Process for purifying high molecular weight hyaluronic acid

Abstract

The present invention relates to a process for purifying high molecular weight hyaluronic acid or a salt thereof, comprising the following steps: a) obtaining an aqueous solution / suspension comprising high molecular weight hyaluronic acid from a biological source; b) subjecting the solution / suspension of step a) to a clarification step, which optionally comprises a dilution step; c) subjecting the solution / suspension of step b) to a sterilization step, which optionally comprises a dilution step; d) optionally concentrating the solution / suspension of step c); e) adjusting the pH of the solution / suspension of step c) or step d) to a pH in the range from 1.7 to 3.3 and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 µm.

Description

[0001] PROCESS FOR PURIFYING HIGH MOLECULAR WEIGHT HYALURONIC ACID

[0002] The present invention relates to an improved process for purifying high molecular weight hyaluronic acid or a salt thereof.

[0003] BACKGROUND OF INVENTION

[0004] Hyaluronic acid is an anionic, non-sulfated glycosaminoglycan that is widely distributed in nature. It is found in connective, epithelial and neural tissues, and is a primary component of the extracellular matrix. Hyaluronic acid plays a key role in tissue regeneration and tissue repair, and hence has therapeutic use in wound healing. Hyaluronic acid is also an important component of cartilage and muscular connective tissue, and can be used to treat osteoarthritis. As a major component of the skin, it also has use in cosmetic applications.

[0005] The use of hyaluronic acid or a salt thereof in cosmetic and in particular in pharmaceutical applications requires a highly pure product.

[0006] Hyaluronic acid can be obtained from various sources such as from animal tissues, from microorganisms and from cell-free systems such as enzymes. Although the extraction of hyaluronic acid from animal tissues has certain economic and environmental benefits (being isolated from animal waste and byproducts that would be otherwise discarded), due to possible contamination concerns and a desire to produce animal-free products, the production of hyaluronic acid from microbial sources has become more attractive.

[0007] Production of hyaluronic acid by Streptococci was first disclosed by in 1937, and it was later demonstrated that the hyaluronic acid produced is identical to that from animal sources, hence does not trigger immune responses and is suitable for use in medical-grade and cosmetic products.

[0008] Microorganisms such as Streptococci have hyaluronic acid as the main component of their protective capsules. The microbial source is first fermented, and the hyaluronic acid or salt thereof is then typically isolated by precipitation using large amounts of organic solvents (such as ethanol, acetone, isopropanol), or using quaternary ammonium salt or surfactants. However, such procedures tend to be complex and expensive.

[0009] WOOO / 44925A1 describes a process in which pharmaceutical grade high molecular weight hyaluronic acid is obtained without the use of organic solvent, where the hyaluronic acid is maintained in aqueous solution during the whole process and hence no step of precipitation is required. This is achieved by using a process involving a step of removing cells, and a step of diafiltration at a pH in a range of 1.7 to 3.3. At this pH, a reversable cross-linkage occurs in which the molecules of hyaluronic acid form a network which may be broken under other pH conditions. At this pH range, the hyaluronic acid network is retained during a diafiltration step by a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 pm, while proteins and other material pass through the filter, thereby making it possible to separate high molecular weight hyaluronic acid and salts thereof from any soluble substances contained in the solution.

[0010] While this process represents a significant advance in the field of hyaluronic acid production and purification, it is desirable to develop a process in which production yields are higher and levels of contaminants (i.e. non-hyaluronic acid substances) are lower.

[0011] SUMMARY OF THE INVENTION

[0012] According to a first aspect of the present invention, there is provided a process for purifying high molecular weight hyaluronic acid or a salt thereof, comprising the following steps: a) obtaining an aqueous solution / suspension comprising high molecular weight hyaluronic acid from a biological source; b) subjecting the solution / suspension of step a) to a clarification step, which optionally comprises a dilution step; c) subjecting the solution / suspension of step b) to a sterilization step, which optionally comprises a dilution step; d) optionally concentrating the solution / suspension of step c); e) adjusting the pH of the solution / suspension of step c) or step d) to a pH in the range from 1.7 to 3.3 and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 pm.

[0013] BRIEF DESCRIPTION OF FIGURES

[0014] Figure 1 is a picture of the infected mare from which the bacterium used in the Examples was isolated.

[0015] Figure 2 shows the microchip identification certificate of the infected mare of Figure 1.

[0016] Figure 3 shows mixed flora growth resulting from inoculation of biological material obtained from the infected mare of Figure 1 . Figures 4 and 5 show colonies of Streptococcus equi. isolated from the mixed flora growth of Figure 3.

[0017] Figure 6 shows the identification document associated with the colonies of Figures 4 and 5. Figures 7A and 7B show a production flow chart of a specific example process of the invention. Figures 8A and 8B show a production flow chart of a further specific example process of the invention.

[0018] DETAILED DESCRIPTION

[0019] Hyaluronic acid (HA) is a polymer comprising alternating and repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, via alternating -(1— >4) and -(1— >3) glycosidic bonds. Polymers of hyaluronic acid can vary in weight from 5,000 Da to 20,000,000 Da in vivo. Native hyaluronic acid is linear, but can be modified to produce a cross-linked form of hyaluronic acid.

[0020] In the meaning of the present invention, high molecular weight hyaluronic acid (HMWHA) is hyaluronic acid having a molecular weight in the range of from about 1 ,000,000 Da to about 3,000,000 Da. “Hyaluronic acid” as used herein is intended to include hyaluronic acid in acid form and in its salt form (e.g. sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate etc).

[0021] An object of the present invention is to provide an improved process for obtaining pharmaceutical grade high molecular weight HA or a salt thereof, in particular a process which provides higher yield and lower level of contaminants compared to currently known processes.

[0022] According to the present invention, this object has been achieved by a process for purifying high molecular weight hyaluronic acid or a salt thereof, comprising the following steps: a) obtaining an aqueous solution / suspension comprising high molecular weight hyaluronic acid from a biological source; b) subjecting the solution / suspension of step a) to a clarification step, which optionally comprises a dilution step; c) subjecting the solution / suspension of step b) to a sterilization step, which optionally comprises a dilution step; d) optionally concentrating the solution / suspension of step c); e) adjusting the pH of the solution / suspension of step c) or step d) to a pH in the range from 1.7 to 3.3 and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 pm. Advantageously, by subjecting the starting material comprising HA to a clarification step and a sterilization step prior to a step of pH adjustment and diafiltration, high molecular weight hyaluronic acid or a salt thereof can be obtained in a higher yield and with lower levels of contamination, compared with equivalent processes involving diafiltration, with no clarification step and sterilization step prior to the diafiltration step.

