WO2002061427A1 - Procede d'analyse d'une composition granulaire par fluorimetrie - Google Patents

Procede d'analyse d'une composition granulaire par fluorimetrie Download PDF

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Publication number
WO2002061427A1
WO2002061427A1 PCT/DK2002/000071 DK0200071W WO02061427A1 WO 2002061427 A1 WO2002061427 A1 WO 2002061427A1 DK 0200071 W DK0200071 W DK 0200071W WO 02061427 A1 WO02061427 A1 WO 02061427A1
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WIPO (PCT)
Prior art keywords
granules
light
granulation
active compound
biologically active
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PCT/DK2002/000071
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English (en)
Inventor
Peter Paasch Mortensen
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Novozymes A/S
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Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to EP02710755A priority Critical patent/EP1358483A1/fr
Priority to JP2002561942A priority patent/JP4198996B2/ja
Publication of WO2002061427A1 publication Critical patent/WO2002061427A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/85—Investigating moving fluids or granular solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B01J2/006—Coating of the granules without description of the process or the device by which the granules are obtained
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Definitions

  • the present invention relates to a method for analyzing a property of a granular composition comprising a biologically active compound by subjecting the granular composition to fluorescence analysis.
  • the invention also relates to a method for producing a particular product comprising subjecting the product to fluorescence analysis.
  • the invention relates to a granulation and/or coating apparatus suitable for preparing a granular composition comprising a biologically active compound said apparatus comprising means for fluorescence analysis.
  • fluorescence analysis Analysis of chemical compounds in samples exploiting that some compounds (fluorophors) exhibit fluorescence when excitated with light (fluorescence analysis) are well known to the art. While fluorescent analysis has its advantages in being sensitive and accurate in well-defined samples it also has serious drawbacks. For example in complex samples the fluorescence of a fluorophor is often altered by other compounds present in the environment surrounding the fluorophor (known as quenching) making it difficult to use a fluorescence analysis quantitatively on complex and/or poorly defined samples. This applies especially in heterogeneous or solid phase samples where also scattering of the light has to be accounted for.
  • Watano et al. has in US 5,497,232 reported imaging of granule growth in a fluid bed or a pan type granulator using a photographic camera such as a CCD camera of the type used in video cameras.
  • the present invention relates to a method for analyzing a property of a granular composition comprising a purified biologically active compound by subjecting the granular composition to fluorescence analysis.
  • Formulation of chemical compounds into finished goods, in particular granulation is usually required to achieve improved properties of the products, thus making them more commercially attractive.
  • granulation is often compulsory to the producers because the active compound must, until being applied in the intentional use, be separated from the surrounding environment to ensure the safe handling of the product.
  • the amount of biologically active compound which can escape from the granulated product, e.g. in the form of dust, must be minimized to ensure that persons handling the product do not suffer any adverse effects from contact with the biologically active compound.
  • the active compound must be protected from the environment outside the granule to remain stabile and active once it is to be used.
  • granules comprising biologically active compound with a coating agent which further suppress the release of active compound from the granule and further improve the stability of the active compound in the granule.
  • a coating agent which further suppress the release of active compound from the granule and further improve the stability of the active compound in the granule.
  • One object of the invention is to provide methods, particularly in the form of an imaging system, for measuring quality parameters of a granular composition comprising a purified biologically active compound.
  • a parameter may be for example the amount of active compound released from granules in the form of active dust, during or after the process for preparing the granular composition.
  • Another parameter is for example the thickness and/or integrity of coating layers applied to the granules to suppress dust formation and increase the stability of the active compound which by the present invention could be measured during or after a process of coating of granules comprising a biologically active compound.
  • Another object of the invention is to design a granulation apparatus and to select method setup, so that the method may be used on-line or in-line in the production of such granular compositions, and that the methods in real may time provide information about levels of dust comprising biologically active compound during processing of the granular composition.
  • compositions comprising a purified biologically active compound both active compound confined in the surface regions of granules and active compound, which is present in the composition in the dust particles, e.g. as a result from release of active compound from the granules or as a result from insufficient granulation, can be evaluated by illuminating the composition with light capable of fluorescence excitation of a fluorescent marker, e.g. the biologically active compound itself, and detecting emitted light from the fluorescent marker.
  • a fluorescent marker e.g. the biologically active compound itself
  • the present invention provides in a first aspect a method for fluorescence analysis comprising illuminating a granular composition comprising a purified biologically active compound with light capable of fluorescence excitation of a fluorescent marker comprised in the granular composition, detecting light emitted from the fluorescent marker and predicting the amount of fluorescent marker in the granular composition accessible to fluorescence excitation.
  • the amount of accessible fluorescent marker may be linked to a property of the granular composition, such as dust levels and/or coating thickness or coating integrity.
  • the invention provides a process for preparing granules comprising a purified biologically active compound, a fluorescent marker and optionally auxiliary granulation agents in a granulation apparatus said process comprising the step of performing fluorescence analysis in accordance with the first aspect of the invention ( vide supra ) .
  • the invention provides a granulation or coating apparatus comprising (a) a granulating or coating device comprising at least one chamber for granulating material or for coating of granulated material,
  • the invention provides use of fluorescence analysis on granules comprising a purified biologically active compound.
  • the present invention relates, as described, to a method for performing fluorescence analysis on a granular composition comprising an active compound.
  • the first step of the method comprises illuminating the granular composition with a beam of light, which can excitate a fluorescent marker comprised in the granular composition.
  • a beam of light which can excitate a fluorescent marker comprised in the granular composition.
  • photons from the illuminating light will be absorbed by the fluorescent marker with the result, that electrons in the fluorescent compound gain energy and are brought into a specific increased energy level.
