WO1990013625A1 - Method of preparing gelatin microcarriers - Google Patents

Method of preparing gelatin microcarriers Download PDF

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Publication number
WO1990013625A1
WO1990013625A1 PCT/DK1990/000112 DK9000112W WO9013625A1 WO 1990013625 A1 WO1990013625 A1 WO 1990013625A1 DK 9000112 W DK9000112 W DK 9000112W WO 9013625 A1 WO9013625 A1 WO 9013625A1
Authority
WO
WIPO (PCT)
Prior art keywords
gelatin
particles
coated
particulate material
microcarrier
Prior art date
Application number
PCT/DK1990/000112
Other languages
French (fr)
Inventor
Kim Jacobsen
Original Assignee
Danochemo A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Danochemo A/S filed Critical Danochemo A/S
Publication of WO1990013625A1 publication Critical patent/WO1990013625A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This invention relates to a method of preparing gelatin microcar ⁇ riers for the cultivation of cells.
  • a microcarrier is a solid particle capable of supporting the growth of anchorage-dependent cells contained in a liquid culture medium.
  • Microcarriers are typically spherical particles having a diameter of 50-500 ⁇ m and preferably 100-400 ⁇ m. Microcarriers may have a porous structure or a dense surface with dents.
  • Cultivation methods based on the use of microcarriers are considered to be some of the most suitable methods for large scale production of cell products.
  • the large surface area per weight unit the possibility of optimizing the microcarrier surface to adapt it to different types of cells, the relative simple up-scaling procedures which are required in microcarrier-based cultivations, the fact that the microcarrier based production technique is similar to ordinary fermentation techniques and can be effected in only slightly odi- fied fermentors are factors which strongly favour microcarrier based production methods.
  • the most critical problem involved in microcarrier based large scale cell production in conventional fermentors is concerned with the stirring of the culture medium in order to continuously expose the cells to fresh culture medium, since the cells are sensitive to the mechanical treatment of the medium.
  • European patent publication No. 066.726 discloses the use of a cross-linked dextran microcarrier, which has been modified by reaction with a tertiary ammonium compound to provide the micro- carrier particles with an outer layer of quaternary amino groups so as to obtain a desired surface charge.
  • an aqueous solution of a colloid e.g. gelatin, pectin, dextran, agarose or gum arabic, and optionally a sugar is emulsified in a liquid which is immiscible with water such as peanut oil, castor oil, and mineral oil or an organic solvent.
  • a colloid e.g. gelatin, pectin, dextran, agarose or gum arabic
  • a sugar is emulsified in a liquid which is immiscible with water such as peanut oil, castor oil, and mineral oil or an organic solvent.
  • the emulsion thus formed is then cooled so as to cause the colloid to form gel par ⁇ ticles.
  • the particles formed are then separated and dried by well known techniques.
  • the above mentioned method suffers form the drawback that the microcarrier particles are not free-flowing but tend to form agglomerates.
  • gelatin particles thus formed are free-flowing but they have been found unsuitable for use as microcarriers primarily because the coating on the gelatin particles prevent the cells to be cultivated from contacting the gelatin surface.
  • the object of the invention is to prepare a free-flowing gelatin microcarrier.
  • a further object of the invention is to provide non-coated gelatin microcarrier particles.
  • a still further object of the invention is to provide gelatin microcarrier particles having a surface which is suitable as a support for anchorage-dependent cells.
  • the method of the invention comprises the steps of atomizing an aqueous gelatin solution in a stream of air while introducing into the atomizing zone a dry particulate material to form gelatin particles coated with said dry particulate material, recovering the coated gelatin particles and treating the coated gelatin particles with a medium capable of removing the coating therefrom without substantially decomposing the gelatin particles.