[0023] In step a) the aqueous solution / suspension comprising high molecular weight hyaluronic acid can be derived from any biological source, but is suitably an aqueous broth containing high molecular weight hyaluronic acid from a microbial source. The microbial source is suitably a Streptococcus species producing high molecular weight hyaluronic acid. In one embodiment, the Streptococcus species producing high molecular weight hyaluronic acid is selected from the group consisting of Streptococcus equi (e.g. Streptococcus zooepidemicus) and Streptococcus pyogenes, and in particular is Streptococcus equi (e.g. Streptococcus zooepidemicus).

[0024] A broth containing high molecular weight hyaluronic acid may be prepared by any well-known process including fermentation of a Streptococcus culture producing high molecular weight hyaluronic acid in an appropriate aqueous medium and under appropriate conditions. An exemplary process is described in General Methods below.

[0025] Once an aqueous solution of HMWHA has been obtained, the solution (or suspension / broth) is subjected to a clarification step. Suitably the solution / suspension is diluted during the clarification step i.e. step b) is a dilution and clarification process. During the dilution, the solution / suspension of step a) is diluted with water (which may be demineralized or purified water), to a total volume which is between about 1.5 times and about 3.5 times that of the volume of the aqueous solution in step a), such as between about 2 times and about 3 times, e.g. about 2.4 times.

[0026] Demineralized water, also known as deionized water, is water that has had its mineral ions removed through a process of deionization or distillation. Purified water is obtained by a purification process that is superior to deionization or distillation. The process reduces the conductivity as well as the total organic carbon (TOC), and microbial content to meet the specified standards. Purified water is commonly used in pharmaceutical and healthcare manufacturing processes. Sterile pyrogen-free water is purified water that has also been sterilized to eliminate viable microorganisms and has been tested to ensure the absence of pyrogens. This kind of water is crucial in pharmaceutical and medical applications where the presence of pyrogens could have adverse effects such as in injectables pharmaceutical products.

[0027] Following the optional dilution step, activated charcoal and / or or celite is suitably added to the solution / suspension of step a). The activated charcoal primarily functions to remove impurities from the microbial broth, having a large surface area and porous structure. The amount of activated charcoal can vary, but will typically be added at a concentration of between about 2 g / L and about 6 g / L, such as about 4 g / L or about 5 g / L (relative to the total volume of the solution). The celite primarily functions to improve the filtration efficiency of the microbial broth, and can enhance the separation of solid particles, such as the microbial cells, from the liquid phase, facilitating the clarification of the broth. The use of celite in conjunction with filtration methods can be beneficial in preparing the broth for subsequent downstream processing steps. The amount of celite can vary, but will typically be added at a concentration of between about 0.5 g / L and about 5 g / L, such as about 1 g / L or about 2 g / L (relative to the total volume of the solution).

[0028] The present inventors have found that the clarification step can be further improved by including salt. Suitable salts include water soluble salts such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride and sodium acetate, or a mixture thereof. Without wishing to be bound by theory, the present inventors believe that the overall ionic strength of the solution / suspension, influenced by salt concentration, can impact the interactions between hyaluronic acid molecules. This, in turn, can affect the entanglement and flow behaviour of the polymer, by decreasing the dynamic viscosity of the solution / suspension.

[0029] Following the addition of activated charcoal and / or or celite, and optionally a salt, the solids are removed by passing the solution / suspension through a filter such as a depth filter, a plate and frame filter such as a filter press, a belt filter or a centrifugal filter. The depth filter suitably has a pore size of less than 1-2 pm. Alternatively, the solids can be removed by centrifugation.

[0030] As an alternative to adding activated charcoal and / or or celite, the clarification step of step b) can instead be carried out by adjusting the pH of the solution / suspension of step a) to a pH in the range from 3.4 to 4.0, and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cutoff to about 0.20 pm. Suitably, the pH of the solution / suspension of step a) is adjusted to about pH 3.5. During this alternative clarification process, a surfactant can also be added, in order to facilitate bacterial decapsulation. Thus, in one embodiment a surfactant is added to the solution / suspension during the clarification process of step b). Any suitable surfactant may be used, such as selected from the group consisting of sodium dodecyl sulphate (SDS), sodium lauryl sulphate (SLS), Tween 20®, Tween 80, Triton X-100 or X-114, octyl beta-glucoside, and combinations thereof. In one embodiment, the surfactant is included in the solution / suspension at a concentration of between about 0.05 wt% and about 0.2 wt%, such as between about 0.05 wt% and 1 wt%.

[0031] The solution / suspension produced in step b) is then sterilized in step c). Suitably, the sterilization is carried out by a filtering sterilization method e.g. by filtering the solution / suspension of step b) through a filter having a pore size of about 0.2 pm.

[0032] In step c), prior to filtering sterilization, the solution / suspension is preferably diluted to a total volume which is between about 4 times and about 10 times that of the volume of the aqueous solution in step a), such as between about 4 times and about 8 times, e.g. about 6 times. The present inventors have found this dilution step to be particularly beneficial for the eventual purity of the high molecular weight hyaluronic acid. Without wishing to be bound by theory, it is believed that the dilution decreases the dynamic viscosity and the flowability of the clarified product in order to facilitate the filtration, and hence increases the yield. The solution / suspension is suitably diluted with water e.g. e.g. demineralized water or purified water.

[0033] Once the solution / suspension has been sterilized, depending on the total volume of solution / suspension and the previous extent of dilution, step c) is typically followed by an optional concentration step (step d)). In one embodiment, in step d) the solution / suspension of step c) is concentrated to a total volume which is between about 1.5 times and 3.5 times that of the volume of the aqueous solution / suspension in step a), such as between about 2 times and about 3 times, e.g. about 2.4 times.

[0034] The solution / suspension can be concentrated by any suitable method, such as by:

[0035] - using tangential ultrafiltration e.g. with a filter having a pore size less than 100,000 Da nominal molecular weight (suitably less than 10,000 Da); or

[0036] - the solution / suspension of step c) is concentrated by tangential ultrafiltration with a filter having a pore size in the range from about 10 kD to 15 kD nominal molecular weight; or

[0037] - using tangential microfiltration with a filter having a pore size less than 0.80 pm (such as less than 0.45 pm); or - the solution / suspension of step c) is concentrated by distillation under vacuum at a temperature less than 100 °C (suitably less than 60 °C, such as at about 40 °C); or

[0038] - distilling the water under vacuum, such that distillation occurs at temperatures below 40 °C. If the step of tangential microfiltration is used, the pH of the solution / suspension of step c) is adjusted to a pH in the range between about 3.3 and about 1 .7 (suitably to about pH 2.75) beforehand.