  • the excitated fluorescent marker will, as the electrons return to their original ground state energy level, subsequently liberate at least some of the gained energy by emitting light photons of a wavelength characteristic for the energy difference between the increased energy level and the original energy level.
  • the second step of the method comprises detecting the emitted light from the granular composition with a detector, capable of converting the emitted light into an electronic signal .
  • the third step of the method comprises processing the electronic signal to correlate the amount of emitted lights to one ore more properties of the granular composition by predicting the amount of fluorescent marker in the granular composition accessible to fluorescence excitation.
  • the illumination of the granular composition may be carried out with any suitable light source delivering light capable of excitating the fluorescent marker in the granular composition.
  • the light source may be e.g. a normal glow lamp, a more specialized xenon lamp or a stroboscope lamp.
  • the optical properties of the fluorescent marker compound may be known and to optimise the excitation of a known fluorescent marker it is preferred to select a light source delivering a substantial portion of light of wavelengths suitable for excitating the fluorescent marker. If it is desired (which it may be when using a known fluorescent marker) to avoid or limit excitation and emission from compounds other than the fluorescent marker, which may interfere with the analysis, it may be desired to filter the beam of light, so that only light of selected wavelengths illuminates the granular composition. This may be done with one or more beam splitters and one or more band pass filters, such as high and/or low band pass filters, or grate monochromators allowing only light with specific wavelengths to pass. These features are normally integrated in commercially available fluorescence analysers, e.g.
  • band pass filters and monochromators will allow passage of light having wavelengths within a narrow ranges, normally within a few nm, such as 0.5-10 nm.
  • monochromatic light is to be understood as light having wavelengths within the narrow range determined by the band pass filter or the grate monochromator .
  • the light illuminating the granular composition consist of 1-10 discrete monochromatic wavelengths, particularly 1-4 discrete monochromatic wavelengths.
  • the light illuminating the granular composition consist of one discrete monochromatic wavelength.
  • an optical arrangement may suitably be employed comprising e.g. mirrors, beam splitters (such as dichroic mirrors) and/or fiber optics to project the illuminating light onto the granular composition.
  • the emitted light may be characteristic for the fluorescent marker or for chemical groups or constituents comprised in the fluorescent marker.
  • the fluorescent marker usually only emit light within one or more narrow ranges of wavelengths it is preferred to filter the emitted light, so that only emitted light within these ranges are allowed to reach the detector. This may be achieved by filtering the emitted light with one or more band pass filters or monochromators as described, supra . This may avoid or limit the amount of emitted light from compounds other than the fluorescent marker from reaching the detector and which may interfere with the analysis. Accordingly, in a particular embodiment only emitted light of 1-10 discrete monochromatic wavelengths are detected, particularly 1-4 discrete monochromatic wavelengths. In particular emitted light reaching the detector consists of one discrete monochromatic wavelength.
  • an optical arrangement comprising e.g. mirrors, beam splitters (such as dichroic mirrors), fiber optics, and or means for focusing the emitted light (such as lenses) to project the emitted light into the detector.
  • the detector may be a photo multiplication type detector, a photo diode or photo diode array, a line scan camera, a CCD camera, an ICCD camera or any other type suitably for detecting the emitted light.
  • a particular detector may be a camera type detector, such as selected from the group of grey scale cameras, line scan cameras, photodiode arrays, CCD (charged Coupled Device) cameras and ICCD (Intensified CCD) cameras.
  • CCD Charged Coupled Device
  • ICCD Intensified CCD
  • detectors may be CCD cameras and ICCD cameras, because they are more sensitive and enable formation of 2 dimensional images, showing the spatial distribution of the light emitting granules or dust particles. This is called fluorescence imaging in terms of the skilled analyst.
  • two or more detectors may be used to record two or more selected wavelengths or two or more ranges of wavelengths simultaneously. This is desired if more than one fluorescent marker is to be measured or if a particular fluorescent marker emits light at different wavelengths.
  • This is particularly achieved using an optical arrangement including one or more beam splitters and two and more band pass filters or monochromators.
  • an optical arrangement including two CCD cameras, dichroic mirrors and band pass filters and lenses as shown schematically in figure 1 is used.
  • Conversion of the emitted light, in the detector, into an electronic signal and converting this signal into a measure, such as a number, from which a prediction of the amount of emitted light and the amount of fluorescent marker accessible to excitation may be inferred, is known to the skilled person, as analysers for making fluorescence analysis are abundantly available.
  • the prediction may suitably be made by comparing the amount of the emitted light from an unknown granular composition with data on emitted light from a granular composition of known properties, and thus predicting in the unknown granular composition the amount of fluorescent marker accessible to excitation.
  • the output of most detectors such as photo multiplication based types or some photo diode based types is an analogue signal.
  • detectors such as many cameras, which comprise numerous single photo diode detectors, may have a build in analogue-digital converter capable of converting the analogue signal into a digital signal, which is more suitable for computerized data processing.
  • the digital data arising from the emitted light may be subjected to processing.
  • This processing is suitably performed in a computer system using software designed for such processes.
  • Such software may be the LabView software as used in the examples herein or any other software providing the necessary capabilities for performing the desired data processing to link the amount of emitted light to a property of the granular composition.
  • the data processing may include operations such as particle counting, gauging, pattern matching (grey scale and colour), statistics, thresholds, multivariate image analysis, AMT, blob analysis, area calculation, edge detection, morphology analysis, convolution, folding and unfolding, FFT, various filtering techniques e.g. median filtering - all techniques known to the skilled analyst, which are data processing functions included in commercially available software.
  • operations such as particle counting, gauging, pattern matching (grey scale and colour), statistics, thresholds, multivariate image analysis, AMT, blob analysis, area calculation, edge detection, morphology analysis, convolution, folding and unfolding, FFT, various filtering techniques e.g. median filtering - all techniques known to the skilled analyst, which are data processing functions included in commercially available software.
