FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"EFFICIENT PROCESS FOR PURIFICATION OF HYALURONIC ACID.”
RELIANCE LIFE SCIENCES PVT.LTD.
an Indian Company having its Registered office at
Chitrakoot, 2n(t Floor,
Ganpatrao Kadam Marg,
Shree Ram Mills Compound,
Lower Parel, Mumbai 400 013,
Maharashtra, India
The following specification describes and ascertains the nature of this invention and the manner in which it is performed.

FIELD OF THE INVENTION:
The present invention relates to a technique of purifying Hyaluronic acid and its salt. The present invention in particular relates to the production and optimization of purification process of Hyaluronic acid and its salt, suitable for biomedical applications
BACKGROUND OF THE INVENTION
Hyaluronic acid (HA) is a naturally occurring biopolymer, having biological functions in bacteria and higher animals including humans. Naturally occurring HA may be found in the tissue of higher animals, in particular as intercellular space filler (Balazs, 1993). It is found in greatest concentrations in the vitreous humour of the eye and in the synovial fluid of articular joints (O'Regan et al., 1994). In gram-positive Streptococci, it is found as a mucoid capsule surrounding the bacterium.
HA is comprised of linear, unbranching, polyanionic disaccharide units consisting of glucuronic acid (GlcUA) an N-acetyl glucosamine (GlcNAc) joined alternately by beta 1-3 and beta 1-4 glycosidic bonds (Fig. 2). It is a member of the glycosaminoglycan family, which includes chondroitin sulphate, dermatin sulphate and heparan sulphate. Unlike other members of this family, it does not covalently bind to proteins.

NHCOCH,
N-acetyl glucosamine
COO

CH2OH

Figure 2 Disaccharide repeating unit of HA comprising GlcUA and GlcNAc.
2

In a neutral aqueous solution, due to the hydrogen bond formation, water molecules and adjacent carboxyl and N-acetyl groups, imparts a conformational stiffness to the polymer, which limits its flexibility. The hydrogen bond formation results in the unique water-binding and retention capacity of the polymer. It also follows that the water-binding capacity is directly related to the molecular weight of the molecule. It is known that up to six liters of water may be bound per gram of HA (Sutherland, 1998).
Hyaluronic acid and its derivatives have been extensively studied for its applications in biomedical practices. The biocompatibility characteristic of this polymer has attracted attention of Orthopedics as it can be used in viscosurgery and allows surgeons to safely create space between tissues. Viscosurgical implants are constructed from HA (Balazs, 1993). Its viscoelastic character has been used to supplement the lubrication in arthritic joints. It has also been used for targeted drug delivery as a microcapsule. Finally, because of its high water retention capacity, this EPS (extracellular polysaccharide) also occupies a niche in the lucrative cosmetics market. Also HA solutions are characteristically viscoelastic and pseudoplastic. This rheology is found even in very dilute solutions of this polymer where very viscous gels are formed. The viscoelastic property of HA solutions which is important in its use as a biomaterial is controlled by the concentration and molecular weight of the HA chains. The molecular weight of HA from different sources is polydisperse and highly variable ranging from 104 to 107 Da. The extrusion of HA through the cell membrane as it is produced permits unconstrained polymer elongation resulting in a very high molecular weight molecule of HA.
HA has more commercial value than other microbial EPS. With an estimated world market value of $US 500 million, it is sold for about $US 100 000 per kilogram.
HA has been conventionally extracted from rooster combs and bovine vitreous humor. However, it is difficult to isolate high molecular weight HA at industrially feasible rate from these sources, because it forms complex with proteoglycans present in animal
3

