BACKGROUND OF THE INVENTION

[0001] Hyaluronic Acid (HA) is a naturally-occurring linear, water-soluble, non-sulfated polymer of a disaccharide with repeating units of D-glucuronic acid and N-acetyl glucosamine (See Figure 1). In nature, there is a family of genes (LINK and non-LINK) that bind these polymers and function to non-covalently cross-linked HA polymers, stabilize extracellular matrices, act as cytoadherins and cell surface receptors for cellular activation (Kohda et al. 1996; Watanabe et al. 1997). HA is ubiquitously found in soft and hard tissues such as hyaline cartilage, skin and organ tissues, and bone, as well as synovial fluid (SF) of mammalian joints. In the latter location, it plays a role in joint lubrication, cushioning and resistance to shear forces, taking advantage of its unique viscoelastic physical properties.

[0002] During the development of osteoarthritis (OA) both the total concentration and average MW of HA diminishes in the SF. Thus, the loss in MW compromises the lubricating, cushioning and resistance to shear characteristics of the SF (viscoelasticity). The loss of these properties has been hypothesized to be a fundamental contributor of the OA symptoms of joint pain and stiffness. Balazs first described the purification of HA for medical use in 1979 (Balazs et al. 1979) and later the covalent chemical cross-linking of HA from animal-derived origins in an attempt to stabilize the higher MW forms of HA and increase residence time for therapeutic use (Balazs et al. 1992; Larsen et al. 1993). Tew (Tew 1984) reviewed the use of HA in horses to treat joint disorders and later the term “viscosupplemetation” was coined by Balazs to describe the treatment of degenerative joint disorders with HA to restore a more normal and healthy SF composition to improve lubrication. The covalent, chemically-cross-linked HA (Hylan) was thought to supplement the SF with a higher MW HA that would perform better in alleviating pain. The specification of the Hylan production utilized animal derived HA even though sources derived from biofermentation were known dating back to 1984 (See US 4801539, EP 694616, WO 03/054163, WO 98/22598). Fermentation-derived or microbiologically-derived HA (hereinafter meant to be interchangeable) often is most commonly derived from strains of Streptococcus and most recently recombinant Gram positive Bacillus hosts.

[0003] HA is biocompatible, biodegradable, and hydrophilic. Due to the abundant hydrophilic carboxyl groups that are part of its chemical structure, HA allows the influx and retention of large amounts of water to the biopolymer. (Allemaan & Baumann, 2008) Naturally-occurring HA has been reported to have a molecular weight ranging from 5 oligo, less than 6x10 Da, to low molecular weight, 0.8 to 8x10 Da, and a high molecular weight of 1x10^ Da (Girish & Kemparaju, 2007, Kogan, et al., 2007; Stern, Kogan, Jedzejas & Soltes, 2007;Day and Sheenan, 2001, Fouissac, Milas, Rinaudo & Borsali, 2002; Lapcik, Lapcik, DeSmedt, Demeester & Charbrecek, 1998; Laurent & Fraser, 1992).

[0004] The larger polymers apparently are the result of the hydrophilic interactions between the aforementioned LINK proteins, e.g. Aggrecan. The hyaluronan synthase genes (HAS) that synthesize HA seem to be limited to polymers of 2x 10^ in MW with the dominant gene expressed in adults being HAS-3 synthesizing 2x 10^ MW polymers (Galloway, et al. 2013).

[0005] Preparations of HA fragments with specific uniform sizes may be achieved by controlling the degradation of high molecular weight HA using acidic, alkaline, ultrasonic and/or thermal degradation (Stem et al., 2007). HA of defined length has been prepared by chemo enzymatic synthesis, as well (DeAngelis, 2008). These techniques provide an advantage in that the microbiological and enzymatic modes of preparation result in the potential absence of immunogenic foreign animal proteins that are often present in animal-derived HA.

[0006] Native non-cross-linked HA has a short half-life after it is injected to a mammalian skeletal joint due to degradation by hyaluronidases and reactive oxygen species (ROS) that can limit the HA utility (Biomacromolecules, 2006 Mar 7(3): 659-68; Soltes L, Mendichi R, Kogan G, Schiller J, Stankovska M, Amhold J). Some HA preparations used for cosmetic, medical and other human in vivo applications comprise a form of HA that has been cross linked to achieve a higher average molecular weight resulting in HA “hydrogels” offering longer residence time after injection (Carbohydrate Polymers, June, 2011). Cross linking may be achieved by physical entanglement of the HA chains, and by the formation of covalent bonds involving functional groups appended to the two monosaccharides comprising the basic HA disaccharide. Alternatively, non-covalent associations such as those due to Van der Waals forces between the saccharide units may be the basis of cross linking. There are various chemistries that are used to achieve covalent cross linking (Chante, Zuber, Vandamme) Carbohydrate Polymers, June, 2011; Khnmanee, Joeng, Park: Journal of tissue Engineering, 8, 2017, 1-16) and generally involve the covalent attachment of a linking agent between functional groups on HA, whether within a single polysaccharide HA strand or between such strands. The glucuronic acid carboxylic acid may be used in a variety of chemistries, both as an electrophile via various carboxyl activating methodologies, or as a nucleophile in the form of its carboxylate anion. The deacetylated amine of N-acetyl glucosamine may be used in a variety of ways including reductive amination with aldehyde terminal linking agents and amidation with carboxylic acid crosslinking agents. The hydroxymethylene of N-acetyl glucosamine is the focus of many covalent crosslinking methodologies. These may involve esters, acetals, for example with glutaraldehyde (Tomahita & Ikada (1977b); Collins & Birkishaw, 2007; Crescenzi et al, 2003a, 2003b) and formaldehyde (US 3713448) , or ethers derived by attack on epoxides such as 1,4-butanediol diglycidyl ether.( US 6921819), and Michael additions to acrylates and addition to vinyl sulfonates (Balazs & Leshchiner (1968); Collins & Birkinshaw (2007); Ramasmurthi & Vesely (2002)). These methodologies are directed away from the water solubility properties of native HA and toward the preparation of “hydrogels” with minimal or no water solubility properties, which may be desirable as a means to inhibit enzymatic or ROS degradation. [0007] Crosslinking using formaldehyde is unique in its simplicity. Aldehyde-based crosslinking using glutaraldehyde under low pH conditions has been reported (Schante, et al). This is likely to occur via the formation of hemiacetals between the aldehyde carbonyl moiety and the primary alcohol appendages of the N-acetyl glucosamines of two different HA strands (See Figure 2). The formation of hemiacetals is reversible under the low pH conditions required to form them, and the glutaraldehyde cross-linked material is found to be unstable unless the material is swelled in a buffer. [0008] A notable exception to the above examples directed to hydrogels with no or minimal water solubility properties, and the instability of aldehyde acetal crosslinks, is a crosslinked HA derived from rooster combs (native rooster comb HA average molecular weight of ca. 600-1200 kDa) prepared via the action formaldehyde in the presence of a lower ketone, such as acetone, and an ionic agent, such as sodium acetate (US 4713448). Several methods have been suggested for recovery and purification of HA from animal tissues and bacterial cultures, such as that found in US 55099013. In this treatment, formaldehyde is used to derivatize proteins native to the rooster comb to enable their separation from the HA while not solubilizing the HA. However, the formaldehyde treatment creates a limited crosslinking of the HA (0.005 to 0.02% by weight elements of formaldehyde content in the resulting cross-linked HA). The formaldehyde treatment medium is removed, the treated rooster combs are dried, then washed with water to extract a water-soluble limitedly formaldehyde cross-linked HA with an average molecular weight in the 3000 to 6000 kDa range. The cross-linked HA may be isolated as a solid material by pH adjustment and precipitation from the aqueous mixture with acetone.

