WO2008133847A1

WO2008133847A1 - Method for making a polysaccharide dialdehyde having high purity

Info

Publication number: WO2008133847A1
Application number: PCT/US2008/005013
Authority: WO
Inventors: Jeffrey David Cohen; Henry Keith Chenault; Rinaldo S. Schiffino
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 2007-04-24
Filing date: 2008-04-18
Publication date: 2008-11-06
Also published as: EP2137219A1; CN101663328A; JP2010525140A

Abstract

A method for making polysaccharide dialdehydes is disclosed, which uses a combination of precipitation and separation steps to purify the polysaccharide dialdehyde formed by oxidation of a polysaccharide with periodate. The method is simple, rapid, and provides a polysaccharide dialdehyde having very low levels of iodine-containing species. The polysaccharide dialdehyde is particularly suitable for preparing hydrogel adhesives for medical applications.

Description

TITLE

METHOD FOR MAKING A POLYSACCHARIDE DIALDEHYDE HAVING HIGH PURITY

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority from U.S. Provisional Application

Serial No. 60/925,948 (filed April 20, 2007), the disclosure of which is incorporated by reference herein for all purposes as if fully set forth.

FIELD OF THE INVENTION The invention relates to the field of medical adhesives. More specifically, the invention relates to a method for making a polysaccharide dialdehyde in a highly pure form which is useful for the preparation of hydrogel adhesives for medical applications.

BACKGROUND OF THE INVENTION Tissue adhesives have many potential medical applications, including wound closure, supplementing or replacing sutures or staples in internal surgical procedures, adhesion of synthetic onlays or inlays to the cornea, drug delivery devices, and as anti-adhesion barriers to prevent post-surgical adhesions. Conventional tissue adhesives are generally not suitable for a wide range of adhesive applications. For example, cyanoacrylate-based adhesives have been used for topical wound closure, but the release of toxic degradation products limits their use for internal applications. Fibrin-based adhesives are slow curing, have poor mechanical strength, and pose a risk of viral infection. Additionally, fibrin- based adhesives do not covalently bind to the underlying tissue. Several types of hydrogel tissue adhesives have been developed, which have improved adhesive and cohesive properties and are nontoxic. These hydrogels are generally formed by reacting a component having nucleophilic groups with a component having electrophilic groups, which are capable of reacting with the nucleophilic groups of the first component, to form a crosslinked network via covalent bonding. Polysaccharide dialdehydes have been used as the component having electrophilic groups for preparing these hydrogel adhesives because they are highly biocompatible and biodegradable (see for example, Kodokian et al., copending and commonly owned U.S. Patent Application Publication No. 2006/0078536, and Goldmann, U.S. Patent Application Publication No. 2005/0002893). The polysaccharide dialdehydes are typically formed by oxidation of the polysaccharide with periodate (Mo et al. J. Biomater. Sci. Polymer Edn. 11 :341-351 , (2000); and Halsall et al., J. Chem. Soc. 1947, 1427-1432). For medical applications of these hydrogel adhesives, the amount of unreacted periodate and iodine-containing byproducts in the polysaccharide dialdehyde preparation must be reduced to low levels to prevent toxic effects. Consequently, the polysaccharide dialdehyde is typically purified by extensive dialysis to remove the iodine containing species (Kodokian et al. supra; Goldmann, supra; Bondarev et al. SU 1541218; and Uraz et al., Carbohydrate Polymers 34:127-130 (1997)). However, dialysis is a slow process requiring many days and therefore, is not well suited to the large scale production of polysaccharide dialdehydes. Alternatively, iodate may be separated from dextran dialdehyde, prepared by the oxidation of dextran with periodate, using an ion exchange resin, as described by Seitz et al. (Journal for Praktishe Chemie (Leipzig) 311(1):141-146 (1969)).

Therefore, the problem to be solved is to provide a method for making polysaccharide dialdehydes that provides a product that has a low level of iodine-containing species and is simple and rapid.

Applicants have addressed the stated problem by discovering a method of making polysaccharide dialdehydes that comprises a combination of precipitation and separation steps to purify the polysaccharide dialdehyde formed by oxidation of the polysaccharide with periodate. The method provides a polysaccharide dialdehyde with very low levels of iodine-containing species.

SUMMARY OF THE INVENTION Disclosed herein is a method for making polysaccharide dialdehydes that is simple, rapid, and produces product with very low levels of iodine-containing species.

Accordingly, in one embodiment the invention provides an improved method for making a polysaccharide dialdehyde by a process that comprises reacting a polysaccharide with at least one periodate salt at a temperature above about 10 ⁰C to produce a product comprising said polysaccharide dialdehyde, iodate in the form of at least one iodate salt, and optionally periodate in the form of at least one unreacted periodate salt, the improvement comprising the steps of: a) adding a source of at least one cation, which is capable of precipitating at least a portion of the iodate, to said product thereby forming a precipitate comprising said at least one cation and iodate, and a supernatant fluid that comprises the polysaccharide dialdehyde; b) separating at least a portion of the precipitate from the supernatant fluid; c) adding at least one iodide salt to the supernatant fluid thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine; d) optionally filtering the mixture to obtain a filtrate; e) adding a first organic solvent to the mixture of (c) or the filtrate of (d) in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture of (c) or the filtrate of (d), thereby forming a first phase comprising the polysaccharide dialdehyde and a second phase comprising the organic solvent; f) separating at least a portion of the first phase from the second phase to provide a separated polysaccharide dialdehyde; g) contacting the separated polysaccharide dialdehyde with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming hardened polysaccharide dialdehyde; h) comminuting the hardened polysaccharide dialdehyde to form particulate polysaccharide dialdehyde; i) contacting the particulate polysaccharide dialdehyde with a cold aqueous liquid at a temperature of less than about 10 ⁰C for a time sufficient to remove at least some iodine-containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde; and j) recovering at least a portion of the purified polysaccharide dialdehyde from the cold aqueous liquid.

