United States Patent c191
`Balazs et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,713,448
`Dec. 15, 1987
`
`[75]
`
`[54] CHEMICALLY MODIFIED HYALURONIC
`ACID PREPARATION AND METHOD OF
`RECOVERY THEREOF FROM ANIMAL
`TISSUES
`Inventors: Enclre A. BallW, Ft. Lee; Adolf
`Leshchiner; Adelya Leshchiner, both
`of Fairview, all of N.J.; Philip Band,
`Brooklyn, N.Y.
`[73] Assignee: Biomatrix, Inc., Ridgefield, N.J.
`[21] Appl. No.: 710,929
`Mar. 12, 1985
`[22] Filed:
`Int. 0.4 •••••.•••••••••••••••••• C08B 37/08; C08F 8/00;
`[51]
`Cl2P 19/04; C12R 1/46
`[52] U.S. a . .................................... 536/55.1; 435/267
`[58] Field of Search ........................ 435/267; 536/55.1
`References Cited
`[56]
`U.S. PATENT DOCUMENTS
`3,396,081 8/1968 Billek ................................. 536/55.1
`4,141,973 2/1979 Balazs .................................. 514/769
`4,272,522 6/1981 Balazs ................................... 424/94
`
`4,303,676 12/1981 Balazs .................................. 514/777
`4,487,865 12/1984 Balazs et al ........................ 525/54.2
`4,500,676 2/1985 Balazs et al ........................... 524/29
`4,517,295 5/1985 Bracke et al ....................... 536/55.1
`4,582,865 4/1986 Balazs et al. .......................... 524/27
`4,605,691 8/1986 Balazs et al ........................... 524/27
`4,629,623 12/1986 Balazs et al ........................... 424/63
`Primary Examiner-Ronald W. Griffin
`Attorney, Agent, or Firm-Sheldon Palmer
`[57]
`ABSTRACT
`Disclosed is hylan, a chemically modified hyaluronic
`acid preparation characterized by the presence of small
`amounts (0.005-0.05% by weight) of aldehyde cross(cid:173)
`linking groups covalently bonded to the hyaluronic acid
`molecular chains. Also disclosed is a method of obtain(cid:173)
`ing hylan comprising treating hyaluronic acid in situ in
`animal tissues containing same with a treatment mixture
`including a reagent (typically an aldehyde) which is
`reactive towards hyaluronic acid and the proteins con(cid:173)
`tained in the animal tissue.
`
`27 Oaims, 8 Drawing Figures
`
`Exhibit 1063
`Prollenium v. Allergan
`
`

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`Dec. 15, 1987
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`US. Patent
`Dec. 15, 1987
`U.S. Patent Dec. 15, 1987
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`

`CHEMICALLY MODIFIED HYALURONIC ACID
`PREPARATION AND METHOD OF RECOVERY
`THEREOF FROM ANIMAL TISSUES
`
`20
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The invention relates to a chemically modified hyalu(cid:173)
`ronic acid preparation characterized by novel chemical,
`physicochemical and rheological properties, and a 10
`novel method for obtaining such preparation.
`2. The Prior Art
`Hyaluronic acid (hereinafter referred to as HA) is a
`naturally occuring high molecular weight glycosami(cid:173)
`noglycan having a repeating disaccharide unit of D- 15
`glucuronic acid and N-acetylglucosamino-2-acetamido-
`2-desoxy-D-glucose joined by /31 -3 glucosidic bond.
`The disaccharides are joined to form an unbranched,
`uncrosslinked polysaccharide chain by /31--+4 gluco-
`sidic bonds.
`HA is found in animal tissues such as umbilical cord,
`vitreous, synovial fluid, rooster combs, skin, etc. The
`molecular weight of purified HA has been reported in
`the literature to be within the range of 50,000 to
`8,000,000 depending on the source, method of isolation 25
`and method of determination of molecular weight
`(Balazs, E. A., Fed. Proceed. 17, 1086-1093 (1958)).
`Several method have been suggested for recovery
`and purification of HA from animal tissues and bacterial
`cultures. Among these are enzymatic digestion of prote- 30
`ins (E. D. T. Atkins, C. F. Phelps and J. K. Sheehan,
`Biochem. J. 128, 1255-1263, (1972); R. Varma, R. S.
