Crosslinked-hyaluronic acid in emulsion

Abstract

The present invention relates to methods of producing crosslinked hyaluronic acid microbeads, as well as the produced microbeads, said method comprising the steps of: (a) mixing an aqueous alkaline solution comprising hyaluronic acid, or a salt thereof, with a solution comprising a crosslinking agent; (b) forming microdroplets having a desired size from the mixed solution of step (a) in an organic or oil phase to form a water in organic or water in oil (W/O) emulsion; (c) continuously stirring the W/O emulsion, whereby the reaction of hyaluronic acid with divinylsulfone takes place to provide crosslinked hyaluronic acid microbeads; and (d) purifying the crosslinked hyaluronic acid microbeads.

1 - 42 . (canceled) 
     
     
         43 . A method of producing crosslinked hyaluronic acid microbeads, said method comprising the steps of:
 (a) mixing an aqueous alkaline solution comprising hyaluronic acid or a salt thereof, with a solution comprising a crosslinking agent;   (b) forming microdroplets having a desired size from the mixed solution of step (a) in an organic or oil phase to form a water in organic or water in oil (W/O) emulsion;   (c) continuously stirring the W/O emulsion, whereby the reaction of hyaluronic acid with divinylsulfone takes place to provide crosslinked hyaluronic acid microbeads; and   (d) purifying the crosslinked hyaluronic acid microbeads.   
     
     
         44 . The method of  claim 43 , wherein the hyaluronic acid or salt thereof is recombinantly produced in a  Bacillus  host cell. 
     
     
         45 . The method of  claim 43 , wherein the hyaluronic acid or salt thereof has a number average molecular weight of between 100 and 3,000 kDa. 
     
     
         46 . The method of  claim 43 , wherein the alkaline solution comprises dissolved hyaluronic acid or a salt thereof, in a concentration of between 0.1%-40% (w/v). 
     
     
         47 . The method of  claim 43 , wherein the alkaline solution comprises dissolved sodium hydroxide in a concentration of between 0.001 -2.0 M. 
     
     
         48 . The method of  claim 43 , wherein the crosslinking agent is divinylsulfone (DVS). 
     
     
         49 . The method of  claim 48 , wherein DVS is comprised in the mixed solution of step (a) in a weight ratio of between 1:1 and 100:1 of HA/DVS (dry weight). 
     
     
         50 . The method of  claim 43 , wherein the reaction of hyaluronic acid with divinylsulfone takes place at a temperature in the range of 5-100° C. 
     
     
         51 . The method of  claim 43 , wherein the stirring in step (c) is continued for a period of between 1-180 minutes. 
     
     
         52 . The method of  claim 43 , wherein the microdroplets of step (b) have an average diameter in the range of about 1 nanometer to 1 millimeter. 
     
     
         53 . The method of  claim 43 , wherein the purifying step comprises dialyzing the crosslinked microbeads against de-ionized water using a dialysis membrane that allows free diffusion of molecules having a size less than 13,000 daltons. 
     
     
         54 . The method of  claim 43 , wherein the purifying step comprises neutralizing the pH of the crosslinked microbeads with a buffer or an acid. 
     
     
         55 . The method of  claim 54 , wherein the buffer comprises a buffer with a pH in the range of 2.0-8.0. 
     
     
         56 . The method of  claim 54 , wherein the buffer comprises a buffer with a pH, which results in that the crosslinked microbeads have a pH between 5.0 and 7.5 after the purifying step. 
     
     
         57 . The method of  claim 54 , wherein the buffer comprises a phosphate buffer and/or a saline buffer. 
     
     
         58 . The method of  claim 43 , wherein the crosslinked microbeads are washed at least once with water, and/or a phosphate and/or saline buffer with a pH in the range of 2.0-8.0. 
     
     
         59 . The method of  claim 43 , wherein a preservative is added as a component to the crosslinked microbeads either before or after the crosslinking reaction.