  • filtering techniques e.g. median filtering - all techniques known to the skilled analyst, which are data processing functions included in commercially available software.
  • the computing unit usually have to be equipped with hardware capable of acquiring the data from the detector for storage in
  • the granular composition of the invention is a composition comprising the biologically active compound, a fluorescent marker, which may be the biologically active compound itself and optionally auxiliary granulation agents and coating agents processed into particles or granules. Accordingly, finished granules are the result of the processes and methods of the invention.
  • the term "granules" are to be understood as a predominantly spherical or near spherical structure of a macromolecular size, particularly having an average size measure in the longest diameter between 20-2000 ⁇ m, more particularly between 100-1000 ⁇ m, most particularly between 200-800 ⁇ m.
  • the spherical granules preferably have a ratio, (a) : (b) , between the diameter in the shortest dimension (a) and the diameter in the longest dimension (b) of the granule of between 1:1 to 1:5, particularly between 1:1 to 1:3.
  • PSD size distribution
  • D50 is the diameter at which 50% of the granules, by mass, have a smaller diameter, while 50% by mass have a larger diameter.
  • D10 and D90 are the diameters at which 10% and 90%, respectively, of the granules, by mass, have a smaller diameter than the value in question.
  • the "SPAN” indicates the breadth of the PSD and is expressed as:
  • the PSD of granules after granulation is normally as narrow as possible.
  • Use of fluorescence analysis, in accordance with the present invention, for controlling the granulation process may aid in lowering of the PSD, and the SPAN of the granular composition after granulation is therefore particularly less than about 2.5, particularly less than about 2.0, more particularly less than about 1.5, and most particularly less than about 1.0.
  • the granules are particularly coated with a coating agent forming a, particularly homogenous, coherent and continuous, layer around the granules.
  • the term coating agent as used herein is to be understood as single coating compound or a mixture of coating compounds.
  • Coated granules thus consist of a granule core and a granule coating.
  • the coating layer is relatively thick in order to further reduce dusting and improve stability of the biologically active compound.
  • the coating thickness may be described by the ratio between the average diameter of a coated granule core and the average diameter of an uncoated granule core (hereinafter abbreviated D G /D C ) , i.e.
  • Coated granules of the invention particularly have a D G /D C of at least 1.1, which means that the thickness of the coating is at least 5% of the average granule core diameter.
  • a more particular D G /D C is at least 1.5, more particularly at least 2, more particularly at least 2.5, more particularly at least 3, most particularly at least 4.
  • D G /D C is however particularly below about 100, particularly below about 50, more particularly below 25, and most particularly below 10.
  • a most particular range for D G /D C is about 4 to about 6.
  • the coating is substantially enzyme-free
  • substantially enzyme free as used herein about a coating means that there is less than 5 mg of enzyme per gram coating agent. Considering the materials making up granules of the invention (vide infra) these are, as opposed to transparent materials such as glass, in general impassable to visual light, e.g. a person will usually not be able to see-through the granules.
  • the content of moisture in the granules is typically be less than 20(w/w)%, particularly less than 15(w/w)%, more particularly less than 10(w/w)%, such as in the range of 4 to 8(w/w)%.
  • the granules may be constructed by any granulation method known in the art.
  • the construction of the granules of the invention may be divided into the following non-exhaustive categories:
  • a) Spray dried granules wherein a liquid solution containing the biologically active compound is atomised in a spray dryer to form small droplets which during their way down the dryer dry to form a granular material comprising the biologically active compound.
  • Very small granules can be produced this way (Michael S. Showell (editor); Powdered detergen ts; Surfactant Science Series; 1998; vol. 71; page 140-142; Marcel Dekker) .
  • the biologically active compound is intimately mixed with any other auxiliary granulation agents present in the liquid solution.
  • Dust particles which may be present in a granular composition, may be characterised in that they are fragments of whole granules, which usually have a considerably smaller size than the granules and do not possess the characteristic spherical shape of the granules. Dust particles typically have an irregular non-spherical and abrupt structure such as rod or flake shaped. Dust particles are typically much smaller than the average size of granules, and most dust particles are, depending on the granular composition less than 20 ⁇ m in diameter . Dust particles being fragments of whole granules possess the same in-transparency and usually have about the same moisture content as intact granules, supra .
  • Biologically active compounds The granular composition of the invention comprises a biologically active compound, in particular a purified biologically active compound.
  • biologically active compound as used herein is to be understood as any compound, which is active in a biological system such as compounds, which interfere with and/or modifies biological pathways or biological reactions.
  • 'purified as used herein is to be understood as biologically active compounds, which before granulation has been subjected to one or more purification step to remove e.g. excess material and/or to concentrate the active compound.
  • the active compound is prepared by a microbiological fermentation process
  • purification particularly includes step selected from filtering, ultra-filtration, flocculation, sedimentation, evaporation, extraction and the like, to remove biomass and other undesired matter including water to yield a mixture which is enriched in the biologically active compound.
  • Biologically active compounds include among others organic compounds such as bio-catalysts, therapeutic agents, herbicides, pesticides and fungicides.
  • the biologically active compound is producible by fermenting a microorganism producing the active compound.
  • compounds are those among proteins and peptides, more particularly catalytic proteins, i.e. enzymes, because proteins such as enzymes are used in vast volumes in the industry and are known to cause adverse allergy reactions in humans or animal when exposed to such proteins.
  • enzymes are widely used in household products such as detergents for removing soil of a biological origin, and many industrial processes involves human handling of the enzymes.
  • the enzyme may be any enzyme for which it is desired to separate the enzyme from the surrounding environment through granulation of the enzymes.
