WO1993013136A1

WO1993013136A1 - Ultra-pure polysaccharide materials for medical use

Info

Publication number: WO1993013136A1
Application number: PCT/US1992/009497
Authority: WO
Inventors: James J. Barry; Paul A. Higham
Original assignee: Howmedica Inc.
Priority date: 1991-12-20
Filing date: 1992-11-13
Publication date: 1993-07-08
Also published as: AU3065192A; PT101140A

Abstract

A process for purifying polysaccharides includes forming a 1% solution of medial grade polysaccharides and passing them through three filters. The first and second filters being nitrocellulose filters whith the third filter being modified with polypeptides to bind hydrophobic impurities. The resulting solution is then dialyzed to remove low molecular weight impurities.

Description

ULTRA-PURE POLYSACCHARIDE MATERIALS FOR MEDICAL USE

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a process for ultra-purifying "medical grade" poly- saccharides so that they may be safely placed internally within the body. More particularly, this invention relates to the prevention of post-surgical fibrin build-up, which ultimately leads to formation of adhesions between adjacent internal body tissues, by the use of highly purified polysaccharides.

Description of the Prior Art

Recently, polysaccharides have gained attention in the medical field, particularly for use in the area of wound dressings. Materials such as cellulose, cellulose deriva¬ tives, calcium alginate, chitin and chitin derivatives have been found in commercially available wound dressing materials. Other materials such as hyaluronic acid are being explored for wound dressing applications.

The advantage of using select polysaccharides in medical applications is that they are biodegradable/biocompatible, bioresorbable, biological in nature, and can be easily modified (e.g. cross-linking) to produce devices that can be internally placed in the body for varying lengths of time. However, from a biocompatibility standpoint, the use of a polysaccharide for an external application, such as a wound dressing, can differ drastically from use internally. Hyaluronic acid is one of the few polysaccharides which has found commercial success as an intra-ocular replacement fluid during select ophthalmic procedures. More recently, methyl cellulose has also been offered commercially for similar applications. Internal applications, such as these, require extensive and costly purification processes (U.S. Patent 4,141 ,973 for Hyaluronic Acid). It has been found that "medical grade" polysaccharide materials such as alginic acid, chitosan, cellulose and derivatives thereof are so impure as to cause severe inflammations when implanted internally in certain sites. There has been a long felt need to provide an inexpensive method of producing highly purified polysaccharides for implantation into the body. The highly purified polysaccharide film or solution produced by the purification process of the present invention is primarily used for prevention of fibrous deposition in orthopedic applications, but is also used in applications such as a lavage fluid, joint lubricant, artificial synovial fluid, anti-inflammatory material, an adjunct to physical therapy and a carrier matrix for pharmacologically active agents including growth factors and other osteo-inductive factors. The solutions or films may be further modified by various chemical methods already recognized in the literature.

