Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
• • 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

• the present invention relates to process for purifying hyaluronic acid salt. More particularly, the present invention provides a process for purification of hyaluronic acid salts, from the fermentation broth obtained by culturing a microorganism capable of producing Hyaluronic acid (HA). BACKGROUND
• Hyaluronic acid is a naturally occurring high molecular weight polysaccharide typically recovered as its salt having an empirical formula of (C 14 3 ⁇ 4o N Na On) n where n >1000. It is well known that hyaluronic acid and its salts, hereafter collectively referred to as HA, can be obtained from at least three sources: human umbilical cords, rooster combs and certain bacterial cultures such as group A and C hemolytic Streptococci. However, certain disadvantages are associated with the former two sources (e.g. relatively low yields, contamination with chondroitin sulfate, and labor intensive processing and purification steps).
• hyaluronic acid salts including a method of extracting from living tissue containing hyaluronic acid, for example, cockscombs and purifying it, and a method of isolating from a culture medium obtained by culturing a microorganism capable of producing hyaluronic acid and purifying it. Since hyaluronic acid salts are polymers of molecular weights of several tens of thousands to several millions, this poses an important problem in purification in terms of the removal of polymeric impurities such as proteins, nucleic acids, pyrogens
• Previous methods for purifying hyaluronic acid or its salt have involved at least two successive precipitation steps, thereby resulting in increased processing costs.
• Previous methods for precipitating hyaluronic acid also involved the use of surfactant or detergents.
• the removal of residual chemicals from the precipitate is complex and cost intensive.
• previously known purification procedures, such as ultrafiltration/diafitration processes are cost intensive and generate high volume of effluent during diafiltation step.
• the present invention provides a process for purification of hyaluronic acid salt from fermentation broth obtained by culturing a microorganism capable of producing Hyaluronic acid.
• the fermentation broth is diluted by distilled or RO water in ratio of about 1 :2 to about 1 :4.
• the diluted hyaluronic acid containing fermentation broth is processed for separation of the microbial cells. Further, protein impurities are separated from the hyaluronic acid containing fermentation broth by using bentonite, followed by separating color impurities, and nucleic acids from hyaluronic acid containing fermentation broth by activated carbon.
• Metallic impurities are separated by passing the hyaluronic acid containing fermentation broth through cation exchange resin bed, followed by forming salt of hyaluronic acid by adding the alkali or alkaline earth metal salt to the hyaluronic acid containing fermentation broth, subjecting the precipitating of hyaluronic acid salt by addition of organic solvent and filtering the precipitate by separating the organic solvent from hyaluronic acid salt; and drying the hyaluronic acid.
• Figure 1 represents the integrated process flow diagram for purification of Hyaluronic acid salt (hereinafter referred as ' ⁇ ') from the fermentation broth.
• Figure 2 illustrates an exemplary system for purification of Hyaluronic acid salt from fermentation broth.
• Figure 1 shows a method (10) for purification of hyaluronic acid (HA) from HA containing fermentation broth (12) obtained by culturing a microorganism capable of producing hyaluronic acid.
• HA containing fermentation broth is diluted (14) using distilled water or RO (Reverse Osmosis) water in a dilution ratio of about 1 :2 to about
• HA containing fermentation broth is then subjected for microbial cell separation (16) by using techniques known in the art such as filtration arid centrifugation. After separation of microbial cells from HA containing fermentation broth, the broth is then subjected to bentonite treatment (18) for separation of protein from the HA containing fermentation broth. Separation of color impurities and nucleic acids (20) from HA containing fermentation broth is carried out by using activated carbon. After removal of said impurities, metallic impurities are separated (22) by passing the HA containing fermentation broth through a cation exchange resin bed.
• the HA containing fermentation broth is subjected to addition of alkali or alkaline earth metal salt to form HA salt (24) followed by addition of organic solvent (26) to form a HA salt precipitate (28).
• the HA salt precipitate is separated (30) from organic solvent by solid -liquid separation techniques such as, filtration, centrifugation and sedimentation techniques.
• the HA salt precipitate is then dried (32) using known drying techniques suph as freeze drying, vacuum drying, and try drying.
• dry white HA salt powder (34) obtained by drying the precipitate is used in cosmetic, medical and food applications.
• the hyaluronic acid producing microorganism are the strains belonging to the family of Streptococcus genus such as Streptococcus equi, Streptococcus zooepidemicus, and Streptococcus pyrogenes.