tissue (O'Regan et al., 1994). It is presently impractical to control the molecular weight of the biopolymer while it is synthesized in animal tissue.
Subsequent improvements in the extraction and purification processes have resulted in an inherent molecular weight reduction. The use of animal-derived biochemicals for human therapeutics has raised ethical issues and this is being met with growing resistance. Besides ethical issues, there is potential infectious disease risk associated with the use of animal-derived biochemicals for human therapeutics
The above drawbacks have forced the Industry to adopt to bacterial fermentation processes with the hope of obtaining commercially viable biopolymer. By the fermentation process, the EPS is released into the growth medium and this helps in controlling the polymer characteristics resulting in improved HA yields. The amount of biopolymer that can be produced by this route is theoretically unlimited. The use of bacterial fermentation does however come with its own disadvantages. The recent trend has been to use Lancefield's group A and C Streptococci which naturally produce a mucoid capsule of HA. The HA capsule is a biocompatibility factor which enables this gram positive bacteria to evade host immune defenses and hence accounts for its characteristically high virulence level.
Streptococcal fermentations have only been able to produce HA with an average molecular weight in the range of 1 to 4 MDa. As previously highlighted, high molecular weight enhances the desirable properties of the biopolymer. Given that 10 MDa chains can be obtained from animal tissue, there is considerable scope for improvement.
Streptococci are nutritionally fastidious, facultative anaerobes, which produce lactic acid as a by-product of glucose catabolism. Hence the energy recovered by these bacteria is lower relative to aerobic bacteria. The yield of HA from bacterial fermentation to date has been characteristically low (0.1 g/g glucose, 0.15 g/lh) and would certainly struggle to meet market demand. The strict nutritional requirements
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influences the fermentation economics by prohibiting the use of chemically defined media for production scale fermentations and limits the choice of complex media that can be employed
Other research focused upon process parameter optimization, selection of process mode, HA yield optimization by complex medium design, and model based fermentation control (Lertwerawat, 1993; Johns et al., 1994; Armstrong et al., 1997; Goh, 1998). As the importance of HA molecular weight became increasingly clear, the researchers have recently turned to the effect of process parameters on the molecular weight properties of HA (Armstrong, 1997; Armstrong and Johns, 1997; Armstrong and Johns, 1995) as a major area of study.
Several fermentation parameters were found to significantly affect the molecular weight of HA produced while others had little influence. Specifically, low growth temperatures (28°C), culture aeration and high initial glucose concentration (40 g/1) resulted in the production of higher molecular weight HA in S. zooepidemicus. Culture pH and agitation did not influence the molecular weight outcome of HA fermentations.
The specific growth rate of the bacteria and the molecular weight of HA produced are inversely proportional. This effect has been explained by a resource-based metabolic model for HA synthesis as illustrated in Figure 3.
Figure 3 HA biosynthetic pathway in streptococci is given below:
5

NTP, NAD PH Organic acid s, ATP
Teichoic acid (walD Fatty acids, L ip ids
A 4
ppp phosphogtucoisom erase
I gffl, EC 5.3.1-9fc
+■ Glucose-6-P *
N**~ Glutamine
Glycolysis Fructose- 6- P
Phosphogucom utase pgm,EC5A22
Po ^saccharide (wall) ^ G1 uc o se-1 -P
Pyrophosphorylase L^"UTP hasC,EC2.7.7.9 k>
Teichoic acid (wall) ^ UDP-Glucose
C/DF- Glucose dehydrogenase V
hasB, EC 1.1.1.22 h*NADH
Teichuroiric acid "^ UDP-Glucuronic acid
Avalll
Hexokinase
r^
ATP
Glucose
Hyaluronic Acid "^~

i
Hyaluronate synlhase hasA, EC 2.4.1.-

lucosami
■
5.42:21
Am idoiransj"erase ghnS, EC 2.6.1.16 ^ rT1ut.«mat.fi G luc o samine-6-P Mutase ghnM,EZS.<
G luc o samine-1-P
Acetyl transferase v
ghiU, EC 2.3.14 N*.
N-A c etyl Glue os am ine-1 - P L^-TTTP Pyrophosphorylase V
gbttU. EC 2.7.723 X*
UDP N-Acetvl Glucosamine
i
Peptidoglycan (wall) Polysaccharide (wall)

