[go: up one dir, main page]

WO2006117001A2 - Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s) - Google Patents

Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s) Download PDF

Info

Publication number
WO2006117001A2
WO2006117001A2 PCT/DK2006/000231 DK2006000231W WO2006117001A2 WO 2006117001 A2 WO2006117001 A2 WO 2006117001A2 DK 2006000231 W DK2006000231 W DK 2006000231W WO 2006117001 A2 WO2006117001 A2 WO 2006117001A2
Authority
WO
WIPO (PCT)
Prior art keywords
polymer
hyaluronic acid
component
molecular sieve
zeolite
Prior art date
Application number
PCT/DK2006/000231
Other languages
English (en)
Other versions
WO2006117001A3 (fr
Inventor
Eric Thwaites
Martin Barfod
Poul Bach
Original Assignee
Novozymes Biopolymer 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 Novozymes Biopolymer A/S filed Critical Novozymes Biopolymer A/S
Priority to CA2606877A priority Critical patent/CA2606877C/fr
Priority to JP2008509304A priority patent/JP5091119B2/ja
Priority to CN2006800153388A priority patent/CN101171265B/zh
Priority to EP06722923A priority patent/EP1879924A2/fr
Publication of WO2006117001A2 publication Critical patent/WO2006117001A2/fr
Publication of WO2006117001A3 publication Critical patent/WO2006117001A3/fr

Links

Classifications

    • 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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • 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
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Definitions

