CLIAMS:1. A fermentation process for production of hyaluronic acid of high molecular weight by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246 in a nutrient medium, characterized in that said nutrient medium comprises;
a. Dextrose: 30.00g
b. Yeast extract: 2.5g
c. Potassium hydrogen 2.5g
phosphate
d. Sodium chloride 2.0g
e. Magnesium sulfate 1.5g

under microaerophilic conditions comprising maintaining dissolved oxygen concentration at 15-20% during growth phase and increasing the DO levels to 40% at harvesting stage to produce enhanced biomass containing hyaluronic acid; and
purifying the supernatant broth, obtained after centrifugation of the treated broth, by diafiltration to remove low molecular weight impurities; recovering sodium hyaluronate from crude supernatant by adding Iso propyl alcohol (IPA) in the ratio 1:2; removing proteins, endotoxins, nuclei acids from crude sodium hyaluronate; concentrating and recovering pure sodium hyaluronate.

2. The fermentation process according to claim 1, wherein, during the growth phase of the microorganism, the back pressure of 0.2 to 0.5 bar is maintained inside the fermenter.

3. The fermentation process according to claim 1, wherein, the fermentation is a batch process.

4. The fermentation process according to claim 1, wherein, the fermentation is carried for 12-14 hours.

5. The fermentation process according to claim 1, wherein, the dissolved oxygen concentration during growth phase and at harvesting phase is maintained by agitating the medium at 150rpm and increasing the agitation to 200rpm with stepwise increase of 25rpm.

6. The fermentation process according to claim 1, wherein, the dissolved oxygen concentration during growth phase and at harvesting phase is maintained by increasing the air flow from 0.5 v.v.m to 1.2v.v.m.

7. The fermentation process according to claim 1, characterized in that, the supernatant of the fermenter broth, after centrifugation, is purified using ultrafiltration membrane with molecular weight cut off from 100,000 to 300,000 Daltons against from 5 to 10 volumes of purified water to remove low molecular weight impurities.

8. The fermentation process according to claim 1, wherein, crude hyaluronic acid pellets are recovered from the supernatant liquid, after ultrafiltration, from IPA in the ratio 1:2.

9. The fermentation process according to claim 1, wherein, the proteins and endotoxins are removed from hyaluronic acid pellets using 3% Sodium acetate with cationic surfactant 0.5% sodium deoxycholate and 25%IPA.

10. The fermentation process according to claim 1, characterized in that, sodium hyaluronate obtained has average molecular weight of 2.5×106 to 4.5×106 Da, a protein content less than 0.2%, nucleotide level of less than 0.15%, UV light absorbance of 1% (w/v) of 0.14AU at 260 nm and 0.1 AU at 280 nm.

11. The fermentation process according to claim 1, wherein, sodium hyaluronate obtained is non-crosslinked.

12. A pharmaceutical composition comprising non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106 Da prepared by the process of claim 1 together with pharmaceutically acceptable excipients for the treatment of osteoarthritis and rheumatoid arthritis.

13. The pharmaceutical composition according to claim 12, wherein said pharmaceutical composition can be administered in the form of solid, liquid or in parenteral dosage forms.

14. The injectable pharmaceutical composition according to claim 13 for the treatment of osteoarthritis and rheumatoid arthritis comprising non-cross linked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da prepared by the process of claim 1 dissolved in physiological buffer.

15. A method for treating the subject with osteoarthritis and rheumatoid arthritis comprising administering an injectable formulation of non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da prepared by the process of claim 1 dissolved in a physiological buffer.

16. Use of injectable formulation comprising non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da prepared by the process of claim 1 dissolved in phosphate physiological buffer for the treatment of osteoarthritis and rheumatoid arthritis. ,TagSPECI:Technical field of invention:
The present invention relates to a process for producing high molecular weight sodium hyaluronate by bacterial fermentation. In particular, the present invention relates to a fermentation process for producing high molecular weight sodium hyaluronate by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246 which is free of hyaluronidase activity.

Background and prior art:
Hyaluronic acid (HA), also known as hyaluronan, is a linear polysaccharide composed of a repeating disaccharide unit of ??(1,4)-glucuronic acid (GlcUA)-??(1,3)-N-acetylglucosamine (GlcNAc). HA is usually present as the sodium salt which has a molecular formula of (C14H2ONNaO11)n where ‘n’ can vary according to the source, isolation procedure and method of determination.

