Methods for Purifying Polysaccharides and Pharmaceutical Compositions and Medical Devices Containing the Same

Abstract

Methods for removing endotoxin from naturally occurring materials, such as polysaccharides (e.g., agarose and/or carrageenan) are described herein. Polysaccharides that are substantially free of endotoxins and uses thereof are also described. The polysaccharide materials can be isolated from microorganisms, multicellular organisms, such as, algae, plants, seaweed, etc. The method involves the use of acidic and basic solutions to hydrolyze the lipid-polysaccharide bond in endotoxins. Cleaving the fatty acid from the polysaccharide reduces the water-solubility of the fatty acid and enables its removal with an organic solvent such as ethanol. The polysaccharide component can also undergo acidic or basic hydrolysis due to the weak glycosidic bond between the sugar rings.

We claim: 
     
         1 . A method for isolating and purifying a naturally occurring agarose or derivative thereof produced from a biological source, the method comprising:
 (i) dispersing the agarose in one or more aliphatic alcohols to disrupt the bacterial wall to solubilize the lipid portion of endotoxins;   (ii) removing the aliphatic alcohol to remove the lipid portion of the endotoxins and obtain the agarose or derivative thereof in solid form;   (iii) dispersing the solid agarose or derivative thereof in a basic solution to hydrolyze lipid-inner core bonds of the endotoxins and solubilize the polysaccharide component of the endotoxins;   (iv) washing the solution from step (iii) with an aliphatic alcohol to remove free lipids;   (v) removing the basic solution in step (iii) to obtain the agarose or derivative thereof in solid form;   (vi) dispersing the solid agarose or derivative thereof in an acidic solution to hydrolyze lipid-inner core bonds of the endotoxins not cleaved in step (iii);   (vii) removing the acidic solution in step (vi) to obtain the agarose or derivative thereof in solid form;   (viii) dispersing the solid agarose or derivative thereof from step (vii) in a second basic solution to further cleave lipid-inner core bonds and to neutralize the acid from step (vi);   (ix) removing the basic solution in step (viii) to obtain the agarose or derivative thereof in solid form; and   (x) dispersing or suspending or dissolving the solid agarose or derivative thereof from step (ix) in sterile water to solubilize the polysaccharide component of the endotoxin and to neutralize any residual acid and base; and   (xi) removing the sterile water in step (x) to obtain the agarose or derivative thereof in solid form,   wherein the agarose or derivative thereof is substantially free of endotoxin.   
     
     
         2 . The method of  claim 1 , wherein aliphatic alcohol in step (i) and/or (iv) is selected from the group consisting of ethanol, n-propanol, isopropanol, glycerol, and combinations thereof. 
     
     
         3 . The method of  claim 1 , wherein the acidic solution is any aqueous solution of an acid selected from the group consisting of hydrochloric acid, sulfuric acid, citric acid, acetic acid, and formic acid. 
     
     
         4 . The method of  claim 3 , wherein the concentration of the acidic solution is from about 0.05 to about 2M. 
     
     
         5 . The method of  claim 3 , wherein the acidic solution is 0.5 M HCl. 
     
     
         6 . The method of  claim 1 , wherein the basic solution in step (iii) and/or (viii) is an aqueous solution of a base selected from the group consisting of sodium hydroxide, potassium hydroxide, and calcium hydroxide. 
     
     
         7 . The method of  claim 6 , wherein the concentration of the base is from about 0.01 m to about 2M, preferably from about 0.1M to about 0.5M. 
     
     
         8 . The method of  claim 1 , wherein the basic solution is 0.25 M NaOH. 
     
     
         9 . The method of  claim 1 , wherein the solid agarose or derivative thereof in step (xi) is dissolved in an autoclavable physiological buffer solution. 
     
     
         10 . The method of  claim 9 , wherein the buffer is Ringer Buffer. 
     
     
         11 . The method of  claim 9 , wherein the buffer is removed to obtain the agarose or derivative thereof in solid form. 
     
     
         12 . The method of  claim 1 , wherein the agarose or derivative thereof is sterilized using a technique selected from the group consisting of steam and gamma irradiation. 
     
     
         13 . The method of  claim 1 , wherein the agarose or derivative thereof is a hydrogel-forming material. 
     
     
         14 . A pharmaceutical composition comprising a purified agarose or derivative thereof, wherein the agarose or derivative thereof is substantially free of endotoxins, and one or more pharmaceutically acceptable carriers. 
     
     
         15 . The pharmaceutical composition of  claim 14 , further comprising one or more therapeutic and prophylactic agents selected from the group consisting of proteins, peptides, growth factors, small molecule drugs, and combinations thereof. 
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein the therapeutic or prophylactic agent is a growth factor. 
     
     
         17 . The pharmaceutical composition of  claim 14 , wherein the agarose or agarose derivative thereof and one or more pharmaceutically acceptable carriers are in the form of a drug delivery system. 
     
     
         18 . A medical device comprising a purified agarose or derivative thereof, wherein the agarose or derivative thereof is substantially free of endotoxins. 
     
     
         19 . The device of  claim 18 , wherein the device is selected from the group consisting of wound dressings, hemostatic materials (dressing or powder), dermal, bone or teeth fillers, implantable gel, implantable devices, and cell carriers.