MXPA01004793A - Biopolymer salts with low endotoxin levels, biopolymer compositions thereof and methods of making the same - Google Patents

Abstract

The present invention is directed towards biopolymer salts and biopolymer compositions comprising a biopolymer salt having an endotoxin content less than about 100 endotoxin units per gram. Because of their low endotoxin content, the biopolymer salts and biopolymer compositions of this invention may be administered parenterally to a patient. The present invention is also directed to methods of preparing the compositions of this invention.

Description

SALTS OF BIOPOLI MERQ WITH ENDOTOXI N IVELES NA LOW, COMPOSITION IS OF BIOPOLI M ERO OF THE M ISMS AND M ETHODS FOR DO THE SAME • 5 BACKGROUND OF THE I NVENTION FIELD OF THE INVENTION This invention relates to biopolymer salts having low levels of endotoxin and to biopolymer compositions thereof. The biopolymer salts of this invention are particularly useful as parenteral implants. The invention also relates to methods for purifying biopolymer salts, such as, alginates and biogomes, to prepare the novel biopolymer salts having low endotoxin content. BACKGROUND OF THE INVENTION Materials that will be used parenterally in the body must be essentially free of pyrogens, which are materials that indicate fever by triggering an immune response. The introduction of pyrogenic materials in the body can produce a sufficiently severe reaction to produce shock or even death. An important pyrogenic material is lipopolysaccharide endotoxin, which exists as a component of the cell walls of gram-negative bacteria. These endotoxins are released in large quantities when the gram-negative cells undergo lysis. Materials that come into contact with water, having high gram-negative bacterial counts, can be expected to contain significant amounts of lipopolysaccharide endotoxin. Although this does not represent a jfl problem for compositions that are administered orally, it is unacceptable for compositions administered parenterally. The lipopolysaccharide endotoxin is not a living material and can not be deactivated by common sterilization techniques, such as autoclaving. Although dry heat and gamma radiation sterilization techniques destroy the endotoxin, these techniques can also destroy or damage many other compounds in the composition. Consequently, many sterile products may contain significant levels of endotoxin unless the endotoxin is specifically removed or deactivated. In addition, because lipopolysaccharide originates from gram-negative bacteria, no sterile material that is originally free of endotoxin can be contaminated with endotoxin as organisms multiply. Endotoxin-free products can also become contaminated after coming into contact with surfaces containing endotoxin; These are mainly surfaces that have had contact with water. Thus, a composition that is to be administered parenterally, must be free of endotoxin and must also be sterile to prevent the regeneration of lipopolysaccharide endotoxin. Biopolymer products, such as alginic acid and its salts, gelatin gum and xanthan gum, are known to be used in a variety of pharmaceutical applications including, for example, in sustained release pharmaceuticals, which are ingested orally. However, these prior biopolymer products have had an endotoxin level that is not suitable for parenteral administration. For parenteral use, the • endotoxin level of biopolymer salts should be less than about 1 00 endotoxin units per gram of biopolymer. It would be highly desirable to provide bipolymer salts having a sufficiently low endotoxin content, so that the biopolymer salts are suitable for parenteral administration. BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to biopolymer salts that are suitable for parenteral use. In particular, this invention relates to biopolymer salts, such as alginates or biogomas, having water-soluble polysaccharides that are biologically produced and having an endotoxin content of less than about 1000 units of endotoxin per gram. The invention is also directed to alginate or biogoma compositions, comprising the biopolymer salt of this invention and a pharmaceutically acceptable solvent. Another embodiment of this invention relates to methods for preparing the biopolymer salts and compositions thereof, which are suitable for parenteral use. In particular, a method comprising the steps of (i) contacting an aqueous solution of a biopolymer salt with a hydrophobic material to adsorb entodotoxin in said material; and (ii) precipitating a biopolymer salt having an endotoxin content of less than about 1000 units of endotoxin per gram from the solution by mixing a water-soluble organic solvent with the solution. In yet another embodiment of the method of this invention, the precipitation step can be replaced by the step of extracting endotoxin from the aqueous solution with an organic solvent immiscible with water. These methods advantageously provide biopolymer salts and biopolymer compositions having an endotoxin content of less than 1000 units of endotoxin per gram. These methods can be applied to a wide variety of biopolymer salts comprising water-soluble polysaccharides, including not only the alginates and biogomes mentioned above, but also chitosan, carrageenan, agar, welana gum, S657 gum, ramsana gum, carboxymethylcellulose and substitutions. of carboxymethylcellulose, among others. The novel biopolymer salts and compositions thereof are highly useful for use as parenteral implants. They can also be used, for example, to supplement natural lubricating fluids, to coat catheters, to thicken parenteral injections, to provide tissue volume and for cell encapsulation techniques.

