EP1349923A2 - Verkapselungsverfahren und damit hergestellte partikel - Google Patents
Verkapselungsverfahren und damit hergestellte partikelInfo
- Publication number
- EP1349923A2 EP1349923A2 EP01986939A EP01986939A EP1349923A2 EP 1349923 A2 EP1349923 A2 EP 1349923A2 EP 01986939 A EP01986939 A EP 01986939A EP 01986939 A EP01986939 A EP 01986939A EP 1349923 A2 EP1349923 A2 EP 1349923A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- molecules
- biologically active
- matrix material
- glucose
- active component
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
Definitions
- the present invention relates to an encapsulation process (inclusion immobilization) for biologically active components and particles produced therewith which contain biologically active components.
- Such methods are used to immobilize bioactive materials, especially cell cultures.
- Such immobilized bioactive materials have several advantages over their cultivation in suspension culture.
- the area of the immobilisation of bioactive materials, especially of cell cultures, offers some .Vormaschine against the cultivation 'suspension culture. Uniform capsule conditions can be found in the capsules, regardless of the reactor volume and that make it much easier to scale up a process. In addition, capsules offer shear-sensitive materials protection against mechanical stress. Furthermore, the establishment of continuous processes is simplified considerably by a simple cell separation.
- bioactive materials When bioactive materials are encapsulated in matrices made of biopolymers, alginate, cellulose derivatives or lipids are conventionally used as biopolymer.
- the bioactive materials can be enzymes, microorganisms, cell cultures and / or parts or organelles from microorganisms and cell cultures.
- the capsules formed can be used for the biosynthesis of metabolites, for biotransformation or for biodegradation. Another point of use is the controlled release of substances, e.g. when used as a tissue replacement.
- the object of the present invention is to provide an encapsulation process and particles produced therewith which are ideally suited for the stated purposes.
- the biologically active components are embedded in a matrix material which is composed of a complex-forming substance.
- a complex-forming substance which is composed of a complex-forming substance.
- the individual sequence of which consists of two glucose molecules and two further non-glucose carbohydrate molecules.
- the two non-glucose carbohydrate molecules ranthose and / or glucuronic acid are particularly advantageous and can also have further side chains, advantageously made of carbohydrates.
- the glucose molecules can, advantageously with glycerate or acetate, be substituted to different degrees and to a preselected degree.
- carbohydrate sequences are gellan or wellan, the properties of which can also be influenced by the degree of acetylation.
- a solution of the carbohydrate sequence is advantageously mixed with the biologically active component to be immobilized and then dripped into a complex-forming liquid. This can also be supported by an additional fluid stream, the speed and strength of which can influence the droplet size and thus the particle size which subsequently form in the complexing liquid. If the process is carried out in a suitable manner, the biologically active components are enveloped or enclosed in the matrix material.
- the capsules or particles hardened in this way can then be transferred to a conventional reaction system such as a stirred reactor and can be cultivated there under the conditions known from classic biotechnology.
- the fields of application extend from the production of native or recombinant proteins (antibodies, enzymes, hormones cytokines and the like) to use as extracorporeal replacement tissue, for example in cytotoxicity measurement, to use as intracorporeal replacement tissue or as intracorporeal tissue for production and / or Release of substances, for example hormones, enzymes, antibodies or pharmaceuticals in general.
- the capsules formed can therefore be used for the biosynthesis of metabolites, for biotransformation, for biodegradation or for the controlled release of substances.
- Gellan is the biopolymer [-3-) -bD-glucose- (1-4) -b- D-glucuronic acid- (1-4-) -bD-glucose- (14) -aL-rhamnose- (l-] n (see Fig. 2), which is mainly used in the food industry as a gelling agent, it is formed by the microorganism Sphingomonas eloedea (formerly: Pseudomonas eleodea) and is available in large quantities in constant quality.
- the glucose subunits can have different degrees of acetylene ,
- Wellan can also be used, the structural formula of which is also shown below:
- the capsule formation takes place through the formation of complexes with cations, preferably divalent cations.
- the Gellan is dissolved at temperatures between 70 ° C and 100 ° C in ultrapure water in a moving bath. '
- the concentration of the gellan is between 0.1% and 10%, preferably 0.75%.
- the encapsulation is carried out with a dropletizer with one nozzle, optionally with two nozzles, which are then advantageously arranged concentrically.
- the component to be encapsulated can be added in two ways: A) directly into the dissolved polymer material.
- the component to be encapsulated is enclosed by the polymer material.