[0009] For HAs that have been previously cross-linked with formaldehyde, an optional further crosslinking can be performed with the use of di vinyl sulfone (DVS) (US 4,582,865). This process results in a material that has some desirable properties while lacking water solubility. It is not apparent that such materials can be prepared by directly treating non-cross-linked HA from any source with divinyl sulfone alone.

[0010] Animal-derived HA, in particular, avian-derived HAs such as those derived from rooster combs, may contain about 0.35 to about 3% protein, depending on a number of variables. There are reports that the protein elements may be covalently linked to HA (Mikuni- Takagaki, 1981 #6424; Swann, 1975 #6425, ) and that the protein content of HA has a correlation to intrinsic viscosity, such that the protein-HA complex has a higher viscosity than HA alone.

[0011] Proteins associated with natural HA may be able to play a more direct role in altered rheology of HA. The apparent crosslinking observed for rooster comb-derived HA following formaldehyde treatment may not be due to formaldehyde alone but may include covalent bonds to functional groups on proteins native to rooster comb. Those proteins may serve as a bridge between two or more molecules of HA (See Figure 3). The crosslinking would comprise, as an example, an acetal bond between a hydroxyethyl moiety on N-acetyl glucosamine with formaldehyde, with the same molecule of formaldehyde bonded to an amine or hydroxyl group on a protein. See Figures 4a and 4b for examples of formaldehyde crosslinking with bridging involving lysine or serine sidechains. Further, other locations on the same protein molecule would be engaged in the same arrangement via the intermediacy of formaldehyde with another molecule of HA (See US 4713448). It is assumed that the production of these “amino bridges” found in the animal-derived HA are necessary for a stable HA-HA cross linking.

[0012] There is a correlation between these proteins in cross linked rooster comb HA and unique safety concerns such as rare localized inflammatory reactions, pseudoseptic arthritis, granulomatous inflammations and severe acute inflammatory reactions (SAIR), in particular with such HA’ s as Hylan G-F 20 or Synvisc-One® . It is important to distinguish local pseudoseptic reactions from systemic anaphylactic reactions that have been rarely reported after intra-articular HA administration. Pseudoseptic arthritis or SAIR may occur with the second or third injection in a cycle or with subsequent courses of Hylan G-F 20 or Synvisc-One administration. Clinically, it may be difficult to immediately differentiate a severe acute inflammatory reaction (SAIR or pseudoseptic arthritis) to Synvisc-One from a septic knee without culture reports. Although the mechanism for SAIR’s is unknown, it has been hypothesized that the chemical cross-linking (using formaldehyde) of the amines of proteins to the HA polymer during the manufacturing process to increase the MW of Hylan A may be responsible for these adverse reactions due to the participation of rooster comb proteins incorporated during the HA extraction process. Indeed, in the U.S. FDA post-marketing safety reports captured in the Manufacturer and User Facility Device Experience (MAUDE) database, the incidence of presumed SAIR reactions is 10-15 fold greater than other HA products.

[0013] As such, there would be an advantage to crosslinking non-animal-derived HA, ostensibly in the absence of immunogenic proteins, to achieve an HA material with an average molecular weight in the approximately 3000 to 6000 kDa range. Those resultant HA’ s should have water soluble properties not offered with more highly crosslinked HA. One such illustration of HA as cross-linked only by an aldehyde is shown in Figure 2. To the extent that proteins in animal-derived HA may play a role in the cross linking with formaldehyde, it has not been obvious that such successful cross linking may take place in the absence of such extraneous proteins. The present invention thus provides a benefit in the search to provide a stable, non-animal -derived, cross-linked HA of an average molecular weight which confers properties of water solubility and that is useful in therapeutic administration. High molecular weight crosslinked HAs are usually not soluble and are usually in gel form. Those cross-linked HA’s that are greater than 500 kDa and less than 10000 kDa, more preferably 6000 kDa, of the invention are not a gel, are stable over time and are water soluble.

SUMMARY OF THE INVENTION

[0014] The present invention provides a non-animal based or derived, cross-linked HA that includes non-animal derived HA, such as microbiologically-derived or fermentation- derived HAs (herein to mean the same thing), as well as chemically or synthetically made HAs, and has a molecular weight of about 500 to about 16000 kDa, preferably about 500 kDa to about 10000 kDa, with a preferred range of about 3000 to about 6000 kDa. Further, the low crosslink HA of the invention is water soluble. Additionally, this HA is cross-linked by covalent bonds which do not hydrolyze under physiologic pH of 7.35-7.45 and is stable over time and does not lose the water solubility beneficial in a medicament. In particular, the HA of the invention is not a hydrogel and is stable over time. This stability over time is evident for about 24 hours to about 144 hours of exposure to aqueous media at pH 11.5, well above and far more erosive of chemical bonds than physiologic pH of 7.35-7.45.

[0015] The HA of this invention is greater than 500 kDa to less than 10000 kDa and is water soluble and non-avian derived. It is preferably about 500 kDa to about 6000 kDa, and even more preferred of about 3000 kDa to about 6000 kDa.

[0016] The HA of the invention is less prone to inducing immunogenic reactions when administered to a subject because of its lack of avian - derived proteins in the formulation. The subject can be an animal or human being. For use in animals and human beings, the HA of the present invention is therefore used to treat musculoskeletal ailments such as to be used for viscosupplementation.

[0017] It is thus another objective of the present invention to provide a HA that is administered to a subject.

[0018] The stability and water solubility of the present cross-linked HAs make them thus able to remain in the body of an animal or human being for a prolonged residence time. Thus, the HAs of the invention are well suited to be administered to subjects for therapeutic purposes.

[0019] It is a further object of the present invention to provide a HA for administration as in viscosupplementation therapy.

[0020] Another object of the present invention is to provide a process for the preparation of the HA of the invention. The process of making the HA herein comprises preparing a mixture of microbiologically-derived HA in an aqueous medium, such as water or PBS. Microbiologically-derived HA includes materials made with various strains Streptococcus zooepidemicus, recombinant Gram positive Bacillus hosts, e. coli hosts, yeast hosts and the like. This preparation of HA is allowed to form a solution. The pH of this solution is adjusted using an aqueous acid, such as a mineral acid or an organic acid, and/or an aqueous base such as sodium hydroxide. This is then mixed with di vinyl sulfone (DVS) in an aqueous medium and incubated.

[0021] Further pH adjustment by using aqueous acid is then done. The preparation is then precipitated by adding an organic solvent, such as acetone. This resultant precipitated material is a solid product and is isolated, for example, by filtration, ion exchange or MW sieving to remove low MW contaminants, and dried to remove volatiles such as water and organic solvents.

[0022] As a further step in the process, after the volatiles are removed, optionally, the product is dissolved in water and lyophilized to form a white powder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGURE 1 : Disaccharide Core for Hyaluronic Acid (HA) shows repeating disaccharide molecular core of hyaluronic acid (HA). [0024] FIGURE 2: Illustration of Acetal Cross Linking represents chemical cross linking of HA via acetal covalent bonding.