DETAILED DESCRIPTION OF THE I NVENTION Endotoxin levels are normally measured using the Lipo Ammocyte Lysate Test Method (LAL). There are several variations of this test method in common use (eg, Clot-gel endpoint, chromogenic LAL, kinetic-chromogenic LAL), which produce a visual or color response in proportion to the amount of endotoxin present. Endotoxin levels are measured in endotoxin units (eu). The biopolymer salts and biopolymer compositions of the present invention have endotoxin levels of less than about 10000 per gram of biopolymer salt in a dry base. Preferably, the biopolymer salts and biopolymer compositions of the present invention have endotoxin levels of less than about 50 eu per gram, and more preferably, less than about 20 eu per gram. The bipolymer salts of this invention are water soluble polysaccharides that are either exuded by, or are extracted from, living organisms. Alginates are salts of alginic acid, which is a copolymer composed of units of D-mannuronic acid and L-guluronic acid. The arrangement and relative amounts of mannuronic and guluronic acid are determined mainly by the source from which the alginate is manufactured. For example, the most commercial algal salts are produced by the extraction of brown marine algae. The alginate produced from Macrfocystis pyrifera has a ratio of manuronic to guluronic unit (M / G ratio) of approximately 1.56: 1, while alginate produced from Laminaria hyperborea has an M / G ratio of approximately 0.45. The monovalent salts (sodium or potassium salts) of alginate are normally soluble in water, although the divalent salts (calcium, barium), polyvalent salts (iron, aluminum, etc.) and alginic acid form water-insoluble solids or gels. Alginates are commercially available from ISP Alginates (San Diego, California). Biogomas are salts of complex organic acids and are produced by fermentation of microorganisms. Gelagne gum refers to the extracellular polysaccharide obtained from microorganisms of the species Sphingomonas elodea, in a suitable nutrient medium. Similarly, xanthan gum is a hydrophilic polysaccharide, which is obtained by fermentation of microorganisms of the genus Xanthomonas, in an appropriate nutrient medium. Gelagne gum and xanthan gum are useful viscosifying agents. Gelane gum is also useful as a gelling agent. Depending on the biogome, the monovalent salts (sodium or potassium salts), in a normal way, but not necessarily, will make the biogoma soluble in water, while the divalent salts (magnesium, calcium, barium) and polyvalent salts (iron, aluminum, etc.) .) in the biogoma can form, but not necessarily, solids or gels insoluble in water. The biopolmer employed in this invention is an alginate or biogoma. The alginate is a salt of algic acid, while the biogoma is a salt of a complex organic acid, usually with a long polymer chain that increases the viscosity. Most preferably, with respect to alginates, the salt is sodium alginate. Alginate will normally have a ratio of mannuronic acid to guluronic acid from about 0.3: 1 to about 2: 1. In general, high mannuronic acid alginates have a ratio higher than 1 while high guluronic acid alginates have a ratio less than 1. An example of a preferred source of alginate, which can be used to prepare the purified alginates of the present invention is "KELTON E", which is available from ISP Alg inates (San Diego, California). KELTON E LVCR is obtained from giant kelp Macrfocystis pyrifera, and is an alginate with a high content of mannuronic acid, having a ratio of mannuronic acid to guluronic acid of approximately 1.56: 1. Normally, commercially available KELTON E LVCR has an endotoxin level in the range from about 30,000 eu per gram to about 60,000 eu per gram. Pharmaceutical compositions for parenteral administration should normally not have more than about 1000 IU per gram. Accordingly, before KELTONE LVCR can be used in a parenteral application, the level of endotoxin must be substantially reduced. This invention provides a method for reducing the level of endotoxin in known salts of algic acid, such as KELTONE LVCR, to below about 100 IU per gram. Two examples of a preferred source of biogoma, which can be used to prepare the purified biogoma of the present invention are gelatin gum "GELRITE", derived from the microorganism Sphingomonas elodea, or xanthan gum "KELTROL T", which is derived from the microorganism Xanthomonas campestris. Both biogomes are available from Kelco Biopolymers (San Diego, California). Normally, GELRITE gelatin gums or KELTROL ™ xanthan gums, which are commercially available, are produced from gram-negative bacteria and, as a result, are found to have endotoxin levels above 1,000 IU per gram. Removing these excessively high endotoxin loads from the biopolymer in a commercially efficient manner can be particularly challenging. As described above with respect to alginates, the pharmaceutical compositions for parenteral administration should normally not have more than 1 000 eu per gram. Consequently, before biogomas can be used in a parenteral application, the level of endotoxin must be dramatically reduced. This invention provides a method for reducing the level of endotoxin in known biogomas below 1 000 eu per gram. The molecular structure of lipopolysaccharide endotoxin consists of a lipid head and a polysaccharide tail. Without being bound by a theory, it is believed that the lipid portion of the polymer induces the pyrogenic response and that removing or breaking the lipid portion can eliminate the induced response. Because the polysaccharide tail of endotoxin is similar in molecular structure to the biopolymer, separation of the endotoxin from the biopolymer salt is not a simple matter. A variety of techniques, which are described in the literature, are used in the pharmaceutical industry to remove endotoxin from materials. However, many of these methods would also destroy or otherwise interact unfavorably with the biopol molecule., making such techniques inappropriate for the depyrogenation of biopolymer compositions.

