EP1438415A2 - Verfahren zur herstellung von heparin durch mastzellen - Google Patents

Verfahren zur herstellung von heparin durch mastzellen

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Publication number
EP1438415A2
EP1438415A2 EP02793179A EP02793179A EP1438415A2 EP 1438415 A2 EP1438415 A2 EP 1438415A2 EP 02793179 A EP02793179 A EP 02793179A EP 02793179 A EP02793179 A EP 02793179A EP 1438415 A2 EP1438415 A2 EP 1438415A2
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EP
European Patent Office
Prior art keywords
heparin
cells
culture
mast
mast cells
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Withdrawn
Application number
EP02793179A
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English (en)
French (fr)
Inventor
Pierre Cans
Jean-Marc Guillaume
Hélène Monique Marie RIGAL
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Aventis Pharma SA
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Aventis Pharma SA
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Publication date
Application filed by Aventis Pharma SA filed Critical Aventis Pharma SA
Publication of EP1438415A2 publication Critical patent/EP1438415A2/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present invention relates to the preparation of heparin from cell cultures.
  • Heparin belongs to the family of glycosaminoglycans (GAG), which groups together the linear polysaccharides containing. a repetition of a disaccharide sequence consisting of an amino sugar (D-glucosamine or galactosamine) and a uronic acid (D- glucuronic or iduronic).
  • GAG glycosaminoglycans
  • the amino sugar is D-glucosamine.
  • Uronic acid is either glucuronic acid (.Glc) or iduronic acid (Ido).
  • Glucosamine can be N-acetylated, N-sulfated or O-sulfated.
  • heparin is used to designate. highly sulfated polysaccharides in which more than 80% of the glucosamine residues are N-sulfated and the number of O-sulfate is greater than that of N-sulfates. The tallow / disaccharide ratio is generally greater than 2 for heparin. However, the structure of heparin is in fact very heterogeneous, and there are chains which may contain very different ratios. Like all GAGs, heparin is synthesized in the form of a proteoglycan. This synthesis preferably takes place in a subpopulation of mast cells, serous or connective mast cells (CTMC).
  • CMC connective mast cells
  • mast cells are abundant in the skin, and the respiratory submucous. Their lifespan is very long (at least 6 months). In addition to heparin, they contain heparan sulfate, and appreciable amounts of histamine (approximately 10 pg / cell, depending on the animal species).
  • the first step in the synthesis of heparin is the formation of the protein nucleus serglycine, consisting serine and glycine residues, regularly alternated.
  • the heparin chain is elongated from a tetrasaccharide, by successive additions of osamine and uronic acids.
  • the proteoglycan thus formed undergoes numerous sequential transformations: N-deacetylation,
  • polysaccharide chains are then cleaved from serglycine by an endoglucuronidase. These chains then have a molecular weight between 5000 and
  • Heparin forms complexes with basic proteases and are thus stored in the granules of mast cells. Heparin is excreted only during the degranulation of mast cells. Heparin plays an important biological role, especially in hemostasis, and is very widely used in therapy, in particular as an anticoagulant and antithrombotic agent.
  • heparin the major part of the heparin used is isolated from the intestinal mucosa of the pig, from where it is extracted by proteolysis, followed by purification on anion exchange resin (for review on the various methods of preparation of l heparin, cf. DUCLOS, "Heparin: fabrication, structure, properties, analysis”; Ed. Masson, Paris, 1984).
  • heparin-type compounds which can be proteoglycans (HEP-PG) or glycosaminoglycans (HEP-GAG) from rat mast cell cells.
  • the compounds are not heparin.
  • the cells thus isolated are not established lines.
  • the applicant recommends the co-cultivation of the isolated cells with fibroblasts.
  • the inventors have found that it is possible to produce in large quantities from cultures of mast cell lines, heparin having properties comparable to those of heparin extracted from porcine intestinal mucus.
  • the use of cell cultures as raw material also makes it possible to control the conditions for the synthesis of heparin, and thus to obtain a product having reproducible characteristics.