[0025] FIGURE 3: Illustration of Formaldehyde Cross Linking Via Protein represents a cross linking between HA subunits via bridging between HA subunits involving formaldehyde and a protein.

[0026] FIGURE 4a: Illustration of Protein Bridge Via Serine Acetal provides a generic chemical representation of cross linking of HA covalently attached via formaldehyde to serine as an example of potential protein bridge attachment points between HA functional groups, formaldehyde, and a protein.

[0027] FIGURE 4b: Illustration of Protein Bridge Via Lysine Hemiaminal provides a generic chemical representation of cross linking of HA covalently attached via formaldehyde to lysine (right) as an example of potential protein bridge attachment points between HA functional groups, formaldehyde, and a protein.

[0028] FIGURE 5: GPC Comparison of Formaldehyde Cross-Linked HA from Rooster Comb (3000- 6000 kDa) with 750-1000 kDa Commercially Available Lifecore Biomedical HA. This is an overlay of GPC chromatography traces for formaldehyde cross-linked rooster comb HA (heavy black curve) with an average MW of 3000-6000 kDa and for commercially available, microbiolgically-dervied Lifecore Biomedical HA, average MW 750-1000 kDa (grey dashed curve), used as a GPC chromatography standard for purposes of laboratory activities reported herein.

[0029] FIGURE 6: Graphic Depiction of the Relationship Between Mn, Mw, Mz and Mv. This generic plot shows the relationship between the various molecular moments used to evaluate different aspects of molecular weight in a polydisperse material such as a biopolymer like HA.

[0030] FIGURE 7: Illustration of Formaldehyde Acetal Cross Linking. This is an illustration of the cross linking of HA via acetal covalent bonding with formaldehyde.

[0031] FIGURE 8: Chemical Structure of Spermine that can be used as a polyamine bridge to aid cross linking via formaldehyde.

[0032] FIGURE 9: Chemical Structure of poly-L-lysine that can be used as a polyamine bridge to aid cross linking via formaldehyde. [0033] FIGURE 10: Illustration of Protein Bridge Cross Link via Poly-L-lysine Hemiaminal. This chemical representation is of HA cross linking as mediated by formaldehyde with a poly-L- lysine bridge. Illustrated are hemiaminal covalent bond linkages between HA, formaldehyde and poly-L-lysine.

[0034] FIGURE 11 : Rheological Comparison of Untreated and Formalin Treated Microbiologically-derived HA. Plots of G’ and G” data for HTL Biotechnology microbiologically-derived HA that has and has not been treated with formalin (Aqueous solution of formaldehyde) shows crossover behavior for untreated material consistent with a freely soluble, minimally interacting polymer, and it shows lack of crossover for treated HA, consistent with a high cross link density (gel). Black diamonds depict G’ for untreated HA. Open diamonds depict G” for untreated HA. Solid grey dots depict G’ for treated HA; Open grey circles depict G” for treated HA.

[0035] FIGURE 12: Rheological Comparison of Microbiologically-derived HA with Various Cross Linking Systems. These are comparative plots of G’ and G” for HTL Biotechnology microbiologically-derived HA, vs the untreated HA control, as treated with formalin alone, or with formalin is in the presence of poly-L-lysine (the protein) or in the presence of spermine. Solid black line depicts G’ for HA control in water; Solid grey curve depicts G” for HA control in water. Black short-dashes curve depicts G’ for HA treated with formalin in water (5: 1 w/w, vs. HA); Grey short-dashes curve depicts G” HA treated with formalin (5: 1 wt/wt, vs. HA) in water. Black long-dashes curve depicts G’ for HA treated with formalin (5: 1 wt/wt vs. HA) and poly-L-lysine in water; Grey long-dashes curve depicts G” for HA treated with formalin (5: 1 wt/wt vs HA) and poly-L-lysine in water. Black sequential long dashes-short dashes curve depicts G’ for HA treated with formalin (5: 1 wt/wt vs. HA) and spermine in water; Grey sequential long dashes-short dashes curve depicts G” HA treated with formalin (5:1 wt/wt vs HA) and spermine in water. This provided a comparison of relative crossover frequencies in view of treatment conditions.

[0036] FIGURE 13: GPC Molecular Weight Distributions for Various Treatments of Microbiologically-derived HA. These over lay traces are for HTL Biotechnology microbiologically-derived HA, vs untreated HA control, as treated with formalin, formalin in the presence of poly-L-lysine, and formalin in the presence of spermine, thus enabling a comparison of the relative molecular weights on the basis of retention times. Dashed vertical grey lines denote center points for individual GPC peaks. Grey solid curve depicts formaldehyde cross-linked rooster comb HA. Black short-dashes curve depicts the untreated HA control, Black long-dashes curve depicts HA treated with formalin (5: 1 wt/wt ratio vs. HA). Black solid curve depicts HA treated with formalin (5 : 1 wt/wt ratio vs HA) and spermine in water. Grey long-dashes curve depicts HA treated with formalin (5: 1 wt/wt ratio vs HA) and poly-L-lysine in water.

[0037] FIGURE 14: Microbiologically-derived HA Treated with a Series of Weight/Weight Ratios of Formalin (50% Solution in Water), vs. the Untreated HA Control. This figure provides comparative plots of G’ and G” for HTL Biotechnology microbiologically- derived HA, vs. the untreated HA control, as treated with various wt/wt ratios of formaline to HA, thus enabling a comparison of relative crossover frequencies in view of treatment conditions. Black sequential long-dashes short-dashes curve depicts G’ for untreated HA control. Grey sequential long-dashes short-dashes curve depicts G” for untreated 750-1000 kDa linear control. Solid black curve depicts G’ for HA treated with 10: 1 wt/wt formalin to HA. Solid gray curve depicts G” for HA treated with 10: 1 wt/wt formalin to HA. Black short-dashes curve depicts G’ for HA treated with 7.5: 1 wt/wt formalin to HA. Grey short-dashes curve depicts G” for HA treated with 7.5:1 wt/wt formalin to HA Black long-dashes curve depicts G’ for HA treated with 5 : 1 wt/wt formalin to HA. Grey long-dashes curve depicts G” for HA treated with 5:1 wt/wt formalin to HA.

[0038] FIGURE 15 : Viscosity Regression Behavior for Mi crobiologically -Derived HA Cross- Linked with Formaldehyde in Water. This provides the comparative viscosities for formaldehyde cross-linked HTL Biotechnology microbiologically-derived HA after exposure to water for various lengths of time thus showing regression in viscosity. This suggests an apparent reversal of cross linking and return to the untreated (native) state. Black sequential long-dashes short dashes curve represents change in viscosity after 24 h. Black short-dashes curve represents change in viscosity after 28 h. Grey long-dashes curve represents change in viscosity after 33 h. Black long-dashes curve represents change in viscosity after 47 h. Grey solid curve represents change in viscosity after 52 h. Black solid curve represents change in viscosity of 73 h. Grey short dashes curve represents change in viscosity after 114 h.