In its most simplistic form two competing processes are identified within the bacterial cell. These two processes are cell growth and the biosynthesis of HA. These two processes compete for the limited resources namely carbon, nitrogen and energy. At low specific growth rates the cell directs more glucose-derived activated precursors (namely UDP-Glc and UDP-GlcNAc) to HA synthesis rather than cell wall synthesis. The higher ATP yields from aerobic glucose catabolism favors the formation of UTP, which is required for the formation of the two activated precursors of HA synthesis, UDP-GlcUA and UDP-GlcNAc. Glucose, which may be used to synthesize HA, is also depleted by lactate production under anaerobic growth.
The specific rate of HA production (g g'1 h"1) was also observed to increase with decreasing specific growth rate. Given the fact that the density of active HA-synthesising enzymes is unlikely to change, the higher rate of production can be attributed to a higher polymerisation rate through each synthase. An increased synthase
6

activity may arise from the higher intracellular substrate concentration resulting from low growth rate.
The molecular weight of the HA produced is determined by the number of precursors that the synthase is able to polymerise during its lifetime. Based upon this theory, overexpression of the synthase has resulted in decrease in the molecular weight of HA synthesised. Indeed it may reduce the mean molecular weight since there are more enzymes competing for the same resources. This effect was reported for the overexpression of polyhydroxybutyrate synthase in recombinant E. coli (Sim et al., 1997).
US patent 4,784,990 has demonstrated a method of obtaining sodium hyaluronate comprising growing with vigorous agitation a microorganism of the genus Streptococcus under appropriate conditions in a suitable nutrient medium containing a sugar component as a carbon source. The microorganism produces large amounts of high molecular weight hyaluronic acid. The sodium hyaluronate is then recovered from the medium by a method comprising treating the medium containing the microorganism so as to remove the microorganism and other materials insoluble in the medium, precipitating the sodium hyaluronate from the medium, e.g. precipitation with organic solvents, and recovering the precipitate. The precipitate can then be ground and dried. Compositions of sodium hyaluronate characterized by an absence of pyrogenicity and inflammatory activity can be produced by these methods. The medium has a substantially constant pH between about 6.0 and 7.5 and the sodium hyaluronate excreted into the medium by the organism is purified using methods involving precipitation, redissolving and reprecipitating the hyaluronate. The sodium hyaluronate can be precipitated from the medium or filtrate by adding a first organic solvent, such as isopropanol, to the medium. The precipitate is redissolved in 3% aqueous sodium acetate and then reprecipitated with a second organic solvent such as ethanol. The second precipitate is redissolved in 3% aqueous sodium acetate and activated charcoal is added to form a suspension. The suspension is filtered and a third organic solvent e.g. acetone is added to produce a precipitate of sodium hyaluronate. The first, second and
7

third organic solvents can each be isopropanol, ethanol or acetone. Alternatively the hyaluronate can be precipitated by the same organic solvent in each step, e.g. sodium hyaluronate is precipitated from the medium by using isopropanol in all three of the precipitation steps.
US patent number 4,946,780 provides a method of producing HA from fermentation by employing surfactants such as cetyl pyridinium chloride and further purification procedure by contacting said solution containing sodium hyaluronate with alumina, activated charcoal or mixtures thereof and then with silica to form a purified solution of sodium hyaluronate, adding alcohol to the purified solution of sodium hyaluronate to precipitate purified sodium hyaluronate and drying said purified sodium hyaluronate.
US patent number 5,563,051 discloses a process wherein after the fermentation process, the biomass is killed with a particularly suitable agent. For killing the biomass, agent used is formaldehyde which may be used as the aqueous solution commonly known as formalin and the HA extracted with an aqueous medium containing an anionic surfactant for extracting the HA from the killed biomass is sodium dodecyl sulphate. The patent further disclosed the use of an ultra filtration membrane with an appropriate molecular weight cut off which is usually from 10,000 to 25,000 Daltons, and preferably 20,000 Daltons nominal molecular weight. The filtered solution containing the dissolved HA is diafiltered with 8 to 20 volumes of purified water, preferably about 10 volumes of purified water and the filtrate is continuously discarded.
US patent number 6,489,467 claims a process for purifying high molecular weight hyaluronic acid from a biological source, including the steps of adjusting the pH of an aqueous solution containing high molecular weight hyaluronic acid from a biological source to a pH in the range from 1.7 to 3.3 and then diafiltering said aqueous solution at the same pH using a filter having a pore size in the range from 100,000 Daltons nominal molecular cut-off to 0.45 \i, and of removing cells from the aqueous solution containing high molecular weight hyaluronic acid from biological source.
8