  • the present invention relates to a process for the production of heparin, characterized in that it comprises the culture of mast cells of porcine origin and the recovery of heparin from the cultures obtained.
  • said mast cell cultures are lines of mast cells of porcine origin.
  • culture here generally designates a cell or a set of cells cultivated in vitro.
  • a culture developed directly from a cell or tissue sample taken from an animal is called "primary culture”.
  • lineage is used from the moment at least one passage, and generally several consecutive passages in subculture have been carried out successfully, and designates any culture which results therefrom. (SCHAEFFER, In Vitro Cellular and Developmental Biology, 26, 91-101, 1990).
  • said mast cells come from cultures and in particular from pig mast cell lines obtained as described in Application FR 0113608, as well as in PCT Application entitled “Pig mast cell cultures and their uses” in the name of INRA, and of the ENVA filed on the same day as this Application.
  • preferred lines for implementing the process according to the invention are:
  • the mast cell line originating from pig fetal liver, and transfected with the T antigen of the SV40 virus deposited by INRA with the CNCM on October 17, 2001, under the number 1-2736; the line of mast cells from bone marrow of pig fetuses and transfected with the T antigen of the SV40 virus, deposited by INRA with the CNCM on October 17, 2001, under the number 1-2734.
  • these mast cells' are serous mast cells.
  • mast cells will preferably be cultured in a defined culture medium (MEM ⁇ / DMEM, RPMI, IMDM, etc.) supplemented with growth factors, used in combination or individually, such as SCF (Stem Cell Factor) at a concentration between 1 ng / ml and 1 ⁇ g / ml, and possibly IL3 (Interleukin 3) at a concentration between 0.1 ng / ml and 100 ng / ml, or PGE2 (prostaglandin E2), at a concentration between 1 nM and 1 ⁇ M.
  • MEM ⁇ / DMEM, RPMI, IMDM, etc. a defined culture medium
  • growth factors used in combination or individually, such as SCF (Stem Cell Factor) at a concentration between 1 ng / ml and 1 ⁇ g / ml, and possibly IL3 (Interleukin 3) at a concentration between 0.1 ng / ml and 100 ng / ml,
  • the media can also be supplemented with bovine serum, at a concentration of between 0.5% and 20% (v / v).
  • bovine serum to the culture media can be replaced by the use of a culture medium without serum such as AIMV (INVITROGEN) so as to reduce the protein concentration of the medium and the risks associated with the use of compounds of animal origin (KAMBE et al., J. Immunol. Methods, 240, 101-10, 200).
  • AIMV AIMV
  • the independence of the cells with respect to the addition of serum and / or the use of growth factors can be obtained by controlled mutation of the cell phenotype by the action of transforming and / or immortalizing agents ( TSUJIMURA, Pathology International, 46, 933-8, 1996; PIAO and BERNSTEIN, Blood, 87 (8), 3117-23, 1996).
  • Mast cells can be cultured using techniques developed for the mass culture of eukaryotic cells, as described for example by GRIFFITHS et al. (Animal Cell Biology,, Eds. Spier and Griffiths, Académie Press, reasonably, vol.3, 179-220, 1986). Bioreactors with a capacity greater than several m 3 can be used as described by PHILIPS et al.
  • the culture can also be carried out in suspension or on micro-support according to the technique described by VAN MEZEL (Nature, 216, 64-65, 1967).
  • the productivity of batch cultures can be advantageously increased by removing part of the bioreactor cells (70% to 90%) for the operations of extracting GAGs and isolating heparin and by retaining the remaining cells within the same bioreactor to initiate a new culture.
  • this so-called repeated batch culture mode it is also possible to distinguish the optimum parameters of the cell growth phase from those allowing a greater accumulation of GAGs and heparin within the cells.
  • Continuous perfusion-type culture systems with or without cell retention can also be used (VELEZ et al., J. Immunol. Methods, 102 (2), 275-278, 1987; CHAUBARD et al., Gen. Eng News, 20, 18-48, 2000).
  • perfused culture systems allowing the retention of the cells inside the reactor, and resulting in growth and a production higher than those obtainable in batch.