[0039] FIGURE 16: Frequency Sweep Data for Cross Linked Rooster Comb HA With and Without Enzyme. The comparative plot of G’ and G” highlight the increase in crossover frequency for formaldehyde cross-linked rooster comb HA in water after exposure to proteinase K vs. the untreated material in the absence of proteinase. This suggests that formaldehyde cross linking of rooster comb HA is aided by linkages with proteins, the disruption of which leads to degradation of cross linking. Black solid curve depicts G’ for formaldehyde cross-linked rooster comb HA without enzyme. Grey solid curve depicts G” for formaldehyde cross-linked rooster comb HA without enzyme. Black dashed curve depicts G’ for formaldehyde cross-linked rooster comb HA with enzyme. Grey dashes curve depicts G” for formaldehyde cross-linked rooster comb HA with enzyme.

[0040] FIGURE 17: Shear Rate Sweep Data for Formaldehyde Cross-Linked Rooster Comb HA with and without Enzyme. This is a comparison of viscosity that highlights the differences in viscosity for formaldehyde cross-linked rooster comb HA that has and has not been treated with proteinase K. This illustrates the lower viscosity, and by implication, the reduced cross linking and average reduced molecular after treatment with the proteinase. Grey curve represents cross-linked rooster comb HA without enzyme. Black curve represents cross-linked rooster comb HA with enzyme.

[0041] FIGURE 18: Illustration of HA Cross-Linked via Divinyl Sulfone (DVS) This figure depicts the covalent bonding of the N-acetyl glucosamine hydroxymethyl locus with DVS, wherein DVS crosslinks two subunits of HA.

[0042] FIGURE 19: GPC Data for Untreated HA and for HA Treated with DVS under a Variety of Conditions, Fitted for Average Molecular Weights. These overlain GPC traces, arranged left to right for increasing average molecular weight, show relative average molecular weights for untreated HTL Biotechnology microbiologically- derived HA (2400-3600 KDa), for untreated Lifecore Biomedical 750-1000 kDa HA GPC standard, and for the HTL Biotechnology HA after treatment with DVS under two conditions, illustrating the relatively higher average molecular weight of cross-linked HA obtained after DVS treatment. Solid grey curve denotes untreated Lifecore Biomedical 750-1000 kDa HA control. Grey dashes curve denotes untreated HTL Biotechnology HA. Black short-dashes curve denotes HTL Biotechnology HA at 0.75 weight % in aqueous media after treatment with 10 wt% DVS (vs. HA) at pH 8. Solid black curve denotes HTL Biotechnology HA at 0.65 weight % in aqueous media after treatment with 10 wt% DVS (vs. HA) at pH 11.5. Black long-dashes curve denotes the reanalysis for HTL Biotechnology HA after treatment with 10 wt% DVS (vs HA) in aqueous media at pH 11.5.

[0043] FIGURE 20: GPC Overlay of DVS Treated Microbiologically-derived HA with Untreated HA Control. These overlay GPC traces are arranged left to right for increasing molecular weight for untreated HTL Biotechnology HA exposed to the reaction medium in the absence of DVS and for HTL Biotechnology HA at 0.65 wt% in aqueous media after treatment with DVS at a 10: 1 HA/DVS wt/wt ratio at pH 11.5. This shows an increase in average molecular weight after treatment. Black curve denotes 2400-3600 kDa HA exposed to reaction media in the absence of DVS. Grey curve denotes treated HA treated with DVS.

[0044] FIGURE 21 : GPC Overlay for DVS Treated Microbiologically-derived HA with Formaldehyde Cross-Linked Rooster Comb HA (MW 3000-6000 kDa), the Untreated Lifecore Biomedical 750-1000 kDa HA GPC chromatography Standard, and the Untreated HTL Biotechnology microbiologically-derived HA (2400-3600 kDa), fitted for Molecular Weight. These overlays are arranged from left to right for increasing molecular weight for untreated Lifecore Biomedical 750-1000 kDa HA (black long- dashes curve), untreated HTL Biotechnology 2400-3600 kDa HA (black short-dashes curve), and for the HTL Biotechnology HA at 0.69 wt% in aqueous media after treatment with 10: 1 wt/wt HA to DVS at pH 11.5 (grey long-dashes curve) and grey curve; reinjected sample for 10: 1 wt/wt HA to DVS at pH 11.5. These are compared with formaldehyde cross-linked rooster comb HA (black curve), showing comparable peak maxima, supporting comparable average molecular weight between DVS treated HA with the formaldehyde cross-linked rooster comb HA. DETAILED DESCRIPTION OF THE INVENTION

[0045] As is seen by the description of the past attempts to cross link and obtain an HA of value in therapeutic uses or medicaments uses (hereinafter referred to as the same), a sample polymer of HA may be comprised of a mixture of materials of differing molecular weights, and thus may be thought of as polydispersable material. In order to study and determine the usefulness of these cross-linked HA’s, their viscoelastic properties are studied and reviewed. For purely elastic solids, the stress is directly proportional to the strain (not the strain rate) and deformation is reversible. In contrast, for purely viscous liquids, the stress is directly proportional to the strain rate (not the strain), and deformation is not reversible. Thus, for viscoelastic materials, such as HA, both solid-like and fluid-like behaviors are observed, and stress is dependent on both strain and strain rate. The cross-linked entities, that are molecular microstructures in the larger HA polymer that can alter viscoelastic behavior of material, are investigated by small amplitude oscillatory shear (SAOS) measurements.

[0046] During SAOS testing, G’, the storage modulus, provides a measure of elasticity, while G”, the loss modulus, provides a measure of viscosity. At low frequencies, when G” is greater than G’, the viscous properties dominate and the material exhibits fluid-like properties. Alternatively, at higher frequencies, when G’ is greater than G”, the elastic properties dominate, and the material exhibits solid-like behavior. Thus, during SAOS measurements of viscoelastic materials, a crossover point, when G’ is equal to G” is observed that marks the transition from a material with more fluid-like behavior to a material with more solid-like behavior.

[0047] Assuming measurements are made on solutions with the same concentration of material, the crossover frequency will decrease as the crosslinking density increases.

[0048] The average molecular weights of HA and cross-linked varieties may be evaluated using gel permeation chromatography (GPC). GPC curves represent the qualitative distribution of molecular weights within a material, and they provide a view of the average molecular weight within a selected number of standard deviations from the apex of the curve. GPC also shows bimodal or further distributions of average molecular weight subpopulations. This methodology is particularly useful for less extensively cross-linked materials, and a standard may thus be used as a comparative measurement (See Figure 5).

[0049] A further evaluation is provided by poly dispersity analysis, which estimates polymer molecular weight by measuring averages associated with categories of mass distributions that contribute to the overall average molecular weight. Each “average” is sensitive to different aspects or “moments” of distribution (RJ Young and PA Lovell, Introduction to Polymers, 1991; Septo, RFT; Gilbert, RG; Hess, M; Jenkins, AD, Jones, RG, Kratochvil, P, 2009) “Disparity inPolymer Science” Pure Appl. Chem. 81(2): 351- 353). This average includes the following:

• Mn, the number average molecular weight moment (derived form the number of molecules above and below the molecular weight in the distribution);

• Mw, the weight average molecular weight moment (derived from the weights of molecules above and below this molecular weight in the distribution);

• Mz, the z-average molecular weight, is the most sensitive to the highest molecular weights

• Mv, the viscosity-average molecular weight, is similar to the Mw, but is modified by the interaction of the polymer with the solvent (through the Mark-Houwink exponent, a [Paul, Hiemenz C., and Lodge P. Timothy. Polymer Chemistry. Second ed. Boca Raton: CRC P, 2007. 336, 338-339; Rubinstein, Michael, and Colby, Ralph H.. Polymer Physics. Oxford University Press, 2003.])