  • enzymes which may appropriately be incorporated in granules of the invention include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).
  • oxidoreductases in the context of the invention are peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)], while particular transferases are transferases in any of the following subclasses :
  • a most particular type of transferase in the context of the invention is a transglutaminase (protein-glutamine ⁇ - glutamyltransferase; EC 2.3.2.13).
  • transglutaminases are described in WO 96/06931 (Novo Nordisk A/S) .
  • hydrolases in the context of the invention are: Carboxylic ester hydrolases (EC 3.1.1.-) such as lipases
  • glycosidases EC 3.2, which fall within a group denoted herein as "carbohydrases” , such as -amylases (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases].
  • carbohydrase is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five- and six-membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six- membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.
  • Carbohydrases of relevance include the following (EC numbers in parentheses) : alpha-amylases (3.2.1.1), beta-amylases (3.2.1.2), glucan 1,4- alpha-glucosidases (3.2.1.3), cellulases (3.2.1.4), endo-
  • oxidoreductases examples include GLUZYMETM (enzyme available from Novo Nordisk A/S) .
  • proteases examples include KANNASETM, EVERLASETM, ESPERASETM,
  • PRO (all available from Novo Nordisk A/S, Bagsvaerd, Denmark) .
  • proteases include MAXATASETM, MAXACALTM, MAXAPEMTM, OPTICLEANTM and PURAFECTTM (available from Genencor International Inc. or Gist-Brocades) .
  • lipases examples include LIPOPRIMETM LIPOLASETM, LIPOLASETM ULTRA, LIPOZYMETM, PALATASETM, NOVOZYMTM 435 and LECITASETM (all available from Novo Nordisk A/S) .
  • lipases include LUMAFASTTM ( Pseudomonas mendocina lipase from Genencor International Inc.); LIPOMAXTM ⁇ Ps . pseudoa lcaligenes lipase from Gist- Brocades/Genencor Int. Inc.; and Bacill us sp. lipase from Solvay enzymes.
  • carbohydrases examples include ALPHA-GALTM, BIO-FEEDTM ALPHA, BIO- FEEDTM BETA, BIO- FEEDTM PLUS BIO-FEEDTM PLUS , NOVOZYMETM 188 , CELLUCLASTTM, CELLUSOFTTM CEREMYLTM, CITROZYMTM, DENIMAXTM, DEZYMETM, DEXTROZYMETM FINI ZYMTM, FUNGAMYLTM, GAMANASETM, GLUCANEXTM, LACTOZYMTM
  • Further carbohydrases are available from other suppliers.
  • the amount of enzyme to be incorporated in a granule of the invention will depend on the intended use of the granule. For many applications, the enzyme content will be as high as possible or practicable.
  • the content of enzyme (calculated as pure enzyme protein) in a granule of the invention will typically be in the range of from about 0.5% to 50% by weight of the enzyme-containing granule .
  • the granules of the invention particularly contains auxiliary granulation agents for purposes such as aiding the formation of granules, controlling density and volume of granules, controlling amount of active compound in the granules, stabilising the active compound and the like.
  • Auxiliary granulating agents may include but is not limited to: a) Fillers such as fillers conventionally used in the field of granulation e.g. water soluble and/or insoluble inorganic salts such as finely ground alkali sulphate, alkali carbonate and/or alkali chloride), clays such as kaolin (e.g. SpeswhiteTM, English China Clay) , bentonites, talcs, zeolites, and/or silicates .
  • Fillers such as fillers conventionally used in the field of granulation e.g. water soluble and/or insoluble inorganic salts such as finely ground alkali sulphate, alkali carbonate and/or alkali chloride), clays such as kaolin (e.g. SpeswhiteTM, English China Clay) , bentonites, talcs, zeolites, and/or silicates .
  • Binders such as binders conventionally used in the field of granulation e.g. binders with a high melting point or no melting point at all and of a non waxy nature e.g. polyvinyl pyrrolidon, dextrins, polyvinylalkohol, cellulose derivatives, for example hydroxypropyl cellulose, methyl cellulose or CMC.
  • a suitable binder is a carbohydrate binder such as Glucidex 21D available from Roquette Freres, France.
  • Fiber materials such as fibers conventionally used in the field of granulation.
  • Pure or impure cellulose in fibrous form can be sawdust, pure fibrous cellulose, cotton, or other forms of pure or impure fibrous cellulose.
  • filter aids based on fibrous cellulose can be used.
  • Several brands of cellulose in fibrous form are on the market, e.g. CEPO and ARBOCELL.
  • Cepo S/20 cellulose the approximate maximum fiber length is 500 ⁇ m, the approximate average fiber length is 160 ⁇ m, the approximate maximum fiber width is 50 ⁇ m and the approximate average fiber width is 30 ⁇ m.
  • CEPO SS/200 cellulose has an approximate maximum fiber length of 150 ⁇ m, an approximate average fiber length of 50 ⁇ m, an approximate maximum fiber width of 45 ⁇ m and an approximate average fiber width of 25 ⁇ m.
  • Cellulose fibers with these dimensions are very well suited for the purpose of the invention.
  • the words "Cepo” and "Arbocel” are Trademarks.
  • Particular fibrous cellulose is ArbocelTM BFC200.
  • synthetic fibres may be used as described in EP 304331 Bl and typical fibres may be made of polyethylene, polypropylene, polyester, especially nylon, polyvinylformat , poly (meth) acrylic compounds.
  • Liquid agents such as conventionally used in the field of granulation.
  • a liquid agent is used in conventional mixer granulation processes for enabling the build up or agglomeration of the conventional granulating component particles into granules.
  • the liquid agent is water and/or a waxy substance.