The present method of this invention uses the combination of two techniques to obtain the purified biopolymer salts hitherto unavailable. In particular, it has been found that when these techniques are used in combination, the level of endotoxin in alginates and biogomas can be reduced to less than about 1000 per g branch. This method for preparing a biopolymer composition comprising, a salt of a biopolymer having an endotoxin content of less than about 1000 units of endotoxin per gram, comprises the steps of (i) contacting an aqueous solution of a salt of biopol number with a hydrophobic material to adsorb endotoxin in said material; and (ii) precipitating the biopolymer salt having an endotoxin content of less than about 100 units of endotoxy per gram of the solution, by mixing a water-miscible organic solvent with the solution. In general, the aqueous start solution will have an alginate or biogoma concentration of from about 0.5 to about 5 percent by weight of the solution. Most preferably, the aqueous solution is a mixture of alginate and water, or biogoma and water. As noted above, an element of the method of this invention includes the adsorption of endotoxin on hydrophobic materials. Without wishing to join a theory, it is believed that the lipid end of the endotoxin molecule is attracted to the hydrophobic material. The biopolymer salts, which are polysaccharide polymers, lack this hydrophobic character. Consequently, it is not believed that they adhere to the hydrophobic material.

Preferred hydrophobic materials for use in this invention include, for example, polystyrene, polypropylene, fluorocarbon polymers, such as "TEFLON" from Dupont and similar. More polypropylene and polystyrene are preferred. If the hydrophobic surface is used in the form of a filtration membrane, it is also possible to physically filter cells or cell fragments from the solution, thereby further reducing the amount of endotoxin. Filtration membranes also advantageously provide an extremely large surface area for the adsorption of endotoxin on the hyperophobic surface. If a hydrophobic filtration membrane is employed, then the membrane will preferably have a pore size of about 1.0 miter to about 0.1 miter. A particularly preferred hydrophobic filtration membrane is a polypropylene membrane having a pore size of about 0.2 microns. Alternatively, the hydrophobic surface may be in the form of hydrophobic resins. Hydrophobic resins can provide efficient contact with high volumes of endotoxin. The hydrophobic resins provide additional advantages over the filtration membranes, since the hydrophobic resins can be regenerated and reused, and can easily be increased in amount to contact larger volumes of biopolymers. Hydrophobic resins are also more suitable for materials, such as biogomas, which are difficult to filter through the hydrophobic filtration membranes due to the viscosity and / or length of the polymer chains. As with the idrophobic filtering membranes, the hydrophobic resins can be varied in size to provide contact with a larger surface area. A particularly preferred hydrophobic resin bead of this invention which is 0.5 mm in diameter and is comprised of polystyrene, dividinyl benzene. If the hydrophobic purification resin method is employed, a preliminary step may be required before contacting the biopolymer salt with the hydrophobic resin. The pH of the solution should be raised first to increase the solubility of the lipopolysaccharide endotoxin before contact with the hydrophobic resin is made. Preferably, the pH is raised to at least 9 by the addition of NaOH, KOH or other bases known to those skilled in the art. After contact, the hydrophobic resin beads are screened from the solution, and the pH is preferably adjusted again to neutral for the second purification technique of the method as described below. Although it is known that the endotoxin lipopolysaccharide binds to hydrophobic materials, such as activated carbon, polypropylene and polystyrene, experiments using this contact technique alone did not successfully produce a biopolymer salt having an endotoxin content of less than 1000. eu per gram. However, it has been discovered that a highly purified biopolymer salt can be obtained by mixing the aqueous solution that had contact with the hydrophobic surface with a water miscible organic solvent. This second step results in the precipitation of the highly purified biopolmer salt from the solution. Without bound to a theory, it is believed that the lipid portion of the endotoxin molecule provides the molecule with solubility in hydrophobic liquids, such as hexane or methyl tertiary butyl ether, or in partially hydrophobic liquids, such as alcohols. In contrast, the biopolymer salts will be precipitated in hydrophobic liquids miscible in water, such as alcohols and ketones, which have suitably low dielectric constants. In this way, the endotoxin in the aqueous solution is separated from the biopolymer salt. The organic precipitation solvent, miscible in water, is selected from the group consisting of alcohols, ketones, aldehydes and mixtures thereof. Preferably, it is a low molecular weight alcohol. More preferably, it is an isopropyl alcohol, methanol, ethanol or acetone. The water-miscible organic solvent is usually mixed with the aqueous solution treated with hydrophobic material at a volume ratio of about 1: 1 to about 6: 1. The mixture is held at a temperature and for a time sufficient to allow precipitation of the purified biopolymer salt. After precipitation, the biopolmer salt can be dried to remove the solvent. A preferred drying technique is drying by low temperature oven (40-80 ° C). Other suitable drying techniques include, but are not limited to, lyophilization and spray drying.