  • the invention is based on the discovery that by providing gelatin particles formed by atomizing a gelatin solution with a coating of a solid particulate material, a build up of material on the walls of the atomizing chamber can be avoided and that the coating on the gelatine particles can be removed so as to form non-coated gelatin particles which are excellently suitable as a substrate for ancho ⁇ rage-dependent cells. Thus it has been found that such cells adhere well and uniformly to the surface of such non-coated gelatin par ⁇ ticles.
  • the gelatin solution is preferably atomized by means of an atomizer or an atomizing wheel mounted for rotation on a spray drying apparatus.
  • the temperature of the gelatin solution is preferably between 20 and 90°C, and the viscosity between 50 and 200 cps.
  • the temperature of the stream of air is preferably between 5 and
  • the dry particulate material such as a powder, is introduced into the atomizing zone so as to bring the solid particulate material in direct contact with the droplets shortly after their formation.
  • the dry particulate material is preferably a powder such as calcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium lactate or magnesium carbonate.
  • a particularly preferred group of particulate materials are compounds which are soluble in acidic aqueous solutions, because gelatin is relatively insoluble in such solutions.
  • starch such as corn starch, rice starch, and wheat starch
  • starch derivatives which are enzymatically degradable.
  • the dry particulate material is preferably used in an amount of from 5 to 50% by weight based on the weight of the gelatin particles.
  • the coated gelatin particles are preferably introduced into a fluid-bed and are dried therein in a conventional way.
  • the tempera ⁇ ture of the fluid-bed is preferably within the range of from 10 to 60°C.
  • the removal of the coating on the gelatin particles is preferably effected by treating the coated particles with a liquid medium capable of dissolving or degrading the coating.
  • the coating material is preferably soluble in acidic solutions and in that case the coated gelatin particles are preferably treated with a solution of a mineral acid such as hydrochloric acid.
  • the particles are preferably treated with an aqueous medium containing a starch degrading enzyme, such as ⁇ -amylase.
  • a starch degrading enzyme such as ⁇ -amylase
  • the gelatin material may be cross-linked either in coated or non- coated state by chemical reaction with e.g. an aldehyde such as formaldehyde or glutaraldehyde or by heating to an elevated tempe- rature e.g. a temperature of from 150 to 200°C for a period of from 30 minutes to 3 hours.
  • an aldehyde such as formaldehyde or glutaraldehyde
  • an elevated tempe- rature e.g. a temperature of from 150 to 200°C for a period of from 30 minutes to 3 hours.
  • the cross-linking may be effected immediately following the forma ⁇ tion of the coated gelatin particles or prior to or subsequent to the removal of the coating from the particles.
  • the removal of the coating may be a step in the conventional pretreatment of a microcarrier before inoculation with cells.
  • microcarrier is ordinarily subjected to a series of washes with different buffers systems, media, etc.
  • the particles thus obtained having a coat of tricalcium phosphate (20-40% w/w) are suspended in 5 1 of water, whereafter 7 1 of IN hydrochloric acid are added in order to dissolve the tricalcium phosphate.
  • the naked gelatine particles thus formed are washed with water until all hydrochloric acid and salts have been removed. Subsequently the particles are filtered off and dried in a fluid- bed.
  • a starch degrading enzyme (Termanyl 120L, N0V0 Industri A/S) is added so as to dissolve the corn starch.
  • the naked gelatin particles thus formed are washed with water until all enzyme and starch residues have been removed and the particles are then filtered off and dried in a fluid-bed.
  • microcarrier was washed with 200 ml PBS buffer, separated by decanting, and re-slurried in 100 ml PBS buffer. Subsequently the microcarrier was sterilized and autoclaved at 121°C for 20 min.
  • a kidney cell line was introduced into 25 ml Erlenmayer flasks to obtain a cell density of 4,4 • 10 cells/ml together with the pretreated microcarrier or a well known Sephadex microcarrier (Cytodex 3 (Pharmacia)) (microcarrier density: 0,1 g/20 ml) and a DME/HAM culture medium.
  • the total volume of the contents of the flasks was 7.5 ml.
  • the flasks were shaken in a water bath at a temperature of 35°C and at 65 rpm.
  • the number of adhering cells will appear from the following table.
  • the adherence of the cells to the microcarrier according to the invention is superior to the adherence of the cells to the known microcarrier.

