CN101171265B - Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s) - Google Patents
Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s) Download PDFInfo
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- CN101171265B CN101171265B CN2006800153388A CN200680015338A CN101171265B CN 101171265 B CN101171265 B CN 101171265B CN 2006800153388 A CN2006800153388 A CN 2006800153388A CN 200680015338 A CN200680015338 A CN 200680015338A CN 101171265 B CN101171265 B CN 101171265B
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- molecular sieve
- mucinase
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- ion
- calcium
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
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- CQBLUJRVOKGWCF-UHFFFAOYSA-N [O].[AlH3] Chemical compound [O].[AlH3] CQBLUJRVOKGWCF-UHFFFAOYSA-N 0.000 description 1
- OBNDGIHQAIXEAO-UHFFFAOYSA-N [O].[Si] Chemical compound [O].[Si] OBNDGIHQAIXEAO-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
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- 229920001222 biopolymer Polymers 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001669 calcium Chemical class 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
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- 210000001520 comb Anatomy 0.000 description 1
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- 239000002537 cosmetic Substances 0.000 description 1
- 239000002178 crystalline material Substances 0.000 description 1
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- 230000006378 damage Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 238000005188 flotation Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229920000550 glycopolymer Polymers 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003035 hyaline cartilage Anatomy 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
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- 239000004615 ingredient Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000001457 metallic cations Chemical class 0.000 description 1
- 230000007483 microbial process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- QVLTXCYWHPZMCA-UHFFFAOYSA-N po4-po4 Chemical compound OP(O)(O)=O.OP(O)(O)=O QVLTXCYWHPZMCA-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 238000000710 polymer precipitation Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 238000012799 strong cation exchange Methods 0.000 description 1
- 230000019635 sulfation Effects 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 238000006277 sulfonation reaction Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- ZNRSXPDDVNZGEN-UHFFFAOYSA-K trisodium;chloride;sulfate Chemical compound [Na+].[Na+].[Na+].[Cl-].[O-]S([O-])(=O)=O ZNRSXPDDVNZGEN-UHFFFAOYSA-K 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- 210000005253 yeast cell Anatomy 0.000 description 1
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
Abstract
The invention provides in a first aspect a method for controlling the content of selected component(s) in one or more polymer(s) by: (a) contacting the polymer(s) with at least one molecular sieve; and optionally (b) isolating the polymer from the molecular sieve(s).
Description
Invention field
The present invention relates to be used for being controlled at the method for the selected component concentration of one or more polymkeric substance.
Background of invention
Polymkeric substance and their verivate are widely used in various application.What cherish a special interest is the XC polymer that can be used in food, makeup, the medicine industry.In order to satisfy these different application, be necessary usually with polymer purification with remove more than component, and further control the balance of component in the finished product.
The abundantest mixed polysaccharide of human body is TGSS C3 (glycosaminoglycan).TGSS C3 is unbranched glycopolymers, forms (only keratan sulfate (keratan sulphate) is a ramose at the core area of sugar) by the multiple disaccharide unit.Said disaccharide unit comprises one of sugar-N-acetylgalactosamine (GalNAc) or N-acetyl-glucosamine (GlcNAc) of two kinds of modifications usually, as first sugar unit.Another unit is uronic acid normally, for example glucuronic acid (GlcUA) or iduronic acid (iduronate).
TGSS C3 is electronegative molecule, and (in solution) has the extension conformation of the high viscosity of giving in solution.TGSS C3 mainly is arranged on the cell surface or extracellular matrix.TGSS C3 also has low compressibility in solution, and is ideal physiology lubricating fluid therefore, for example joint (joint).The rigidity of TGSS C3 is that cell provides the globality of structure and the passage that allows cell migration between the cell is provided.Having, the TGSS C3 of high physiological significance is hyaluronan (hyaluronan), CHS, heparin, Suleparoid, LMWDS and keratan sulfate.Most TGSS C3s are covalently bond to the proteoglycan core protein through special oligosaccharide structure.Hyaluronan and some proteoglycan form big aggregate (large aggregate), form non-covalent complex body but exception is free sugar chain and proteoglycan.
Identified in vivo many effects of hyaluronan (referring to, Laurent T.C.and Fraser J.R.E., 1992, FASEB is J.6:2397-2404; With Toole B.P, 1991, " Proteoglycans andhyaluronan in morphogenesis and differentiation. " In:Cell Biology of theExtracellular Matrix; Pp.305-341, Hay E.D., ed.; Plenum, New York).Hyaluronan is present in hyaline cartilage, synovial joint fluid (synovial joint fluid) and corium and the epidermis skin histology.Guess that also hyaluronan has effect in many physiological functions, for example adhere to, growth, cell mobility, cancer, blood vessel take place and wound healing.Because physics and biological nature that hyaluronan is unique are used it in eye and joint surgery, and just in other medical approaches, it are being assessed.
HA plays the part of important role in organism, as many tissues, and the for example machinery support of the cell of skin, tendon, muscle and cartilage, it is the staple of intercellular stroma.HA also plays the part of other important role in bioprocess, for example wetting the and lubrication of tissue.
HA can extract from above-mentioned natural tissues, although preferably prepare HA minimizing the potential risk of transmitting infectious agent (infectious agent) through microbial process now, and improves homogeneity, quality and the operability of product.
The level of HA and its differing mol size has been divided with their salt separately to be used as medicine, especially aspect the treatment joint disease; As the auxiliary and/or surrogate of natural organ and tissue, especially aspect ophthalmology and cosmetic surgery; And as the reagent in the cosmetic formulations (agent).The product of also having developed hyaluronan is used for plastic surgery (orthopaedics), rheumatology (rheumatology) and dermatology (dermatology).
HA also can be used as the multiple polymeric materials that is used for health and surgical article, the additive of urethane, polyester etc. for example, and it has makes the biocompatible effect of these materials.