  • TITLE Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s).
  • the present invention relates to a method for controlling the content of selected component(s) in one or more polymer(s).
  • Polymers and their derivatives are used in a wide range of applications. Of particular interest are biopolymers which can be applied in the food, cosmetic, medical and pharmaceutical industries. In order to meet these diverse applications it is often necessary to purify the polymer to remove unwanted components and, further, to control the balance of components in the final product.
  • glycosaminoglycans are unbranched carbohydrate polymers, consisting of repeating disaccharide units (only keratan sulphate is branched in the core region of the carbohydrate).
  • the disaccharide units generally comprise, as a first saccharide unit, one of two modified sugars - N-acetylgalactosamine (GaINAc) or N-acetylglucosamine (GIcNAc).
  • the second unit is usually an uronic acid, such as glucuronic acid (GIcUA) or iduronate.
  • Glycosaminoglycans are negatively charged molecules, and have an extended conformation that imparts high viscosity when in solution. Glycosaminoglycans are located primarily on the surface of cells or in the extracellular matrix. Glycosaminoglycans also have low compressibility in solution and, as a result, are ideal as a physiological lubricating fluid, e.g., joints. The rigidity of glycosaminoglycans provides structural integrity to cells and provides passageways between cells, allowing for cell migration.
  • glycosaminoglycans of highest physiological importance are hyaluronan, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Most glycosaminoglycans bind covalently to a proteoglycan core protein through specific oligosaccharide structures. Hyaluronan forms large aggregates with certain proteoglycans, but is exceptional as free carbohydrate chains form non-covalent complexes with proteoglycans.
  • Hyaluronan is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis.
  • Hyaluronan is also suspected of having a role in numerous physiological functions, such as adhesion, development, cell motility, cancer, angiogenesis, and wound healing. Due to the unique physical and biological properties of hyaluronan, it is employed in eye and joint surgery and is being evaluated in other medical procedures.
  • HA plays an important role in the biological organism, as a mechanical support for the cells of many tissues, such as the skin, tendons, muscles and cartilage, it is a main component of the intercellular matrix. HA also plays other important parts in the biological processes, such as the moistening of tissues, and lubrication.
  • HA may be extracted from the above mentioned natural tissues, although today it is preferred to prepare it by microbiological methods to minimize the potential risk of transferring infectious agents, and to increase product uniformity, quality and availability.
  • HA and its various molecular size fractions and the respective salts thereof have been used as medicaments, especially in treatment of arthropathies, as an auxiliary and/or substitute agent for natural organs and tissues, especially in ophtalmology and cosmetic surgery, and as agents in cosmetic preparations.
  • Products of hyaluronan have also been developed for use in orthopaedics, rheumatology, and dermatology.
  • HA may also be used as an additive for various polymeric materials used for sanitary and surgical articles, such as polyurethanes, polyesters etc. with the effect of rendering these materials biocompatible.
  • HA usage and derivatives thereof Due to the wide diversity of biopolymer usage particularly HA usage and derivatives thereof, some of which are mentioned above, and the frequent use of HA in pharmaceutical compositions or surgical articles as well as tailored for specific applications, it is often necessary to provide HA products of high purity which should be substantially absent of other contaminating components in the end product.
  • the non-polymer content and ionic composition of the polymer, particularly of HA, is also important.
  • the sodium salt of the HA is preferred as the most biocompatible form and other HA salts (Fe, Ca, Cu, Zn, Al,
  • Mg, Mn Ca++ salts of HA.
  • controlled levels of calcium are actually desirable in some wound care applications, controlled zinc levels have been proposed for combating foot ulcers (ref diabetologia Croatica 30-3, 2001) and for antibacterial properties (Acta Pharm Hung. 2002;72(1): 15-24); and controlled iron levels are sometimes used to control the rheological properties in some types of hyaluronic acid gel (CN 1473572A; WO 95/04132), whereas, low iron levels are often desired to reduce HA polymer susceptibility to degradation.
  • polymers including HA, having predominantly the sodium salt form and low or no Ca++ and low or no other metal ion content have been provided by carefully avoiding the presence of calcium and other unwanted ions during the fermentation process step and during subsequent purification steps.
  • the desired ion balance is conventionally achieved by first creating an environment of high sodium ion concentration from, for example, addition of sodium salt(s) such as sodium acetate, sodium chloride, sodium sulphate, etc.
  • the high sodium ion concentration is used to competitively displace calcium from the HA molecule.
  • the calcium ions liberated from the HA molecule can then be removed from the HA molecule, or vice versa, by any of a wide range of conventional polymer isolation processes.