In another embodiment, the invention provides an improved method for making a polysaccharide dialdehyde by a process that comprises reacting a polysaccharide with at least one periodate salt at a temperature above about 10 ⁰C to produce a product comprising said polysaccharide dialdehyde, iodate in the form of at least one iodate salt, and optionally periodate in the form of at least one unreacted periodate salt, the improvement comprising the steps of: a) cooling said product to a crystallization temperature below about 5 ⁰C and maintaining said product at the crystallization temperature for a time sufficient to form a precipitate comprising at least one periodate salt and a first supernatant fluid that comprises polysaccharide dialdehyde; b) separating at least a portion of the precipitate from the first supernatant fluid; c) adding a source of at least one cation, which is capable of precipitating at least a portion of the iodate, to the first supernatant fluid thereby forming a second precipitate comprising said at least one cation and iodate, and a second supernatant fluid that comprises the polysaccharide dialdehyde; d) separating at least a portion of the second precipitate from the second supernatant fluid; e) adding at least one iodide salt to the second supernatant fluid thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine; f) optionally filtering the mixture to obtain a filtrate; g) adding a first organic solvent to the mixture of (e) or the filtrate of [T) in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture of (e) or the filtrate of (T), thereby forming a first phase comprising the polysaccharide dialdehyde and a second phase comprising the organic solvent; h) separating at least a portion of the first phase from the second phase to provide a separated polysaccharide dialdehyde; i) contacting the separated polysaccharide dialdehyde with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming hardened polysaccharide dialdehyde; j) comminuting the hardened polysaccharide dialdehyde to form particulate polysaccharide dialdehyde; k) contacting the particulate polysaccharide dialdehyde with a cold aqueous liquid at a temperature of less than about 10 ⁰C for a time sufficient to remove at least some iodine containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde; and I) recovering at least a portion of the purified polysaccharide dialdehyde from the cold aqueous liquid.

DETAILED DESCRIPTION OF THE INVENTION Disclosed herein is a method for making polysaccharide dialdehydes, which uses a combination of precipitation and separation steps to purify the polysaccharide dialdehyde formed by oxidation of a polysaccharide with periodate. The method is simple, rapid, and provides a polysaccharide dialdehyde having very low levels of iodine-containing species, specifically, less than about 0.03% by weight elemental iodine. The low level of iodine-containing species makes the polysaccharide dialdehydes particularly useful for preparing hydrogel adhesives for medical and veterinary applications, including, but not limited to, wound closure, supplementing or replacing sutures or staples in internal surgical procedures such as intestinal anastomosis and vascular anastomosis, tissue repair, ophthalmic procedures, drug delivery, and in anti-adhesive applications. Additionally, the resulting polysaccharide dialdehyde product has a higher bulk density than polysaccharide dialdehydes that are purified by dialysis and subsequently lyophilized. The higher bulk density facilitates the transportation and storage of the polysaccharide dialdehyde. The following definitions are used herein and should be referred to for interpretation of the claims and the specification. The terms "polysaccharide dialdehyde" or "oxidized polysaccharide" are used interchangeably herein to refer to a polysaccharide which has been reacted with periodate to introduce aldehyde groups into the molecule. The term "water-dispersible, multi-arm polyether amine" refers to a branched polyether, wherein at least three of the branches ("arms") are terminated by a primary amine group, which is water soluble or able to be dispersed in water to form a colloidal suspension capable of reacting with a second reactant in aqueous solution or dispersion. The term "polyether" refers to a polymer having the repeat unit [-O-

R]-, wherein R is a hydrocarbylene group having 2 to 5 carbon atoms.

The term "tissue" refers to any tissue, both living and dead, in humans or animals.

The term "hydrogel" refers to a water-swellable polymeric matrix, consisting of a three-dimensional network of macromolecules held together by covalent or non-covalent crosslinks, that can absorb a substantial amount of water to form an elastic gel.

By medical application is meant medical applications as related to humans and animals. In the method disclosed herein, a polysaccharide is oxidized to produce a polysaccharide dialdehyde using periodate. The polysaccharide dialdehyde is then separated from excess periodate and iodine-containing byproducts using a series of precipitation and separation steps, as described below. Polysaccharides useful in the invention include, but are not limited to, dextran, starch, cellulose, hemicellulose, methyl cellulose, ethyl cellulose, chondroitin sulfate, dextran sulfate, and hyaluronic acid. These polysaccharides are available commercially from sources such as Sigma Chemical Co. (St Louis, MO). In one embodiment, the polysaccharide is dextran. Typically, polysaccharides are a heterogeneous mixture having a distribution of different molecular weights, and are characterized by an average molecular weight, for example, the weight-average molecular weight (M_w), or the number average molecular weight (M_n), as is known in the art. Suitable polysaccharides have a weight-average molecular weight from about 1 ,000 to about 1 ,000,000 Daltons, preferably from about 3,000 ' to about 250,000 Daltons. In one embodiment the polysaccharide is dextran having a weight-average molecular weight of about 8,500 to 11 ,500 Daltons. In another embodiment, the polysaccharide is dextran having a weight-average molecular weight of about 60,000 to 90,000 Daltons.