Because the extensive variety of XC polymer purposes; The extensive variety of HA purposes and verivate thereof particularly; Wherein some is mentioned hereinbefore; And, be necessary the HA product that provides highly purified, should in finished product, not have other pollution components basically usually owing to the frequent use of HA in medicinal compsns or surgical article and the HA that is suitable for application-specific.It also is important that (particularly HA) the non-polymer content (non-polymer content) of said polymkeric substance and ion are formed (ionic composition).Usually, the sodium salt of HA is preferably as the most biocompatible form, and avoid other HA salt (Fe, Ca, Cu, Zn, Al, Mg, Mn), the Ca++ salt of HA particularly.Although the controlled balance of in some applications, having a preference for other salt of HA or creation gegenion (counter ion) possibly expected.For example; Controlled calcium level is expected in some Wound care is used really; Proposed controlled zinc level is used to resist ulcer of foot (foot ulcer) (ref diabetologia Croatica 30-3,2001) and is used for antibacterial properties (Acta Pharm Hung.2002; 72 (1): 15-24); Sometimes controlled iron level is used for controlling rheological property (the CN 1473572A of the hyaluronic acid derivatives of some type; WO 95/04132), however low iron level is normally expected the susceptibility of degraded for reduction HA polymkeric substance.
With regard to conventional; Mainly having sodium salt and be form, low or do not have Ca++ content and low or do not have the polymkeric substance (comprising HA) of other metal ion content, is through avoiding calcium and other undesired ion to provide modestly during the fermentative processing step with during the follow-up purification step.The ionic equilibrium of expectation is reached through at first creating high Na ion concentration environment usually, for example, and through adding sodium salt such as sodium acetate, sodium-chlor, sodium sulfate etc.High Na ion concentration is used for from HA molecule competition displacement calcium.Can will remove from the HA molecule from the calcium ion that the HA molecule discharges through any of multiple conventional polymer separation method then, vice versa.The most general situation is; With chemical process and/or through adding deposition or crystallizations such as organic solvent such as ethanol, Virahol, acetone, chloroform, CETAB, will discharge thus and undesired ion stays in the supernatant that (CZ 9700350 with said polymkeric substance (or particularly HA); WO 84/03302; EP0694616A2 and EP 0144019 be some instances of this method just).Can the ion that discharge be separated also that (WO 95/04132 through the ultrafiltration process of diafiltration technology (dia-filtration techniques) from polymkeric substance; GB2249315A).Also can use water extraction (aqueous extraction) and other general polymer stripping technique.
Do not expect that from polymer displacement other method of component (normally Ca++) comprises use sequestrant for example EDTA, phosphatic chelating (CN 85103674A) etc., or Application of ion exchange polymeric adsorbent (EP0694616A2).
Depend on selected method and used promoting agent, the inherent shortcoming is in the above-mentioned technology: need repeated application to reach desired components balance (for example repeating deposition and resuspended or extensive diafiltration (extensive dia-filtration)) usually; And/or component is optionally poor; And/or the poorness of the ability of component; And/or displacement or chelating efficient are low; And/or method has been introduced follow-up other component that be difficult to remove and/or toxic; And/or processing stream (process stream) condition need be handled to reach the desired components balance.
Therefore, because above-mentioned difficulty for conventional means component balanced in the final polymeric articles of control is used to operate the component balanced interchangeable method relevant with polymkeric substance and expects.The present invention provides such method, and several kinds of advantages that are described below are provided in addition.
The invention summary
The present invention is in the method that is provided for being controlled at selected component concentration in one or more polymkeric substance aspect first, and it passes through:
(a) polymkeric substance is contacted with at least a molecular sieve; Randomly
(b) from the molecular sieving polymkeric substance.
Detailed Description Of The Invention
The present invention is provided for not expecting in the controlling polymers method of component or selected component concentration, and it is through removing said component or with this component of displacement of desired components more and/or through operation component balanced carry out relevant with said polymkeric substance.Remove or the operation component is enough to remove or the time of exchange or operational group balance-dividing carries out through polymeric articles is contacted one section with suitable molecular sieve.In some applications, molecular sieve being stayed in the polymeric articles possibly be easily together; Yet, expectation be can molecular sieve be separated from polymkeric substance.
Term " polymer " " be by many multiple less chemical unit or molecular material in this context.Polymkeric substance can be natural or synthetic.
In this context, term " polymer " " also comprise the suspension-s of liquid polymers or polymkeric substance.
Wait that " component " of removing or operating comprises atom, molecule, ion or compound.
In this context, the meaning of statement " control " is the component concentration in component concentration in removal (completely or partially), exchange and/or the balance polymer or the liquid with polymkeric substance.
Exist and to control ideally, to reduce or selection and polymkeric substance and/or many application with the relevant component of the liquid of polymkeric substance:
I) for example, for biocompatibility, mucinase is preferably na form.In addition; The qualification of concrete application is normally expected; For example; Calcic polymkeric substance itself can be insoluble (a for example Protanal TXF 200 (calcium alginate)) or can in common phosphoric acid buffer, produce problematic deposition (problematicprecipitation), or produces adverse reaction (adverse reaction) with activeconstituents or preparation chemical (formulation chemical).On the contrary, specify controlled in some applications calcium level, for example, specified alginate to be used for Wound care products.Similarly, Fe, Ca, Cu, Zn, Al, Mg, other ion and other component.
Viscosity and other character that ii) ion content and ionic type can impact polymers; For other group categories seemingly.Ion content must receive careful and accurate control, for example, and for the generation that contains the zinc hydrogel.
Iii) can polymkeric substance be gone that stable molecule (polymer destabilising molecule) such as Fe and Cu remove, control or by other more harmless ionic replacement.
Iv) polymeric articles need be removed smell or color molecule usually.
The present invention provides the advantage of the method for many operational group balance-dividings of using compared to routine.Can be applied in according to molecular sieve of the present invention on any point of manufacturing processed, comprise being applied to raw material; Use during manufacture or as the aftertreatment (post-treatment) of polymeric articles.For example pH, temperature, polymer concentration etc. are unlike in other conventional method of using equally crucial the method condition in addition.