Abstract

Method of preparing gelatin microcarriers for the cultivation of cells comprising the steps of atomizing an aqueous gelatin solution while adding a dry particulate anti-clogging material to form coated gelatin particles and treating the coated particles with a medium capable of removing the coating from the particles.

Description

Method of preparing gelatin microcarriers
This invention relates to a method of preparing gelatin microcar¬ riers for the cultivation of cells.
A microcarrier is a solid particle capable of supporting the growth of anchorage-dependent cells contained in a liquid culture medium. Microcarriers are typically spherical particles having a diameter of 50-500μm and preferably 100-400 μm. Microcarriers may have a porous structure or a dense surface with dents.
Cultivation methods based on the use of microcarriers are considered to be some of the most suitable methods for large scale production of cell products. Thus the large surface area per weight unit, the possibility of optimizing the microcarrier surface to adapt it to different types of cells, the relative simple up-scaling procedures which are required in microcarrier-based cultivations, the fact that the microcarrier based production technique is similar to ordinary fermentation techniques and can be effected in only slightly odi- fied fermentors are factors which strongly favour microcarrier based production methods.
Ordinary fermentors find wide spread use in the biochemical industry and by using existing conventional fermentors or slightly modified versions thereof the production costs can be kept at a relatively low level.
The most critical problem involved in microcarrier based large scale cell production in conventional fermentors is concerned with the stirring of the culture medium in order to continuously expose the cells to fresh culture medium, since the cells are sensitive to the mechanical treatment of the medium.
This problem, however, can be solved by using specially shaped stirrers and by constantly pumping fresh culture medium into the fermentor at its bottom and removing spent medium from the top thereof.
By modifying the ordinary fermentation technique in this manner a plant that produces microcarrier based cell cultures can be run continuously for months.
A.L. Van Wezel , Nature, 216, pp. 64-65, (1967) described the use of DEAE-Sephadex microcarriers for the cultivation of two different cell lines. Other workers also concentrated on the use of DEAE Sephadex microcarriers, cf. D.W. Levine et al., "Optimizing Parameters for Growth of Anchorage-Dependent Mammalian Cells in Microcarrier Culture", in "Cell Culture and its Applications", Academic Press, New York, 1977, pp. 191-216 and US patent specification No. 4.036.693.
Other materials than DEAE Sephadex have been employed as microcarriers, see e.g. US patent specification No. 3.717.551 which discloses the cultivation of cells on porous silica spherules. The use of gelatin, collagen, and related materials in the cultivation of cells is also known, cf. US patent specification No. 4.169.761 which discloses the cultivation of cells on collagen fibres and the use of collagen in the form of gel particles and EP patent publication No. 0.058.689 which discloses a cell culture medium comprising cross-linked gelatin particles which can be totally degraded by proteolytic enzymes.
European patent publication No. 066.726 discloses the use of a cross-linked dextran microcarrier, which has been modified by reaction with a tertiary ammonium compound to provide the micro- carrier particles with an outer layer of quaternary amino groups so as to obtain a desired surface charge.
According to a well known method for the preparation of micro- carriers an aqueous solution of a colloid e.g. gelatin, pectin, dextran, agarose or gum arabic, and optionally a sugar is emulsified in a liquid which is immiscible with water such as peanut oil, castor oil, and mineral oil or an organic solvent. The emulsion thus formed is then cooled so as to cause the colloid to form gel par¬ ticles. The particles formed are then separated and dried by well known techniques.
The above mentioned method suffers form the drawback that the microcarrier particles are not free-flowing but tend to form agglomerates.