[0039] Following concentration, the solution / suspension may be subjected to tangential ultrafiltration with a filter having a pore size in the range from about 10 kD to 15 kD nominal molecular weight until the conductivity of the solution is less than 30 pS / cm (more or less 7 equivalent volumes).

[0040] Step e) of the process of the invention involves a pH adjustment step followed by a diafiltration step. Firstly, the pH of the solution of step c) (or step d) if a concentration step has taken place) is adjusted to a pH in the range from 1.7 to 3.3, e.g. in the range from about 2.4 to about 3.0 (e.g. about 2.75). The pH can be adjusted using any suitable aqueous acid (e.g. an HCI aqueous solution or a phosphoric acid aqueous solution, e.g. 1 M HCI aqueous solution). As discussed above, at this pH the aqueous hyaluronic acid solution is transformed into a viscoelastic solution containing high molecular weight hyaluronic acid in a network arrangement. When diafiltered, this high molecular weight hyaluronic acid network is retained when a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 pm (e.g. about 0.45 pm) is used, thereby allowing the HA network to be purified. In one embodiment, the filter used for the diafiltration of step e) has a pore size in the range from about 300,000 Da nominal molecular weight cut-off to about 0.45 pm (e.g. about 0.2 pm).

[0041] Following pH adjustment, the solution / suspension is diafiltered, preferably according to a cross flow diafiltration process. The pH of the solution / suspension is maintained in the range from 1.7 to 3.3 during the whole diafiltration process i.e. is kept at the same pH.

[0042] Diafiltration is carried out using water (e.g. purified water or sterile pyrogen free water) which is added to the tank containing the viscoelastic solution / suspension containing high molecular weight hyaluronic acid, at the same flow rate as the outgoing filtrate, in order to maintain a constant volume. In one embodiment, during the diafiltration of step e), water is added at the same rate as filtrate is removed, in order to maintain a constant volume, and diafiltration is carried out until 5-20, such as 5-11 , 12-20, 5-7, 12-18, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 equivalent volumes of water have been added. Suitably, the filter used for the diafiltration of step e) has a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.75 pm (e.g. about 0.70 pm, about 0.65 pm, about 0.60 pm, about 0.55 pm, about 0.50 pm, about 0.45 pm, about 0.40 pm, about 0.35 pm, about 0.30 pm, about 0.25 pm or about 0.20 pm). In a preferred embodiment, the filter has a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.45 pm (e.g. about 0.20 pm).

[0043] In a further embodiment, the filter used for the diafiltration of step e) has a pore size in the range from about 300,000 Da nominal molecular weight cut-off to about 0.80 pm (e.g. to about 0.75 pm, about 0.70 pm, about 0.65 pm, about 0.60 pm, about 0.55 pm, about 0.50 pm, about 0.45 pm, about 0.40 pm, about 0.35 pm, about 0.30 pm, about 0.25 pm or about 0.20 pm). In a preferred embodiment, the filter has a pore size in the range from about 300,000 Da nominal molecular weight cut-off to about 0.45 pm (e.g. about 0.20 pm).

[0044] The diafiltration process is suitably carried out until 12-18 equivalent volumes of water have been added.

[0045] The present inventors have found that the protein content of the resulting hyaluronic acid or salt thereof product can be lowered (i.e. the purity further improved) by adding a salt to the diafiltration solution (the solution containing the high molecular weight hyaluronic acid network), part way through the diafiltration process e.g. after 3-10 equivalent volumes (e.g. after 7 volumes) of water have been added. The salt is suitably a water soluble salt e.g. an inorganic salt such as a sodium salt (such as NaCI) or a calcium salt (e.g. Ca(OH)2), which is suitably added at a concentration of between about 0.01 M and about 3 M (typically a minimum concentration of 10-15 mM, with a preferable range of 100-300 mM, e.g. about 150 mM). If a sodium salt is added during this step, the resulting purified high molecular weight hyaluronic acid will be in the form of sodium hyaluronate. If a calcium salt is added during this step, the resulting purified high molecular weight hyaluronic acid will be in the form of calcium hyaluronate. As such, this step allows the user to produce a desired hyaluronate salt type, and facilitates ion exchange if necessary (e.g. the hyaluronate salt type can be changed at this stage). The salt can be added as a solution in sterile, pyrogen-free water. Alternatively, the salt can be added in solid form, but the recirculation should be temporarily halted to allow sufficient time for the salt to dissolve completely. The present inventors have found that addition of the salt at this stage of the diafiltration process (when 3-10 equivalent volumes have been added), when most of the proteins have already been washed out, allows the salt to displace the remaining proteins attached to the hyaluronic acid. This attachment is the result of the proteins being protonated at the maintained pH in the range from 1.7 to 3.3. Adding the salt at this stage facilitates the detachment of the proteins from the hyaluronic acid through a competitive displacement phenomenon between the cation of the salt and the positively charged protein.

[0046] Once diafiltration step e) has been completed, preferably the pH of the solution / suspension is adjusted to a pH of between about 6 and about 8, e.g. about 7 (step e-1 )). Suitably, the pH is adjusted using an aqueous base (e.g. NaOH aqueous solution or Ca(OH)2 aqueous solution). At this pH, the network of high molecular weight hyaluronic acid is broken down, and when filtered using a pore size of 0.2 pm, the high molecular weight hyaluronic acid will pass through the filter. Prior to the adjustment to pH 6-8, the pH can be optionally adjusted to pH 13 to facilitate molecular weight degradation of the hyaluronic acid. However, if a high molecular weight hyaluronic acid is desired, the pH can be adjusted directly to pH 6-8.

[0047] Depending on the total volume of solution / suspension obtained from step e) and the previous extent of dilution, step e) or step e-1) can be followed by a concentration step (step e-2)).

[0048] The solution can be concentrated in step e-2) by any suitable method, such as by:

[0049] - using tangential diafiltration e.g. through a filter having a pore size less than 100,000 Da nominal molecular weight (suitably less than 10,000 Da); or

[0050] - by evaporation under vacuum, suitably at a temperature of less than 100 °C (e.g. at about 40 °C).