Molecular sieve can be a highly selective for the set of concrete component or component.Molecular balance reaches rapidly usually, means to handle faster and control according to the component balanced accurate product performance relevant with polymkeric substance.Molecular sieve demonstrates for the ability of waiting to operate component (capacity), and thereby only needs the individual molecules sieve to handle usually.Molecular sieve simply adds, contact and follow-up removal through conventional solid-liquid separation method, means to need not specific equipment and need not soluble reactant of follow-up removal or additive.Can molecular sieve be stayed in the polymers soln, this has further simplified method.Because method of the present invention makes that containing the raw material of not expecting component in the finished product can be used in the step upstream, for example (contains Ca with tap water
++) but not deionized water is used for fermentation and dilution, therefore can also processing cost be reduced.In addition, can direct interpolation of molecular sieve be need not pre-equilibration or pre-treatment usually.Molecular sieve is generally hypotoxicity.
High molecular weight contaminants/impurity
Therefore even can in the upstream process step, add component and do not cause any problem because method of the present invention is applied to remove component from polymeric articles, for the downstream purification step.
Owing to method of the present invention is conveniently used in removing calcium from polymeric articles, so even can in the upstream process step, adds Ca
++And do not cause any problem for the downstream purification step.
Remove high molecular weight contaminants/impurity through throwing out (flocculation) in the feasible purification step in early days of the interpolation of calcium or other divalent salts and become possibility; Maybe these impurity be removed from the acellular prepared product of interested TGSS C3 equally.This has further description at WO2004/001054.During the upstream process step, add the possibility of component, the possibility of particularly adding calcium has constituted another advantage that the present invention compares with traditional method.
Above advantage is provided by method of the present invention, and said method is used for being controlled at one or more polymkeric substance selected (normally not expecting) components contents, and it comprises the steps:
(a) polymkeric substance is contacted with molecular sieve; Randomly
(b) from the molecular sieving polymeric articles.
The meaning of in context of the present invention, " controlling selected components contents " is to remove (completely or partially) said component and/or replace (exchange) this component and/or operation and said polymkeric substance or have the balance of the relevant component of the liquid of polymkeric substance with desired components more.
" molecular sieve " meaning in context of the present invention is the material with hole of molecular size, can be used in bigger molecule and less separating.They include but not limited to zeolite, carbonaceous molecular sieve, silica gel, activated alumina (activated alumina).Usually, the crystalline network that molecular sieve has produces the spline structure (cage like structure) that comes out of steamer, and it has the window (window) of only allowing less than the molecule of specific size.Through using the material of different sources and different creating conditions, might produce the different a series of molecular sieves that get into size (differing access dimensions).The size of concrete molecular sieve can be accurate usually, because they are derived from the crystalline structure of said sieve.
Some can be used for the instance of the molecule of (admitted by) differing mol sieve, and together with other details of relevant molecular sieve, from Chemical Engineering, Volume 2,4th Edition; JM Coulson andJF Richardson; Pergamon Press provides in the table 17.3 of 1991 " Classification of Some MolecularSieves ".The DB more comprehensively of the dependency structure of molecular sieve and character is provided on (http://www.iza-structure.org/databases/) by International Zeolite Association (http://www.iza-online.org/).The basic material that molecular sieve is relevant comprises: DW Breck:Zeolite Molecular Sieves, Wiley, New York, 1974; RM Barrer:Hydrothermal Chemistry of Zeolites, Academic Press, 1982; Intro to ZeoliteScience and Practice, H van Bekkum, EM Flannigen, PA Jacobs and JC Jansen, Studies in Surface Science, Vol 137,1-1060, (2001) Elsevier, Amsterdam.
Molecular sieve is a zeolite in concrete embodiment.The classics definition of zeolite is a kind of crystalline, porous aluminosilicate (aluminosilicate).Yet some relevant recent discovery is the same with classical zeolite in fact material, but said material is made up of the oxide structure with the element outside silicon and the aluminium, and this has expanded definition.In fact present most investigators are included in all types of porous oxide structures in their definition to zeolite, and said porous oxide structure is owing to highly crystalline property has the clearly pore structure of definition.In context of the present invention, all be included in the term " zeolite " above.We are called in the crystalline material of zeolite at these, and atoms metal (being silicon or aluminium under the classical case) is surrounded by four oxygen anions and is similar to tetrahedral structure to form, and said tetrahedron is made up of the metallic cation of central authorities and the oxygen anion on four summits.Abbreviate said tetrahedron metal as the T atom, these tetrahedrons pile up with regularly arranged then, form passage thus.It is unlimited that the possible mode of piling up comes down to, and known hundreds of particular structure.
Zeolite channels (or hole) is little at microscopically, and in fact it has the size of molecular size, and they are represented with term " molecular sieve " usually thus.The size of passage and shape have unusual influence for the character of these material adsorption processes, and this character causes their purposes in sepn process.Through with component possible relevant shape and big or small effect of direction in the hole, and/or the difference through adsorption strength can be separated them.Thereby can component optionally be removed.
Because silicon exists with the 4+ oxidation state usually, silicon-oxygen tetrahedron is an electroneutral.Yet in zeolite, aluminium exists with the 3+ oxidation state usually; Therefore aluminium-oxygen tetrahedron forms the center that lacks an electronics.Therefore, zeolite framework (framework) is generally anionic, and the positively charged ion of compensation charge is gathered in (populate) hole to keep electroneutral.
In specific embodiments of the present invention, molecular screening is from the group of zeolite, and wherein the porousness of material conforms to component to be removed, and in other embodiment, the porousness of material conforms to Ca++; In other embodiment, being gathered in zeolite pore is those ions in the polymeric articles desired to keep electroneutral ion; In other embodiment, sodium ion is to be gathered in zeolite pore to keep electroneutral ion.
