Patent Publication Number: US-2023158127-A1

Title: Compositions and methods for treating collagen-mediated diseases

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 17/062,372 filed Oct. 2, 2020, which is a continuation of U.S. application Ser. No. 15/160,392, filed May 20, 2016, now abandoned, which is a continuation of U.S. application Ser. No. 14/613,882, filed Feb. 4, 2015, now abandoned, which is a continuation of U.S. application Ser. No. 13/713,019, filed Dec. 13, 2012, now abandoned, which is a continuation of U.S. application Ser. No. 13/463,142, filed on May 3, 2012, now abandoned, which is a continuation of U.S. application Ser. No. 13/084,722, filed on Apr. 12, 2011, now abandoned; which is a continuation of U.S. application Ser. No. 12/871,159, filed on Aug. 30, 2010, now abandoned; which is a continuation of U.S. application Ser. No. 11/699,302, filed on Jan. 29, 2007, now U.S. Pat. No. 7,811,560, issued on Oct. 12, 2010, which claims the benefit of U.S. Provisional Application No. 60/763,470 filed on Jan. 30, 2006, and U.S. Provisional Application No. 60/784,135, filed Mar. 20, 2006, the entire contents of which are incorporated herein by reference in their entirety 
    
    
     PARTIES TO A JOINT RESEARCH AGREEMENT 
     Auxilium Pharmaceuticals Inc. and BioSpecifics Technologies Corp. are parties to a “joint research agreement” as defined in 35 USC 103(c)(3). 
     BACKGROUND OF THE INVENTION 
     Collagen is the major structural constituent of mammalian organisms and makes up a large portion of the total protein content of skin and other parts of the animal body. In humans, it is particularly important in the wound healing process and in the process of natural aging. Various skin traumas such as burns, surgery, infection and accident are often characterized by the erratic accumulation of fibrous tissue rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfigured deposits of new tissue sometimes form during the healing process. The excess collagen deposition has been attributed to a disturbance in the balance between collagen synthesis and collagen degradation. 
     Numerous diseases and conditions are associated with excess collagen deposition and the erratic accumulation of fibrous tissue rich in collagen. Such disease and conditions are collectively referred to herein as “collagen-mediated diseases”. Collagenase has been used to treat a variety of collagen-medicated diseases. Collagenase in an enzyme that has the specific ability to digest collagen. 
     Collagenase for use in therapy may be obtained from a variety of sources including mammalian (e.g. human), crustacean (e.g. crab, shrimp), fungal, and bacterial (e.g. from the fermentation of  Clostridium, Streptomyces, Pseudomonas , or  Vibrio ). Collagenase has also been genetically engineered. One common source of crude collagenase is from a bacterial fermentation process, specifically the fermentation of  C. histolyticum  ( C. his ). The crude collagenase obtained from  C. his  may be purified using any of a number of chromatographic techniques. 
     One drawback of the fermentation process from  C. his  is that it yields uncertain ratios or the various collagenases such as collagenase and collagenase II, often used in therapeutic compositions to treat collagen mediated conditions. Further, the culture has historically required the use of meat products. This meat culture was originally derived from the H4 strain of  Clostridium histolyticum , Dr. I. Mandl&#39;s laboratory in Columbia University in  1956  and deposited with the ATCC. Lyophilized vials were made out of the cooked meat culture and named as ABC  Clostridium histolyticum  master cell bank. 
     Various ratios of collagenase I to collagenase II in a therapeutic collagenase preparation have different biological effects. Therefore, a therapeutic collagenase preparation in which the ratio of collagenase I to collagenase II in the preparation can be easily and efficiently determined and controlled to obtain superior, and consistent enzyme activity and therapeutic effect, would be desirable. 
     SUMMARY OF THE INVENTION 
     The present invention provides a collagenase composition comprising a combination of highly purified collagenase I and collagenase II. Preferably, the collagenase I and collagenase II are present in a mass ratio of about 1 to 1. When used as a pharmaceutical composition for treating collagen-mediated disease, the composition of the invention provides improved and consistent therapeutic effect while lowering the potential for side effects. 
     The invention further provides methods for preparing a collagenase composition of the invention, pharmaceutical formulations comprising a composition of the invention and methods for treating patients suffering from a collagen-mediated disease using a collagenase composition of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference character refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 
         FIG.  1    depicts growth curves (OD vs time) of  C. histolyticum  in 5 L DCFT24a,b fermentations. 
         FIG.  2    depicts net growth curves (Net OD vs time) of  C. histolyticum  in 5 L DCFT24a,b fermentations. 
         FIG.  3    is a 8% Tris-glycine SDS PAGE gel from the second fermentation:
         Lane 1: High Molecular Weight Marker   Lane 2: Collagenase I—0.27 μg   Lane 3: Collagenase II—0.29 μg   Lane 4: 20 h (6.12 μL of sample)—Harvest point   Lane 5: 19 h (6.12 μL of sample)   Lane 6: 17 h (6.12 μL of sample)   Lane 7: 16 h (6.12 μL of sample)   Lane 8: 15 h (6.12 μL of sample)   Lane 9: 14 h (6.12 μL of sample)   Lane 10: 13 h (6.12 μL of sample)   Lane 11: 11.6 h-19 h (6.12 μL of sample)   Lane 12: 10.5 h (6.12 μL of sample).       

         FIG.  4    is a 8% Tris-glycine SDS PAGE gel from the first fermentation:
         Lane 1: High Molecular Weight Marker   Lane 2: Collagenase I—0.27 μg   Lane 3: Collagenase II—0.29 μg   Lane 4: 20 h (6.12 μL of sample)—Harvest point   Lane 5: 19 h (6.12 μL of sample)   Lane 6: 17 h (6.12 μL of sample)   Lane 7: 16 h (6.12 μL of sample)   Lane 8: 15 h (6.12 μL of sample)   Lane 9: 14 h (6.12 μL of sample)   Lane 10: 13 h (6.12 μL of sample)   Lane 11: 11.4 h (6.12 μL of sample)   Lane 12: 10.4 h (6.12 μL of sample).       

         FIG.  5    is a Semi-quantitative SDS PAGE gel for the second fermentation, harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.87 μL of sample (1/7 dilution of fermentation sample)   Lane 3: 1.22 μL of sample (1/5 dilution of fermentation sample)   Lane 4: 1.53 μL of sample (1/4 dilution of fermentation sample)   Lane 5: 2.04 μL of sample (1/3 dilution of fermentation sample)   Lane 6: 0.27 μg collagenase I   Lane 7: 0.18 μg collagenase I   Lane 8: 0.135 μg collagenase I   Lane 9: 0.29 μg collagenase II   Lane 10: 0.193 μg collagenase II   Lane 11: 0.145 μg collagenase II.       

         FIG.  6    represents fermentation strategy used for DCFT26a and DCFT26b. 
         FIG.  7    depicts growth curves (OD vs time)  C. histolyticum  in 5 L DCFT26a,b fermentations. 
         FIG.  8    depicts net growth curves (Net OD vs time) of  C. histolyticum  in 5 L DCFT26a,b fermentations. 
         FIG.  9    is a SDS PAGE gel for DCFT26a:
         Lane 1: High Molecular Weight Marker   Lane 2: Collagenase I—0.67 μg   Lane 3: Collagenase II—0.72 μg   Lane 4: 20 h (6.12 μL of sample)—Harvest point   Lane 5: 19 h (6.12 μL of sample)   Lane 6: 18 h (6.12 μL of sample)   Lane 7: 17 h (6.12 μL of sample)   Lane 8: 16 h (6.12 μL of sample)   Lane 9: 14 h (6.12 μL of sample)   Lane 10: 13 h (6.12 μL of sample)   Lane 11: 11 h (6.12 μL of sample).       

         FIG.  10    is a SDS PAGE gel for DCFT26a:
         Lane 1: High Molecular Weight Marker   Lane 2: 20 h (6.12 μL of sample)—Harvest point   Lane 3: 19 h (6.12 μL of sample)   Lane 4: 18 h (6.12 μL of sample)   Lane 5: 17 h (6.12 μL of sample)   Lane 6: 16 h (6.12 μL of sample)   Lane 7: 15 h (6.12 μL of sample)   Lane 8: 14 h (6.12 μL of sample)   Lane 9: 13 h (6.12 μL of sample)   Lane 10: 11 h (6.12 μL of sample)   Lane 11: Collagenase I—0.67 μg   Lane 12: Collagenase II—0.72 μg.       

         FIG.  11    is a semi-quantitative SDS PAGE gel for DCFT26a, harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I   Lane 3: 0.18 μg collagenase I   Lane 4: 0.135 μg collagenase I   Lane 5: 0.29 μg collagenase II   Lane 6: 0.193 μg collagenase II   Lane 7: 0.145 μg collagenase II   Lane 8: 0.87 μL of sample (1/7 dilution of fermentation sample)   Lane 9: 1.22 μL of sample (1/5 dilution of fermentation sample)   Lane 10: 1.53 μL of sample (1/4 dilution of fermentation sample)   Lane 11: 2.04 μL of sample (1/3 dilution of fermentation sample).       

         FIG.  12    is a Semi-quantitative SDS PAGE gel for DCFT26b, harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I   Lane 3: 0.18 μg collagenase I   Lane 4: 0.135 μg collagenase I   Lane 5: 0.29 μg collagenase II   Lane 6: 0.193 μg collagenase II   Lane 7: 0.145 μg collagenase II   Lane 8: 2.04 μL of sample (1/3 dilution of fermentation sample)   Lane 9: 1.53 μL of sample (1/4 dilution of fermentation sample)   Lane 10: 1.22 μL of sample (1/5 dilution of fermentation sample)   Lane 11: 0.87 μL of sample (1/7 dilution of fermentation sample).       

         FIG.  13    is a SDS PAGE gel for post-dialysed ammonium sulphate precipitated (100 g/L and 150 g/L) samples, DCFT26a, harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 4: 6.12 μL of supernatant sample from SC11   Lane 5: post dialysed sample—100 g/L AS (Neat)   Lane 6: post dialysed sample—100 g/L AS (1/5)   Lane 7: post dialysed sample—100 g/L AS (1/10)   Lane 8: post dialysed sample—150 g/L AS (Neat)   Lane 9: post dialysed sample—150 g/L AS (1/5)   Lane 10: post dialysed sample—150 g/L AS (1/10).       

         FIG.  14    is a SDS PAGE gel for post-dialysed ammonium sulphate precipitated (200 g/L and 250 g/L) samples, DCFT26a, harvest point:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 4: 6.12 μL of supernatant sample from SC11   Lane 5: post dialysed sample—200 g/L AS (Neat)   Lane 6: post dialysed sample—200 g/L AS (1/5)   Lane 7: post dialysed sample—200 g/L AS (1/10)   Lane 8: post dialysed sample—250 g/L AS (Neat)   Lane 9: post dialysed sample—250 g/L AS (1/5)   Lane 10: post dialysed sample—250 g/L AS (1/10).       

         FIG.  15    is a SDS PAGE gel for post-dialysed ammonium sulphate precipitated (300 g/L and 400 g/L) samples, DCFT26a, harvest point:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 4: 6.12 μL of supernatant sample from SC11   Lane 5: post dialysed sample—300 g/L AS (Neat sample)   Lane 6: post dialysed sample—300 g/L AS (1/5 dilution)   Lane 7: post dialysed sample—300 g/L AS (1/10 dilution)   Lane 8: post dialysed sample—400 g/L AS (Neat)   Lane 9: post dialysed sample—4000 g/L AS (1/5 dilution)   Lane 10: post dialysed sample—400 g/L AS (1/10 dilution).       

         FIG.  16    depicts a Growth curves (OD vs time and net OD vs time) of  C. histolyticum  in PBFT57 fermentation. 
         FIG.  17    is a Semi-quantitative SDS PAGE gel, harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I   Lane 3: 0.18 μg collagenase I   Lane 4: 0.135 μg collagenase I   Lane 5: 0.29 μg collagenase II   Lane 6: 0.193 μg collagenase II   Lane 7: 0.145 μg collagenase II   Lane 8: 2.04 μL of sample (1/3 dilution of fermentation harvest sample)   Lane 9: 1.53 μL of sample (1/4 dilution of fermentation harvest sample)   Lane 10: 1.22 μL of sample (1/5 dilution of fermentation harvest sample)   Lane 11: 0.87 μL of sample (1/7 dilution of fermentation harvest sample).       