[0051] The solution / suspension of step e) or step e-1 ) is suitably concentrated to a total volume which is between about 0.5 times and about 1.5 times that of the volume of the aqueous solution / suspension in step a), such as between about 0.75 times and about 1.25 times, e.g. a total volume which is about equal to the volume of the aqueous solution / suspension in step a).

[0052] If part way through the diafiltration process salt is added, it must be removed e.g. by carrying out a further diafiltration step (step e-3)) at the pH of between about 6 and about 8, e.g. about 7. This additional diafiltration step is suitably carried out by exchanging 5 to 7 volumes of permeate with water, with the same filter having a cut-off of less than 100,000 Da e.g. less than 10,000 Da. The step is carried out following step e-1) (pH adjustment), and any optional concentration step (step e-2)). Step e-3) is suitably carried out using sterile, pyrogen-free water. This step must be carried out at neutral pH because any further pH adjustment will bring further salts into the solution. In one embodiment, step e), step e-1), step e-2) or step e-3) is followed by step e-4), of adding aluminium oxide, activated charcoal and sodium chloride to the solution / suspension. The present inventors have found that adding these components to the solution / suspension as a final, or near final step before final processing such as packaging absorbs the residues of endotoxins and proteins, ensuring that the hyaluronic acid produced is of pharmaceutical grade. The aluminium oxide suitably has an average surface area (defined using the BET method) of between about 50 m2 / g and about 200 m2 / g, preferably 100 m2 / g, and an average bead diameter of around 2 pm. Suitably, the aluminium oxide is included in the solution / suspension at a concentration of between about 0.1 wt% and about 4 wt% (preferably about 1.5 wt%). In one embodiment, for every 1 g of hyaluronic acid, 1 g-40 g, such as about 15 g of aluminium oxide is added. Suitably, the activated charcoal is included in the solution / suspension at a concentration of between about 0.1 wt% and about 4 wt% (preferably about 1 wt%). In one embodiment, for every 1 g of hyaluronic acid, 1 g-40 g, such as about 10 g of activated charcoal is added. Suitably, the sodium chloride is included in the solution / suspension at a concentration of between about 0.05 wt% and about 3 wt% (preferably about 0.3 wt%).

[0053] In one embodiment, in step e-4) the solution / suspension is agitated at a temperature between about 4 °C and about 50 °C, such as between about 20 °C and about 25 °C, e.g. about 22 °C. In one embodiment, in step e-4) the solution / suspension is agitated for a period of at least 5 hours up to 3 days, preferably for about 5 hours. In one embodiment, following step e-4) the solution / suspension is depth filtered or centrifuged and subsequently filtered again through a filter having a pore size in the range of 0.1 pm to 2 pm, preferably 0.45 pm, in order to retain all the aluminium oxide and charcoal residues.

[0054] The solution of high molecular weight hyaluronic acid or salt thereof following step e), step e- 1), step e-2) or step e-3) meets the specifications of the European and American pharmacopoeias and therefore can be packaged immediately. Thus, in one embodiment, step e), step e-1), step e-2) or step e-3) is followed by step f) of packaging the solution of high molecular weight hyaluronic acid or salt thereof. Additional components can be added to the solution of HMWHA prior to packaging, i.e. to the solution of step e), step e-1), step e-2) or step e-3). Additional components include a salt, a buffer, a stabilizer (such as trehalose), one or more active pharmaceutical ingredients, an antifibrinolytic substance (such as tranexamic acid), a coagulation activator (such as calcium gluconate), a silk protein (such as silk fibroin), vitamin K2, and mixtures thereof. Prior to packaging, a further concentration step (e.g. using tangential filtration or distillation under vacuum) may be required to prepare a pharmaceutical formulation according to its specifications.

[0055] The HMWHA or salt thereof can be packaged into any suitable container, but is particularly suited for packaging in sterile bags, which facilitate easy use of the solution.

[0056] While the solution of HMWHA or salt thereof can be used directly, the hyaluronic acid therein can also be converted to a solid form e.g. for a smaller storage volume and easier transportation.

[0057] In one embodiment, step e), step e-1), step e-2) or step e-3) is followed by a step g) of drying the solution / suspension of step e), step e-1), step e-2) or step e-3) to obtain a dry powder of hyaluronic acid or salt thereof. Drying can be carried out by any suitable method e.g. by freeze drying, by sublimation or by spray drying. During freeze drying, the substance is first frozen, and then the water is removed by sublimation, with no liquid phase. In spray drying, the substance is dissolved in a liquid and then atomized into small droplets and introduced into a hot air chamber. The water evaporates, leaving behind dry particles. A spray dryer can be equipped with a rotary nozzle or with a twin fluid nozzle.

[0058] In another embodiment, step e), step e-1), step e-2) or step e-3) is followed by a step h) of adding a salt (e.g. sodium acetate, suitably at a concentration of between about 0.1 M and about 1 M e.g. about 0.3 M) to the solution / suspension, followed by precipitation with an organic solvent (e.g. comprising ethanol, acetone, methanol, or isopropanol, or a mixture thereof).

[0059] In another embodiment, step e), step e-1), step e-2) or step e-3) is followed by a step i) of drying the solution / suspension of step e), step e-1), step e-2) or step e-3) to obtain a dry powder of hyaluronic acid or salt thereof though needleless electrospinning or electrospraying techniques to obtain dried nanofibers or nanoparticles respectively.

[0060] Alternatively, the hyaluronic acid can be used as a biocompatible polymer in a tissue engineered construct comprising a non-woven network of electrospun fibres (e.g. as described in W02004 / 000915A2).

[0061] Processes of the invention are, at least in some embodiments, expected to have one or more of the following merits or advantages:

[0062] • high hyaluronic acid yield (with regard to the HA content in the crude solution); low protein content; low nucleic acid content; low neutral sugar content.

[0063] It should be noted that in the context of the present application, when referring to a range of between about “AA” and about “BB”, the point values of AA and BB are intended to be included as possible values in the range.

[0064] ABBREVIATIONS

[0065] BET Brunauer-Emmett-Teller

[0066] HA hyaluronic acid

[0067] HMWHA high molecular weight hyaluronic acid rpm revolutions per minute

[0068] SDS sodium dodecyl sulphate

[0069] SLS sodium lauryl sulphate

[0070] TOC total organic carbon

[0071] EXAMPLES

[0072] General Methods

[0073] Preparation of an aqueous broth containing high molecular weight hyaluronic acid from microbial source

[0074] High molecular weight HA-producing cells were isolated from nature in the pus extracted from an infected horse. A highly mucoid colony was isolated from the cell mixture and characterized, and was found to contain a bacterium of the Streptococcus genus, equi species, subspecies zooepidemicus. A culture on Columbia CAP Selective Agar with Sheep Blood plates showed negative results in terms of beta-hemolytic activity. Further details of the isolation are set out below.