The polysaccharide is oxidized to introduce aldehyde groups by reaction with periodate in an aqueous solution using any suitable periodate salt, for example, sodium periodate or potassium periodate, as is known in the art (see for example, Mo et al. J. Biomater. Sci. Polymer Edn. 11 :341-351 , (2000); Halsall et al., J. Chem. Soc. 1947, 1427-1432); Kodokian et al. supra; and Goldmann, supra). The polysaccharide may be reacted with different amounts of periodate to give polysaccharides with different degrees of oxidation and therefore, a different dialdehyde content. Specifically, a polysaccharide is reacted with at least one periodate salt at a temperature above about 10 ⁰C to produce a product comprising a polysaccharide dialdehyde, and iodate in the form of at least one iodate salt. Additionally, if an excess of periodate is used in the reaction, the product will also contain periodate in the form of at least one unreacted periodate salt. The temperature used for the oxidation reaction is typically from about 11 ⁰C to about 70 ⁰C, preferably from about 20 ⁰C to about 60 ⁰C. It should be noted that the oxidation reaction is an exothermic process that causes the temperature of the solution to rise. Consequently, the solution may need to be cooled to attain the desired temperature. The time required for the reaction will vary depending on a number of factors, including the temperature and the concentration of the polysaccharide and periodate used. Typical reaction times range from about 30 minutes to about 6 hours. In one embodiment, an aqueous dextran solution is reacted with an aqueous solution of sodium periodate at a temperature of about 20 ⁰C to 30 ⁰C for a period of about 3.5 to about 5 hours.

In one embodiment, the product of the oxidation reaction is cooled to a crystallization temperature below about 5 ^CC, preferably between about 2 ⁰C and about -8 ⁰C and maintained at a crystallization temperature for a time sufficient to form a precipitate comprising at least one periodate salt and a first supernatant fluid that comprises the polysaccharide dialdehyde. The time required will vary depending on the particular conditions used (e.g., the volume of the product and the temperature), but is typically from about 5 minutes to about 60 minutes. Preferably, the time is not long enough for the entire product to freeze. This cooling may be carried out in two separate steps wherein the reaction product is rapidly cooled to one crystallization temperature, specifically below about 5 ⁰C for a period of time, typically from about 5 minutes to about 40 minutes, to form the precipitate. Then, the reaction mixture is maintained at a second crystallization temperature, specifically below about 5 ⁰C, which is different from the first crystallization temperature, for a time sufficient to crystallize the periodate salt, typically from about 20 minutes to about 55 minutes, preferably about 30 minutes. The cooling step is optional, but is particularly beneficial when an excess of periodate is used in the oxidation reaction. Then, at least a portion of the precipitate is separated from the first supernatant fluid using methods known in the art, for example, filtering, decanting, centrifuging, or siphoning off the first supernatant fluid. Ideally, substantially all of the precipitate is separated from the first supernatant fluid.

Next, iodate is precipitated out as an insoluble salt by adding a source of at least one cation, which is capable of precipitating at least a portion of the iodate, to the first supernatant fluid thereby forming a second precipitate comprising the cation(s) and iodate, and a second supernatant fluid that comprises the polysaccharide dialdehyde. Ideally, the cation(s) is /are capable of precipitating substantially all of the iodate. The source of the cation(s) is at least one soluble salt comprising a cation that forms a salt with iodate that has a low solubility in aqueous solution. Suitable cations include, but are not limited to, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺ , Cu²⁺,

Zn²⁺ , Pb²⁺, Ag⁺ , Cd²⁺, and Hg²⁺. If the polysaccharide dialdehyde is to be used for medical applications, a cation with low toxicity is preferred, for

2+ 2+ 2+ 2+ example, Ca , Mn , Zn , or Cu . Suitable soluble salts include, but are not limited to, calcium chloride, calcium acetate, calcium bromide, strontium chloride, strontium acetate, barium chloride, barium acetate, manganese chloride, manganese acetate, cupric chloride, cupric acetate, zinc chloride, zinc acetate, silver nitrate, cadmium chloride, cadmium acetate, lead chloride, lead acetate, mercuric chloride, and mercuric acetate. Preferred soluble salts for the preparation of polysaccharide dialdehydes to be used for medical applications include calcium chloride, calcium acetate, calcium bromide, manganese chloride, manganese acetate, zinc chloride, zinc acetate, cupric chloride, and cupric acetate. In one embodiment, the salt is calcium chloride. This step is typically done at room temperature (e.g., 20 ⁰C to 25 ⁰C) for a period of at least about 30 minutes. Then, at least a portion of the second precipitate is separated from the second supernatant fluid using methods known in the art, for example, filtering, decanting, centrifuging, or siphoning off the second supernatant fluid. Ideally, substantially all of the second precipitate is separated from the second supernatant fluid.