         FIG.  18   a    is a quantitative SDS PAGE gel for post-dialysed 500 mL sample from fermentation PBFT57, harvest point sample 400 g/L of ammonium sulphate added:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.272 μg collagenase I and 0.286 μg collagenase II   Lane 3: 0.181 μg collagenase I and 0.190 μg collagenase II   Lane 4: 0.136 μg collagenase I and 0.142 μg collagenase II   Lane 5: 0.109 μg collagenase I and 0.114 μg collagenase II   Lane 6: post dialysed sample—400 g/L AS (1/15 dilution)   Lane 7: post dialysed sample—400 g/L AS (1/20 dilution)   Lane 8: post dialysed sample—400 g/L AS (1/25 dilution)   Lane 9: post dialysed sample—400 g/L AS (1/30 dilution)   Lane 10: post dialysed sample—400 g/L AS (1/35 dilution)   Lane 11: High Molecular Weight Marker.       
         FIG.  18   b    is a SDS PAGE of the supernatants after centrifugation of the ammonium sulphate precipitated samples:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg Col I and 0.29 μg Col II   Lane 3: Supernatant (neat) of post ammonium sulphate precipitated sample (400 g/L slow addition)   Lane 4: Supernatant (neat) of post ammonium sulphate precipitated sample (400 g/L fast addition)   Lane 5: Supernatant (neat) of post ammonium sulphate precipitated sample (440 g/L slow addition)   Lane 6: Supernatant (neat) of post ammonium sulphate precipitated sample (480 g/L slow addition)   Lane 7: Supernatant (neat) of post ammonium sulphate precipitated sample (520 g/L slow addition)   Lane 8: Supernatant (neat) of post ammonium sulphate precipitated sample (440 g/L pH 6)   Lane 9: Supernatant (neat) of post ammonium sulphate precipitated sample (440 g/L oxygenated).       
         FIG.  19    is a Semi-quantitative SDS PAGE gel showing diluted samples from the harvest point supernatant and the post dialysed ammonium sulphate (with 400 g/L—fast addition) precipitated sample:
         Lane 1: High Molecular Weight Marker   Lane 2: Fermentation sample—harvest (neat)   Lane 3: Fermentation sample—harvest (1/1 dilution)   Lane 4: Fermentation sample—harvest (1/2 dilution)   Lane 5: Fermentation sample—harvest (1/3 dilution)   Lane 6: Fermentation sample—harvest (1/4 dilution)   Lane 7: Post dialysed sample—harvest (1/17.54 dilution) corresponds to lane 1   Lane 8: Post dialysed sample—harvest (1/35.08 dilution) corresponds to lane 2   Lane 9: Post dialysed sample—harvest (1/52.62 dilution) corresponds to lane 3   Lane 10: Post dialysed sample—harvest (1/70.16 dilution) corresponds to lane 4   Lane 11: Post dialysed sample—harvest (1/87.70 dilution) corresponds to lane 5       

         FIG.  20    is a semi-quantitative SDS PAGE gel for PBFT57 showing diluted samples from the harvest point supernatant and the post dialysed ammonium sulphate (with 520 g/L) precipitated sample:
         Lane 1: High Molecular Weight Marker   Lane 2: Fermentation sample—harvest (neat)   Lane 3: Fermentation sample—harvest (1/1 dilution)   Lane 4: Fermentation sample—harvest (1/2 dilution)   Lane 5: Fermentation sample—harvest (1/3 dilution)   Lane 6: Fermentation sample—harvest (1/4 dilution)   Lane 7: Post dialysed sample—harvest (1/15.63 dilution) corresponds to lane 1   Lane 8: Post dialysed sample—harvest (1/31.26 dilution) corresponds to lane 2   Lane 9: Post dialysed sample—harvest (1/46.89 dilution) corresponds to lane 3   Lane 10: Post dialysed sample—harvest (1/62.52 dilution) corresponds to lane 4   Lane 11: Post dialysed sample—harvest (1/78.15 dilution) corresponds to lane 5.       

         FIG.  21    depicts growth curves (Net OD vs time) of  C. histolyticum  strains 004 and 013 in PBFT58c,d fermentations. 
         FIG.  22    is a SDS PAGE gel for PBFT58c (Strain 004):
         Lane 1: High Molecular Weight Marker   Lane 2: Collagenase I—1.00 μg   Lane 3: Collagenase I—0.67 μg   Lane 4: Collagenase II—1.08 μg   Lane 5: Collagenase II—0.72 μg   Lane 6: 16.25 h (6.12 μL of sample)   Lane 7: 17 h (6.12 μL of sample)   Lane 8: 18 h (6.12 μL of sample)   Lane 9: 19 h (6.12 μL of sample)   Lane 10: 20.5 h (6.12 μL of sample).       

         FIG.  23    is a SDS PAGE gel for PBFT58d (Strain 013):
         Lane 1: High Molecular Weight Marker   Lane 2: Collagenase I—1.00 μg   Lane 3: Collagenase I—0.67 μg   Lane 4: Collagenase II—1.08 μg   Lane 5: Collagenase II—0.72 μg   Lane 6: 16.25 h (6.12 μL of sample)   Lane 7: 17 h (6.12 μL of sample)   Lane 8: 18 h (6.12 μL of sample)   Lane 9: 19 h (6.12 μL of sample)   Lane 10: 20.5 h (6.12 μL of sample).       

         FIG.  24    is a semi-quantitative SDS PAGE gel for PBFT58c (strain 004), harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II   Lane 5: 0.108 μg collagenase I and 0.116 μg collagenase II   Lane 6: 6.12 μL of sample   Lane 7: 3.06 μL of sample   Lane 8: 2.04 μL of sample   Lane 9: 1.53 μL of sample   Lane 10: 1.22 μL of sample.       

         FIG.  25    is a semi-quantitative SDS PAGE get for PBFT58d (strain 013), harvest point sample:
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II   Lane 5: 0.108 μg collagenase I and 0.116 μg collagenase II   Lane 6: 6.12 μL of sample   Lane 7: 3.06 μL of sample   Lane 8: 2.04 μL of sample   Lane 9: 1.53 μL of sample   Lane 10: 1.22 μL of sample.       

         FIG.  26    is SDS PAGE gel for post-dialysed harvest point sample (520 g/L ammonium sulphate of PBFT58c fermentation (strain 004):
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II   Lane 5: 0.108 μg collagenase I and 0.116 μg collagenase II   Lane 6: post dialysed harvest point sample—Neat   Lane 7: post dialysed harvest point sample—(1/5 dilution)   Lane 8: post dialysed harvest point sample—(1/10 dilution)   Lane 9: post dialysed harvest point sample—(1/15 dilution)   Lane 10: post dialysed harvest point sample—(1/20 dilution).       

         FIG.  27    is a SDS PAGE gel for post-dialysed harvest point sample (400 g/L ammonium sulphate) of PBFT58d fermentation (strain 013):
         Lane 1: High Molecular Weight Marker   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II   Lane 5: 0.108 μg collagenase I and 0.116 μg collagenase II   Lane 6: post dialysed harvest point sample—Neat   Lane 7: post dialysed harvest point sample—(1/5 dilution)   Lane 8: post dialysed harvest point sample—(1/10 dilution)   Lane 9: post dialysed harvest point sample—(1/15 dilution)   Lane 10: post dialysed harvest point sample—(1/20 dilution).       

         FIG.  28    is illustrates a flow chart of the Experimental procedure used for screening the alternative vegetable peptones. 
         FIG.  29    illustrates a fed-batch strategy for DCFT27a,b fermentations. 
         FIG.  30    depicts growth curves (Net OD vs time) of  C. histolyticum  in 5 L DCFT27a and DCFT27b fed-batch fermentations. 
         FIG.  31    depicts growth curves (NET OD vs time) of  C. histolyticum  in 5 L PBFT59a,b,c batch fermentations. 
         FIG.  32    depicts growth curves (NET OD vs time) of  C. histolyticum  in 5 L DCFT27d fed-batch fermentations. 
         FIG.  33   a    is a SDS PAGE gel for DCFT27d (Phytone supplemented with amino acids):
         Lane 1: High Molecular Weight Marker   Lane 2: 18 h (6.12 μL of sample)   Lane 3: 17 h (6.12 μL of sample)   Lane 4: 15 h (6.12 μL of sample)   Lane 5: 14 h (6.12 μL of sample)   Lane 6: 13 h (6.12 μL of sample)   Lane 7: 11.3 h (6.12 μL of sample)   Lane 8: 0.27 μg Collagenase I and 0.29 μg Collagenase II       
         FIG.  33   b    represents a schematic diagram of the inoculation procedure. 
         FIG.  33   c    represents a flow chart of an approximately 200 L fed batch inoculation process. 
         FIG.  34    shows a chromatogram after hydroxyapatite chromatography. 
         FIG.  35    shows a chromatogram after a fractogel TMAE anion exchange. 
         FIG.  36    is an 8% Tris-Glycine SDS PAGE analysis of Pre Ha, Post HA and POST TMAE material from 5 L scale process: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                   1 
                   High Molecular Weight Marker 
                   20 
                 
                 
                   2 
                   Collagenase ABC I reference 1 μg 
                   20 
                 
                 
                   3 
                   Collagenase ABC II reference 1 μg 
                   20 
                 
                 
                   4 
                   Pre HA-1 μg 
                   20 
                 
                 
                   5 
                   Post HA-1 μg 
                   20 
                 
                 
                   6 
                   Post TMAE-1 μg 
                   20 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  37    shows a chromatogram after a fractogel TMAE anion exchange. 
         FIG.  38    is an 8% Tris-Glycine SDS-PAGE analysis of Q Sepharose IEX chromatography of post TMAE material run in the presence leupeptin: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                   1 
                   High Molecular Weight marker 
                   20 
                 
                 
                   2 
                   Collagenase ABC I reference 1 μg 
                   20 
                 
                 
                   3 
                   Collagenase ABC II reference 1 μg 
                   20 
                 
                 
                   4 
                   Post TMAE/Post Dialysis-1 μg 
                   20 
                 
                 
                   5 
                   Fraction D6-neat 
                   20 
                 
                 
                   6 
                   Fraction E6-neat 
                   20 
                 
                 
                   7 
                   Fraction F7-1 μg 
                   20 
                 
                 
                   8 
                   Fraction F6-1 μg 
                   20 
                 
                 
                   9 
                   Fraction F5-1 μg 
                   20 
                 
                 
                   10  
                   Fraction F4-neat 
                   20 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  39    is an 8% Tris-Glycine SDS-PAGE analysis of Q Sepharose IEX chromatography of post TMAE material run in the presence of leupeptin. Gel 2—Peak 2 (ABCI): 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   SampleLoad volume, μl 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                   1 
                   High Molecular Weight marker 
                   20 
                 
                 
                   2 
                   Collagenase ABC I reference 1 μg 
                   20 
                 
                 
                   3 
                   Collagenase ABC II reference 1 μg 
                   20 
                 
                 
                   4 
                   Fraction B2-neat 
                   20 
                 
                 
                   5 
                   Fraction C1-1 μg 
                   20 
                 
                 
                   6 
                   Fraction C2-1 μg 
                   20 
                 
                 
                   7 
                   Fraction C3-1 μg 
                   20 
                 
                 
                   8 
                   Fraction C4-1 μg 
                   20 
                 
                 
                   9 
                   Fraction C5-neat 
                   20 
                 
                 
                   10  
                   Fraction C6-neat 
                   20 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  40    shows a chromatogram after a Q Sepharose HP anion exchange with modified gradient. 
         FIG.  41    shows a chromatogram after a Superdex 75 Gel Permeation chromatography of ABCII. 
         FIG.  42    is a 12% Bis-Tris SDS-PAGE analysis of Superdex 75 GPC of concentrated ABCII run in the presence of arginine: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                   1 
                   Mark 12 marker 
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 1 μg 
                   15 
                 