The biopolymer salt can also be reconstituted in an acceptable solvent. A particularly preferred solvent is water. In another embodiment of this invention, the method can be performed by replacing the precipitation step with a liquid-liquid extraction, using a solvent immiscible with water. An exemplary water-immiscible solvent includes hexane or methyl tert-butyl ether. The method of this invention may include additional steps as desired. For example, with regard to alginates, it is preferable to treat the aqueous starting solution with an oxidizing agent, such as 1 00 ppm NaOCI, to destroy the polyphenols and thereby remove the color of the alginate. Alternatively, activated carbon may come into contact with the aqueous solution in place of NaOCI. Most preferably, the activated carbon is added after the NaOCI to adsorb the polyphenols and remove any residual NaOCI, which can decompose the alginates in storage. In addition, the step of contacting a hydrophobic material and / or precipitation can be conducted multiple times if desired. If a filtration membrane is used, it may be preferable to pass the aqueous solution through membranes having different pore sizes. For example, it may be preferred to employ a hydrophobic membrane of 10 microns followed by a membrane of 0.2 microns. Using this sequence, it will tend to improve the capacity of the smaller pore size filter. Similarly, if hydrophobic resins are used, the size and amount of the hydrophobic resin beads can be varied, or formed into layers, to maximize the available surface area and obtain the desired level of contact.

The method of this invention is particularly advantageous because the purified biopolymer salt can be prepared using a commercial scale manufacturing process. (The biopolymer salts of this invention can be used for prepare the biopolymer compositions which are suitable for parenteral administration to a patient. This can be achieved by dissolving a biopolymer salt of this invention having an endotoxin level of less than or equal to about 1 000 eu per gram, in a pharmaceutically acceptable solvent. Preferably, the solvent Pharmaceutically acceptable is water for injection. The water for injection is a deionised water of pharmaceutical grade, free of particles, free of endotoxin, sterile, certified. The concentration of biopolymer salt in the composition can vary from about 0.5 weight percent to about 5 weight percent. percent by weight, based on the total weight of the solution. Preferably, the concentration of biopolmer salt is between about 2 percent by weight and about 4 percent by weight. ^ weight. Many of the biopolymer compositions of this invention also include gels prepared by adding a gelling agent to the biopolymer composition described above. These gels are suitable for parenteral administration. The gels can be made in any desired way. For example, the gels can be made in the form of beads, sheets or filaments, which can be administered to a patient. Preferred gelling agents include divalent or trivalent cations. It is also possible to incorporate a pharmaceutically active component into the biopolymer gel before it is administered to a patient. The amount of gelling agent that can be added to the biopolymer solution to form a suitable gel can vary depending on the concentration of biopolymer in the solution, as well as on the particular gelling agent employed. Preferably, the gelling agent is added as an aqueous solution, in which the gelling agent is present at a concentration range of from about 0.5% to about 10%. The following examples are intended to illustrate certain embodiments of the invention and no limitation of the invention is implied.

EXAM PLO 1 Preparation of an alginate suitable for parenteral use Three liters of a 3 weight percent alginate solution were prepared using Keltone LVCR and water for injection. The endotoxin level of this alginate was approximately 61, 500 eu per gram of dry alginate. All equipment that had contact with the alginate solution was depirogenated, either by heating at 250 ° C for at least one hour or by treatment with 0.1 M NaOH for at least one hour. The alginate solution was treated with 200 ppm NaOCI, which destroys the colored polyisolenic compounds and therefore produces a colorless alginate solution.