[0075] A culture broth with the following composition in demineralized water was prepared:

[0076] • Soytone (15 g / L)

[0077] • Yeast extract (5 g / L)

[0078] . KH2PO4 (2 g / L)

[0079] . MgSO4*7H2O (1 g / L) Glucose (60 g / L)

[0080] 150 mL of the culture broth was incubated overnight with the high molecular weight HA- producing cells (grown on plates containing the same culture medium with the addition of agar- agar). The incubation took place at 37 °C for approximately 20 hours in a glass flask at a stirring speed of 250 rpm. When the culture is in the exponential phase, the content of the flask is inoculated into 3.5 L of the same culture broth. The pH was maintained at around 7.3, the temperature at around 37 °C. The agitation was kept constant throughout the fermentation process at a speed of 250 rpm.

[0081] The incubation has been successfully carried out on a 500 mL scale, using the same broth components.

[0082] Details of hyaluronic acid isolation

[0083] The bacterium used for this experiment was selected from nature, isolated from the furuncle of an infected mare named Gina (see Fig. 1) (microchip number 941000012004179, UELN number 380011082009054) (see Fig. 2). The mare belongs to a private owner in the municipality of Choex (Valais - Switzerland), who kindly permitted the sample collection. The mare exhibited a furuncle under her left eye, with small amounts of purulent material oozing from it.

[0084] Using a sterile syringe (without a needle), a few microliters of pus were aspirated. The syringe containing the biological material was placed inside a 50 ml tube, which was then capped. This tube was subsequently sealed in a box to ensure safe transportation to the laboratory. The furuncle was disinfected after sample collection.

[0085] Under a biohazard laminar flow hood, the collected material was inoculated onto an agar plate and incubated at 37 °C in a ventilated incubator. A mixed flora growth was observed (see Fig. 3). Among the various colonies, some appeared shiny and transparent, resembling those typically formed by Streptococcus equi.

[0086] One of these colonies was aseptically picked under the biohazard laminar flow hood and inoculated onto a new agar plate (see Fig. 4 and Fig. 5).

[0087] The strain has been identified by Mabritec AG in Riehen (Switzerland) as being Streptococcus equi subsp. Zooepidemicus (see Fig.6). The strain was plated on blood agar plates and identified as being non-p-haemolytic, which implies that it is less virulent compared to a or p-haemolytic streptococci, which are often associated with more severe infections. For example, Streptococcus pneumoniae (alpha- hemolytic) can cause pneumonia, meningitis, and otitis media, while Streptococcus pyogenes (P-haemolytic) is associated with conditions like strep throat, scarlet fever, and necrotizing fasciitis, p-haemolytic streptococci can cause more aggressive infections partly due to their ability to lyse red blood cells, which contributes to tissue damage and inflammation, aiding in the spread of infection. Non p-haemolytic streptococci lack this capability, which might limit their ability to cause such extensive tissue damage and invasive disease.

[0088] The strain was sequenced by Microsynth AG (Switzerland, Schiitzenstrasse 15 • CH-9436 Balgach, Phone +41 71 722 83 33, www.microsynth.com) and has the sequence set out in SEQ ID N0:1. In particular, SEQ ID NO: 1 encodes the genomic sequence of Streptococcus equi subsp. Zooepidemicus.

[0089] Example 1 - Purifying process according to the present invention

[0090] After 24 hours of fermentation, 3.5 L of viscous broth obtained as set out in General Methods was collected into a suitable vessel and diluted 2.4 times with purified water (to 8.4 L). 4 % of activated charcoal and 2 % of Celite 545 were added to the diluted broth. The suspension was stirred for a period of 2 hours, after which 76 g of sodium chloride was added. The suspension was then passed through a depth filter of brand Sartorius.

[0091] The clarified product was then further diluted up to 6 times the original volume of the original broth with purified water (to 21 L) and filtered through a filter with a pore size of 0.2 pm, so as to sterilize the solution, according to a standard filtering sterilization method.

[0092] Following filtration, the diluted clarified product was concentrated until it reached the original volume of the fermentation broth after being diluted 2.4 times (8.4 L). The operation was carried out through tangential filtration with a filter of brand Sartorius, type Sartocon slice, with a cut-off of 10kD.

[0093] 8.4 L of concentrated product was introduced into a diafiltration system, and the pH adjusted to between 2.4 and 3 with the addition of HCI 1 M. During pH adjustment, the permeate was recirculated together with the retentate inside the container containing the product. When the pH reached a value between 2.4 and 3, the permeate was then excluded from the recirculation and sent to another container. The process was carried out through tangential filtration with a cut-off of 0.2 m, continuously replacing the permeate volume with sterile pyrogen free water. Throughout the process, the pH was maintained between 2.4 and 3. After 10 to 15 volumes of permeate, the diafiltration process was stopped. The permeate was again recirculated with the retentate in the container containing the diafiltered product, and the pH is adjusted to 7.0 with the addition of NaOH 1 M.

[0094] The resulting 8.4 L of product was further concentrated until it reached the original volume of the fermentation broth (3.5 L). The operation was carried out through tangential filtration with a filter of brand Sartorius, type Sartocon slice, with a cut-off of 10kD.

[0095] Final processing - Embodiment 1

[0096] 3.5 L of diafiltered and concentrated product (high molecular weight sodium hyaluronate) were precipitated in ethanol according to standard procedures, and the precipitate was dried in a vacuum oven at a pressure of less than 1 mbar, at a temperature of 40 °C for a period of 6-8 hours.

[0097] Final processing - Embodiment 2

[0098] 3.5 L of diafiltered and concentrated product were freeze dried according to a standard freeze dried process in order to obtain a dry powder of high molecular weight sodium hyaluronate.

[0099] In this Example, the yield in hyaluronic acid was about 87-88% with regard to the hyaluronic acid content in the crude solution. The analysed product was found to meet the specifications of the European and American pharmacopoeias. The resulting high molecular weight HA may therefore be used directly as a pharmaceutical composition.