  • the retention can be carried out by means of retention systems of the spin filter type, hollow fibers, or solid matrix (WANG et al., Cytotechnology, 9, 41-49, 1992; VELEZ et al., J. Immunol, Methods, 102 (2), 275-278, 1987).
  • the cell densities obtained are generally between 10 7 and 5 x 10 7 cells / ml.
  • the culture in bio-reactors allows, through the use of online measurement sensors, better control of the physico-chemical parameters of cell growth as well as of the accumulation of GAGs and heparin within cells: pH, p02, Red / Ox, growth substrates such as vitamins, amino acids, carbon substrates (e.g. glucose, fructose, galactose), metabolites such as lactate or ammonia, etc.
  • growth substrates such as vitamins, amino acids, carbon substrates (e.g. glucose, fructose, galactose), metabolites such as lactate or ammonia, etc.
  • From 3 to 30 days of culture generally from 3 to 10 days of culture under these conditions, the cells can be harvested and separated from the culture medium, generally by centrifugation or iltration. Different centrifugation systems can be used, for example those described by VOGEL and TODARO (Fermentation and Biochemical
  • separation can be carried out by tangential microfiltration, using membranes whose porosity is less than the average diameter of the cells (5 to 20 ⁇ m) while allowing the passage of the other compounds in solution. /suspension.
  • the speed of the tangential flow and the pressure applied to the membrane will be chosen so as to generate little shear force (Reynolds number less than 5000 sec "1 ) in order to reduce clogging of the membranes and preserve the integrity of the cells during l separation operation.
  • membranes can be used, for example, spiral membranes (AMICON, MILLIPORE), flat membranes or hollow fibers (AMICON, MILLIPORE, SARTORIUS, PALL, GF).
  • GAGs and heparin can also be harvested from the culture medium after lysis or degranulation of the cells.
  • Degranulation can be caused by the binding of specific ligands to receptors present on the surface of mast cells, for example the binding of allergen-like agents (such as Fc fragment of IgE or 'analogs of this fragment) on mast cell IgE receptors.
  • allergen-like agents such as Fc fragment of IgE or 'analogs of this fragment
  • the use of membranes of more porosity reduced can also be considered.
  • the separation of the cells is combined with an ultrafiltration step on one or more membranes whose arrangement and porosity makes it possible to concentrate the heparin and to separate it from the other species present in the medium, depending on the size and molecular weight, and possibly electrical charge, or biological properties.
  • the cut-off threshold of the membranes is preferably between 1000 and 5 KDa.
  • Membrane systems similar to those used for microfiltration can be used, for example, spiral membranes, flat membranes, or hollow fibers. Can be advantageously used for performing membrane separation and purification of heparin, by virtue of their charge properties, or grafting ligands having an affinity for heparin (e.g. antibody, ATIII, lectin, peptides, nucleotides, etc.). Other agents can also induce mast cell clégranulation.
  • cytotoxic agents can be classified into several categories such as cytotoxic agents, enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (ionophore A23187, ionomycin, etc. ).
  • cytotoxic agents enzymes, polysaccharides, lectins, anaphylatoxins, basic compounds (compound 48/80, substance P, etc.), calcium (ionophore A23187, ionomycin, etc. ).
  • degranulating agent can be carried out repeatedly on the same cells maintained in culture. In this mode of production productivity is significantly increased by simplifying the harvesting process from the supernatant and by maintaining the cells in culture.
  • the degranulation of the mast cells can be induced for example by treatment of 2.10 6 cells / ml of mast cells with the isophore A23187 at concentrations between 1 to 100 ⁇ g / ml and times of action varying from 1 minute to 4 hours.
  • Mast cell lysis can be induced, for example, by osmotic shock using hypotonic or hypertonic solutions, by thermal shock (freezing / thawing), by mechanical shock (for example sonication or pressure variation), by the action of chemical agents (NaOH, THESIT TM, NP40 TM, TWEEN 20 TM, BRIJ- 58 ⁇ ' ⁇ TRITON X TM -100, ...), or by enzymatic lysis (papam, trypsin, %), or by a combination of two or many of these methods.
