FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION
(SECTION 10)
"A PROCESS FOR THE PREPARATION AND PURIFICATION OF
SODIUM HYALURONATE OF PHARMACEUTICAL GRADE"
UNICHEM LABORATORIES LIMITED,
A COMPANY REGISTERED UNDER THE INDIAN COMPANIES ACT, 1956,
HAVING ITS REGISTERED OFFICE LOCATED AT UNICHEM BHAVAN,
PRABHAT ESTATE, OFF S. V. ROAD, JOGESHWARI (WEST), MUMBAI -
400 102, MAHARASTRA, INDIA.
The following specification describes the invention and the manner in which it is to be performed.

A PROCESS FOR THE PREPARATION AND PURIFICATION OF SODIUM HYALURONATE OF PHARMACEUTICAL GRADE
FIELD OF THE INVENTION:
The present invention relates to a process to obtain purified sodium hyaluronate of pharmaceutical grade by bacterial fermentation which is useful for pharmaceutical and biomedical applications.
BACKGROUND OF THE INVENTION:
Hyaluronic acid, whose structure is depicted in Figure 1, is a high molecular weight polyanionic, glycosaminoglycan. It is a linear polymer of repeating disaccharide units of D-Glucuronic acid and N-Acetyl Glucosamine linked via (1→3) glycosidic bonds. Within each disaccharide unit, D-Glucuronic acid is linked to N-Acetyl Glucosamine by (1→4) glycosidic bond.
The synthesis of Hyaluronic acid as depicted in Figure 2 (Marcovitz et al, 1959) is carried out enzymatically by hyaluronate synthase using the precursors UDP-Glucuronic acid and UDP-N Acetyl Glucosamine.
Structurally, the molecule exists as a twisted ribbon in solution, exhibiting a highly hydrophilic nature and exhibits unusual rheological behavior at low and high concentrations in solution. This coil structure is responsible for binding high amount of water (Necas, J. et al, 2008). Its viscoelastic property is attributed to the high molecular weight along with the extended polymer conformation and rigidity in solution.
Hyaluronic acid occurs naturally in body tissues like the vitreous humor of the eye, cartilage, umbilical cord, roosters comb and synovial fluid. In the body. Hyaluronic

acid occurs as hyaluronate salt. The primary structure of Hyaluronic acid is the same; rather the extent of polymerization of the chain defines its applicability in different tissues.
Hyaluronic acid is a versatile molecule having properties like high viscoelasticity, high water retention capacity and high biocompatibility. Additionally it is non-immunogenic and non-toxic. Therefore, it has widespread application in biomedicals, cosmetics and nutraceuticals (Chong et al, 2005). The major criterion that governs its use in either of the field is its molecular weight (Armstrong, G.C. et al, 1997). Hyaluronic acid obtained from rooster combs and umbilical cords have molecular weight in the range of 106 to 107 KDa. Hyaluronic acid obtained from the humor of the eye has molecular weight in the range of 104 to 105 KDa.
Balaz et al, 2004, has categorized the biomedial applications as follows; Viscosurgery, Viscoaugmentation, Viscoseparation, Viscosupplementation and viscoprotection. Some commercially available products of sodium hyaluronate are Synvisc, Hyalgan (Fidia), Healon.
Balaz et al, 1979 has described the extraction and purification of high molecular weight Hyaluronic acid from the roosters comb. The rooster comb-based extraction process is facing a growing concern over the use of animal-derived components in biomedical and pharmaceutical applications. Hence, microbial fermentation has emerged as a new alternative for hyaluronic acid production. (Liu, L., 2011)
Hyaluronic acid is produced by Streptococcus sp belonging to Lancefield A and C group. The extracellular capsule produced by the bacteria consists of Hyaluronic acid. More recently, Hyaluronic acid is also produced by Bacillus subtilis (Widner, B. et al, 2005), Agrobacterium sp (Mao Z et al, 2007) and E.coli (Yu, H.M., 2008) using recombinant DNA technology.

Most Streptococci species produce potential toxins, which is of major concern in the biomedical field. A non β-hemolytic strain, Streptococcus equi subsp zooepidemicus ATCC 39920 is now widely used for the production of Hyaluronic acid.
Streptococcal cultures are facultative anaerobes. It has been reported that high yields of Hyaluronic acid require aerobic conditions. Hence, the fact remains that both the growth and production are inversely related. It is reported by Aroskar et al (IIOAB, 2012 Letters, 2:7-15) that Hyaluronic acid production by the said culture is dependent upon different physical parameters like temperature, aeration, fermentation run period and pH. The initial conditions for cultivation of the culture are reported to have a noteworthy effect on the production of Hyaluronic acid, which includes inoculum growth and transfer parameters. Likewise, the medium components also influence the yield and molecular weight of Hyaluronic acid. Most inventors have specified the use of glucose, sucrose and lactose as the source of energy for growth and as monomer required for the synthesis of Hyaluronic acid. Maintaining a balance between optimum growth and production conditions is difficult and critical.
Yvas et al (EP 2, 216,412) describe a method wherein Streptococcus zooepidemicus is cultured in sucrose containing media. The broth is diluted 1:1 and centrifuged to recover hyaluronic acid in the supernatant. Precipitation with isopropanol is carried out and 3% sodium acetate is used for re-dissolution of the precipitate. Removal of impurities is carried out by the use of silica gel and filtration using 0.45u charcoal adsorbed filter. Finally, a diafiltration step is carried out, wherein the solution is aseptically filtered and lyophiiized to obtain purified hyaluronic acid powder. The reported yields are about 5-6 g/L. The said process involves steps like centrifugation which may not be preferred as a scalable step. The fermentation period in the process is quite long i.e. 24hrs. Silica gel and activated charcoal is used for protein and color removal, respectively. The process generates huge volumes of hyaluronic acid solution for lyophilization. These steps