                 
                   4 
                   GPC load 1 μg 
                   15 
                 
                 
                   5 
                   Fraction D4 
                   15 
                 
                 
                   6 
                   Fraction D3 
                   15 
                 
                 
                   7 
                   Fraction D2 
                   15 
                 
                 
                   8 
                   Fraction D1 Neat 
                   15 
                 
                 
                   9 
                   Fraction E1 
                   15 
                 
                 
                   10  
                   Fraction E2 
                   15 
                 
                 
                   11  
                   Fraction E3 
                   15 
                 
                 
                   12  
                   Fraction E4 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  43    shows a chromatogram after a Superdex 75 Gel Permeation chromatography of ABCI. 
         FIG.  44    is a 4-12% Bis-Tris SDS-PAGE analysis of Superdex 75 GPC of concentrated ABC I run in the presence of arginine: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                   1 
                   Mark 12 marker 
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 1 μg 
                   15 
                 
                 
                   4 
                   GPC load 1 μg 
                   15 
                 
                 
                   5 
                   Fraction D4 
                   15 
                 
                 
                   6 
                   Fraction D3 
                   15 
                 
                 
                   7 
                   Fraction D2 
                   15 
                 
                 
                   8 
                   Fraction D1 
                   15 
                 
                 
                   9 
                   Fraction E1 
                   15 
                 
                 
                   10  
                   Fraction E2 
                   15 
                 
                 
                   11  
                   Fraction E3 
                   15 
                 
                 
                   12  
                   Fraction E4 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  45    represents a flow chart of one proposed manufacturing process. 
         FIG.  46    represents a flow chart of the fermentation procedure for process 3. 
         FIG.  47    represents a flow chart of the purification procedure for process 3. 
         FIG.  48    is a SDS-PAGE (reduced) Coomasie stained for Intermediates AUXI and AUXII:
         Lane   1. High Molecular Weight Makers   2. 0.132 mg/ml ABC-I Reference   3. 0.0265 mg/ml ABC-I Reference   4. 0.132 mg/ml AUX-I Intermediate   5. 0.0265 mg/ml AUX-I Intermediate   6. 0.132 mg/ml ABC-I Intermediate   7. 0.265 mg/ml ABC-II Reference   8. 0.132 mg/ml AUX-II Intermediate   9. 0.0265 mg·ml AUX-II Intermediate.       

         FIG.  49    is a SDS-PAGE (reduced) Coomasie stained for Drug Substance:
         Lane   1. High Molecular Weight Makers   2. 0.132 mg/ml Mixed BTC Reference   3. 0.0265 mg/ml Mixed BTC Reference   4. 0.132 mg/ml Drug Substance   5. 0.0265 mg/ml Drug Substance.       

         FIG.  50    is SDS-PAGE (reduced) Silver stained Drug Substance:
         Lane   1. HMW marker   2. 0.132 mg/ml Mixed BTC Reference   3. 0.0265 mg/ml Mixed BTC Reference   4. 0.132 mg/ml Drug Substance   5. 0.0265 mg/ml Drug Substance.       

         FIG.  51    depicts a comparison of  C. histolyticum  grown to Proteose Peptone #3 in a batch fermentation to the existing fermentation process using Phytone peptone during fed-batch cultivation;  GCFT03b PP3 batch  GCFT03d Phytone fed-batch 
         FIG.  52    is a SDS-PAGE analysis of the collagenase product at the harvest point (20 h) of a 5 L Proteose Peptone #3 batch fermentation (GCFT03b) (8% Tris-Glycine): 
       
         
           
             
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
              
                 
                   1 
                   High Molecular Weight Marker 
                 
                 
                   2 
                   0.27 μg AUXI 
                 
                 
                   3 
                   0.18 μg AUXI 
                 
                 
                   4 
                   0.135 μg AUXI 
                 
                 
                   5 
                   0.29 μg AUXII 
                 
                 
                   6 
                   0.193 μg AUXII 
                 
                 
                   7 
                   0.145 μg AUXII 
                 
                 
                   8 
                   0.87 μL of sample (1/7 dilution of fermentation sample) 
                 
                 
                   9 
                   1.22 μL of sample (1/5 dilution of fermentation sample) 
                 
                 
                   10 
                   1.53 μL of sample (1/4 dilution of fermentation sample) 
                 
                 
                   11 
                   2.04 μL of sample (1/3 dilution of fermentation sample) 
                 
                 
                     
                 
                 
                   Estimates 
                 
                 
                   AUXI~176 mg/L 
                 
                 
                   AUXII~190 mg/L 
                 
              
             
           
         
       
         FIG.  53    is a SDS-PAGE analysis of the collagenase product at the harvest point (20 h) of a 5 L Phytone fed-batch fermentation (GCFT03d) (8% Tris-Glycine): 
       
         
           
             
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
              
                 
                   1 
                   High Molecular Weight Marker 
                 
                 
                   2 
                   0.27 μg AUXI 
                 
                 
                   3 
                   0.18 μg AUXI 
                 
                 
                   4 
                   0.135 μg AUXI 
                 
                 
                   5 
                   0.29 μg AUXII 
                 
                 
                   6 
                   0.193 μg AUXII 
                 
                 
                   7 
                   0.145 μg AUXII 
                 
                 
                   8 
                   0.87 μL of sample (1/7 dilution of fermentation sample) 
                 
                 
                   9 
                   1.22 μL of sample (1/5 dilution of fermentation sample) 
                 
                 
                   10 
                   1.53 μL of sample (1/4 dilution of fermentation sample) 
                 
                 
                   11 
                   2.04 μL of sample (1/3 dilution of fermentation sample) 
                 
                 
                     
                 
                 
                   Estimates 
                 
                 
                   AUXI~88 mg/L 
                 
                 
                   AUXII~142 mg/L 
                 
              
             
           
         
       
         FIG.  54    illustrates three fermentations of  Clostridium histolyticum  grown on 50 g/L PP3 demonstrating a reproducible growth profile:  CCFT03b GCFT04c GCFT05d 
         FIG.  55    is a SDS-PAGE analysis showing the time course of GCFT05d (batch fermentation with Proteose Peptone #3), 8% Tris Glycine gel, colloidal stained): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   Reference, AUXI and AUXII 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   3 
                   hours 
                 
                 
                     
                   4 
                   4 
                   hours 
                 
                 
                     
                   5 
                   5 
                   hours 
                 
                 
                     
                   6 
                   6 
                   hours 
                 
                 
                     
                   7 
                   7 
                   hours 
                 
                 
                     
                   8 
                   8 
                   hours 
                 
                 
                     
                   9 
                   9 
                   hours 
                 
                 
                     
                   10 
                   10 
                   hours 
                 
                 
                     
                   11 
                   11 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  56    is a SDS-PAGE analysis showing the time course of GCFT05d (batch fermentation with Proteose Peptone #3), (8% Tris Glycine gel, colloidal stained): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   Reference, AUXI and AUXII 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   3 
                   hours 
                 
                 
                     
                   4 
                   4 
                   hours 
                 
                 
                     
                   5 
                   5 
                   hours 
                 
                 
                     
                   6 
                   6 
                   hours 
                 
                 
                     
                   7 
                   7 
                   hours 
                 
                 
                     
                   8 
                   8 
                   hours 
                 
                 
                     
                   9 
                   9 
                   hours 
                 
                 
                     
                   10 
                   10 
                   hours 
                 
                 
                     
                   11 
                   11 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  57    is a SDS-PAGE analysis showing the time course of DCFT24b (fed-batch fermentation using Phytone peptone), (8% Tris Glycine gel, colloidal stained): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   AUXI - 0.27μ 
                 
                 
                     
                   3 
                   AUXII - 0.29μ  
                 
                 
                     
                   4 
                   20 hours - Harvest point 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   5 
                   19 
                   hours 
                 
                 
                     
                   6 
                   17 
                   hours 
                 
                 
                     
                   7 
                   16 
                   hours 
                 
                 
                     
                   8 
                   15 
                   hours 
                 
                 
                     
                   9 
                   14 
                   hours 
                 
                 
                     
                   10 
                   13 
                   hours 
                 
                 
                     
                   11 
                   11.6 
                   hours 
                 
                 
                     
                   12 
                   10.5 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  58    illustrates a comparison of growth curves from  C. histolyticum  fermentations using different lots of PP3: 
       
         
           
             
                 
                 
                 
                 
                 
               
                 
                     
                     
                 
               
              
                 
                     
                       GCFT05d 
                       GCFT06b 
                       GCFT07c 
                       GCFT07d 
                 
                 
                     
                       GCFT08c 
                       GCFT06d 
                       GCFT09C 
                       GCFT09D 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  59    illustrates a small scale comparison of three lots of PP3 and evaluation of 100 g/L PP3: 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
               
              
                 
                     
                   ▪batch 5354795 (50 g/L) 
                   □batch 5332395 50 g/L) 
                 
                 
                     
                   ▪batch 5325635 (50 g/L) 
                   □batch 5392398 (10-g/L) 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  60    depicts a grown profiles of two 5 L fermentations utilizing PP3 at 100 g/L:  PBFT70c lot #5354796  PBFT70d lot #5325635 
         FIG.  61    is a SDS-PAGE analysis of the time course of PBFT70c, 100 g/L (lot #5354796) fermentation (8% Tris-Glycine): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   Reference, 0.4 μg AUXI and AUXII 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   3.1 
                   hours 
                 
                 
                     
                   4 
                   4.4 
                   hours 
                 
                 
                     
                   5 
                   5.5 
                   hours 
                 
                 
                     
                   6 
                   6.6 
                   hours 
                 
                 
                     
                   7 
                   8.0 
                   hours 
                 
                 
                     
                   8 
                   9.1 
                   hours 
                 
                 
                     
                   9 
                   10.1 
                   hours 
                 
                 
                     
                   10 
                   11.4 
                   hours 
                 
                 
                     
                   11 
                   13.0 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  62    is a SDS-PAGE analysis of the timecourse of PBFT70d, 100 g/L PP3 (lot #5325635) fermentation (8% Tri-Glycine): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   Reference, 0.4 μg AUXI and AUXII 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   3.1 
                   hours 
                 
                 
                     
                   4 
                   4.3 
                   hours 
                 
                 
                     
                   5 
                   5.5 
                   hours 
                 
                 
                     
                   6 
                   6.6 
                   hours 
                 
                 
                     
                   7 
                   7.8 
                   hours 
                 
                 
                     
                   8 
                   9.1 
                   hours 
                 
                 
                     
                   9 
                   10.1 
                   hours 
                 
                 
                     
                   10 
                   11.3 
                   hours 
                 
                 
                     
                   11 
                   12.0 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  63    represents a densitometry analysis of SDS-PAGE to compare cell growth to product formation from 5 L fermentation of PBFT70c:  Precursor  Aux1 Aux 2 OD. 
         FIG.  64    illustrates a Comparison of 100 g/L PP3 process at 5 L and 200 L scale.  PBFT70c  pbft70d  200 L scale-up fermentation 
         FIG.  65    is a SDS-PAGE analysis of the time course of the 200 L fermentation (8% Tris-Glycine): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   AUXI and AUXII mixed reference (1.2 μg) 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   4 
                   hours 
                 
                 
                     
                   4 
                   6 
                   hours 
                 
                 
                     
                   5 
                   8 
                   hours 
                 
                 
                     
                   6 
                   9.4 
                   hours 
                 
                 
                     
                   7 
                   12 
                   hours 
                 
                 
                     
                   8 
                   14 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  66    represents a densitometry analysis of SDS-PAGE to compare cell growth to product formation from 200 L fermentation:  Precursor Aux1 Aux 2 OD. 
         FIG.  67    is a SDS-PAGE analysis of the time course of the 200 L fermentation (4-12% Bis-Tris): 
       
         
           
             
                 
                 
                 
               
                 
                     
                     
                 
                 
                     
                   Lane 
                   Sample 
                 
                 
                     
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                     
                   1 
                   High Molecular Weight Marker 
                 
                 
                     
                   2 
                   AUXI and AUXII mixed reference (1.2 μg) 
                 
              
             
             
                 
                 
                 
                 
              
                 
                     
                   3 
                   4 
                   hours 
                 
                 
                     
                   4 
                   6 
                   hours 
                 
                 
                     
                   5 
                   8 
                   hours 
                 
                 
                     
                   6 
                   9.4 
                   hours 
                 
                 
                     
                   7 
                   12 
                   hours 
                 
                 
                     
                   8 
                   14 
                   hours 
                 
                 
                     
                     
                 
              
             
           
         