[0100] Example 2 - Purifying process according to the present invention, including salt addition step during diafiltration

[0101] In this example, the fermentation, clarification and sterilization steps are as described above for Example 1. The diafiltration process is executed analogously to Example 1, with a notable modification introduced following the fifth occurrence of permeate volume. This variation involves the introduction of a salt into the process to ensure the displacement of all counterions bound to the carboxylic group of hyaluronic acid by an excess of cations derived from the added salt. Consequently, all residual proteins that are protonated due to the acidic pH — rendering them positively charged — are detached from the hyaluronic acid chains and subsequently washed away. This same modification can be applied to produce a distinct hyaluronic acid salt, differing from the original sodium one.

[0102] After 24 hours of fermentation, 3.5 L of viscous broth obtained as set out in General Methods was collected into a suitable vessel and diluted 2.4 times with purified water (to 8.4 L). 4 % of activated charcoal and 2 % of Celite 545 were added to the diluted broth. The suspension was stirred for a period of 2 hours, after which 76 g of sodium chloride was added. The suspension was then passed through a depth filter of brand Sartorius.

[0103] The clarified product was then further diluted up to 6 times the original volume of the original broth with purified water (to 21 L) and filtered through a filter with a pore size of 0.2 pm, so as to sterilize the solution, according to a standard filtering sterilization method.

[0104] Following filtration, the diluted clarified product was concentrated until it reached the original volume of the fermentation broth after being diluted 2.4 times (8.4 L). The operation was carried out through tangential filtration with a filter of brand Sartorius, type Sartocon slice, with a cut-off of 10kD.

[0105] 8.4 L of concentrated product was introduced into a diafiltration system, and the pH adjusted to between 2.4 and 3 with the addition of HCI 1 M. During pH adjustment, the permeate was recirculated together with the retentate inside the container containing the product. When the pH reached a value between 2.4 and 3, the permeate was then excluded from the recirculation and sent to another container. The process is carried out through tangential filtration with a cut-off of 0.2 pm, continuously replacing the permeate volume with sterile pyrogen free water. Throughout the process, the pH was maintained between 2.4 and 3. After 5 to 7 volumes of permeate, NaCI was added up to a minimum concentration of 10-15 mM in the product solution, with a preferable target of 150 mM. This was accomplished by introducing the salt as a solution in sterile, pyrogen-free water. The retentate recirculation proceeded seamlessly without any interruptions.

[0106] In an alternative process, the salt was introduced in its solid form. In this case, it was imperative to temporarily halt the recirculation to allow sufficient time for the salt to dissolve completely before resuming the diafiltration process.

[0107] In a further alternative process, the salt was incorporated gradually throughout the remaining diafiltration process. This was achieved by substituting the sterile, pyrogen-free water with a solution containing salt. This versatile approach allows for flexibility in adapting the method to specific process requirements.

[0108] In all cases, after a further 5-10 volumes of permeate, the diafiltration process was stopped. The permeate was again recirculated with the retentate in the container containing the diafiltered product, and the pH is adjusted to 7.0 with the addition of NaOH 1 M.

[0109] The resulting 8.4 L of product was further concentrated until it reached the original volume of the fermentation broth (3.5 L). The operation was carried out through tangential filtration with a filter of brand Sartorius, type Sartocon slice, with a cut-off of 10kD. Due to the presence of salt in the solution, another diafiltration process was carried out at pH 7.0, by exchanging 5 to 7 volumes of permeate with sterile pyrogen free water, with the same filter having a cut-off of 10 kD.

[0110] Final processing - Embodiment 1

[0111] 3.5 L of diafiltered and concentrated product (high molecular weight sodium hyaluronate) were precipitated in ethanol according to standard procedures, and the precipitate was dried in a vacuum oven at a pressure of less than 1 mbar, at a temperature of 40 °C for a period of 6-8 hours.

[0112] Final processing - Embodiment 2

[0113] 3.5 L of diafiltered and concentrated product were freeze dried according to a standard freeze dried process in order to obtain a dry powder of high molecular weight sodium hyaluronate.

[0114] In this Example, the yield in hyaluronic acid was about 87-88% with regard to the hyaluronic acid content in the crude solution. The analysed product was found to meet the specifications of the European and American pharmacopoeias. The resulting high molecular weight HA may therefore be used directly as a pharmaceutical composition.

[0115] Example 3 - Purifying process according to the present invention with alternative clarification method

[0116] In this example, the fermentation, sterilization and reduced pH diafiltration process steps are as described above for Example 1. Following fermentation, rather than adding activated charcoal and celite as for Examples 1 and 2, an alternative clarification process involving pH adjustment followed by diafiltration was carried out.

[0117] After 24 hours of fermentation, 3.5 L of viscous broth obtained as set out in General Methods was collected into a suitable vessel and diluted 2.4 times with purified water to reach a volume of 8.4 L. The pH was adjusted to 3.5 by adding HCI 1 M solution. Diafiltration was then performed using tangential flow filtration with a cut-off of 0.2 pm, continuously replacing the permeate volume with demineralized water, approximately 7 nominal volumes in total.

[0118] The permeate was collected in a container and pumped through a 0.2 pm filter cartridge to sterilize the solution according to a standard filtering sterilization method.

[0119] The sterilized permeate was transferred to another container and continuously concentrated via tangential flow filtration with a cut-off of 10 kDa. Upon completing the first diafiltration, when all the permeate had been sterilized and concentrated back to the initial volume of 8.4 liters, a second diafiltration was conducted using the same tangential flow filtration with a cut-off of 10 kD, again continuously replacing the permeate volume with approximately 7 nominal volumes of demineralized water.

[0120] This intermediate was subjected to another diafiltration process. The pH was adjusted to between 2.4 and 3 with the addition of HCI 1 M. During pH adjustment, the permeate was recirculated together with the retentate inside the container containing the product and the permeate valve closed. When the pH reached a value between 2.4 and 3, the permeate valve opened. The process was carried out through tangential filtration with a cut-off of 0.2 pm, continuously replacing the permeate volume with sterile pyrogen free water. Throughout the process, the pH was maintained between 2.4 and 3. After 7 volumes of permeate, the diafiltration process was stopped. The permeate was again recirculated with the retentate in the container containing the diafiltered product, and the pH is adjusted to 7.0 with the addition of NaOH 1 M.