  • the liquid agent is always used in a liquid phase in the granulation process but may later on solidify; the waxy substance if present, therefore, is either dissolved or dispersed in the water or melted.
  • waxy substance as used herein is meant a substance which possesses all of the following characteristics 1) the melting point is between 30 and 100°C, particularly between 40 and 60°C, 2) the substance is of a tough and not brittle nature, and 3) the substance possesses a certain plasticity at room temperature. Both water and waxy substance are liquid agents, i.e. they are both active during the formation of the granules; the waxy substance stays as a constituent in the finished granules, whereas the majority of the water is removed during a drying step.
  • waxy substances are polyglycols, fatty alcohols, ethoxylated fatty alcohols, mono-, di- and triglycerolesters of higher fatty, acids, e.g. glycerol monostearate, alkylarylethoxylates, and coconut monoethanolamide .
  • the liquid agent can be either water alone, waxy substance alone or a mixture of water and waxy substance.
  • the water and the waxy substance can be added in any sequence, e.g. first the water and then the waxy substance, or first the waxy substance and then the water or a solution or suspension of the waxy substance in the water.
  • the waxy substance can be soluble or insoluble (but dispersible) in water. If water is used a liquid agent it may not be a part of the finished mixer granule as usually most of the water is dried off at a subsequent drying of the mixer granules.
  • Enzyme stabilizing or protective agents such as conventionally used in the field of granulation.
  • Stabilizing or protective agents may fall into several categories: alkaline or neutral materials, reducing agents, antioxidants and/or salts of first transition series metal ions. Each of these may be used in conjunction with other protective agents of the same or different categories.
  • alkaline protective agents are alkali metal silicates, -carbonates or bicarbonates, which provide a chemical scavenging effect by actively neutralizing e.g. oxidants.
  • reducing protective agents are salts of sulfite, thiosulfite or thiosulfate
  • antioxidants are methionine, butylated hydroxytoluene (BHT) or butylated hydroxyanisol (BHA).
  • BHT butylated hydroxytoluene
  • BHA butylated hydroxyanisol
  • Most particular agents are salts of thiosulfates, e.g. sodium thiosulfate.
  • enzyme stabilizers may be borates, borax, formates, di- and tricarboxylic acids and reversible enzyme inhibitors such as organic compounds with sulfhydryl groups or alkylated or arylated boric acids.
  • Cross linking agents such as conventionally used in the field of granulation.
  • Cross linking agents may be enzyme-compatible surfactants e.g. ethoxylated alcohols, especially ones with 10 to 80 ethoxy groups.
  • suspension agents, mediators (for boosting bleach action upon dissolution of the granule in e.g. a washing application or mediators for enzymes) and/or solvents may be incorporated as auxiliary granulating agents.
  • Coating agents The coating comprises one or more conventional coating agents components such as described in WO 89/08694, WO 89/08695, EP 270 608 Bl and/or WO 00/01793.
  • Other examples of coating agents may be found in US 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, US 5,324,649, US 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229 A, DD 263790, JP 61162185 A and/or JP 58179492.
  • the coating agent may be selected from the list of auxiliary granulation agents described, supra .
  • Further coating agents may be selected the following non-limiting list of polymers, chlorine scavengers, plasticizers, pigments, lubricants (such as surfactants or antistatic agents) and fragrances .
  • Polymers useful in coating layers include vinyl polymers or vinyl co-polymers such as polyvinyl alcohol (PVA) and/or polyvinyl pyrrolidone or derivatives thereof. Also included are isoptalic acid polymers.
  • Plasticizers useful in coating layers in the context of the present invention include, for example: polyols such as sugars, sugar alcohols, or polyethylene glycols (PEGs) having a molecular weight less than 1000; urea, phthalate esters such as dibutyl or dimethyl phthalate; and water.
  • polyols such as sugars, sugar alcohols, or polyethylene glycols (PEGs) having a molecular weight less than 1000
  • PEGs polyethylene glycols having a molecular weight less than 1000
  • urea, phthalate esters such as dibutyl or dimethyl phthalate
  • water water
  • Suitable pigments include, but are not limited to, finely divided whiteners, such as titanium dioxide or kaolin, coloured pigments, water soluble colorants, as well as combinations of one or more pigments and water soluble colorants .
  • lubricant refers to any agent, which reduces surface friction, lubricates the surface of the granule, decreases tendency to build-up of static electricity, and/or reduces friability of the granules.
  • Lubricants can also play a related role in improving the coating process, by reducing the tackiness of binders in the coating.
  • lubricants can serve as anti- agglomeration agents and wetting agents.
  • suitable lubricants are polyethylene glycols (PEGs) and ethoxylated fatty alcohols.
  • the coating may also optionally comprise functional components selected for their specific use in the, pharmaceutical industry, agriculture, foodstuffs industry, baking industry, additives industry, feed industry, detergents industry or other industries where granules comprising a biologically active compound can be used.
  • the granule of the invention is coated with a protective coating having a high constant humidity of at least 81% such as described in WO 89/08694, which is hereby incorporated by reference.
  • the coating should, in certain embodiments, act as a moisture and/or bleach barrier to stabilise the biologically active compound in the core.
  • the coating unit acts as a mechanical barrier during mechanical processes such as dosing or tabletting.
  • the coating unit is sufficiently compressible and flexible for the core to withstand a tabletting process, both in a structural sense and with regards to biological activity of the active compound. This is potentially most applicable for detergent formulations .
  • the coating agent absorbs light from the excitation source and/or emitted from a fluorescent marker in the granule, so that when an area of the granule surface is coated with the coating agent a reduction in detected light emitted from this area is achieved.
  • the fluorescent marker
  • the fluorescent marker comprised the granular composition of the invention may be any compound exhibiting fluorescence when being illuminated.