At least one iodide salt is then added to the second supernatant fluid, thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine. Any suitable iodide salt may be used including, but not limited to, sodium iodide, potassium iodide, lithium iodide, calcium iodide, or ammonium iodide. In one embodiment, the iodide salt is potassium iodide. This step is typically done at room temperature (e.g., 20 ⁰C to 25 ⁰C) for a period of at least about 30 minutes. The mixture is optionally filtered to remove the iodine and obtain a filtrate that comprises the polysaccharide dialdehyde. Alternatively, the iodine may be removed by allowing it to sublime.

Next, a first organic solvent is added to either the mixture or the filtrate in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture or the filtrate, thereby forming a first phase comprising the polysaccharide and a second phase comprising the organic solvent. Ideally, substantially all of the polysaccharide dialdehyde is separated from the mixture or the filtrate. Suitable first organic solvents include, but are not limited to, acetone, methyl ethyl ketone, tetrahydrofuran (THF), methanol, ethanol, and isopropanol. In one embodiment the first organic solvent is acetone. The amount of the first organic solvent will vary depending on the conditions, and the optimum amount can be readily determined using routine experimentation by one skilled in the art. Typically, the amount of first organic solvent used is about three times the mass of the reaction mixture or the filtrate. This step is typically done at room temperature (e.g., 20 ⁰C to 25 °C) for a time of at least about 30 minutes. At least a portion of the first phase is separated from the second phase using methods known in the art (e.g., filtering, decanting, centrifuging, or siphoning) to provide a separated polysaccharide dialdehyde. Ideally, substantially all of the first phase is separated from the second phase.

The separated polysaccharide dialdehyde is then contacted with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming a hardened polysaccharide dialdehyde. Suitable second organic solvents include, but are not limited to, methanol, ethanol, isopropanol, butanol, and acetonitrile. In one embodiment the second organic solvent is methanol. The amount of the second organic solvent will vary depending on the conditions, and the optimum amount can be readily determined using routine experimentation by one skilled in the art. Typically the amount of second organic solvent used is from about 25% to about 100% of the volume of the first organic solvent, as described above.

The hardened polysaccharide dialdehyde is comminuted to form a particulate polysaccharide dialdehyde. The comminuting reduces the particulate polysaccharide to a powder and can be done using methods known in the art, for example, grinding, milling, or crushing with a mortar and pestle. Optionally, the particulate polysaccharide dialdehyde may be dried using any suitable method, for example, using heat, vacuum, a combination of heat and vacuum, or flowing a stream of dry air or a dry inert gas such as nitrogen over the particulate polysaccharide dialdehyde. The particulate polysaccharide dialdehyde is then contacted with a cold aqueous liquid at a temperature of less than about 10 ⁰C, preferably from about -15 ⁰C to about 5 ⁰C, for a time sufficient to remove at least some of the iodine-containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde. Ideally, substantially all of the iodine-containing species are removed from the particulate polysaccharide. The cold aqueous liquid may be pure water or comprise a mixture of water and a water-soluble organic solvent such as methanol, ethanol, or isopropanol. If a mixture of water and a water- miscible organic solvent is used, the mixture contains less than or equal to about 50% of the water-miscible organic solvent by volume, preferably from about 5% to about 50% by volume. In one embodiment, the cold aqueous liquid is pure water at a temperature of about 0 ⁰C to about 5 ⁰C. In another embodiment, the cold aqueous liquid is a mixture comprising water and 30 to 40% methanol by volume at a temperature of about -15 ⁰C to about 5 ⁰C. The time of contacting needs to be optimized to maximize the purity of the polysaccharide dialdehyde while minimizing its loss due to dissolution in the cold aqueous liquid. The solubility and the rate of dissolution of the polysaccharide dialdehyde in the cold aqueous liquid depend on several factors such as its molecular weight and degree of oxidation. Typically, the contact time is from about 30 seconds to about 4 hours, preferably from about 10 minutes to about 60 minutes. At least a portion of the purified polysaccharide dialdehyde is then recovered from the cold aqueous liquid using filtering, decanting, centrifuging, siphoning, or the like. Ideally, substantially all of the purified polysaccharide dialdehyde is recovered from the cold aqueous liquid. Optionally, the contacting with cold aqueous liquid may be repeated one or more times. Optionally, the purified polysaccharide dialdehyde may be dried using any suitable method, for example, using heat, vacuum, a combination of heat and vacuum, or flowing a stream of dry air or a dry inert gas such as nitrogen over the purified polysaccharide dialdehyde. In one embodiment, the purified polysaccharide dialdehyde is dried in a vacuum oven at 20 °C for about 16 to 24 hours. In another embodiment, the product of the oxidation reaction is not cooled to a temperature below about 5 ⁰C, i.e., the optional first step is omitted, and the method is followed as described above from the step that comprises adding a source of at least one cation that is capable of precipitating at least a portion of the iodate. Ideally, the cation(s) is/are capable of precipitating substantially all of the iodate. Specifically, a source of at least one cation is added to the product of the oxidation reaction, thereby forming a precipitate comprising the cation(s) and iodate, and a supernatant fluid. Then, at least a portion of the precipitate is separated from the supernatant fluid using methods as described above. Ideally, substantially all of the precipitate is separated from the supernatant fluid. Next, at least one iodide salt is added to the supernatant fluid thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine. The mixture is optionally filtered to obtain a filtrate. A first organic solvent is added to the mixture or the filtrate in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture or the filtrate, thereby forming a first phase comprising the polysaccharide dialdehyde and a second phase comprising the organic solvent. Ideally, substantially all of the polysaccharide dialdehyde is separated from the mixture or the filtrate. At least a portion of the first phase is separated from the second phase using methods as described above, to provide a separated polysaccharide dialdehyde. Ideally, substantially all of the first phase is separated from the second phase. The separated polysaccharide dialdehyde is then contacted with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming a hardened separated polysaccharide dialdehyde. The hardened polysaccharide dialdehyde is comminuted to form particulate polysaccharide dialdehyde. Optionally, the particulate polysaccharide dialdehyde may be dried as described above. The particulate polysaccharide dialdehyde is then contacted with a cold aqueous liquid at a temperature below about 10 ⁰C for a time sufficient to remove at least some iodine containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde. Ideally, substantially all of the iodine containing species are removed from the particulate polysaccharide dialdehyde. The contacting with cold aqueous liquid may be repeated one or more times. Then at least a portion of the purified polysaccharide dialdehyde is recovered as described above. Ideally, substantially all of the purified polysaccharide dialdehyde is recovered. Optionally, the purified polysaccharide dialdehyde may be dried, as described above.