`Varma, W. S. Alten and A.H. Wardi, Carbohydr. Res.
`32, 386-395, (1974); treatment with ion exchange resins
`(T. C. Laurent, J. Biol. Chem. 216, 263-271, (1955); E. 35
`R. Berman, Biochim. Biophys. Acta 58, 120-122
`(1962)); precipitation with cationic surfactants (T. C.
`Laurent, M. Ryan, and A. Pietruszkiewicz, Biochem.
`Biophys. Acta 42, 476-485 (1960)); treatment with tri(cid:173)
`chloroacetic acid (H. Hofmans, 0. Schmut, H. Sterk, 40
`and H. Koop, Naturforsch. 34c, 508-511 (1979); D.
`Schmut, and H. Hofmans, Biochim Biophys. Acta 673,
`192-196 (1981)); preparative density gradient sedimen(cid:173)
`tation (P. Silpanata, J. R. Dunstone, A. G. Ogston,
`Biochem. J. 109, 43-50 (1968)); and electrodeposition 45
`(S. Roseman, D. R. Watson, J. F. Duff, and W. D.
`Robinson, Annals Rheumatic Diseases, 15, 67-68
`(1955)). One can also use a method which contains
`several different treatments, e.g., enzymatic digestion
`and precipitation with cetylpyridinium chloride (J. E. 50
`Scott, Biochem. J., 62, 31 (1956)).
`The principal problem encountered in recovering
`HA from any biological source involves separating the
`polymer (HA) from proteins and other biological poly(cid:173)
`mers which are extracted from the tissue along with the 55
`HA. Depending upon the raw material, the amount of
`undesirable polymers can be very large, exceeding by
`many times the amount of HA. The methods of HA
`recovery cited above are all used for the preparation of
`HA in laboratories but can· hardly be used for large 60
`scale production of HA because of various drawbacks
`inherent in each of those methods.
`The most advanced method for HA recovery and
`purification on an industrial scale is described in U.S.
`Pat. No. 4,141,973 (E. A. Balazs). According to this 65
`method, an ultrapure HA with a protein content of less
`than 0.5% by weight and a molecular weight more than
`1,200,000 is obtained by water extraction from rooster
`
`1
`
`4,713,448
`
`2
`combs or human umbilical cord. Proteins and other
`substances are removed with several chloroform extrac(cid:173)
`tions at varying pH values. In a chloroform extraction
`of the water extract an interface layer is formed in
`5 which denatured proteins and other substances are col(cid:173)
`lected. Some substances, e.g., fats, are solubilized, prob(cid:173)
`ably in the chloroform phase. The process can also
`include a treatment with a proteolytic enzyme, e.g.
`pronase. By combining several quite elaborate treat(cid:173)
`ments a process has been developed which allows one
`to obtain a pyrogen-free, non-inflammatory fraction of
`HA. This product is presently being marketed as a 1 %
`solution under the trademark Healon ® and is used in
`viscosurgery where it protects tissues against mechani(cid:173)
`cal damage, provides space and permits manipulation of
`tissues during surgery (E. A. Balazs, Healon, J. Wiley
`and Son, NY, 1983, pp 5-28).
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a graph showing viscosity vs. shear rate
`dependence for a 1 % (wt.) solution of HY in aqueous
`0.15 M NaCl (V =viscosity; S=shear stress);
`FIG. 2 is a graph giving the results of an oscillation
`test for a 1% (wt.) solution of HY in aqueous 0.15 M
`NaCl (V =viscosity; F=phase angle; G' =dynamic
`storage moduli; G"=loss moduli);
`FIG. 3 is a graph showing the relaxation curve for a
`1 % (wt.) solution of HY in aqueous 0.15 M NaCl;
`FIG. 4 is a graph showing the distribution ofESD for
`a 1 % (wt.) solution of HY (limiting viscosity number
`4,300 cc/g.) in aqueous 0.15 M NaCl;
`FIG. 5 is a graph showing the distribution ofESD for
`a 1 % (wt.) solution of HA (limiting viscosity number
`3,562 cc/g.) in aqueous 0.15 M NaCl;
`FIG. 6 is a graph showing viscosity vs. shear rate
`dependence for a jelly-type product according to the
`invention;
`FIG. 7 is a graph showing the results of an oscillation
`test for a jelly-type product according to the invention
`(V=viscosity; F=phase angle, G'=dynamic storage
`moduli; G"=loss moduli); and
`FIG. 8 is a graph showing the relaxation curve for a
`jelly-type product according to the invention.