  • osmotic shock using hypotonic or hypertonic solutions
  • thermal shock freezing / thawing
  • mechanical shock for example sonication or pressure variation
  • chemical agents NaOH, THESIT TM, NP40 TM, TWEEN 20 TM, BRIJ- 58 ⁇ ' ⁇ TRITON X TM -100, .
  • enzymatic lysis papam, trypsin,
  • Cell ivsat separate the polysaccharide chains from no ⁇ to ser-glycine, and separate the heparin chains from the other GAGs present in the extraction medium, we can use methods similar to those used in the context of extraction and heparin purification from animal tissues, which are known in themselves, and described in general works, such as the manual of DUCLOS, cited above).
  • the cell lysate can be subjected to one or more enzymatic digestions (pronase, trypsin, papamus, etc.); the heparin-protein bonds can be hydrolysed in an alkaline medium, in the presence of sulfates or chlorides;
  • heparin preparations capable of being obtained from mast cell cultures by implementing a method according to the invention.
  • heparin preparations according to the invention which have biological properties comparable to those of the heparin preparations obtained in the prior art from animal tissues, can be used in all the usual applications of heparin.
  • the present invention will be better understood with the aid of the additional description which follows, which refers to examples of heparin preparation from mast cell cultures and characterization of the heparin obtained.
  • a pig fetal liver mast cell line and a pig fetal liver mast cell line transfected with the SV40 virus T antigen (lines
  • the cells are seeded at a rate of 10 5 to 5:. 10 r cells / ml, in complete MEM ⁇ medium in the presence of porcine IL3 (2 ng / ml) and porcine SCF (80 ng / ml).
  • Cultures are carried out in a culture dish or in suspension in a 1-liter spinner-type bottle. Cell growth is monitored daily for 4 to 12 days. The heparin production is followed in parallel, by the analysis of the glycosaminoglycans produced in culture. The results are presented in Figures 1 to 5.
  • Figures 1, 2 and 3 illustrate the growth of liver mast cells in static culture in a dish ( Figure 1; initial seeding:: 1 x 10 5 cells; H: 2 x 10 5 cells) and in suspension in a bottle ( Figure 2) , and the growth of transfected liver mast cells in vial suspension ( Figure 3).
  • the cultures in suspension in a bottle have a maximum cell density ranging from approximately 8 ⁇ 10 5 (for the non-transfected cells) to approximately 1.5 ⁇ 10 6 cells / ml (for the transfected cells).
  • the doubling time, calculated during the exponential growth phase, is between 24 and 48 hours.
  • the cells undergo hydrolysis in an alkaline medium in the presence of salt in order to break the proteoglycans and avoid interactions GAGs / proteins of ionic types.
  • This treatment includes the following stages:
  • Treatment with sodium hydroxide in a saline environment aims to destroy the cells and cut the links between heparin and its mother protein.
  • the step includes adding 100 ⁇ l of 1 M NaOH and 800 ⁇ l of 0.5 M NaCl to a pellet of 10 6 cells.
  • the mixture thus obtained is heated in a water bath at 80 ° C for 30 minutes and then sonicated for 5 minutes before being neutralized with 1N HCl.
  • Desalting / Lyophilization the elimination of sodium chloride (necessary to be able to apply some of the analysis methods which are described below) is carried out by steric exclusion chromatography on SEPHADEX G10 gel, followed by conductimetry. The collected heparin fractions are then lyophilized to concentrate the sample.
  • This technique makes it possible to separate the GAGs according to their size and their charge, and constitutes a test making it possible to rapidly verify the presence or absence of heparin.
  • the purified preparation obtained as described above is deposited on Tris / tricine polyacrylamide gel (gradient from 10 to 20%) making it possible to separate molecules from 30 to 1 kDa, at the rate of 20 ⁇ il of preparation by deposition. 25 ng of dermatan, 25 ng of SPIM standard porcine heparin (4 th international standard for porcine heparin, intestinal mucus), and for heparin extracted from porcine mucus and purified by treatment with sodium hydroxide and purification on anion exchange resin under the same conditions as those described above.