  • the polymer, or particularly HA is precipitated or crystallised chemically and/or by organic solvent addition, such as ethanol, iso- propylalcohol, acetone, chloroform, CETAB etc, thereby leaving the liberated and unwanted ions in the supernatant (CZ 9700350; WO 84/ 03302; EP 0694616A2 and EP 0144 019 being just some examples of this process).
  • the liberated ions can also be separated from the polymer by ultra-filtration of dia-filtration techniques (WO 95/04132; GB2249315A). It is also possible to use aqueous extraction and other common polymer separation techniques.
  • sequestration agents such as, EDTA, phosphates (CN 85103674A), etc or application ion exchange adsorbent resins (EP 0694616A2).
  • the invention provides in a first aspect a method for controlling the content of selected component(s) in one or more polymer(s) by:
  • the present invention provides a method for controlling the content of undesirable or selected components from a polymer by removing or replacing the component(s) with a more desirable component and or manipulating the balance of components associated with the polymer.
  • the removal or manipulation of the component(s) is performed by contacting the polymer product with an appropriate molecular sieve(s) for a time period sufficient to remove or exchange or manipulate the component balance.
  • an appropriate molecular sieve(s) for a time period sufficient to remove or exchange or manipulate the component balance.
  • polymer in the present context is a substance which is made up of many repeating smaller chemical units or molecules. Polymers can be natural or synthetic.
  • polymer also comprises liquid polymers or suspensions of polymers, in this context.
  • component to be removed or manipulated comprises atoms, molecules, ions, or compounds.
  • control means remove (completely or partially), exchange and / or balance the content of component(s) from the polymer or polymer bearing liquid.
  • control means remove (completely or partially), exchange and / or balance the content of component(s) from the polymer or polymer bearing liquid.
  • components associated with the polymer and / or polymer bearing liquid i)
  • hyaluronic acid is preferred in the sodium form.
  • limitations for specific applications are often desired, for example, calcium containing polymers may be insoluble themselves, (e.g. calcium alginate) or may create problematic precipitation in common phosphate buffers, or create adverse reactions with active ingredients or formulation chemicals.
  • a controlled level of calcium in some applications has been specified in, for example, alginate for wound care products.
  • Ionic content and ion type can affect the viscosity and other properties of the polymer; similarly for other components. Ion content has to be carefully and accurately controlled, for example, for the creation of hydrogels containing zinc.
  • Polymer destabilising molecules, such as Fe, and Cu can be removed, controlled or replaced by another less harmful ion.
  • Polymer products often require removal of odour or colour molecules.
  • the present invention provides a number of advantages over conventionally applied methods for manipulation of the component balance.
  • the MOLECULAR SIEVE(S) according to the invention can be applied at any point in the manufacturing process, including to raw materials; during manufacture or as a post-treatment of the polymer product. Furthermore the process conditions such as pH, temperature, polymer concentration etc. are not as critical as in other conventionally applied processes.
  • Molecular sieves can be highly selective for particular components or groups of components. Reaction equilibrium is normally achieved rapidly meaning faster processing and closer control of the product characteristics in terms of the component balance associated with the polymer. The molecular sieves demonstrate a high capacity for the component(s) to be manipulated and therefore there is generally only the need for a single MOLECULAR SIEVE(S) treatment.
  • the simple addition, contact and subsequent removal of the MOLECULAR SIEVE(S) by conventional solid-liquid separation methods means there is no need for dedicated equipment and no need to subsequently remove soluble reactants or additions. It is possible to leave the MOLECULAR SIEVE(S) in the polymer solution which further simplifies the process. Process costs can also be reduced since the present method allows raw materials containing components which are undesirable in the end product, to be used in upstream steps, e.g. tap water (Ca ++ containing) for fermentation and dilution instead of de-ionized water. Furthermore the MOLECULAR SIEVE(S) can often be added directly and without the requirement for pre-equilibrium or pre-treatment. Molecular sieves are generally low in toxicity.
  • component(s) may even be added in an upstream process step without causing any problems for the downstream purification steps. Since the method according to the invention is conveniently applied for the removal of calcium from a polymer product, Ca ++ may even be added in an upstream process step without causing any problems for the downstream purification steps.
  • controlling the content of selected components means removing (completely or partially) the component(s) and or replacing (exchanging) the component(s) with a more desirable component and or manipulating the balance of components associated with the polymer or polymer bearing liquid.