`SUMMARY OF THE INVENTION
`In one aspect, the present invention provides a novel
`method for the in situ chemical modification of HA in
`animal tissues prior to its extraction therefrom.
`In another aspect, the invention provides a novel
`method for recovery and purification of the chemically
`modified HA from animal tissues.
`In still another aspect, the invention provides a novel
`ultra-pure, pyrogen-free, non-inflammatory chemically
`modified HA containing about 0.005 to 0.05% by
`weight of aldehyde cross-linking groups covalently
`bonded to the hyaluronic acid polymer chains.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention is based on our discovery that
`HA can be chemically modified in situ before it is ex(cid:173)
`tracted from animal tissues by treatment of the tissue
`with a substance that reacts with proteins and HA in
`aqueous media. Among these substances are formalde(cid:173)
`hyde, glutaraldehyde, glyoxal, etc. It has been found
`that this in situ chemical modification produces substan(cid:173)
`tial changes in the primary structure of the HA macro-
`
`

`

`4,713,448
`
`3
`molecule, in molecular size, inter- and intramolecular
`interactions and in the resulting rheological properties
`of solutions made from the modified product. It is,
`therefore, warranted to give this chemically modified
`HA a new name. We have chosen to call this material 5
`Hylan (hereinafter referred to as HY). When HA is
`extracted from an animal tissue, usually, a large amount
`of proteins go into solution along with it. The amount of
`protein can vary substantially depending upon the na(cid:173)
`ture of the tissue and the parameters of the extraction. 10
`In extraction of HA from rooster combs, the weight
`ratio of HA to proteins can vary from 1 :0.5 up to 1 :4
`(U.S. Pat. No. 4,303,676, E. A. Balazs). As a result, the
`first problem in recovery of pure HA from animal tissue
`is the removal of proteins. We have found that the 15
`above mentioned preliminary treatment of the tissue
`provides a water extract of HY with a substantially
`lower protein content than in the absence of such treat(cid:173)
`ment.
`The precise nature of the chemical events occurring 20
`during the treatment of the tissue is not fully understood
`and, therefore, the invention should not be limited by
`any specific chemical reaction. It is believed that, as a
`result of chemical reactions between proteins in the
`tissue and a reagent in the treating mixture, the proteins 25
`become denatured and immobilized in the tissues and
`are therefore insoluble in the subsequent aqueous ex(cid:173)
`traction.
`Any substance which reacts with proteins in a water(cid:173)
`containing medium can be used for the purpose of the 30
`present invention. We have found that the most advan(cid:173)
`tageous substance is formaldehyde. Other aldehydes
`such as glutaraldehyde or glyoxal also can be used in
`the method according to the present invention.
`The treatment of the tissue can be carried out in a 35
`water solution of the reagent. However, if this is done,
`there will be a substantial loss of HY because of its good
`solubility in water. For this reason, it is preferable to
`carry out the treatment of the tissue in a mixture of
`water and a water-miscible organic solvent. The solvent 40
`should not react with the reagent used for the protein
`immobilization. Among these solvents are lower ke(cid:173)
`tones such as acetone or methyl ethyl ketone, alcohols
`such as ethanol or isopropanol, aprotic solvents such as
`dimethylformamide, dimethylacetamide or dimethyl- 45
`sulfoxide and others. When such a solvent is mixed with
`a tissue which contains a large amount of water, usually
`80-90% or more by weight, a water-solvent mixture is
`formed. The water-solvent ratio in the mixture can be
`adjusted to any desired level by changing the solvent/- 50
`tissue ratio or by adding water to the mixture. The
`preferable water/solvent ratio is determined by HY
`solubility in the sense that the HY should not be soluble
`in the mixture used for the treatment of the tissue. The
`HY solubility depends upon the type of solvent used, 55
`water/solvent ratio, the presence and concentration of
`any electrolyte in the mixture and the pH of the mix(cid:173)
`ture. The HY solubility can be substantially reduced by
`introducing an electrolyte into the mixture. Any type of
`electrolyte can be used which is soluble in the water- 60
`solvent mixture and which provides the desired pH of
`the mixture. For example, when acetone is used as a
`solvent sodium acetate can be conveniently used as a
`soluble electrolyte.