are not easy to handle on large scale. Hence, these steps and the process as a whole take longer durations for completion and to achieve the end product which may not be conforming to pharmacopoeial requirements.
Nimrod et al (US 4,780,414) teaches a process to produce hyaluronic acid using a mutant of S. zooepidemicus, with glucose as the carbon source. The broth is pre-filtered or centrifuged to achieve cell separation. Hyaluronic acid is precipitated from the solution using ethanol. The precipitate is re-dissolved in 0.15M NaCl and treated with activated charcoal. The subsequent steps involve precipitants like cetylpyridinium chloride and treatment with adsorbent resin Florisil to obtain purified Hyaluronic acid solution which is air dried with a stream of sterile nitrogen. Repeated steps of alcohol precipitation each for about 10 minutes are performed for purification of hyaluronic acid. The process uses huge volumes of alcohol. The process takes long time to complete and adds to cost of production, besides risk of carry over of traces of cetylpyridinium chloride.
Similarly, Ellwood et al (US 5,411,874) discloses a process using Streptococcus equi, wherein continuous fermentation is carried out and the broth generated is treated with 0.025% SDS and 1% formalin. Purification steps involve the use of cetylpyridinium chloride for nucleic acid precipitation followed by steps involving hyaluronic acid precipitation with isopropanol. There is a possibility that cetylpyridinium chloride may form a complex with hyaluronic acid and may be retained in the final product. The process uses formalin. Human Exposure to formalin is harmful. There is high chance that formalin remains in hyaluronic acid solution during process, which is not suitable for biomedical purpose. The process uses repeated depth fdtration steps for removal of cells which is cumbersome technology, difficult to scale up besides being costly.
Carlino et al (US 6,489,467) describes a process wherein cells along with hyaluronic acid at pH of 2.5 are diafiltered using three 0.2u filter cartridges stacked together so as to remove the impurities, followed by adjustment of the pH

to 4.0 and centrifugation of the broth. The pH of the supernatant is then adjusted to 7.0 and filter sterilized using 0.2μ filtration. Use of very low pH below 4.0 has reported to bring about the degradation of hyaluronic acid. Besides, the diaftltration of cells using 0.2μ filter creates problems of rapid fouling of the membranes and would demand stringent regeneration procedures. The process uses 0.45 μ pore size for clarification resulting into less flux, more duration and requires larger filter area.
Cazzola, F. et al (EP 0,716,688) disclose a process for purification of hyaluronic acid produced by S. equi wherein the broth obtained after fermentation is treated with formalin and a detergent like Sodium dodecyl sulfate or Tween 60 to facilitate the release of capsular hyaluronic acid. The cells are then separated using a prefilter in the range of 0.1-1Oμ.. This filtrate is subjected to 0.45μ microflltration. An ultra filtration step is used to remove the bactericide and detergents used. The limit specified for protein is <1.5%, which does not satisfy the European Pharmacopoeia (EP) specification for a biomedical grade product. Method used for cell separation and clarification involves two steps. Pre-filtration with 0.1-10μ filter followed by micro filtration followed ultra filtration. The process takes more time. The process uses formalin. Human Exposure to formalin is harmful. There is high chance that formalin remains in hyaluronic acid solution during process, which is not suitable for biomedical purpose.
Prior art is full of some inappropriate practices such as the use of quaternary salts, cetylpyridinium chloride (Nimrod et al US 4,780,414; Ellwood et al US 5,411,874; US 4, 517, 295) or use of formalin, which may be retained in Hyaluronic acid salt. Prior art mainly teaches use of activated charcoal and alumina for removal of remnant color in the Hyaluronic acid solution (Han et al US 7,575,914; US 4,784,990). US 4,782,046 teaches repeated steps of solvent precipitations for removal of color and other impurities Inconveniently longer durations, difficulties of scalability of processes continue to be the problems to be resolved. Prior art teaches Cell separation as a separate step before purification step. Process to