  • the gels are then analyzed by a scanner (BIO-RAD) to quantify the different GAGs.
  • the limit of quantification of heparin is 10 ng per band.
  • Figure 4 illustrates the production of heparin during the growth of liver mast cells in static dish culture.
  • heparin concentrations generally observed are between 2 and 14 ⁇ g for 10 6 cells, in static culture and in suspension.
  • EXAMPLE 2 CHARACTERIZATION OF THE PREPARATION OF HEPARIN OBTAINED FROM MASTOCYTE CULTURES
  • the disaccharide composition makes it possible to differentiate heparin from other glycosaminoglycans.
  • the disaccharide profile of the glycosaminoglycans produced by the mast cells in culture was determined according to the method described by LINHARDT et al. (Biomethods, 9, 183-97, 1997).
  • the preparation of GAGs obtained as described in Example 1 above was depolymerized by a mixture of heparinases from Flavobacterium heparinium (heparinases I, II, and III, GRAMPIAN ENZYMES). The conditions used are described in the publication by LINHARDT et al., Cited above.
  • Figure 6 representing the disaccharide profile of the heparin preparation produced by a flask culture of mast cells derived from fetal liver (B), compared to the disaccharide profile of standard heparin (D).
  • the separation is followed by a post-column derivatization, to form a fluorescent complex with guanidine.
  • the trisulfated disaccharide IS which has the strongest response factor by this technique, is detected and quantified compared to a standard heparin solution of known concentration.
  • the detection limit of the method is of the order of 5 ng / ml of heparin in cell culture samples.
  • Table 2 illustrates the IS / IIS ratio of cell cultures over time.
  • the reaction takes place in three stages:
  • the amount of paranitroaniline (pNA) released is measured at 405 nm. It is inversely proportional to the amount of heparin.
  • the anti-Xa or anti-IIa activity is evaluated with respect to a calibration line established with the SPIM standard.
  • the sensitivity of the method is 0.006 IU / l.
  • the electrophoresis is carried out on a 2.8 "agarose gel in a solution at pH 3 (acetic acid / lithium hydroxide). To 100 ⁇ l of sample to be tested, add
  • the gels are scanned and interpreted with QUANTITY ONE software (BIO-RAD).
  • a line of mast cells from the non-transfected pig fetal liver was used.
  • the cells are seeded at the rate of 2.0 to 4.0 ⁇ 10 5 cells per ml in complete DMEM / F12 medium supplemented with porcine IL3 (2ng / ml) of porcine SCF (80ng / ml).
  • the bioreactor used has a capacity of 2 liters of culture media, the oxygen tension of the culture is maintained between 20% and 40% of saturation, the pH between 7.0 and 7.4, the temperature is maintained at 37 ° C + / - 0.5 ° C by circulation of thermostatically controlled water in the jacket of the bioreactor.
  • the culture is stirred by a marine type propeller with a speed between 80 to 150 revolutions / minute.
  • the cell density is 1.3 x 10 6 cells / ml, corresponding to a doubling time of between 24 and 48 hours.
  • 80% of the culture is taken for heparin extraction, the rest of the culture is kept in the bio-reactor and diluted with fresh medium to a concentration of between 2.0 and 3.0 ⁇ 10 5 cells / ml as described for a repeated batch production operation.
  • the cell density obtained is 9.0 x 10 5 cells / ml, corresponding to a doubling time of between 24 and 48 hours and comparable to the first cultivation (Figure 8).
  • the purified heparin is then analyzed by HPLC, as described in Example 2, using standard SPIM heparin as a control.
  • Table 5 and Figure 9 represent the disaccharide profile and the proportion of the protein nucleus serglycine (Gly-Ser) of the heparin preparation produced by the culture of suspended mast cells derived from porcine fetal liver (M), compared to the profile obtained for standard heparin SPIM (D).
  • Table 6 shows the N-acetylation, N-sulfation and O-sulfation profile of heparin disaccharides produced by the suspension mast cell culture derived from porcine fetal liver compared to that of SPIM heparin disaccharides standard.