`The composition of the mixture for treating the tissue 65
`can vary over a broad range depending upon the nature
`of the tissue, the type of solvent used, the type of elec(cid:173)
`trolyte, etc. As mentioned above, HY from the tissue
`
`4
`should not be soluble in the treating mixture but the
`latter should contain enough water to allow the tissue to
`swell so as to facilitate the reaction between the reagent
`and the tissue polymers. We have found that in the case
`ofrooster combs as a source of HY, the composition of
`the mixture, taking into account the water from the
`combs, can be in the following range, % by weight:
`water 10-50, solvent 40-85, electrolyte 0-20, reagent
`0.2-10. Several different solvents can be used in the
`same treating mixture, if desired. We have found it to be
`advantageous to use small amounts of water-immicsible
`solvents, such as chloroform, in the treating mixture.
`The content of these solvents in the mixture can be from
`0.5 to 10% by weight.
`The pH of the treating mixture can be varied depend(cid:173)
`ing upon the nature of the reagents, composition of the
`mixture, temperature and time of the treatment. In re(cid:173)
`covery of HY from animal tissues according to the
`present invention, the following considerations are very
`important. Some of the reagents, formaldehyde, for
`example, can react with hydroxyl groups of HA macro(cid:173)
`molecules at low pH to give a cross-linked polymer,
`insoluble in water. A prolonged treatment in a medium
`with a relatively high pH leads to the degradation of
`HA and only a low molecular weight polymer can be
`recovered. We have found that when an aldehyde type
`of reagent is used according to the present invention the
`best results are obtained with pH close to neutral, for
`example, in the range from 4 to 10.
`The ratio of the treating mixture to the tissue can
`vary over broad limits. The lower limit is determined,
`usually, by the provision that the tissue, which is quite
`bulky in the case of rooster combs, should at least be
`fully covered with the mixture. The upper limit can be
`chosen based on economical consideration. In treatment
`of rooster combs according to the present invention, the
`ratio of the treating mixture to the tissue (calculated on
`dry weight of the tissue) is usually more than 10:1.
`The temperature affects the efficiency of the treat(cid:173)
`ment according to the present invention. However,
`since HY is susceptible to hydrolysis at elevated tem(cid:173)
`perature, it is preferable to carry out the treatment at
`room temperature or below in order to obtain a high
`molecular weight product.
`The time needed to perform the treatment depends
`upon many factors including the composition of the
`mixture, the nature of the tissue, temperature, etc. It is
`assumed that the limiting factor in the treatment is the
`diffusion of the reagent into the tissue slices. For this
`reason, the size of these slices is an important parameter.
`We have found that in treatment of rooster combs sliced
`into pieces of 1-3 mm thickness, the time of the treat(cid:173)
`ment can be in the range of 4-24 hours.
`The tissue treated as described above is then washed
`with a solvent or a solvent/water mixture to remove the
`excess treating mixture from the tissue. It is convenient
`to use the same solvent as is used in the mixture for the
`tissue treatment. One can use any number of washings
`but, it has been found that one washing gives satisfac(cid:173)
`tory results.
`The washed tissue is then directly extracted with
`water to recover the HY. We have found that the effi(cid:173)
`ciency of the extraction depends upon the water/tissue
`ratio, pH of the extracting media, temperature and time.
`We have also found that the efficiency of the extraction
`of the treated tissue can be substantially increased by
`first drying the treated tissue to remove the solvent
`which was used in the treating and washing steps. The
`
`

`

`4,713,448
`
`6
`5
`tions of HY diffuse more easily from the tissue into the
`best results are obtained when the tissue is dried to ¼ to
`extract and the product precipitated from the first ex-
`½ of its original weight.