Then one liter of the colorless alginate solution was successively passed through three separate filters: a 1 0 μm pore size polypropylene filter, an impregnated activated carbon filter of pore size of 3 μm, and a filter of polypropylene with a pore size of 0.2 μm. All filter cartridges were manufactured by Meissner, Camarillo, California. Polypropylene filter cartridges were first activated by soaking them in reagent grade isopropanol for 1 5 minutes before installing them in the filter cartridge housings and washing them with water jet for injection. The filter cartridge impregnated with activated carbon was washed with water jet for injection to wet it and remove the carbon powder before use. After the alginate solution was passed through the filter cartridges, the aiginate in the filtrate was precipitated with isopropyl alcohol. The precipitation was achieved by mixing one volume of the alginate solution with two volumes of isopropyl alcohol for 5 seconds at high speed in an Oster blender jar. The isopropanol used for precipitation was of reactive grade and was obtained from a previously unopened container to avoid contamination with endotoxin. The precipitated alginate fibers were drained on a 40 mesh stainless steel screen, depirogenated. The alginate fibers were squeezed against the screen with aluminum foil and depirogenated to remove excess solvent from the alginate. The resulting alginate fibers were then dried in an oven at 60 ° C for 1-4 hours to remove the remaining solvent. The dried fibers were ground and then tested by endotoxin levels. The results of these measurements are shown in Table 1.

EXAMPLE 2 Preparation of an alginate suitable for parenteral use A second liter of the colorless alginate solution of Example 1 was filtered and precipitated as described in Example 1. The filters used in this Example were the same as those previously used in Example 1. After filtration, adsorption, drying and grinding as in Example 1, the alginate fibers were tested by endotoxin levels. The results of these measurements are shown in Table 1.

EXAM PLO 3 Preparation of an alginate suitable for parenteral use A third liter of the colorless alginate solution of Example 1 was filtered and precipitated as described in Example 1. The filters used in this Example were the same as those previously used in Examples 1 and 2. After filtration, precipitation, drying and milling as in Example 1, the alginate fibers were tested by endotoxin levels. The results of these measurements are shown in Table 1.

TABLE 1 . Levels of endotoxin in alginates As shown in Table 1, alginates that are suitable for parenteral use can be prepared from alginates that have high levels of endotoxin by adsorption and precipitation techniques. It is believed that the somewhat higher but still pharmaceutically acceptable endotoxin levels in Example 3 are due to the endotoxin adsorption limits of one or more of the filters used in these examples. This is supported by the levels of endotoxin found in the alginates prepared in the following examples, where only 2 liters of alginate solution were passed through the same filters.

EXAMPLE 4 Preparation of an alginate suitable for parenteral use A two-liter sample of the 3 percent by weight alginate solution, prepared in Example 1, was treated as described in Example 1, except that this two-liter sample was filtered through two activated carbon filters. 3 μm, instead of just one before filtering through the 0.2 μm polypropylene filter. After filtration through previously unused filters, precipitation, drying and milling as in Example 1, the alginate fibers were tested by endotoxin levels. The results of these measurements are shown in Table 2.

EJ EM PLO 5 Preparation of an alginate suitable for parenteral use A second two-liter sample of the 3 weight percent alginate solution prepared in Example 1 was treated as described in Example 4. After filtration through previously unused filters, precipitation, drying and milling as in Example 1, the alginate fibers were tested for endotoxin levels. The results of these measurements are shown in Table 2.

EXAMPLE 6 Preparation of an alginate suitable for parenteral use A third sample of two liters of the 3 weight percent alginate solution prepared in Example 1 was treated as described in Example 4. After filtration through previously unused filters, precipitation, drying and milling as in Example 1, the alginate fibers were tested by endotoxin levels. The results of these measurements are shown in Table 2.

TABLE 2. Levels of endotoxin in alginates As shown in Table 2, the methods of this invention can be used to prepare alginates with extremely low levels of endotoxin. This can be achieved by avoiding overloading the filters with endotoxin. Table 2 shows that when filters previously not used in the filtration process are used, the level of endotoxin in the alginate can be reduced to less than 5 eu per gram.

EXAMPLE 8 Preparation of an alginate suitable for parenteral use Three liters of a solution of alginate at 3 percent by weight were prepared using Keltone LVCR and water for injection. The endotoxin level of this alginate was approximately 46,400 eu per gram of dry alginate. All the equipment that was to have contact with the alginate solution was depirogenated, either by heating at 250 ° C for at least one hour or by treatment with 0. 1 M NaOH for at least one hour. The alginate solution was treated with NaOCI 1 00 ppm, which destroys the colored polyphenolic compounds, and therefore, produces a colorless alginate solution. The solution was then passed successively through three separate filters: two activated carbon filters of pore size of 3 μm in series and a polypropylene filter of pore size of 0.2 μm. After the alginate solution was passed through the filter cartridges, a 300 ml portion of the alginate solution was mixed in the filtrate with the water immiscible solvent, methyl tert-butyl ether (MTBE). The extraction was achieved by mixing one volume of the alginate solution with two volumes of MTBE at high speed in an Oster blender jar. When mixing was stopped, two layers were formed: a layer of aqueous alginate in the bottom and a layer of MTBE in the upper part. Then the two layers were carefully separated. The MTBE used for extraction was of reactive grade and was obtained from a previously unopened container to avoid contamination with endotoxin. The extracted alginate layer was then tested by endotoxin levels. The results of these measurements are shown in Table 3.