US patent number 7,002,007 discloses the methods which include contacting a hyaluronate-containing source with an acid to make an acidic hyaluronate suspension, contacting that suspension with an anionic exchange medium in the presence of an acidic buffer, and thereafter contacting the medium with an acidic buffer having a higher salt content to desorb the hyaluronate from the medium.
The teaching in most of aforesaid patents involves the step of atleast thrice repeated solvent precipitation, resulting in an increased process steps and an overload on the lyophiliser.
Further, the conventional precipitation of the fermentation broth involves the use of surfactants or detergents such as cety1 pyridinium chloride/hexadecyltrimethy1 ammonium bromide. However, the removal of residual detergent or surfactant posed a limitation as the number of processing steps increased.
Further to this, the purification procedures of HA such as diafiltration steps employed to achieve the high molecular weight HA involved the use of large volumes of organic solvent, which has the disadvantage of handling problems in scale up operations resulting in the environmental hazards.
Looking to the need of the hour, the inventors of the present invention have developed a purification process of hyaluronic acid that includes silica gel filtration combined with active carbon treatment and subsequent diafiltration with less amount of solvent yielding a better quality of hyaluronic acid having biomedical applications. The present invention is commercially viable purification process of hyaluronic acid and its salts thereof, having industrial applications.
OBJECT OF THE INVENTION
It is the object of the present invention to provide purification process of hyaluronic acid, yielding a better quality of hyaluronic acid having biomedical applications.
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It is the object of the present invention to provide a commercially viable process for Hyaluronic acid and its salt.
It is the object of the present invention to provide an efficient process for removal of protein impurities from Hyaluronic acid by employing two steps of silica gel purification and carbon treatment.
It is the object of the present invention to provide an efficient process for diafiltration using less amount of solvent in the purification of hyaluronic acid.
It is the object of the present invention to provide a high molecular weight hyaluronic acid of medical grade.
It is the object of the present invention to provide a process that is free from surfactants or detergents.
It is the object of the present invention to provide a single cycle process for purification of hyaluronic acid.
It is the object of the present invention to provide a process, which comprises minimal amount of solvents.
It is the object of the present invention to remove at least 90% of the protein impurities employing silica gel and activated carbon treatment.
It is the object of the present invention to provide a process for constant volume diafiltration resulting in high molecular weight HA.
It is also the object of the present invention to provide HA as per specifications of British Pharmacoepia 2003
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SUMMARY OF THE INVENTION
The present invention provides an purification process of Hyaluronic acid of high molecular weight, yielding a better quality of hyaluronic acid, having biomedical applications, free from surfactants or detergents.
In one embodiment, the present invention uses fermentation technique for the production of high molecular weight Hyaluronic acid using bacteria. The preferred bacterium for the present invention is Streptococcus zooepidemicus
In one embodiment, the present invention provides an efficient process for removal of protein impurities from Hyaluronic acid. The present invention employs silica gel filtration and active carbon treatment for efficient removal of protein impurities.
In one embodiment the present invention provides an efficient process for purification of Hyaluronic acid by diafiltration. The present invention employs less amount of solvent in the process.
The present invention comprises the following steps for the production of medical grade Hyaluronic acid.
a) Preparation of HA by fermentation
b) Removal of protein impurities
c) Diafiltration
The preparation of HA is done by initial culturing of the microorganism Streptococcus zooepidemicus, in a suitable medium and subsequent fermentation of the broth in a 1-10 Litre fermentor at about 30-40 deg C, with agitation at about 300-500 rpm and 1-3 wm aeration for about 15-28 hours by maintaining the pH at a neutral range.
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After incubation, the fermentation broth is suitably diluted with water and clarified. The HA present in the clarified broth is reprecipitated with equal volumes of suitable solvent including but not limited to isopropanol.
The precipitated high molecular weight HA is then converted to its salt wherein it can be then solubilised for subsequent purification procedure. The present invention converts the HA into its sodium salt prepared by addition of 3% sodium acetate and homogenized till complete dissolution.