- the drop that forms at the end of the nozzles falls into a bath containing a complex-forming substance.
- the complexing agent can be a cation, for example sodium, preferably a multivalent cation, for example calcium or barium.
- the complexing agent is in a buffer such as PBS (0.2 g / L KC1, 0.2 g / L KH 2 P0 4 , 8.0 g / L NaCl and 1.15 g / L Na 2 HP0 4 in H 2 0 ) or preferably Tris buffer (300 mM in H 2 0) dissolved in a concentration between 0.5 and 10%, preferably 1%.
- the capsules can be immobilized, for example, using an encapsulation apparatus as shown in FIGS. 1 and 2.
- FIG. 1 shows an encapsulation apparatus 1 with a.
- Container 2 which contains a precipitation bath 3 with polyvalent cations.
- This container 2 is arranged on a stirrer or base 4, with which the precipitation bath is kept in motion.
- a nozzle 5 which here contains two concentric outlets 8 '9'.
- the outlet 8 ' surrounds the central axial outlet 9'.
- the outlet 8 ' is connected to a fluid supply channel 8 and the outlet 9' is connected to a component supply channel 9.
- the channel 9 is now a mixture 23 of capsule-forming
- Fig. 2 shows a corresponding encapsulation apparatus 1, wherein the same or similar reference numerals designate the same or similar elements.
- three nozzle openings 8 ', 9', 13 ' are provided, the outermost nozzle opening 8' again containing a fluid.
- the innermost axial nozzle opening 13 ' is connected to a feed 13, via which the ' bioactive material 22 is fed to the nozzle opening 13 '.
- the nozzle opening 9 'located between the nozzle opening 13' and the nozzle opening 8 ', which concentrically surrounds the nozzle opening 13', is connected to the feed 9, via which the capsule-forming component 21 is fed to the nozzle 5.
- the capsule-forming material is enclosed as a core 22 in a jacket 21 made of capsule-forming component when spraying out through the nozzle 5 and droplets. These drops 10 then again fall into the precipitation bath 3 and harden there to produce capsules 11.
- the bioactive component is now surrounded by a jacket made of capsule-forming component.
- the individual substances to be injected are supplied with the aid of a pump or with the aid of a pressure-overlaid storage vessel. The following are some examples of inventive Encapsulation process given.
- Gellan is dissolved 1% (w / v) in ultrapure water.
- 0.4 g of gellan (low acetylated) is dissolved in 40 ml of ultrapure water and heated to 90 ° C. with stirring.
- Calcium chloride is made up to 10% (w / v) in 20 mm Tris buffer, the pH value of which was previously determined
- 0.1 N HCI is set to 6.3.
- the Gellan and the dropping apparatus. are heat sterilized in an autoclave, the calcium chloride is sterile filtered.
- a cell suspension of SF21 cells is added to the gellan, the cell concentration of which is 5x107 cells / ml.
- the suspension formed in this way is sent with the aid of a pump through the dropping apparatus shown in FIG. 1.
- the resulting drops fall into the calcium chloride bath located under the dropletizer and moved by a magnetic stirrer and are stirred there for 30 minutes.
- the stirring speed is 60 rpm so that undeformed capsules can form.
- the capsules are then separated from the calcium chloride and washed three times with sodium / potassium buffer (100 mm, pH 6.3). This is followed by a washing step with medium (SF900II).
- the cells immobilized in the capsules are cultivated in SF900II medium at 27 ° C. Vitality tests with MTT and EZ4U confirm the growth of the cells in the
- the solutions are heat sterilized. After sterilization, the glucose solution (II) is added to the gellan solution (I) so that 50 ml of a solution (IV) is removed . stand, which is 1% (w / v) on both the gellan and the glucose.
- 95 ml of cell culture medium (V) are mixed with the sodium chloride solution (III), so that a 1% (w / v) sodium chloride solution in 100 ml medium is obtained (VI).
- a cell suspension (VII) of CHO cells with a living cell count of 1x106 cells with a vitality of> 90% are centrifuged at 150 rcf (+/- 50 rcf) and taken up in 1 ml of medium.
- the concentrated cell suspension is added to the gellan-glucose solution (IV), mixed and heated to 27 ° C. for dripping in a water bath (VIII).
- the cell suspension (VIII) is sent through a droplet apparatus according to FIG. 1 with a hose of 3 mm diameter by means of a hose pump at 5 ml / min.
- the drops fall into the medium (VI), in which they harden for 1 h.