  • a "molecular sieve” in the present context means materials having molecule-sized pores that can be used in separating larger molecules from smaller ones. They include, but are not limited to, zeolites, carbon molecular sieves, silica gels, activated alumina. Typically, molecular sieves have a lattice structure creating a cage like structure with windows which admit only molecules of less than a certain size. By using different source materials and different conditions of manufacture, it is possible to produce a range of molecular sieves of differing access dimensions. The dimensions can often be precise for a particular molecular sieve(s) because they derive from the crystal structure of that sieve.
  • the molecular sieve(s) is a zeolite.
  • the classical definition of a zeolite is a crystalline, porous aluminosilicate.
  • oxide structures with elements other than silicon and aluminium have stretched the definition.
  • zeolites In these crystalline materials we call zeolites, the metal atoms (classically, silicon or aluminum) are surrounded by four oxygen anions to form an approximate tetrahedron consisting of a metal cation at the centre and oxygen anions at the four apexes.
  • the tetrahedral metals are called T-atoms for short, and these tetrahedra then stack in, regular arrays such that channels form.
  • the possible ways for the stacking to occur is virtually limitless, and hundreds of unique structures are known.
  • the zeolitic channels (or pores) are microscopically small, and in fact, have molecular size dimensions such that they are often termed "molecular sieves".
  • the size and shape of the channels have extraordinary effects on the properties of these materials for adsorption processes, and this property leads to their use in separation processes.
  • Components can be separated via shape and size effects related to their possible orientation in the pore, and or by differences in strength of adsorption. Therefore, components can be selectively removed.
  • silicon-oxygen tetrahedra are electrically neutral.
  • aluminium typically exists in the 3+ oxidation state; so that aluminium-oxygen tetrahedra form centres that are electrically deficient one electron.
  • zeolite frameworks are typically anionic, and charge compensating cations populate the pores to maintain electrical neutrality.
  • the molecular sieve is chosen from the group of zeolites where the porosity of the material is compatible with the component to be removed, in a further embodiment the porosity of the material is compatible with Ca++; in a further embodiment ions populating the zeolite pores to maintain electrical neutrality are those desired in the polymer product; in a further embodiment sodium ions are the ions populating the zeolite pores to maintain electrical neutrality.
  • Type 4 Those molecular sieves classically grouped as "Type 4" have molecular sieve(s) dimensions appropriate for the sequestration of calcium ions having Linde sieve 4A dimensions of approximately 0.4 nm. A number of these molecular sieves have sodium ions populating the zeolite pores to maintain electrical neutrality.
  • the zeolite is therefore a Type 4 zeolite.
  • the pores of the molecular sieve contains sodium ions.
  • the polymer is a biopolymer.
  • a "biopolymer” is any polymeric substance (examples being, but not limited to, polysaccharides, proteins nucleic acids, etc,) formed in a biological system. Many examples of common biopolymers exist, including, but not limited to: Chitosan, glucan, keratin, cellulose, gelatine, glycosaminoglycans and derivatives of all these polymers.
  • the biopolymer is a polysaccharide, and in a further particular embodiment the polysaccharide is a glycosaminoglycan.
  • a glycosaminoglycan may be any carbohydrate polymer having a molecular weight of at least 700 Daltons; preferably a molecular weight of at least 10,000 Daltons; more preferably a molecular weight of at least 20,000 Daltons, even more preferably a molecular weight of at least 30,000 Daltons.
  • Preferred glycosaminoglycans are hyaluronic acid, chondroitin sulphate, chondroitin
  • non-sulphated heparin, heparin sulphate, dermatan sulphate, and keratin sulphate.
  • Hyaluronic acid is constituted by alternating and repeating units of D-glucoronic acid and N- acetyl-D-glucosamine, to form a linear chain having a molecular weight of up to 15,000,000 Daltons.
  • Preferred Glycosaminoglycans according to the invention are Glycosaminoglycans having a molecular weight of from 700 Daltons to 15,000,000 Daltons.
  • hyaluronic acid in the present application and claims may mean indifferently hyaluronic acid in its acidic form or in its salt form such as for example sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or others.
  • hyaluronan or "hyaluronic acid” are used in literature to mean acidic polysaccharides with different molecular weights constituted by residues of D-glucuronic and
  • N-acetyl-D-glucosamine acids which occur naturally in cell surfaces, in the basic extracellular substances of the connective tissue of vertebrates, in the synovial fluid of the joints, in the endobulbar fluid of the eye, in human umbilical cord tissue and in cocks' combs.
  • hyaluronic acid is in fact usually used as meaning a whole series of polysaccharides with alternating residues of D-glucuronic and N-acetyl-D-glucosamine acids with varying molecular weights or even the degraded fractions of the same, and it would therefore seem more correct to use the plural term of "hyaluronic acids".