Those molecular sieves that classics are grouped into " 4 type " (" Type 4 ") have the molecular sieve size that is suitable for the calcium ion chelating, and it has the Linde sieve 4A size of about 0.4nm.Many these molecular sieves all have the zeolite pore of being gathered in to keep electroneutral sodium ion.
Thereby in the concrete embodiment of the present invention, zeolite is 4 type zeolites.In other embodiment, sodium ion is contained in the hole of molecular sieve.
In one embodiment of the invention, polymkeric substance is XC polymer (biopolymer)." XC polymer " is any polymeric material (instance does, but is not limited to polysaccharide, protein, nucleic acid etc.) that in living things system, forms.There is the instance of many common XC polymers, includes but not limited to: the verivate of chitosan (Chitosan), VISOSE, Keratin sulfate, Mierocrystalline cellulose, gelatin (gelatine), TGSS C3 and all these polymkeric substance.
In concrete embodiment, XC polymer is a polysaccharide, and in other specific embodiments, polysaccharide is a TGSS C3.
TGSS C3
According to the present invention, TGSS C3 can be any carbohydrate polymer, and it has at least 700 daltonian molecular weight; Preferred at least 10,000 daltonian molecular weight; More preferably at least 20,000 daltonian molecular weight; Even more preferably at least 30,000 daltonian molecular weight.
Preferred TGSS C3 is mucinase, CHS, chrondroitin (not sulfation), heparin (heparin), heparin sulfate, LMWDS and keratin sulfate (keratin sulphate).Mucinase has as many as 15,000 with formation, the linear chain of 000 daltonian molecular weight by replacing and multiple D-glucuronic acid (D-glucoronic acid) and N-acetyl-D-glucosamine units formation.
Preferred TGSS C3 is to have from 700 dalton to 15 TGSS C3 of 000,000 Dalton molecular weight according to the present invention.
What should be noted that is the mucinase that term " mucinase " in the application and claims can indistinguishably refer to its sour form or its salt form, for example hyaluronate sodium, potassium hyaluronate, mucinase magnesium, Calcium hyaluronate or other.
The meaning that term " hyaluronan (hyaluronan) " or " mucinase (hyaluronic acid) " are used for document is the acidic polysaccharose with different molecular weight; Its residue by D-glucuronic acid and N-acetyl-D-glycosamine constitutes; Its natural cell surface that is present in, the outer material of the basic born of the same parents of vertebrates reticular tissue, the synovia in joint; Intraocular ball liquid (endobulbar fluid), human umbilical tissue (human umbilicalcord tissue) and cockscomb are (among the cocks ' comb).
Term " mucinase " in fact is commonly used to refer to have the complete series polysaccharide that has alternative D-glucuronic acid and N-acetyl-D-glycosamine residue of different molecular weight; Or even the level of its degraded divide, and look that therefore to use plural term " mucinase (hyaluronic acids) " more correct.Yet this singular references will still be used in this manual; In addition, will use abbreviation " HA " to replace this collective term continually.
XC polymer is TGSS C3 (glucosaminoglucan) for example, can provide from animal tissues, and perhaps more preferably the host cell through the said XC polymer of culture expression provides.
Fermented liquid (fermentation broth)
TGSS C3 can obtain from any fermented liquid.TGSS C3 also can be through comprising the TGSS C3 of the method generation of cultivating host cell in addition.
Host cell preferably can be a mikrobe.Said mikrobe can be unicellular microorganism, for example prokaryotic organism; Or non-unicellular microorganism, for example eukaryote.Useful single celled cell is a bacterial cell; Gram positive bacterium for example; It includes but not limited to bacillus (Bacillus) cell; For example, Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillusamyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacilluscirculans), gram Lloyd's genus bacillus (Bacillus clausii), Bacillus coagulans (Bacilluscoagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), bacillus lentus (Bacillus lentus), Bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacstearothermophilus (Bacillus stearothermophilus), subtilis (Bacillus subtilis) and bacillus thuringiensis (Bacillus thuringiensis); Or streptomyces (Streptomyces) cell; For example; Shallow Streptomyces glaucoviolaceus (Streptomyces lividans) or squirrel streptomycete (Streptomyces murinus), or gram negative bacterium, for example intestinal bacteria and Rhodopseudomonas bacterial classification (Pseudomonas sp.).In concrete embodiment, bacterial host cell is bacillus lentus cell, Bacillus licheniformis cell, bacstearothermophilus cell or bacillus subtilis mycetocyte.Being particularly suitable for recombinant expressed sudden change bacillus subtilis mycetocyte describes in WO 98/22598.
Host cell can be an eukaryote, for example mammalian cell, insect cell, vegetable cell or fungal cell.Useful mammalian cell comprises Chinese hamster ovary (Chinese hamster ovary; CHO) cell, HeLa cell, immature hamster kidney (baby hamster kidney; BHK) cell, COS cell, or any many other available immortalized cell systems are for example from American type culture collection (American Type Culture Collection).Any other part of method for transformation, selectable marker gene and expression construct can be selected from that those skilled in the art know and can obtain those.
Host cell can be the fungal cell." fungi " is used for this paper and comprises Ascomycota (Ascomycota), Basidiomycota (Basidiomycota), chytrid door (Chytridiomycota) and Zygomycota (Zygomycota), and oomycetes door (Oomycota) and all mitospore fungies (mitosporic fungi).The representative monoid (group) of Ascomycota comprises, for example Neurospora (Neurospora), Eupenicillium sp (Eupenicillium) (=penicillium (Penicillium)), Emericella (=Aspergillus (Aspergillus)), Eurotium (Eurotium) (=Aspergillus) and below listed true yeats (true yeast).The instance of Basidiomycota comprises mushroom (mushroom), rest fungus (rust) and ustilago (smut).The representative monoid of chytrid door comprises, for example Allomyces (Allomyces), little Blastocladia (Blastocladiella), Coelomomyces (Coelomomyces) and aquatic fungi (aquatic fungi).The representative monoid of oomycetes door comprises that for example Saprolegniomycetous aquatic fungi (water mold) is such as Achyla (Achlya).The instance of mitospore fungi comprises Aspergillus, penicillium, Candida (Candida) and Alternaria (Alternaria).The representative monoid of Zygomycota comprises, for example Rhizopus (Rhizopus) and Mucor (Mucor).
