FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(Section 10 and Rule 13)

PROCESS FOR THE PURIFICATION OF HYALURONIC ACID SALTS (HA) FROM FERMENTATION BROTH

PRAJ INDUSTRIES LIMITED of Praj House, Bavdhan,
Pune – 411021, Maharashtra, India

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION

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 (C14 H20 N Na O11)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).

Methods are known for producing 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 (endotoxin), and the like. This has resulted in known methods for preliminarily removing impurities before obtaining hyaluronic acid salts, such as a method of adding CETAB (a mixed trimethylammonium bromide), cetyl pyridium chloride; a method of adding surfactants; a method of treating with anionic exchange resin; a method of treating with hydrophobic polymers and alumina; a method of use of ultrafiltration, redissolving precipitate and treating with alumina and silica gel; a method of adding formalin, surfactants and use of ultrafiltration and diafiltration; a method of adding EDTA, alumina to ultrafiltered broth; a method of redissolving the precipitate and use of diafiltration, and the like.

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. However, the removal of residual chemicals from the precipitate is complex and cost intensive. In addition, previously known purification procedures, such as ultrafiltration/diafitration processes are cost intensive and generate high volume of effluent during diafiltation step.

Therefore there exist a need for an efficient and inexpensive procedure to obtain high molecular weight hyaluronic acid suitable by microbiological fermentation, which gives high yields of a relatively uncontaminated high molecular weight hyaluronic acid and a recovery procedure that does not create unnecessary purification problems and does not adversely affect the molecular weight of the hyaluronic acid.

SUMMARY OF THE INVENTION

The following is a summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not intended to identify key or critical elements of the invention or to delineate the scope of the invention. The sole purpose of this section is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

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.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, become better understood with reference to the following description, appended claims, and accompanying drawings in which like characters represent like parts throughout the drawings.

Figure 1 represents the integrated process flow diagram for purification of Hyaluronic acid salt (hereinafter referred as ‘HA’) from the fermentation broth.

Figure 2 illustrates an exemplary system for purification of Hyaluronic acid salt from fermentation broth.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description and explanation of the present invention with some examples thereof. In order to better appreciate the invention, it is described with reference to the figures which illustrate various embodiments of the invention.

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 1:4. Further, HA containing fermentation broth is then subjected for microbial cell separation (16) by using techniques known in the art such as filtration and 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. After separation of metallic impurities, 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 such as freeze drying, vacuum drying, and try drying. In certain exemplary embodiments, 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 150C to 450C 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 Fe2+ or Mg2+ 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. However, other suitable liquid separation processes may be employed.

Partially purified fermentation broth after bentonite treatment is subjected to the separation of nucleic acid and color impurities using activated carbon. As used herein, the term “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. The 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.

As used herein, 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.

Figure 2 illustrates an exemplary system 50 for purification of Hyaluronic acid salt from the fermentation broth. As illlustrated, 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. In this embodiment, an overhead stirrer (not shown) is employed to faciliatte the mixing during the dilution process. Moreover, HA containing fermenation broth 60 from the broth holding tank 56 is subjected for microbial cell separation in a tubular bowl centrifuge 62. Further, 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. Subsequently, 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. Next, 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.

Moreover, 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. Subsequently, 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.

According to the present invention, the downstream process for the purification of HA formed from a bacterial fermentation broth is provided. The method 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 invention is described hereinafter, with reference to the following examples, which are illustrative only and should not be construed to the limit of the scope of present invention.

Example 1

Preparation of seed culture medium:
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 37ºC and at 200 rpm.

Preparation of fermentation medium:
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 37ºC and then glucose was added to the fermenter.

Main fermentation
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 36ºC to 38ºC 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.

Example 2

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.

To the bentonite treated clarified broth, about 30 grams per liter charcoal was added to the solution obtained from the bentonite treatment step. The resulting mixture was stirred for about 15 min to adsorb proteins, color impurities, nucleic acids, endotoxins etc. Further, pad filtration is used to obtain an aqueous solution of hyaluronic acid aqueous solution from which the carbon is completely removed.

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 (Indion 225H). The flow rate of the filtrate through the bed was maintained at 300 ml/min at 26C - 30C.

To the hyaluronic acid aqueous solution, about 15 grams per liter sodium acetate trihydrate is added and the solution is filtered using 0.2 m filter. Then, ethanol is added in a volume of 1.5 to 2.5 times that of the solution to precipitate hyaluronic acid and its salt, and precipitate obtained is washed with 70% ethanol and followed by 95% ethanol. The precipitate was dried to obtain high purity sodium hyaluronate.

Example 3

Hyaluronic acid salt is purified by the same method as in Example 2, except that the precipitation was carried using isopropyl alcohol.

Example 4

The process in example 1 was scaled up to 60 L. Recovery and purification of hyaluronic acid and its salt profile could be reproduced. The final product resulted in a product that had 0.06% protein with respect to HA and the recovery of HA was 99.3%.

Example 5:
The table provided below shows examples of purification steps along with material specification for each of the step.