  • the singular term will, however, be used all the same in this description; in addition, the abbreviation "HA" will frequently be used in place of this collective term.
  • the biopolymer e.g. a glucosaminoglucan
  • the biopolymer can be provided from animal tissues or more preferably by culturing a host cell expressing the biopolymer.
  • the glycosaminoglycan may be obtained from any fermentation broth.
  • the glycosaminoglycan may furthermore be one which is producible by a method comprising cultivating a host cell.
  • the host cell may preferably be a micro-organism.
  • the micro-organism may be a unicellular micro-organism, e.g., a prokaryote, or a non-unicellular micro-organism, e.g., a eukaryote.
  • Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E.
  • a Bacillus cell e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus cla
  • the bacterial host cell is a Bacillus lentus cell, a Bacillus licheniformis cell, a Bacillus stearothermophilus cell or a Bacillus subtilis cell. Mutant Bacillus subtilis cells particularly adapted for recombinant expression are described in WO 98/22598.
  • the host cell may be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.
  • Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
  • the transformation method, selectable marker gene and any other parts of the expression construct may be chosen from those well known and available to one skilled in the art.
  • the host cell may be a fungal cell.
  • "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi.
  • Examples of Basidiomycota include mushrooms, rusts, and smuts.
  • Chytridiomycota include, e.g., Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.
  • Representative groups of Oomycota include, e.g., Saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Alternaria.
  • Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • the fungal host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes).
  • the ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera Kluyveromyces, Pichia, and Saccharomyces).
  • Schizosaccharomycoideae e.g., genus Schizosaccharomyces
  • Nadsonioideae e.g., Lipomycoideae
  • Saccharomycoideae e.g.,
  • the basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella.
  • Yeast belonging to the Fungi lmperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida).
  • the fungal host cell is a filamentous fungal cell.
  • "Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota.
  • the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides.
  • Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic.
  • vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
  • the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma.
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known perse.
  • the micro-organism producing the glycosaminoglycan of interest is cultivated in a nutrient medium suitable for production of the glycosaminoglycan using methods known in the art.
  • the micro-organism may be cultivated by shake flask cultivation, small- scale or large-scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art.
  • Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection).
  • a glycosaminoglycan produced in a micro-organism WO2003/054163 describes the production of hyaluronic acid in a Bacillus host cell.
  • -Precipitation of the unwanted component for example, with phosphate and application of a sodium ion rich environment; -Dialysis or Dia-filtration in a sodium ion rich environment; -Sequestration and application of a sodium rich environment; and -Other conventional means for polymer purification.
  • reaction equilibrium changes as the concentration of the soluble(s) components change
  • achievement of reaction equilibrium is often relatively slow
  • capacity for the component to be manipulated is often relatively low
  • the techniques only work for a narrow range of process conditions (e.g. pH, ionic strength, polymer concentration, etc).
  • process conditions e.g. pH, ionic strength, polymer concentration, etc.
  • a method/process comprising the steps of contacting the polymer or polymer bearing liquid to be modified with the appropriate type(s) of molecular sieve(s) under appropriate conditions; and separating the MOLECULAR SIEVE(s) from the polymer, if necessary.
  • the type, amount and contacting conditions of the Molecular sieve(s) required to achieve the desired component balance in the polymer product can be simply determined by someone skilled in the art.
  • the component to be removed or manipulated according to the invention comprises atoms, molecules, ions, or compounds.
  • Examples of components which can be removed by particular molecular sieve types include, but are not limited to, those given in Table 17.3 "Classification of Some Molecular Sieves" from Chemical Engineering, Volume 2, 4 th Edition ; JM Coulson and JF Richardson; Pergamon Press, 1991.
  • a more comprehensive database of relevant structures and the properties of molecular sieves is hosted by the International Zeolite Association (http://www.iza-online.org/) at (http://www.iza-structure.org/databases/).
  • Relevant basic texts for molecular sieves include: • DW Breck: Zeolite Molecular Sieves, Wiley, New York, 1974
  • zeolites can be used to control the content of (remove or manipulate) organic solvents (including but not limited to: alcohols, aldehydes, ketones, etc.), metal ions, anions, quaternary ammonium compounds, SDS, EDTA, CETAB, TCA, cetyl pyrimidine chloride, etc.
  • organic solvents including but not limited to: alcohols, aldehydes, ketones, etc.