[0050] Molecular weight moments and poly dispersity indices (PDI) are calculated as per the following equations:

[0051] Mv is most closely related to the Intrinsic Viscosity (IV) of the system, which is a measure of the contribution of the solute to the viscosity of the system. The ratio of the weight average molecular weight and the number average molecular weight provides the poly dispersity index, a value which represents the breadth of molecular weight distribution. The larger the PDI, the broader the distribution. A graphic representation of the range of weights represented by each average is provide in Figure 6.

[0052] With this information and explanation of measurements made pursuant to the present invention, the following examples are provided to assist in exemplifying the invention. These examples are provided to help describe the invention and are not meant to be limitative thereof.

[0053] Example 1 - This example isolates a 3000 to 6000 kDa cross-linked HA from commercially available (Genzyme Biosurgery) rooster comb derived Synvisc-One. Synvisc-One consists of an aqueous mixture of 80%, by weight, of a limited formaldehyde cross-linked water-soluble HA and 20%, by weight, of a more extensively cross-linked, water-insoluble HA component. The more extensively crosslinked component is prepared by initial cross-link with formaldehyde, followed by treatment with DVS. The formaldehyde-only cross-linked HA and the formaldehyde then DVS crosslinked HA are formulated in a combination that provides 8 mg/ml of total HA in aqueous media.

[0054] Specifically, an 8.9 g sample of Synvisc-One, presumed ca 71,2 mg total HA sample content) is placed in a nylon mesh bag (McMaster Carr 9318T48 Nylon 207 Mesh - 83 microns opening). The entire assembly is placed in an excess of water, and the water- soluble material is allowed to diffuse out. After a period of time, the bag is removed, and the dissolved, freed material is precipitated from the filtrate by adding 1 L acetone, thus providing 65.5 mg of a solid material (ca. 92%% recovery of the total HA in the product). This material is presumed to comprise largely water soluble 3000-6000 kDa crosslinked HA with some fraction of more extensively HA that traversed the membrane. The recovered water-soluble cross-linked HA when compared using gel filtration chromatography (GPC) against Lifecore Biomedical 750-1000 kDa HA GPC standard is shown to have a relatively higher average molecular weight (See Figure 5). An illustration of HA as cross-linked only by formaldehyde is shown in Figure 7.

As previously indicated, HA’s in the 3000 to 6000 kDa MW range confer certain benefits over lower MW HA’s. Presently, marketed products that utilize HAs in this MW range in their manufacturing process, such as Synvisc-One, utilize avian derived HA as its starting material. Here we are describing a technique that instead utilizes microbiologically-derived or otherwise nonanimal derived HA as its starting material. The intent of using microbiologically-derived or other non-animal derived HA as a starting material is to avoid incorporation of avian antigens into the final product and consequently reduce the likelihood of triggering a Severe Acute Immune Response (SAIR) to the HA product. [0055] Example 2 - This example is a formaldehyde treated microbiologically-derived HA with and without, separately, poly-L-lysine and spermine. HTL Biotechnology microbiologically-derived HA is exposed to formaldehyde (50% concentrated formalin in a 10: 1 ratio formaldehyde to HA) in water and acetone at ambient temperature (4- 35°C). Then, it is treated with dilute aqueous sodium hydroxide, followed by dilution with acetone to precipitate the treated material in an attempt to achieve limited cross linking of this microbiologically- derived HA. This procedure is also conducted in the presence of spermine (Figure 8), and separately, it is conducted in the presence of poly- L-lysine (Figure 9). These would become involved in cross linking with HA via formaldehyde as a hemiaminal as depicted, for example, in Figure 10 for poly-L-lysine.

[0056] Specifically, HTL Biotechnology microbiologically-derived HA (average MW of 2400-3600) (10 mg) is treated with 50% wt/wt concentrated formalin (200 volumes 10: 1 ratio formaldehyde vs. HA) in water (1000 volumes) and acetone (1200 volumes) at ambient temperature (ca. 4-35°C) for 24 hours. It is then treated with 0.04 M aqueous sodium hydroxide, followed by acetone (10 volumes vs. the volume of the HA solution in water) to precipitate the treated material. This procedure also is conducted in the presence of, separately, approximately 5 mg spermine and approximately 6.5 mg of poly-L-lysine.

[0057] The HA in this example treated with formaldehyde alone or in combination with spermine or poly-L-lysine is exposed to frequency sweeps at 1% stain from 100 Hz to 0.01 Hz oscillatory shear (logarithmic 10 points per decade). For the formaldehyde cross-linked HA, G’ remains above G”, indicating a predominantly elastic fluid and highly cross-linked material (See Figure 11) (i.e., a gel). By contrast, a crossover point is observed for the untreated HA control (see Figure 11).

[0058] The treatment in the presence of spermine and poly-L-lysine affords small chunks of gel within the aqueous mixture, indicative of cross linking to a greater degree than observed using formaldehyde alone (not depicted in Figure 11).

[0059] Example 3 - Cross linking of HA using reduced weight/weight ratios of formalin, spermine or poly-L-lysine is studied:

[0060] Specifically, to 50 mg HTL Biotechnology microbiologically-derived HA (MW 2400- 3600) dissolved in 5 ml water (100 volumes) is added a mixture comprising 50% concentrated formalin (0.5 ml, 10 volumes vs. HA, 5:1 weight ratio formalin: HA) and 6 mL acetone (120 volumes). The mixture is shaken briefly and incubated for 25 hours at ambient temperature (ca. 4-35°C). Subsequently, 6 mL of 0.04 M aqueous NaOH is added to this ambient temperature mixture. The mixture is shaken to ensure complete dissolution and aged one hour at ambient temperature. Then, 1 mL 0.1 M aqueous NaOH/Ac is added, and the mixture is shaken to ensure complete mixing. This mixture is diluted with 200 mL (4000 volumes) acetone, and the HA is collected by filtration and dried under vacuum at ambient temperature. The dried product is prepared as a solution in water at 10 mg/mL for purposes of rheological and GPC evaluation.

[0061] The above procedure is repeated in the presence of spermine and poly-L-lysine. In each experiment, a spatula tip of each additive is added to the HA before any solvents or formalin solution. The respective products are isolated, dried, and prepared for analysis in the same manner as described above where HA is treated using only formalin.

[0062] Crossover occurs at lower frequencies for the samples prepared with spermine and poly-L-lysine relative to the untreated HA control and vs. the sample prepared with formalin alone (See Figure 12). The samples prepared with spermine and poly-L-lysine also have higher moduli, indicating a higher deformation energy storage capacity consistent with a cross-linked material. GPC overlay (See Figure 13) of treated samples vs. the untreated HA control and vs. formaldehyde cross-linked rooster comb HA show relatively higher molecular weights approaching cross-linked rooster comb material when spermine and poly-L-lysine are included, but show little change vs control using formalin alone.