`tract is enriched with these fractions.
`The water/tissue ratio in the extraction step is chosen
`based on several considerations. First of all, there
`One can use more than two extractions to achieve as
`should be enough of the liquid phase to cover the tissue 5 high a yield as possible, but the concentration of HY in
`during extraction. On the other hand, the amount of
`an extract decreases with each consecutive extraction.
`water should not be too large in order to have as high a
`HY can be recovered from the filtrates by any
`concentration of HY in the extract as possible so as to
`method known in the prior art. The most convenient
`reduce the amount of precipitant in the next step of the
`method is precipitation with a water-miscible solvent
`process. We have found that in the case of rooster 10 such as acetone, ethanol, isopropanol, etc. The precipi-
`combs the preferred ratio of water tissue is from 2 to 5
`tation can be done in the presence of an acid such as
`based on the weight of the untreated combs.
`hydrochloric, sulfuric, phosphoric, etc., or in the pres-
`The pH of the extracting media can be kept neutral,
`ence of neutral electrolytes such as sodium acetate,
`acidic or alkaline depending upon the desired quality of
`sodium chloride and their salts. HY and its salts are
`the end product. We have found that in order to obtain 15 usually precipitated as a white fiber-like material or a
`ultra high molecular weight product, the pH of the
`powder. The precipitate can be washed with the same
`extracting media should be in the range of 6-8.5. A
`solvent which is used for precipitation, or with any
`higher pH leads to an increase in the HY concentration
`other solvent mixture which will not dissolve the prod-
`in the extract but, at the same time, to a decrease in the
`uct, for example, ether. The washed product can be
`molecular weight of the product and to changes in 20 dried with any conventional means or can be stored
`under a layer of a solvent such as acetone, ethanol, etc.
`other polymer properties which will be discussed below
`in more detail. In any case, by controlling the pH during
`Alternately, the filtrate can be freeze-dried.
`It is clear that any additional steps known in the prior
`the extraction step, one can conveniently regulate the
`properties of the end product in a desired direction.
`art and used in HA purification can be included in the
`It is preferable to extract the treated tissue at a tern- 25 process according to the present invention without
`perature less than 25° C. because the degradation of HY
`limiting its scope. For example, to remove pyrogens or
`at higher temperatures can substantially decrease the
`inflammatory agents, extraction by well known sol-
`molecular weight of the polymer.
`vents, such as chloroform, in which HY is insoluble but
`The time needed for extracting the maximum amount
`lipoproteins, glycolipids or glycolipoproteins are solu-
`of HY from the treated tissue varies substantially with 30 ble or separated, can be used.
`other parameters of the extraction, such as pH, liquid/-
`The process according to the present invention al-
`tissue ratio and intensity of stirring. We have found that
`lows one to obtain HY products with properties vary-
`in the extraction of rooster combs with water, good
`ing over a broad range. The following properties were
`results can be obtained when the treated combs are
`evaluated. The cited methods were used to characterize
`extracted for from 6 hours to several days.
`35 the products obtained according to the present inven-
`The mixture of treated tissue and extracting media
`tion.
`can be stirred during the extraction or left without any
`The HY concentration in solutions was determined
`stirring. Clearly, stirring will increase the diffusion rate
`by hexuronic acid assay using the automated carbazole
`method (E. A. Balazs, K. 0. Berntsen, J. Karossa and
`of HY molecules from the tissue into the extract. On the
`other hand, vigorous stirring can lead to degradation of 40 D. A. Swann, Analyt. Biochem. 12, 547-558 (1965)).
`HY and, hence, to a decrease in the molecular weight.
`·The hexosamine content was determined by the auto-
`In addition, we have found that vigorous stirring causes
`mated colorimetric method (D. A. Swan and E. A.
`tissue disintegration which makes it difficult to separate
`Balazs, Biochem. Biophys. Acta 130, 112-129 (1966)).
`the tissue from the extract. Therefore, it is preferable to
`Protein content of HY solutions was determined by the
`carry out the extraction step without or with very slow 45 phenol reagent method (Lowry et al., J. Biol. Chem
`193, 265-275 (1951)).
`and gentle stirring.