  • metal ions, anions, quaternary ammonium compounds SDS, EDTA, CETAB, TCA, cetyl pyrimidine chloride, etc.
  • MOLECULAR SIEVE(S) can be used to remove or manipulate the balance of ion, more particularly cations.
  • the ion is a divalent ion, more particularly Ca ++ .
  • the invention relates to a method for controlling the content of calcium ions from a polymer using a MOLECULAR SIEVE(s).
  • the invention relates to a method for controlling the content of calcium and sodium ions from a polymer using a Molecular sieve(s).
  • the component(s) to be removed or partly removed can be suspended in the liquid comprising the polymer or the component(s) can be associated or be present on the polymer.
  • the component(s) is exchanged for another component.
  • Ca- ions can e.g. be exchanged for Na-ions.
  • the component(s) balance in the product is controlled.
  • the molecular sieve(s) is a zeolite.
  • “Appropriate conditions” in this context mean those of the process stream and those conditions which can be easily determined by one skilled in the art for effecting the component removal or manipulation.
  • the appropriate conditions can include but are not limited to; molecular sieve type, molecular sieve dosing, temperature, pH, polymer concentration, ionic strength, solvent concentration, mixing, incubation time, etc.
  • the polymer is a glycosaminoglycan.
  • the Molecular sieve(s) can be removed from the polymer by any of a number of means, for example, but not limited to: filtration, centrifugation, floatation, sedimentation, phase exclusion, etc. In some cases, it may not be necessary to remove, or may be desirable to leave, the Molecular sieve(s) in the polymer bearing liquid.
  • the particular component removal or manipulation process according to the invention can be optimised or improved through, for example, but not limited to, manipulation of: pH, temperature, viscosity, concentration, mixing, ionic strength, additives, ingredients, etc. More than one polymer may be treated in the same polymer bearing liquid. More than one type of Molecular sieve(s) may be contacted with the polymer bearing liquid. More than one treatment with Molecular sieve(s) may be used to manipulate the polymer(s) bearing liquid.
  • the method of the invention provides control (removal, exchange and/ or balancing) of ions in the polymer bearing liquid using an appropriate zeolite.
  • the polymer is a glycosaminoglycan.
  • the polymer is hyaluronic acid.
  • the process of the invention provides control of the content of ions on the polymer itself using an appropriate zeolite.
  • the polymer is hyaluronic acid.
  • the process of the invention provides control of the content of calcium and sodium ions on the polymer itself using an appropriate molecular sieve.
  • the polymer is hyaluronic acid.
  • Example 2 Hyaluronic acid purification with a conventional calcium removal stage by dia- filtration against a sodium salt.
  • the hyaluronic acid solution was dia-filtered against an excess of sodium ions.
  • the liberated calcium ions were thereby removed from the solution by passage through the dia- filtration membrane.
  • the liberated calcium ions from the hyaluronic acid could equally have been removed by polymer precipitation.
  • the hyaluronic acid solution was dia-filtered against 3 x volumes of a 10 wt% sodium sulphate solution at constant volume followed by extensive dialysis against deionised water to remove excess sulphate and sodium ions.
  • the resulting product contained 1.2 wt% calcium relative to the hyaluronic acid.
  • Example 3 Control of calcium content of hyaluronic acid using a cation exchange resin.
  • Hyaluronic acid was produced as described in Example 1 above.
  • a strong cation exchange resin with SO 3 " (> 2 eq/l) in the sodium ion form was used to manipulate the calcium ion content of the polymer.
  • the performance of the exchange resin was characterised for the range of conditions likely to be met throughout hyaluronic acid manufacturing and purification processes described earlier.
  • 1.0 wt% exchange resin was added to a 0.5 wt% solution of hyaluronic acid containing 2wt% calcium relative to the hyaluronic acid. After 120 minutes of incubation with stirring the resin was filtered from the solution. The resulting filtrate contained hyaluronic acid with a calcium content of 0.5 wt% calcium relative to the hyaluronic acid. After an incubation of 240 minutes under the same conditions the calcium content relative to the hyaluronic acid was below detection.
  • Example 4 Control of calcium content of Hyaluronic acid using a cation exchange gel.
  • Hyaluronic acid was produced as described in Example 1 above.
  • a strong cation exchange gel having a sulphonated functional group (2.05 eq/l) was used to manipulate the calcium ion content of the polymer.
  • the performance of the exchange resin was characterised for the range of conditions likely to be met throughout hyaluronic acid manufacturing and purification processes described earlier.
  • 0.25 wt% exchange gel was added to a 0.5 wt% solution of hyaluronic acid containing 2 wt% calcium relative to the hyaluronic acid.
  • the mixture was incubated with stirring and allowed to come to equilibrium which was achieved in 1 hour.
  • the gel was filtered from the solution.
  • the resulting filtrate contained hyaluronic acid with a calcium content of 0.3 wt% calcium relative to the hyaluronic acid.
  • 1wt% of the exchange gel in the sodium form reduced the calcium content relative to the hyaluronic acid to below detection.