The removal of protein impurities involves the initial treatment with Silica gel, which removes about 68-70% protein. After removal of Silica gel by centrifugation, the high molecular weight HA is then treated with active carbon, which then removes 85-90% of the protein. The process involves passing the silica treated sample through a carbon impregnated on cellulose cartridge.
The solution obtained after removal of protein impurities is further purified by diafiltration. The present invention provides a continuous mode of diafiltration, which involves the dilution with sterile, pyrogen, free water preferably five times
Finally, the process of isolating sterile, purified hyaluronic acid is done by filtering through a 0.22 u filter.
The final presentation of the sodium hyaluronate product for biomedical purposes can be obtained by aseptic filtration.
Optionally, the sodium hyaluronate can be reprecipitated with isopropanol to yield the purified high molecular weight HA. The HA obtained can be lyophilized till moisture content is less than 5 %.
The main features of the present invention is highlighted below 1. The precipitation process does not employ any acidic pH.
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2. The precipitation process does not employ any detergents nor surfactants
3. The removal of protein impurities step does not employ any hazardous chemical such as formalin.
4. The purification process employs less solvent dilutions and thus reduces the load on the lyophiliser
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Fig.1: Illustrates the flow sheet of the process for preparation of high molecular weight Hyaluronic acid of medical grade.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term " hyaluronic acid" or "HA" as used herein indicates Hyaluronic acid obtained
from any biological source.
The term " hyaluronate" as used herein refers to any salt form of HA, however, the present invention relates to the sodium salt of HA.
The term high molecular weight as used herein refers to HA or a salt of HA having a molecular weight not less than about one million.
The microbial source is preferably a Streptococcus species producing high molecular weight hyaluronic acid, and more preferably the microbial source is selected from Streptococcus zooepidemicus, Streptococcus equi and Streptococcus pyogenes.
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The term "solvent" used herein indicates any solvent organic or inorganic solvents. The preferred solvent of the present invention is isopropanol.
The present invention provides a cost effective process for purification of HA which comprises less cumbersome steps.
The present invention provides the removal of protein impurities, which comprise of two steps including silica gel treatment followed by active carbon treatment. The subsequent diafiltration step ensures a highly purified salt of HA. The product thus obtained can be then aseptically filtered for biomedical applications.
The conventional processes employed the precipitation of HA by using detergents such as cety1 pyridinium chloride/hexadecyltrimethy1 ammonium bromide. However this posed a limitation of multiple steps required for removing the residual detergent. The present invention has provided an efficient method, which does not employ detergents or surfactants.
Further conventional diafiltration techniques adopted for purification of HA involves high dilutions with solvent, which posed a limitation in handling at a larger scale. Further, the problems of processes involving large volumes of solvents is suitably addressed by the present invention, which involves very less dilutions.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
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EXAMPLE 1: FERMENTATION
Streptococcus zooepidemicus ATCC 39920 was cultured in a medium composed of Yeast extract, 1 %; Casein enzyme hydrolysate, 1 %; K2HP04, 0.2 %; NaCl, 0.15 %; MgS04.7H20, 0.04 % and sucrose, 5 %. The organism was grown in 1 L medium at 37°C at 400 rpm for 24 h at 1 wm agitation. The pH of the medium was maintained at 7.0 by continuous addition of NaOH aqueous solution (5). After the incubation, the broth was diluted with 1 volume of water and clarified by centrifugation at 12,000 rpm for 30 min at 4°C. The HA in the clarified broth was precipitated with equal volume of isopropanol. The precipitate is suspended in 1 L of 3 % sodium acetate using a mechanical homogenizer (Kinematica- A.G., polytron P T 2100) at 15000 rpm for 10 min X 3 cycles.
EXAMPLE 2: REMOVAL OF PROTEIN IMPURITIES
Silica gel treatment
A portion of the homogenized suspension, 100 ml, was treated in batch mode, with silica gel at 2 % final concentration for adsorption of protein. This step removes 68-70 % protein. The silica gel is separated from the sodium hyaluronate solution by centrifugation at 12,000 rpm for 20 min at 4oC.
Active carbon treatment
Further treatment of high molecular weight hyaluronic acid solution was done with charcoal adsorbed 0.45u filter assembly (Millipore, Millistak +, Activated Carbon minicapsule M40AC23HH3). The flow rate was 14ml / min. This step removed 85-90 % of the remaining protein.
EXAMPLE 3 : PURIFICATION Continuous Mode Diafiltration
The flow through from carbon filter was further purified by using continuous mode diafiltration process. Diluted HA solution was pumped (Flow rate 15ml-20ml /min) in
15