An adhesion results from the organization of fibrinous exudate on tissue surfaces due to the infliction of trauma or process of inflammation. Tissues such as blood vessels or organs including kidney, liver and intestines are coated with mucous membranes or serous membranes so that they can function independently of each other. Examples of the mucous or serous membranes are the body wall pleura and organ pleura in the thoracic cavity, and the parietal peritoneum and mesentery in the abdominal cavity, each protecting the corresponding organs. Surgical trauma or inflammation in those portions of the body coated with serous membranes may result in the adhesion regardless of the size of the affected part. Such adhesions between tissues may be observed in all tissues of the body, not just those mentioned above. Adhesions between tissues can lead to severe pain, decreased function, and even permanent loss of motility. In the orthopedics field, conditions such as acute or chronic arthritis (e.g. suppurative arthritis, rheumatoid arthritis, gonorrheal arthritis, tuberculous), or traumatic injuries at the joint (e.g. fracture or sprain), would result in aklotic disease wherein the surface of the bones, as well as the effected soft tissues constituting the joint, adhere to each other and thereby restrict the mobility of the joint. Another adhesion condition, congenital radioulnar syntosis, wherein a spoke bone and an ulna adhere together, is difficult to remedy by a surgical operation since the separated bones frequently re-adhere. While complications of the patella-femoral joint following total knee replacement are rare, the dysfunction of the patella-femoral articulation has been found to be secondary to intra-articular fibrous bands (Thorpe et al, Journal of Bone and Joint Surgery (JBJS) Vol. 72A, No. 6, p. 811, 1990). Intra-articular fibrosis in anterior cruciate ligament (ACL) reconstruction has also been noted (K. Shelbourne et al, Am. J. Sports Med., Vol. 19, No. 4, p. 332, 1991). Adhesions are also prominent in tendon surgery. In this instance, there is a general tendency towards adhesions between the tendon and the tendon sheath and other surrounding tissue during an immobilization period following the operation (P. Matthews et al, JBJS Vol. 58B, No. 2, P. 230, 1976; Matthews, The Hand, Vol. 11 , No. 3, P. 233, 1979; Gelberman et al, Hand Clinics, Vol. 1 , No. 1 , P. 35, 1985). More recently, there has been increased interest in the prevention of the "laminectomy membrane" formed following various spinal procedures. This membrane is a well organized mass of fibrinous tissue which replaces the bone that was removed at the laminectomy. This fibrinous mass binds the dura to overlying muscles (H. LaRocca and I. McNab, JBJS, Vol. 56B, No. 3, P. 545, 1974). This causes narrowing of the spinal canal which places pressure on the cauda equina or nerve roots. This scar tissue formation may require reoperation which is tedious and dangerous, leading to the possibility of dural tears and damage to the emergent nerve roots resulting in motor weakness, sensory change, and painful paresthesia. Numerous papers have been published on various treatments to prevent adhesion formation. Treatments such as liquid paraffin, camphor oil, chondroitin sulfate and urea exhibit an insufficient effect since they function only temporarily. Other prophylactic treatments such as silicone membranes, gutta percha, or poly (tetrafluoroethylene) membranes have been used to serve as barriers to adhesion formation. However, these materials remain in the body and many times are recognized by the body as foreign bodies. Therefore, a second operation may be necessary to remove the barrier material.

The highly purified polysaccharide material of the present invention may be used for orthopedic applications such as prevention of intra-articular adhesion, flexor tendon adhesions, spinal scarring, frozen shoulder, rotator cuff injury and others. Furthermore, this highly purified material may be used as a matrix for growing chondrocytes or other cells for re-implantation and regeneration of natural tissues such as cartilage. As a matrix, the material may also serve as a carrier for growth factors, pharmacologically active agents which might induce the regeneration of selected tissues. The thus highly purified biodegradable polysaccharide gel, solution or film of the present invention would be placed between tissues to affect adhesion formation, localized in contact with the affected tissue(s) for other applications referenced, and affixed to cover the affected tissue for regeneration. The application of these materials may occur before, during or post operatively. Co-pending application 07/644,758, filed January 24, 1991 and assigned to the assignee of the present invention, relates to the use of derivatives of chitosan for adhesion prevention.

Several patents have been issued revolving around the use of hyaluronic acid for anti-adhesion applications (Balasz, U.S. Patent 4,141,973 and DeBelder, WO 86/00912). While these patents focus on the use of gels and films for the prevention of adhesion formation, they do not address the fabrication of highly purified films which can be easily manufactured, manipulated, and applied for various anti-adhesion applications. Other articles and patents have been issued involving the use of polysac¬ charides as carriers of therapeutic agents to prevent fibrin deposition (Mohler, WO 89/00049; Sheffield, U.S. Patent 4,889,722 and Dizegra, EP 0234887). Still other polysaccharides such as xanthan gum (Higham, U.S. Patent 4,994,277), oxidized regenerated cellulose (Linsky, EP 0262890 and EP 0213563), and sodium/calcium alginate (G. Blaine, Medical Press, August 20, 1947, p. 166).