The amount of iodine-containing species remaining in the purified polysaccharide dialdehyde can be determined using methods known in the art. For example, the total amount of elemental iodine in any form remaining in the purified polysaccharide dialdehyde can be determined using X-ray fluorescence spectrometry. Typically, the amount of elemental iodine remaining in the polysaccharide dialdehyde produced by the method disclosed herein is less than about 0.03 wt%. After the purified polysaccharide dialdehyde is recovered and optionally dried, it may be used to prepare hydrogel adhesives for medical applications by reacting it in an aqueous medium with a polyamine having three or more amine groups. For example, the purified polysaccharide dialdehyde may be reacted with a water-dispersible, multi-arm polyether amine to form a hydrogel tissue adhesive as described by Kodokian et al. (copending and commonly owned U.S. Patent Application Publication No. 2006/0078536, which is incorporated herein by reference). The resulting hydrogel may be used for medical and veterinary applications, including, but not limited to, wound closure, supplementing or replacing sutures or staples in internal surgical procedures such as intestinal anastomosis and vascular anastomosis, tissue repair, ophthalmic procedures, drug delivery, and in anti-adhesive applications.

EXAMPLES The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

The meaning of abbreviations used is as follows: "min" means minute(s), "h" means hour(s), "sec" means second(s), "d" means day(s), "mL" means milliliter(s), "L" means liter(s), "μl_" means microliter(s), "cm" means centimeter(s), "mm" means millimeter(s), "μm" means micrometer(s), "cm³" means cubic centimeter, "mol" means mole(s), "mmol" means millimole(s), "g" means gram(s), "kg" means kilogram(s), "mg" means milligram(s), "meq" means milliequivalent(s), "M_w" means weight-average molecular weight, "wt%" means percent by weight, and "NMR' means nuclear magnetic resonance spectrometry.

EXAMPLE 1 Preparation of Dextran Dialdehyde Having an Average Molecular Weight of 8,500 to 11 ,5000

The purpose of this Example was to prepare dextran dialdehyde having an average molecular weight of 8,500 to 11 ,500 Daltons and a total iodine content of less than 0.03 wt%.

In a 1 L glass reactor vessel equipped with a mechanical stirrer and an addition funnel, 37.5 g of sodium periodate and 350 ml_ of deionized water were added. The reactor contents were stirred until all the solids dissolved. The reactor was cooled to 20 ⁰C. Using the addition funnel, a solution containing 37.5 g of dextran (average molecular weight 8,500 - 11 ,500; Sigma Chemical Co, St Louis, MO, catalog no. D9260) dissolved in 300 mL of deionized water was added to the periodate solution. After completing the addition, the reactor contents were stirred for 5 h at 20 ⁰C. At the end of the reaction period, the reactor contents were transferred to a beaker and the beaker was chilled in an ice/acetone bath until a precipitate comprising a periodate salt was formed. The mixture was then transferred back to the reactor and stirred for 30 min at 2 ⁰C. Then, the reactor contents were filtered, yielding 17.13 g of solids. The filtrate was transferred to a beaker and 18.75 g of CaCI₂.2H₂O was added to the filtrate. The mixture was stirred for 30 min at room temperature forming a precipitate comprising calcium iodate. The precipitate was filtered, yielding 21.45 g of solids. The filtrate was transferred to a beaker and 15.0 g of potassium iodide was added to the filtrate. The mixture was stirred for 30 min at room temperature. The mixture was filtered, but did not result in any detectable amount of solids being collected. The filtrate was transferred to a plastic jug and 2 L of acetone was slowly added to separate the dextran dialdehyde from the filtrate. The contents in the jug were stirred for 30 min, after which time the acetone was decanted off to collect the precipitated solid. The solids were washed with methanol (1 L) and transferred to a blender to be chopped at high speed in the presence of methanol. The solids were filtered and dried under vacuum. Next, 10 g of the solids was mixed in a blender with 200 ml_ of water and 200 cm³ of ice. The blender was stirred for 1 min; then, the mixture was filtered to collect about 8.51 g of solids. The dialdehyde content of the product was determined to be about 51% using proton NMR.