[0063] Based on comparative rheologic and GPC analyses, neither formaldehyde alone or with the additives of spermine or polylysine provide material with average molecular weight matching that of a cross-linked rooster comb HA, with a crossover frequency of 0.0155 Hz

[0064] Example 4 - In this example HTL Biotechnology microbiologically-derived HA treated with formalin at pH 7, 8, 9 and 10 is compared with formaldehyde cross-linked rooster comb HA (See Table 1). Among the pH levels tested, the crossover frequency is lowest at pH 7, at 0.07084 HZ, vs. the untreated HA control, at 0.1699 Hz. However, the crossover data do not closely approach that for the rooster comb material, at 0.0155 Hz. The crossover modulus increases with decreasing pH, which is consistent with an increasing degree of crosslinking. To the extent that crossover frequency may be correlated with average molecular weight, these conditions for preparing the HA using formaldehyde crosslinking do not produce cross-linked HA with an average molecular weight comparable to the cross-linked rooster comb derived HA.

[0065] Specifically, in each of separate four vials, 25 mg of HTL Biotechnology microbiologically-derived HA (MW 2400-3600) prepared as a solution in 2.5 mL water, (ie - concentration of 10 mg/mL) is treated with 0.5 mL Formalin Fixx (50% weight /weight formaldehyde in water) and 3 mL acetone, agitated, and then allowed to incubate overnight at ambient temperature (ca. 4-35°C). After overnight incubation, each sample is then individually treated with dilute aq. NaOH to achieve a pH of 10 (0.4 M aq. NaOH), 9 (0.004 M aq. NaOH), 8 (0.0004 M aq. NaOH) and 7 (0.000004 M aq NaOH and allowed to age for about 1 hour. After the aging period, each sample is treated with 0.5 mL of 0.1 M sodium acetate and diluted with acetone (10 volume vs. the starting volume of HA in water) to precipitate the product that is then collected by filtration and dried under vacuum at ambient temperature. The dried product is prepared as a solution in water at 10 mg/ml for purposes of rheological evaluation. (See Table 1)

TABLE 1

Crossover Frequency and Crossover Modulus as Function of Formaldehyde Treatment pH

[0066] Example 5 - In yet this example HTL Biotechnology microbiologically-derived HA is treated with formalin (50% formaldehyde weigjht/weight in aq solution) alone in a weight/weight ratio to HA of 10: 1, 7.5: 1 and 5:1.

[0067] Specifically, HTL Biotechnology microbiologically-derived HA (average MW of 2400-3600) in water (10 volumes) and acetone (120 volumes) is treated with Formalin Fixx (50% wt/wt formaldehyde in water) alone with ratios by volume of Formalin Fixx to HA of 10:1, 7.5: 1 and 5: 1.. Each vial is stirred 25 hours at ambient temperature (ca. 4-35°C), then individually treated with 0.04 M aqueous sodium hydroxide (120 volumes), followed with 0.1 M sodium acetate (20 volumes). The resulting materials in each vial is then precipitated with acetone (10 volume vs. the starting volume of HA in water), and the isolated materials are vacuum dried without applying heat. The resulting materials after this treatment, in each case, are dissolved in water at a concentration of 10 mg/mL in IX PBS, and then are examined for rheological properties (See Figure 14).

[0068] Ratios of 10: 1 and 7.5: 1 yield gels, as indicated by the rheology (no crossover point), and the ratio of 5: 1 yields a material that is Theologically similar to the untreated HA control (Figure 14).

[0069] These data in combination suggest that formaldehyde treatment results in some degree, of crosslinking of HA, an effect that increases with formaldehyde alone at or near pH 7, and in the presence of polyamines. Analyses using GPC and comparative rheology with regard to crossover point vs. cross-linked HA as stabilized by proteins in the rooster comb suggest that the target 3000 to 6000 kD molecular weight is not achieved.

[0070] Example 6 - In addition to failing to achieve a cross-linked microbiologically-derived HA comparable with formaldehyde cross-linked rooster comb HA, upon further examination it is apparent that formaldehyde treated microbiologically-derived HA stored as a solution in aqueous media is not physically stable over time and appears to revert to the untreated state. Examples of data demonstrating this behavior are shown in Figure 15, which illustrates that an aqueous solution of formaldehyde cross-linked microbiologically-derived HA undergoes a decrease in viscosity over 24-144 hours. In Table 2, it is seen that a decrease in the modulus and an increase in crossover frequency occurs over the same period, both indicative of a degradation in crosslinking.

[0071] This example evaluates the physical stability of microbiologically derive HA treated with formaldehyde.

[0072] Specifically, HTL Biotechnology microbiologically-derived HA treated with Formalin Fixx as in Example 5, above, at the 10: 1 ratio is stored unstirred at ambient temperature in water at about pH 7. The mixture is evaluated at intervals of 28, 33, 47, 73 and 144 hours. At each time point, the aqueous mixture is exposed to frequency sweeps at 1% strain from 100 Hz to 0.01 Hz oscillatory shear (logarithmic 10 points per decade). Viscosity regression behavior data are plotted in Figure 15. Data for crossover frequency, crossover modulus and zero sheer rate viscosity are provided in Table 2 and demonstrate changes over time consistent with a degradation of crosslinking. TABLE 2

Rheological Evaluation of Reversal in Formaldehyde Cross-linked hyaluronic acid

[0073] This observed behavior is consistent with cross linkages via hydrolytically unstable acetals. Further, these data support the possibility that stable cross links in HA from rooster combs treated with formaldehyde may involve proteins in the rooster combs. The change over time in the rheology after formaldehyde treatment also suggests that behaviors attributed to covalent cross linking may instead be due to physical phenomena. This contrast in behavior between microbiologically-derived HA treated with formaldehyde and formaldehyde treated rooster comb HA highlights a challenge in order to achieve a cross-linked HA, stable in aqueous media, using formaldehyde, or any aldehyde, in the absence of proteins in the material matrix.

[0074] Example 7 - The involvement of proteins in the cross linking observed in formaldehyde-treated rooster combs HA is further supported by enzymatic digestion. A sample of the formaldehyde cross-linked rooster comb HA is isolated above by bag filtration (See Example 1) and is digested in an aqueous solution in Proteinase K. This enzyme reduces the moduli of the solution, moves the crossover to higher frequencies and reduces the viscosity of the mixture, consistent with disruption of cross linking and supporting a role for proteins in the cross linking result observed for formaldehyde treated rooster comb HA (Figure 16). Figure 17 depicts the comparative loss in viscosity observed for formaldehyde cross-linked rooster comb HA, with and without exposure to Proteinase K.

[0075] Specifically, a 1.5 mL sample of formaldehyde cross-linked rooster comb derived HA as isolated in Example 5 as a solution in IX PBS at 10 mg/mL concentration is treated with 15 uL of proteinase K at 15- 30°C. The mixture is initially shaken gently, then allowed to incubate overnight at 15-30°C.

[0076] The mixture is then exposed to frequency sweeps at 1% strain from 100 Hz to 0.01 Hz oscillatory shear (logarithmic 10 points per decade). The enzyme reduces the moduli of the solution, moves the crossover point to higher frequencies and causes viscosity to degrade (See Figures 16 and 17). This is consistent with disruption of covalent bonds with protein, and supports a role for proteins in the cross linking observed when formaldehyde treatment is used to crosslink rooster comb derived HA.