S.No. Recovery/ purification step Volume
liter Dry Powder
gm/lit HA yield
gm/lit Protein
mg/lit Total HA gm Total protein mg % Protein w.r.t dry powder
1. Cell removal 10.0 6.0 3.90 39.9 39.0 399 6.5%
2. Bentonite treatment 9.0 5.9 3.98 17.4 35.8 157 2.6%
3. Activated carbon treatment 7.8 5.8 4.10 3.71 32.0 29 0.50%
4. Cation exchange resin treatment 7.8 4.12 4.08 0.26 31.8 2.0 0.05

Example 6:

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, 6th edition, 2008 and results are tabulated below:

S.No. Test Specification Result
1. Character A white to almost white Complies
2. Appearance of solution Solution clear, absorbance of solution measured at 600 nm is not greaten than 0.01 Solution clear; absorbance of solution measured at 600 nm is 0.008
3. IR spectrophotometry The spectrum of the test substance corresponds to the reference spectrum of sodium hyaluronate Complies
4. pH In the range of 5.0 – 8.5 6.8
5. Nucleic acids Absorbance at 260 nm does not exceed 0.5 0.052
6. Protein Not more than 0.3%. If intended for use in parenteral preparations, not more than 0.1% 0.06%
7. Loss on drying Not more than 20% by weight 8.5%
8. Assay Not less than 95.0% and not more than 105.0% of sodium hyaluronate calculated with reference to the dried substance. 99.31%
9. Chlorides 0.5% (max) Complies
10. Sodium To pass test Complies
11. Iron 80 ppm (max) Complies
12. Heavy metals 20 ppm (max) Complies
13. Total bacteria count ≤ 100 cfu/gm < 10 cfu/gm
14. Total fungal count ≤ 100 cfu/gm < 10 cfu/gm
Additional advantages and modification will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and illustrative examples shown and described herein. Accordingly various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.

We claim:

1. A process for purifying hyaluronic acid salt formed from fermentation broth, comprising:
diluting the fermentation broth obtained by culturing a microorganism capable of producing hyaluronic acid and to form a diluted fermentation broth;
separating the microbial cells from the diluted fermentation broth;
adding bentonite to the diluted fermentation broth for separating protein impurities from the diluted fermentation broth;
separating at least one of color impurities and nucleic acids from the diluted fermentation broth using activated carbon;
separating metallic impurities from the diluted fermentation broth by passing the fermentation broth through a cation exchange resin bed;,
adding an alkali or an alkaline earth metal salt to the fermentation broth to form hyaluronic acid salt;
precipitating the hyaluronic acid salt by addition of an organic solvent;
separating hyaluronic salt precipitate from the organic solvent; and
drying the hyaluronic acid salt to obtained dry salt.

2. A process as claimed in claim 1, in wherein the microorganism capable of producing hyaluronic acid comprises a microorganism belonging to the genus Streptococcus.

3. A process as claimed in claim 2, wherein the microorganism is selected from a group consisting of Streptococcus zooepidemicus, Streptococcus equi and Streptococcus pyogenes.

4. A process as claimed in claim 3, wherein the microorganism comprises Streptococcus zooepidemicus.

5. A process as claimed in claim 1, wherein fermentation broth is diluted with water in a ratio of about 1:2 to about 1:4.

6. A process as claimed in claim 1, wherein the microbial cells are separated from the diluted fermentation broth by using at least one of filtration and centrifugation processes.

7. A process as claimed in claim 1, wherein a concentration of added bentonite is about 10 grams per liter to about 50 grams per liter.

8. A process as claimed in claim 1, wherein a concentration of activated carbon added to the diluted fermentation broth is about 10 grams per liter to about 30 grams per liter.

9. A process as claimed in claim 1, wherein a concentration of cation exchange resin used for separating the metallic impurities is about 10 grams per liter to about 100 grams per liter of activated carbon treated broth.

10. A process as claimed in claim 1, wherein the alkali or alkaline earth metal salts are selected from the group consisting of sodium chloride, sodium sulfate, sodium acetate, potassium chloride, and potassium acetate.

11. A process as claimed in claim 10, wherein a concentration of the alkali or alkaline salts is about 10 grams per liter to about 100 gram per liter.

12. A process as claimed in claim 1, wherein the organic solvent is selected from the group consisting of acetone, methanol, ethanol, propanol, isopropyl alcohol and acetonitrile.

13. A process as claimed in claim 12, wherein the organic acid is added at a ratio of about 1:1 to 1:3.

14. A process as claimed in claim 1, wherein the solvent obtained after precipitate filtration is further distilled and recovered for reuse.

15. A process as claimed in claim 1, wherein the HA salt precipitate is dried using at least one of a freeze dryer, a vacuum dryer and a tray dryer.

16. A system for purifying hyaluronic acid salt formed from fermentation broth, the system comprising:
a broth holding tank configured to receive fermentation broth and to dilute the fermentation broth to form a diluted fermentation broth;
a tubular bowl centrifuge configured to separate microbial cells from the diluted fermentation broth;
a supernatant collection tank configured to introduce bentonite to the diluted fermentation broth and to separate protein impurities from the diluted fermentation broth;
a collection tank configured to introduce activated carbon to the diluted broth and to separate at least one of color impurities and nucleic acids from the diluted fermentation broth;
a resin column configured to separate metallic impurities from the diluted fermentation broth;
a mixing unit to add an alkali or an alkaline earth metal salt to the fermentation broth to form hyaluronic acid salt;
a precipitate settling tank configured to add an organic solvent to precipitate the hyaluronic acid and to form hyaluronic salt precipitate;
a vacuum filtration assembly configured to separate hyaluronic salt precipitate from the organic solvent; and
a drying unit configured to dry the hyaluronic acid salt to obtained dry salt.

17. The system as claimed in claim 16, further comprising a tubular bowl centrifuge to separate the bentonite after separation of the protein impurities from the diluted fermentation broth.

18. The system as claimed in claim 16, further comprising a sparkler filter to separate the activated carbon after separation of the color impurities and the nucleic acid from the diluted fermentation broth.

19. The system as claimed in claim 16, wherein the vacuum filtration assembly comprises at least one of a filter paper, a buchner funnel, a vacuum flask and a vacuum pump.

20. The system as claimed in claim 16, wherein the drying unit comprises a vacuum oven.