The solid product was analyzed using X-ray fluorescence spectrometry and was found to contain 0.215 wt% chlorine, 0.017 wt% iodine, 0.198 wt% calcium, 0.0272 wt% sodium and 0.237 wt% potassium, and an overall purity of 99.02 wt%. EXAMPLE 2

Preparation of Dextran Dialdehvde Having an Average

Molecular Weight of 60.000 to 90,000

The purpose of this Example was to prepare dextran dialdehyde having an average molecular weight of 60,000 to 90,000 Daltons and a total iodine content of less than 0.03 wt%.

The procedure described in Example 1 was used except that the dextran had an average molecular weight of 60,000 to 90,000 Daltons (Sigma Chemical Co, St Louis, MO, catalog no. D3759) and the amount of sodium periodate used was 18.75 g. The dialdehyde content of the product was determined to be 28% using proton NMR. The solid product was analyzed using X-ray fluorescence spectrometry and was found to contain 0.225 wt% chlorine, 0.019 wt% iodine, 0.0475 wt% calcium, and 0.185 wt% potassium, and an overall purity of 99.98 wt%. EXAMPLE 3

Preparation of Dextran Dialdehvde Having an Average Molecular Weight of 60.000 to 90,000 and an Extent of Oxidation of About 27% without the

Optional Initial Cooling Step The purpose of this Example was to prepare dextran dialdehyde having an average molecular weight of 60,000 to 90,000 Daltons, an extent of oxidation of about 27%, and a total iodine content of less than 0.03 wt%. The purification process did not involve the optional initial cooling to a crystallization temperature below about 5 °C to form a precipitate comprising at least one periodate salt.

A 20-L reactor equipped with a mechanical stirrer, addition funnel, internal thermocouple, and nitrogen purge was charged with 1.00 kg of dextran and 9.00 L of water. The mixture was stirred at ambient temperature to dissolve the dextran, and the solution was cooled to 10-15 ⁰C. A solution of 500 g of sodium periodate in 9.00 L of water was added to the reactor over about an hour while keeping the temperature of the reactor contents below 25 ⁰C. After the sodium periodate solution was added, the mixture was stirred at 20-25 °C for 4 h. At the end of the reaction period, 350 g of CaCI₂-2H₂O was added to the reaction mixture and the mixture was stirred at ambient temperature for 1 h, forming a precipitate comprising calcium iodate. The mixture was filtered to remove the precipitate, and 200 g of potassium iodide was added to the filtrate. The potassium iodide-containing filtrate was stirred at ambient temperature for 30 min and then filtered, giving a second filtrate. A 5- gallon, plastic pail equipped with a mechanical stirrer was charged with 9.0 L of acetone. About 3.0 L of the second filtrate was added to the acetone over about 15 min with stirring, producing a soft, rubbery precipitate. The supernatant liquid was decanted, and the rubbery precipitate was collected. The rest of the second filtrate was processed similarly, adding it in portions to acetone, stirring, and decanting to collect the precipitate. The combined precipitate from acetone was broken up into pieces and washed in portions with methanol in a large stainless steel blender. The precipitated dextran dialdehyde was collected by filtration, dried under vacuum with a nitrogen blanket, and hammer-milled to a fine powder. A 20-L reactor was charged with 18.0 L of deionized water and cooled to about 0 ⁰C. The hammer-milled dextran dialdehyde was added and stirred vigorously for 1 h. The slurry of dextran dialdehyde in cold water was discharged in portions and filtered to collect the solid, which was immediately washed with methanol to remove water from the product. The solid was dried under vacuum with a nitrogen purge to give 789 g of white, granular dextran dialdehyde. The dialdehyde content of the product was determined to be 27% using proton NMR.

The solid product was analyzed using X-ray fluorescence spectrometry and was found to contain 0.271 wt% sodium, 0.0922 wt% chlorine, 0.0723 wt% potassium, 0.0327 wt% calcium and 0.0068 wt% iodine, and an overall purity of 99.2 wt%.

Claims

CLAIMS What is claimed is:

1. In a method for making a polysaccharide dialdehyde by a process that comprises reacting a polysaccharide with at least one periodate salt at a temperature above about 10 ⁰C to produce a product comprising said polysaccharide dialdehyde, iodate in the form of at least one iodate salt, and optionally periodate in the form of at least one unreacted periodate salt, the improvement comprising the steps of: a) adding a source of at least one cation, which is capable of precipitating at least a portion of the iodate, to said product thereby forming a precipitate comprising said at least one cation and iodate, and a supernatant fluid that comprises the polysaccharide dialdehyde; b) separating at least a portion of the precipitate from the supernatant fluid; c) adding at least one iodide salt to the supernatant fluid thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine; d) optionally filtering the mixture to obtain a filtrate; e) adding a first organic solvent to the mixture of (c) or the filtrate of (d) in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture of (c) or the filtrate of (d), thereby forming a first phase comprising the polysaccharide dialdehyde and a second phase comprising the organic solvent; f) separating at least a portion of the first phase from the second phase to provide a separated polysaccharide dialdehyde; g) contacting the separated polysaccharide dialdehyde with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming hardened polysaccharide dialdehyde; h) comminuting the hardened polysaccharide dialdehyde to form particulate polysaccharide dialdehyde; i) contacting the particulate polysaccharide dialdehyde with a cold aqueous liquid at a temperature of less than about 10 ⁰C for a time sufficient to remove at least some iodine-containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde; and j) recovering at least a portion of the purified polysaccharide dialdehyde from the cold aqueous liquid.