Hyaluronic acid is non-immunogenic, and has a molecular weight ranging from 5000 Da to 20million daltons depending upon the source and possesses visco-elastic properties. Hyaluronic acid or its sodium salt, having multiple functions in the human body being involved in creating flexible and protective layers in tissues and in many signalling pathways during embryonic development, wound healing, inflammation, cancer and has proved to be an exceptional material for use in medical, pharmaceutical and cosmetic applications.

Traditionally, hyaluronic acid is isolated from connective tissues of animal such as skin and cartilage. Some organs rich in hyaluronic acid such as the umbilical cord, synovial fluid, vitreous humor and rooster combs are used as a source for production of hyaluronic acid on large scale. A solution of ultrapure hyaluronic acid from rooster combs was first disclosed in US4141973 of E.A. Balazs (1979) for human application as a supportive material in ophthalmic surgery.

The industrial production of hyaluronan from animal tissues or avian sources, however, is associated with certain drawbacks such as degradation of hyaluronan due to the endogenous hyaluronidase activity during extraction and purification process, breaking down the polymer chain through enzymatic hydrolysis, the harsh conditions of extraction, low yield of hyaluronan and the potential risk of contamination with proteins and viruses which demands for extensive purification thus making the process economically not feasible.

Production of hyaluronic acid by bacterial fermentation has evolved a more viable alternative and has been commercially exploited since the early 1980s through fermentation of microorganisms belonging to the genus Streptococcus which include S. pyogenes, S. faecalis, S. dysgalactiae, S. equi, S. equisimilis, Streptococcus equi subsp. equi and S. equi subsp. zooepidemicus which are classified as Lansfield serum group A or C and as hemolytic chain-formed cocci having ß hemolytic action.

Extracting hyaluronan from bacterial fermentation is a relatively simple process and is advantageous over the avian or animal tissue extraction process since the microbial cells can be physiologically and/or metabolically adapted to produce more hyaluronan of high molecular weight which is suitable for medical/pharmaceutical applications.

There are numerous Patents/Patent Applications, viz. US2975104, US4517295, US4801539, US4780414, US5023175, US4897349, US4946780, US5496726, EP0694616, US5563051 and others which discloses the bacterial fermentation for the production of hyaluronic acid under aerobic or anaerobic conditions, batch or continuous from the microorganism of genus Streptococcus and its mutated strains.

US4780414 disclose the production of sodium hyaluronate by culturing the microorganism Streptococcus zooepidemicus HA-116 ATTC No. 39920 and mutants derived therefrom in the culture medium consisting of casein hydrolysate, yeast extract, NaCl, 2MgSO4.7H2O, K2HPO4 and glucose under aeration. Sodium hyaluronate precipitated is further filtered, purified to obtain sodium hyaluronate of cosmetic grade with molecular weight between about 700,000 and 1,500,000 daltons as well as clinical grade of an average molecular weight from about 2 to about 3.5×106 daltons. The separation of sodium hyaluronate by filtration and purification involves use of excess solvents, costly surfactant such as cetyl-pyridinium chloride, and other rigorous operating conditions. Multiple purification steps are required to produce clinical grade sodium hyaluronate. US’414 further disclose fermentation with aeration yields from about 4 to about 6 g/l of hyaluronic acid with an average M.W of about 2.2 to about 3.3 x 106.

US5563051 relate to continuous process for the production of hyaluronic acid by fermentation of Streptococcus in a nutrient medium containing an assimilable source of carbon; a source of nitrogen; sources of phosphorus, sodium, potassium, magnesium, iron, zinc and manganese; sources of growth factors; and a source of sulphur. The hyaluronic acid produced has a molecular weight ranging from 1.6 to 2.5 million.

An article titled, “Hyaluronic acid production by Streptococcus zooepidemicus in marine by-products media from mussel processing wastewaters and tuna peptone viscera” by José A Vázquez, et.al published in Microbial Cell Factories 2010, 9:46 disclose the production of hyaluronic acid using the strain Streptococcus equi subsp. zooepidemicus ATCC 35246 grown in the complex culture medium consisting Mussel processing wastewater (MPW) as a sugar source and tuna peptone (TP) from viscera residue as a protein substrate in batch fermentation. The article discloses the production of 2.46g/l of hyaluronic acid with mol. wt. of approx.2500kDa.