[0077] The experiments and examples above suggest that while it is possible to treat HA in the absence of animal proteins with formaldehyde alone to obtain a material that exhibits decreased crossover frequency and other evidence cross linking (e.g. GPC retention time vs. control), the effect appears reversible over time if the treated HA is exposed to aqueous media. The instability of such cross-linked HA is thus contrary to its use in a water-based medicinal preparation. Further, it appears that is has not been possible to obtain a cross-linked HA with the target average molecular weight of 3000 to 6000 kDa.

[0078] Alternatively, HA may be cross-linked with divinyl sulfone (DVS). Cross linking using DVS occurs under basic conditions thereby allowing the two electrophilic vinyl groups of DVS to react with the nucleophilic hydroxymethyl moi eties on HA to form covalent ether bonds that are not hydrolysable at physiologic pH (pH 7.35-7.45). One possible structure of such a cross-linked system is shown in Figure 18. Previous reports regarding the cross linking of HA with DVS begin with mildly cross-linked HA (US 4713448) to form insoluble gels or entail direct treatments of non-cross-linked HA directed toward gels. Thusly prepared cross-linked HA’s need extensive mechanical manipulation to produce physically flowable particles and lack water solubility (US 4636524).

[0079] Example 8 - Microbiologically-derived HA is treated with aqueous DVS. [0080] Solutions of HTL Biotechnology microbiologically-derived HA (2400-3600 kDa) are prepared in in 1 X PBS in the concentrations shown Table 3 in 20 mL scintillation vials. Aqueous NaOH (IN) and aqueous HC1 (IN) are added to adjust pH as per Table 3. Then, DVS dissolved in one volume IX PBS (vs DVS) is added to give the HA DVS concentrations shown in Table 3. The mixtures are briefly vortexed then incubated for 24 hours at ambient temperature (4-35°C). Then, the mixtures are treated with dilute aqueous HC1 (IN) to adjust pH to neutral at ambient temperature (4- 35°C). After that, the resultant mixtures are diluted with acetone (10 volume vs. the starting volume of HA in 1 X PBS) to precipitate the materials. The precipitated materials are re-dissolved in IX PBS at a concentration of 10 mg/mL and compared by GPC (See Figures 19 and 20). They are tested for their various comparable molecular moments by using the Agilent GPC/SEC Software (v A.02.01).

[0081] Various pHs, concentrations of HA and relative quantities of DVS are explored. The general procedures in these experiments involve preparing an aqueous solution of HA, adjusting the solution pH toward basic conditions; adding an aqueous DVS solution; vortexing the mixture; then incubating at ambient temperature ( 4-35°C) for up to several days. These mixtures are then neutralized using dilute aqueous acid and treated with a water-soluble organic solvent to precipitate the cross-linked HA. The resulting product may optionally then be re-dissolved in water and reprecipitated by diluting with a water-soluble organic solvent to remove residual salts. As an alternative, the solid product may optionally be dissolved as an aqueous mixture and lyophilized. Samples of HA obtained after DVS treatment are then analyzed for their comparative molecular moments using Agilent GPC/SEC Software (v A.02.01).

[0082] Specifically, treatment at about pH 8 and about pH 9 with HTL Biotechnology microbiologically-derived HA at 0.65 wt% in PBS and a HA to DVS weight ratio at 10: 1 affords little molecular weight gain during the 24 hour incubation period. At pH 8 with HA at 0.75 wt% in PBS and a HA to DVS weight ratio of about 10: 1, weight increase is observed during the 24-hour incubation period. At pH 11.5 with a HA at 0.65 wt% in PBS and a HA to DVS weight ratio of about 10: 1 significant weight gain is observed during the 24-hour incubation period. [0083] Treatment conditions of HTL Biotechnology microbiologically-dervied HA at 6.5 mg/mL in aqueous media with DVS in a weight ratio of 10: 1 (HA:DVS) at pH around 8 and 9 for approximately 24 hours (See Table 3) produce little increase in average molecular weight vs. untreated HA control as estimated using poly-dispersity indices (See Table 4). Treatment of the HA at 7.5 mg/mL and at the 10: 1 weight/weight ratio of HA to DVS at pH 8 for about 24 hours show a marked increase in average molecular weight (Table 4). Likewise, treatment of the HA at 6.5 mg/mL and at the 10:1 weight/weight ratio of HA to DVS at pH 11.5 for about 24 hours show a marked increase in average molecular weight (Table 4) Figures 19 and 20 provide GPC overlays for DVS-treated microbiologically-derived HTL Biotechnology HA according to conditions in Table 3, along with the substrate (2400-3600 kDa HA, HTL Biotechnology) exposed to the cross coupling media in the absence of DVS (Control 1) , untreated substrate as purchased, (Control 2) and untreated Lifecore Biomedical 750-1000 kDa HA .

TABLE 3

Experimental Conditions for DVS Cross-linked Microbiologically-derived Hyaluronic Acid TABLE 4

Molecular Moments and Poly Dispersity Indices for DVS Cross-linked Hyaluronic Acid As Per Table 1

[0084] Example 9 - This example further evaluates favorable cross linking conditions for microbiologically-derived HA using DVS from the previous example (Example 8) and compares the product with Lifecore HA (750-1000 kDa), untreated HTL Biotechnology microbiologically derived HA (2400-3600 kDa) and formaldehyde cross-linked rooster comb HA.

[0085] Specifically, HTL Biotechnology microbiologically-derived HA (2400-3600 kDa) (1.04 g) is taken up in 1XPBS (117.2 mL). It is allowed to dissolve for about 24 hours. The pH is adjusted using 1 N aqueous HC1 and 1 N aqueous NaOH to a pH of 11.5. Then, 8.9 pL (10.5 mg) of DVS in 33mL of IX PBS is added, and the mixture (1.04 g/150.2 mL = 0.69 wt %) is incubated for 96 hours at ambient temperature (ca. 4-35°C). The mixture is then neutralized using 1 NHC1. Then, the resulting cross-linked material is precipitated as a solid by adding approximately 1 liter of acetone and isolated by filtration. The resulting material is re-dissolved in water at approximately 10 mg/mL and lyophilized to afford a white powder. The cross-linked HA obtained by this procedure is re-dissolved in water at a concentration of 10 mg/mL and analyzed by GPC (See Figure 21). The GPC analysis for thusly treated HA shows a higher average molecular weight vs. the Lifecore Biomedical 750-1000 kDa GPC standard and vs. the untreated HTL Biotechnology HA control, and co-elutes with the formaldehyde crosslinked rooster comb HA isolated from commercial product (See Example 1 above).

[0086] The GPC maxima for the material prepared after approximately 4 days of incubation in Example 9 (see Figure 21) is comparable with the GPC maxima for HA treated under the same conditions for approximately a day in Example 8 (see Tables 3 and 4, RB2- 9-4; see Figure 19). HA in both cases is used in vast molar excess to DVS. Because the GPC maxima are comparable in both cases, no further crosslinking is observed after the extended incubation to approximately four days, indicating the reaction between HA and DVS reaches completion within about a day and that DVS is substantially depleted in that period, with little or none remaining for further reaction with HA. The approximately 4-day incubation period extends well beyond this apparent depletion of DVS. The comparability of the GPC maxima for HA treated for about a day and for about four days indicates that after extended exposure to the aqueous reaction media at pH 11.5 in the presence of little or no DVS the cross-linked HA remains stable. Under the milder physiologic condition of pH 7.35 - 7.45 the cross-linked HA of this example is comparably or more stable vs. at pH 11.5. This is in sharp contrast to Example 6 wherein formaldehyde cross-linked microbiologically-derived HA showed substantial loss of crosslinking at approximate pH 7 within as little as 33-47 hours.