produce Hyaluronic acid of pharmacopoeial grade provides an immense scope for improvement.
Therefore there is urgent need to provide a simpler, improved, commercially viable process that is devoid of procedures that would use resins or charcoal for color removal or is devoid of use of chemicals such as formalin, cetylpyridinium chloride. The process described in present invention does not employ centrifugation or prefiltration for cell separation. Similarly, the present invention has Lyophilization as the final step for producing sodium hyaluronate powder, with significantly less amount of the volume generated for lyophilization. It was surprisingly noticed that use of significant quantity of starch as a carbon source in the fermentation media does not impact the yield of high molecular weight hyaluronic acid.
OBJECT OF THE INVENTION:
The present invention relates to a simpler and an improved process which is suitably scalable for industrial purpose to produce high molecular weight hyaluronic acid of pharmaceutical grade conforming to pharmacopoeial criteria and to European Pharmacopoeia with high yields.
The main object of the invention is to provide a simpler and an improved process to produce high molecular weight hyaluronic acid of pharmaceutical grade conforming to pharmacopoeial criteria and to European Pharmacopoeia with high yields.
Another object of the present invention is to provide an improved process to carry out simultaneous cell separation and clarification using cross flow filtration employing 0.65u open channeled microfiltration membrane.

Yet another object of the present invention is to provide an improved scalable process to remove proteins and other impurities using SP207 adsorption resin.
Yet another object of the present invention is to provide an improved process for color removal using diafiltration using 50KDa ultra filtration membrane.
Yet another object of the present invention is to provide a simpler process to produce high molecular weight hyaluronic acid of pharmaceutical grade conforming to pharmacopoeia! criteria and to European Pharmacopoeia with high yields using polysaccharide such as soluble starch as carbon source with or without monosaccharide or disaccharide.
SUMMARY OF THE INVENTION
The present invention relates to a simpler and an improved process which is suitably scalable for industrial purpose to produce high molecular weight hyaluronic acid of pharmaceutical grade conforming to pharmacopoeial criteria and to European Pharmacopoeia with high yields.
In one aspect the present invention describes a process for the preparation of hyaluronic acid by bacterial fermentation comprising:
a. culturing Streptococcus equi subsp zooepidemicus ATCC 39920 in a seed
medium;
b. producing hyaluronic acid by Streptococcus equi subsp zooepidemicus ATCC
39920 in presence of carbon source;
wherein carbon source comprising of a polysaccharide optionally in combination with a monosaccharide or a disaccharide.
In one more aspect the invention describes a process for the preparation of hyaluronic acid by bacterial fermentation comprising culturing Streptococcus equi subsp zooepidemicus ATCC 39920 in a seed medium comprising 1% yeast extract,

1% casein hydrolysate, 1.5% K2HPO4 and glucose followed by aerobic fermentation in presence of carbon source with 1-2 vvm aeration for 12- 14hrs in a suitable media at an agitation of 900-1 OOOrpm, at pH 6.8 to 7.2, at temperature of 33°C to 40°C and dissolved oxygen is controlled at 50-60%, wherein:
a) carbon source is comprising of a polysaccharide optionally in combination with a monosaccharide or a disaccharide;
b) culture broth has viscosity in the range of 300O-4500cP;
c) the process yields at least 5.5g/L of Hyaluronic acid having average molecular weight of 3000-4500 KDa.
In another aspect the present invention describes a process for the purification of hyaluronic acid in solution as a salt, comprising the steps:
a. Clarifying the acidified broth by 0.65μ open channel microfiltration
membrane to give clarified cell free broth preferably by carrying out
microfiltration at constant permeate flow;
b. Ultra filtering the clarified cell free broth obtained in step a) using ultra
filtration membrane upto 500KDa MWCO, more preferably 50KDa MWCO at
neutral pH with continuous diafiltration to remove colorants and to obtain
hyaluronic acid retained solution;
c. Adding sodium salt solution in to the retained hyaluronic acid solution
obtained in step b) and treating with an aromatic adsorbent resin selected from
HP10, HP20, HP21, HP30, SP800, SP205, SP206, SP207, more preferably
SP207 to remove proteins, endotoxins and nucleic acids and to obtain purified
sodium hyaluronate solution;
d. Precipitating the sodium hyaluronate from purified sodium hyaluronate
solution obtained in step c) with a solvent, re-dissolving the precipitate in a
sodium salt solution and then filtering to obtain a clear sterile sodium
hyaluronate solution;
e. Re-precipitating the sterile sodium hyaluronate from sterile sodium hyaluronate
solution obtained in d) with a solvent, dissolving the precipitate in pyrogen free
water preferably cooled water and then lyophilizing it to obtain final powder.