Molecular weight is an important parameter for a commercial hyaluronic acid as it determines the quality, the rheological properties, affects physiological response and defines its suitable applications. Hyaluronic acid of high molecular weight greater than 10kDa has high viscoelasticity, moisture retention, mucoadhesion which are desirable in ophthalmology, in orthopaedics, wound healing and cosmetics.

There are various factors which influence the production of medical grade hyaluronic acid during fermentation process such as medium composition, pH, temperature, aeration and agitation. Moreover, the batch fermentation process is observed to produce low yield of hyaluronic acid due to high viscosity of the broth and mass transfer constraints.

Although, various strategies are disclosed in the art for increasing microbial hyaluronic acid production such as controlling the pH and temperature parameter, aeration and agitation conditions, continuous culturing, medium optimisation, the present inventors observed that the initial concentration of the mineral ions and carbon source have a significant effect on microbial HA production.

Further, since Streptococcus equi subsp. zooepidemicus ATCC 35246 are facultative anaerobes, are aerotolerant, is free of hyaluronidase activity and have fastidious nutrient requirements, improving upon the medium composition, optimising the aeration and agitation conditions, maintaining the dissolved oxygen concentration during both growth phase and at harvesting phase, sodium hyaluronate of high molecular weight and purity can be obtained which can find application in medicine and in pharmaceuticals.

The invention further improves upon the separation and recovery of hyaluronic acid from a highly viscous broth by overcoming the problems of the processes.

The harvest of fermentation broth is highly viscous in nature. The separation of cell from the broth is very difficult because of its nature. If the cells are not completely removed then sodium hyaluronate obtained contains high level of impurities like protein and nucleic acid. It is very difficult to remove such impurities during purification process. The present invention provides an improved process for production of sodium hyaluronate and separation of impurities such as cells, protein or nuclei acids, thus yielding sodium hyaluronate in high purity.

Summary of the invention
It is therefore primary object of the present invention to provide fermentation process for production of high molecular weight sodium hyaluronate and medical grade purity.

The other object of the invention is to provide a fermentation process by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246, free of hyaluronidase activity, for production of high molecular weight sodium hyaluronate and purity.

Yet another object of the invention is to develop a fermentation process, which is free from the usual disadvantages of contamination having the multiple media components and longer duration of cultivation age, with lower cost in which HA having a high molecular weight can be produced.
In an aspect, the present invention provides a fermentation process for production of hyaluronic acid of high molecular weight by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246 in a nutrient medium characterized in that said nutrient medium comprises;
a. Dextrose: 30.00g
b. Yeast extract: 2.5g
c. Potassium hydrogen phosphate 2.5g
d. Sodium chloride 2.0g
e. Magnesium sulfate 1.5g

The fermentation process is carried under microaerophilic conditions comprising maintaining dissolved oxygen concentration at 15-20% during growth phase and increasing the DO levels to 40% at harvesting stage by increasing the agitation speed from 150rpm to 200rpm with stepwise increase of 25rpm to produce enhanced biomass containing hyaluronic acid.

Further, pure sodium hyaluronate is obtained by purifying the supernatant broth, obtained after centrifugation of the treated crude broth, by diafiltration to remove low molecular weight impurities followed by further diafiltration to obtain high molecular weight sodium hyaluronate; recovering sodium hyaluronate from crude supernatant adding isopropyl alcohol (IPA) in the ratio 1:2; removing proteins, endotoxins, nuclei acids from crude sodium hyaluronate; concentrating and recovering pure sodium hyaluronate.

In an aspect, the fermentation process is a batch process.

In another aspect, the inoculum comprises Streptococcus equi subsp. zooepidemicus ATCC 35246 which is used for fermentation on achieving an optical density, OD>1. The growth of Streptococcus equi subsp. zooepidemicus ATCC 35246 is carried under microaerophilic conditions.