`After extraction, the tissue is separated from the ex-
`The formaldehyde content in the product was deter-
`tract using any of several conventional methods includ-
`mined by hydrolysis of about 0.1 g of the sample in 10
`ing filtration, centrifugation, decantation and the like.
`ml of 10% aqueous sulfuric acid for 2 hours with boiling
`We have found that the most simple and economical 50 followed by steam distilling free formaldehyde off the
`way is filtration. Depending upon the kind of tissue used
`solution obtained and determing formaldehyde in the
`as a starting meterial, it may be preferable to use a two-
`distillate using a colormetric method with chromo-
`tropic acid (M. J. Boyd and M. A. Logan, J. Biol.
`step filtration. Thus, in the case of rooster combs, large
`pieces of tissue can be easily separated by filtration
`Chem., 146, 279 (1942)).
`through a nylon mesh and the fine purification of the 55
`The limiting viscosity number (intrinsic viscosity)
`extract can be achieved by filtration through any dense
`defined as lim (n/no-1)/c where n and no are the viscos-
`ities of the solution and the solvent respectively, and c
`filter media, e.g., a cellulosic material.
`The HY concentration in the extract depends upon
`the concentration of HY in g/cc. The measurements
`were carried out in aqueous 0.20 M NaCl solutions in an
`many factors including pH during extraction, time,
`liquid/tissue ratio, and intensity of stirring. Usually it is 60 Ubbelohde capillary type dilution viscosimeter. The
`in the range of from 0.3 to 3.0 mg/ml and, sometimes,
`viscosity-average molecular weight is calcµlated by the
`equation [n]=0.0228 MO.SI (R. C. Cleland and J. L.
`even higher. In some cases, when the HY concentration
`in the extract is on the lower side, we have found it Wang, Biopolymers 9, 799 (1970)).
`The weight-average molecular weight is determined
`desirable to run a second extraction of the tissue. We
`have also found that the product precipitated from the 65 by the low angle laser light scattering method using a
`Chromatix KMX-6 instrument equipped with a helium-
`second extract usually has a higher molecular weight as
`neon laser set at 632.8 nm. The weight-average molecu-
`compared to the HY from the first extract. This can be
`explained by the fact that low molecular weight frac-
`Jar weight is also calculated from the sedimentation and
`
`

`

`4,713,448
`
`8
`7
`agents which are known to break the glucosidic bond of
`diffusion constants determined in an analytical ultra-
`the polysaccharide chain. Such well known agents are
`centrifuge.
`specific enzymes, .e.g, hyaluronidase, free radical gen-
`The dynamic light scattering method is used to evalu-
`erating systems, shear forces, heat, strong alkalies and
`ate the aggregation of the molecules in relatively con-
`centrated solutions of HY. This method gives the distri- 5 acids, etc.
`bution of equivalent spherical diameters (ESD) in solu-
`The partial specific volume (psv) of HY in solution
`tion.
`depends upon the ionic strength of the solution. It was
`Rheological properties were evaluated with the Boh-
`determined by densitometry for HY solution in water
`lin Rheometer System which is a computerized rheome-
`containing 0.15 M of NaCl in the concentration range
`ter.which can operate in three modes: viscometry, oscil- 10 from zero to 0.5 mg/ml and was found to be 0.627 cc/g.
`lation and relaxation. The following parameters are
`The distribution ofESD for a sample of HY dissolved
`in 0.15 M NaCl solution in 1 % solution is presented in
`measured for the HY solution: viscosity for the broad
`range of shear rates, dynamic viscosity, dynamic stor-
`FIG. 4. It is apparent from the data presented that HY
`age moduli and dynamic loss moduli for various oscilla-
`exists in a very highly aggregated form, although these
`15 aggregates are stable and do not sediment.
`tion frequencies and relaxation time.
`As mentioned above, the product according to the
`The rheological properties of a typical ultra-high
`present invention (HY) is a new polymer, obtained as a
`molecular weight product prepared according to the
`result of an in situ chemical reaction between HA and a
`present invention are presented in FIGS. 1-3. We have
`cross-linking agent, such as formaldehyde. We have
`found that this product forms solutions (0.5 wt % and
`found, by chemical analysis of HY, that the content of 20 higher) with remarkable viscoelastic properties.