While these patents and publications all address the uses for the polysaccha¬ rides, the issue of purification and fabrication has been limited.

SUMMARY OF THE INVENTION

An object of this invention is to provide an inexpensive and efficient method of preparing highly purified biodegradable polysaccharides for internal biomedical applications. It is another object of this invention to provide a filtration process for purifying polysaccharides so that a more manageable and easily manipulated film composed of highly purified polysaccharide material may be manufactured.

The materials of choice in this invention include, but are not limited to, methyl cellulose and derivatives thereof, chitin/chitosan and derivatives, alginic acid, xanthan gum, and low molecular weight hyaluronic acid (> 750,000 daltons). Other materials such as collagen, polyamino acids, and others may also successfully be employed utilizing the purification method of this invention. While there have been patents that addressed the purification of naturally occurring hyaluronic acid for medical applications (Balazs, U.S. 4,141 ,973; Hildesheim, EP 0239335; Delia Valle, EP 0138572 and Brown, EP 0144019), this invention provides for a simple filtering procedure which can be employed for all polysaccharides to be used for internal applications. While many "medical grade" polysaccharides can be purchased from various suppliers, most are unfit for internal implantation in several tissues and result in severe inflammatory responses. What are referred to as "medical grade" are, for the most part, materials approved for food substitutes or for external use. In the present invention, these materials are rendered free of all excess inflammatory agents such as protein, nucleic acids, pyrogen, lipids, hydrophobic impurities, low molecular weight impurities and others. The resulting products may provide high molecular weight materials, if desired, which are protein, peptide and pyrogen free and whose concentrated solutions do not cause an inflammatory reaction when implanted in animal or human connective tissue spaces. This inflammatory reaction, or lack thereof, may be characterized intra-articularly in the highly sensitive stifle joint of the New Zealand white rabbit. Common methods of analyzing inflammation of this joint would include gross evaluation, cytological evaluation, histopathology, and standard assays for inflammatory mediators in synovial the fluid. It should also be noted that these highly purified materials can be further modified (e.g. cross-linked or blended) to provide final products with varying /n vivo residence times.

It is important to place the materials purified by the process of the present invention into the appropriate physical form to be effective in each application. In two related uses of the present invention, a lavage solution or arthroscopic replacement fluid, a purified dilute solution of polysaccharide may be employed. This solution ranges in concentration anywhere from 0.1 %-2.0% depending on the molecular weight of the polysaccharide. In other related applications such as adhesion prevention, artificial synovia! jluid, or joint lubrication, a more viscous solution or gel of the highly purified polysaccharide may be employed. This type of gel may have a concentration anywhere from 0.1% to 10.0% depending on the molecular weight. in yet other applications, such as spinal scarring, flexor tendon adhesions, and intra-articular adhesions, a lyophilized film offers an excellent form for use. The physical form of this film can be manipulated to provide the desired flexibility and thickness which will provide a film with better application qualities as well as efficacy qualities. The biodegradable polysaccharides to be used in the various orthopedic appli¬ cations indicated would eventually be degraded into smaller biocompatible products which could be easily removed from the body through natural excretory routes.

These and other objects and features of the present invention wiH become apparent from the following detailed description, which discloses several examples of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In the process of the present invention it is preferred to use water soluble polysaccharides, however, the invention is not limited to a water-soluble polymer. Examples of these water soluble materials include, but are not limited to: low molecular weight hyaluronic acid (750,000 daltons), carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium alginate, chitin, chitosan, chitosan lactate, chitosan glutamate, chitosan acetate, methyl chitosan, N -carboxymethyl chitosan, O-carboxymethyl chitosan, N,O-carboxymethyl chitosan, N,O-carboxymethyl chitosan acetate, N-carboxybutyl chitosan, O-carboxybutyl chitosan, N,O-carboxybutyl chitosan and xanthan gum. AH of these products are prepared from natural products or may be obtained through well known fermentation methods and are for the most part commercially available in a medical grade.