[0121] Aluminium oxide (NORIT C Extra USP, particle size D10 (4 pm), D50 (20 pm), D90 (80 pm), B.E.T. 1100 m2 / g), activated charcoal, and sodium chloride were added to the solution in quantities of 1.5%, 1%, and 0.3% respectively, relative to the hyaluronic acid content. The suspension was agitated for 5 hours and then filtered through a 0.45 pm cut-off filter.

[0122] A final diafiltration was performed using tangential flow filtration to remove residual salts, employing a cut-off of 10 kD, continuously replacing the permeate volume with sterile pyrogen free water. 3.5 liters of diafiltered and concentrated product were freeze-dried following a standard freeze- drying process to obtain a dry powder of high molecular weight hyaluronic acid.

[0123] In this example, the yield of hyaluronic acid was approximately 92% relative to the product content in the crude solution. The analyzed product met the specifications of the European and American pharmacopoeias, with an endotoxin level of 0.01 lU / mg of HA and a protein content of 0.05%. Thus, the resulting product is suitable for use as an injectable pharmaceutical composition.

[0124] The keys steps of the process are shown in representative processes set out in Figures 7A, 7B, 8A and 8B.

Claims

CLAIMS1. A process for purifying high molecular weight hyaluronic acid or a salt thereof, comprising the following steps: a) obtaining an aqueous solution / suspension comprising high molecular weight hyaluronic acid from a biological source; b) subjecting the solution / suspension of step a) to a clarification step, which optionally comprises a dilution step; c) subjecting the solution / suspension of step b) to a sterilization step, which optionally comprises a dilution step; d) optionally concentrating the solution / suspension of step c); e) adjusting the pH of the solution / suspension of step c) or step d) to a pH in the range from 1.7 to 3.3 and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.80 pm.

2. The process according to claim 1 , wherein the aqueous solution / suspension comprising high molecular weight hyaluronic acid from a biological source is an aqueous broth containing high molecular weight hyaluronic acid from a microbial source.

3. The process according to claim 2, wherein the microbial source is a Streptococcus species producing high molecular weight hyaluronic acid.

4. The process according to claim 3, wherein the Streptococcus species producing high molecular weight hyaluronic acid is selected from the group consisting of Streptococcus equi (e.g. Streptococcus zooepidemicus) and Streptococcus pyogenes, and in particular is Streptococcus equi (e.g. Streptococcus zooepidemicus).

5. The process according to any one of claims 1 to 4, wherein the clarification step of step b) is a dilution and clarification process.

6. The process according to claim 5, wherein the dilution process involves diluting the solution / suspension of step a) to a total volume which is between about 1.5 times and about 3.5 times that of the volume of the aqueous solution / suspension in step a), such as between about 2 times and about 3 times, e.g. about 2.4 times.

7. The process according to claim 5 or claim 6, wherein the solution / suspension is diluted with water, e.g. demineralized water or purified water.

8. The process according to any one of claims 1 to 7, wherein the clarification step of step b) comprises adding activated charcoal and / or celite to the solution / suspension of step a).

9. The process according to claim 8, wherein the clarification step further comprises adding a salt such as sodium chloride to the solution / suspension.

10. The process according to any one of claims 1 to 9, wherein the clarification step comprises passing the solution / suspension through a filter e.g. a depth filter, a plate and frame filter such as a filter press, a belt filter or a centrifugal filter.

11. The process according to any one of claims 1 to 9, wherein the clarification step comprises a step of centrifugation.

12. The process according to any one of the claims 1 to 7, wherein the clarification step of step b) comprises adjusting the pH of the solution / suspension of step a) to a pH in the range from 3.4 to 4.0, and then diafiltering the solution / suspension at the same pH using a filter having a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.20 pm.

13. The process according to claim 12, wherein the pH of the solution / suspension of step a) is adjusted to about pH 3.5.

14. The process according to claim 12 or claim 13, wherein a surfactant is added to the solution / suspension during the clarification process of step b).

15. The process according to claim 14, wherein the surfactant is selected from the group consisting of sodium dodecyl sulphate (SDS), sodium lauryl sulphate (SLS), Tween 20®, Tween 80, Triton X-100 or X-114, octyl beta-glucoside, and combinations thereof.

16. The process according to claim 15, wherein the surfactant is included in the solution / suspension at a concentration of between about 0.05 wt% and about 0.2 wt%, such as between about 0.05 wt% and 1 wt%.

17. The process according to any one of claims 1 to 16, wherein the sterilization step c) is a filtering sterilization step comprising filtering the solution / suspension of step b) through a filter having a pore size of about 0.2 pm.

18. The process according to claim 17, wherein prior to the filtering sterilization step, the solution / suspension of step b) is diluted to a total volume which is between about 4 times and about 10 times that of the volume of the aqueous solution / suspension in step a), such as between about 4 times and about 8 times, e.g. about 6 times.

19. The process according to claim 18, wherein the solution / suspension is diluted with water e.g. demineralized water or purified water.

20. The process according to any one of claims 1 to 19, wherein in step d):- the solution / suspension of step c) is concentrated by tangential ultrafiltration with a filter having a pore size less than 100,000 Da nominal molecular weight; or- the solution / suspension of step c) is concentrated by tangential ultrafiltration with a filter having a pore size in the range from about 10 kD to 15 kD nominal molecular weight; or- the pH of solution / suspension of step c) is adjusted to a pH in the range between about 3.3 and about 1.7 (suitably to about pH 2.75), and the resulting solution / suspension is then concentrated by tangential microfiltration with a filter having a pore size less than 0.80 pm (e.g. less than 0.45 pm); or- the solution / suspension of step c) is concentrated by distillation under vacuum at a temperature less than 100 °C (suitably less than 60 °C, such as at about 40 °C).

21. The process according to any one of claims 1 to 20, wherein in step d), the solution / suspension of step c) is concentrated to a total volume which is between about 1.5 times and 3.5 times that of the volume of the aqueous solution / suspension in step a), such as between about 2 times and about 3 times, e.g. about 2.4 times.

22. The process according to claim 20 or claim 21 , wherein following concentration, the solution / suspension is subjected to tangential ultrafiltration with a filter having a pore size in the range from about 10 kD to 15 kD nominal molecular weight until the conductivity of the solution is less than 30 pS / cm (more or less 7 equivalent volumes).

23. The process according to any one of claims 1 to 22, wherein in step e), the pH of the aqueous solution / suspension containing the high molecular weight hyaluronic acid is adjustedto a value in the range from about 1.7 to about 3.3, e.g. in the range from about 2.4 to about 3.0 (e.g. about 2.75) and then the solution / suspension is diafiltered at the same pH.