2. The method according to Claim 1 further comprising drying the purified polysaccharide dialdehyde after step (j).

3. The method according to Claim 1 wherein the polysaccharide is selected from the group consisting of dextran, starch, cellulose, hemicellulose, methyl cellulose, ethyl cellulose, chondroitin sulfate, dextran sulfate, and hyaluronic acid.

4. The method according to Claim 1 wherein the polysaccharide is dextran.

5. The method according to Claim 1 wherein the source of at least one cation in step (a) is at least one soluble salt comprising a cation selected from the group consisting of Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Ag⁺, Cd²⁺, and Hg²⁺.

6. The method according to Claim 5 wherein the at least one soluble salt is selected from the group consisting of calcium chloride, calcium acetate, calcium bromide, strontium chloride, strontium acetate, barium chloride, barium acetate, manganese chloride, manganese acetate, cupric chloride, cupric acetate, zinc chloride, zinc acetate, silver nitrate, cadmium chloride, cadmium acetate, lead chloride, lead acetate, mercuric chloride, and mercuric acetate.

7. The method according to Claim 5 wherein the at least one soluble salt is calcium chloride.

8. The method according to Claim 1 wherein the separating of (b) is by filtering, decanting, centrifuging, or siphoning.

9. The method according to Claim 1 wherein the at least one iodide salt of (c) is selected from the group consisting of sodium iodide, potassium iodide, lithium iodide, calcium iodide, and ammonium iodide.

10. The method according to Claim 1 wherein the at least one iodide salt of (c) is potassium iodide.

11. The method according to Claim 1 wherein the first organic solvent of (e) is selected from the group consisting of acetone, methyl ethyl ketone, tetrahydrofuran, methanol, ethanol, and isopropanol.

12. The method according to Claim 1 wherein the first organic solvent of (e) is acetone.

13. The method according to Claim 1 wherein the separating of (T) is by filtering, decanting, centrifuging, or siphoning.

14. The method according to Claim 1 wherein the second organic solvent of (g) is selected from the group consisting of methanol, ethanol, isopropanol, butanol, and acetonitrile.

15. The method according to Claim 1 wherein the second organic solvent of (g) is methanol.

16. The method according to Claim 1 wherein the comminuting of (h) is by grinding, milling, or crushing.

17. The method according to Claim 1 wherein step (i) is repeated one or more times.

18. The method according to Claim 1 wherein the recovering of (j) is by: filtering, decanting, centrifuging, or siphoning.

19. The method according to Claim 1 wherein the cold aqueous liquid of (i) is pure water at a temperature of about 0 ⁰C to about 5 ⁰C.

20. The method according to Claim 1 wherein the cold aqueous liquid of (i) is a mixture of water and from about 5% to about 50% methanol by volume at a temperature of about -15 ⁰C to about 5 ⁰C.

21. In a method for making a polysaccharide dialdehyde by a process that comprises reacting a polysaccharide with at least one periodate salt at a temperature above about 10 ⁰C to produce a product comprising said polysaccharide dialdehyde, iodate in the form of at least one iodate salt, and optionally periodate in the form of at least one unreacted periodate salt, the improvement comprising the steps of: a) cooling said product to a crystallization temperature below about 5 ⁰C and maintaining said product at the crystallization temperature for a time sufficient to form a precipitate comprising at least one periodate salt and a first supernatant fluid that comprises polysaccharide dialdehyde; b) separating at least a portion of the precipitate from the first supernatant fluid; c) adding a source of at least one cation, which is capable of precipitating at least a portion of the iodate, to the first supernatant fluid thereby forming a second precipitate comprising said at least one cation and iodate, and a second supernatant fluid that comprises the polysaccharide dialdehyde; d) separating at least a portion of the second precipitate from the second supernatant fluid; e) adding at least one iodide salt to the second supernatant fluid thereby forming a mixture comprising the polysaccharide dialdehyde and molecular iodine; f) optionally filtering the mixture to obtain a filtrate; g) adding a first organic solvent to the mixture of (e) or the filtrate of (T) in an amount sufficient to cause at least some of the polysaccharide dialdehyde to separate from the mixture of (e) or the filtrate of (f), thereby forming a first phase comprising the polysaccharide dialdehyde and a second phase comprising the organic solvent; h) separating at least a portion of the first phase from the second phase to provide a separated polysaccharide dialdehyde; i) contacting the separated polysaccharide dialdehyde with an amount of a second organic solvent sufficient to harden the separated polysaccharide dialdehyde, thereby forming hardened polysaccharide dialdehyde; j) comminuting the hardened polysaccharide dialdehyde to form particulate polysaccharide dialdehyde; k) contacting the particulate polysaccharide dialdehyde with a cold aqueous liquid at a temperature of less than about 10 ⁰C for a time sufficient to remove at least some iodine containing species from the particulate polysaccharide dialdehyde, thereby forming a purified polysaccharide dialdehyde; and

I) recovering at least a portion of the purified polysaccharide dialdehyde from the cold aqueous liquid.