When processing the raw material into the purified material suitable for implan¬ tation, it is desired to initially proceed through several dead end filtration steps. For dead end filtration, it is preferred to prepare a relatively dilute solution less than 1% and preferably 0.25% in purified water, (this concentration may be varied depending on the material and the molecular weight) with the higher molecular weight material being diluted further in purified water if necessary to facilitate filtering. It is preferred to conduct the serial dead end filtration via nitrocellulose filters which have a high affinity for protein and nucleic acids. Such filters are available commercially from Schleicher and Schuell.

Following dead end filtration it is desired to process the resulting solution by a modified method of ultra-filtration. In this method, a derivatized ultra-filtration

(UF) membrane is utilized. A membrane of this type is available through AlerCHEK

(Portland, Maine). The membrane as purchased is modified with polypeptides which specifically bind lipopolysaccharides, and other hydrophobic impurities. It is further preferred that the modified ultra-filtration membrane have a pore size that would allow material to not only pass over the membrane, but also through the membrane so that the impurities discussed above are removed in a more efficient manner. This offers exposure of the materials to a greater surface area of the modified filter. It is also desirable, although not necessary, that the ultra-filtration apparatus have a tangential flow. This would allow for a continual circulation of material rather than a single passage as would occur with an axial flow. The pore size of the modified membrane can range anywhere from 0.2 microns to 12 microns depending on the material and molecular weight. If a tangential flow apparatus is used, it is also possible to stack several modified membranes one on the other to reduce the filtering time and at the same time increase the surface area for treatment.

In the next step, it is then preferred to dialyze the resulting solution via a standard ultra-filtration technique utilizing a lower molecular weight cut-off membrane. Once again, the molecular weight cut-off will depend upon the molecular weight of the material being purified and the chemical make-up of the material. In this step, it is desired to use a membrane having the highest possible molecular weight cut-off while still retaining the material with the desired molecular weight. This step will remove any low molecular weight materials which were not removed by absorption onto the modified ultra-filtration membrane in the preceding step. This retained material would then be rendered high in purity.

The highly purified polysaccharide solution made by the method of the present invention may then be placed in the appropriate form for various orthopedic and other medical applications. For example, a lavage or arthroscopic fluid may be prepared by taking the highly pure material and dialyzing against water, physiological saline, PBS, Ringers, or other physiological solutions to get the desired Ph and ion make-up for the desired application. The appropriate concentration can be obtained by diluting the solution with the appropriate aqueous solution, or concentrating by ultra-filtration against the appropriate solution. The final concentration should be approximately 0.1%-4.0%, although this may vary according to the material and its characteristics. Alternatively, the highly purified material can be formed into films for applications such as anti-adhesion of the flexor tendon, intra-articular adhesions, spinal scarring, and intra and extra-articular shoulder afflictions. While there are several ways to prepare films, the desired way is pouring a solution into a shallow flat container, such as a petri dish, and lyophilizing the solution. The resulting product is a highly purified film with outstanding handling characteristics. The physical characteristics of the films may be manipulated by varying the solution concentration to produce a stronger, less flexible film with a more concentrated solution, or a weaker, more fibrous film with better flexi¬ bility with a less concentrated solution. Again, depending on the application, the film may be varied in thickness by varying the amount of solution initially placed in the shallow container. The other advantage to utilizing a lyophilized film in these appli¬ cations, other than facilitated handling, is that the film does not require plasticizers to produce flexibility, and the lyophilized film will have a longer residence time in the specific location due to the initial time needed to hydrate the material prior to the material entering the gelatinous state.