  • the fluorescent marker is particularly organic and exhibiting fluorescence when illuminated with light in the X-ray, ultra violate and/or visible regions of the electromagnetic spectrum, e.g. light having wavelengths between 10-700 nm, more particular light in the ultra violate region, i.e. 10-380 nm.
  • the fluorescent marker comprised the granular composition of the invention is capable upon excitation to emit light in the ultra violate, visible and/or near infrared regions of the electromagnetic spectrum, i.e. suitably between 185-2600 nm.
  • the fluorescent marker may belong to the group of biologically active compounds, auxiliary granulation agents and coating agents or it may be a compound added to the granular composition with the sole purpose of performing the fluorescence analysis of the invention. It is however from a cost saving point of view preferred that the fluorescent marker is the biologically active compound itself or it is an auxiliary granulation agent.
  • fluorescent marker may be chosen depending on the property of the granulated composition to be evaluated by the fluorescence analysis, e.g. dust properties or coating thickness.
  • different fluorescent marker may be chosen.
  • the biologically active compound as the fluorescent marker because it is necessary to assess the amount of potentially damaging active compound in dust particles present in the granular composition
  • coating thickness it may be possible to choose a suitable auxiliary granulation agent as fluorescent marker.
  • the biologically active compound is the fluorescent marker, especially those belonging to the particular group of proteins and/or peptides, in which it is aromatic amino acids residues such as tyrosine and tryptophan that are responsible for the fluorescence emission
  • a light source delivering ultra violet light (UV-light) , particularly delivering a substantial portion of UV-light having wavelengths between 10-380 nm, more particularly between 200-400 nm or 200-300 nm, most particularly between 260-280 nm.
  • UV-light ultra violet light
  • emitted light in the range of 200-700 nm, such as from 400-700 nm, or from 300-400 nm, especially light from 280-360 nm or from 325-375 nm. Also, in this case it is required to choose a detector, such as a CCD camera, capable of detecting emitted light having these wavelengths.
  • the fluorescent marker is one of the auxiliary granulation agents
  • a detector such as a CCD camera, capable of detecting emitted light having these wavelengths.
  • suitable fluorescent compound are available e.g. from Molecular Probes, USA.
  • Fluorescence analysis in granulation and coating processes also encompass processes for preparing granular compositions comprising a biologically active compound and optionally auxiliary granulation agents in a granulation apparatus using the above mentioned method of fluorescence analysis to predict properties of the granular composition and control and improve the preparation process.
  • the present invention provides a process for preparing granules comprising a biologically active compound and optionally auxiliary granulation agents in a granulation apparatus said process comprising the step of performing fluorescence analysis on a fluorescent marker comprised in the granular composition as described, supra , on the granules forming in the granulator.
  • the fluorescence analysis is carried out during the formation of granules in the granulation process, particularly on-line, meaning that the fluorescence analysis is performed more than one time in real time during the granulation process with a suitable rate of repetition.
  • the repetition rate will, in ter alia , depend on the data processing of data from the detector (s).
  • about 15 measurements of the emitted light per second is recorded in form of two-dimensional images in the granulation process.
  • the term "formation of granules" includes also coating granules with a coating layer.
  • the process also particularly comprises the step of changing at least one process parameter as a result of the fluorescence analysis.
  • the process parameter to be changed may be any parameter influencing the granulation process and/or the properties of the formed granules. These parameters may be supply of granulation material, i.e. biologically active compound and/or auxiliary granulation agents and/or coating agent to the granulator, supply of gas to the granulator, temperature in the granulator, pressure in the granulator, pH in the granulator and mechanical force conferred to the granulation material.
  • the process parameter may be changed manually or though an automated system, cf. granulation apparatus.
  • fluorescence analysis in accordance with the invention may also suitably be used to control dusting properties of finished granular compositions after granulation.
  • the invention further provides a method for fluorescence analysis of active dust in a granular composition comprising a biologically active compound. Using this method, granular compositions, which do not meet the desired quality with respect to dust, may be discarded or reprocessed.
  • fluorescence analysis in accordance with the invention may also suitably be used to control coating thickness and/or homogeneity of finished granular compositions after granulation. Accordingly, the invention further provides a method for fluorescence analysis of coating thickness in a composition of coated granules comprising a biologically active compound. Using this method granular compositions, which do not meet the desired quality with respect to coating thickness may be discarded or reprocessed.
  • the imaging system can, as said be used for evaluation of a quality parameter, such as active dust values or coating thickness.
  • a quality parameter such as active dust values or coating thickness.
  • the evaluation is performed on- line during for example a coating process.
  • the evaluation is performed by recording, in a particular by means of a camera, images of emitted light from granules subjected to an excitation light source and comparing the recorded images with images recorded of samples (reference samples) with known values of a quality parameter.
  • the fluorescent images of the reference samples and the corresponding quality parameters are used to provide a calibration model, such as a Partial Least Square model or any other modelling system suitable for making calibration models of image data.
  • the model may then used to predict estimates of a quality parameter from the fluorescent images of an unknown sample.
  • the present invention also includes a method for estimating a quality parameter of a granular composition comprising a purified biologically active comprising the steps of: a) providing a calibration model by illuminating a granular composition comprising a purified biologically active compound having a known quality parameter with light capable of fluorescence excitation of a fluorescent marker comprised in the granular composition, recording one or more images of the light emitted from the granular composition of a known quality and subjecting recorded images to data processing, particularly in the form of partial least squares data processing, to form a calibration model, b) illuminating a unknown granular composition comprising a purified biologically active compound with light capable of fluorescence excitation of a fluorescent marker comprised in the granular composition, recording at least one image of the light emitted from the unknown granular composition, c) comparing at least one image of the unknown granular composition with the calibration model and d) estimating the quality parameter of the unknown granular composition.