to cross flow filter holder equipped with 50 kDa cut off polyether sulphone cassette. (Sartocon slice cassette mod. No. 305 1465001E-SG). At the beginning of the feeding of the aqueous broth in to filter, the permeate valve was closed in order to recirculate the aqueous broth for several times until the system was stable and no bubbles seen in retentate. The permeate valve was opened after 10 min and the aqueous broth was recirculated in the feed reservoir for additional 10 min to ensure no leakage of HA occurs across the membrane through the permeate valve. After 15 min. permeate and retentate were collected separately. Five equivalent volumes of sterile pyrogen free distilled water were added continuously in the reservoir containing HA solution. This addition of water to HA solution was done at same flow rate as the outflow rate of the filtrate. The inlet pressure of the cross flow filter holder was maintained around 0.5-1.5 bar. The retentate was used for further recovery of Hyaluronic acid and permeate was discarded. Essentially, the permeate contained no HA. The retentate was concentrated to original volume and analyzed for purity. The diafiltration step removes 80-85 % of the remaining protein.
ASEPTIC FILTRATION
The concentrated hyaluronic acid solution from diafiltration process is finally subjected
to 0.22 pin filtration aseptically. (Millipore stericup: SCGPU02RE , PVDF filter
material) which renders the product that qualifies for biomedical application. The
product thus formed was lyophilized to obtain high molecular wt. medical grade
sodium hyaluronate.
EXAMPLE 4: RELATIVE ANALYSIS
The following table indicates the relative analysis