  • EXAMPLE 5 PRODUCTION OF HEPARIN IN THE CULTURE SURNANTANT BY USE OF A DEGRANULATION AGENT.
  • the experiments were carried out on a line of non-transfected fetal liver mast cells.
  • the mast cell concentration has been adjusted to 2 ⁇ 10 6 cells / ml, and the culture is incubated for one hour in MEM medium comprising 4 ⁇ g / ml of 1 lonophore A23187, which induces degranulation of mast cells.
  • the mast cells for which the harvest of GAGs was carried out on the 762nd day of culture were returned to culture. No loss of viability and growth rate was observed.
  • mast cells 21 days later, these mast cells are subjected to a new degranulation, and the GAGS are assayed as described above.
  • FIG. 10 shows that the percentages of GAGs secreted is comparable to that obtained during the first degranulation and also comparable to that obtained with control cells of the same age.

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EP02793179A 2001-10-22 2002-10-22 Verfahren zur herstellung von heparin durch mastzellen Withdrawn EP1438415A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0113606 2001-10-22
FR0113606A FR2831186B1 (fr) 2001-10-22 2001-10-22 Production d'heparine a partir de cultures de mastocytes
PCT/FR2002/003617 WO2003035886A2 (fr) 2001-10-22 2002-10-22 Preparation d'heparine a partir de cultures de mastocytes

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US (1) US20050042733A1 (de)
EP (1) EP1438415A2 (de)
JP (1) JP2005506092A (de)
KR (1) KR20040071127A (de)
CN (1) CN1575341A (de)
AR (1) AR036915A1 (de)
BR (1) BR0213478A (de)
CA (1) CA2462714A1 (de)
CO (1) CO5570711A2 (de)
FR (1) FR2831186B1 (de)
HU (1) HUP0401794A2 (de)
IL (1) IL161066A0 (de)
MX (1) MXPA04003740A (de)
NO (1) NO20041633L (de)
NZ (1) NZ532414A (de)
PL (1) PL368599A1 (de)
WO (1) WO2003035886A2 (de)
ZA (1) ZA200402304B (de)

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FR2853663B1 (fr) * 2003-04-14 2007-08-31 Aventis Pharma Sa Procede d'obtention de lignees de mastocytes a partir de tissus de porcs et procede de production de molecules de type heparine
FR2876386B1 (fr) * 2004-10-12 2007-04-06 Aventis Pharma Sa Lignees de mastocytes porcins produisant des molecules de type heparine
KR100688553B1 (ko) * 2005-06-22 2007-03-02 삼성전자주식회사 코어 사이즈를 감소시킨 반도체 메모리 장치
KR101447123B1 (ko) * 2014-02-27 2014-10-06 박상협 헤파린의 추출 방법
KR102104367B1 (ko) 2019-09-02 2020-04-24 팜앤바이오 주식회사 헤파린나트륨의 제조장치 및 제조방법
CN111979193A (zh) * 2019-09-27 2020-11-24 云南洛宇生物科技有限公司 大鼠骨髓源肥大细胞培养方法
CN110592165B (zh) * 2019-10-18 2021-04-27 福州大学 燕窝中硫酸乙酰肝素/肝素的提取方法与结构解析

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MXPA04003740A (es) 2005-06-20
JP2005506092A (ja) 2005-03-03
CA2462714A1 (fr) 2003-05-01
FR2831186A1 (fr) 2003-04-25
WO2003035886A3 (fr) 2004-02-26
HUP0401794A2 (hu) 2004-11-29
US20050042733A1 (en) 2005-02-24
ZA200402304B (en) 2004-10-07
CN1575341A (zh) 2005-02-02
NZ532414A (en) 2006-12-22
WO2003035886A2 (fr) 2003-05-01
KR20040071127A (ko) 2004-08-11
AR036915A1 (es) 2004-10-13
FR2831186B1 (fr) 2004-06-18
PL368599A1 (en) 2005-04-04
IL161066A0 (en) 2004-08-31
BR0213478A (pt) 2004-11-03
NO20041633L (no) 2004-04-21

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