- the cells are cultivated at 37 ° C. in a roller bottle at 6 rpm.
- the number of living cells is determined by means of an MTT test and a previously recorded calibration line. 3 shows the course of cultivation.
- the Gellan and the dropping apparatus are heat sterilized.
- the calcium chloride solution is sterile filtered.
- 220 ml of a cell suspension (III) of CHO cells with a living cell count of 1x106 cells / ml with a vitality of> 85% are centrifuged off at 200 rcf (+/- 50 rcf) and taken up in 5 ml of medium.
- the concentrated cell suspension is added to the Gellan solution (I) (VI).
- the cell suspension (VI) is sent with a tube of 1 mm diameter by means of a peristaltic pump at 5 ml / min through the inner capillary nozzle of a dropping apparatus according to FIG. 1.
- the gellan (I) is passed through the jacket capillary at 5 ml / min.
- a Cell suspension drop forms in a Gellan drop at the exit of the nozzles.
- the drops fall into the calcium chloride bath (II), where they harden for 1 min.
- the cells are cultivated at 37 ° C. in a roller bottle at 6 rp i.
- the living cell number is determined using an MTT test and a previously recorded calibration line. 4 shows the course of a cultivation of these CHO cells enclosed in gellan capsules.
- Wellan is a linear polymer consisting of repeated sequences of glucose, rhamnose and glucuronic acid in a ratio of 2: 1: 1. There are one-molar side chains made of L-rhamnose or L-mannose on this chain. Wellan is 0.25% (w / v) in WFI.
- the Wellan is conveyed at 5 ml / min through a dropping apparatus according to FIG. 1 via a hose with an inner diameter of 3 mm.
- Calcium chloride solution 1% (w / v) in WFI, serves as a hardening bath. The drops remain in the calcium chloride bath for 30 min.
- the harmlessness of the welan for cell cultures is determined. A cytotoxicity test is used for this.
- the capsules have a strength of approx. 10 N against mechanical destruction and are 2 mm in size on average.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Sustainable Development (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10065843 | 2000-12-29 | ||
DE2000165843 DE10065843A1 (de) | 2000-12-29 | 2000-12-29 | Einschlussimmobilisierung |
PCT/EP2001/015335 WO2002053725A2 (de) | 2000-12-29 | 2001-12-27 | Verkapselungsverfahren und damit hergestellte partikel |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1349923A2 true EP1349923A2 (de) | 2003-10-08 |
Family
ID=7669504
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01986939A Withdrawn EP1349923A2 (de) | 2000-12-29 | 2001-12-27 | Verkapselungsverfahren und damit hergestellte partikel |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1349923A2 (de) |
AU (1) | AU2002238485A1 (de) |
DE (1) | DE10065843A1 (de) |
WO (1) | WO2002053725A2 (de) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH563807A5 (en) * | 1973-02-14 | 1975-07-15 | Battelle Memorial Institute | Fine granules and microcapsules mfrd. from liquid droplets - partic. of high viscosity requiring forced sepn. of droplets |
DE3877411T2 (de) * | 1987-05-07 | 1993-06-24 | Merck & Co Inc | Welan-gummi in zementzusammensetzungen. |
CA1319632C (en) * | 1988-04-22 | 1993-06-29 | Carol Louise Nolan | Method for microbial immobilization |
-
2000
- 2000-12-29 DE DE2000165843 patent/DE10065843A1/de not_active Ceased
-
2001
- 2001-12-27 WO PCT/EP2001/015335 patent/WO2002053725A2/de not_active Application Discontinuation
- 2001-12-27 AU AU2002238485A patent/AU2002238485A1/en not_active Abandoned
- 2001-12-27 EP EP01986939A patent/EP1349923A2/de not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO02053725A3 * |
Also Published As
Publication number | Publication date |
---|---|
DE10065843A1 (de) | 2002-07-11 |
AU2002238485A1 (en) | 2002-07-16 |
WO2002053725A3 (de) | 2003-04-10 |
WO2002053725A2 (de) | 2002-07-11 |
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Legal Events
Date | Code | Title | Description |
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
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17P | Request for examination filed |
Effective date: 20030729 |
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AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR |
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AX | Request for extension of the european patent |
Extension state: AL LT LV MK RO SI |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KAISER, DANIEL Inventor name: SCHROEDTER, MATHIAS |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: SIEGFRIED BIOLOGICS GMBH |
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17Q | First examination report despatched |
Effective date: 20060704 |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
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18D | Application deemed to be withdrawn |
Effective date: 20080425 |