It has been attempted to prepare gelatin particles for use as microcarriers by a spray-drying process comprising atomizing a gelatin solution in a spray drying chamber so as to form droplets while supplying to the atomizing zone a stream of air and a dry particulate absorbent serving to form a coating on the gelatin particles, thus preventing them from adhering to the walls of the spray drying chamber.
The gelatin particles thus formed are free-flowing but they have been found unsuitable for use as microcarriers primarily because the coating on the gelatin particles prevent the cells to be cultivated from contacting the gelatin surface.
The object of the invention is to prepare a free-flowing gelatin microcarrier.
A further object of the invention is to provide non-coated gelatin microcarrier particles.
A still further object of the invention is to provide gelatin microcarrier particles having a surface which is suitable as a support for anchorage-dependent cells.
These and other objects which will appear form the following description are achieved by the method of the invention, which method comprises the steps of atomizing an aqueous gelatin solution in a stream of air while introducing into the atomizing zone a dry particulate material to form gelatin particles coated with said dry particulate material, recovering the coated gelatin particles and treating the coated gelatin particles with a medium capable of removing the coating therefrom without substantially decomposing the gelatin particles.
The invention is based on the discovery that by providing gelatin particles formed by atomizing a gelatin solution with a coating of a solid particulate material, a build up of material on the walls of the atomizing chamber can be avoided and that the coating on the gelatine particles can be removed so as to form non-coated gelatin particles which are excellently suitable as a substrate for ancho¬ rage-dependent cells. Thus it has been found that such cells adhere well and uniformly to the surface of such non-coated gelatin par¬ ticles.
The gelatin solution is preferably atomized by means of an atomizer or an atomizing wheel mounted for rotation on a spray drying apparatus. The temperature of the gelatin solution is preferably between 20 and 90°C, and the viscosity between 50 and 200 cps.
The temperature of the stream of air is preferably between 5 and
95°C.
The dry particulate material, such as a powder, is introduced into the atomizing zone so as to bring the solid particulate material in direct contact with the droplets shortly after their formation.
The dry particulate material is preferably a powder such as calcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium lactate or magnesium carbonate.
A particularly preferred group of particulate materials are compounds which are soluble in acidic aqueous solutions, because gelatin is relatively insoluble in such solutions.
Another preferred type of particulate materials are starch, such as corn starch, rice starch, and wheat starch, and starch derivatives which are enzymatically degradable.
The dry particulate material is preferably used in an amount of from 5 to 50% by weight based on the weight of the gelatin particles.
The coated gelatin particles are preferably introduced into a fluid-bed and are dried therein in a conventional way. The tempera¬ ture of the fluid-bed is preferably within the range of from 10 to 60°C. The removal of the coating on the gelatin particles is preferably effected by treating the coated particles with a liquid medium capable of dissolving or degrading the coating. As mentioned above the coating material is preferably soluble in acidic solutions and in that case the coated gelatin particles are preferably treated with a solution of a mineral acid such as hydrochloric acid.
When the coating material is starch or a starch derivative, the particles are preferably treated with an aqueous medium containing a starch degrading enzyme, such as α-amylase.
The gelatin material may be cross-linked either in coated or non- coated state by chemical reaction with e.g. an aldehyde such as formaldehyde or glutaraldehyde or by heating to an elevated tempe- rature e.g. a temperature of from 150 to 200°C for a period of from 30 minutes to 3 hours.
The cross-linking may be effected immediately following the forma¬ tion of the coated gelatin particles or prior to or subsequent to the removal of the coating from the particles.