The process uses minimum resources primarily solvent used for precipitation. In accordance with the present invention, the process does not necessitate a particular ceil separation step before clarification and purification of hyaluronic acid but deals in a single step of cross flow filtration using 0.65μ open channel microfiltration membrane, thereby significantly minimizing process time. Subsequently, performing continuous diafiltration singly undertakes color removal and partial purification of hyaluronic acid solution. Additional step of color removal is not required. The process as described in present invention has significantly reduced the handling of volume for further purification steps of hyaluronic acid.
BRIEF DESCRIPTION OF DRAWINGS:
The illustrations provided are for the better understanding of the process described
herein.
Figure 1: Repeating units of D-Glucuronic acid and N-Acetyl Glucosamine.
Figure 2: Biosynthesis of Hyaluronic acid in Streptococcus.
Figure 3: Process of present invention for the production of sodium hyaluronate of
Pharmaceutical grade,
DETAILED DESCRIPTION:
The present invention describes a simpler and an improved process which is suitably scalable for industrial purpose to produce high molecular weight hyaluronic acid of pharmaceutical grade conforming to pharmacopoeia] criteria and to European Pharmacopoeia with high yields.
The term "High yields'" as used in the invention is meant to cover consistent yields of 5g/L or above.

The term "simpler" and/or "improved process" as used in the invention is meant to cover a process wherein cell separation and clarification of diluted culture broth is carried out in unison. It is also means a process in which a polysaccharide is used as carbon source optionally with a mono or disaccharide. "Shorter fermentation run times" is another interpretation of simpler and or improved process as described herein. Simpler and/or improved process is also to be interpreted as a process wherein cross flow filtration membrane as a system is used for cell separation, clarification and partial purification of hyaluronic acid. Shorter run times are to be interpreted as run times of about 12 hrs to 14 hrs as described in this invention.
The term "minimum amount of pyrogen free water" as used in the invention is meant to cover dissolving Ig of precipitate (wet weight) in 30-70ml of pyrogen free water.
The term cooled water as used in this invention is meant to cover pyrogen free water having a temperature in the range of 10-20°C.
The term "purification" as used in the invention is meant to cover a process to separate nucleic acids, endotoxins, coloring matter, protein impurities and other unrequired materials from the hyaluronic acid solution.
The term "total recovery" as used in the invention is meant to cover the quantity of hyaluronic acid recovered at the end of the process with respect to the amount begun within the broth and is given in %.
In an embodiment the present invention provides a process for the preparation of hyaluronic acid by bacterial fermentation comprising:
a. culturing Streptococcus equi subsp zooepidemicus ATCC 39920 in a seed
medium;
b. producing hyaluronic acid by Streptococcus equi subsp zooepidemicus ATCC
39920 in presence of carbon source;

wherein carbon source comprising of a polysaccharide optionally in combination with a monosaccharide or a disaccharide.
In yet another embodiment the present invention provides the process for the preparation of hyaluronic acid by bacterial fermentation comprising culturing Streptococcus equi subsp zooepidemicus ATCC 39920 in a seed medium comprising 1% yeast extract, 1% casein hydrolysate, 1.5% K2HPO4 and glucose followed by aerobic fermentation in presence of carbon source with 1-2 vvm aeration for 12- 14hrs in a suitable media at an agitation of 900-1 OOOrpm, at pH 6.8 to 7.2, at temperature of 33°C to 40°C and dissolved oxygen is controlled at 50-60%, wherein:
a. carbon source is comprising of a polysaccharide optionally in combination
with a monosaccharide or a disaccharide;
b. culture broth has viscosity in the range of 3000 - 4500cP;
c. the process yields at least 5.5g/L of Hyaluronic acid having average molecular
weight of 3000- 4500 KDa.
In another embodiment, the pH of the seed was selected at 6.6-7.0, when the transfer was to be carried out into the fermentation media.
In yet another embodiment, the present invention describes the production of high molecular weight sodium hyaluronate. Fermentation is preferably carried out at 33-40°C most preferred temperature being 37°C. The cultivation of seed for the fermentation was carried out, wherein the culture is grown for at least 8-10 hrs in a medium suitable for its growth.
EP 221 641 2 teaches production of hyaluronic acid having molecular weight of 200-400KDa using glucose as carbon source whereas with sucrose the molecular weight increased to ~800KDa. In the present invention, it was surprisingly found that use of soluble starch optionally in combination with monosaccharide or

disaccharide further increases the viscosity leading to better hyaluronic acid yield of at least 5.5g/L preferably between 5.8-6.3g/L.
Varying the concentration and type of carbon source of the fermentation medium greatly influenced the viscosity of the broth.
In yet another embodiment, the present invention provides, the process of producing hyaluronic acid by Streptococcus equi subsp zooepidemicus ATCC 39920 in presence of carbon source, wherein carbon source comprising of a polysaccharide optionally in combination with a monosaccharide or a disaccharide which surprisingly increased broth viscosity containing high molecular weight hyaluronic acid with high yields of 5.8-6.3g/L.
Polysaccharide used according to present invention is selected from raw starch, soluble starch, white dextrin, maltodextrin or mixtures thereof.
Monosaccharide used according to present invention is selected from glucose, fructose or mixtures thereof.
Disaccharide used according to present invention is selected from sucrose, lactose, maltose or mixtures thereof.
Combination of carbon source used according to present invention is selected from soluble starch and sucrose or soluble starch and glucose, more preferably soluble starch and sucrose.
In yet another embodiment, concentration of polysaccharide used according to present invention is in the range of 2%w/v to 3%w/v, more preferably 2.5% w/v.
In yet another embodiment, concentration of monosaccharide used according to present invention is up to 5%w/v, more preferably 2.5% w/v.