In an aspect, the oxygen level during fermentation is maintained by manually increasing the agitation speed from 150rpm to 200rpm with stepwise increase of 25rpm.
Accordingly, the process steps for obtaining pure hyaluronic acid include the following steps:
a. inactivating the fermentation broth;
b. removing the cells by centrifugation;
c. purifying the supernatant broth by diafiltration to remove low molecular weight impurities;
d. recovering sodium hyaluronate from crude supernatant by adding isopropyl alcohol (IPA) in the ratio of 1:2;
e. removing proteins and endotoxins from crude sodium hyaluronate by adding 3% sodium acetate, 0.5% sodium deoxycholate and 25% IPA;
f. removing nuclei acids by carbon filtration followed by diafiltration;
g. concentrating the sodium hyaluronate followed by diafiltration using 0.9% sodium chloride solution and filtering to obtain clarified supernatant;
h. recovering purified sodium hyaluronate by adding IPA to the clarified supernatant in the ratio of 1:3 ratio in the form of pellets;
i. lyophilizing and storing under cool temperature.

In another aspect, the medical grade HA is prepared by dissolving pure sodium hyaluronate in 0.15M sterile saline solution buffered with the phosphate buffer at pH 7.3.

Description of the figure
Fig 1 depicts comparative Growth Curve for different Batches.

Detailed description of the invention
The present invention relates to an improved process for production of hyaluronic acid in high purity by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246 acquired from the collection.
In an embodiment, the present invention discloses a fermentation process for production of hyaluronic acid of high molecular weight by culturing Streptococcus equi subsp. zooepidemicus ATCC 35246 in a nutrient medium, characterized in that said nutrient medium comprises;
a. Dextrose: 30.00g
b. Yeast extract: 2.5g
c. Potassium hydrogen 2.5g
phosphate
d. Sodium chloride 2.0g
e. Magnesium sulfate 1.5g

under microaerophilic conditions comprising maintaining dissolved oxygen concentration at 15-20% during growth phase and increasing the DO levels to 40% at harvesting stage by increasing the agitation speed from 150rpm to 200rpm with stepwise increase of 25rpm to produce enhanced biomass containing hyaluronic acid; and
purifying the supernatant broth, obtained after centrifugation of the treated broth, by diafiltration to remove low molecular weight impurities; diafiltering further to obtain high molecular weight sodium hyaluronate; recovering sodium hyaluronate from crude supernatant adding Iso propyl alcohol (IPA) in the ratio 1:2; removing proteins, endotoxins, nuclei acids from crude sodium hyaluronate; concentrating and recovering pure sodium hyaluronate.

In an embodiment, the nutrient medium is prepared by dissolving dextrose in 100ml of water, dissolving magnesium sulfate in 50ml of water and dissolving salts/sodium chloride/potassium hydrogen phosphate in 850ml of water, sterilizing separately by autoclaving at 121°C, for 15 minutes prior to mixing to obtain chemically defined medium for inoculation of the microorganism.

The inoculum comprising Streptococcus equi subsp. zooepidemicus ATCC 35246 is used for fermentation on achieving an optical density, OD>1.

The growth of Streptococcus equi subsp. zooepidemicus ATCC 35246 is carried under microaerophilic conditions. Accordingly, the fermentation is initiated by passing air or sterile oxygen containing gas into the culture medium at a rate sufficient to maintain dissolved oxygen tension in the fermentation medium of less than 30% saturation and preferably in the range of 15 to 20% saturation during growth phase. If air is used, a flow rate of 0.5 to 1.2 v.v.m (volume per fermenter volume per minute) is maintained.

Since the fermentation medium is viscous, thorough mixing of the medium is necessary to eliminate “dead zones” in which growth of undesired non-HA producing strains of Streptococcus equi can occur. Further, the dissolved oxygen (DO) levels are maintained at 40% at harvesting stage which is achieved by continuous agitation of the fermentation medium with an impeller speed of 40-200 rpm. The agitation is manually increased from 150rpm to 200rpm with stepwise increase of 25 rpm to produce enhanced biomass containing hyaluronic acid.

The temperature of the fermentation medium is maintained in the range of 35°C to 40°C; preferably at 37°C.

During the growth phase of the microorganism, set back pressure of 0.2 to 0.5 bar is maintained inside the fermenter.

The pH of the nutrient medium is maintained within the range of 6.0 to 7.0, preferably in the range 6.6 to 7.0 and most preferably at pH 6.8 by addition of 20%sodium hydroxide during the fermentation process.