  • Example 5 Reduction of iron in fermentation broth using a cation exchange gel.
  • Gel type was a strong cation exchanger having functional groups: sulphonates (2.05 eq/l).
  • the original clarified broth and that treated to remove iron were subsequently heat treated.
  • the exchange gel treated material was found to be substantially more heat stable with regard to molecular weight than the untreated clarified broth material under the same conditions.
  • Example 6 Control of calcium content of Hyaluronic acid and replacement of ions on the hyaluronic acid with sodium ions using the sodium form of powdered aluminium silicate Type 4A Zeolite
  • Hyaluronic acid was produced as described in Example 1 above.
  • a powdered Type 4A zeolite in the sodium form was used to manipulate the calcium ion content of the polymer.
  • the performance of the exchange resin was characterised for the range of conditions likely to be met throughout hyaluronic acid manufacturing and purification processes described earlier. The characteristics were reproduced at both bench and pilot scale and diverse hyaluronic acid batches, produced as in Experiment 1 , were tested.
  • the zeolite was found to be able to remove around O.O ⁇ mg calcium from the hyaluronic acid bearing solutions per mg zeolite when the two were contacted. This ratio was found to be largely independent of the process conditions used (eg pH, temperature, HA concentration, etc). This made it very simple to control the calcium level in the hyaluronic acid bearing solution by simple dosed addition of zeolite. Equilibrium was achieved in less than 15 minutes in all cases.
  • Hyaluronic acid starting material and after treatment with 0.3wt% of the powdered zeolite were analysed. It was found that the ions on the hyaluronic acid, following treatment with 0.3wt% of the zeolite, had been replaced by sodium ions.
  • Example 7 Reduction of iron in fermentation broth using the sodium form of powdered aluminium silicate Type 4A Zeolite Ions such as iron, copper and others have been demonstrated to reduce the stability of hyaluronic acid towards degradation.
  • An excess of the sodium form of a powdered aluminium silicate Type 4A Zeolite was used to reduce the level of iron in fermentation broth.
  • Raw fermentation broth obtained from fermentation of a recombinant Bacillus subtilis was diluted with ordinary tap water and filtered to remove host cells to give a 3.5 g/l hyaluronic acid solution containing 24 ppm iron ion. This clarified broth was then incubated and stirred with an excess (3 wt%) of powdered zeolite for 1 hour. The zeolite was then filtered from the solution. The iron content of the filtrate was found to be below detection ( ⁇
  • the original clarified broth and that treated to remove iron were subsequently heat treated.
  • the exchange gel treated material was found to be substantially more heat stable with regard to molecular weight than the untreated clarified broth material under the same conditions.
  • Example 8 Control of calcium content of Hyaluronic acid and replacement of ions on the hyaluronic acid with sodium ions using a sodium form of a granular Type 4A zeolite
  • the performance of the granular zeolite for calcium ion removal and replacement with sodium ion was characterised for the range of conditions likely to be met throughout hyaluronic acid manufacturing and purification processes described earlier. The characteristics were reproduced at both bench and pilot scale and diverse hyaluronic acid batches, produced as in Experiment 1 , were tested. Typically, this granular Type 4A zeolite was able to remove around 0.1 mg calcium from the hyaluronic acid bearing solutions per mg zeolite when the two were contacted. This ratio was found to be largely independent of the process conditions used. This made it very simple to control the calcium level in the hyaluronic acid bearing solution. Equilibrium was achieved in less than 10 minutes in all cases.
  • 0.2 wt% of the granular zeolite was contacted with a hyaluronic acid solution containing 220 ppm calcium. After 20 minutes incubation with stirring, the zeolite was filtered from the solution. The resulting filtrate contained 20 ppm calcium.
  • Hyaluronic acid from the starting material and from the filtrate treated with 0.25 wt% of the granular zeolite was analysed. It was found that the ions on the hyaluronic acid, following treatment with 0.25 wt% of the zeolite had been replaced by sodium ions.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Polymers & Plastics (AREA)
  • Sustainable Development (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Dans un premier aspect, l'invention concerne un procédé de contrôle du contenu de composant(s) sélectionné(s) dans un ou plusieurs polymère(s), ledit procédé consistant (a) à mettre en contact le(s) polymère(s) avec au moins un tamis moléculaire, et éventuellement (b) à isoler le polymère du (des) tamis moléculaire(s).
PCT/DK2006/000231 2005-05-04 2006-04-28 Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s) WO2006117001A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA2606877A CA2606877C (fr) 2005-05-04 2006-04-28 Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s)
JP2008509304A JP5091119B2 (ja) 2005-05-04 2006-04-28 分子篩を用いてポリマーからの選択された成分の含有率を調節するための方法
CN2006800153388A CN101171265B (zh) 2005-05-04 2006-04-28 使用分子筛控制聚合物中所选组分含量的方法
EP06722923A EP1879924A2 (fr) 2005-05-04 2006-04-28 Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200500661 2005-05-04
DKPA200500661 2005-05-04