In yet another embodiment, concentration of disaccharides used according to present invention is up to 5%w/v, more preferably 2.5% w/v.
In yet another embodiment, the present invention provides a scheme for the purification of the sodium hyaluronate. Novelty and the inventive step reside in the process, wherein cell separation and clarification of diluted culture broth is carried out in unison. Inventive ingenuity also resides in concentration and subsequent continuous diafiltration of the hyaluronic acid solution with at least 10 volumes of pyrogen free water to remove colorants and other impurities, besides reducing the handling volume.
Additionally, the present invention includes 0.2u filter sterilization of sodium hyaluronate solution. Still further, the solution is re-precipitated in the said hydrophilic solvent, and re-dissolved suitably in a minimum volume of solvent. followed by lyophilization of the purified solution of sodium hyaluronate to obtain final powder.
In another aspect the present invention describes a process for the purification of hyaluronic acid in solution as a salt, comprising the steps:
a. Clarifying the acidified broth by 0.65u open channel microfiltration
membrane to give clarified cell free broth preferably by carrying out
microfiltration at constant permeate flow;
b. Ultra filtering the clarified cell free broth obtained in step a) using ultra
filtration membrane upto 500KDa MWCO, more preferably 50KDa MWCO at
neutral pH with continuous diaflltration to remove colorants and to obtain
hyaluronic acid retained solution;
c. Adding sodium salt solution in to the retained hyaluronic acid solution
obtained in step b) and treating with an aromatic adsorbent resin selected from
HP 10, HP20, HP21, HP30, SP800, SP205, SP206, SP207, more preferably

SP207 to remove proteins, endotoxins and nucleic acids and to obtain purified sodium hyaiuronate solution;
d. Precipitating the sodium hyaiuronate from purified sodium hyaiuronate
solution obtained in step c) with a solvent, re-dissolving the precipitate in a
sodium salt solution and then filtering to obtain a clear sterile sodium
hyaiuronate solution;
e. Re-precipitating the sterile sodium hyaiuronate from sterile sodium hyaiuronate
solution obtained in d) with a solvent, dissolving the precipitate in pyrogen free
water preferably cooled water and then lyophilizing it to obtain final powder.
Conditions for culturing of hyaluronic acid as disclosed herein imply the production of a highly viscous culture broth with high hyaluronic acid yields. Viscosity is a parameter associated with the molecular weight of hyaluronic acid. Increased viscosity of hyaluronic acid in the broth relates to an increase in molecular weight therein.
Mark-Houwink gives the relation of viscosity with respect to the molecular weight (when measured in a particular solvent system), where it can be conclusively said that an increase in viscosity directly influences the increase in molecular weight
[η]=KMa
where [Η] is intrinsic viscosity, M is the molecular weight, K and a are Mark-Houwink's constants.
In yet another embodiment cross flow filtration according to present invention uses 0.65μ open channel microfiltration membrane and 50KDa MWCO ultra-filtration membrane which reduces the handling volume, aids color removal and provides cell separation while simultaneously carrying out clarification of the diluted broth. Use of 0.65u open channeled microfiltration membrane and 50KDa membrane is not only novelty but also an inventive step of the present invention.

Novelty and inventive step resides in conducting cross flow filtration as described herein using 0.65μ membrane and not 0.2μ or 0.45μ as taught by prior art (Carlino et al US 6,489,467).
Subsequent concentration of the clarified broth on a 50KDa Molecular weight cut . off (MWCO) membrane (Millipore, Pellicon) followed by continuous mode diafiltration with pyrogen free water is beneficial, in that, the handling volume is reduced and colorants and other impurities are washed off, rendering the solution partially purified. The feed solution applied to the membrane is filtered to obtain a permeate and a retentate, such that the membrane with a particular cutoff, will allow only the low molecular weight substances to pass through it and will retain the rest, therefore allowing those in the art to select the retention of a particular range of molecules.
In yet another embodiment, concentration of the adsorbent resin used according to present invention is in the range of 5-20% w/v, preferably 15% w/v.
In yet another embodiment, present invention provides a process for removal of proteins and other impurities by resin treatment with an aromatic adsorbent.
Resin used according to present invention is selected from HP10, HP20, HP21, HP30, SP800, SP205, SP206, SP207 more preferably SP207. Process as described in the present invention eliminates use of silica gel or alumina.
Addition of sodium chloride to the hyaluronic acid solution before addition of resin helps in adsorption of impurities. The concentration of sodium chloride changes the manner in which the precipitate is formed.
In yet another embodiment, the sodium salt is selected from sodium acetate and sodium chloride more preferably sodium chloride which produces sodium