       
         FIG.  68    shows a standard curve for densitometry quantification of collagenase concentration. 
         FIG.  69    represents a schematic illustration of the fermentation and harvest of  Clostridium histolyticum.    
         FIGS.  70  ( a ) and ( b )  are chromatograms resulting from Hydrophobic interaction chromatography using Phenyl Sepharose FF (low sub): (a) is full scale chromatogram and (b) is an expanded chromatogram showing fraction collection. 
         FIG.  71    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the MUSTANG Q filter step to the TFF1 step. The gel is stained with Colloidal blue and overloaded (2.5 μg total protein/lane) to show contaminant bands: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   12 
                 
                 
                   2 
                   Post MUSTANG Q filtrate 
                   15 
                 
                 
                   3 
                   Pre HIC Bag 1 
                   15 
                 
                 
                   4 
                   Pre HIC Bag 2 
                   15 
                 
                 
                   5 
                   HIC flow-through Bag 1 
                   15 
                 
                 
                   6 
                   HIC flow-through Bag 2 
                   15 
                 
                 
                   7 
                   HIC Peak 1 (0.3M AS wash) 
                   15 
                 
                 
                   8 
                   Post HIC pool (peak 2) 
                   15 
                 
                 
                   9 
                   Pre TFF (post HIC pool + 2 day hold) 
                   15 
                 
                 
                   10 
                   Post TFF 
                   15 
                 
                 
                   11 
                   Pre Q-AEX (post TFF + overnight hold) 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  72    is an Ion exchange chromatogram (Q Sepharose HP) of the post HIC material after concentration and diafiltration into 10 mM Tris, 200 μM leupeptin pH 8. 
         FIG.  73    is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 1 (AUXII) eluted during the ion exchange column ( FIG.  5   ). Gel 1: the gel is stained with Colloidal blue: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                     
                     
                     
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   4 
                   Load 
                     
                     
                   1 μg 
                   15 
                 
                 
                   5 
                   Fraction 1 
                     
                     
                   neat 
                   15 
                 
                 
                   6 
                   Fraction 2 
                     
                     
                   neat 
                   15 
                 
                 
                   7 
                   Fraction 3 
                     
                     
                   neat 
                   15 
                 
                 
                   8 
                   Fraction 4 
                     
                   AUXII 
                   1 μg 
                   15 
                 
                 
                   9 
                   Fraction 5 
                    {close oversize brace}  
                   fractions 
                   1 μg 
                   15 
                 
                 
                   10 
                   Fraction 6 
                     
                     
                   1 μg 
                   15 
                 
                 
                   11 
                   Fraction 7 
                     
                     
                   1 μg 
                   15 
                 
                 
                   12 
                   Fraction 8 
                     
                     
                   1 μg 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  74    is a 4-12 Bis Tris SDS-PAGE analysis of fractions from peak 1 (AUXII) eluted during the ion exchange columns ( FIG.  5   ). Gel 2: the gel is stained with Colloidal blue: 
       
         
           
             
                 
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                     
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                     
                     
                     
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   4 
                   Fraction 9 
                     
                     
                   1 μg 
                   15 
                 
                 
                   5 
                   Fraction 10 
                     
                   AUXII 
                   neat 
                   15 
                 
                 
                   6 
                   Fraction 11 
                   {close oversize brace}  
                   fractions 
                   neat 
                   15 
                 
                 
                   7 
                   Fraction 12 
                     
                     
                   neat 
                   15 
                 
                 
                   8 
                   Peak 1 Tail 
                     
                     
                   1 μg 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  75    is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 2 (AUXI) eluted during the ion exchange column ( FIG.  5   ). Gel 3: the gel is stained with the Colloidal blue: 
       
         
           
             
                 
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                     
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                     
                     
                     
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   4 
                   Fraction 13 
                     
                     
                   neat 
                   15 
                 
                 
                   5 
                   Fraction 14 
                     
                     
                   1 μg 
                   15 
                 
                 
                   6 
                   Fraction 15 
                     
                     
                   1 μg 
                   15 
                 
                 
                   7 
                   Fraction 16 
                     
                   AUXI 
                     μg 
                   15 
                 
                 
                   8 
                   Fraction 17 
                   {close oversize brace}  
                   fractions 
                     μg 
                   15 
                 
                 
                   9 
                   Fraction 18 
                     
                     
                   1 μg 
                   15 
                 
                 
                   10 
                   Fraction 19 
                     
                     
                   1 μg 
                   15 
                 
                 
                   11 
                   Fraction 20 
                     
                     
                   1 μg 
                   15 
                 
                 
                   12 
                   Fraction 21 
                     
                     
                   1 μg 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  76    is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 2 (AUXI) eluted during the ion exchange column ( FIG.  5   ). Gel 4: the gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                     
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                     
                     
                     
                   10 
                 
                 
                   2 
                   Collagenase ABC I reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   3 
                   Collagenase ABC II reference 
                     
                     
                   1 μg 
                   15 
                 
                 
                   4 
                   Fraction 22 
                     
                     
                   1 μg 
                   15 
                 
                 
                   5 
                   Fraction 23 
                     
                     
                   1 μg 
                   15 
                 
                 
                   6 
                   Fraction 24 
                     
                     
                   1 μg 
                   15 
                 
                 
                   7 
                   Fraction 25 
                   {close oversize brace}  
                   AUXI 
                   1 μg 
                   15 
                 
                 
                   8 
                   Fraction 26 
                     
                   fractions 
                   1 μg 
                   15 
                 
                 
                   9 
                   Fraction 27 
                     
                     
                   1 μg 
                   15 
                 
                 
                   10 
                   Peak 2 Tail 
                     
                     
                   1 μg 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  77    is a 4-12% Bis Tris SDS-PAGE analysis of in-process samples from the anion exchange step to final product. The gel is stained with Colloidal blue. Gel 1: 1 μg/lane loading: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   10 
                 
                 
                   2 
                   ABC I Reference 
                   15 
                 
                 
                   3 
                   ABC II Reference 
                   15 
                 
                 
                   4 
                   Post IEX AUX I Pool 
                   15 
                 
                 
                   5 
                   Post IEX AUX II Pool 
                   15 
                 
                 
                   6 
                   AUX I Intermediate (DOM: 12 MAY 2006) 
                   15 
                 
                 
                   7 
                   AUX II Intermediate (DOM: 10 MAY 2006) 
                   15 
                 
                 
                   8 
                   AUX I Intermediate (Pre Mix) 
                   15 
                 
                 
                   9 
                   AUX II Intermediate (Pre Mix) 
                   15 
                 
                 
                   10 
                   Drug Substance (DOM: 15 MAY 2006) 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  78    is a 4-12% Bis Tris SDS-PAGE analysis of in-process samples from the anion exchange step to final product. The gel is stained with Colloidal blue. Gel 2: 2.5 μg/lane loading: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Maker 
                   10 
                 
                 
                   2 
                   ABC I Reference 
                   15 
                 
                 
                   3 
                   ABC II Reference 
                   15 
                 
                 
                   4 
                   Post IEX AUX I Pool 
                   15 
                 
                 
                   5 
                   Post IEX AUX II Pool 
                   15 
                 
                 
                   6 
                   AUX I Intermediate (DOM: 12MAY06) 
                   15 
                 
                 
                   7 
                   AUX II Intermediate (DOM: 10MAY06) 
                   15 
                 
                 
                   8 
                   AUX I Intermediate (Pre Mix) 
                   15 
                 
                 
                   9 
                   AUX II Intermediate (Pre Mix) 
                   15 
                 
                 
                   10 
                   Drug Substance (DOM: 15MAY06) 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  79    is a SDS-PAGE with 8% Tris Glycine: (NB Ref AS/1640/020):
         1. High Molecular Weight Markers   2. Blank Lane   3. 1 μg Fermentation Filtrate Day 4   4. 1 μg Fermentation Filtrate Day 5   5. 1 μg Post Mustang Q Day 4   6. 1 μg Post HIC Day 3   7. 1 μg Post HIC Day 6   8. 1 μg Post TFF Day 2   9. 1 μg Post TFF Day 4       

         FIG.  80    is a SDS-PAGE with 8% Tris Glycine:
         1. High Molecular Weight Markers   2. 1 μg AUX-I Reference   3. 1 μg AUX-II Reference   4. 1 μg Post IEX AUX-I Day 5   5. 1 μg Post IEX AUX-I Day 12   6. 1 μg Post IEX AUX-II Day 5   7. 1 μg Post IEX AUX-II Day 12       

         FIG.  81    is a SDS-PAGE gel:
         1. High Molecular Weight Markers   2. 1 μg AUX-I Reference   3. 1 μg AUX-II Reference   4. 1 μg Post AUX-I Intermediate Day 5   5. 1 μg Post AUX-I Intermediate Day 12   6. 1 μg Post AUX-II Intermediate Day 5   7. 1 μg Post AUX-II Intermediate Day 12       

         FIG.  82    represents analytical chromatography analysis. 
         FIG.  83    shows protein concentration determination by UV. 
         FIG.  84    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the 20 L demonstration run-through taken at the point of manufacture and stored at −20° C. The gel is stained with Colloidal blue. 1 μg loading: 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   10 
                 
                 
                   2 
                   Post Mustang Q filtrate 
                   15 
                 
                 
                   3 
                   Pre HIC: Bag 1 
                   15 
                 
                 
                   4 
                   Pre HIC: Bag 2 
                   15 
                 
                 
                   5 
                   HIC flow-through Bag 1 
                   15 
                 
                 
                   6 
                   HIC flow-through Bag 2 
                   15 
                 
                 
                   7 
                   HIC Peak 1 (0.3M AS wash) 
                   15 
                 
                 
                   8 
                   Post HIC pool 
                   15 
                 
                 
                   9 
                   Pre TFF1 (post HIC pool + weekend hold) 
                   15 
                 
                 
                   10 
                   Post TFF1 
                   15 
                 
                 
                   11 
                   Pre Q-AEX (post TFF + overnight hold) 
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  85    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the 20 L demonstration run-through after 22 hrs at Room Temperature. The gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   10 
                 
              
             
             
                 
                 
                 
                 
                 
              
                 
                   2 
                   Pre Mustang Q filtrate 
                     
                     
                   15 
                 
                 
                   3 
                   Post Mustang Q filtrate 
                     
                     
                   15 
                 
                 
                   4 
                   Pre HIC Bag 1 
                     
                     
                   15 
                 
                 
                   5 
                   Pre HIC Bag 2 
                     
                     
                   15 
                 
                 
                   6 
                   Post HIC Pool 
                     
                     
                   15 
                 
                 
                   7 
                   Pre TFF1 
                    {close oversize brace}  
                   1 μg loading 
                   15 
                 
                 
                   8 
                   Post TFF1 
                     
                     
                   15 
                 
                 
                   9 
                   Post IEX Aux I Pool 
                     
                     
                   15 
                 
                 
                   10 
                   Post IEX Aux II Pool 
                     
                     
                   15 
                 
                 
                   11 
                   AUX I Intermediate 
                     
                     
                   15 
                 
                 
                   11 
                   AUX II Intermediate 
                     
                     
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  86    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the 20 L demonstration run-through after 22 hrs at 37° C. The gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   12 
                 
              
             
             
                 
                 
                 
                 
                 
              
                 
                   2 
                   Pre Mustang filtrate 
                     
                     
                   15 
                 
                 
                   3 
                   Post Mustang Q filtrate 
                     
                     
                   15 
                 
                 
                   4 
                   Pre HIC Bag 1 
                     
                     
                   15 
                 
                 
                   5 
                   Pre HIC Bag 2 
                     
                     
                   15 
                 
                 
                   6 
                   Post HIC Pool 
                     
                     
                   15 
                 
                 
                   7 
                   Pre TFF1 
                    {close oversize brace}  
                   1 μg loading 
                   15 
                 
                 
                   8 
                   Post TFF1 
                     
                     
                   15 
                 
                 
                   9 
                   Post IEX AUX I Pool 
                     
                     
                   15 
                 
                 
                   10 
                   Post IEX AUX II Pool 
                     
                     
                   15 
                 
                 
                   11 
                   AUX I Intermediate 
                     
                     
                   15 
                 
                 
                   12 
                   AUX II Intermediate 
                     
                     
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  87    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the 20 L demonstration run-through after 94 hrs at Room Temperature. The gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   12 
                 
              
             
             
                 
                 
                 
                 
                 
              
                 
                   2 
                   Pre Mustang Q filtrate 
                     
                     
                   15 
                 
                 
                   3 
                   Post Mutstang Q filtrate 
                     
                     
                   15 
                 
                 
                   4 
                   Pre HIC Bag 1 
                     
                     
                   15 
                 
                 
                   5 
                   Pre HIC Bag 2 
                     
                     
                   15 
                 
                 
                   6 
                   Post HIC Pool 
                     
                     
                   15 
                 
                 
                   7 
                   Pre TFF1 
                    {close oversize brace}  
                   1 μg loading 
                   15 
                 
                 
                   8 
                   Post TFF1 
                     
                     
                   15 
                 
                 
                   9 
                   Post IEX Aux I Pool 
                     
                     
                   15 
                 
                 
                   10 
                   Post IEX Aux II Pool 
                     
                     
                   15 
                 
                 
                   11 
                   AUX I Intermediate 
                     
                     
                   15 
                 
                 
                   12 
                   AUX II Intermediate 
                     
                     
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  88    is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples from the 20 L demonstration run-through after 94 hrs at 37° C. The gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
              
                 
                   1 
                   Mark 12 Molecular Weight Marker 
                   12 
                 
              
             
             
                 
                 
                 
                 
                 
              
                 
                   2 
                   Pre Mustang Q filtrate 
                     
                     
                   15 
                 
                 
                   3 
                   Post Mutstang Q filtrate 
                     
                     
                   15 
                 
                 
                   4 
                   Pre HIC Bag 1 
                     
                     
                   15 
                 
                 
                   5 
                   Pre HIC Bag 2 
                     
                     
                   15 
                 
                 
                   6 
                   Post HIC Pool 
                     
                     
                   15 
                 
                 
                   7 
                   Pre TFF1 
                    {close oversize brace}  
                   1 μg loading 
                   15 
                 
                 
                   8 
                   Post TFF1 
                     
                     
                   15 
                 
                 
                   9 
                   Post IEX Aux I Pool 
                     
                     
                   15 
                 
                 
                   10 
                   Post IEX Aux II Pool 
                     
                     
                   15 
                 
                 
                   11 
                   AUX I Intermediate 
                     
                     
                   15 
                 
                 
                   12 
                   AUX II Intermediate 
                     
                     
                   15 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  89    is a 8% Tris-Glycine SDS-PAGE analysis of selected post IEX AUX I and post IEX AUX II fractions. Fractions were selected from the 20 L demonstration run which were enriched for the required contaminate protein. The gel is stained with Colloidal blue. 
       
         
           
             
                 
                 
                 
               
                 
                     
                 
                 
                   Lane 
                   Sample 
                   Load volume, μl 
                 
                 
                     
                 
               
              
                 
                     
                 
              
             
             
                 
                 
                 
                 
                 
              
                 
                   1 
                   Post IEX AUX I Fraction 16 
                     
                     
                   20 
                 
                 
                   2 
                   Post IEX AUX I Fraction 16 
                     
                     
                   20 
                 
                 
                   3 
                   Post IEX AUX 1 Fraction 16 
                    {close oversize brace}  
                   eluted with 
                   20 
                 
                 
                   4 
                   Post IEX AUX I Fraction 16 
                     
                   AUXI 
                   20 
                 
                 
                   5 
                   Post IEX AUX I Fraction 16 
                     
                     
                   20 
                 
                 
                   6 
                   Post IEX AUX II Fraction 2 
                     
                     
                   20 
                 
                 
                   7 
                   Post IEX AUX II Fraction 2 
                     
                     
                   20 
                 
                 
                   8 
                   Post IEX AUX II Fraction 2 
                    {close oversize brace}  
                   eluted with 
                   20 
                 
                 
                   9 
                   Post IEX AUX II Fraction 2 
                     
                   AUXII 
                   20 
                 
                 
                   10 
                   Post IEX AUX II Fraction 2 
                     
                     
                   20 
                 
                 
                     
                 
              
             
           
         
       
         FIG.  90    is a 8% Tris-Glycine SDS-PAGE analysis of selected post IEX AUX I and post IEX AUX II fractions. Fractions were selected from purified material generated from fermentation 20 L PP3 and enrich for the ˜90 kDa contaminant protein. The gel is stained with Colloidal blue. 
     
    
    
       
     
       
         
           
               
               
               
             
               
                   
               
               
                 Lane 
                 Sample 
                 Load volume, μl 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 High Molecular Weight marker 
                 20 
               
            
           
           
               
               
               
               
               
            
               
                 2 
                 Post IEX Fraction B7 R2 
                   
                   
                 20 
               
               
                 3 
                 Post IEX Fraction B7R2 
                   
                 eluted with 
                 20 
               
               
                 4 
                 Post IEX Fraction B7R2 
                  {close oversize brace}  
                 AUXI 
                 20 
               
               
                 5 
                 Post IEX Fraction B7R2 
                   
                   
                 20 
               
               
                 6 
                 Post IEX Fraction D1 
                   
                   
                 20 
               
               
                 7 
                 Post IEX Fraction D1 
                   
                   
                 20 
               
               
                 8 
                 Post IEX Fraction D1 
                  {close oversize brace}  
                 eluted with 
                 20 
               
               
                 9 
                 Post IEX Fraction D1 
                   
                 AUXII 
                 20 
               
               
                 10 
                 Post IEX Fraction D1 
                   
                   
                 20 
               
               
                   
               
            
           
         
       
     
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention provides a novel collagenase drug substance comprising a mixture of highly purified collagenase I and collagenase II in a mass ratio of about 1 to 1. It has been discovered that a composition comprising a mixture of collagenase I and collagenase II in an artificial mass ratio of 1 to 1 provides highly reproducible and optimal enzymatic activity and imparts superior therapeutic effect while lowering the potential for side effects. It is understood that the terms “drug substance”, “drug product” or “collagenase composition” can be used interchangeably. 
     The one embodiment, the present invention provides a drug substance consisting of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, having a mass ratio of about 1 to 1 with a purity of at least 95% by area, and preferably a purity of at least 98% by area. 
     In another embodiment, the present invention provides a drug substance, wherein the drug substance having at least one specification selected from table A below: 
     
       
         
           
               
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                   
                 Specification 
               
            
           
           
               
               
               
            
               
                 Test 
                 AUX-I 
                 AUX-II 
               
               
                   
               
            
           
           
               
               
            
               
                 Appearance 
                 Clear colourless and essentially free from 
               
               
                   
                 particulate matter 
               
               
                 Potentiometric Measure of 
                 7.5 to 8.5 
               
               
                 pH of Solution 
                   
               
               
                 Endotoxin 
                 &lt;10 EU/mL 
               
            
           
           
               
               
               
            
               
                 Identity (and purity) by 
                 Major collagenase band 
                 Major collagenase  
               
               
                 SDS-PAGE (Reduced 
                 between 98-188 kDa  
                 band between  
               
               
                 conditions, Coomasie  
                 MW markers 
                 97-200 kDa; MW 
               
               
                 and silver stained) 
                   
                 markers; major  
               
               
                   
                   
                 bands comparable  
               
               
                   
                   
                 to reference  
               
               
                   
                   
                 standard 
               
            
           
           
               
               
            
               
                 Total Protein by 
                 0.8-1.2 mg/mL 
               
               
                 Absorbance Spectroscopy 
                   
               
            
           
           
               
               
               
            
               
                 SRC assay (AUX-I) 
                 13,000-23,000 fSRC 
                 NA 
               
               
                   
                 units/mg 
                   
               
               
                 GPA assay (AUX-II) 
                 NA 
                 230,00-430,000  
               
               
                   
                   
                 fGPA units/mg 
               
            
           
           
               
               
            
               
                 Residual host cell protein 
                 Comparable to refereme standard; no  
               
               
                   
                 individual impurity band exhibiting greater  
               
               
                   
                 intensity than 1% BSA intensity marker 
               
               
                 Residual host cell DNA 
                 ≤10 pg/dose 
               
               
                 Analysis of Proteins using the 
                 ≥98% main peak, ≤ 2% aggregates  
               
               
                 Agilent 1100 HPLC System 
                 by area 
               
               
                 (Aggregation by size 
                   
               
               
                 exclusion chromatography) 
                   
               
               
                 Analysis of Proteins using 
                 2 major peaks (AUX I &amp; AUX II),  
               
               
                 the Agilent 1100 HPLC 
                 combined ≥97% by area; Retention 
               
               
                 System (Identity and purity 
                 times of AUX-I and AUX-II within 5% 
               
               
                 by reverse phase liquid 
                 of reference 
               
               
                 chromatography) 
                   
               
               
                 Analysis of Proteins using 
                 ≤1% by area 
               
               
                 the Agilent 1100 HPLC 
                   
               
               
                 System (Residual clostripain 
                   
               
               
                 by reverse phase liquid 
                   
               
               
                 chromatography 
                   
               
               
                 Analysis of Proteins using 
                 ≤1% by area 
               
               
                 the Agilent 1100 HPLC 
                   
               
               
                 System (Residual gelatinase 
                   
               
               
                 by anion exchange 
                   
               
               
                 chromatography) 
                   
               
               
                 Residual leupeptin by 
                 ≤1 ng/mg w/w 
               
               
                 reverse phase 
                   
               
               
                 chromatography 
                   
               
               
                 Bioburden 
                 ≤1 cfu/mL 
               
               
                   
               
            
           
         
       
     
     In one aspect, the invention provides a process for producing a drug substance consisting of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, having a mass ratio of about 1 to 1 with a purity of at least 95% by area, comprising the steps of:
         a) fermenting  Clostridium histolyticum;      b) harvesting a crude product comprising collagenase I and collagenase II;   c) purifying collagenase I and collagenase II from the crude harvest via filtration and column chromatography; and   d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.       

     In one preferred embodiment, the fermentation step is conducted in the presence of a porcine derived, a phytone peptone or a vegetable peptone medium. More preferably, the porcine derived medium is proteose peptone #3. 
     In one embodiment, the invention provides a fermentation procedure comprising the steps of:
         a) inoculating the medium in a first stage; with  Clostridium histolyticum  and agitating the mixture;   b) incubating the mixture from step (a) to obtain an aliquot;   c) inoculating the medium in a second stage with aliquots resulting from step (b) and agitating the mixture;   d) incubating mixtures from step (c);   e) inoculating the medium in a third stage with aliquots resulting from step (d) and agitating;   f) incubating mixtures from step (e);   g) inoculating the medium in a fourth stage with an aliquot resulting from step (f) and agitating;   h) incubating mixtures from step (g); and   i) harvesting culture resulting from step (h) by filtration.       

     In a preferred embodiment, the fermentation procedure comprises the steps of:
         a) Inoculating 3×25 mL PP3 (proteose peptone) medium with 3×250 μL of WCB (25 mL cultures in 3×125 mL shake flasks, contained within Anaerobe gas jar) at a temperature set point of 37° C., and agitating the mixture at 125 rpm;   b) incubating the mixture from step (a) for 12 hours;   c) inoculating Inoculate 4×200 mL pp3 medium with 4×5 mL aliquots from 1 of the above 25 mL cultures (200 mL cultures in 4×500 mL shake flasks, contained within Anaerobe gas jar) at a temperature set point of 37° C., and agitating the mixture at 125 rpm;   d) incubating mixtures from step (c) for 12 hours;   e) inoculating 14.4 L of PP3 medium with 3×200 mL culture (15 L culture in 20 L fermenter) at a temperature set point of 37° C., and pH set point of 7.00 and agitating the mixture at 125 rpm;   f) incubating mixtures from step (e) for 12 hours;   g) inoculating 192 L of PP3 medium with 8 L of 15 L culture (200 L culture in 270 L fermenter) at a temperature set point of 37° C. and pH set point of 7.00, and agitating the mixture at 125 rpm;   h) incubating mixtures from step (g) for 14 hours; and   i) harvesting 200 L culture by filtration (depth followed by 0.2 μm) via Millipore Millistak 4 m 2  and 0.2 μm filter (2×Millipore Express XL 10 filters) at a flow rate of 200 L/h.       

     In one embodiment, the invention provides a purification procedure comprising the steps of:
         a) filtering the crude harvest through a MUSTANG Q anion-exchange capsule filter;   b) adding ammonium sulphate; preferably to a final concentration of 1M;   c) filtering the crude harvest; preferably through a 0.45 μm filter;   d) subjecting the filtrate through a HIC column; preferably a phenyl sepharose 6FF (low sub);   e) adding leupeptin to the filtrate; preferably to a final concentration of 0.2 mM to post HIC eluted product;   f) removing the ammonium sulfate and maintaining leupeptin for correct binding of collagenase I and collagenase II with buffer exchange by TFF; preferably with buffer exchange by TFF;   g) filtering the mixture of step (f) preferably through a 0.45 μm filter;   h) separating collagenase I and collagenase II using Q-Sepharose HP;   i) preparing TFF concentration and formulation for collagenase I and collagenase II separately; wherein TFF is a tangential flow filtration using 10 and/or 30K MWCO (molecular weight cut off) PES or RC-polyethersulfone or regenerated cellulose filter membranes. Provides means to retain and concentrate select protein and exchange the protein from one buffer solution into another; and   j) filtering through a 0.2 μm filtration system.       

     The drug substance of the present invention includes both collagenase I and collagenase II. A preferred source of crude collagenase is from a bacterial fermentation process, specifically the fermentation of  C. histolyticum  ( C. his ). In one embodiment of the invention, a fermentation process is described. The crude collagenase obtained from  C. his  may be purified by a variety of methods known to those skilled in the art, including dye ligand chromatography, heparin affinity chromatography, ammonium sulfate precipitation, hydroxyapatite chromatography, size exclusion chromatography, ion exchange chromatography, and metal chelation chromatography. Crude and partially purified collagenase is commercially available from many sources including Advance Biofactures Corp., Lynbrook, N.Y. 
     Both collagenase I and collagenase II are metalloproteases and require tightly bound zinc and loosely bound calcium for their activity (Eddie L. Angleton and H. E. Van Wart,  Biochemistry  1988, 27, 7406-7412). Both collagenases have broad specificity toward all types of collagen (Steinbrink, D; Bond, M and Van Wart, H; (1985),  JBC,  260 p2771-2776). Collagenase I and Collagenase II digest collagen by hydrolyzing the triple-helical region of collagen under physiological conditions (Steinbrink, D; Bond, M and Van Wart, H; (1985),  JBC,  260 p2771-2776). Even though each collagenase shows different specificity (e.g. each have a different preferred amino sequence for cleavage), together, they have synergistic activity toward collagen [Mandi, I., (1964),  Biochemistry,  3: p.1737-1741; Vos-Scheperkeuter, G H, (1997),  Cell Transplantation,  6: p.403-412]. Collagenase II has a higher activity towards all kinds of synthetic peptide substrates than collagenase I as reported for class II and class I collagenase in the literatures. [Bond, M. D. (1984),  Biochemistry,  23: p.3085-3091. Hesse, F, (1995), Transplantation Proceedings, 27: p.3287-3289]. 
     Examples of collagen mediated-diseases that may be treated by the compositions and methods of the invention include but are not limited to: Dupuytren&#39;s disease; Peyronie&#39;s disease; frozen shoulder (adhesive capsulitis), keloids; hypertrophic scars; depressed scars such as those resulting from inflammatory acne; post-surgical adhesions; acne vulgaris; lipomas, and disfiguring conditions such as wrinkling, cellulite formation and neoplastic fibrosis. U.S. Pat. Nos. 6,086,872 and 5,589,171 incorporated herein by reference disclose the use of collagenase preparations in the treatment of Dupuytren&#39;s disease. U.S. Pat. No. 6,022,539 incorporated herein by reference discloses the use of collagenase preparations in the treatment of Peyronie&#39;s disease. 
     In addition its use in treating collagen-mediated diseases, the composition of the invention is also useful for the dissociation of tissue into individual cells and cell clusters as is useful in a wide variety of laboratory, diagnostic and therapeutic applications. These applications involve the isolation of many types of cells for various uses, including microvascular endothelial cells for small diameter synthetic vascular graft seeding, hepatocytes for gene therapy, drug toxicology screening and extracorporeal liver assist devices, chondrocytes for cartilage regeneration, and islets of Langerhans for the treatment of insulin-dependent diabetes mellitus. Enzyme treatment works to fragment extracellular matrix proteins and proteins which maintain cell-to-cell contact. Since collagen is the principle protein component of tissue ultrastructure, the enzyme collagenase has been frequently used to accomplish the desired tissue disintegration. In general, the composition of the present invention is useful for any application where the removal of cells or the modification of an extracellular matrix, are desired. 
     Collagenase compositions of the invention may also be prepared by mixing either a specific number of activity units or specific masses of the preferably purified enzymes. Collagenase activity can be measured by the enzyme&#39;s ability to hydrolyze either synthetic peptide or collagen substrate. Those skilled in the art will recognize that enzyme assays other than those disclosed herein may also be used to define and prepare functionally equivalent enzyme compositions. 
     Another aspect of the present invention is the reproducible optimization of the 1 to 1 mass ratio of collagenase I to collagenase II in the composition of the invention. The reproducibility of the ratio of collagenase I to collagenase II has previously been a challenge because of several factors. First, commercial fermentation of  Clostridium  generally results in a 1 to 2 ratio of collagenase I and collagenase II. Second, the purification procedures are known to alter this ratio significantly resulting in inconsistent ratios of purified product. The optimized fixed mass ratio of the composition of the present invention maximizes the synergistic activity provided by the two different collagenases resulting in superior therapeutic benefit. 
     The invention also provides pharmaceutical formulations of the compositions of the invention. The pharmaceutical formulations of the present invention comprise a therapeutically effective amount of a collagenase composition of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients. 
     As used herein, the term “pharmaceutically acceptable carrier or excipient” means a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer&#39;s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. 
     The pharmaceutical compositions of this invention may be administered parenterally, topically, or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, infrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In a preferred embodiment, the composition is injected into the disfiguring tissue. In the case of Peyronie&#39;s or Duputyren&#39;s diseases or adhesive capsulitis, the composition is injected into the cord or plaque. The term “local administration” is defined herein to embrace such direct injection. 
     Furthermore, particularly good results can be obtained by immobilizing the site of injection after administration. For example, the site of administration can be immobilized for 4 or more hours. 
     Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer&#39;s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. 
     The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. The sterile solutions may also be lyophilized for later use. 
     Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. 
     The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. 
     Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. 
     Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. 
     In one preferred embodiment, the drug substance of the invention is a lyophilized injectable composition formulated with lactose. In one embodiment each milligram of injectable collagenase is formulated with 1.9 mg of lactose. In another embodiment, each milligram of injection collagenase preferably has approximately 2800 SRC units and 51000 units measured with a potency assay using a synthetic substrate, pzGPGGPA. 
     In another preferred embodiment, the collagenase composition of the invention is a lyophilized injectable composition formulated with Sucrose, Tris at a pH level of about 8.0. Most preferably, 1.0 mg of the drug substance of the invention is formulated in 60 mM Sucrose, 10 mM Tris, at a pH of about 8.0 (this equates to 20.5 mg/mL of sucrose and 1.21 mg/mL of Tris in the formulation buffer). Examples of some of the formulations include, but not limited to for a 0.58 mg of the drug substance dose, 18.5 mg of sucrose and 1.1 mg of Tris are added in each vial, where the targeting a vial fill volume is 0.9 mL; and for a 0.58 mg of the drug substance dose, 12.0 mg sucrose (multicompendial) and 0.7 mg of Tris (multicompendial). 
     In accordance with the invention, methods are provided for treating collagen-mediated diseases comprising the step of administering to a patient in need thereof, a therapeutically effective amount of a composition of the invention, or a therapeutically effective amount of a pharmaceutical formulation of the invention. By a “therapeutically effective amount” of a compound of the invention is meant an amount of the compound which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. 
     The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts. 
     The drug substance for injectable collagenase consists of two microbial collagenases, referred to as Collagenase AUX I and Collagenase ABC I and Collagenase AUX II and Collagenase ABC II. It is understood that the terms “Collagenase I”, “ABC I”, “AUX I”, “collagenase AUX I”, and “collagenase ABC I” mean the same and can be used inter-changeably. Similarly, the terms “Collagenase II”, “ABC II”, “AUX II”, “collagenase AUX II”, and “collagenase ABC II” refer to the same enzyme and can also be used interchangeably. These collagenases are secreted by bacterial cells. They are isolated and purified from  Clostridium histolyticum  culture supernatant by chromatographic methods. Both collagenases are special proteases and share the same EC number (E.C 3.4.24.3). 
     Collagenase AUX I has a single polypeptide chain consisting of approximately 1000 amino acids with a molecular weight of 115 kDa. Collagenase AUX II has also a single polypeptide chain consisting of about 1000 amino acids with a molecular weight of 110 kDa. 
     Even though the literature indicates that there are sequence homologies in regions of collagenase AUX I and AUX II, the two polypeptides do not seem to be immunologically cross reactive as indicated by the western blot analysis. 
     The drug substance (collagenase concentrate) has an approximately 1 to 1 mass ratio for collagenase AUX I and AUX II. The collagenase concentrate has an extinction coefficient of 1.528. 
     All references cited herein, whether in print, electronic, computer readable storage media or other form, are expressly incorporated by reference in their entirety, including but not limited to, abstracts, articles, journals, publications, texts, treatises, internet web sites, databases, patents, and patent publications. 
     EXAMPLES 
     The compositions and processes of the present invention will be better understood in connection with the following examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the processes, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims. 
     Process 2: 
     Fermentation Process 
     This work was set out to develop a fermentation process that aimed at delivering a target yield of 250 mg/L of total collagenases ABC I &amp; II from the 5 L fermentation scale process in an animal free component growth media. Various potential alternative nitrogen sources were screened to see if they had any affect on collagenase expression over and the above the phytone component currently used in the growth media. An experiment comparing productivities from two strains of  C. histolyticum,  004 and 013, was to determine any differences between the two strains with respect to growth kinetics, collagenase productivity and production of contaminating proteases grown in an animal derived media. This comparison highlighted significant differences between growing the  C. histolyticum  strain in animal derived media as opposed to animal free growth media. 
     Previous results described that increased concentrations of phytone and yeast extract were shown to support higher biomass concentrations and hence higher levels of collagenase expression. In an attempt to further increase biomass concentrations and total collagenase productivity of the optimised batch fermentation media, a fed-batch fermentation strategy was designed. Two 5 L fermentations were performed, one with a high concentration of media in the batch phase followed by a low concentration feeding phase, the second with a low concentration of media in the batch phase. Both fermentations produced high biomass concentrations, however the high concentration batch phase showed relatively low levels of collagenase expression. The low concentration batch fermentation showed very high levels of collagenase expression (˜280 mg/L), however, this culture also produced significant quantities of the contaminating protease, clostripain. 
     Although the low concentration batch fermentation gave very good results with respect to expression of the collagenase, the highly concentrated phytone and yeast extract feed solution was very difficult to prepare. Two additional fermentations were performed, the first was a repeat of the previous successful fed-batch fermentation the second had a slightly higher concentration batch phase media composition with a lower concentrated feeding solution. Both fermentations achieved similar biomass concentrations and showed the same expression profile of the collagenase and clostripain. The quantity of collagenase produced was again estimated at approximately 280 mg/L in both fermentations. However, these fermentations produced significant quantities of contaminating protease clostripain. 
     A selection of alternative nitrogen sources were assessed for their ability to replace the phytone peptone used in the fed-batch fermentation strategy. The  C. histolyticum  grew extremely well on the vegetable peptones reaching optical densities (600 nm) of 4 to 5 units. However, SDS-PAGE analysis of these fermentations showed no expression of either collagenase or clostripain. Due to the luxuriant cell growth observed on these peptones it was thought that the concentration of complex nitrogen source was too high resulting in an inhibition of protease expression. A second set of fermentations was therefore carried out using the alternative peptones at 50 g/L in a batch strategy. When the fermentations were analyzed by SDS-PAGE no expression of collagenase or clostripain was seen again. A fed-batch fermentation using phytone peptone was supplemented with three amino acids, glutamine, tryptophan and asparagine. These amino acids were identified as being present in lower amounts in the non-animal media. The growth profile of the fermentation was very similar to that of the fed-batch fermentation without amino acid supplementation. SDS-PAGE analysis showed a similar yield of collagenase but a slightly lower level of clostripain. The clostripain assay showed reduced activity in the amino supplemented when compared to the control fed-batch fermentation. The reduction in clostripain activity whilst still significant was not as great as the difference between animal and non-animal media. 
     The assessment of the primary recovery step of the collagenases using ammonium sulphate precipitation was carried out on 0.2 μm filtrates of the crude fermentation supernatants. The aim here was to help increase the collagenase yield and ideally decrease the quantity of clostripain that was carried through the process. Initially ammonium sulphate concentrations of 100-400 g/L were assessed. Ammonium sulphate at 400 g/L resulted in significant recovery of collagenase. A further study was carried out with a higher range of ammonium sulphate (400-520 g/L). in addition, the effect of decreasing the pH to 6.0 and oxygenating the media prior to precipitation were also investigated. No difference was observed in either quantity of the collagenases or clostripain recovered from the supernatant under any of these conditions. The pellet generated from the 400 g/L ammonium sulphate was the easiest to resuspend. 
     The study to compare the two strains of  C. histolyticum  (004 and 013) showed that the productivity of the collagenases from the animal derived media was lower than that of the optimal non-animal derived media. SDS-PAGE analysis, supported by an enzymatic assay for clostripain activity, highlighted that there were significantly lower quantities of clostripain in the material produced from the animal derived media than the non-animal media. This highlighted the fact that the feedstock produced from the non-animal derived media fermentation was significantly different feedstock material from the fermentation using animal derived media with respect to the production of contaminating proteases. 
     1 st  Set of Fed-Batch Fermentation—DCFT24 
     The results from the process development work showed that the use of an enriched media (100 g/L phytone peptone and 50 g/L yeast extract) resulted in the expression of higher amounts of collagenases compared to the original media (50 g/L phytone peptone and 8.5 g/L yeast extract). In addition, it initially appeared that it reduced the amounts of clostripain produced. 
     Two 5 L fermentations were then performed. Firstly the strategy consisted of a long batch phase/short fed-batch phase, whereas the second consisted of a short batch phase/long fed-batch phase. In both strategies at the end of the fermentation (after 20 h) the concentrations of phytone peptone and yeast extract were 100 g/L and 50 g/L, respectively, as in the case of the batch fermentations. Table 1 and 2 detail the media recipes and strategies used. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Media recipe and fed-batch strategy 
               
            
           
           
               
               
            
               
                   
                 Long batch − short fed-batch 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Concentrations at 
               
               
                 Component 
                 Batch phase 
                 Feed 
                 harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phytone Peptone 
                 100 
                 g/L 
                 100 
                 g/L 
                 100 
                 g/L 
               
               
                 Yeast extract 
                 50 
                 g/L 
                 50 
                 g/L 
                 50 
                 g/L 
               
               
                 Glucose 
                 10 
                 g/L 
                 10 
                 g/L 
                 10 
                 g/L 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Filtered 
                   
                   
               
               
                   
                   
                   
                 sterilised 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
                   
                   
                   
                   
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
                   
                   
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
                   
                   
                   
                   
               
               
                 NaCl 
                 2.5 
                 g/L 
                   
                   
                   
                   
               
               
                 Magnesium 
                 0.08 
                 g/L 
                   
                   
                   
                   
               
               
                 Vitamin solution 
                 10 
                 mL/L 
                   
                   
                   
                   
               
               
                 Volume 
                 4 
                 L 
                 1 
                 L 
                 ~5 
                 L 
               
               
                   
               
            
           
           
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Media recipe and fed-batch strategy 
               
            
           
           
               
               
            
               
                   
                 Short batch − long fed-batch 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Concentrations at 
               
               
                 Component 
                 Batch phase 
                 Feed 
                 harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phytone Peptone 
                 40 
                 g/L 
                 254 
                 g/L 
                 100 
                 g/L 
               
               
                 Yeast extract 
                 10 
                 g/L 
                 153 
                 g/L 
                 50 
                 g/L 
               
               
                 Glucose 
                 7.5 
                 g/L 
                 17.8 
                 g/L 
                 10 
                 g/L 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Filtered 
                   
                   
               
               
                   
                   
                   
                 sterilised 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
                   
                   
                   
                   
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
                   
                   
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
                   
                   
                   
                   
               
               
                 NaCl 
                 2.5 
                 g/L 
                   
                   
                   
                   
               
               
                 Magnesium 
                 0.08 
                 g/L 
                   
                   
                   
                   
               
               
                 Vitamin solution 
                 10 
                 mL/L 
                   
                   
                   
                   
               
               
                 Volume 
                 4 
                 L 
                 1 
                 L 
                 ~5 
                 L 
               
               
                   
               
            
           
           
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
       FIG.  1    shows the growth curves (OD 600 nm  vs time) from the two fermentations, whereas  FIG.  2    shows the net growth curves (Net OD 600 nm  vs time). It was observed that cells from the first fermentation grew very fast and reached their maximum OD after approximately 10 hours. This was due to the fact that the media in the batch phase was very rich. During the fed-batch phase the cells did not appear to grow. The OD values decreased slightly, which could be partly attributed to the fact that the cells were dying and to the dilution effect of the feed to the fermenter. 
     For the second fermentation, the fed-batch phase was started after 6 hours. At that point the OD value would have been low, as suggested by the growth curve in  FIG.  1   . The cells continued to grow slowly up to approximately 18 hours. 
     It was noted that the net growth curves in  FIG.  2    suggested that the cell densities in DCFT24b fermentation were higher than in DCFT24a fermentation. The OD 600 nm  of the media prior to inoculation was approximately 1.7, whereas in DCFT24b it was approximately 0.4. These differences are due to the fact when the fermenters are autoclaved a precipitate is formed. For DCFT24a, higher amounts were formed compared to DCFT24b. SDS PAGE gels: 
     SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samples were carried out for each for the two fermentations. The gels are shown in  FIGS.  3  and  4   . A semi-quantitative SDS PAGE gel was also produced for the harvest point sample of the second fermentation. 
     The SDS PAGE gel analysis in  FIG.  4    indicated that very low amounts of the collagenases were expressed. This could be due to the fact that the cells grew very fast during the batch phase and as a result the maximum cell concentration was reached after approximately 10 hours. In contrast, very high level of collagenase expression was observed in the second fermentation, probably due to the fact that the cells grew more slowly during the short batch phase and continued to grow during the fed-batch phase. Thus the invention relates to an improved fermentation method for  C. his  wherein cell growth is controlled and slow during the short batch phase and continuing growth during the fed-batch phase. Slow growth is defined to mean that the rate of growth during the short batch phase does not result in a maximum cell concentration prior to the fed-batch phase, such as within about 10 hours of the beginning of the fermentation process. In a preferred embodiment, the rate of growth is approximately that resulting from the second fermentation cycle described herein. 
     Estimated collagenase productivities from the semi-quantitative SDS PAGE gel at the harvest point of the second fermentation cycle ( FIG.  5   ), were 132 mg/L for collagenase ABC I and 158 mg/L for collagenase ABC II. Comparing these values with those previously obtained, there is approximately a 3-fold increase in the expression levels using the fed-batch strategy. 
     The next step was to perform an additional set of fed-batch fermentations using slightly modified fed-batch strategies and media. The aim was to improve the scalability and robustness of the fermentation process. 
     The media recipe for this fermentation was the same as above, with the exception that the phytone peptone and the yeast extract in the batch phase were filter sterilised instead of being autoclaved. This was done in order to avoid autoclaving the yeast extract and phytone, which can potentially affect their composition by heat and denaturation of proteins in the media. For fermentation DCFT26b, the amount of yeast extract and phytone peptone was increased. This was done so that the concentration of yeast extract and peptone in the feed was less than that in DCFT26a and thus easier to make up and filter sterilise. For both fermentations the strategy followed was the same, a 6 h batch phase followed by a 14 h fed-batch phase. Tables 3 and 4 present the media recipes, whereas  FIG.  6    the strategy used for both fermentations. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Media recipe and fed-batch strategy for DCFT26a 
               
            
           
           
               
               
            
               
                   
                 DCFT26a-(short batch-long fed-hatch) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Concentrations  
               
               
                 Component 
                 Batch phase 
                 Feed 
                 at harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phytone Peptone 
                 40  
                 g/L 
                 254 
                 g/L 
                 100  
                 g/L 
               
               
                 Yeast extract 
                 10  
                 g/L 
                 153 
                 g/L 
                 50  
                 g/L 
               
               
                 Glucose 
                 7.5  
                 g/L 
                 17.8 
                 g/L 
                 10 
                 g/L 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Filtered sterilised 
                   
               
               
                 KH 2 PO 4   
                 1.92  
                 g/L 
                   
                   
               
               
                 K 2 HPO 4   
                 1.25  
                 g/L 
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5  
                 g/L 
                   
                   
               
               
                 NaCl 
                 2.5  
                 g/L 
                   
                   
               
               
                 Magnesium 
                 0.08  
                 g/L 
                   
                   
               
               
                 Vitamin solution  
                 10  
                 mL/L 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Volume 
                 3.6  
                 L 
                 1.4 
                 L 
                 ~5 
                 L 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Media recipe and fed-batch strategy for DCFT26b 
               
            
           
           
               
               
            
               
                   
                 DCFT26b-(short batch-long fed-hatch) 
               
            
           
           
               
               
               
               
            
               
                   
                 Batch  
                   
                 Concentrations  
               
               
                 Component 
                 phase 
                 Feed 
                 at harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phytone Peptone 
                 60  
                 g/L 
                 151.4 
                 g/L 
                 100  
                 g/L 
               
               
                 Yeast extract 
                 20  
                 g/L 
                 127.1 
                 g/L 
                 50  
                 g/L 
               
               
                 Glucose 
                 7.5  
                 g/L 
                 17.8 
                 g/L 
                 10 
                 g/L 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Filtered sterilised 
                   
               
               
                 KH 2 PO 4   
                 1.92  
                 g/L 
                   
                   
               
               
                 K 2 HPO 4   
                 1.25  
                 g/L 
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5  
                 g/L 
                   
                   
               
               
                 NaCl 
                 2.5  
                 g/L 
                   
                   
               
               
                 Magnesium 
                 0.08  
                 g/L 
                   
                   
               
               
                 Vitamin solution  
                 10  
                 mL/L 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Volume 
                 3.6 
                 L 
                 1.4 
                 L 
                 ~5 
                 L 
               
               
                   
               
            
           
         
       
     
       FIG.  7    shows the growth curves (OD 600 nm  vs time) from the two fermentations, whereas  FIG.  8    shows the net growth curves (Net OD 600 nm  vs time). 
     The growth curves for DCFT26a and DCFT26b were very similar to that of DCFT24b shown in  FIG.  2   . The cells grew slowly during the fed-batch phase and reached a final net OD 600 nm  of approximately 3.5. 
     SDS PAGE Gels of Fermentation Samples: 
     SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samples was carried out for each of the two fermentations ( FIG.  9    and  FIG.  10   ). In addition, in order to have a better estimate of the amount of collagenases, a semi-quantitative SDS PAGE gel was conducted for the harvest sample point of DCFT26a ( FIG.  11   ) and DCFT26b ( FIG.  12   ). 
     In both fermentations the levels of collagenases were similar to those in DCFT24b ( FIG.  3   ). The semi-quantitative SDS PAGE gel shows that very similar levels to DCFT24b (between 280 mg/L to 300 mg/L total collagenase) were obtained for both DCFT26a and DCFT26b. The harvest point of the DCFT26a fermentation cycle ( FIG.  11   ) were ˜142 mg/L for collagenase I and ˜132 mg/L for collagenase II. The harvest point of the DCFT26b fermentation cycle ( FIG.  12   ) were ˜147 mg/L for collagenase I and ˜158 mg/L for collagenase II. The levels of clostripain, as in the case of DCFT24b, were still high. 
     Study of the Ammonium Sulphate Precipitation Step: 
     The results from these fermentations indicated that although the levels of collagenases were high using the fed-batch strategy, the levels of clostripain were also significantly high. Therefore, a small scale experimental study was set up to investigate the effect of the ammonium sulphate concentration on the recovered amounts of clostripain and collagenases in the precipitated pellet from the filtered fermentation supernatant. 
     In order to evaluate the efficiency of the ammonium sulphate precipitation step, 6×100 mL supernatant samples were harvested from fermentation DCFT26a. These samples were precipitated with 6 different ammonium sulphate concentrations as detailed in the following table. The pellets were re-suspended in 3.3 mL of WFI and dialysed against 100 mM of K 2 HPO 4  (pH 6.7). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Ammonium sulphate concentrations 
               
               
                 that were used to precipitate 100 mL  
               
               
                 supernatant samples from DCFT26a. 
               