A third example of an internal application for the highly purified film is to take the lyophilized films and modify them to further increase the residence time in an anti-adhesion or spinal scarring application. This can be accomplished by using well known modification techniques such as complexing of sodium alginate films with calcium chloride, or treatment of carboxymethyl chitosan derivatives with an acid solution as discussed in co-pending application 07/644,758. There are, of course, other methods of modifying the various other polysaccharides mentioned which are well known.

It is also possible in this invention to first produce a modified gel with the highly purified solution, and then fyophilize the gel into a film form.

The invention will now be described in further detail with reference being made to the following examples. It should, however, be recognized that the examples are given as being illustrative of the present invention and are not intended to define the spirit and scope thereof.

Example 1

Atwo liter solution of 0.5% high glucuronic acid content (>50%) sodium alginate was prepared in pyrogen free water (saline, PBS, or Ringer's are alternative solvents). The resulting solution was passed through a 12 micron nitro-cellulose membrane filter (Schneider and Schuller). This solution was then passed through a 0.45 micron nitro¬ cellulose filter (it may be necessary to go to a higher pore size, such as 8 microns, prior to the 0.45, depending on the degree of insoluble impurities in the materials and the molecular weight). Also, if necessary, up to a 45 micron filter may be used as a first step and a 1 micron filter may be used as a final step. A 0.5 micron Endotoxin Affinity Membrane (AlerCHEK, Portland, ME Cat # 4200) was installed in a tangential flow ultra-filtration apparatus (Filtron Part Number

FS021K01) and the resulting setup was totally dehydrogenated by passage of a dilute alkaline solution or a dilute sodium dodecylphosphate detergent. This was then flushed out with a dilute methanol solution (10%) followed by exhaustive washing with pyrogen- free water. Removal of trace detergent can be determined by UV analysis at 270 nm.

The alginate solution was then allowed to pass over and through the membrane, constantly recirculating for approximately one hour (this could be reduced if two or more membranes, or larger membranes with greater surface area, were utilized). After this filtration, the pale yellow color in the starting solution was gone and a crystal clear alginate solution remained.

Next, the solution was exhaustively dialyzed on a 300,000 molecular weight cutoff membrane to remove any low molecular weight materials which were not removed by the modified ultra-filtration membrane. This was done against water. For this example, a final 1 liter solution was dialyzed against 7 liters of purified water. As noted above, the molecular weight cutoff for the membrane may be varied depending on the material molecular weight. It is most desirable to use the highest molecular weight cutoff membrane to remove as much low molecular weight material without compromising the desired molecular weight range for the end product. The solution can be dialyzed against any appropriate aqueous solution depending on the application.

Finally, the solution was concentrated via standard ultra-filtrations technique to a 1 % concentration.

The resulting highly purified alginate solution was then run through the standard in vivo biocompatibility tests (sterility, cytotoxicity, pyrogen, hemolysis). Results were negative for each of these tests. The material was then implanted, via injection, into the stifle joint of an SPF New Zealand white rabbit for a period of 2-4 days. The alginate did not produce a gross inflammatory response, or any abnormal cytological or histo- pathological responses. This demonstrates that the material had been purified to a level suitable for internal orthopedic applications.

The use of the rabbit stifle joint for biocompatibility testing is superior to intra¬ muscular testing. Standard intra-muscular implantation of "medical grade" alginate has oh several occasions indicated a biocompatible material. However, when this "medical grade" material was implanted into the stifle joint of a rabbit, it produced a severe infiammatory reaction. Therefore, the stifle joint, being highly vascular, provides a conservative site for biocompatibility testing. Furthermore, the stifle joint is an area where these purified materials may be used for several orthopedic applications.

Example 2

A one liter solution of 0.5% alginate was prepared and purified as indicated above in Example 1. Following the dialysis step against water to remove low molecular weight materials, the solution was dialyzed against 7 liters of Ringer's Lactate on an ultra-filtration membrane with a 30,000 molecular weight cutoff. This solution was then concentrated on the same membrane to half the original volume, thus giving a 1 % solu¬ tion of alginate in Ringer's. This resulting solution was sterile filtered through a 0.22 micron disposable sterile filter system (Falcon). This may then be utilized as an infrequently applied lavage solution.

