CA2146938C - Method of controlling structure stability of collagen fibers produced from solutions or dispersions treated with sodium hydroxide for infectious agent deactivation - Google Patents

Abstract

In accordance with the present invention, it has been discovered that the stability of collagen fibers is altered upon treatment of a solution or dispersion of the collagen fibers with a chemical reagent for the inactivation of infectious agents. This fiber destabilization makes it difficult to formulate the collagen fibers to final product when the formulation contains an ingredient which alters the destabilized collagen fibers.
Collagen fiber stability in a collagen dispersion can be controlled using either a physical fiber-stabilizing agent or a chemical fiber-stabilizing agent. The physical fiber-stabilizing agent is used to protect the stability of the assembled fibers, without providing for covalent bonding between fibrils which make up the fibers. The chemical fiber-stabilizing agent is used to provide covalent bonding between fibrils, whereby the fibers are stabilized.
A stabilized dispersion of collagen fibers is achieved by the presence of a physical fiber-stabilization means comprising a polymeric material of the kind which is capable, at sufficient concentration, of causing collagen fibers to precipitate from solution. Preferred polymeric materials are water soluble or water miscible. Preferably the water soluble or water miscible polymeric material is biocompatible, such as a polyethylene glycol or derivatives thereof.
A stabilized dispersion of collagen fibers is also prepared by using a chemical fiber-stabilizing agent which crosslinks fibrils within the fiber, stabilizing the fiber.
A preferred method of stabilizing the collagen fiber produced from a solution ora dispersion of collagen treated with an agent for the inactivation of infectious agents is to stabilize the collagen fiber subsequent to the treatment, by using one of the physical or chemical fiber-stabilizing means.

Description

214fi93~

METHOD O~ COI~ROLLING STRUCTURE STABILlTY OF COLLAGEN
FIBERS PRODUCED FROM SOLUTIONS OR DISPERSIONS TREATED
WlTH SOD~UM HYDROXIDE FOR INFECTIOUS AGENT DEACTIVATION

BACKGROUND OF THE INVENTION

1. Field of the Invention 2 The present invention relates to a method of controlling the structure stability of 3 collagen ~Ibers produced from solutions or suspensions of collagen which have been 4 treated with a chemical reagent for the deactivation of infectious agents.

2. Description of the Back,~round Art 6 There are pùblic health issues pertaining to the use of animal and human tissues 7 in medical devices which are implanted into human beings, to the use of such tissues in 8 pharmaceuticals, and to a lesser degree, to use in cosmetics. Of particular concern are 9 slow acting viruses present in such tissues, which slow viruses are particularly dif~lcult to deactivate.
11 U.S. Patent No. 4,511,653 to Play et al., issued April 16, 1985, describes a 12 process for the industrial preparation of human collagenous material from human 13 placental tissue. This process includes subjecting the placental tissue to an alkaline 14 treatment with a 0.5 M solution of NaOH, a 0.5 M solution of potassium hydroxide (KOH), or a saturated lime water solution at a temperature of less than or equal to 10 16 ~C, for purposes of inactivation of viruses.
17 In June of 1986, "Concise Comrnunications", The Journal of Lnfectious Diseases, 18 Vol. 153, No. 6, there is a description of the inactivation of slow acting viruses, such 19 as scrapie virus and CJD virus present in 20% brain homogenates, using various 21~6938 concentrations of sodium hydroxide (NaOH) for one hour at room temperature.
2 In September of 1991, "Recomrnendations for Minimizing the Risk of Infection 3 by Agents Causing Zoonoses and Other Anirnal Infections in Manufacture of Medicinal 4 Products", Federal Journal of Offlcial Publications (BAnz., Germany), No. 164, p 6120, there is the description of the treatment of medical materials with a solution of lN (lM) 6 NaOH for one hour at 20~C for the purpose of inactivation of infectious agents. This 7 treatment was recommended particularly for application to bovine spongiform 8 encephalopathy (BSE) and materials of bovine origin.
9 In 1992, "Public Health Issues Related to Animal and Human Spongiform Encephalopathies: Memorandum from a WHO Meeting, Bulletin of the World Health 11 Organization, 70(2): pp 183-190, a discussion is presented regarding BSE, a member 12 of the group of transmissible spongiform encephalopathies (THE) whose prototype is 13 scrapie. Treatment of medicinal products derived from bovine tissues with NaOH, 14 preferably lM, for 1 hour at 20~C is recommended as a manufaçtl-ring process for removal or reduction of BSE infectivity.
16 Collagen Corporation, assignee of the present application, produces a variety of 17 products having bovine collagen as a principal component. The source of Collagen's 18 bovine collagen is a closed herd which is controlled to reduce the potential of 19 con~~ ation by a source of THE (or other infectious agent). It is questionable whether THE is present within the U.S.; however, due to the problem in foreign countries and 21 the possibilit,v of cont~min~tion of U.S. bovine supplies, it is desirable to have a method 22 of treating animal tissues used in the pf~lion of implantable medical devices, 23 medicinal products, and cosmetics. The sodium l~yd~o~ide (NaOH) tlc~tl~el~l of such 24 tissues has been demonstrated to be particularly effective in the reduction of infectious agents in generaL With this in mind, the evaluation of a processes for NaOH treatm-ont 26 of collagen solutions and suspensions used to pl~dfe collagen-based products was '~ 214693~

carried out.
2 Collagen may be obtained in commercially useful amounts &om the connective 3 tissues of a variety of domesticated ~nim~ls, such as cattle and swine, for example. The 4 native collagen is most conveniently obtained &om tendons or skin and is &eed from S extraneous matter such as lipids, saccharides and non collagen protein, so as to leave the 6 collagen protein free or substantially &ee of other connective tissue materials. Native 7 collagen fibers are composed of regularly arranged subunit structures referred to as 8 collagen molecules. Each collagen molecule is about 3000 A long and 15 A in 9 diameter. This long rigid rod-like structure consists of three polypeptide chains wound together in a triple helical configuration. Typically two of the constituent chains are 11 identical in composition and the third is diLfelell~ A characteristic distribution of amino 12 acid residues along the length of any given polypeptide strand, wherein repeating triplets 13 contain glycine at every third position, favors the formation of a helical configuration.
14 The individual collagen units form fibrils which associate to form fibers.
The nonhelical terminal portions of the native collagen molecule, the telopeptides, 16 exhibit a preferred coil conformation extending &om the amino and carboxy ends of the 17 molecule. These telopeptides appear to serve a number of functions in the formation of 18 the native collagen fiber. The telopeptides serve as the pli-~-a,y sites for crosslinking 19 intramolecularly (between the three con~tihlent polypeptide chains in the native collagen molecule) and intermolecularly (be~een two or more native collagen molecules). In 21 addition, the telopeptides facilitate the arrangement of the individual collagen molecules 22 in a pattern which provides for the regular structure of native fibrous collagen.
23 However, the telopeptide portions of native (heterogenic) collagen are believed to be the 24 major sites of its immunogenicity. Therefore, in order to minimi7~ the immunogenicity of heterogenic collagen, it is desirable that the telopeptides be removed. This leaves the 26 collagen fibers in a less stable, more &agile condition, and in need of protection when - 2146~38 protection when exposed to prooessing conditions, which can disturb the arrangement 2 (association) of collagen molecules within collagen fibers.3 Typically collagen is obtained from bovine hides. The initial stage is to clean 4 the hide physically so as to remove some of the noncollagen materials, such as hair, fat, carbohydrates, mucopolysaccharides and the like. See, for example, U.S. Patent 6 Nos. 2,934,446 and 3,121,049, as well as Chvapil et al., "Medical and Surgical 7 Applications of Collagen", Connective Tissue Research 4 (1973).
8 To enhance the ease of purification and facilitate the enzymatic removal of the 9 telopeptides, the collagenous material is subjected to various mechanical treatments, such as dissection, grinding, high speed shearing, milling and the like. Depending upon the 11 particular treatrnent, the collagen may be wet or dry, fro~n or cooled, with grinding and 12 high speed shearing preferably being wet processes, and milling being a dry process.
13 Coarsely divided connective tissues are swollen in aqueous acidic solutions under 14 nondçn~hlring conditions. Further dispersion is achieved through extensive wet grin(ling, to facilitate enzyme access to the native collagen. Preferably dilute acid solutions at low 16 temperatures are employed to minimi7e denaturation. Suitable acids are acetic, malonic 17 or lactic acids, or other lyotropic carboxylic acids having pK values from about 2 to 18 about 5 at 25~C. Concentlalions of acid in the dispersion mediurn range from about 19 0.01 M to 1.0 M, and t~ln~ Lures may vary from about 4~C to about 25~C. The dispersion which is obtained by tre~ ont with acid is a viscous dispersion cont; ini 21 native collagen microaggregates and a small amount of native collagen in solution.
22 The viscous product is subjected to enzymatic lre~ nL to remove the 23 telopeptides and to produce soluble atelopeptide collagen. Various proteolytic ~n~,yn~es 24 rnay be employed which prer~ Lially attack the telopeptidcs, while lcaving the rnajor portion of the molecule intact. Illus~ative en~yn,~s include pepsin, ~ypsin and p ona~, 26 for example. See U.S. Patent Nos. 3,131,130 and 3,530,037.

2l~69~8 The preferred enzyme is pepsin, which is used in combination with an acidic 2 solution, generally at a pH of about 2 to 4. The concentration of the enzyme varies 3 from about 0.001 to about 10 weight percent based on the weight of collagen present.
4 The collagen concentration generally varies from 0.5 g/l. to about 10 g/l. Preferably, the acidity is provided by a carboxylic acid in a concentration of about 0.01 M to about lM.
6 If necessary, the pH can be adjusted by the addition of a mineral acid, e.g. hydrochloric 7 acid. The enzymatic treatment is generally carried out over temperatures ranging from 8 about 0~ C to about 30 ~C over a time period ranging between two days and two weeks, 9 with progress monitored periodically until substantially complete solubilization of the collagen is achieved.
11 The resulting solution is treated to separate the soluble atelopeptide collagen from 12 insoluble collagen, enzymes, residual amino acids, and the telopeptide units which have 13 been separated from the collagen molecules. Primarily, the treatment involves 14 separations, precipitations and dialysis against various solutions of different ionic strength. Moderate tempela~urcs are employed, normally from about 4 ~C to about 16 30 ~C, and salt solutions of various ionic strength or concentration are employed, 17 generally from about 0.01 M to 3.5 M, depending upon the particular salt. Ionic 18 strengths are usually about 0.01 to 3.5.
19 Conveniently, the solution is treated with an aLkaline material, e.g., sodium hydroxide, to raise the pH of the solution to at least about seven, to inactivate the 21 ellz~llle. After inactivating the enzyme, non-solubilized contaminants which have been 22 ~rc~ ated during the inactivation tre~tm-ont are filtered off to yield a filtrate which 23 contains collagen in solution.
24 The collagen in solution is passed through a bed of celite and subsequently processed via ultrafiltration to provide a purified, clear solution con~;l;.. i.-g about 3 26 mg/ml of atelopeptide collagen. This concen~ed solution of collagen is relatively free ~ 21469~8 of higher aggregates, and is referred to as concentrated submicron filtrate (CSF).
2 From a virus deactivation/inactivation point of view, it is preferable to treat a 3 solution of collagen with sodium hydroxide for virus inactivation prior to the formation 4 of the fibrous micropolymers, since the collagen molecules and any beginning fibrils contained in the solution are dissociated to permit maximum availability of any 6 infectious agents which may reside in or be trapped within fiber structures. The 7 collagen triple helix is too tightly wound (1.5 nm diameter) for a virus to reside within 8 the collagen molecule. Therefore, such virus would be present either in the solution or 9 absorbed onto the surface of a collagen molecule. In the soluble environment, where collagen fibers are dissociated into collagen molecules, there is no mass transfer barrier, 11 which requires the sodium hydroxide to diffuse through solids (assembled fibers) to 12 reach the infectious agents on the surface of collagen molecules.
13 During the evaluation of NaOH treatment of collagen solutions and suspensions, 14 it was discovered that the ~llu~;lur~ of the collagen fibers formed was affected by the NaOH treatment. The fibrillar collagen produced from solutions and suspensions of 16 NaOH treated collagen exhibited significantly diLr~,~cnl polymeric characteristics.
17 To be able to use the NaOH tre~tm~nt of collagen solutions for deactivation of 18 infectious agents, it was n~ssaly to find a method to compensate for the effects of the 19 treatment, enabling production of the desired fibrillar collagen product The fibrous collagen product had to be biocompalible and sufficiently stable that it was relatively 21 insensitive to contact with chemi-~l additives and to processing necessary to achieve 22 final product form~ tion.

24 - In accordance with the present invention, it has been discovered that the st~bility of collagen Sbers is altered upon tre~fmPnt of a solution or dispersion of the collagen 21~6938 '_ fibers with a chemical reagent for the inactivation of infectious agents. This fiber 2 destabilization makes it difficult to formulate the collagen fibers to final product when 3 the formulation contains an ingredient which alters the destabilized collagen fibers.
4 Collagen fiber stability is controlled using either a physical means or a chemical means. The physical means is used to protect the stability of the assembled fibers, 6 without providing for covalent bonding between fibrils. The chemical means is used to 7 provide covalent bonding between fibrils, whereby the fibers are stabilized.
8 A stabilized collagen fiber is achieved by the presence of a physical fiber-9 stabilizing means (agent) comprising a water soluble or water miscible polymeric material of the kind which is capable, at sufficient concentration, of causing collagen 11 fibers to precipitate from solution. These water soluble or water miscible polyrneric 12 materials are typically polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, 13 polyacrylamide, polyethylene glycol or derivatives thereof, polypropylene glycol or 14 derivatives thereof, polyvinyl methylether, maleic anhydride copolymers, h~o~yel~lyl starches, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, 16 hydro~y~rl)pyl cellulose, agarose, dextrins, dextrans, pectins and ~lgin~tes~ for example.
17 Preferably the water soluble or water miscible polymeric m~ten~l is biocompatible, such 18 as a polyethylene glycol (PEG) or derivatives thereof.
19 A stabilized collagen fiber is also prepared by using a chemical agent which crosslink~ the fiber, stabilizing the fiber. Preferably the cros~linking agent is also 21 bioco,ll~aLible, such as a glutaraldehyde, hex~methyl diisocyanate, dimethyl 22 subçrimi~ate, carbo liirri~e or an activated PEG.
23 One method of stabili ing the collagen fiber produced from a solution or a 24 ~1ispersion of collagen treated with a chemic~l reagent for the inactivation of infectious agents is to stabilize the collagen ~lber subse luel~l to the tre~tm~.nt by using at least one 26 of the physical or chemical fiber-st~bili7ing agénts described above.

2 ~ 3 ~ ~
A second, less preferable, method of stabilizing the collagen fiber is to use 2 a physical fiber-stabilizing agent prior to or simultaneously with treatment using 3 the agent for deactivation of infectious agents.
4 Preferably, sodium hydroxide is the chemical reagent used to inactivate S infectious agents in the method of the present invention.
6 According to a first aspect of the invention, there is provided a dispersion of 7 stabilized collagen fibers prepared from a solution or a dispersion of collagen, 8 said collagen having been treated with a chemical reagent to inactivate infectious 9 agents and said collagen fibers having been destabilized as a result of treatment with said chemical reagent, comprising: a dispersion of said treated collagen in11 the form of fibers, said fibers having been stabilized by a physical fiber-stabilizing 12 agent, said agent comprising a polymeric material capable of causing collagen 13 fibers to precipitate from solution.
14 The polymeric material may be water soluble or water miscible and may be selected from the group consisting of: polyvinyl alcohol, polyethylene oxide, 16 polyvinylpyrrolidone, polyacrylamide, polyethylene glycol, polypropylene glycol, 17 polyvinyl methyl ether, maleic anhydride copolymers, hydroxyethyl starches, 18 methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl 19 cellulose, agarose, dextrins, dextrans, pectins and alginates.
The water soluble or water miscible polymeric material may be 21 biocompatible and may be selected from the group consisting of polyethylene 22 glycols and derivatives thereof. The polyethylene glycol may have a molecular 23 weight ranging from about 200 to about 20,000, or from about 200 to 8,000.
24 According to a second aspect of the invention, there is provided a dispersion of stabilized collagen fibers prepared from a solution or dispersion of 26 collagen, said collagen having been treated with a chemical reagent to inactivate 27 infectious agents and said collagen fibers having been destabilized as a result of 28 treatment with said chemical reagent, comprising: a dispersion of said treated 2 ~ 3 8 -collagen in the form of fibers said fibers having been stabilized by a chemical 2 fiber-stabilizing agent, wherein said agent comprises a crosslinker reacted with 3 said collagen fiber either prior to or subsequent to the treatment of said collagen 4 solution or dispersion by said chemical reagent.
The crosslinker may be reacted with said collagen fibers subsequent to the 6 treatment of said solution or dispersion. The crosslinker may be selected from the 7 group consisting of glutaraldehyde, hexamethyl diisocyanate, dimethyl 8 subermidate, carbodiimide and activated polyethylene glycol.
9 The crosslinker may be biocompatible. The biocompatible crosslinker may be an activated polyethylene glycol.
11 The activated polyethylene glycol may be selected from the group 12 consisting of polyethylene glycol succinimidyl glutarate, polyethylene glycol 13 succinimidyl, polyethylene glycol succinimidyl carbonate, polyethylene glycol 14 propion aldehyde and polyethylene glycol glycidyl ether, wherein at least two active sites are present on each polyethylene glycol molecule prior to crosslinking 16 with collagen fibers of said collagen dispersion.
17 According to a third aspect of the invention, there is provided a method of 18 stabilizing a dispersion of collagen fibers prepared from a solution or a dispersion 19 of collagen, said collagen having been treated with a chemical reagent to inactivate infectious agents and said collagen fibers having been destabilized as a 21 result of treatment with said chemical reagent, the method comprising: stabilizing 22 said collagen fibers subsequent to said treatment by reacting said collagen fibers 23 with a physical fiber-stabilizing agent, said agent comprising a polymeric material 24 capable of causing collagen fibers to precipitate from solution.
The polymeric material may be water soluble or water miscible. The water 26 soluble or water miscible polymeric material may be selected from the group27 consisting of polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, 28 polyacrylamide, polyethylene glycol, polypropylene glycol, polyvinyl methyl ether, -8a -maleic anhydride copolymers, hydroxyethyl starches, methyl cellulose, 2 hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, agarose, 3 dextrins, dextrans, pectins and alginates.
4 The water soluble or water miscible polymeric material may be biocompatible, selected from the group consisting of polyethylene glycols and 6 derivatives thereof.
7 The water soluble or water miscible polymeric material may be a 8 polyethylene glycol having a molecular weight ranging from about 200 to about 9 20,000 or ranging from about 1,000 to about 6,000.
Preferably, the concentration of said polyethylene glycol ranges from about 11 2.3 mg/ml to about 2.6 mg/ml and the concentration of said collagen fiber ranges 12 from about 10 mg/ml to about 100 mg/ml or the concentration of polyethylene13 glycol ranges from about 2.3 mg/ml to about 2.6 mg/ml and the concentration of 14 said collagen fiber ranges from about 10 to about 50 mg/ml.
The chemical reagent used to inactivate infectious agents may be sodium 16 hydroxide.
17 According to a fourth aspect of the invention, there is provided a method of 18 stabilizing a dispersion of collagen fibers prepared from a solution or a dispersion 19 of collagen, said collagen having been treated with a chemical reagent to inactivate infectious agents and said collagen fibers having been destabilized as a 21 result of treatment with said chemical reagent, the method comprising:
22 crosslinking said collagen fibers by reacting said collagen fibers with a chemical 23 fiber-stabilizing agent prior to or subsequent to said treatment with a chemical 24 reagent of said solution or dispersion containing said collagen fibers.
The crosslinking may be carried out subsequent to said treatment.
26 The chemical fiber-stabilizing agent may be selected from the group 27 consisting of: glutaraldehyde, hexamethyl diisocyanate, dimethyl subermidate, 28 carboimide and activated polyethyleneglycol.
- 8b -r. ~

2 ~ 3 ~ ~
The crosslinking may be accomplished using a biocompatible chemical 2 fiber-stabilizing agent. The chemical fiber-stabilizing agent may be an activated 3 polyethylene glycol or glutaraldehyde.
4 Preferably, the concentration ratio of said glutaraldehyde to said collagen fiber ranges from about 0.0005:1 to about 0.02:1.
6 The chemical reagent used to inactivate infectious agents may be sodium 7 hydroxide.

Figure 1 shows a process flow diagram for a typical treatment of collagen in 11 a solution or suspension with a chemical reagent for deactivation/inactivation of 12 infectious agents. In this instance, the collagen is in the form of a solution, 13 concentrated submicron filtrate (CSF), at the beginning of treatment, and the 14 chemical reagent is sodium hydroxide.
Figure 2A, 2B, and 2C show differential scanning calorimetry (DSC) 16 thermograms for collagen dispersions prepared using the methods described in 17 Example 1C. Figure 2A shows DSC Curve 200 for a collagen dispersion prepared 18 from a solution of collagen without sodium hydroxide treatment of the solution, 19 where the collagen dispersion was formulated to a final product containing 35 mg/ml collagen at a pH of 7.0 to 7.4, using a water base solution containing 3 21 mg/ml lidocaine, 0.13 M sodium chloride, and 0.02 M disodium phosphate. Figure 22 2B shows DSC Curve 220 for a final product containing 35 mg/ml collagen at a pH
23 of 7.0 to 7.4 , prepared from a collagen solution treated with sodium hydroxide, 24 and formulated to final product using a water base solution containing 0.13 M
sodium chloride and 0.02 M disodium phosphate. Figure 2C shows DSC Curve 26 230 for a final product containing 35 mg/ml collagen at a pH of 7.0 to 7.4 prepared 27 from a collagen solution treated with sodium hydroxide, and formulated to final - 8c -2 ~
product using a water base solution containing 3 mg/ml lidocaine, 0.13 M sodium 2 chloride, and 0.02 M disodium - 8d -~ 21469~8 phosphate.
2Figure 3 shows DSC Curves 310 and 320 for collagen dispersions produced from 3a collagen solution treated with sodium hydroxide in the manner described in Example 4lA. Curve 310 illustrates the control dispersion of collagen, where no PEG was added 5for purposes of fiber stabilization. Curve 320 illustrates the collagen dispersion 6formulated to final product where S0 g/l of PEG (8kD) was present during the sodium 7hydroxide treatment, with no PEG added at time of formulation to final product.8Figure 4 shows a series of DSC curves for collagen dispersions produced from 9a collagen solution treated with sodium hydroxide in the manner described in Example 10lB, where various arnounts of PEG were added only during formulabon to final product.
11Figure SA shows the rheological characteristics, measured at 20 ~C and 121 rad/sec, for a dispersion of collagen fibers containing 35 mg/ml of collagen, produced 13from a sodium hydroxide treated solution and formulated to final product in the presence 14of lidocaine (localized anesthetic) and a salt. Curves S10 and 512 show storage modulus 15vs. % strain, for collagen fibers formulated to final product without a physical fiber-16stabilizing agent, and in the presence of a physical fiber-stabilizing agent, respectively.
17Figures SB and SC show gel elasticity data measured at 37 ~C for dispersions 18con~ g 35 mg/ml of collagen. Flgure SB, Curve S10 shows the ger frequency 19response data for a dispersion of collagen fibers prepared from a sodium hydroxide-20treated solution; Curve 512 shows the gel L~uellcy response data for the same 21dispersion when 10 mg/ml of PEG physical fiber-stabilizing agent is added to the 35 22mg/ml collagen dispersion during formulation to final product. Figure SC, Curve 520 23shows gel frequency response data for another dispersion cont~ining 35 mg/ml of 24collagen fibers, prepared from a sodium hydroxide-treated solution; Curve 522 shows 25the gel frequency response data for the same dispersion when 2.5 m~/ml of PEG
26physical fiber-st~bili7ing agent is added during form~ tion to final produc~

'I Q
Z~~9~8 Figure 6A shows the DSC thermogram for a dispersion containing 35 mg/ml of 2 collagen, prepared from a sodium hydroxide - treated collagen solution. In one instance, 3 the collagen fibers were formulated to final product with no fiber-stabilizing agent 4 present, in the other instance a physical fiber - stabilizing agent (PEG) was present.
S Figure 6B shows the rheological characteristics, measured at 20 ~C and 1 rad/sec 6 for a dispersion of collagen fibers containing 35 mg/ml of collagen corresponding with 7 the DSC curves are illustrated in Figure 6A.
8 Figure 7 provides the DSC thermograms for dispersions containing 35 mg/ml 9 of collagen, prepared from a sodium hydroxide - treated collagen solution, where a differing amount of a chemical fiber - stabilizing agent (glutaraldehyde) was present in 11 each instance.
12 Figure 8 shows the rheological characteristics, measured at 20 ~C; and13 1 rad/sec for forrnulated to final product under specific conditions. Curve 810 represents 14 a dispersion of collagen fibers prepalcd from a non-sodium hydroxide - treated collagen solution (Control). Curve 812 represents a dispersion of collagen fibers prepared from 16 a sodium hydroxide - treated solution without the presence of a fiber - stabilizing agent.
17 Curve 814 r~ sel,ts collagen fibers p~a~ed from a sodium hydroxide - treated 18 solution where a physical fiber - stabilizing agent was present during formulation to final 19 product. And, curves 816 rcpresents a dispersion of collagen fibers plepa,~ from sodium hydroxide - treated solutions, where a chemical fiber - st~bili7ing agent was 21 present during formulation to final product.

23 DETAILED DESCRIPTION OF THE PREFERRED EMBODIl\~ENTS
24 In accordance with the present invention, collagen solutions or suspensions are treated with sodium hydroxide (NaOH) to deactivate infec~ous agents. The stabili7~tion ~1~693~

of collagen fibers formed from a treated solution of collagen molecules; from a treated 2 suspension of collagen fibrils and fibers; or from a mixture containing both collagen 3 molecules and collagen fibrils and fibers, is controlled using either at least one physical 4 fiber - stabilizing agent, at least one chemical fiber - stabilizing means, or a combination thereof. The physical fiber - stabilization means are used to control fiber stability 6 without providing for covalent bonding between fibrils within assembled (associated) 7 fibers. The chemical fiber - stabilizing means are used to provide covalent bonding 8 between fibrils, whereby the fibers formed from the fibrils are stabilized.

Example 1: Treatment of Collagen Solution with Sodium Hydroxide.
11 Concentrated Submicron Filtrate (CSF) collagen solution, prepared in the manner 12 previously described, was treated with sodium hydroxide to develop an acceptable 13 process for scaling up to a m~nllf~cturing process.

14 A. 600 ml of soluble collagen as CSF (3.0 mg/ml protein, pH 2.0) was brought up to lM sodium hydroxide (NaOH) by the addition to 300 ml of 3 M NaOH (in a 16 volurnetric ratio of 2:1 of CSF: NaOH) over a 15 rninute time period. The addition of 17 sodium hydroxide was done using a peristalitic pump and the mixture was stirred 18 throughout the addition. The ~ ule was then incubated at 20 ~C for 63 + 3 "~il~ules 19 (not a critical ~me period). The ~u~ e was then cooled by recirculating it for 5 minutes through 5 ft. of 316 L stainless steel tubing (OD = 0.125 in., ID = 0.097 in.) 21 immersed in a cold water bath. Siml-l~neously, during the recirculation, 600 ml of 22 chilled water (~6 ~C) was pumped into the ~n~ e over a S minute period. The 23 addition of chilled water helped cool the ~ lure and also provided a 40% reduction in 24 NaOH concentration within 5 1ninllte~s. The sodium hydroxide in the ~ lu~'e was then 21~6938 neutralized by the addition of approximately 300 ml of 3M HCI over 15 minutes using 2 a peristalitic pump. During the neutralization, the mi~ e was recirculated through the 3 stainless steel tubing immersed in the cold water bath to remove the heat generated by 4 the exothem~ic acid/base reaction. The le-l.yelature of the ~ e during the addition S of the 3M HCI was maintained below 17 ~C. The pH of the neutralized Il~L~ e was 6 then adjusted to 2.0 using lM HCI; 600 ml of water was also added to reduce the salt 7 concentration in the mixture. The diluted mixture (~2400 rnl) was then concentrated to 8 approximately 600 ml (2.5 - 3.0 mg/ml protein, essentially collagen) using the Filtron(~

9 Ultrasette Omega series Polyethersulfone membrane (MWCO 100kD), available from Filtron Corp., Northborough, MA. The concentrated ~-~ e was then diafiltered at 11 constant volume with 1800 ml of 0.013 M HCI using the same membrane described 12 above. The diaf~tration reduced the residual salt concentration to less than 20 mM. The 13 resulting material was ~ filtered to provide a CSF as nearly equivalent as possible to 14 the CSF prior to tre~tment with NaOH.
16 B. In addition to the above three processes, a smaller scale laboratory process, 17 about 200 ml in volume, was employed to independently evaluate the effects of the 18 sodium hydroxide tre~tn~l-t on the stability of fibers formed from treated solutions or 19 suspensions.
Due to the reduced vol~lmetric scale of this process, the addition of sodium 21 hydroxide was done over a 10 minute time period. After incuba~on at about 20 ~C for 22 about 60 - 65 .. i.lu~es, cold water (~ 4 ~C) was added to quickly quench any sodium 23 hydroxide reaction. The volume of cold water added was about 8 tirnes the CSF
24 volume. The diluted mi~ was neutralized with cold hydrochloric acid ( ~ 4 ~C) over a 10 minute period. The m~u~e~ having a pH of about 2.0, was conce"tl~l to 26 approxim~t~o,ly 3.0 mg/ml of collagen and then diafiltered at constant volume against at 2 ~
least 4.5 volumes of 0.013 M hydrochloric acid. The addition of a large volume of 2 cold water prior to neutralization with hydrochloric acid reduced the sodium 3 hydroxide concentration in the mixture to approximately 0.15 M, and the 4 temperature to about 6~C. As a result, the process temperature after the sodium hydroxide treatment was maintained below about 6~C and the effects observed 6 due to sodium hydroxide treatment were attributed to the exposure to 1 M sodium 7 hydroxide at 20~C for about 60-65 minutes.
8 While the majority of laboratory trials were done using the processes 9 described in Example 1A and illustrated in Figure 1, a few of the experiments were carried out using process 1B. The Example 1A process was used in all 11 examples from which collagen fiber stability was determined, unless otherwise 12 specified.
13 With reference to Figure 1, in step 100, clarified submicron filtrate (CSF) 14 bovine collagen, comprising atelopeptide collagen at a concentration of about 3.0 mg/ml in protein, having a pH of about 1.9 - 2.2 was treated with 3 M sodium 16 hydroxide (NaOH), which was mixed into the CSF, to bring the CSF solution to a 17 concentration of 1 M NaOH. The addition of the NaOH to the CSF solution was 18 carried out at atmosphere pressure (exposed to air) and at a temperature of about 19 20~C. The 3 M NaOH was added to the CSF over a time period of about 10 minutes, followed by incubation at about 20~C for a period of about 60-65 21 minutes. Contact of the CSF with the NaOH produced a solution which was 1 M in 22 NaOH, with a resulting pH of slightly less than about 14.
23 We have previously described the use of pH manipulation and/or addition 24 of a salt to control fiber size during precipitation of fibrous collagen from solution.
In particular, the pH of the starting collagen suspension in an aqueous medium 26 was adjusted to 5 or less to permit fiber disassembly (permit the collagen to go . ~, .

-into solution); subsequently the pH of the medium was adjusted over a range of 6 to 9 2 to produce various fiber si~ populations. At a pH above about 10, the fibers begin to 3 disassemble and return to solution. An increase in pH, up to about 10, resulted in an 4 increase in fiber si~, i.e., produced a fiber population having an increased number of S larger fibers (indicated by light scattering techniques). Further, addition of a salt (NaCl) 6 to the collagen solution produced a fiber population which was shown by dir~e~ential 7 scanning calorimetry to have multiple transition temperatures with two major 8 endotherms, indicating the disruption of the collagen fiber structure and partial 9 disassembly of the collagen fibers due to the presence of the salt.
In the present instance, as shown in Figure 1, step 110, after the sodium 11 hydroxide treatment, cold water (~= 8 ~C) was added over a time period of about S
12 minutes. The volume of water added was about equivalent to the original CSF volume.
13 The diluted mi~ e was neutrali~d using 3M hydrochloric acid (HCl) added to the 14 CSF/NaOH l~ to adjust the pH of the I~ lu~'e to about 2.0, enabling the formation of a collagen solution at this pH. The HCl was added over a 15 minute time period, and 16 the process Illi~c~e was circulated through a heat exchanger to remove the heat 17 generated by the exothermic acid/base reaction, m~int~ining a ten~ alul~ for the 18 ~ lulG of around 16~C. The pH 2 solution produced contained about 0.75 mg/ml of 19 collagen molecules and a NaCI concentration of about 375 mM.
The mL~ e produced in step 110 was concenllaled in step 120 to 3.0 mg/ml 21 of collagen by Ultraf;ltration/Diafiltration, standard membrane filtering techniques known 22 in the industry. The diafiltration was done at constant volume with at least 3 volumes 23 of 0.013 M hydrochloric acid, to ensure that the filtered solution would remain at a pH
24 of about 2Ø The filtered solution exhibited a residual salt concentration of less than 20 mM NaCl.
26 The collagen solution produced in step 120 was further purified through a batch 21~fi938 cation exchange column (a 2,400 ml batch column), as shown in Figure 1, step 130, and 2 eluted in a two stage elution at about 1,660 ml/stage in combination with HCl at an 3 elution pH of 2.0, to produce a collagen solution containing about 0.3 mg/ml protein.
4 The eluate was then concentrated in step 140 to about 2.5 to 3.0 mg/ml protein by Ultraflltering and then Diafitering in combination with at least 2.5 volumes of 0.013 6 M HCl (pH 2.0), using the Filtron(~ membrane previously described. This diaf~tration 7 produced a ten-fold reduction in acetate in the column eluates, decreasing residual 8 acetate concentration to less than 1 rnM. The resulting collagen in solution was 9 expected to be equivalent to that produced in a process where the CSF was not treated with sodium hydroxide. This concentrated, purified collagen solution, having been 11 treated for deactivation of infectious agents, was then ready for final processing into 12 desired end products.

13 Example 2: Preparation of a Non-crosslinked Fibrous Collagen Suspension.
14 The concentrated, purified collagen solution processed as described in Example lA and illustrated in Figure 1, is typically used to prepare an injectable water based 16 dispersion of fibrous, non-crosslinked collagen of the kind sold by Collagen Corporation 17 under the tradçm~rk Zyderm~. A solution, ple~al~d in the manner described in 18 Example lA, at 3.0 mg/ml protein was l"~ipila~d at about 17 ~C by the addition of 0.2 19 M Disodium Phosphate buffer adjusted to a pH of 11.2 using sodium hydroxide. The volumetric ratio of collagen solution to buffer was 9:1. The preci~ilale produced 21 contained a fibrous collagen concentration of approximately 2.7 mg/ml. The precipitate 22 w~ concentrated by cen~ ugation to a protein content in excess of 35 mg/ml.
23 Formulation to final product was made by fliluting the centrifugate with a water-based 24 solution comprising 0.02 M disodium phosphate, 3 mg/ml lidocaine (localized ~nesthetiC)~ and i.3 M sodium chlori-le (NaCl), at a pH of 6.3. The reslllting product 2146g~8 comprised an aqueous dispersion containing about 35 mg/ml of fibrous collagen, 2 3 mg/ml of lidocaine, 0.02 M disodium phosphate, and 0.13 M sodium chloride, at a pH
3 of 7.0-7.4.
4 The "Zyderm", non-crosslinked, fibrous collagen product produced was compared S with a Zyderm(~ control product prepared without an NaOH treatment for inactivation 6 of infectious agents. The control was prepared from CSF purified and concentrated 7 using the cation exchange column, with subsequent ultra~lltration and diafiltration as 8 described above, with reference to Figure 1 steps 130 and 140. It was discovered that 9 the fibrous collagen product prepared from an NaOH - treated collagen solution marginally passed or failed Zyderm(~ product specifications for Differential Sc~nning 11 Calo~ elly and Opacity. Differential Sc~nning Calorimetry and Opacity are used as 12 indicators of collagen fiber size population distribution. See, for example, Wallace et 13 al., Journal of Biopolymers, Vol. 25, p. 1875 (1986) and McPherson et al., Collagen 14 Related Research, Vol. S, pp. 119-135 (1985), respectively.
Figure 2A shows the differential scanning calorimetry (DSC) Curve 200 for 16 collagen fibers prepared from a collagen solution without sodium hydroxide treatment, 17 formnl~t~l to a water based dispersion containing about 35 mg~ml of collagen fibers, 3 18 mg/ml of lidocaine (localized ~nest~letic)~ 130 mM of sodium chloride, and 0.02 M
19 disodium phosphate, at a pH of about 7.0 - 7.4. The DSC curve for the collagen dis~ersion exhibits two peaks, a minor peak 210 at about 46 ~C, and major peak 212 at 21 about 54 ~C This in~ic~tes the forrnation of two fiber size populations, with the minor 22 population at 210 r~resenting some portion of the collagen fibers having a smaller 23 average fiber size (which have appa~e~ y been produced as a result principally of the 24 addition of the lidocaine and secondarily of the salt to the suspension of collagen fibers) and a myor population at 212, r~l~sen~ g a larger average fiber size (of the kind 26 typically observed for collagen fibers produced from a solution of collagen not treated 21469~

with sodium hydroxide).
2 Figure 2B shows DSC Curve 220 for a collagen dispersion prepared with sodium 3 hydroxide treatment, but formulated with 0.13 M sodium chloride and 0.02 M sodium 4 phosphate, onlv (without lidocaine), at a pH of 7.0 - 7.4. Only one peak 222is evident, at about 57 ~C. This represents the formation of a single fiber size population with an 6 average fiber size approximately equal to the average fiber size observed for the larger 7 collagen fibers shown in Figure 2A.
8 Figure 2C shows DSC Curve 230 for a collagen dispersion prepared with sodium 9 hydroxide treatment, which was formulated to a 35 mg/ml fibrillar collagen concentration, with 3 mg/ml of lidocaine and 130 mM of sodium chloride, at a pH of 11 7.0 - 7.4. Again, when the lidocaine and sodium chloride are present in the diluting 12 medium, two peaks are evident, a first peak 231 at about 45 ~C, representing the fiber 13 population having a smaller average fiber size, and a second peak 231 at about 55 ~C, 14 represent ng the larger average fibers size. The peaks 231 and 232 shown in Curve 220 are approximately equal in size, indicating approximately equal quantities of collagen 16 in each fiber size population.
17 A comparison of DSC curves 200 and 230 indicates that the sodium hydroxide 18 tre~ ent of the collagen in solution prior to its processing to form fibers altered the 19 stability of the collagen fibers produced. Curve 200 shows a collagen dispersion ~r~ aled from non-sodium hydroxide - treated collagen solution, which exhibits a first, 21 larger average fiber size population 212, and a second, smaller average fiber size 22 population 210 (after forrn~ tion to final product in the presence of lidocaine). The 23 collagen dispersion prepared from a sodium hydroxide - treated collagen solution, 24 shown in Curve 230, exhibits a grossly increased amount of the second, smaller average fiber size population 231 after the same form~ tion to final product.
26 Although not shown in Figure 2, the DSC curve for a collagen dispersion Attomey Docket No.: 214 6 9 ~ 8 prepared without sodium hydroxide treatment, and without the addition of lidocaine and 2 sodium chloride upon dilution, also shows a single peak, basically the same at that of 3 curve 220, for the collagen fiber population distribution.
4 A comparison of Figure 2B, Curve 220, with Figure 2C, Curve 230, con~lrms the fact that it is the- addition of the lidocaine (localized anesthetic) which disrupts the 6 stability of the collagen fibers, leading to the formation of a second fiber population 7 having a smaller average fiber size.
8 The increased instability of the collagen fibers produced using sodium hydroxide 9 treatment for deactivation of infectious agents is further supported by light transmission data indicating the same alteration of average fiber size populations. For example, the 11 opacity at 410 nm, 0.1 path length, measured using a 0.1 cc quartz cuvette on a 12 Beckman Model DU 650 spectrophotometer, for the collagen dispersion shown in Figure 13 2A is 1.85 absorbance; the opacity measurement for the collagen dispersion shown in 14 Figure 2B is 2.1; and the opacity measurement for the collagen dispersion shown in Figure 2C is 0.8. For a given concentration of collagen fibers in the dispersion16 measured, the larger the average fiber size, the higher the opacity measurement. Thus, 17 opacity for the single, large average fiber size population of Figure 2B is the highest, 18 followed by the opacity for the collagen fibers of Figure 2A, which have the dual fiber 19 size population with the smaUest quantity of collagen fibers having the smaller average fiber size; followed by the opacity for the coll~gen fibers of Figure 2C, which have the 21 dual fiber size population with the largest quantity of collagen fibers having the smaller 22 average fiber size.

23 Example 3: Modification of the Process for Preparation of a Non-cros~linked Fibrous 24 Collagen Suspension: Physical Process.
The collagen fibers produced from sodium hydroxide treated solutions (produced Attorney Docket No.: 214 6 9 3 8 by the method described in Example lA, for example) can be stabilized to prevent2 alteration upon exposure to chemicals and/or processes necessary to formulate to final 3 product. The physical process for such stabilization involves addition of an agent which 4 protects the fibers from dissociation upon contact with chemicals or exposure to S processing-conditions.
6 In the present instance, it was desired to provide a fibrous collagen which would 7 be stable in the presence of chernical additives and potentially adverse processing 8 conditions used in ~l~al"lg a final forrnulation cont~ining the fibrous collagen. It was 9 apparent that formulation to final product by the addition of a diluent containing salts and lidocaine (during the preparation of Zyderm, for exarnple) was causing the collagen 11 fibers to disassociate. It was unknown whether the collagen fibers would have to be 12 stabilized prior to precipitation, or whether precipitated fibers could be stabilized 13 subsequent to precipitation, simultaneously with formulation to final produc~
14 The most pl~f~lled stabilizing agents are those which are biocompa~ible, nonimmunogenic and otherwise suitable to remain in the finished formulation, because 16 these agents do not have to be removed from the finished fonnnl~ion. With this in 17 mind, the stabilizing agent used to demonstrate the concept of fiber stabilization was a 18 biocompatible molecule, polyethylene glycol (PEG).
19 PEG is a biocompdtible molecule which has been widely used in ph~. n~ceuLical industry formnl~tion~ There are a wide array of co",~ ially available polyethylene 21 glycols and derivatives thereof. The more commo~ly used polyethylene glycols range 22 in weight average molecular weight from about 200 to about 20,000, with 200 to about 23 8,000 being prtife~lcd, and 300 to 6,000 being most l)l,f~,llcd. D~livali~res of 24 polyethylene were not evaluated in the pl~fc~l~ embo l;~ nls~ but would be expected to work as stabilizing agents provided they have surface fim~tion~l ch~r~ct~ i~tics which 26 are s.ll,~ lly the same as PEG. Other bioco",palible, water soluble or water miscible Attorney Docket No.: 21 ~ 6 9 ? 8 polymers which tend to cause precipitation of collagen fibers from collagen solutions 2 (such as those described in U.S. Patent No. 4,980,403), are expected to work as fiber-3 stabilizing additives.
4 U.S. Patent No. 4,980,403 describes the use of water-soluble or water-miscible polymers to cause precipitation of collagen fibers &om collagen solutions. Among the 6 polymers listed as useful in achieving precipitation are polyvinyl alcohol, polyethylene 7 oxide, polyvinylpyrrolidone, polyacrylamide, polyethylene glycol, polypropylene glycol, 8 polyvinyl methyl ether, and maleic anhydride copolymers. Other natural polymeric 9 materials listed include hydroxyethyl starches, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, agarose, dextrins, dextrans, pectins and 11 ~lgin~tes. The use of such water-soluble or water-miscible polymers to aid in 12 precipitation of collagen fibers &om collagen solutions is said to be preferable to the 13 addition of a neutral salt or a decrease in pH in the presence of a neutral salt, which is 14 said to cause denaturation and disruption of the natural rod-like character of collagen.
In the present instance it was discovered that the addition of a physical fiber-16 stabilizing agent to the collagen solution during treatment of the solution with sodium 17 hydroxide did not fully provide the degree of Sber st~bili7~tion desired. An 18 improvement in fiber stability upon fonmll~tion to final product was observed for 19 collagen dispersions pr~ared with a physical st~bili7ing agent present during sodium hydroxide l,~ nt, however. Optimi7~tion of the arnount of physical fiber-stabili~ing 21 agent present and of process conditions should even further improve this method of 22 fiber st~bili7~tion.
23 The effect of the presence of a physical fiber-st~ili7in~ agent during sodium 24 hy~o~ide ~reatment is illustrated in Figure 3. Figure 3 shows DSC curves for collagen dispersions form~ te~ to final product con~ ing lidocaine and salts, where the collagen 26 solution was treated with sodium hydroxide in the manner described in Example lA.

Attomey Docket No.:

Curve 310 shows a collagen dispersion prepared from a collagen solution treated with 2 sodium hydroxide with no physical fiber-stabilizing agent present. Curve 320 shows a 3 collagen dispersion prepared from a collagen solution treated with sodium hydroxide in 4 the presence of 50 g/l of PEG (8 kD), with no additional PEG added upon formulation to final product. The CSF - NaOH mL~ was puri~led and concentrated as previously6 described, and subsequently forrn~ ted to final product cont~ining 3 mg/ml of lidocainç, 7 0.13 M sodium chloride, and 0.02 M sodium phosphate, to a pH of 7.0-7.4 to provide 8 the collagen dispersion tested, in each instance. It is readily apparent from DSC curves 9 310 and 320, that the presenc~ of PEG during the sodium hydroxide treatment reduced the amount of the fiber population having a smaller average fiber size, as that population 11 decreased from the amount shown as 314, in Curve 310 to the amount shown as 324, 12 in Curve 320. However, the amount of smaller average fiber size population shown at 13 324, in Curve 320 subs~n~i~lly exceeds the amount present in collagen dispersions 14 ~lepared from non-sodium hydroxide-lreated solutions.
Since it is desired to maintain the collagen molecules in solution during tre~tment 16 of the solution with sodium hydroxide (to provide maximum exposure of the infectious 17 agents to sodium hydroxide, as previously described), the amount of PEG which should 18 be added to the collagen solu.*on is less than the amount which will cause a subst~n*~l 19 amount of collagen fiber forma*on. It was determined that at a sodium hydroxide concentration of lM (pH ~14) and a collagen co~cer-tration of about 2.0 mg/mL the 21 following amounts of PEG can be added to the collagen solution, depending on the 22 molecular weight of the PEG, without c~lsin~ subs~nti~l fiber form~*on for a PEG
23 of 3.3 kD, 0 - 130 g/l of collagen containing solu-*on; for a PEG of 8 Id), 24 9 - 75 g/l of collagen~on~ g solution; for a PEG of 20 kD, 0 - 45 g/l of collagen-co~ ing solution.
26 It was subseguently disco~ ,d, unexpec~dly, that addition of a fiber-st?~kili7in~

Attorney Docket No.: 214 6 9 ~ 8 agent during formulation to final product alone stabilized the collagen fibers, enabling 2 the preparation of collagen dispersions equivalent to those produced from collagen 3 solutions which had not been treated with sodium hydroxide. This was unexpected, 4 since one skilled in the art would believe that stabilization of the fibers during the sodium hydroxide tre~ent step should provide a fiber which remains stable during6 formulation to final product, and that such stabilization would indicate that addition of 7 the stabilizing agent to the buffers used in formulation to final product would likely be 8 less successful.
9 Figure 4 shows the DSC curves for collagen dispersions produced from a collagen solution treated in the manner described in Example lB; purified and 11 concentrated as previously described; and, formulated to final product with a solution 12 containing lidocaine, salts and various arnounts of PEG. The amount of lidocaine and 13 salts were those adequate to produce a final product containing 35 mg/ml of protein, 3 14 mg/ml of lidocaine, 0.13 M sodium chloride and 0.02 M sodium phosphate at a combined pH of 7.0-7.4. The amount of PEG (3.4 kD) used ranged from no PEG to 16 about 10 mg/ml PEG. Figure 4, curve 410, shows the effect of the absence of PEG
17 during formulation to final product; Curve 412 shows the effect of 1 mg/ml of PEG;
18 curve 414 shows the effect of the presence of 2.5 mg/ml of PEG; Curve 416 shows the 19 effect of the presence of 5.0 mg/ml of PEG; and Curve 418 shows the effect of the presence of 10 mg/ml of PEG. It is readily a~dreQt that the addition of PEG during 21 fo~ tion to final product stabilizes the collagen fibers and prevents tlica~sembly of 22 a first fiber population of larger sized fibers into a second fiber population of smaller 23 fiber size.
24 However, the pr~nce of PEG in the final product formul~tion affected the rheological ~io~l(ies of the m~terinl, At high PEG concent~tions (10 mg/ml), thc26 m~t~ l showed a s~rong strain-thinning behavior. Flgure S shows the plot of mo~nlll~

21469~8 Anorney Docket No.:

v. % stress for product formulations. The measurements were carried out using a Model 2 8400 Fluid Spectrometer, parallel plate configuration of 25 mrn diameter plates at 20 ~C
3 and 1 rad/sec, available from Rheometrics, Inc., Piscataway, N.J.. Figure 5A, Curves 4 510 and 512, show storage modulus versus % strain, where Curve 510 represents the S formula without PEG and Curve 512 represents the formulation cont~ining 3.3 kD-PEG~
6 at a concentration of 10 mg/ml. The decrease in modulus with increasing strain when 7 PEG is present translates into dif~erc,lces in the extrusion properties of the m~tçri~l 8 through an injection needle, for example. In addition, at 37 ~C the collagen dispersion 9 formulated to final product with PEG present forms a weaker gel than the dispersion formulated to final product without PEG. Figure SB shows Curve 510, illustrating the 11 gel elasticity data for a dispersion of collagen fibers forrnulated to final product. Flgure 12 SC, Curve 520 shows the gel elasticity for a second collagen dispersion formulated to 13 final product, where the preceding collagen solution was sodium hydroxide treated.
14 Curve 512 shows the gel elasticity for the same dispersion when 10 mg/ml of PEG
physical fiber-stabilizing agent is added to the dispersion during formulation to final 16 product where the preceding collagen solution was sodium hydroxide treated. Curve 522 17 shows the gel elasticity data for the sarne dispersion when 2.5 mg/ml of PEG physical 18 fiber-stabilizing agent is added during form~ tion to final product. Thus, there is a 19 prGr~llcd PEG content for a product, depending on the end use application for the product. ~ the case of a non-crosslinkel, injectable collagen dispersion for use in soft 21 tissue augmentation, such as Zyderm~, the PEG content of the dispersion should range 22 from about 1 mg/ml to about 4 mg/ml, preferably from about 2 mg/ml to about 4 mg/ml, 23 and most preferably from about 2.4 mg/ml to 2.6 mg/ml. 'Ihis presumes a PEG
24 molecular weight of about 3 to 3.5 kD. One sl~lled in the art, with minim~l exp~;,nç~ ion can clet~--n;nç an ol,~ni~ed PEG content for particular product, 26 depending on the PEG molecular weight and the end use application.

;'~ 2146938 Attorney Docket No.:

Further investigation indicated that addition of PEG as a stabilizing-agent during 2 final formulation at a concentration providing about 2.5 mg/ml in the final formulation 3 provided sufficient stabilization of the collagen fibers to meet normal Zyderm(~
4 le luirw~lents for fiber size population distributiont while only marginally affecting the S rheological behavior of the product. Figure 6A shows the DSC curves-for collagen- -6 dispersions produced from a sodium hydroxide-treated solution of collagen and 7 formulated to final product with lidocaine and salt; Curve 610 represents the absence of 8 PEG from additives during forrnulation to final product, and Curve 612 shows the effect 9 due to the presence of 2.5 mg/ml of PEG in the formulation. It is apparent that the 2.5 mg/ml of PEG fiber-stabilizing agent prevented the formation of a second, smaller sized I l fiber population. In addition, Figure 6B shows the rheological properties for these 12 m~tlori~l.s, where storage modulus as a function of % strain for the control (non-sodium 13 hydroxide treated collagen) formulation without PEG, shown in curve 614, is only 14 "~d~gihlally dirrelcnt from the curve 616 storage modulus as a function of % strain for the sodium hydroxide treated collagen formulated to final product with 2.5 mg/ml of 16 PEG. The Figure 6B storage modulus curves were generated at 20 ~C and at a 17 frequency of 1 rad/s.
18 One skilled in the art, in view of the present disclosure, can adjust the amount 19 of fiber-stabilizing agent to provide the rheological plopellies desired in a given product while l"~ ining other physical prope.lies desired in the collagen fibers them~elves.

21 l~xample 4: Use of Crosslinking to Stabilize the Collagen Fiber for Formulation 22 to Fmal Product. Chernical Process.
23 A. To dot~ .. ;ne whether crosslinking with tracc amounts of a clos.slinking agent 24 would be useful in st~bili7in~ thc collagen fibers for fonnul~tion to final product, for exarnple, glutaraldehyde was used as the cros~linking agent.

'- 21~6338 Attomey Doc~cet No.:

The prooedure used to prepare a collagen suspension similar to Zyderm(~) was as 2 follows:
3 The prooess described in Example lA and i~lustrated in Figure 1 was used to 4 prepare sodium hydroxide treated CIS (NCIS).
The NCIS was used to prepare three different batches of material treated 6 with trace amounts of glutaraldehyde. In each batch, 60 g of NCIS was plaoed in a 500 7 ml oentrifuge bottle. The centrifuge bottle was plaoed in a 17 ~C water bath. A
8 0.2 M disodium phosphate buffer (adjusted to a pH of 11.2 using sodium hydroxide) 9 was added in a ratio of 1:9 (buffer: ~CIS)~ and the mixture was incubated for 8-12 hours. The bottles were shaken well and then a glutaraldehyde buffer was added at a 11 conoentration of about 0.5 - 2.0 mg glutaraldehyde per g of protein (essentially collagen) 12 to provide a mi~ule having a pH of about 7 - 7.4, while stirring of the mixture was 13 continued. In the first batch of m~teri~l, the amount of glutaralehyde added was 0.5 mg 14 glutaralehyde / g prole;n; 1.0 mg glutaraldehyde / g protein was used for the second batch; and 2.0 mg glutaraldehyde / g protein was used for the third batch. In each case, 16 the ~ ur~ was permitted to incubate at 25 + 5 ~C for a n~ini~ l, period of 13 hours.
17 For each batch, the contents of the beaker were transferred to a centrifuge bottle and the 18 bottle was spun at about 9000 rpm for about 30 Il~i~lul~S in a Sorvall oentrifuge (GS -19 3 rotor). The clear liquid was decanted from each bottle, leaving a pellet in the bottom of the bottle. Each pellet was transferred to a syringe, and mixing to obtain a 21 homogeneous composition was accomplished by syringe-to-syringe exchange. The 22 protein content of the homogeneous composition was about 40 to 80 mg/ml.
23 The ~mpulalion of ingredients to be added to reach final product formulation 24 was based on 35 mg/ml of protein in the final fo~nnl~t;on Lidocaine buffer (30 mg/ml 25 - lidoca ne, 1.3 M NaCl and 0.02 M disodium phosphate at a pH of 6.3) was added to the 26 homogeneous composition pr~alcd above to produce a final formulation which 21~6938 , Attorney Docket No.~

contained 10% b~ ~lume of this buffer. The remainder of ingredients added was made 2 up of 0.02 M disodium phosphate buffer, pH 7.2. This ~ ule was mixed thoroughly 3 by syringe to syringe exchange. The final formulation contained 35 mg/ml of protein, 4 0.13 M sodium chloride, 0.02 M sodium phosphate, and 3 mg/ml lidocaine in a water-based dispersion at a pH of-7.0 - 7.4.
6 The DSC profiles for the collagen dispersions from the three m~teri~l~ produced 7 are shown in Figure 7. Figure 7, curve 710, shows the collagen fibers crosslinked using 8 0.5 mg glutaraldehyde / g protein; curve 712 shows the collagen dispersion DSC for 9 fibers crosslinked using 1.0 mg glutaraldehyde / g protein; and curve 714 shows the collagen dispersion DSC for fibers crosslinked using 2.0 mg glutaraldehyde / g protein.
11 It is readily apparent that crosslinking with 2.0 mg glutaraldehyde / g protein has 12 stabilized the fibers in a manner which prevents destabilization upon formulation to final 13 product. However, rheological mea~ure~l~en~" illustrated in Figure 8, showed that the 14 collagen m~teri~l behaves in a distinctly crosslinked manner even when such low concentrations of glutaraldehyde are used. Thus, the use of low concentrations of 16 crosslink.or, the chemical means of fiber stabilization, is not equivalent to the use of a 17 physical fiber-stabilizing agent.
18 Figure 8A shows the storage modulus as a function of % strain at 20 ~C and 19 1 rad/sec for various collagen dispersions formulated to final product conlailling the amounts of lidocaine and salts previously described as present in the Zyderm~ product.
21 Curve 810 l~epresellt, a collagen dispersion prepdred without sodium hydroxide treatment 22 (the control). Curve 812 represents a collagen dispersions p~ d from sodium 23 hydroxide treated solution, without use of any means of fiber stabilization during 24 formnl~tion to final product. Curve 814 l~rese.'~7 a collagen dispersion pç~alc~ using sodium hydroxide l~- ~f ~ I ,ent, with form~ tion to final product carried out in the presence 26 of P~EG, with the amount of PEG being such that the PEG content in the final product Attorney Docket No.:

was 2.5 mg PEG / ml product. Curve 816 represents a collagen dispersion prepared2 using sodium hydroxide treatrnent and crosslinked with 1.0 mg glutaraldehyde / g protein 3 just prior to formulation to final product.
4 It is readily apparent from Figure 8 that the use of the physical fiber-stabilizing agent, PEG, during formlllation to final product, to stabilize collagen fibers produced 6 from a sodium hydroxide - treated solution, results in st~bili7~1 fibers which have 7 rheological properties very closely approaching those of collagen fibers produced from 8 a solution which has not been treated with sodium hydroxide (the control). The use of 9 a chemical means, i.e. a crosslinker, glutaraldehyde, just prior to formulation to final product, to stabilize the collagen fibers produced from a sodium hydroxide-treated 11 solution, results in stabilized fibers whose fiber si_e population is not affected by 12 formulation to final product; however, the rheological properties of the fibers and the 13 product produced ther~liolll will be different from the control. In some product 14 applications, the chemically stabilized, crosslinked collagen fibers offer advantages and in other applications, the physically stabilized collagen fibers offer advantages.
16 B. In a single trial, glutaraldehyde was added to a collagen solution (NCIS), ie.
17 after treatment of the solution with sodium hydroxide, but before p~ ion (to fibers) 18 and formulation to final product. 1.0 mg glutaraldehyde per g of collagen was used.
19 The DSC curve of a collagen dispersion formnl~ted to final product using the collagen from this trial (where no fùr~er glutaraldehyde was added upon fc-rrm-l~*on to final 21 product) indir~t~1 that the collagen fibers had not been stabilized. Further investigation 22 is to be carried out related to addition of a chemical st~bili7.ing agent simultaneously 23 with sodium hydroxide treatment of collagen solutions.
24 There are a number of crosslink.qrs which can be used to chemically stabilize the collagen fibers. Such crosslink~ inclllde, but are not limited to, glutaraldehyde, 26 h~oY~me~hyl diisocyanate, dimethyl suberimid~te, carbimide, and activated polyethylene ~ Attorney Docket No.: 21 4 6 9 ~ 8 glycol. The most preferred are those which are biologically compatible. There are 2 numerous derivatives of PEG which are biologically compatible and which have 3 activated functional sites along the polymer chain or at the ends of the polymer chain 4 which can be used to crosslink collagen. In particular, polyethylene glycol is modified to provide functional groups so that covalent bonding can occur between the activated 6 PEG and the primary amino groups on a collagen molecule. The term PEG, as used in 7 describing these activat~ ten~l~, represents polyrners having the repeating structure 8 (OCH2CH2)D. The activated PEGS preferred in the present invention include PEG
9 succinimidyl glutarate, having a succir~imidyl ~,lu~ate group ~N~-co~cH2)3~C-o-at least at two sites on the PEG molecule; PEG succinimidyl, having a succinimidyl 11 group ~ N-O-OC-(CH2)2-0- ~I' ~ N~(CH2)3-O- at least at two sites of the PEG
o o 12 molecule; PEG succinimidyl carbonate, having a succinimidyl carbonate 13 group ~N-O~c-o at least at two sites on the PEG
14 molecule; PEG propion aldehyde, having an OHC-(CH2)2-O- functional group at least at two sites on the PEG molecule; and PEG glycidyl ether, having an O-CH2-CH-CH2-O-16 functional group at least at two sites on the PEG molecule.
17 One skilled in the art will recognize other biocompatible crosslink~rs which can 18 readily be used in the stabilization of collagen fibers.
19 The above-described pr~r~lled embodiments of the present invention are not inten.1~d to limit the scope of the present invention, as demonstrated by the claims which 21 follow, as one skilled in the art can, with minim~l experimentation, extend the disclosed ~ concepts of the invention to the cl~imeA scope of the invention.

Claims (9)

1. A dispersion of stabilized collagen fibers prepared from a solution or a dispersion of collagen, said collagen having been treated with a chemical reagent to inactivate infectious agents and said collagen fibers having been destabilized as a result of treatment with said chemical reagent, comprising: a dispersion of said treated collagen in the form of fibers, said fibers having been stabilized by a physical fiber-stabilizing agent, said agent comprising a polymeric material capable of causing collagen fibers to precipitate from solution.

2. The dispersion of stabilized collagen fibers of Claim 1, wherein said polymeric material is water soluble or water miscible.

3. The dispersion of stabilized collagen fibers of Claim 2, wherein said water soluble or water miscible polymeric material is selected from the group consisting of: polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, polyacrylamide, polyethylene glycol, polypropylene glycol, polyvinyl methyl ether, maleic anhydride copolymers, hydroxyethyl starches, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, agarose, dextrins, dextrans, pectins and alginates.

4. The dispersion of stabilized collagen fibers of Claim 2, wherein said water soluble or water miscible polymeric material is biocompatible.

5. The dispersion of stabilized collagen fibers of Claim 4, wherein said water soluble or water miscible polymeric material is selected from the group consisting of polyethylene glycols and derivatives thereof.

6. The dispersion of stabilized collagen fibers of Claim 5, wherein said water soluble or water miscible polymeric material is a polyethylene glycol having a molecular weight ranging from about 200 to about 20,000.

7. The dispersion of stabilized collagen fibers of Claim 6, wherein said polyethylene glycol has a molecular weight ranging from about 200 to 8,000.

8. A dispersion of stabilized collagen fibers prepared from a solution or dispersion of collagen, said collagen having been treated with a chemical reagent to inactivate infectious agents and said collagen fibers having been destabilized as a result of treatment with said chemical reagent, comprising: a dispersion ofsaid treated collagen in the form of fibers said fibers having been stabilized by a chemical fiber-stabilizing agent, wherein said agent comprises a crosslinker reacted with said collagen fiber either prior to or subsequent to the treatment of said collagen solution or dispersion by said chemical reagent.

9. The dispersion of Claim 8, wherein said crosslinker is reacted with said collagen fibers subsequent to the treatment of said solution or dispersion.10. The dispersion of stabilized collagen fibers of Claim 8 or 9, wherein said crosslinker is selected from the group consisting of glutaraldehyde, hexamethyl diisocyanate, dimethyl subermidate, carbodiimide and activated polyethylene glycol.
11. The dispersion of stabilized collagen fibers of Claim 8 or 9, wherein said crosslinker is biocompatible.
12. The dispersion of stabilized collagen fibers of Claim 11, wherein said biocompatible crosslinker is an activated polyethylene glycol.
13. The dispersion of stabilized collagen fibers of Claim 12,wherein said activated polyethylene glycol is selected from the group consisting of polyethylene glycol succinimidyl glutarate, polyethylene glycol succinimidyl, polyethylene glycol succinimidyl carbonate, polyethylene glycol propion aldehyde and polyethylene glycol glycidyl ether, wherein at least two active sites are present on each polyethylene glycol molecule prior to crosslinking with collagen fibers of said collagen dispersion.

14. A method of stabiiizing a dispersion of collagen fibers prepared from a solution or a dispersion of collagen, said collagen having been treated with achemical reagent to inactivate infectious agents and said collagen fibers havingbeen destabilized as a result of treatment with said chemical reagent, the method comprising: stabilizing said collagen fibers subsequent to said treatment by reacting said collagen fibers with a physical fiber-stabilizing agent, said agent comprising a polymeric material capable of causing collagen fibers to precipitate from solution.
15. The method of Claim 14, wherein said polymeric material is water soluble or water miscible.
16. The method of Claim 15, wherein said water soluble or water miscible polymeric material is selected from the group consisting of polyvinyl alcohol, polyethylene oxide, polyvinylpyrrolidone, polyacrylamide, polyethylene glycol, polypropylene glycol, polyvinyl methyl ether, maleic anhydride copolymers, hydroxyethyl starches, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, agarose, dextrins, dextrans, pectins and alginates.
17. The method of Claim 15, wherein said water soluble or water miscible polymeric material is biocompatible.
18. The method of Claim 17, wherein said biocompatible polymeric material is selected from the group consisting of polyethylene glycols and derivatives thereof.
19. The method of Claim 18, wherein said water soluble or water miscible polymeric material is a polyethylene glycol having a molecular weight ranging from about 200 to about 20,000.
20. The method of Claim 19, wherein said water soluble or water miscible polymeric material is a polyethylene glycol having a molecular weight ranging from about 1,000 to about 6,000.

21. The method of Claim 20, wherein the concentration of said polyethylene glycol ranges from about 2.3 mg/ml to about 2.6 mg/ml and the concentration of said collagen fiber ranges from about 10 mg/ml to about 100 mg/ml.
22. The method of Claim 21, wherein the concentration of polyethylene glycol ranges from about 2.3 mg/ml to about 2.6 mg/ml and the concentration of said collagen fiber ranges from about 10 to about 50 mg/ml.
23. The method of any one of Claims 14 to 22, wherein said chemical reagent used to inactivate infectious agents is sodium hydroxide.
24. A method of stabilizing a dispersion of collagen fibers prepared from a solution or a dispersion of collagen, said collagen having been treated with achemical reagent to inactivate infectious agents and said collagen fibers havingbeen destabilized as a result of treatment with said chemical reagent, the method comprising: crosslinking said collagen fibers by reacting said collagen fibers with a chemical fiber-stabilizing agent prior to or subsequent to said treatment with a chemical reagent of said solution or dispersion containing said collagen fibers.25. The method of Claim 24, wherein said crosslinking is carried out subsequent to said treatment.
26. The method of Claim 24 or 25, wherein said chemical fiberstabilizing agent is selected from the group consisting of: glutaraldehyde,hexamethyl diisocyanate, dimethyl subermidate, carboimide and activated polyethyleneglycol.
27. The method of Claim 24, wherein said crosslinking is accomplished using a biocompatible chemical fiber-stabilizing agent.
28. The method of Claim 27, wherein said chemical fiber-stabilizing agent is an activated polyethylene glycol.
29. The method of Claim 25, wherein said chemical fiber-stabilizing agent is glutaraldehyde.

30. The method of Claim 29, wherein the concentration ratio of said glutaraldehyde to said collagen fiber ranges from about 0.0005:1 to about 0.02:1.
31. The method of any one of Claims 24 to 30, wherein said chemical reagent used to inactivate infectious agents is sodium hydroxide.