EXAM PLO 9 Preparation of an alginate suitable for parenteral use A second portion of 300 ml of the 3 weight percent alginate solution that was prepared, treated and filtered in Example 8, it was precipitated with methanol. The precipitation was achieved by mixing a volume of the alginate solution with two volumes of methanol for 30 seconds at high speed in an Oster blender jar. The methanol used for precipitation was of reactive grade and was obtained from a previously unopened container to avoid contamination with endotoxin. The precipitated alginate fibers were drained on a 40 mesh stainless steel screen, depirogenated. The alginate fibers were pressed against the screen with depirogenated aluminum foil to remove the excess solvent from the alginate. The resulting alginate fibers were then dried in an oven at 60 ° C for 1-4 hours, to remove the remaining solvent. The dried fibers were ground and then tested by endotoxin levels. The results of these measurements are shown in Table 3.e.

EXAM PLO 1 0 Preparation of an alginate suitable for parenteral use A third portion of 300 ml of the 3 weight percent alginate solution, which was prepared, treated and filtered in Example 8, was precipitated as per the method described in Example 9, but with ethanol instead of methanol. The results of these measurements are shown in Table 3.

EXAMPLE 1 1 Preparation of an alginate suitable for parenteral use A fourth portion of 300 ml of the 3 percent by weight alginate solution which was prepared, treated and filtered in Example 8 was determined as per the method described in Example 9, but with isopropanol instead of methanol. The results of these measurements are shown in Table 3.

EXAM PLO 1 2 Preparation of an alginate suitable for parenteral use A fifth portion of 300 ml of the 3 weight percent alginate solution that was prepared, treated and filtered in Example 8 was precipitated as per the method described in Example 9, but with acetone instead of methanol. The results of these measurements are shown in Table 3.

TABLE 3. Levels of endotoxin in some inmates As shown in Table 3, alginates that are suitable for parenteral use, can be prepared by the method described above, using either water-miscible or water-immiscible solvents.

EXAMPLE 1 3 Preparation of a gellana gum suitable for parenteral use A solution of 20 g of gelatin GELRITE gum and 0.5 g of sodium citrate, which sequesters polyvalent cations and improves the ability to flow, was dissolved in 986 g of water for injection. The endotoxin level of this gellan gum solution was greater than 1,000,000 eu per gram of unpurified, dry gelatin gum. All the equipment that was in contact with the gum solution was depirogenated by heating at 250 ° C for at least one hour, or by treatment with 0. 1 M NaOH for at least one hour. In particular, the resin beads had been previously cleaned of endotoxin and activated by soaking overnight in a 0. 1 NaOH solution, rinsing with water for injection, soaking in isopropanol for 1 5 minutes and again rinsing with water for injection. To this solution was added 4 g of NaOH to raise the pH to at least 9 and increase the solubility of the endotoxin. Then 1 00 g of polystyrene divinyl benzene resin beads H P-20 were added to the gellana solution and mixed overnight. All the resin beads were manufactured by Supelco-Diainon.

The resin beads were then sieved from the solution with a screen. The pH of the solution was then adjusted back to 7 using 0. 1 M HCl. GELRITE gellana gum was then precipitated from the solution by the addition of 2 volumes of isopropanol, followed by mixing in an Oster blender at high speed. The precipitated fibers were sieved from the solution, dried at 50 ° C overnight and then tested by endotoxin levels. The results of these measurements are shown in Table 4.

EXAMPLE 14 Preparation of a xanthan gum suitable for parenteral use A solution of 14 g of KANTROL T xanthan gum in 2 liters of water for injection was dissolved. The endotoxin level of this xanthan gum solution was greater than 1,000,000 eu per gram of dry, unpurified xanthan gum. All the equipment that was to have contact with the gum solution was depirogenated by heating at 250 ° C for at least one hour, or by treatment with 0.1 M NaOH for at least one hour. In particular, the resin beads had been previously cleaned of endotoxin and activated by soaking overnight in a 0. 1 NaOH solution, rinsing with water for injection, soaking in methanol for 1 hour and again rinsing with water for injection. To this solution, 8 g of NaOH was added to raise the pH to at least 9 and increase the solubility of the endotoxin. Then 384 g of polystyrene divinyl benzene resin beads H P-20 were added to the xanthan solution and mixed overnight. The resin beads were sieved from the solution with a sieve and the pH of the solution was again adjusted to 7. The xanthan gum KELTROL T was then precipitated from the solution by the addition of 2 volumes of isopropanol, followed by mixing in an Oster blender at high speed. The precipitated fibers were sieved from the solution, dried at 60 ° C for 1-4 hours, and then tested by endotoxin levels. The results of these measurements are shown in Table 4.

