processed and for emitting light emitted from said particles to a detector is selected from one or more of fiber optics, mirrors, lenses and beam splitters. Item 30. The apparatus according to Item 28. 29. The apparatus of claim 28, wherein the filtering device is positioned to filter the emitted light and reach the detector after the emitted light passes through the filter. 35. The device of claim 34, wherein at least two filtering devices are positioned to filter both illumination light and emitted light. The optical arrangement includes a stroboscope light source, two CCD camera detectors, one bandpass filter for filtering illumination light, two bandpass filters for filtering emitted light, a lens, and two dichroic beam splitters. 29. The device of claim 28, wherein the device comprises: 29. The device of claim 28, wherein a portion of the granules to be processed reside in the chamber. 29. The apparatus of claim 28, further comprising means for providing a purge stream of particulates from the chamber, and wherein the optical arrangement is positioned to enable fluorescence analysis of the particulates in the purge stream. apparatus. 39. The apparatus of claim 38, wherein the means for providing a purge flow includes means for forming a monolayer of particulate. 40. The apparatus of claim 39, wherein the means for forming a single layer of granules comprises a tilted vibrating surface. 41. The apparatus of claim 40, wherein the optical arrangement is positioned to perform a fluorescence analysis on a single layer of the granules after falling onto one or more rims of the vibrating surface. 29. The device of claim 28, further comprising one or more elements selected from a computational unit and a control unit. Use of fluorescence analysis on granules comprising a purified bioactive compound. Process:
(A) illuminating a granule composition comprising a purified bioactive compound having known quality parameters with light capable of fluorescently exciting a fluorescent marker contained in the granule composition; Recording one or more images of light emitted from the body composition and preparing a calibrated model by subjecting the recorded images to data processing, particularly in the form of least squares data processing;
(B) illuminating the unknown particulate composition comprising the purified bioactive compound with light capable of fluorescence-exciting a fluorescent marker contained in the particulate composition, and radiating from the unknown particulate composition Recording at least one image of the applied light;
(C) comparing at least one image of the unknown granular composition with a calibrated model; and (d) estimating a quality parameter of the unknown granular composition.
The method of claim 1, comprising: