Dilute filtration sterilization process for viscoelastic biopolymers

Abstract

Manufactured hyaluronic acid products are used in numerous surgical applications including viscoelastic supplementation for the treatment of osteoarthritis, however, traditional sterilization techniques result in the breakdown of such high molecular weight viscoelastic biopolymers and are thus unsuitable. Disclosed are processes for obtaining concentrated sterile solutions of high molecular weight biopolymers such as hyaluronic acid. The processes include filter sterilization with a dilute preparation of the biopolymer, and concentration of the dilute filter sterilized biopolymer by ultrafiltration to a desired concentration.

1 . A process for formulating a soluble viscoelastic biopolymer comprising:
 (i) sterile-filtering soluble bulk manufactured biopolymer by passage through a membrane suitable for sterile filtration; and   (ii) concentrating the biopolymer by ultrafiltration to a desired final concentration.   
     
     
         2 . The process according to  claim 1 , wherein the biopolymer is selected from the group consisting of a homopolysaccharide, a heteropolysaccharide and mixtures thereof. 
     
     
         3 . The process according to  claim 2 , wherein the homopolysaccharide is selected from the group consisting of carboxymethylcellulose, chitin, polymannuronic acid, curdlan gum and dextran. 
     
     
         4 . The process according to  claim 2 , wherein the heteropolysaccharide is selected from the group consisting of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, agar, alginate, carrageenan, gellan, guar gum, locust bean gum, and xanthan gum. 
     
     
         5 . The process according to  claim 1 , wherein the biopolymer is obtained from a source selected from the group consisting of a biological source, an in vitro enzymatic synthesis, a chemical synthesis, and combinations of two or more such sources. 
     
     
         6 . The process according to  claim 5 , wherein the biological source is selected from the group consisting of a bacterium, a yeast, a plant, an amphibian, an avian and a mammal. 
     
     
         7 . The process according to  claim 5 , wherein the biopolymer obtained from a biological source further comprises a chemical modification. 
     
     
         8 . The process according to  claim 7 , wherein the chemical modification comprises a modification selected from the group consisting of addition of sulfate groups, addition of carboxyl groups, addition of hydroxyl groups, addition of acetyl groups, esterification and cross-linking. 
     
     
         9 . The process according to  claim 1 , wherein the viscoelastic biopolymer has an average molecular weight in the range from 1×10 4  to 1×10 7  daltons. 
     
     
         10 . The process according to  claim 9 , wherein the viscoelastic biopolymer has an average molecular weight of 3×10 6 ±0.6×10 6  daltons. 
     
     
         11 . The process according to  claim 6 , wherein the bacterium is a strain of the genus  Streptococcus.    
     
     
         12 . The process according to  claim 11 , wherein the bacterium is a  Streptococcus  species selected from the group consisting of  Streptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis, Streptococcus dysgalactiae  and  Streptococcus zooepidemicus.    
     
     
         13 . The process according to  claim 11 , wherein the  Streptococcus  strain is non-hemolytic and non-pathogenic. 
     
     
         14 . The process according to  claim 1 , wherein the bulk manufactured biopolymer is isolated from the culture broth of a fermented  Streptococcus  strain. 
     
     
         15 . The process according to  claim 14 , wherein the bulk manufactured biopolymer is hyaluronic acid. 
     
     
         16 . The process according to  claim 15 , wherein the bulk manufactured hyaluronic acid is substantially free of impurities. 
     
     
         17 . The process according to  claim 16 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial endotoxin. 
     
     
         18 . The process according to  claim 17 , wherein the level of bacterial endotoxin is <0.25 EU/ml. 
     
     
         19 . The process according to  claim 16 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial cells. 
     
     
         20 . The process according to  claim 19 , wherein the viable count of bacterial cells is <100 CFU/g. 
     
     
         21 . The process according to  claim 20 , wherein the viable count of bacterial cells is <50 CFU/g. 
     
     
         22 . The process according to  claim 21 , wherein the viable count of bacterial cells is <10 CFU/g. 
     
     
         23 . The process according to  claim 16 , wherein the bulk manufactured hyaluronic acid is substantially free of protein. 
     
     
         24 . The process according to  claim 23 , wherein the level of protein is <1 mg/g. 
     
     
         25 . The process according to  claim 1 , wherein the concentration of the soluble bulk manufactured biopolymer in step (i) is <0.2%. 
     
     
         26 . The process according to  claim 25 , wherein the concentration is 0.10-0.13%. 
     
     
         27 . The process according to  claim 1 , wherein the concentrating is carried out by ultrafiltration. 
     
     
         28 . The process according to  claim 27 , wherein the ultrafiltration is carried out using a ceramic membrane. 
     
     
         29 . The process according to  claim 1 , wherein the desired final concentration is in the range of 0.8 to 3.0% w/v. 
     
     
         30 . A process according to  claim 29 , wherein the desired final concentration is about 1.0% w/v. 
     
     
         31 . A process according to  claim 29 , wherein the desired final concentration is about 1.2% w/v. 
     
     
         32 . A process according to  claim 29 , wherein the desired final concentration is about 2.0% w/v. 
     
     
         33 . A process according to  claim 1 , further comprising aseptic filling of a suitable packaging device with the biopolymer. 
     
     
         34 . A process according to  claim 33 , wherein the packaging device is selected from the group consisting of a syringe, a vial, a catheter and a nebulizer. 
     
     
         35 . A process according to  claim 1 , wherein the formulated viscoelastic biopolymer has a pseudo-plasticity index in the range from 500 to 4000. 
     
     
         36 . A process according to  claim 35 , wherein the pseudoplasticity index is in the range from 600 to 1200. 
     
     
         37 . A process according to  claim 36 , wherein the pseudoplasticity index is in the range from 600 to 800. 
     
     
         38 . A process according to  claim 1 , wherein the sterile-filtering is carried out using a membrane of absolute pore size 0.2 micron. 
     
     
         39 . A process for formulating a viscoelastic biopolymer comprising:
 (i) dissolving bulk manufactured biopolymer in a suitable buffer medium to achieve a dilute concentration for sterile-filtering;   (ii) sterile-filtering the biopolymer by passage through a membrane suitable for sterile filtration; and   (iii) concentrating the biopolymer by ultrafiltration to a desired final concentration.   
     
     
         40 . The process according to  claim 39 , wherein the biopolymer is selected from the group consisting of a homopolysaccharide, a heteropolysaccharide and mixtures thereof. 
     
     
         41 . The process according to  claim 40 , wherein the homopolysaccharide is selected from the group consisting of carboxymethylcellulose, chitin, polymannuronic acid, curdlan gum and dextran. 
     
     
         42 . The process according to  claim 40 , wherein the heteropolysaccharide is selected from the group consisting of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, agar, alginate, carrageenan, gellan, guar gum, locust bean gum, and xanthan gum. 
     
     
         43 . The process according to  claim 39 , wherein the biopolymer is obtained from a source selected from the group consisting of a biological source, an in vitro enzymatic synthesis, a chemical synthesis, and combinations of two or more such sources. 
     
     
         44 . The process according to  claim 43 , wherein the biological source is selected from the group consisting of a bacterium, a plant, an amphibian, an avian and a mammal. 
     
     
         45 . The process according to  claim 43 , wherein the biopolymer obtained from a biological source further comprises a chemical modification. 
     
     
         46 . The process according to  claim 45 , wherein the chemical modification comprises a modification selected from the group consisting of addition of sulfate groups, addition of carboxyl groups, addition of hydroxyl groups, addition of acetyl groups, esterification, and cross-linking. 
     
     
         47 . The process according to  claim 39 , wherein the viscoelastic biopolymer has an average molecular weight in the range from 1×10 4  to 1×10 7  daltons. 
     
     
         48 . The process according to  claim 47 , wherein the viscoelastic biopolymer has an average molecular weight of 3×10 6 ±0.6×10 6  daltons. 
     
     
         49 . The process according to  claim 44 , wherein the bacterium is a strain of the genus  Streptococcus.    
     
     
         50 . The process according to  claim 49 , wherein the bacterium is a  Streptococcus  species selected from the group consisting of  Streptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis, Streptococcus dysgalactiae  and  Streptococcus zooepidemicus.    
     
     
         51 . The process according to  claim 49 , wherein the  Streptococcus  strain is nonhemolytic and non-pathogenic. 
     
     
         52 . The process according to  claim 39 , wherein the bulk manufactured biopolymer is isolated from the culture broth of a fermented  Streptococcus  strain. 
     
     
         53 . The process according to  claim 52 , wherein the bulk manufactured biopolymer is hyaluronic acid. 
     
     
         54 . The process according to  claim 53 , wherein the bulk manufactured hyaluronic acid is substantially free of impurities. 
     
     
         55 . The process according to  claim 54 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial endotoxin. 
     
     
         56 . The process according to  claim 55 , wherein the level of bacterial endotoxin is <0.25 EU/ml. 
     
     
         57 . The process according to  claim 53 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial cells. 
     
     
         58 . The process according to  claim 57 , wherein the viable count of bacterial cells is <100 CFU/g. 
     
     
         59 . The process according to  claim 58 , wherein the viable count of bacterial cells is <50 CFU/g. 
     
     
         60 . The process according to  claim 59 , wherein the viable count of bacterial cells is <10 CFU/g. 
     
     
         61 . The process according to  claim 54 , wherein the bulk manufactured hyaluronic acid is substantially free of protein. 
     
     
         62 . The process according to  claim 61 , wherein the level of protein is <1 mg/g. 
     
     
         63 . The process according to  claim 39 , wherein the dissolving in step (i) yields soluble bulk manufactured biopolymer at a concentration of <0.2%. 
     
     
         64 . The process according to  claim 63 , wherein the concentration is in the range of 0.10-0.13%. 
     
     
         65 . The process according to  claim 39 , wherein the concentrating in step (ii) is carried out by ultrafiltration. 
     
     
         66 . The process according to  claim 65 , wherein the ultrafiltration is carried out using a ceramic membrane. 
     
     
         67 . The process according to  claim 1 , wherein the desired final concentration in step (iii) is in the range of 0.8 to 3.0% w/v. 
     
     
         68 . A process according to  claim 67 , wherein the desired final concentration is about 1.0% w/v. 
     
     
         69 . A process according to  claim 67 , wherein the desired final concentration is about 1.2% w/v. 
     
     
         70 . A process according to  claim 67 , wherein the desired final concentration is about 2.0% w/v. 
     
     
         71 . The process according to  claim 39 , further comprising aseptic filling of a suitable packaging device with the biopolymer. 
     
     
         72 . The process according to  claim 33 , wherein the packaging device is selected from the group consisting of a syringe, a vial, a catheter and a nebulizer. 
     
     
         73 . The process according to  claim 39 , wherein the formulated viscoelastic biopolymer has a pseudoplasticity index in the range from 500 to 4000. 
     
     
         74 . The process according to  claim 39 , wherein the pseudoplasticity index is in the range from 600 to 1200. 
     
     
         75 . The process according to  claim 39 , wherein the pseudoplasticity index is in the range from 600 to 800. 
     
     
         76 . The process according to  claim 39 , wherein the sterile-filtering is carried out using a membrane of absolute pore size 0.2 micron. 
     
     
         77 . The process according to  claim 39 , wherein the buffer medium comprises a metal salt. 
     
     
         78 . A process for formulating a viscoelastic preparation of hyaluronic acid comprising:
 (i) dissolving bulk manufactured hyaluronic acid in a suitable buffer medium to achieve a dilute concentration for sterile-filtering;   (ii) sterile-filtering the dissolved hyaluronic acid by passage through a 0.2 micron absolute membrane; and   (iii) concentrating the hyaluronic acid by ultrafiltration to a desired final concentration.   
     
     
         79 . The process according to  claim 78 , wherein the bulk manufactured hyaluronic acid is obtained from a source selected from the group consisting of a biological source, an in vitro enzymatic synthesis, a chemical synthesis, and combinations of two or more such sources. 
     
     
         80 . The process according to  claim 79 , wherein the biological source is selected from the group consisting of a bacterium, a yeast, a plant, an amphibian, an avian and a mammal. 
     
     
         81 . The process according to  claim 80 , wherein the bulk manufactured hyaluronic acid obtained from a biological source further comprises a chemical modification. 
     
     
         82 . The process according to  claim 81 , wherein the chemical modification comprises a modification selected from the group consisting of addition of sulfate groups, addition of carboxyl groups, addition of hydroxyl groups, addition of acetyl groups, esterification, and cross-linking. 
     
     
         83 . The process according to  claim 78 , wherein the bulk manufactured hyaluronic acid has an average molecular weight in the range from 1×10 4  to 1×10 7  daltons. 
     
     
         84 . The process according to  claim 83 , wherein the bulk manufactured hyaluronic acid has an average molecular weight of 3×10 6 ±0.6×10 6  daltons. 
     
     
         85 . The process according to  claim 80 , wherein the bacterium is a species of the genus  Streptococcus.    
     
     
         86 . The process according to  claim 85 , wherein the bacterium is a  Streptococcus  species selected from the group consisting of  Streptococcus equi, Streptococcus pyogenes, Streptococcus equisimilis, Streptococcus dysgalactiae  and  Streptococcus zooepidemicus.    
     
     
         87 . The process according to  claim 85 , wherein the  Streptococcus  strain is non-hemolytic and non-pathogenic. 
     
     
         88 . The process according to  claim 79 , wherein the bulk manufactured hyaluronic acid is isolated from the culture broth of a fermented  Streptococcus  strain. 
     
     
         89 . The process according to  claim 78 , wherein the bulk manufactured hyaluronic acid is substantially free of impurities. 
     
     
         90 . The process according to  claim 89 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial endotoxin. 
     
     
         91 . The process according to  claim 90 , wherein the level of bacterial endotoxin is <0.25 EU/ml. 
     
     
         92 . The process according to  claim 89 , wherein the bulk manufactured hyaluronic acid is substantially free of bacterial cells. 
     
     
         93 . The process according to  claim 92 , wherein the viable count of bacterial cells is <100 CFU/g. 
     
     
         94 . The process according to  claim 93 , wherein the viable count of bacterial cells is <50 CFU/g. 
     
     
         95 . The process according to  claim 94 , wherein the viable count of bacterial cells is <10 CFU/g. 
     
     
         96 . The process according to  claim 89 , wherein the bulk manufactured hyaluronic acid is substantially free of protein. 
     
     
         97 . The process according to  claim 96 , wherein the level of protein is <1 mg/g. 
     
     
         98 . The process according to  claim 78 , wherein the concentration of the dissolved bulk manufactured biopolymer obtained in step (i) is <0.2%. 
     
     
         99 . The process according to  claim 98 , wherein the concentration is 0.10-0.13%. 
     
     
         100 . The process according to  claim 78 , wherein the concentrating is carried out by ultrafiltration. 
     
     
         101 . The process according to  claim 100 , wherein the ultrafiltration is carried out using a ceramic membrane. 
     
     
         102 . The process according to  claim 78 , wherein the desired final concentration is in the range of 0.8 to 3.0% w/v. 
     
     
         103 . A process according to  claim 102 , wherein the desired final concentration is about 1.0% w/v. 
     
     
         104 . A process according to  claim 102 , wherein the desired final concentration is about 1.2% w/v. 
     
     
         105 . A process according to  claim 102 , wherein the desired final concentration is about 2.0% w/v. 
     
     
         106 . A process according to  claim 78 , further comprising aseptic filling of a suitable packaging device with the biopolymer. 
     
     
         107 . A process according to  claim 106 , wherein the packaging device is selected from the group consisting of a syringe, a vial, a catheter and a nebulizer. 
     
     
         108 . A process according to  claim 78 , wherein the formulated hyaluronic acid has a pseudoplasticity index in the range from 500 to 4000. 
     
     
         109 . A process according to  claim 108 , wherein the pseudoplasticity index is in the range from 600 to 1200. 
     
     
         110 . A process according to  claim 109 , wherein the pseudoplasticity index is in the range from 600 to 800. 
     
     
         111 . A process according to  claim 78 , wherein the sterile-filtering is carried out using a membrane of absolute pore size 0.2 micron. 
     
     
         112 . A process according to  claim 78 , wherein all steps are performed under clean room conditions. 
     
     
         113 . A process according to  claim 78 , wherein the bulk manufactured hyaluronic acid is obtained by a process comprising:
 (i) precipitating with ethanol a culture broth of a non-hemolytic nonpathogenic hyaluronic acid-producing fermented  Streptococcus  strain;   (ii) dissolving the precipitate obtained in step (i) in sodium chloride/ethanol/charcoal;   (iii) precipitating the dissolved material obtained in step (ii) with cetylpyridinium chloride;   (iv) dissolving the precipitate obtained in step (iii) in sodium chloride/ethanol;   (v) treating the dissolved material obtained in step (iv) with magnesium silicate;   (vi) filtering the treated material obtained in step (v) through a 0.65 micron absolute membrane; and   (vii) precipitating the filtrate obtained in step (vi) with ethanol.   
     
     
         114 . A formulation of viscoelastic hyaluronic acid suitable for injection during surgery to mammals, obtained by the process according to  claim 78 . 
     
     
         115 . The formulation according to  claim 114 , substantially free of impurities and having a pseudoplasticity index greater than 600. 
     
     
         116 . The formulation according to  claim 115 , having an average molecular weight of 3×10 6 ±0.6×10 6  daltons. 
     
     
         117 . The formulation according to  claim 114 , further comprising a drug. 
     
     
         118 . The formulation according to  claim 114 , wherein the hyaluronic acid is chemically cross-linked. 
     
     
         119 . The formulation according to  claim 114 , wherein the hyaluronic acid is complexed with a metal. 
     
     
         120 . The process of  claim 1 , wherein the resulting viscoelastic biopolymer is stable and sterile for at least about one year. 
     
     
         121 . The process of  claim 120 , wherein the resulting viscoelastic biopolymer is stable and sterile for at least about two years. 
     
     
         122 . The process of  claim 120 , wherein the resulting viscoelastic biopolymer is stable and sterile for at least about five years. 
     
     
         123 . The process of  claim 1 , wherein no preservative of the viscoelastic biopolymer is used.