TABLE 4. Levels of endotoxin in biogomas As shown in Table 4, the methods of this invention can be used to remove concentrations of high charge endotoxins from biogomes. This can be achieved by adsorption through contact with hydrophobic resins and by precipitation techniques with various solvents. Other variations and modifications of this invention will be obvious to those skilled in the art. This invention is not limited except as set forth in the art.

Claims (69)

1. REVIVAL NAME IS 1 . A suitable biopolymer salt for parenteral use, which e) comprises a biopolymer salt having an endotoxin content 5 less than about 1000 endotoxin units per gram.
2. 2. The biopolmer salt according to claim 1, wherein said endotoxin content is less than about 50 endotoxin units per gram.
3. 3. The biopolmer salt according to claim 1, wherein said bipolymer salt is an alginate.
4. 4. The biopolymer salt according to claim 3, wherein said biopolmer salt is sodium alginate.
5. 5. The biopolymer salt according to claim 3, wherein said alginate is a product of giant kelp Macrocystis pyrifera.
6. 6. The biopolmer salt according to claim 3, wherein said alginate is a kelp product Laminaria hyperborea.
7. 7. The biopolymer salt according to claim 1, wherein said biopolymer salt is gellane gum.
8. 8. The biopolmer salt according to claim 7, wherein said gellane gum is derived from Sphingomonas elodea.
9. 9. The biopolmer salt according to claim 1, wherein said biopolmer salt is xanthan gum.
10. 1 0. The biopolmer salt according to claim 9, wherein said xanthan gum is derived from Xanthamonas campestris.
11. 11. A biopolymer composition suitable for parenteral use comprising a biopolymer salt, having an endotoxin content of less than about 100 endotoxin units per gram and a pharmaceutically acceptable solvent.
12. 12. The biopolymer composition according to claim 11, wherein said biopolymer salt is an alginate.
13. 13. The biopolymer composition according to claim 11, wherein said biopolymer salt is a biogome.
14. The biopolymer composition according to any of claims 11, 12 and 13, wherein said endotoxin content is less than about 50 endotoxin units per gram.
15. 15. The biopolymer composition according to claim 14, wherein said biopolymer salt is present in an amount from about 0.5 percent to about 5 percent by weight of the composition.
16. 16. The biopolymer composition according to claim 15, wherein the pharmaceutically acceptable solvent.
17. 17. The biopolymer composition according to claim 12, wherein said alginate is sodium alginate.
18. 18. The biopolymer composition according to claim 13, wherein said biogoma is gellane gum.
19. 19. The biopolymer composition according to claim 13, wherein said biogoma is xanthan gum.
20. 20. The biopolymer composition according to any one of claims 1 1, 12 and 13, further comprising a gelling agent in an amount effective to gel said biopolymer. twenty-one .
21. The biopolymer composition according to claim 20, wherein said gel is in the form of beads, sheets or filaments.
22. 22. The biopolymer composition according to claim 20, wherein said gelling agent is selected from the group consisting of divalent or polyvalent cations.
23. 23. The biopolymer composition according to claim 22, wherein said gelling agent are calcium cations.
24. 24. A method for preparing a biopolymer salt having an endotoxin content of less than about 1000 endotoxin units per gram, said method comprising the steps of: (i) contacting an aqueous solution of a biopolymer salt with a hydrophobic material to adsorb endotoxin in said material; and (ii) precipitating said biopolymer salt having an endotoxin content of less than about 100 endotoxin units per gram of said solution, by mixing a water-miscible organic solvent with said solution.
25. 25. The method according to the re-excitation 24, wherein said contact step comprises passing said aqueous solution through a filter of the hydrophobic material or a packed column containing the hydrophobic material.
26. 26. The method according to claim 24, wherein said hydrophobic material are hydrophobic resins.
27. 27. The method according to claim 26, which further comprises raising the pH level of said aqueous solution before contacting said aqueous solution with said hydrophobic resins.
28. 28. The method according to claim 27, wherein said pH level is at least 9.
29. 29. The method according to claim 24, wherein said hydrophobic material is selected from the group consisting of polypropylene, polystyrene or a Fluorocarbon polymer.
30. 30. The method according to the re-identification 29, wherein said hydrophobic material is polypropylene.
31. 31 The method according to claim 25, wherein said idrophobic material has an average pore size of about 1 μm to about 0.1 μm.
32. 32. The method according to the re-fractionation 30, wherein said average pore size is approximately 0.2 μm.
33. 33. The method according to claim 26, wherein said hydrophobic resins are less than 5 μm in diameter.