hyaluronate that complies with European Pharmacopoeia with respect to Infra-Red spectroscopy.
In yet another embodiment, concentration of sodium salt used before resin treatment according to present invention is from l%w/v - 6%w/v, preferably 3%w/v to 6%w/v and more preferably 5%w/v-6%w/v.
In yet another embodiment the concentration of sodium salt used before re-precipitating sodium hyaluronate according to present invention is from l%w/v -6%w/v, preferably 1 % w/v to 5% w/v and more preferably 1.0%w/v - 2%w/v.
Solvent used for precipitation and reprecipitation of sodium hyaluronate according to present invention is selected from methanol, isopropanol, acetonitrile, ethanol, acetone or mixture thereof more preferably acetone in equal quantity. This is strikingly different from prior art where in larger amount of solvent volumes are used to precipitate sodium hyaluronate. The precipitate is separated by either centrifugation or by allowing the precipitate to settle and then siphoning off the supernatant.
The sodium hyaluronate solution is filter sterilized using 0.2u membrane filtration to obtain a solution of high purity and clarity which is then re-precipitated with solvent, the precipitate is re-dissolved in pyrogen free water and then lyophilized to obtain a powder as per European Pharmacopoeia specification.
The foregoing description of the invention has been presented for purposes of illustration and explanation and is not intended to be exhaustive or in no manner limits the invention to the precise form disclosed and obviously many modifications and variations are possible in the light of the given details by those skilled in the art.

Examples and descriptions provided herein are disclosed for reference and include description of hyaluronic acid production and purification.
EXAMPLE 1: Seed preparation/ Culturing
The growth of S. equi subsp. zooepidemicus (ATCC 39920) was carried out in media comprising of 1% yeast extract 1% casein enzyme hydrolysate, 1.5% K2HPO4, 0.15% NaCl, 0.04% MgSO4. The carbon source used was glucose. The medium suitably provided pH stability. The medium provided optimal growth, with optical density of 5.3-5.7 when measured at 600nm on a spectrophotometer.
EXAMPLE 2: Effect of carbon source on hyaluronic acid yield and viscosity:
The production of Hyaluronic acid by S. equi subsp. zooepidemicus (ATCC 39920) was carried out in media comprising of 1% yeast extract, 1% casein enzyme hydrolysate, 0.15% NaCl, 0.2% K2HPO4, 0.04% MgSO4. A combination of 2.5% sucrose and 2.5% starch was used as the carbon source. Fermentation at 3L scale was carried out at 37°C for 14hrs. Viscosity of about 3000-4500cP on Brookfield viscometer was observed.
EXAMPLE 3: Colorimetric analysis of hyaluronic acid
Analysis of hyaluronic acid in broth samples as well as purification stage samples was routinely carried out by a modified assay using m-hydroxydiphenyl in place of carbazole (Filisetti-Cozzi, T.M., Carpita, N.C., Measurement of Uronic acids without interference from neutral sugars, Analytical Biochemistry, 1991, 197(l):157-62)
EXAMPLE 4: Clarification by microfiltration
The step describes a simpler way of carrying out cell separation and simultaneous clarification of the culture broth. Dilution of the broth was carried out with 0.15M NaCl and 0.02% sodium dodecyl sulfate to a final concentration. pH was maintained in the range of 4.0 to 5.5, preferably 4.0. Addition of minimum amount of detergent, aids in removing hyaluronic acid attached to the bacterial cell.

The diluted broth was stirred for at least lhr. Microfiltration using 0.65u. open channeled microfiltration membrane (Millipore, Prostak module: PSGVAG021) was carried out by cross flow filtration. A constant permeate flow was maintained. The retentate was diafiltered in continuous mode for maximum recovery of the hyaluronic acid in the permeate. More than 90% hyaluronic acid was recovered in the permeate.
EXAMPLE 5: Ultra filtration of hyaluronic acid solution
Partially clarified hyaluronic acid solution is concentrated at least 7 times on 50KDa membrane (Millipore, Pellicon cassette), to reduce the handling volume. Peristaltic pump at a flow rate of 300ml/min to 500ml/min was used to feed the solution. The present invention prefers the use of a 50KDa membrane; maximum feed pressure being 15 psig and no pressure should be maintained on the retentate side.
The retentate is then diafiltered in continuous mode at the said parameters, giving at least 10 washes with cooled, pyrogen free water. The retentate and permeate are collected separately. More than 80% to about 90% hyaluronic acid is recovered. Consequently, a more purified solution of hyaluronic acid is recovered. The said process step primarily removes colorants and impurities and reduces the volume of hyaluronic acid solution and further handling becomes simpler and easy.
EXAMPLE 6: Treatment with aromatic adsorbent resin
The process described herein employs aromatic adsorbent resins like SP207 and HP20, preferably SP207 (Sepabeads, Mitsubishi Chemicals), to remove impurities from the solution. Resins as stated above, have a higher mesh size with a minimum diameter of 250μ, are highly hydrophobic, and have a high settling density. Resins are used in the concentrations of range of 5-20%, preferably 15% by weight.