`The following parameters characterize the elastic
`the combined formaldehyde in products obtained from
`properties of the polymer solutions in the best way:
`the rooster combs treated with a formaldehyde-contain-
`ing mixture, was in the range of from 0.005 to 0.02 wt.
`dynamic storage moduli (G'), the frequency of "cross-
`% calculated on the weight of polymer, depending
`upon the various parameters of the treatment.
`25 over point" (a point at which dynamic storage module
`The presence of the combined formaldehyde in the
`G' becomes greater than dynamic loss moduli G"),
`product was also proved by an experiment in which the
`phase angle and relaxation time.
`treatment was carried out with radiolabeled formalde-
`HY forms very viscous solutions in water or water
`hyde 14CHzO (see example 12 below). The results of the
`solution of electrolytes. The viscosity of the solution
`experiment show that the product obtained according 30 depends upon the polymer concentration, electrolyte
`to the invention contained combined formaldehyde
`content, temperature and decreases with shear rate, i.e.,
`the HY solutions have substantial pseudoplasticity.
`which could not be removed by repeated precipitations
`or by exhaustive dialysis of the polymer solutions This
`When the rheological properties of HY solutions are
`is strong evidence for the evidence of covalent bonding
`considered, one should understand that these properties
`of formaldehyde to the polymeric molecules of the 35 are greatly dependent upon the molecular weight of the
`product. In order to find out whether formaldehyde is
`product, as in the case of any other polymer. It was
`specifically combined with HA, the latter was treated
`mentioned above that HY can be obtained with a mo-
`with bacterial or leech hyaluronidases, enzymes specifi-
`lecular weight varying over a broad range and the rheo-
`cally degrading HA. The results of this treatment
`logical properties will vary accordingly. Thus, we have
`showed that a noticable amount of formaldehyde was 40 found that for the ultra-high molecular weight product
`(limiting viscosity number higher than 4500 cc/g) the
`covalently attached directly to the HY macromole-
`cules.
`viscosity of 1 wt% solution in 0.15 M NaCl solution in
`The protein content in the product obtained accord-
`water is up to 1000 Pa.s and even higher at shear rate
`ing to the present invention is usually not more than
`0.055, whereas it is only about 2 Pa.s for 1 wt % solution
`0.5% as calculated on the weight of a dry polymer and 45 of polymer with limiting viscosity number about 1000
`can be as little as 0.1 % and even less.
`cc/g. We have found that the elastic properties of HY
`The chemical modification of hyaluronic acid by
`solutions also depend upon the polymer molecular
`covalent attachment of a cross-linking agent to its mac-
`weight. Thus, the dynamic storage module G' for a 1 wt
`% solution of ultra-high molecular weight HY in 0.15
`romolecules, in other words the changes in the primary
`structure of the polymer, substantially affect its physi- so' M NaCl is about 40 Pa at a frequency 0.01 Hz, whereas
`it is only about 0.2 Pa for a solution of HY with limiting
`cochemical parameters such as molecular weight and
`molecular size, intermolecular interaction, and rheolog-
`viscosity number about 1000 cc/ g. The frequency of the
`ical properties of polymer solutions, as well.
`"cross-over point" which characterizes in a very good
`HY obtained according to the present invention can
`way the ratio between elastic and viscous properties of
`have a very high molecular weight. Thus, the limiting 55 polymer solutions is usually less than 0.025 Hz for 1 wt
`viscosity number can be higher than 7,000 cc/g which
`% HY solutions in 0.15 M NaCl at 25° C. when molecu-
`corresponds to a viscosity-average molecular weight of
`lar weight of the polymer is in the range from about
`1.5 X 106 to 8 X 106 and higher.
`around 6 X 106. The weight-average molecular weight
`from the light-scattering data can reach a value of
`The physicochemical and rheological properties of
`13 X 1Q6, We have found that this discrepancy between 60 an HY sample and its solution were compared with the
`same;! properties of an HA proquct obtained according
`the weight-average and the viscosity-average molecular
`weights to be quite meaningful as is discussed below. It
`to a well known method and are presented in Table 1
`and in FIGS. 4 and 5. The product us