Any foaming resulting during fermentation is controlled by the addition of non-toxic foaming agent used such as 10% polypropylene glycol 2000.

In an embodiment, stable culture conditions are maintained for a period of over 12 to 14 hours wherein, under the aforesaid conditions, as much as 3.0 g of HA for every litre of fermentation medium was obtained.

The further procedural method includes harvesting of crude hyaluronic acid from the fermentation broth and purification to obtain pure and medical grade hyaluronic acid.

Accordingly, the product from the fermentation broth is harvested on attaining a constant OD=3.5 after two consecutive absorbance readings. The objective of the harvest is to precipitate the crude product from the fermentation culture. This is followed by inactivating the fermentation broth either by heating or using killing agent such as antibacterial agent. The killing agent used is aqueous solution of formaldehyde, i.e. formalin at a concentration ranging from 0.5 to 1.0% (v/v). Preferably, the inactivation is carried by heating the fermentation broth at a temperature in the range of 65-75°C for about 20-30 min followed by chilling to a temperature of 10°C.

After inactivation of the fermentation broth, the dead cells are removed by centrifugation. Since the fermentation broth is highly viscous the formation of biomass is very difficult. To overcome this, the fermented broth is diluted with distilled water and centrifuged at 8000± 50 rpm and 2-8°C for about 30 minutes.

The supernatant of the fermenter broth is purified by diafiltration to remove low molecular weight impurities derived from the production organism’s metabolism, the residual components of the nutrient medium, residual killing agent, using ultrafiltration membrane with an appropriate molecular weight cut off ranging from 100,000 to 300,000 Daltons, and preferably 100,000 Daltons nominal molecular weight. The ultrafiltration membranes are based on polysulphones and are available commercially. The diafiltration is performed using purified water and the filtrate is discarded continuously.

Sodium hyaluronate is recovered from crude supernatant by adding Iso propyl alcohol (IPA) in the ratio of 1:2 respectively. The pellets are collected and the supernatant is discarded by decantation.

The crude sodium hyaluronate pellets contain the impurities like protein, nucleic acid and endotoxin. The proteins and endotoxin impurities are further removed by dissolving crude sodium hyaluronate in distilled water under continuous stirring at a temperature of 2 to 8 °C until complete dissolution. This is followed by addition of 3% sodium acetate, 0.5% of anionic surfactant such as sodium deoxycholate. IPA is then added to a final concentration of 25% for the removal of protein and endotoxins. The nucleic acids can be removed by the activated carbon filtration. Any traces of IPA, excess of chemicals and traces of carbon are further removed by diafiltration using 300,000 dalton nominal molecular weight membranes using purified water.

The sodium hyaluronate solution is further kept for incubation at 2 to 8°C for 12 to 14 hours by addition of IPA and centrifuged for about 30 min. The supernatant is clarified through 53SLP filter and kept for recirculation till the OD reaches =0.01 at 260nm.

The clarified supernatant is concentrated to the initial volume followed by diafiltration using 0.9% sodium chloride solution. The supernatant is harvested and clarified using 0.45µ filter. The purified sodium hyaluronate is further recovered by adding IPA to the clarified supernatant in the ratio of 1:3 respectively. The pellets are collected and supernatant is discarded by decantation. The pellets are air dried to evaporate traces of IPA, lyophilized and stored under cool temperature.

The medical grade hyaluronic acid is prepared by dissolving hyaluronic acid obtained by the process described above in 0.15M sterile saline solution buffered with the phosphate buffer at pH 7.3 to give a 1% (w/V) solution of hyaluronic acid.

In an embodiment, the sodium hyaluronate so prepared is characterized by an average molecular weight 2.5×106 to 4.5×106 Da determined by high pressure liquid chromatography with gpc column and viscometry; protein content of less than 0.2% and a nucleotide level of less than 0.15%; the 1% (w/v) solution shows a U.V absorption of 0.14AU at 260 nm and 0.1 AU at 280 nm and the viscosity of 6850cPs for 10mg/ml hyaluronic acid.

In another embodiment, the sodium hyaluronate so prepared is non-crosslinked.

In yet another embodiment, the present invention disclose pharmaceutical composition comprising non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106 Da prepared by the present process along with pharmaceutically acceptable excipients useful in the treatment of osteoarthritis and rheumatoid arthritis.