34. 34. The method according to claim 24, wherein said organic solvent soluble in water is selected from the group consisting of alcohols, ketones, aldehydes and mixtures thereof.
35. 35. The method according to claim 34, wherein said water-miscible organic solvent is an alcohol.
36. 36. The method according to claim 35, wherein said alcohol is selected from the group consisting of isopropyl alcohol, methanol and ethanol.
37. 37. The method according to claim 34, wherein said ketone is acetone.
38. 38. The method according to claim 34, wherein the) volume ratio of said organic solvent to aqueous solution is 5 about 1: 1 to about 6: 1.
39. 39. The method according to claim 24, further comprising the step of passing said aqueous solution over activated carbon to said precipitation step.
40. 40. The method according to claim 24, wherein the concentration of the biopolymer salt in said aqueous solution is about 0.5 to about 5 percent by weight of the solution.
41. 41 The method according to any of claims 24 and 25, wherein said biopolmer salt is an alginate.
42. 42. The method according to any of claims 24 and 26, wherein said biopolymer salt is a biogoma.
43. 43. The method according to claim 24, which further comprises the step of drying said precipitate.
44. 44. The method according to claim 43, wherein said drying step 20 comprises drying in a low temperature oven (40-80 ° C).
45. 45. A method for preparing a biopolymer composition, comprising the steps of forming a biopolymer salt according to claim 43, and reconstituting said precipitate with a parenterally acceptable solvent.
46. 46. The method according to claim 45, wherein the parenterally acceptable solvent is water.
47. 47. A method for preparing a biopolymer salt having an endotoxin content of less than about 1000 units of endotoxin per gram, said method comprising the steps of: (i) contacting an aqueous solution of a biopolymer salt with a material hydrophobic to adsorb endotoxin in said material; and (ii) extracting endotoxin from the aqueous solution treated in step (i) with an organic solvent immiscible with water, to form the biopolymer salt having an endotoxin content of less than about 1000 units of endotoxin per g branch.
48. 48. The method according to claim 47, wherein said contacting step comprises passing said aqueous solution through a filter of hydrophobic material or a packed column containing the hydrophobic material.
49. 49. The method according to claim 47, wherein said hydrophobic material are hydrophobic resins.
50. 50. The method according to claim 49, which further comprises raising the pH level of said aqueous solution before contacting said aqueous solution with said hydrophobic resins.
51. 51 The method according to claim 50, wherein said pH level is at least 9.
52. 52. The method according to claim 47, wherein said idrophobic material is selected from the group consisting of polypropylene, polystyrene or a Fluorocarbon polymer.
53. 53. The method according to claim 52, wherein said hydrophobic material is polypropylene.
54. 54. The method according to claim 48, wherein said hydrophobic material has an average pore size of about 1 μm to about 0.1 μm.
55. 55. The method according to claim 54, wherein said average pore size is about 0.2 μm.
56. 56. The method according to claim 49, wherein said hydrophobic resins are less than 5 mm in diameter.
57. 57. The method according to claim 47, wherein said organic solvent immiscible with water, is selected from the group consisting of hydrocarbons, ethers, chloroform and mixtures thereof.
58. 58. The method according to claim 57, wherein said solvent immiscible in water is a hydrocarbon.
59. 59. The method according to claim 58, wherein said hydrocarbon is hexane.
60. 60. The method according to claim 58, wherein said hydrocarbon is methyl tert-butyl ether.
61. 61 The method according to claim 57, wherein the volume ratio of said water-immiscible organic solvent to said aqueous solution is about 0.5: 1 to about 5: 1.
62. 62. The method according to claim 47, further comprising a step of passing said aqueous solution over activated carbon before said extraction step.
63. 63. The method according to claim 47, wherein the concentration of the biopolmer salt in said aqueous solution is about 0.5 to about 5 percent by weight of the solution.
64. 64. The method according to claim 47, further comprising the step of drying said biopolymer composition.
65. 65. The method according to claim 64, wherein said drying step comprises drying at low temperature (40-80 ° C).
66. 66. A method for preparing a biopolymer composition, comprising the steps of forming a biopolymer salt according to claim 64, and reconstituting said biopolymer composition with a parenterally acceptable solvent.
67. 67. The method according to claim 66, wherein said parenterally acceptable solvent is water.
68. 68. The method according to any of cla47 and 48, wherein said biopolymer salt is an alginate.
69. 69. The method according to any of cla47 and 49, wherein said biopolymer salt is a biogome.