24. The process according to any one of claims 1 to 23, wherein the filter used for the diafiltration of step e) has a pore size in the range from about 100,000 Da nominal molecular weight cut-off to about 0.75 pm (e.g. about 0.70 pm, about 0.65 pm, about 0.60 pm, about 0.55 pm, about 0.50 pm, about 0.45 pm, about 0.40 pm, about 0.35 pm, about 0.30 pm, about 0.25 pm or about 0.20 pm).

25. The process according to any one of claims 1 to 23, wherein the filter used for the diafiltration of step e) has a pore size in the range from about 300,000 Da nominal molecular weight cut-off to about 0.45 pm (e.g. about 0.2 pm).

26. The process according to any one of claims 1 to 25, wherein during the diafiltration of step e), water is added at the same rate as filtrate is removed, in order to maintain a constant volume, and diafiltration is carried out until 5-20, such as 5-11 , 12-20, 5-7, 12-18, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 equivalent volumes of water have been added.

27. The process according to claim 26, wherein after 3-10 equivalent volumes (e.g. after 7 volumes) of water have been added, a salt is added to the diafiltration solution / suspension.

28. The process according to claim 27, wherein the salt is a water soluble salt e.g. a sodium salt (such as NaCI) or a calcium salt (e.g. Ca(OH)2).

29. The process according to claim 27 or claim 28, wherein the salt is added at a concentration of between about 0.01 M and about 3 M, e.g. between about 100 mM and about 300 mM, e.g. about 150 mM.

30. The process according to any one of claims 1 to 29, wherein step e) is followed by step e-1) of adjusting the pH of the solution of step e) to a pH of between about 6 and about 8, e.g. about 7.31 . The process according to claim 30, wherein the pH is adjusted using an aqueous base (e.g. NaOH aqueous solution or Ca(OH)2aqueous solution).

32. The process according to any one of claims 1 to 31 , wherein step e) or step e-1) is followed by step e-2) of concentrating the solution / suspension of step e) or step e-1).

33. The process according to claim 32, wherein the concentration is carried out using tangential diafiltration e.g. through a filter having a pore size less than 100,000 Da nominal molecular weight (suitably less than 10,000 Da).

34. The process according to claim 32, wherein the concentration is carried out by evaporation under vacuum, suitably at a temperature of less than 100 °C (e.g. at about 40 °C).

35. The process according to any one of claims 32 to 34, wherein the solution / suspension of step e) or step e-1) is concentrated to a total volume which is between about 0.5 times and about 1.5 times that of the volume of the aqueous solution / suspension in step a), such as between about 0.75 times and about 1.25 times, e.g. a total volume which is about equal to the volume of the aqueous solution / suspension in step a).

36. The process according to any one of claims 32 to 35, wherein step e-1) is followed by step e-3) of a further diafiltration step at pH of between about 6 and about 8.

37. The process according to claim 36, wherein the diafiltration step is carried out by exchanging 5 to 7 volumes of permeate with water, with the same filter having a cut-off of 100,000 Da e.g. less than 10,000 Da.

38. The process according to any one of claims 1 to 37, wherein step e), step e-1), step e- 2) or step e-3) is followed by step e-4), of adding aluminium oxide, activated charcoal and sodium chloride to the solution / suspension.

39. The process according to claim 38, wherein the aluminium oxide has an average surface area (BET) of between about 50 m2 / g and about 200 m2 / g, preferably 100 m2 / g, and an average bead diameter of around 2 pm.

40. The process according to claim 38 or claim 39, wherein the aluminium oxide is included in the solution / suspension at a concentration of between about 0.1 wt% and about 4 wt% (preferably about 1.5 wt%).41 . The process according to any one of claim 38 to 40, wherein the activated charcoal is included in the solution / suspension at a concentration of between about 0.1 wt% and about 4 wt% (preferably about 1 wt%).

42. The process according to any one of claims 38 to claim 41 , wherein the sodium chloride is included in the solution / suspension at a concentration of between about 0.05 wt% and about 3 wt% (preferably about 0.3 wt%).

43. The process according to any one of claims 38 to 42, in step e-4) the solution / suspension is agitated at a temperature between about 4 °C and about 50 °C, such as between about 20 °C and about 25 °C, e.g. about 22 °C.

44. The process according to any one of claims 38 to 43, wherein in step e-4) the solution / suspension is agitated for a period of at least 5 hours up to 3 days, preferably for about 5 hours.

45. The process according to any one of claims 38 to 44, wherein the solution / suspension is depth filtered or centrifuged and subsequently filtered again through a filter having a pore size in the range of 0.1 pm to 2 pm, preferably 0.45 pm, in order to retain all the aluminium oxide and charcoal residues.

46. The process according to any one of claims 1 to 45, wherein step e), step e-1), step e- 2), step e-3) or step e-4) is followed by step f) of packaging the solution of high molecular weight hyaluronic acid or salt thereof.

47. The process according to claim 46, wherein prior to being packaged, an additional component is added to the solution of step e), step e-1), step e-2), step e-3) or step e-4).

48. The process according to claim 47, wherein the additional component is selected from the group consisting of a salt, a buffer, a stabilizer (such as trehalose), one or more active pharmaceutical ingredients, an antifibrinolytic substance (such as tranexamic acid), a coagulation activator (such as calcium gluconate), a silk protein (such as silk fibroin), and vitamin K2 ; and mixtures thereof.

49. The process according to any one of claims 46 to 48, wherein the solution of high molecular weight hyaluronic acid or salt thereof is packaged in sterile bags.

50. The process according to any one of claims 1 to 49, wherein step e), step e-1), step e- 2), step e-3) or step e-4) is followed by a step g) of drying the solution of step e), step e-1), step e-2), step e-3) or step e-4) to obtain a dry powder of hyaluronic acid or salt thereof.51 . The process according to claim 50, wherein the drying is freeze drying, sublimation or spray drying.

52. The process according to claim 51 , wherein the freeze drying is carried out using a spray dryer equipped with a rotary nozzle or with a twin fluid nozzle.

53. The process according to any one of claims 1 to 52, wherein step e), step e-1), step e- 2), step e-3) or step e-4) is followed by a step h) of adding a salt (e.g. sodium acetate, suitably at a concentration of between about 0.1 M and about 1 M e.g. about 0.3 M) to the solution / suspension, followed by precipitation with an organic solvent (e.g. comprising ethanol, acetone, methanol, or isopropanol, or a mixture thereof).

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