The present invention uses at least 1.0-6.0% salt solution, preferably 5.8% during resin treatment of salts like sodium acetate and sodium chloride, preferably sodium chloride. pH is varied between pH 4.0 to 8.5, preferable being neutral pH. Temperature is maintained between 4 C to 30 C, preferably 28 C.
Sodium hyaluronate solution is stirred with the resin at a concentration of 5-20% by weight, preferable being 15%. The residence/adsorption time for the resin during stirring is between 3-12hrs, the least being 6hrs at 200rpm.
Resin is allowed to settle, and the solution poured out to separate the resin. Any fine remnants of the resin can be removed by filtration with a glass filter of 1.5u. 75-80% of the proteins are removed in this step.
EXAMPLE 7: Precipitation of Sodium hyaluronate
The process describes the use of solvents like isopropanol, ethanol, methanol and acetone for recovery of sodium hyaluronate from solution. The process herein demonstrates the use of equal volume of acetone for precipitation of sodium hyaluronate followed by centrifugation at 6000rpm for lOmin at 15°C to obtain the pellet. Alternatively, the precipitate can be allowed to settle down for at least 20-30min, followed by siphoning off the supernatant. The residual solution can then be centrifuged at 6000rpm for lOmin at 15 C, thereby reducing handling volume and time. Pellet can then be re-dissolved in 0.9% to 3%, of salt solution.
EXAMPLE 8: 0.2μ filtration of hyaluronic acid solution
Filter sterilization of sodium hyaluronate can be carried out after complete re-dissolution. 0.2μ filtration is carried out under sterile conditions by membrane filtration under vacuum. The final solution obtained is clear visually and has quality suitable for a pharmaceutical grade product. The filtrate is re-precipitated with the said solvent in the same ratio to obtain a pellet which is re-dissolved in minimum amount of pyrogen free water before Lyophilization.

EXAMPLE 9: High molecular weight hyaluronic acid -production and purification parameters
With carbon source as 2.5% soluble starch and 2.5% sucrose, and other medium components as 1% yeast extract, 1% casein enzyme hydrolysate, 0.15% NaCl, 0.2% K2HPO4, 0.04% MgSO4 growth and production of Streptococcus equi subsp zooepidemicus ATCC 39920 was carried out at 3L scale, with batch mode fermentation for 14hrs, pH 7.0 and dissolved oxygen concentration of 50-60%. A yield of 6mg/ml is achieved by the end of the fermentation run. Dilution of the broth is carried out wherein 10 volumes of pyrogen free water are added to the broth along with 0.15M NaCl and 0.02% SDS at pH 4.0. Microfiltration of the diluted broth was carried out using a 0.65u open channeled membrane, with a permeate pressure generated by a constant permeate flow. The retentate was left in open circulation at all times. More than 90% hyaluronic acid is recovered in the permeate, which is concentrated 7 times on a 50KDa MWCO membrane at a feed flow of 400ml/min, with the retentate in open circulation. The retentate is then diafiltered in continuous mode at the said parameters, giving at least 10 washes with cooled, pyrogen free water.
The recovered hyaluronic acid solution is stirred with an aromatic adsorbent resin, SP207 (Sepabeads), in the presence of 1-6% NaCl at neutral pH for at least 6hrs. Resin separation is carried out by allowing the resin to settle and filtering the solution by 1.5μ glass filter. The filtrate is precipitated with an equal volume of acetone and the precipitate is centrifuged at 6000rpm for l0min at 15°C. Pellet is re-dissolved in at least 1.75% aqueous NaCl solution and filtered using 0.2μ membrane fdter to obtain a clear sodium hyaluronate solution which was then re-precipitated with equal volume of acetone. The obtained precipitate was re-dissolved in minimum amount of pyrogen free water and lyophilized for at least 18-20hrs.
Sodium hyaluronate obtained by the said process conforms to European Pharmacopoeia specifications, with a total recovery between 50-60%.

EXAMPLE 10: Hyaluronic acid specification compliance with European Pharmacopoeia
Lyophilized powder obtained as per process of the present invention was analyzed as per European Pharmacopoeia specifications. The tests included % protein weight by weight (w/w) with respect to sodium hyaluronate, nucleic acid, appearance, pH, IR spectroscopy, loss on drying, assay and chlorides. Findings are presented in
Table No. 1
Table no. 1

Sr. no. Test Limit specified as per European Pharmacopoeia Observation /Result
1 Appearance of powder White powder or fibrous aggregate Complies
2 Appearance
of solution Clear solution, optical density at 600 nm not more than 0.01 Complies
3 pH 5.0-8.5 Complies
4 IR spectroscopy Spectrum of the test substance matches with that of the reference Complies
5 % Protein Not more than 0.1% Less than 0.1%
6 Nucleic acid Absorbance not more than 0.5 0.1 to 0.2
7 Loss on
drying Not more than 20% Less than 10%
8 Assay In the range of 95.0-105.0% 98.24%
9 Chloride Not more than 0.5%) Complies