Publications (2)

Publication Number Publication Date
WO2006117001A2 true WO2006117001A2 (fr) 2006-11-09
WO2006117001A3 WO2006117001A3 (fr) 2006-12-28

Family

ID=36735931

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2006/000231 WO2006117001A2 (fr) 2005-05-04 2006-04-28 Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s)

Country Status (5)

Country Link
EP (1) EP1879924A2 (fr)
JP (1) JP5091119B2 (fr)
CN (1) CN101171265B (fr)
CA (1) CA2606877C (fr)
WO (1) WO2006117001A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113121721B (zh) * 2021-04-21 2022-02-15 山东焦点福瑞达生物股份有限公司 一种乙酰化透明质酸钠的制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2684385B1 (fr) * 1991-11-28 1997-08-01 Sanofi Elf Heparosanes-n,o-sulfates de haute masse moleculaire, leur procede de preparation et les compositions pharmaceutiques qui les contiennent.
FR2697023B1 (fr) * 1992-10-16 1994-12-30 Roquette Freres Polymère soluble hypocalorique du glucose et procédé de préparation de ce polymère .
CA2310422A1 (fr) * 1997-11-20 1999-06-03 Ikuo Yamashina Modification de l'heparine de faible masse moleculaire et remede contre l'ulcere de la peau

Also Published As

Publication number Publication date
WO2006117001A3 (fr) 2006-12-28
JP5091119B2 (ja) 2012-12-05
JP2008540702A (ja) 2008-11-20
CN101171265B (zh) 2012-02-01
CA2606877A1 (fr) 2006-11-09
EP1879924A2 (fr) 2008-01-23
CA2606877C (fr) 2013-09-03
CN101171265A (zh) 2008-04-30

Similar Documents

Publication Publication Date Title
KR102611970B1 (ko) 히알루론산의 정제 공정
KR20190039099A (ko) 히알루론 산의 소듐 염의 제조 및 정제를 위한 공정
EP1951394A1 (fr) Procede chromatographique utilisant des ligands de l'heparine semi-synthetiques
KR20120089772A (ko) 정제 히알루론산류의 제조 방법
KR101639105B1 (ko) 히알루론산의 정제 방법 및 제조 방법
CA2606877C (fr) Procede de controle du contenu de composant(s) selectionne(s) a partir de polymere(s) au moyen de tamis moleculaire(s)
US20100190212A1 (en) Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s)
Xu et al. Chain structure and immunomodulatory activity of a fructosylated chondroitin from an engineered Escherichia coli K4
JP5053512B2 (ja) 非常に大きい硫酸化度を持つｋ５多糖体のエピマー化誘導体
CA2486985C (fr) Floculation au moyen d'un sel divalent
US20040014179A1 (en) Flocculation with divalent salt
WO2020085903A1 (fr) Production de composés biomédicaux par enrichissement de cultures de micro-organismes
KR0149793B1 (ko) 고분자량 히알우론산의 정제방법
US20040126853A1 (en) Flocculation with divalent salt
Ozcan et al. Microbial Production of Hyaluronan
JP5758878B2 (ja) ヒアルロン酸の精製方法
AU778945B2 (en) Hyaluronan product and process for manufacturing thereof
JPH02103203A (ja) ヒアルロン酸の精製法
RU2333222C2 (ru) Эпимеризованные производные полисахарида к5 с высокой степенью сульфатирования

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2006722923

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2008509304

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2606877

Country of ref document: CA

Ref document number: 4946/CHENP/2007

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 200680015338.8

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Ref document number: DE

NENP Non-entry into the national phase

Ref country code: RU

WWW Wipo information: withdrawn in national office

Ref document number: RU

WWP Wipo information: published in national office

Ref document number: 2006722923

Country of ref document: EP