Pharmaceutical compositions suitable for use in context of the present invention include compositions where the active ingredients are contained in an amount effective to achieve the intended purpose.
The pharmaceutical composition may be administered in the form of solid, liquid or in parenteral dosage forms.

In an embodiment, the present invention relates to an injectable pharmaceutical composition for the treatment of osteoarthritis and rheumatoid arthritis comprising non-cross linked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da dissolved in physiological buffer comprising sodium chloride; Disodium hydrogen phosphate dedecahydrate; Sodium dihydrogen phosphate dehydrate and water for injection.

In another embodiment, the present invention relates to a method for treating the subject with osteoarthritis and rheumatoid arthritis comprising administering an injectable formulation of non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da dissolved in phosphate physiological buffer.

In yet another embodiment, the present invention relate to the use of injectable formulation comprising non-crosslinked sodium hyaluronate of average molecular weight 2.5×106 to 4.5×106Da dissolved in phosphate physiological buffer for the treatment of osteoarthritis and rheumatoid arthritis.

Examples:
Example 1: Fermentation process (Batch process)
1.1 Inoculum development:
Seed development for 10 litre fermenter with 7.0 litres working volume was carried out in three stage using Todd Hewitt broth (THB) media.

Preparation of Streptococcus selective medium (SSM) agar plats:

1.53gm of SSM was weighed and transferred into glass bottle containing 40ml of WFI and 1% of agar was added, mixed well and final volume was made up to 50ml. Sterilization was carried in pressure cooker for three whistles and after completion of sterilization, media was poured in pre-sterilized plates aseptically under LAFU and plates was left for solidifying.
Preparation of seed media [THB (Todd hewitt broth) Media]:
19.8 gm of Todd Hewitt broth was transferred to WFI and made up to 1000 ml with WFI water and sterilized at 121.5° C for 15 minutes.

In first stage streak plates were prepared from working cell bank (WCB) in SSM agar plates and incubated at 37°C for 48± 2 hrs.

In second stage pure colony from SSM plate was transferred to 10 ml THB media in test tubes and incubated at 37° C for 12± 2 hrs in the shaker incubator at agitation speed of 100± 5 RPM.

In third stage 2 ml of inoculum from 10 ml THB seed media was inoculated into the 210 ml of THB media and incubated at 37° C for 12± 2 hrs in the shaker incubator at agitation speed of 100± 5 RPM.

The process parameters are described in table 1 below:
Process parameter Observed value Process Limit
HA016/14 HA017/14 HA018/14
Final OD600nm 1.5 1.41 1.5 1.2 -1.6*
Inoculum pH 6.09 6.05 6.06 6.0±0.1
Agitation (RPM) 100 100 100 100±5

1.2 Media preparation:

a. Preparation of Dextrose solution (50%):
Weighed about 75 grams of dextrose and added into 250 ml of hot WFI and made the volume to 350 ml using WFI water and sterilized at 121.5? C for 20 minutes.

b. Preparation of MgSO4 solution:
Weighed about 10.5 grams of magnesium sulphate into WFI water and made up to 100 ml using WFI water and sterilized at 121.5? C for 20 minutes.

c. Preparation of 20% NaOH solution:
Weighed about 100 grams of sodium hydroxide into WFI water and made up to 500 ml using WFI water and sterilized at 121.5° C for 20 minutes.

d. Preparation of 10 % Antifoam solution:
Measured about 30 ml of poly propylene glycol 2000 and made up to 300 ml using WFI water and sterilized at 121.5° C for 20 minutes.

Fermentation Media Preparation

Fermentation media component for 10 litre fermenter with 7.0 litre of working volume is as follows:
a) Yeast Extract : 70 grams
b) Di potassium hydrogen phosphate : 17.5 grams
c) Sodium Chloride : 14 grams

Dissolved above mentioned components into 5.0 litres of water and made up to 6.4 litres with WFI water and added into the fermenter and sterilized at 121.5? C for 30 minutes. After sterilization, allowed it to cool up to 50?C and added 50% dextrose and MgSO4 solution using peristaltic pump. Then temperature was brought to 37? C and 210 ml of seed was inoculated using peristaltic pump.