Example 3

A nine liter batch of alginate was prepared as indicated in Example 1 with the exception of the medium, in which Ringer's was substituted throughout the procedure instead of water. After sterile filtration through a 0.22 μm disposable sterile filter setup, the solution was ready for use as an arthroscopy solution for distention of the joint.

[Example 4

A solution was prepared as indicated in Example 1. The dialysis step was conducted against 7 liters of IN. saline. The final step was concentration on the 30K membrane to 1/4 the original volume giving a 2% final solution. This solution could subsequently be used for prevention of intra-articular adhesions, spinal scarring, flexor tendon adhesion, intra-articular injections for inflamed joints, or a lubricant for frozen shoulder.

Example 5

As an alternative to concentrating the purified solution via ultra-filtration, a 0.5% solution as prepared in Example 1 was sterile filtered and subsequently freeze dried aseptically. This freeze dried material was then rehydrated with an appropriate aqueous medium to any desired concentration. For example, 5.25 g of purified, sterile, freeze dried alginate could be rehydrated in 50 cc of Ringer's to give a solution of 10.5%. This could be aseptically placed in a syringe and injected into a 1 liter IN. bag of Ringer's for an arthroscopic procedure, giving a final concentration in the bag of 0.5%.

Example 6

Approximately 50 cc of the solution prepared in Example 1 was placed into a standard 90 mm diameter petri dish. The petri dish was then placed in a tray drier at -50°C for 3 days (until the material was fully dehydrated). The resulting film was cut into 2.5 cm² and sterilized by exposure to 0.5 mRad of gamma irradiation. The resulting film was very flexible and may be applied to prevent flexor tendon adhesions, spinal scarring intra-articular adhesions, frozen shoulder, and rotator cuff repairs.

Example 7 A. film was prepared identical to the one illustrated in Example 6, with the exception that the solution was filter sterilized following the dialysis step and the lyophilization of the film was done aseptically. (Preparation via this method will lead to a material with characteristics more closely aligned with the raw material as degradation created by gamma irradiation would not be evident, as might be the case in Example 6).

Example 8

A solution was prepared as outlined in Example 1. Following the dialysis against the 7 liters of purified water, the solution was concentrated to 1/4 the original volume producing a 2% solution. A 50 cc sample of this was placed in a petri dish and placed in a tray dryer for three days at -50°C. The resulting film was less fibrous and less flexible, but retained more integrity than that of the film prepared in Example 6. This type of film could be effectively utilized in a spinal scarring application where the laminectomy defect is relatively more substantial. Example 9

A film was prepared as outlined in Example 6. The sterile alginate film was subsequently submerged in a sterile solution of 2% calcium chloride for 30 minutes. The resulting film was rendered insoluble in water. The film was exhaustively washed in sterile water to remove excess calcium chloride. The film was tested for sterility, cytotoxicity, and hemolysis. All tests were negative. This film may be applied in areas where a longer in vivo residence time of the film would be required. This would include spinal scarring, flexor tendon adhesions, and intra-articular adhesions.

Example 10

A solution of sodium alginate was prepared as described in Example 1. . Approximately 100 cc were sterile filtered through a 0.22 micron sterile membrane apparatus (Falcon). Under a laminar flow hood, 35 cc of the solution was placed into a sterile container. Approximately 0.158 grams of calcium carbonate (sterilized by steam autoclave) was added to the solution and it was stirred. After about 30 minutes to allow the calcium carbonate to totally disperse, a freshly made solution of D-glucono- delta-lactone (GDL) (0.187 g in 10 cc water) was sterile filtered into the solution. This was allowed to be stirred for about 20 minutes at which point a sample of 30 cc was poured into a 90 mm dish. After about 24 hours a smooth optical clear gel was obtained. The gel was then extensively washed against water and tested for sterility, cytotoxicity (direct and extraction), and hemolysis (direct and extraction). All tests were negative. This material may be used to prevent spinal scarring, intra-articular adhesions, and may have some potential as an artificial cartilage.