The removal of the coating may be a step in the conventional pretreatment of a microcarrier before inoculation with cells.
Thus, during such pretreatment the microcarrier is ordinarily subjected to a series of washes with different buffers systems, media, etc.
The invention will now be described in further detail with reference to the following examples.
Example 1
1.0 kg of 70 bloom gelatin is dissolved in 2.9 kg of water at 65-70°C. The solution thus formed is atomized in a cold or hot airstrea , which causes the atomized particles to gelatinize or solidify. During the ato ization tricalcium phosphate is introduced into the spray zone, and the coated particles formed are dried in a fluid-bed. Following the drying step the gelatin is cross-linked by addition of 200 g of a 10% solution of glutaraldehyde, and the particles are dried.
The particles thus obtained having a coat of tricalcium phosphate (20-40% w/w) are suspended in 5 1 of water, whereafter 7 1 of IN hydrochloric acid are added in order to dissolve the tricalcium phosphate. The naked gelatine particles thus formed are washed with water until all hydrochloric acid and salts have been removed. Subsequently the particles are filtered off and dried in a fluid- bed.
Example 2
1.0 kg of 70 bloom gelatin is dissolved in 2.9 kg of water at 65-70°C. The solution thus formed is atomized in a cold or hot airstream, which causes the atomized particles to gelatinize or solidify. During the atomization corn starch is introduced into the spray zone, and thereafter the coated particles formed are dried in a fluid-bed. Following the drying step the gelatin is cross-linked by addition of 200 g of a 10% solution of glutaraldehyde, and the particles are dried.
The gelatin particles thus obtained and having a coat of corn starch, (20-40% w/w), are suspended in 5 1 of water, whereafter a starch degrading enzyme (Termanyl 120L, N0V0 Industri A/S) is added so as to dissolve the corn starch. The naked gelatin particles thus formed are washed with water until all enzyme and starch residues have been removed and the particles are then filtered off and dried in a fluid-bed.
Example 3
1.0 kg 70 of bloom gelatin is dissolved in 2.9 kg of water at 65-70°C. The solution thus formed is atomized in a cold or hot airstream, which causes the atomized particles to gelatinize or solidify. During the atomization corn starch is introduced into the spray zone, and thereafter the coated particles formed are dried in a fluid-bed. The dried gelatin particles thus obtained and having a coat of corn starch (20-40% w/w) are suspended in 5 1 of cold (8-12°C) water and after 5 minutes 90 ml of a 25% solution of glutardialdehyde are added so as to cause the gelatin to cross-link. After the gelatin has been cross-linked the starch is washed off with water and the gelatin particles are filtered off and dried in a fluid-bed.
Example 4
Biological test.
1. Pretreatment
2 g of a microcarrier prepared as described in example 3 were swelled in 200 ml PBS buffer at room temperature for 3 hours, and then the buffer was removed by decanting.
The microcarrier was washed with 200 ml PBS buffer, separated by decanting, and re-slurried in 100 ml PBS buffer. Subsequently the microcarrier was sterilized and autoclaved at 121°C for 20 min.
2. Determination of adherence to microcarrier
A kidney cell line was introduced into 25 ml Erlenmayer flasks to obtain a cell density of 4,4 • 10 cells/ml together with the pretreated microcarrier or a well known Sephadex microcarrier (Cytodex 3 (Pharmacia)) (microcarrier density: 0,1 g/20 ml) and a DME/HAM culture medium. The total volume of the contents of the flasks was 7.5 ml. The flasks were shaken in a water bath at a temperature of 35°C and at 65 rpm.
After 20, 40 and 80 minutes of shaking the number of cells adhering to the microcarrier was determined by removing the free cells by washing and by treating the cells adhering to the microcarrier with a solution of crystal violet. The stained cells were counted in a Fuchs-Rosenthal counting chamber. 3. Results
The number of adhering cells will appear from the following table.
Time, min Gelatin microcarrier "Cytodex 3" microcarrier
20 2.5 • 104 2.1 • 104
40 3.0 • 104 2.4 • 104
80 3.3 • 104 2.9 • 104
As will appear from the above table the adherence of the cells to the microcarrier according to the invention is superior to the adherence of the cells to the known microcarrier.