  • a granulation and/or coating apparatus comprising means for performing fluorescence analysis on granular compositions in accordance with the invention. Accordingly, the invention provides a granulation or coating apparatus comprising:
  • a granulation or coating device comprising at least one chamber for processing material into granules or coated granules
  • an optical arrangement for performing fluorescence analysis comprising a light source for illumination of material being processed, at least one detector capable of detecting light emitted from the material being processed, means for projecting illuminating light onto a portion of the material being processed, means for projecting light emitted from illuminated material to the detector and at least one device for filtering light.
  • the granulation or coating device may be any conventional granulation device, in particular it may be selected from fluid bed granulators or coaters, high shear mixer granulators, spray dryers, a spray coolers and extruders.
  • the light source is particularly a normal glow lamp, a more specialized xenon lamp or a stroboscope lamp, particularly capable of delivering light having wavelengths between 10-700 nm, more particularly light in the ultra violate region, i.e. 10-380 nm.
  • the detector is particularly a camera type detector, more particularly a line-scan camera, a CCD or an ICCD camera.
  • the optical arrangement comprises means for focusing emitted light, such as lenses.
  • the means for projecting illuminating light onto the material being processed and projecting emitted light from said material to the detector includes one or more of fiber optics, mirrors, lenses, beam splitters and the like.
  • the optical arrangement includes at least one filtering device for filtering the illuminating and/or emitted light.
  • this device is a band pass filter or a grate monochromator .
  • one or more filtering devices are positioned so than only the emitted light is filtered and so that the emitted light must pass the filter (s) before reaching the detector (s).
  • at least two filtering devices are positioned so that both the illuminating light and the emitted light is filtered. Most preferred filters are those selecting wavelength as described for the particular fluorescent markers, supra .
  • the optical arrangement also includes at least one beam splitter such as dichroic mirrors.
  • the optical arrangement includes a stroboscope light source, 2 CCD camera detectors, one band pass filter for filtering illumination light, two band pass filters for filtering emitted light, lenses and two dichroic mirror beam splitters as depicted in figure 1.
  • the projecting means include means for projecting illuminating light through an opening in the chamber onto a portion of material being processed in the chamber and projecting emitted light from this material in the chamber to the detector.
  • the granulation apparatus further comprise means for providing a purge stream of material from the chamber.
  • the optical arrangement is positioned to allow fluorescence analysis of material in the purge stream rather than on material present in the chamber.
  • granulation processes usually involves considerable wear and tear of the granulation equipment.
  • some auxiliary granulation agents may be clays or other inorganic substances. These substances may have a significant sanding effect on the granulation equipment. Accordingly, when granulating in e.g. a mixer it is not unusual to observe that several millimetres of steel is sanded of the surfaces on the interior parts of the granulation equipment per year.
  • the granules in the purge stream may suitably be recycled and this way the fluorescence analysis does not interfere with the granulation process.
  • the purge stream may suitably lead the granules past a part of the purge stream transparent to light wherein the projection of light may occur.
  • the purge stream may be transported from the granulation chamber, through a transporting system, which e.g. may include one or more elements selected from chutes, pumps, pipe, conveyor belts, cyclones and the like, to the optical arrangement.
  • the fluorescence analysis may suitably occur at a point in the transporting system, where the granulated product can be accessed by the illuminating light, and from which point the emitted light may reach the detector (s) .
  • a point may be a point where part of e.g. a pipe material is replaced with a transparent material such as glass, quartz or a polymeric material.
  • the means for providing the purge stream includes means for forming a single layer of granules, so that no or little overlapping occurs in the detection from granules, which superimpose each other at the moment of detection.
  • a tilted (non- horizontal) vibrating surface e.g. a vibrating chute
  • the granules may particularly also leave the vibrating surface as a single layer as they fall over one or more edges of the vibrating surface (comparable to a waterfall) and in order to avoid reflections from the vibrating surface the fluorescence analysis particularly take place at some point after the granules leaves the vibrating surface, but while they still maintain a single layer of granules.
  • By measuring on granules forming a single layer of granules overlapping is avoided and the emitted light from the granules may be more precisely focused as the granules is primarily distributed in only two dimensions. This way, sharply focused images of nearly all individual granules passing the point of fluorescence analysis may be obtained using a two-dimensional detector such as a CCD camera .
  • the optical arrangement is suitably connected to the granulation or coating device to enable online or at-line fluorescence analysis of granular compositions.
  • On-line analysis is to be understood as analysis performed on granules as they are actually being granulated, e.g. by analyzing granules in the granulator or in a recycled purge stream.
  • At-line analysis is to be understood as analysis performed down stream after the granulation process (e.g. at the outlet) or on non-recycled samples taken from the granulator during granulation.
  • the granulation apparatus may comprise other elements such computing units for processing data from detectors, optionally equipped with specialised data handling hardware and software.
  • the granulation apparatus may also comprise control units linked to the computing units for controlling and adjusting the granulation process based on the results of the fluorescence analysis.
  • a control unit may be a PLC or other equipment capable of receiving data from a computing unit and converting these data into output controlling one or more hardware devices influencing the granulation process, such as feed streams, speed, temperature, airflows etc.
  • Fluorescence analysis of eight different raw materials for enzyme granulation and enzyme concentrate where measured on a LS50B (Perkin Elmer) instrument.
  • the raw materials were excitated with light of the internal source of different wavelengths from 230-500 nm in 10 nm steps and emission from the materials was recorded from 270-700 nm in steps of 1 nm. Slit gap in the Perkin Elmer instrument for both excitation and emission was 4 nm.