Example 11

A gel was prepared as indicated in Example 10. The gel was allowed to dehydrate under laminar flow for 24 hours. The gel was then rehydrated in a 0.1% solution of heparin. This gel could then be used as a method of delivering an anti- thrombogenic material to further assist in reducing fibrinous exudate build up in the process of adhesion formation. This example also indicates the possibility of delivering enzymes such as streptokinase to break down adhesions in areas where they have already formed, or delivery of Tissue Plasminogen Activator (TPA) also known to break down fibrinous tissue. Example 12

A solution was prepared as indicated in Example 1. Further, heparin (from Sigma) was added to the solution to give an alginate solution 0.1% in heparin. This was then placed into a film form as indicated in Example 6.

Example 13

A 0.5% solution of N.O-CM Chitosan (NOVA Chem) was prepared by dissolving the material in purified pyrogen free water. The solution was initially filtered through a 12 micron nitro-cellulose filter, followed by filtration through an 8 micron nitro-cellufose filter, and a 0.45 micron nitro cellulose filter. The resulting material was passed over and through the 0.5 micron modified ultra-filtration membrane (AlerCHEK) for 8 hours via tangential flow ultrafiitration set (Filtron). The resulting solution was then dialyzed against 7 liters of water on a 30K molecular weight cut off membrane utilizing the same ultra-filtration setup without the modified 0.5 micron filter. This solution was then sterile filtered through a 0.45 micron sterile filter (Falcon). 0.5 cc of this solution was placed intra-articularly into a New Zealand white rabbit. After 2 days the rabbit was sacrificed and the joint evaluated. There was no abnormal finding at necropsy, nor on cytological evaluation of the synovial fluid, nor on histology of the various tissues, including the articular cartilage, and the synovial membrane.

Example 14

A sample of NOCC prepared in Example 13 was taken and processed exactly as the alginate in Example 6. The resulting film was extremely flexible and could be utilized for all the applications noted with the alginate films.

Example 15

A 30cc sample of the solution prepared in Example 13 was cast in a 90 mm petri dish and allowed to evaporate for 24 hours. The resulting thin film was easily removed from the plate and may be utilized as described above.

While several examples of the present invention have been described, it is obvious that many changes and modifications may be made thereunto, without departing from the spirit and scope of the invention.

Claims

CLAIMS:

1. A process for purifying polysaccharides comprising: filtering a polysaccharide solution having a concentration of less than 1% through a first nitrocellulose filter having a pore size of at least 45 microns; filtering the resulting solution through a second nitrocellulose filter having a pore size of less than 12 microns; filtering the resultant solution through a membrane having a pore size less than 12 microns, said membrane modified with polypeptides to bind hydrophobic impurities; and dialyzing the resulting solution with a membrane having a lower molecular weight cut-off than the first ultra-filtration membrane.

2. The process for purifying polysaccharides as set forth in claim 1 wherein said first filter has a pore size of 8 to 12 microns and said second filler has a pore size of .22 to .45 micron.

3. A polysaccharide material for biomedical application made by the process of claim 1.

4. A process for purifying polysaccharides comprising the steps of: forming a less than 1% by weight aqueous solution of polysaccharides; passing this solution through at least one filtration step with a nitrocellulose filter having a pore size of less than one micron; filtering the resultant solution with a membrane having a pore size less than 12 microns, said membrane modified with polypeptides to bind hydrophobic impurities; and dialyzing the second resultant solution against an aqueous solution.

5. A polysaccharide material for biomedical applications made by the process of claim 4.