Claims

C 1 a i m s
1. A method of preparing gelatin microcarriers for the cultivation of eelIs, c h a r a c t e r i z e d in that it comprises the steps of atomizing an aqueous gelatin solution in a stream of air while introducing into the atomizing zone a dry particulate material to form gelatin particles coated with said dry particulate material, recovering the coated gelatin particles and treating the coated gelatin particles with a medium capable of removing the coating therefrom without substantially decomposing the gelatin particles.
2. A method as in claim 1, c h a r a c t e r i z e d in spray drying a gelatin solution of a temperature of between 20 and 90 C.
3. A method as in claim 2, c h a r a c t e r i z e d in that the temperature of the stream of air is between 5 and 95°C.
4. A method as in any of the claims, c h a r a c t e r i z e d in using a dry particulate material which is soluble in acidic solu- tions and treating the coated gelatin particles with a solution of an acid.
5. A method as in any of claims 1-3, c h a r a c t e r i z e d in using a dry particulate material in the form of a starch or a starch derivative and treating the coated gelatin particles with a medium containing a starch degrading enzyme.
6. A method as in any of the proceeding claims, c h a r a c ¬ t e r i z e d in using the particulate material in an amount of from 5 to 50% by weight based on the weight of the gelatin par¬ ticles.
7. A method as in any of the preceeding claims, c h a r a c ¬ t e r i z e d in cross-linking the gelatin of the gelatin particles in coated or non-coated state.
8. A method as in Claim 7, c h a r a c t e r i z e d in cross- linking the gelatin with an aldehyde.
9. A method as in claim 7, c h a r ac t e r i z e d in cross- linking the gelatin by heating to an elevated temperature.
PCT/DK1990/000112 1989-05-01 1990-05-01 Method of preparing gelatin microcarriers WO1990013625A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK2109/89 1989-05-01
DK210989A DK210989D0 (en) 1989-05-01 1989-05-01 MICROCARRIERS MANUFACTURED BY SPRAYING TECHNIQUES

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WO1990013625A1 true WO1990013625A1 (en) 1990-11-15

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012510A1 (en) * 1994-10-25 1996-05-02 Boehringer Mannheim Gmbh Biomaterial containing epithelial cells and use thereof as a transplant
US6224629B1 (en) * 1998-12-09 2001-05-01 Purzer Pharmaceuticals Co. Ltd. Bone substitute composition and process of preparation thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1392135A (en) * 1972-03-24 1975-04-30 Hoechst Ag Vinyl acetate/ethylene polymer powders
WO1982000660A1 (en) * 1980-08-20 1982-03-04 Mosbach K Immobilization of animal cells
EP0239648A1 (en) * 1985-10-03 1987-10-07 Nauchno-Proizvodstvennoe Obiedinenie "Biolar" Method of obtaining microcarriers for cultivation of cells and microcarriers obtained by that method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1392135A (en) * 1972-03-24 1975-04-30 Hoechst Ag Vinyl acetate/ethylene polymer powders
WO1982000660A1 (en) * 1980-08-20 1982-03-04 Mosbach K Immobilization of animal cells
EP0239648A1 (en) * 1985-10-03 1987-10-07 Nauchno-Proizvodstvennoe Obiedinenie "Biolar" Method of obtaining microcarriers for cultivation of cells and microcarriers obtained by that method

Non-Patent Citations (1)

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Title
DIALOG INFORMATION SERVICES, File 351, World Patent Index 81-90, Dialog accession no. 89-209258/29, (LION CORP): "Improving fluidity of powdered carotene compsn. - by covering with impalpable powder of a least one of calcium and magnesium carbonate(s)"; & JP,A,01 144 953, 07-06-1989, 8929. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012510A1 (en) * 1994-10-25 1996-05-02 Boehringer Mannheim Gmbh Biomaterial containing epithelial cells and use thereof as a transplant
US6224629B1 (en) * 1998-12-09 2001-05-01 Purzer Pharmaceuticals Co. Ltd. Bone substitute composition and process of preparation thereof

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Publication number Publication date
AU5654090A (en) 1990-11-29
DK210989D0 (en) 1989-05-01

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