The fermentation was started at 150 rpm agitation, at 37°C ± 1?C temperature, passed 0.5 v.v.m of air and 100% pO2. The fermentation process pH was maintained at 6.8 to 7.0 using 20% NaOH solution. To maintain the 30-40% DO during the process, agitation was manually increased from 150 rpm to 200rpm (stepwise increase of 25 rpm); air was increased from 0.5 v.v.m to 1.2 v.v.m; and the oxygen enrichment was given manually to maintain DO. The duration of batch phase was observed to be 12?2 hr.

The batch fermentation phase (output) is given in Table 2 below:
Process Parameter Observation Process Limit
Batch 1 Batch 2 Batch 3
Inoculum % 3 3 3 3±1
pH Max 7.2 7.2 7.2 7.0±0.2
pH Min 6.81 6.8 6.72
Temperature Max 37.5 37.5 37.6 37±0.5
Temperature Min 36.3 35.9 36.5
DO 20 20 20 10 to 25
Agitation (RPM) 150 150 150 150±50
Batch Cultivation Age 13.5 12 13.25 12?3 hr
Batch Harvest OD600nm 4 4.7 4 >3

The batch phase duration and OD600nmobserved has been consistent in the three batches.
1.3 Harvesting;
Harvesting was carried to precipitate the crude product from the fermentation culture. The product from the fermentation broth was harvested on attaining a constant OD=3.5 after two consecutive absorbance readings.

Table 3: Harvesting (Out-put)
Process Parameter Observation Process Limit
Batch 1 Batch 2 Batch 3
Cultivation Age (H) 13.5 12 13.5 13±1
DO at Harvest 45 50 49.0 =40
Different between two conjugative OD 0.1 0.14 0 0.2
OD 600 at the time of Harvest 4 4.7 4 NLT 3.0±1
Harvest volume (L) 7.4 7.45 7.4 7.0? 0.5 L

1.4 Purification Process:
After OD600nm is constant and OD started rising, broth was collected followed by centrifugation at 8000± 50 RPM and 2-8°C for 30 minutes. The cell pellets were discarded and supernatant was taken for purification process.

Recovery of Sodium hyaluronate:
• Sodium Hyaluronate was recovered from crude supernatant by adding Iso propyl alcohol in the ratio of 1:2 respectively;
• Pellet was collected and supernatant was discarded by decantation
Dissolution of crude Sodium hyaluronate:
• The crude sodium hyaluronate pellet was dissolved in 7000ml of WFI under continuous stirring in 2 to 8 °C
Removal of impurities:
• After complete dissolution of crude sodium hyaluronate pellet, 3% of sodium acetate and 0.5% of sodium deoxycholate was added to the crude sodium hyaluronate solution
• Iso propyl alcohol was then added to a final concentration of 25%
• After addition of IPA crude sodium hyaluronate solution was kept for incubation in 2 to 8 °C for 12? 2 hrs.
Centrifugation:
• Centrifugation was done at 4500rpm for 30min to separate precipitate and supernatant
• Supernatant was collected into a separate bottle and pellet was discarded
Clarification:
• The supernatant was clarified through 53SLP filter and kept for recirculation till the OD reaches =0.01 at 260nm
Concentration and diafiltration:
• The clarified supernatant was concentrated to 7000ml (initial volume) using 300KDa Cassettes
• 5 Volumes of di filtration was done using 0.9% Sodium chloride solution using 300KDa Cassettes
• After completion of diafiltration the diafiltered supernatant was harvested into a separate glass bottle
Clarification:
• The diafiltered supernatant was clarified using 0.45µ filter
Recovery of purified sodium hyaluronate:
• The purified sodium hyaluronate was recovered by adding IPA to the clarified supernatant in the ratio of 1:3 ratio respectively
• The pellet was collected and supernatant was discarded by decantation
• The pellet was air dried under the LAFU to evaporate traces of IPA
Lyophilzation and Storage:
The purified sodium hyaluronate pellet was lyophilized and stored in cold room (2 to 8 °C).

Example 2: Formulation
Sr. No. Content Quantity
Each 1ml of Formulated Bulk contains:
1. Sodium Hyaluronate 10mg
2. Sodium Chloride 8.5mg
3. Disodium hydrogen phosphate dedecahydrate 0.56mg
4. Sodium dihydrogen phosphate dehydrate 0.05mg
5. Water for injection q.s

Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.