[0087] The data suggest that the DVS treatment as provided herein produces a water soluble, limitedly cross-linked HA, stable in aqueous media, having an average molecular weight closely matching that for the rooster comb-derived material, 3000-6000 kDa. However, it is achieved without reliance on immunogenic foreign proteins or polyamine additives, an unexpected result based on experiences with formaldehyde and the technology previously described (See US 4713448). This is even more unexpected given that in US 4636524 when the concentration of HA in the reaction mixture is less than 1% by weight, a cross-linked gel cannot be obtained even at low HA/DVS ratios (See US 4636524).

[0088] This DVS cross-linked material thus is useful for medical uses in a manner that provides HA without the immunogenic liability and reactions caused by animal proteins in the HA materials currently used.

[0089] Sources of Materials for Example - HTL Biotechnology microbiologically derived solid powdered HA, average molecular weight 2400-3600 was used for cross linking examples. Lifecore Biomedical microbiologically derived, solid powdered HA with average molecular weight of 750-1000 kDa is used as the linear HA GPC chromatography standard. Poly-L- lysine is obtained from Sigma Aldrich. Spermine is obtained from Sigma Aldrich.

[0090] Formalin Fixx is obtained as a 50% wt/wt solution in water from Fisher Scientific. DVS is obtained from Van Waters, and Rogers (VWR).

[0091] Analytical Methodology - Rheometry General Setup A TA instrument AR-G2 shear rheometer is used for the testing (CPG ID No. 11460) with a 2-inch stainless steel cone, 60 truncation (CPG ID No. 11490). Use of the instrument follows (CPGSOP0012). Before use each day, the instrument inertia is determined (19.01 to 19.05 microN.m to the second), the geometry is added, and the geometry inertia determined (8.27-8.36 microN.m squared). Then, the “zero” position is determined for the upper geometry relative to the lower geometry. On each day of testing, a standard reference fluid (Cannon S600 lot# 13301) with a nominal viscosity at 37.78 degrees C of 486.4 mPas.s is run. The viscosity standard is run under the same conditions as the example samples. The selected geometry is chosen to provide a balance between sensitivity (a function of diameter) and sample volume under the plate. This geometry requires 0.69 ml of fluid and therefore uses approximately half of the total volume available in the smallest samples.

[0092] Small Amplitude Oscillatory Shear (SAOS) relies on the material under test being in its linear viscoelastic regime to generate meaningful complex modulus values. For simple fluids, this requirement is generally not an issue, but for more complex fluids, it is important to verify that the testing is being performed in this regime.

[0093] Rheometry experimental procedures - Before each run, the geometry is removed from the instruments, and both the geometry and lower plate of the instrument are cleaned with lint-free “Kimwipes” and isopropyl alcohol, with a follow up wipe clean with water. The geometry is place back on the instrument, and then the gap is zeroed. The temperature is set to 37 degrees C throughout the testing. The geometry is then backed off from the surface by approximately 5 cm, and then it is measured and scaled by the known volume. The information about the volume is used to determine the length of plunger travel required to obtain 0.7 ml. This length is then marked on the syringe using an indelible marker. The liquid is then expelled directly from the syringe by depressing the plunger until the mark is reached. The geometry is immediately lowered into place and rotated slowly to distribute the liquid evenly around the geometry. Then, the example experiment is started. During the initial equilibrium phase, excess fluid is scraped away if required, and a thin layer of 5 mPa.s silicone oil (Sigma Aldrich PN 317667) is deposited onto the exposed fluid surface to prevent evaporation.

[0094] The following example experimentation steps are as follows:

• Example experimental step

• Frequency sweep

• Using 5% strain unless noted 100 Hz to 0.01 Hz oscillatory shear; logarithmic 10 points per decade Post example experimentation Hold at 37 degrees C Size Exclusive Chromatography (SEC) Gel Permeation Chromatograph (GPC, also known as SEC) testing is performed using an Agilent PL-GPC 220 system equipped with a 1200 infinity pump/de-gasser, a refractor index detector, a G7821A viscometer and a G7822A multi angle laser light scattering detector (MALLS; two angles at 15 and 90°). Sample preparation is performed using an Agilent PL-SP 260 VS Sample Prep Station.

[0095] The column chosen for separation is the Waters Ultrahydrogel Linear Column 10 micron, 7.8 mm X 300 mm. This column has a nominal molecular weight separation range from about 500 g/mol to 10,000,000 g/mol. A single column is used to minimize band broadening and overall run time. The Ultrahydrogel column is packed with a stationary phase consisting of about 10-micron gel particles composed of a cross-linked hydroxylate polymethacrylate, that contains some residual carboxyl functionality. The Ultrahydrogel column provides stability in a pH range of 2 to 12 and a temperature range of 10°C to 8°C. Compatible mobile phases include pure aqueous phases, as well as aqueous solutions of organic solvents, such as methanol, ethanol, acetonitrile, formic acid and dimethylsulfoxide.

[0096] During initial method optimization, sample concentrations and method flow rate are reduced until no adverse chromatographic behavior is observe (poor peak shape, evidence of poor material diffusion through the mobile phase, light scattering date suggesting prolong elution of high molecular weight material, and the like). Samples are ultimately analyzed at a concentration of approximately 0.028 mg/mL Samples are allowed to dissolve overnight in the mobile phase prior to loading on the instrument auto sampler. The mobile phase selected consists of 0.1 NaNO3 with 0.02% sodium azide. The sodium nitrate is added to mitigate aggregation effects while the sodium azide is added to mitigate bacterial growth.

[0097] Samples are analyzed in triplicate using the conditions described in Table 4. An injections volume of 200 pL is used to maximize signal to noise under the dilute sample concentrations employed. A slow draw up speed of 52 pL/sec is used to improve injection reproducibility and mitigate the potential for sample shear degradation. For similar purposes, samples are not filtered prior to analysis, and the flow rate through the column is minimized to prevent potential shear degradation of the high molecular weight material. The mobile phase is triple filtered through a 0.02 um inorganic membrane filter, and a 0.2 um post column PES (polyethersulfone) filter is used to reduce background light scattering noise.

[0098] A summary of the sample preparation conditions is described in Table 5. No evidence of an insoluble fraction is observed for any of the tested samples.

TABLE 5

GPC test parameters

[0099] Data collected by refractive index and dual angle light scattering is used for characterization of the molecular weight distribution, molecular weight moments and poly dispersity of the samples. The data is analyzed using the Agilent GPC/SEC software v A.02.01. The system is calibrated with a single narrow polyethylene oxide standard (Mw + 148,000 g/mole, American Polymer Standards.).

[0100] The system is verified with a single injection of a certified Agilent EasiVial PEG/PEO standard, and triplicate injections of two certified HA standards (LifeCore).Molecular weight moments and poly dispersity indices (PDI) are calculated by Agilent GPC/SEC Software (v A.02.01).:

[0101] The examples provided herein are meant to be illustrative of the invention and not limitative thereof.