• the medium used for culturing Streptococcus zooepidemicus is Tryptone Soya broth or Brain Heart Infusion Broth.
• the culture grown in shake flasks also called as prefermentor in present invention is used for fermentation.
• the fermented broth is the broth obtained by culturing the HA producing strain. Fermentation is performed under aerobic conditions by providing aeration and agitation at a temperature of about 15°C to 45°C for about 15 hours to about 60 hours. The pH is maintained from about 4 to about 9 by using an inorganic or organic acid, alkaline solution and calcium hydroxide.
• the fermented broth contains cells, hyaluronic acid, dissolved salts, proteins, nucleic acids and other media components. Dilution of the fermented broth is performed by diluting the fermentation broth with suitable diluent that is added to the broth to reduce the viscosity of broth in order to facilitate centrifugation.
• Distilled water or RO water is used as a diluent for diluting the fermented broth in a ratio from about 1 :2 to about 1 :4.
• Separation of cells from diluted fermented broth is carried out by solid-liquid separation techniques such as centrifugation and filtration and the separated cell mass is subjected to sterilization or chemical treatment before being discarded.
• the diluted cell free fermented broth is subjected to Bentonite treatment for protein separation from the fermented broth.
• Bentonite is an adsorbent mainly composed of montmorillonite, that consists of two tetrahedral sheets (Si-O) separated by an octahedral sheet (Al-O-OH).
• Divalent Fe 2+ or Mg 2+ ions sometimes replace the tetravalent silicon in the tetrahedral sheet. This replacement, referred to as the isomorphous substitution, results in a net negative charge on the clay surfaces.
• the ions or groups with positive charge can be adsorbed onto the surface of bentonite owing to the interaction between the negative and positive charges.
• Bentonite is added to the cell free fermentation broth at a concentration of about 10 grams per liter to about 50 grams per liter.
• Bentonite is removed after adsorption of protein impurities from the fermentation broth by solid - liquid separation methods by using at least one of filtration, centrifugation and sedimentation processes.
• suitable liquid separation processes may be employed.
• activated carbon refers to charcoal or coal which is a form of carbon that has been processed to make it extremely porous to have a very large surface area for adsorption of color and nucleic acid impurities.
• concentration of added activated carbon is in a range of about 5 gram per liter to about 30 grams per liter.
• Activated carbon is removed from the fermentation broth by solid -liquid separation methods by using at least one of centrifugation, filtration (e.g., sparkler filter) and sedimentation processes. After separation of activated charcoal a clear and partially purified fermentation broth is obtained.
• Clear and partially purified broth is further subjected to cation exchange chromatography in order to separate metallic impurities from the fermentation broth.
• Strong or weak cationic resins can be used for the removal of metallic impurities from the fermentation broth.
• Cation resins are used at a concentration of about 10 grams per liter to about 100 grams per liter.
• Partially purified fermentation broth is further processed to form HA salts.
• An alkali or alkaline earth metal salts selected from the group consisting of sodium chloride, sodium sulfate, sodium acetate, potassium chloride and potassium acetate is added to the fermentation broth to form HA salts.
• the alkali or alkaline earth metal salts are added at a concentration of about 10 grams per liter to 100 grams per liter.
• HA salt precipitate herein refers to the precipitate formed by addition of organic solvent to HA salt.
• Organic solvents are selected from the group consisting of ethanol, propanol, isopropanol and acetone.
• Organic solvent is added to the broth in a ratio of about 1 :1 to about 1 :3 to obtain a precipitate of HA Salt.
• HA salt precipitate is separated by solid -liquid separation methods by using at least one of centrifugation, filtration and sedimentation processes.
• the residual organic solvent obtained after solid -liquid separation may contain water and other impurities, therefore organic solvent is separated by using at least one of distillation (single, multiple or vacuum), molecular sieve distillation, pervaporation and osmotic membrane distillation processes. Such processes are known in the art.
• the recovered solvent by distillation may be reused for HA salt precipitation step.
• the term 'Drying' refers to removal of traces of solvent and moisture from the HA precipitate obtained after solid- liquid separation.
• the drying of HA precipitate is carried out by using at least one of freeze dryer, vacuum dryer and tray dryer to form a white, dry powder of HA salt.
• FIG. 2 illustrates an exemplary system 50 for purification of Hyaluronic acid salt from the fermentation broth.
• HA containing fermnetation broth 52 received from a fermentation unit 54 is diluted in a broth holding tank 56 using distilled or RO water 58.
• an overhead stirrer (not shown) is employed to faciliatte the mixing during the dilution process.
• HA containing fermenation broth 60 from the broth holding tank 56 is subjected for microbial cell separation in a tubular bowl centrifuge 62.
• supernatant 64 is collected in a supernatant collection tank 66 and bentonite 68 is added to the supernatant 64 for separation of protein from the broth.
• bentonite is separated using a tubular bowl centrifuge 70 and the supernatant 72 is directed to a collection tank 74 where it is mixed with activated carbon 76 for separating color impurities and nucleic acids.
• carbon separation is performed using a sparkler filter 78 and filtrate 80 from the sparkler filter 78 is collected in a filtrate collection tank 82.
• metallic impurities are separated by passing filtrate 80 through a resin column 84 and resin treated broth 86 is collected in a broth collection tank 88.
• the resin treated broth 86 is then mixed with alkali or alkaline earth metal salt such as sodium salt 90 in a mixing unit 92 using an over head stirrer (not shown) and an organic solvent 94 is added to form a HA salt precipitate 96 in a precipitate settling tank 98.
• HA salt precipitate 96 is separated from organic solvent using a vacuum filtration assembly 100 and the precipitate 96 is collected in a precipitate collection unit 102.
• the HA salt precipitate 96 is then dried in a drying unit 104 using known drying techniques such as freeze drying, vacuum drying, and try drying.
• the downstream process for the purification of HA formed from a bacterial fermentation broth comprising diluting and clarifying the fermentation broth; adding bentonite to the clarified broth obtained after centrifugation and then removing the bentonite by centrifugation; treating the HA containing broth from previous step with an activated carbon followed by removing the charcoal by using pad filtration; removing the metallic impurities from the by passing through cation exchange resin bed; adding inorganic salts to the cation exchange resin treated solution; adding an organic solvent to the solution from the previous step to precipitate HA salt, and drying the precipitate.
• the Streptococcus zooepidemicus is grown in a media containing about 3% Tryptone Soya Broth and 1% of glucose for 24 h in a shaking incubator at 37°C and at 200 rpm.
• a medium containing 5% glucose, 2% Tryptone soya broth, about 1% yeast extract, 0.2% sodium chloride, 0.15% magnesium sulfate and 0.25% di-potassium hydrogen phosphate was prepared.
• Glucose was dissolved in another flask and sterilized. The other components were sterilized in a 30L fermenter, cooled to 37°C and then glucose was added to the fermenter.
• a 30L fermenter containing 13L of the medium is inoculated with 0.75 L of seed culture (step 1.1), and the fermentation is carried at 36°C to 38°C at 300 rpm until the concentration of glucose reaches less than 0.1%.
• the pH of the fermentation broth is maintained at 7.0, and air flow during the process is maintained at 1 VVM.
• the hyaluronic acid culture solution was diluted with distilled or RO water such that the concentration of hyaluronic acid becomes 0.1 to 0.15%.
• the bacteria cells were removed using high speed centrifuge (Pennwalt Ltd, Model No. AS 16).
• the clarified broth was treated in a batch mode, with a bentonite of about 10 grams per liter concentration for adsorption of protein. This step removes about 60% protein from the clarified broth.
• the bentonite is separated from the sodium hyaluronate solution by using continuous centrifuge at 16000 rpm at 26°C to 30°C.
• the clear filtrate obtained after activated carbon treatment is passed through the cation exchange resin bed column.
• the column is filled with about 9 liter of cation exchange resin (Inchon 225H).
• the flow rate of the filtrate through the bed was maintained at 300 ml/min at 26°C - 30°C.
• Hyaluronic acid salt is purified by the same method as in Example 2, except that the precipitation was carried using isopropyl alcohol. 5
• Example 4
• the table provided below shows examples of purification steps along with material specification for each of the step.
• the final product was tested for its protein, nucleic acid, appearance, pH, glucuronic acid content, molecular weight, IR spectra, chloride content, and moisture content.
• the tests mentioned above and the material specifications are as set forth as per European Pharmacopoeia, 6 edition, 2008 and results are tabulated below:

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• 2012
  • 2012-03-28 WO PCT/IN2012/000203 patent/WO2013132506A1/fr not_active Ceased

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