S.No HA purified / treated with Volume ml HA yield mg/ml Protein mg/ml Total HA mg Total Protein mg % Protein w.r.t. HA
1 IPA 100 3.376 0.56 337.6 56 14.22
2 Silica gel 90 3.22 0.15 289.8 13.5 4.45
3 Carbon 90 3.114 0.0187 280.26 1.68 0.6
4 Dia-filtration (5X) 128 1.716 0.0014 219.6 0.179 0.075
5 0.22 nm filtration 128 1.96 0.0011 250.88 0.14 0.056
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EXAMPLE 5: PRODUCT SPECIFICATION
The final product is tested for its protein, nucleic acid, appearance, pH, glucuronic acid
content, molecular weight, ER spectra, chloride content, and moisture content. The present invention provides a process for HA having a molecular weight of 3.0-3.5 million daltons. The tests mentioned above and the material specifications are as set forth in British Pharmacopoeia 2003.
The comparative results is tabulated below:

SR.NO TEST SPECIFICATION RESULT
1. Appearance of solution Solution clear; absorbance of solution measured at 600 nm is not greater than 0.01 Solution clear; absorbance of soution measured at 600 nm is 0.004
2. IR spectrophotometry The spectrum of the test substance corresponds to the reference spectrum of sodium hyaluronate Complies
3. pH In the range 5.0-8.5 6.65
4. Nucleic acids Absorbance at 260 nm does not exceed 0.5 0.033
5. Protein Not more than 0.3%. If intended for use in parenteral preparations, not more than 0.1% 0.056 %
6. Chlorides 0.5 % (max) Complies
7. Loss on Drying Not more than 20% by weight 18.2%
8. Assay Not less than 95.0% and not more than 105.0% of sodium hyaluronate calculated with reference to the dried substance 99.2 %
Molecular weight determination
Molecular weight of HA was determined by size exclusion chromatography on Shodex OH Pak SB 804-805HQ column by HPLC. The mobile phase used was 0.1 M NaN03 at a flow rate of lm1/min using a RI detector.
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References
Armstrong, D. (1997). The Molecular Weight Properties of Hyaluronic Acid produced Streptococcus zooepidemicus. Chemical Engineering (Brisbane: University of Queensland).
Armstrong, D. C, and Johns, M. R. (1997). Effect of Culture Conditions on Molecular Weight of Hyaluronic Acid Produced by Streptococcus zooepidemicus. Applied and Environmental Microbiology 63,2759-2764
Armstrong, D. C, and Johns, M. R. (1995). Improved molecular weight analysis of streptococcal hyaluronic acid by size exclusion chromatography. Biotechnology Techniques 9,491-496.
Armstrong, D. L., Cooney, M. J., and Johns, M. R. (1997). Growth and amino acid requirements of hyaluronic acid -producing Streptococcus zooepidemicus. Applied Microbiology and Biotechnology 47, 309-312.Crater, D. L., Dougherty, B. A., and van de Rijn, I. (1995). Molecular characterization of hasC from an operon for hyaluronic acid synthesis in group A streptococci - demonstration of UDP-glucose pyrophosphorylase activity. Journal of Biological Chemistry 270, 28676-28680.
DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993). Molecular cloning, identification, and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. Journal of Biological Chemistry 268, 19181-19184.
Dougherty, B. A., and van de Rijn, I. (1994). Molecular characterization of hasA from an operon for hyaluronic acid synthesis in group A streptococci. Journal of Biological Chemistry 269,169-175.
Dougherty, B. A., and van de Rijn, I. (1994). Molecular characterization of hasB from an operon for hyaluronic acid synthesis in group A streptococci - demonstration of
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UDP-glucose dehydrogenase activity. Journal of Biological Chemistry 268, 169-175.
Goh, L.-T. (1998). Effect of culture conditions on rates of intrinsic hyaluronic acid production by Streptococcus equi subsp. zooepidemicus. Chemical Engineering (Brisbane: University of Queensland).
Johns, M. R., Goh, L.-T., and Oeggerli, A. (1994). Effect of pH, agitation and aeration on hyaluronic acid production by Streptococcus zooepidemicus. Biotechnology Letters 16, 507-512.Kitchen, J. r., and Cysyk, R. L. (1995). Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts. Biochemical Journal 309, 649-656. Kumari, K., and Weigel, P. H. (1997). Molecular cloning, expression and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. Journal of Biological Chemistry 272, 32539-32546.
Lertwerawat, Y. (1993). Hyaluronic acid production and its instability in Streptococcus zooepidemicus. Chemical Engineering (Brisbane: University of Queensland).
O'Regan, M., Martini, I., Crescenzi, F., De Luca, C, and Lansing, M. (1994). Molecular mechanisms and genetics of hyaluronan biosynthesis. International Journal of Biological Macromolecules 16,283-286.
Weigel, P. H., Hascall, V. C, and Tammi, M. (1997). Hyaluronan synthases. The Journal of biological chemistry 272,13997-14000.
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.
Dated this o day of July , 2006

K. V. Subramaniam President
For Reliance Life Sciences Pvt. Ltd.

ABSTRACT
The present invention provides an efficient process for purification of high molecular weight Hyaluronic acid. The invention in particular relates to the process of purification of HA which comprises treatment with silica gel and then treatment with active carbon and subsequent diafiltration with less volume of solvent.

RELIANCE LIFE SCIENCES PVT. LTD

Page 1 of 1

Figure 1: Flow Sheet of the process of purified preparation of HA and its salts.

Fermentation broth

Clarification of the broth

Dilution with water 1-1

Isopropanol precipitation

I

Filtration through a carbon cartridge

Treatment with silica gel

Dissolution of ppt in 3 % Na-acetate

Diafiltration through a 50 kDa cassette at constant volume at 5X dilution

0.22 u filtration -> Isopropanol precipitation

Lyophilisation

For Reliance Life Sciences Pvt. Ltd. K. V. Subramaniam
President