22. The method according to Claim 21 wherein the cooling of (a) is at a temperature between about 2 ⁰C and about -8 ⁰C.

23. The method according to Claim 21 further comprising: drying the purified polysaccharide dialdehyde after step (I).

24. The method according to Claim 21 wherein the polysaccharide is selected from the group consisting of dextran, starch, cellulose, hemicellulose, methyl cellulose, ethyl cellulose, chondroitin sulfate, dextran sulfate, and hyaluronic acid.

25. The method according to Claim 21 wherein the polysaccharide is dextran.

26. The method according to Claim 21 wherein the separating of (b) is by filtering, decanting, centrifuging, or siphoning.

27. The method according to Claim 21 wherein the source of at least one cation in step (c) is at least one soluble salt comprising a cation selected from the group consisting of Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Ag⁺ , Cd²⁺, and Hg²⁺.

28. The method according to Claim 27 wherein the at least one soluble salt is selected from the group consisting of to calcium chloride, calcium acetate, calcium bromide, strontium chloride, strontium acetate, barium chloride, barium acetate, manganese chloride, manganese acetate, cupric chloride, cupric acetate, zinc chloride, zinc acetate, silver nitrate, cadmium chloride, cadmium acetate, lead chloride, lead acetate, mercuric chloride, and mercuric acetate.

29. The method according to Claim 27 wherein the at least one soluble salt is calcium chloride.

30. The method according to Claim 21 wherein the separating of (d) is by filtering, decanting, centrifuging, or siphoning.

31. The method according to Claim 21 wherein the at least one iodide salt of (e) is selected from the group consisting of sodium iodide, potassium iodide, lithium iodide, calcium iodide, and ammonium iodide.

32. The method according to Claim 21 wherein the at least one iodide salt of (e) is potassium iodide.

33. The method according to Claim 21 wherein the first organic solvent of (g) is selected from the group consisting of acetone, methyl ethyl ketone, tetrahydrofuran, methanol, ethanol, and isopropanol.

34. The method according to Claim 21 wherein the first organic solvent of (g) is acetone.

35. The method according to Claim 21 wherein the separating of (h) is by filtering, decanting, centrifuging, or siphoning.

36. The method according to Claim 21 wherein the second organic solvent of (i) is selected from the group consisting of methanol, ethanol, isopropanol, butanol, and acetonitrile.

37. The method according to Claim 21 wherein the second organic solvent of (i) is methanol.

38. The method according to Claim 21 wherein the comminuting of Q) is by grinding, milling, or crushing.

39. The method according to Claim 21 wherein step (k) is repeated one or more times.

40. The method according to Claim 21 wherein the recovering of (I) is by: filtering, decanting, centrifuging, or siphoning.

41. The method according to Claim 21 wherein the cold aqueous liquid of (k) is pure water at a temperature of about 0 ⁰C to about 5 ⁰C.

42. The method according to Claim 21 wherein the cold aqueous liquid of (k) is a mixture of water and from about 5% to about 50% methanol by volume at a temperature of about -15 ⁰C to about 5 ⁰C.

43. The method according to Claim 21 wherein after the cooling of (a), the product is maintained at a second crystallization temperature below about 5 ⁰C, which is different from the crystallization temperature of (a), to crystallize the precipitate.

44. In a method for making dextran dialdehyde by a process that comprises reacting dextran with sodium periodate at a temperature about 20 ⁰C to produce a product comprising said dextran dialdehyde, iodate in the form of at least one iodate salt, and optionally periodate in the form of at least one unreacted periodate salt, the improvement comprising the steps of: a) cooling said product to a crystallization temperature between about 2 ⁰C and -8 °C, and maintaining said product at the crystallization temperature for a time sufficient to form a precipitate comprising at least one periodate salt and a first supernatant fluid that comprises dextran dialdehyde; b) separating at least a portion of the precipitate from the first supernatant fluid; c) adding calcium chloride to the first supernatant fluid thereby forming a second precipitate comprising calcium and iodate, and a second supernatant fluid that comprises the dextran dialdehyde; d) separating at least a portion of the second precipitate from the second supernatant fluid; e) adding potassium iodide to the second supernatant fluid thereby forming a mixture comprising the dextran dialdehyde and molecular iodine; f) optionally filtering the mixture to obtain a filtrate; g) adding acetone to the mixture of (e) or the filtrate of (f) in an amount sufficient to cause at least some of the dextran dialdehyde to separate from the mixture of (e) or the filtrate of (T), thereby forming a first phase comprising the dextran dialdehyde and a second phase comprising acetone; h) separating at least a portion of the first phase from the second phase to provide a separated dextran dialdehyde; i) contacting the separated dextran dialdehyde with an amount of a methanol sufficient to harden the separated dextran dialdehyde, thereby forming hardened dextran dialdehyde; j) grinding the hardened polysaccharide dialdehyde to form particulate polysaccharide dialdehyde; k) contacting the particulate dextran dialdehyde with cold water at a temperature of about 0 ⁰C to about 5 ⁰C for a time sufficient to remove at least some iodine containing species from the particulate dextran dialdehyde, thereby forming a purified dextran dialdehyde;

I) recovering at least a portion of the purified dextran dialdehyde from the cold water; and m) drying the purified dextran dialdehyde.