We claim,
1. A process for the preparation of hyaluronic acid by bacterial fermentation
comprising:
a. culturing Streptococcus equi subsp zooepidemicus ATCC 39920 in a seed
medium;
b. producing hyaluronic acid by Streptococcus equi subsp zooepidemicus
ATCC 39920 in presence of carbon source;
wherein carbon source is comprising of a polysaccharide optionally in combination with a monosaccharide or a disaccharide.
2. A process for the preparation of hyaluronic acid by bacterial fermentation
comprising culturing Streptococcus equ subsp zooepidemicus ATCC 39920 in
a seed medium comprising 1% yeast extract, 1% casein hydrolysate, 1.5%
K2HPO4 and glucose followed by aerobic fermentation in presence of carbon
source with 1-2 vvm aeration for 12 - 14hrs in a suitable media at an agitation
of 900-100rpm, at pH 6.8 to 7.2, at temperature of 33°C to 40°C and dissolved
oxygen is controlled at 50-60%, wherein;
a. carbon source is comprising of a polysaccharide optionally in combination
with a monosaccharide or a disaccharide;
b. culture broth has viscosity in the range of 3000-4500cP;
c. the process yields at least 5.5g/L of hyaluronic acid having average
molecular weight of 3000 - 4500 KDa.
3. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2,
wherein the carbon source is polysaccharide selected from raw starch, soluble
starch, white dextrin, maltodextrin or mixtures thereof, more preferably soluble
starch.

4. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2, wherein the carbon source is polysaccharide in combination with monosaccharide selected from glucose and fructose or disaccharide selected from sucrose, lactose, and maltose.
5. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2 wherein carbon source is selected from raw starch, soluble starch, white dextrin, maltodextrin or mixtures thereof in combination with disaccharide selected from lactose, maltose, sucrose or mixtures thereof, more preferably combination of soluble starch with sucrose.
6. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2 wherein carbon source is selected from raw starch, soluble starch, white dextrin, maltodextrin or mixtures thereof in combination with monosaccharide selected from glucose, fructose or mixtures thereof, more preferably combination of soluble starch with glucose.
7. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2, wherein the polysaccharide is used in the range of 2% w/v to 3% w/v.
8. The process for the preparation of hyaluronic acid as claimed in claim 1 and 2, wherein the monosaccharide or disaccharide is used upto 5% w/v.
9. The process for the preparation of hyaluronic acid as claimed in claim 8. wherein the concentration of disaccharide is 2.5%.

10. The process for the preparation of hyaluronic acid as claimed in claim 9, wherein the disaccharide is sucrose.
11. The process for the preparation of hyaluronic acid as claimed in claim 7, wherein the concentration of polysaccharide is 2.5%.

12. The process for the preparation of hyaluronic acid as claimed in claim 11 wherein the polysaccharide is soluble starch.
13. A process for the purification of hyaluronic acid in solution as a salt, comprising the steps:
a. Clarifying the acidified broth by 0.65u open channel microfiltration
membrane to give clarified cell free broth preferably by carrying out
microfiltration at constant permeate flow;
b. Ultra filtering the clarified cell free broth obtained in step a) using ultra
filtration membrane upto 500KDa MWCO, more preferably 50KDa
MWCO at neutral pH with continuous diaflltration to remove colorants and
to obtain hyaluronic acid retained solution;
c. Adding sodium salt solution in to the retained hyaluronic acid solution
obtained in step b) and treating with an aromatic adsorbent resin selected
from HP10; HP20, HP21, HP30, SP800, SP205, SP206, SP207, more
preferably SP207 to remove proteins, endotoxins and nucleic acids and to
obtain purified sodium hyaluronate solution;
d. Precipitating the sodium hyaluronate from purified sodium hyaluronate
solution obtained in step c) with a solvent, re-dissolving the precipitate in a
sodium salt solution and then filtering to obtain a clear sterile sodium
hyaluronate solution;
e. Re-precipitating the sterile sodium hyaluronate from sterile sodium
hyaluronate solution obtained in d) with a solvent, dissolving the precipitate
in pyrogen free water preferably cooled water and then lyophilizing it to
obtain final powder.
14. A process for the purification of hyaluronic acid as claimed in 13c), wherein
the adsorbent resin is used at a concentration of 5-20% by weight, more
preferably 15% by weight.

15. The process of claim 13c), wherein the salt is selected from sodium acetate, sodium chloride more preferably sodium chloride to produce sodium hyaluronate complying with European Pharmacopoeia with respect to Infra-Red spectroscopy.
16. The process of claim 13c), wherein the salt concentration is l-6%w/v more preferably 5-6%w/v.
17. The process of claim 13c), wherein the resin is stirred with the sodium hyaluronate solution, for 3-12hrs, preferably 5-6 hours.
18. The process of claim 13d) and e), wherein the solvent used is selected from methanol, isopropanol, acetonitrile, ethanol, acetone, more preferably acetone.
19. The process of claim 13d). wherein the salt concentration is 0.9-3.0%w/v, preferably 1.5 -2%w/v.