Fungal host cells can be a yeast cell." yeast " is used for the yeast that this paper comprises ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), product load yeast (basidiosporogenous yeast) and belongs to imperfect fungi (Fungi Imperfecti) (gemma guiding principle (Blastomycetes)).Ascosporogenous yeast is divided into Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter is made up of four subfamilies; (for example be respectively Schizosaccharomycoideae (Schizosaccharomycoideae); Schizosaccharomyces (Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), Lipomycetoideae (Lipomycoideae) and Saccharomycoideae (for example, genus kluyveromyces (Kluyveromyces), Pichia (Pichia) and yeast saccharomyces cerevisiae belong to (Saccharomyces)).Producing the load yeast comprises Leucosporidium (Leucosporidim), Rhodosporidium (Rhodosporidium), locks and throw yeast belong (Sporidiobolus), net spore Pseudomonas (Filobasidium) and Filobasidiella (Filobasidiella).The yeast that belongs to imperfect fungi is divided into two sections, Sporobolomycetaceae (Sporobolomycetaceae) (for example Sporobolomyces (Sporobolomyces) and cloth are reined in and played spore yeast belong (Bullera)) and Cryptococcaeceae (Cryptococcaceae) (for example, Candida).
In other embodiment, fungal host cells is a filamentous fungal cells." filamentous fungus " comprises all thread forms of fungi (Eumycota) and oomycetes (Oomycota) subphylum.Filamentous fungus is characterised in that the mycelia body wall of being made up of chitin (chitin), Mierocrystalline cellulose, VISOSE, chitosan, mannosans (mannan) and other complicated polysaccharide.Nourish and grow and carry out, and carbon katabolism is obligate aerobic through the mycelia prolongation.In contrast, nourishing and growing of yeast such as yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) then carried out through the budding (budding) of unicellular thalline (unicellular thallus), and carbon katabolism can ferment.In a more particular embodiment; Filamentous fungal host cell is, but is not limited to the cell of following Pseudomonas bacterial classification: the branch mould genus of top spore (Acremonium), Aspergillus, fusarium (Fusarium), Humicola (Humicola), Mucor, myceliophthora (Myceliophthora), Neurospora, penicillium, Thielavia (Thielavia), the curved mould genus of neck (Tolypocladium) and Trichoderma (Trichoderma).
Can the fungal cell be transformed through relating to protoplastis formation, protoplast transformation and cell walls regenerated method in a manner known way.
Using means known in the art to cultivate being suitable for the mikrobe that produces interested TGSS C3 produces in the nutritional medium of said TGSS C3.For example, can with said mikrobe through in the laboratory or the shake-flask culture that carries out in the industrial fermentation jar, small-scale or large scale fermentation (include but not limited to continuously, in batches, fed-batch or solid state fermentation) cultivate.In comprising the suitable nutritional medium of carbon source and nitrogenous source and inorganic salt, use methods known in the art to cultivate.Suitable medium can obtain from commercial provider, or can prepare according to disclosed composition (for example, in the catalogue of American type culture collection).As the instance that is created in the TGSS C3 that produces in the mikrobe, WO2003/054163 has described hyaluronic generation in the genus bacillus host cell.
As stated, being used to produce traditional method or process with the component balanced purifying TGSS C3 (the for example mucinase of sodium-salt form) of expectation comprises:
-precipitation polymers in being rich in the environment of sodium ion;
-crystallization in being rich in the environment of sodium ion;
-precipitate undesired component, for example use phosphoric acid salt (phosphate) and use the environment that is rich in sodium ion.
-dialysis or diafiltration in being rich in the environment of sodium ion;
The environment of sodium is rich in-chelating and application; With
-be used for other conventional means of polymer purification.
With a relevant problem of currently known methods that is used to remove calcium is that they almost always introduce soluble contaminants, and this pollutent need be removed by other treatment step.These methods are often used and follow-uply are difficult to from product, remove and maybe be to the poisonous or deleterious chemical precipitation agent of final application or other auxiliary (aids) in addition.This meets the for example competition metathetical situation of calcium in the presence of the excess sodium ion.Need metathetical calcium and excessive sodium gegenion be removed.Same situation is to use sequestrant for example during EDTA.Must subsequently EDTA be removed.Also need the deposition of excessive precipitation agent and formation be removed with said calcium phosphate precipitation calcium ion.
These treatment steps are not easy to control; Because molecular balance changes along with the change of soluble constituent concentration; It is relatively slow usually to reach molecular balance; Component relatively low usually by operational capability, said technology only works under treatment condition (for example pH, ionic strength, polymer concentration etc.) among a small circle.They are normally time-consuming and need be more than a kind of method (application) in addition, and it makes the influence that product is subject to degrade, and and non-selective or effectively control method component balanced in the product.
Comprise the steps: polymkeric substance to be finished or liquid with polymkeric substance are contacted under conditions suitable with the molecular sieve of suitable type according to method/process provided by the invention; If necessary, sieve from the polymkeric substance isolated molecule.
Liquid with polymkeric substance to be finished can be accomplished through in the several different methods any with the contact between the molecular sieve, and said method includes but not limited to:
● simply suspend or mix
● the liquid with polymkeric substance is passed through: (packed) of filling, settled (settled), expansible (expanded), fluidizing (fluidised) molecular sieve bed (bed).
● contact with molecular sieve as body feed (body feed), precoated layer (pre-coat) or filtration auxiliary (aid offiltration).
The type, quantity and the contact conditions that in polymeric articles, reach the required molecular sieve of desired components balance can be confirmed by those skilled in the art simply.
The component of removing or operating of waiting according to the present invention comprises atom, molecule, ion or compound.The instance of the component that can remove through concrete molecular sieve type includes but not limited to that at ChemicalEngineering, Volume 2,4
ThEdition; JM Coulson and JF Richardson; PergamonPress, those that provide in the table 17.3 of 1991 " Classification of Some Molecular Sieves ".The DB more comprehensively of the dependency structure of molecular sieve and character is provided on (http://www.iza-structure.org/databases/) by International Zeolite Association (http://www.iza-online.org/).The basic material that molecular sieve is relevant comprises:
●DW?Breck：Zeolite?Molecular?Sieves，Wiley，New?York，1974
●RM?Bafrer：Hydrothermal?Chemistry?of?Zeolites，Academic?Press，1982
●Intro?to?Zeolite?Science?and?Practice，H?van?Bekkum，EM?Flannigen，PAJacobs?and?JC?Jansen，Studies?in?Surface?Science，Vol?137，1-1060，(2001)Elsevier，Amsterdam
Particularly, zeolite can be used in the content of control (removing or operation) organic solvent (including but not limited to: alcohol, aldehyde, ketone etc.), metals ion, negatively charged ion, quaternary ammonium compound, SDS, EDTA, CETAB, TCA, TMBEA (cetyl pyrimidine chloride) etc.
Particularly, molecular sieve can be used in and removes or the operation ionic equilibrium more specifically cationic balance.
In one embodiment, ion is a divalent ion, more specifically is Ca
++
In concrete embodiment, the present invention relates to use molecular sieve to come the method for calcium ion content in the controlling polymers.
In concrete embodiment, the present invention relates to use molecular sieve to come calcium and the method for sodium ions content in the controlling polymers.
Can with the ingredients in suspension of waiting to remove or partly remove in comprise the liquid of polymkeric substance, maybe can component be combined or be present on the polymkeric substance.
In other embodiment, component is exchanged into other component.For example can Ca IX be become the Na ion.
In other embodiment, component balanced being controlled in the product.
In concrete embodiment, molecular sieve is a zeolite.
" appropriate condition " meaning in this article be used to realize component is removed or those conditions of the processing stream (process stream) of operation with can be by those conditions of the simple decision of those skilled in the art.Appropriate condition can include but not limited to: molecular sieve type, molecular sieve dosage (molecular sievedosing), temperature, pH, polymer concentration, ionic strength, solvent strength, mixing, incubation time etc.
In a more particular embodiment, polymkeric substance is a TGSS C3.
Can molecular sieve be removed from polymkeric substance through in the several different methods any, said method such as but not limited to: filter, centrifugal, flotation (floatation), sedimentation, phase exclusion (phase exclusion) etc.In some cases, possibly not need to remove, or molecular sieve stayed in the liquid with polymkeric substance expect.
Can optimization or improvement be carried out in concrete component removal of the present invention or working method, it is through grading such as but not limited to operation: pH, temperature, viscosity, concentration, mixing, ionic strength, additive, one-tenth.Can in identical liquid, handle more than a kind of polymkeric substance with polymkeric substance.Can the molecular sieve more than a type be contacted with the liquid with polymkeric substance.Can be with using the processing of molecular sieve to be used to operate liquid with polymkeric substance more than a kind of.
In concrete embodiment, method of the present invention provides the control (removal, exchange and/or balance) of the liquid intermediate ion of the suitable zeolite of use to having polymkeric substance.Particularly, said polymkeric substance is a TGSS C3.
In a more particular embodiment, said polymkeric substance is a mucinase.
In other specific embodiments, method of the present invention uses suitable zeolite that the control to the ion content of polymkeric substance own is provided.Particularly, said polymkeric substance is a mucinase.
In other embodiment, method of the present invention provides uses the control of suitable molecular sieve to calcium of polymkeric substance own and sodium ions content.Particularly, said polymkeric substance is a mucinase.
Embodiment
Embodiment 1 no calcium is removed the mucinase purifying in stage
In the present embodiment, will dilute with ordinary tap water by the 5g/l hyaluronic acid solution that the recombined bacillus subtilis fermentation obtains, and filter to remove host cell.Subsequently with filtrate with respect to deionized water widely (extensively) dialysis to remove great majority not in HA molecule on one's body free ca originally.The gained dialysis product contains the calcium with respect to mucinase quality 4.4wt%.
Embodiment 2 has the conventional mucinase purifying that passes through the stage that the calcium of sodium salt diafiltration is removed
Described in embodiment 1, obtain mucinase in the present embodiment.Introduce calcium controlled step (not relating to molecular sieve of the present invention), said step relates to the competition displacement of calcium in the presence of the excess sodium ion, as follows:
With hyaluronic acid solution with respect to the diafiltration of excess sodium ion.Thus the calcium ion that discharges is removed from solution through flowing through diafiltration membrane.The calcium ion that discharges from mucinase possibly be equal to ground (equally) removal through polymer precipitation.In present case, with hyaluronic acid solution with respect to the 10wt% metabisulfite solution of 3x volume with constant volume (at constant volume) diafiltration, extensively dialyse to remove excess sulfates and sodium ion with respect to deionized water then.Products obtained therefrom contains the calcium with respect to mucinase 1.2wt%.
Embodiment 3 uses Zeo-karb to control hyaluronic calcium contents
As above produce mucinase described in the embodiment 1.The SO that has with the sodium ion form
3 -The strong cation-exchanging resin of (＞2eq/l) is used to operate the calcium ion content of polymkeric substance.
The performance of exchange resin is come qualitative by a series of situation that in said whole mucinase manufacturing of preamble and purification process, run into probably.
In an example, the 1.0wt% exchange resin is added into the hyaluronic acid solution of 0.5wt%, said solution contains the calcium with respect to mucinase 2wt%.After stirring incubation 120 minutes, resin is filtered from solution.It is the calcium with respect to mucinase 0.5wt% that gained contains the calcium contents that hyaluronic filtrate has.Under the same conditions after the incubation 240 minutes, with respect to hyaluronic calcium contents (below detection) below the value of detecting.
Embodiment 4 uses the cationic exchange gel to control hyaluronic calcium contents
As above produce mucinase described in the embodiment 1.The strong cation exchange gel that will have sulfonation functional group (2.05eq/l) is used to operate the calcium ion content of polymkeric substance.
The performance of exchange gel is come qualitative by a series of situation that in said whole mucinase manufacturing of preamble and purification process, run into probably.
In an example, 0.25wt% is exchanged gel be added into the 0.5wt% hyaluronic acid solution, said solution contains the calcium with respect to mucinase 2wt%.Mixture is stirred incubation and makes it in 1 hour, reach balance.Gel is filtered from solution.It is the calcium with respect to mucinase 0.3wt% that gained contains the calcium contents that hyaluronic filtrate has.Under the same conditions, the exchange gel of 1wt% na form will be reduced to below the value of detecting with respect to hyaluronic calcium contents.
In other pilot scale instance (pilot scale example), the exchange gel of 0.5wt% na form is added in the 0.7wt% hyaluronic acid solution without pre-treatment, said solution contains 211ppm calcium., said mixture was stirred incubation 2 hours, then gel is filtered from solution.The gained filtrate contains the calcium with respect to mucinase 14ppm, and it accurately tests (benchscale experiment) corresponding to bench scale under the same conditions.Under the same conditions, the exchange gel of 1wt% will be reduced to below the value of detecting with respect to hyaluronic calcium contents.
To use the parent material and the filtrate of the exchange Gel Treatment of 1wt% na form to dialyse to remove dissociated ion with respect to deionized water.Find that by analyzing the gained mucinase ion on the mucinase is replaced by sodium ion after the exchange Gel Treatment with the 1wt% na form.
Embodiment 5 uses the cationic exchange gel to reduce the iron in the fermented liquid
Gel type is a strong cation exchanger, and it has (2.05eq/l) functional group of sulphonate (sulphonate).
Excess amount of ions is exchanged the iron level that gel is used for reducing fermented liquid.Proved such as iron, copper plasma and reduced the stability of mucinase degraded.
To contain hyaluronic thick fermented liquid (raw fermentation broth) through clarifying with the filtration of ordinary tap water dilution and removal mikrobe.Subsequently with the exchange gel incubation and stirring 1 hour of this clarifying meat soup with excessive (10wt%).To exchange gel then filters from solution.Compare with the 0.4wt% (with respect to mucinase) in the clarification nutrient solution, find the iron level (＜1ppm is with respect to mucinase) below the value of detecting of filtrate.
Then primary is clarified nutrient solution and heat-treat through handling with the nutrient solution of removing iron.Consider molecular weight, find that undressed clarification nutrient solution material is compared more thermally-stabilised basically under the material of exchange Gel Treatment and the same terms.
Processing to the clarification nutrient solution does not change MWD or contained hyaluronic concentration.
Application and the method shown in other treatment liq and component ability enough embodiment of being similar to 3 and 4 of finding controlled.Removing component from clarified broth has stablized hyaluronic to thermal destruction.
Embodiment 6 uses na form powdered silicate aluminium 4A type zeolite to control hyaluronic calcium contents and usefulness
Ion on the sodium ion displacement mucinase
As above embodiment 1 produces mucinase.The calcium ion content that powdery sodium form 4A type zeolite is used to operate polymkeric substance.
The performance of exchange resin is come qualitative by a series of situation that in said whole mucinase manufacturing of preamble and purification process, run into probably.The two reappears with bench scale and pilot scale with said characteristic, and has tested many batches of mucinases that produce as among the embodiment 1.
When the two is contacted, find that every milligram of zeolite can remove about 0.06mg calcium from have hyaluronic solution.Think that this ratio does not rely on used treatment condition (for example pH, temperature, HA concentration etc.) to a great extent.This makes that the calcium level that has in the hyaluronic solution through the control of simple dosage interpolation zeolite is very simple.In all cases, be equilibrated to be less than in 15 minutes and reach.
In common instance, only with the powdery zeolite of 0.2wt% through no pre-equilibration or pretreated direct interpolation and contact with the hyaluronic acid solution that contains 142ppm calcium.Stirred after the incubation, zeolite is filtered from solution in 20 minutes.The gained filtrate contains 14ppm calcium.Under identical flow process, contact calcium is reduced to below the detection limit with the 0.3wt% zeolite.
Analyze the mucinase parent material with its with 0.3wt% powdery zeolite treatment after.Ion on the discovery mucinase is replaced by sodium ion after with the 0.3wt% zeolite treatment.
Hyaluronic processing does not change MWD or concentration, and the detailed sign of processed transparent matter acid is shown not have harmful the change.
Discovery is removed the degree of calcium and can be predicted and control on circulation ratio ground from contain hyaluronic solution.Ion on the mucinase is replaced through sodium ion.There is not detrimental effect (adverseeffect) for mucinase.
Embodiment 7 uses na form powdered silicate aluminium 4A type zeolite to reduce the iron in the fermented liquid
Proved such as iron, copper plasma and reduced the stability of mucinase degraded.The iron level that the powdered silicate aluminium 4A type zeolite of excess sodium form is used for reducing fermented liquid.
The thick fermented liquid that will obtain from the fermentation of recombined bacillus subtilis dilutes with ordinary tap water, and filters and remove host cell, so that the 3.5g/l that contains the 24ppm iron ion to be provided hyaluronic acid solution.Subsequently with clarifying nutrient solution with the powdery zeolite incubation of excessive (3wt%) and stirred 1 hour.Subsequently zeolite is filtered from solution.Find the iron level (＜1ppm is with respect to mucinase) below the value of detecting of filtrate.
Then primary is clarified nutrient solution and heat-treat through handling with the nutrient solution of removing iron.Find that undressed clarification nutrient solution material is compared more thermally-stabilised basically under the material of exchange Gel Treatment and the same terms.
Processing to the clarification nutrient solution does not change contained hyaluronic MWD or concentration.
Embodiment 8 uses the granular 4A type of na form zeolite to control hyaluronic calcium contents and use sodium ion
Ion on the displacement mucinase
Granular zeolite removes and uses sodium ion metathetical performance to come qualitative by a series of situation that in said whole mucinase manufacturing of preamble and purification process, run into probably for calcium ion.The two reappears with bench scale and pilot scale with said characteristic, and has tested many batches of mucinases that produce as among the embodiment 1.
Usually, when with granular 4A type zeolite with have hyaluronic solution when contacting, every milligram of this granular 4A type zeolite can be from having the about 0.1mg calcium of hyaluronic solution removal.Think that this ratio does not rely on used treatment condition to a great extent.This calcium level that makes control have in the hyaluronic solution is very simple.Be equilibrated in all cases to be less than in 10 minutes and reach.
In common instance, the granular zeolite of 0.2wt% is contacted with the hyaluronic acid solution that contains 220ppm calcium.Stirred after the incubation, zeolite is filtered from solution in 20 minutes.The gained filtrate contains 20ppm calcium.Contact with 0.25wt% zeolite (II) under the same conditions calcium is reduced to below the detection limit.
Analysis is from the mucinase of parent material and the mucinase of the filtrating of the granular zeolite treatment of the 0.25wt% that uses by oneself.Ion on the discovery mucinase is replaced by sodium ion after with the 0.25wt% zeolite treatment.
Hyaluronic processing is not changed MWD or concentration, and treated hyaluronic detailed sign is shown not have harmful the change.
Discovery is removed the degree of calcium and can be predicted and control on circulation ratio ground from contain hyaluronic solution.Ion on the mucinase is replaced through sodium ion.There is not detrimental effect for mucinase.
Claims (13)
1. method that is used for being controlled at the selected component concentration of mucinase, it passes through:
(a) said mucinase is contacted with at least one molecular sieve; Randomly
(b) from the said mucinase of molecular sieving,
Wherein said component is an ion.
2. according to the process of claim 1 wherein that said ion is a divalent ion.
3. according to the method for claim 2, wherein said divalent ion is Ca
++
4. according to each method of claim 1-3, wherein said component is removed from comprise said hyaluronic liquid or part is removed.
5. according to the process of claim 1 wherein said component is removed from said mucinase or the part removal.
6. according to the process of claim 1 wherein said component is exchanged into other component.
7. according to the method for claim 3, wherein with Ca
++Be exchanged into other component.
8. according to the method for claim 7, wherein with Ca
++Be exchanged into sodium.
9. according to Claim 8 method is wherein with Ca
++Be exchanged into sodium, and the ratio of said hyaluronic calcium form and na form is controlled.
10. according to the process of claim 1 wherein that said molecular sieve is a zeolite.
11. according to the method for claim 10, wherein said zeolite comprises 4 type zeolites.
12. according to the process of claim 1 wherein that the hole of said molecular sieve comprises sodium ion.
13. according to the process of claim 1 wherein that said contact between mucinase and molecular sieve provides through following: i) suspension or mixing; Ii) make have the filling of said hyaluronic liquid through molecular sieve, settled, expansible or fluidizing bed; Or iii) contact with the molecular sieve that is used as body feed, precoated layer or filtration auxiliary.
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| DKPA200500661 | 2005-05-04 | ||
| PCT/DK2006/000231 WO2006117001A2 (en) | 2005-05-04 | 2006-04-28 | Method of controlling the content of selected component(s) from polymer(s) using molecular sieve(s) |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5384398A (en) * | 1991-11-28 | 1995-01-24 | Elf Sanofi | High molecular mass N,O-sulphated heparosans, process for their preparation and the pharmaceutical compositions which contain them |
| US5424418A (en) * | 1992-10-16 | 1995-06-13 | Roquette Freres | Low-calorie soluble glucose polymer and process for preparing this polymer |
| EP1033375A1 (en) * | 1997-11-20 | 2000-09-06 | Ikuo Yamashina | Low-molecular heparin modification and remedy for skin ulcer |
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2006
- 2006-04-28 JP JP2008509304A patent/JP5091119B2/en not_active Expired - Fee Related
- 2006-04-28 CA CA2606877A patent/CA2606877C/en not_active Expired - Fee Related
- 2006-04-28 WO PCT/DK2006/000231 patent/WO2006117001A2/en not_active Application Discontinuation
- 2006-04-28 EP EP06722923A patent/EP1879924A2/en not_active Withdrawn
- 2006-04-28 CN CN2006800153388A patent/CN101171265B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5384398A (en) * | 1991-11-28 | 1995-01-24 | Elf Sanofi | High molecular mass N,O-sulphated heparosans, process for their preparation and the pharmaceutical compositions which contain them |
| US5424418A (en) * | 1992-10-16 | 1995-06-13 | Roquette Freres | Low-calorie soluble glucose polymer and process for preparing this polymer |
| EP1033375A1 (en) * | 1997-11-20 | 2000-09-06 | Ikuo Yamashina | Low-molecular heparin modification and remedy for skin ulcer |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006117001A3 (en) | 2006-12-28 |
| JP5091119B2 (en) | 2012-12-05 |
| CN101171265A (en) | 2008-04-30 |
| CA2606877C (en) | 2013-09-03 |
| CA2606877A1 (en) | 2006-11-09 |
| WO2006117001A2 (en) | 2006-11-09 |
| EP1879924A2 (en) | 2008-01-23 |
| JP2008540702A (en) | 2008-11-20 |
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