  • the fluorescence was measured by illuminating the raw material placed in a quartz container (cuvette) and measuring fluorescence emission from the raw materials at an angle displaced 22.5 degrees from the direction of the illuminating light as required by the instrument design.
  • Several raw materials fluoresced significantly but the enzyme concentrate had a uniquely a very intensive emission at about 350 nm.
  • Fluorescence analysis of uncoated enzyme granules as well as coated granules were measured on a LS50B (Perkin Elmer) instrument.
  • the granules were excitated with light of the internal source of different wavelengths from 230-500 nm in 10 nm steps and emission from the materials was recorded from 270- 700 nm in steps of 1 nm.
  • Slit gap in the Perkin Elmer instrument for both excitation and emission was 4 nm.
  • the fluorescence was measured by illuminating the raw material placed in a quartz container (cuvette) and measuring fluorescence emission from the raw materials at an angle displaced 22.5 degrees from the direction of the illuminating light as required by the instrument design.
  • the results showed a peak of emission around 350 nm corresponding to the enzyme concentrate. Another strong peak occurred between 450-500 nm due to fluorescent compounds among granulation auxiliaries. The results show the feasibility of detecting enzymes in the granules.
  • Fluorescence analysis of uncoated enzyme granules as well as coated granules was measured on using a optical arrangement as shown in figure 1.
  • Two camera detectors of the type Donpisha "Progressive Camera Module” XC-8500CE 1/2" ITCCD 782(H) x 582 (V) , with a CCIR Additional 3 ' 5 mm lens at sample introduction point was used.
  • the light source was an Oriel
  • Illuminating light was filtered using a band pass filter to produce a beam of light having a wavelength of 450 nm. Before reaching the cameras the emitted light was split into two beams by a dichroic mirror beam splitter and each beam was filtered by a band pass filters.
  • One filter allowed passage of 530 nm light (green filter) and the other filter allowed passage of 620 nm light (red filter) .
  • the illumination and detection of emitted light was carried out by loading enzyme granules into a funnel having an outlet of proportion allowing passage of the granules and performing the analysis on granules leaving the funnel immediately after the funnel outlet.
  • the signal from the detectors was transferred to a computing unit (ordinary personal computer) equipped with data acquisition hardware, DAQ and SCB-68 breadboard and image processing software, Labview 4.1.1 IMAQ vision and All-purpose Labview all from National Instruments to produce instantaneous digital images of fluorescent granules passing the beam of illuminating light.
  • a computing unit ordinary personal computer
  • data acquisition hardware DAQ and SCB-68 breadboard and image processing software
  • Labview 4.1.1 IMAQ vision and All-purpose Labview all from National Instruments to produce instantaneous digital images of fluorescent granules passing the beam of illuminating light.
  • Illuminating light was filtered using a band pass filter to produce a beam of light in the region 300-400 nm.
  • the emitted light was detected with a 3-CCD camera from JAI (M-90) .
  • the camera was mounted with a Computar 55 Telecentric lens.
  • the signal from the detectors was transferred to a computing unit (ordinary personal computer) equipped with IFC51 frame grabber and Image-Pro Plus version 4.5. Fluorescent images recorded of the granules showed visually 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 (PEG4000) containing kaolin and titan dioxide. Fluorescent images recorded of the coated granules showed visually that the average intensity of emitted light increased as a function of increasing concentrations of purified protease. This indicates that the biologically active, in this experiment a protease, may also be the fluorescent marker.
  • PEG4000 PEG-coating
  • the samples were excitated with an Oriel Xenon Flash Lamp 60000 w/ Oriel attachment 60008 and Oriel Power Supply 68826. Illuminating light was filtered using a band pass filter to produce a beam of light in the region from 300-400 nm. The emitted light was detected with a 3-CCD camera from JAI (M- 90). The camera was mounted with a Computar 55 Telecentric lens. The signal from the detectors was transferred to a computing unit (ordinary personal computer) equipped with IFC51 frame grabber and Image-Pro Plus version 4.5. Fluorescent images recorded of the coated granules showed visually that the average intensity of emitted light decreased for increasing concentrations of coating (i.e. for 10%, 25% and 50% of coating administered) . This indicates that the intensity of fluorescent emitted light can be used to predict the thickness of the coating.
  • the samples were excitated with an Oriel Xenon Flash Lamp 60000 w/ Oriel attachment 60008 and Oriel Power Supply 68826. Illuminating light was filtered using a band pass filter to produce a beam of light in the region from 300-400 nm. The emitted light was detected with a 3-CCD camera from JAI (M- 90). The camera was mounted with a Computar 55 Telecentric lens. The signal from the detectors was transferred to a computing unit (ordinary personal computer) equipped with IFC51 frame grabber and Image-Pro Plus version 4.5. The samples were conveyed to the imaging system in a distance of approximately 10 cm.
  • Fluorescent images recorded of the granular composition showed visually that the average intensity of emitted light increased for compositions with increasing amounts of protease active dust values.

Abstract

La présente invention concerne un procédé d'analyse par fluorimétrie consistant à éclairer une composition granulaire contenant un composé purifié biologiquement actif avec une lumière pouvant provoquer l'excitation de fluorescence d'un marqueur fluorescent contenu dans la composition granulaire, à détecter la lumière émise par le marqueur fluorescent et à prévoir la quantité de marqueur fluorescent, pour la composition granulaire, dont l'excitation de fluorescence peut être provoquée.
PCT/DK2002/000071 2001-01-31 2002-01-31 Procede d'analyse d'une composition granulaire par fluorimetrie WO2002061427A1 (fr)

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CN100445745C (zh) 2008-12-24
CN1613013A (zh) 2005-05-04
JP2004518140A (ja) 2004-06-17
JP4198996B2 (ja) 2008-12-17

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