            
           
           
               
               
               
            
               
                   
                 Percentage  
                 Ammonium sulphate  
               
               
                   
                 saturation  
                 Concentration (g/L) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                   15% 
                 100 
                 g/L 
               
               
                   
                 22.5% 
                 150 
                 g/L 
               
               
                   
                   30% 
                 200 
                 g/L 
               
               
                   
                 37.5% 
                 250 
                 g/L 
               
               
                   
                   45% 
                 300 
                 g/L 
               
               
                   
                   60% 
                 400 
                 g/L 
               
               
                   
                   
               
            
           
         
       
     
     The post-dialysed samples were then analysed by SDS PAGE analysis. 
       FIG.  13   : post-dialysed harvest point sample precipitated with 15% and 22.5%. 
       FIG.  14   : post-dialysed harvest point sample precipitated with 30% and 37.5%. 
       FIG.  15   : post-dialysed harvest point sample precipitated with 45% and 60%. 
     The gels show that in the case where the ammonium sulphate used was between 15% to 45% saturation, the levels of collagenases in the posy-dialysed samples were very low. The recovery in these cases seemed to be less than 5%. 
     In the case where 60% saturation of ammonium sulphate was used (440 g/L) the levels of collagenases in the post-dialysed sample were very high ( FIG.  15   ). By comparing the intensity of the bands (sample versus references) it can be estimated that approximately 70 mg/L for each of the collagenases were present in the post-dialysed sample. This suggests a recovery of about 50% to 60%, since according to the semi-quantification gel for DCFT26a ( FIG.  11   ) there were approximately 130 mg/L of each of the collagenases in the harvest point sample. 
     Thus, the invention relates to the use of the media recipe (of course, amounts set forth therein are approximated) set forth above in DCFT26b and the use of ammonium sulphate to precipitate collagenase wherein about 400 g/liter of ammonium sulphate is added to the collagenase-containing medium. 
     3 rd  Set of Fed-Batch Fermentations 
     Here the primary aim was to assess the reproducibility of the developed fed-batch strategy. A fed-batch fermentation was performed which was a replicate fermentation of DCFT26b. In addition, the ammonium sulphate/precipitation steps were investigated in more detail compared to the previous small-scale study. More specifically, the aim was to examine the effect of various ammonium sulphate concentrations, from 60% (400 g/L) up to 80% (530 g/L) on the recovery of collagenases and clostripain in the post precipitated/dialysed samples. In addition, two methods of treating the harvested supernatant samples were also assessed, i.e., shifting the pH and oxygenating the media. 
     Growth Curve: 
     The media and fed-batch strategy used was exactly the same as DCFT26b.  FIG.  16    shows the growth curve (OD 600 nm  vs time) and the net growth curve (Net OD 600 nm  vs time) from fermentation. The growth curve was very similar to that of DCFT26b, indicating the good reproducibility of the process. 
     SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samples taken throughout the fermentation indicated that the levels of collagenases and clostripain were very similar to those of DCFT26b (SDS PAGE gel not shown). A semi-quantitative SDS PAGE gel (8% Tris-Glycine gel) was performed for the harvest point sample ( FIG.  17   ). The gel suggests that there is higher than 120 mg/L of each of the collagenases present, similar to the levels observed in the DCFT26b. 
     Ammonium Sulfate Precipitation of Fermentation Harvest Samples: 
     In order to evaluate the efficiency of the ammonium sulphate precipitation step, 7×500 mL supernatant samples were harvested. These were precipitated using the following six methods. 
     In all cases, the pellets were re-suspended in 16.5 L of WFI and dialysed against 100 mM of K 2 HPO 4  (pH 6.7), with the exception of method 4, where the pellet was re-suspended in 16.5 L of 100 mM of K 2 HPO 4  (pH 6) and dialysed against the same buffer. SDS PAGE gels were then performed in order to estimate the amounts of collagenases in the post-dialysed samples and evaluate the recovery of the precipitation/dialysis steps. 
     The methods for precipitation/dialysis followed are the following:
     1 Precipitation with 400 g/L of ammonium sulphate added all at once in the supernatant sample. Dialysis against 100 mM of K 2 HPO 4 , pH 6.7.   2 Precipitation with 400 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample. Dialysis against 100 mM of K 2 HPO 4 , pH 6.7.   3 Precipitation with 400 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample, which was pre-oxygenated. This was done by oxygenating for approximately 10 minutes 500 mL of cell culture harvested from the fermenter. The culture was then filter sterilised. The pellet formed after ammonium sulphate precipitation was dialysed against 100 mM of K 2 HPO 4 , pH 6.7.   4 Precipitation with 400 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample, the pH of which was changed to pH 6 by adding 5N HCl. The pellet formed after was dialysed against 100 mM of K 2 HPO 4 , pH 6.   5 Precipitation with 440 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample. Dialysis against 100 mM of K 2 HPO 4 , pH 6.7.   6 Precipitation with 480 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample. Dialysis against 100 mM of K 2 HPO 4 , pH 6.7.   7 Precipitation with 520 g/L of ammonium sulphate added slowly (about 30 min) into the supernatant sample. Dialysis against 100 mM of K 2 HPO 4 , pH 6.7.   

     The ammonium sulphate did not completely dissolve when added at 480 g/L and 520 g/L in the supernatant samples, whereas it completely dissolved when added at 400 g/L and 440 g/L. 
     The results from the SDS PAGE indicated that the different levels of ammonium sulphate used for the precipitation step (400 g/L, 440 g/L, 480 g/L, 520 g/L) or the other methods used (oxygenation, pH shift) did not seem to have an obvious effect on the amounts of collagenases present in the post dialyzed samples. In all cases, the concentration of each of the collagenases in the post dialyzed samples ranged between 50 mg/L and 60 mg/L.  FIG.  18   a    shows a representative SDS PAGE gel, such as that of the post dialyzed sample precipitated with 400 g/L ammonium sulphate. Since all the gels were very similar the other SDS PAGE gels are not presented in this report. 
     Taking into account the estimated concentrations of collagenases in the harvest point sample ( FIG.  17   ) and in the post dialyzed samples, the recovery of the collagenase after the precipitation/dialysis steps was approximately 50%. In order to investigate whether the value of 50% recovery was accurate, since the error in the estimation of collagenase concentration by SDS gel is in general high, the following SDS PAGE gels were carried.
         An SDS PAGE gel of all the supernatants after centrifugation of the ammonium sulphate precipitated samples ( FIG.  18   a   ). The aim was to assess whether any amount of collagenases is lost into the supernatant.   An SDS PAGE gel in which the harvest point supernatant sample and the post dialysed ammonium sulphate (400 g/L) precipitated sample were appropriately diluted to contain equal amounts of collagenases and loaded on the same gel ( FIG.  19   ).   An SDS PAGE gel in which the harvest point supernatant sample and the post dialysed ammonium sulphate sample (520 g/L) were appropriately diluted to contain equal amounts of collagenases and loaded on the same gel ( FIG.  20   ).       

     It can be seen from  FIG.  18   b    that the amount of collagenases present in the supernatants after centrifugation of the ammonium sulphate precipitated samples was very low. In  FIG.  19    and  FIG.  20    that amount of collagenases after the precipitation/dialysis steps appeared to be very similar to that in the supernatant harvest sample. It was therefore likely that the recovery value that was derived by comparing the semi-quantitative SDS PAGE gels of the supernatant and the post-dialyzed samples was actually higher. 
     Benchmarking Fermentation Experiments with Animal Derived TSB/Proteose. 
     Fermentations of  C. histolyticum  013 and 004 strains in the media containing animal derived components were performed. The aim was to compare strain 013 to strain 004 and evaluate the effect of the animal components on cell growth, collagenase expression and on the levels of contaminants. 
       C. histolyticum  013: 
     The lyophilized strain was re-constituted in PBS and plated out onto TSB/Proteose agar plates (30 g/L TSB, 10 g/L proteose peptone, 12 g/L agar. The plates were incubated in an anaerobic jar in the presence of anaerobic gas packs. Single colonies were picked and used to inoculate 5 mL TSB/Proteose media. After 15 hours of incubation at 37° C. and the OD 600 nm  of the culture was approximately 1.0 unit. 5 mL of culture was then mixed with 1 mL of sterile and stored below −70° C. 
     PBFT58 Fermentations 
     Growth Curves: 
     Two 5 L batch fermentations were carried out, PBFT58c (strain 004) and PBFT58d (strain 013). Table 6 presents the recipe of the TSB/Proteose media used.  FIG.  21    shows the growth curves obtained (Net OD 600 nm  vs time). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Recipe for TSB/Proteose media 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Concentration 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Proteose peptone 
                 50 
                 g/L 
               
               
                   
                 TSB 
                 15  
                 g/L 
               
               
                   
                 MgSO 4  7H 2 O 
                 0.08 
                 g/L 
               
               
                   
                 KH 2 PO 4   
                 1.92 
                 g/L 
               
               
                   
                 Na 2 HPO 4   
                 3.5 
                 g/L 
               
               
                   
                 Vitamin solution 
                 10 
                 mL/L 
               
               
                   
                 (Sterile filtered) 
               
               
                   
                   
               
            
           
         
       
     
     It was seen from  FIG.  21    that the strain 013 grew to a higher OD than strain 004. In both cases however the final OD 600 nm  was higher than 2.5, indicating that the animal derived media supported good growth for both strains. 
     It was noted that strain 013 continued to grow slowly up to the harvest point (20 hours) whereas strain 004 grew up to a net OD 600 nm  of approximately 2.7 and then stopped growing. Compared to the fed-batch fermentations presented previously, using the non-animal derived media, the final OD obtained using the animal derived TSP/Proteose media was lower. 
     SDS Page Analysis: 
     The SDS PAGE gels (8% Tris-Glycine gels) of the supernatant samples taken throughout the fermentations are shown in  FIG.  22    and  FIG.  23   . 
     There did not seem to be any clostripain in the fermentation supernatants, especially in the case of strain 013. This was a very important finding since it could explain the fact that the originator may not have had issues or reduced issues during the purification of collagenases. In contrast, significant problems with degradation of the collagenases had been previously experienced during the purification process. This could be partly attributed to the presence of clostripain in the fermentation. 
     In order to obtain a better estimate of the amount of collagenases present in the fermentations, a semi-quantitative SDS PAGE gel was conduced for the harvest point samples ( FIG.  24    and  FIG.  25   ). The gels suggest that lower amount of collagenases was produced in the batch fermentations with the TSB/Proteose media (PBFT58c) compared to the fed-batch fermentation with the vegetable media (PBFT57). This could be attributed to the fact that higher cell densities were obtained in the latter case (OD 600 nm  ˜4 to OD 600 nm  ˜2.7). Table 7 summarizes the results from the semi-quantitative gels. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Results from semi-quantitative SDS PAGE 
               
               
                 gels for PBFT57 and PBFT58c, d 
               
            
           
           
               
               
               
               
            
               
                   
                 PBFT57  
                 PBFT58c 
                 PBFT58d 
               
               
                   
                 (Animal-free, 
                 (Animal-derived, 
                 (Animal-derived, 
               
               
                   
                 strain) 
                 strain 004) 
                 strain 013) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 AUX I (mg/L) 
                 132 
                 88 
                 59 
               
               
                 AUX II (mg/L) 
                 142 
                 95 
                 95 
               
               
                 Total 
                 274 
                 183 
                 154 
               
               
                   
               
            
           
         
       
     
     Ammonium Sulphate Precipitation of Fermentation Harvest Samples: 
     For each fermentation, 2×500 mL harvest point samples were precipitated with 400 g/L (60%) and 520 g/L (80%) ammonium sulphate. The pellets were re-suspended in 16.5 mL of WFI and dialyzed against 100 mM of K 2 HPO 4  (pH 6.7). SDS PAGE analysis (8% Tris-Glycine gels) of the post-dialyzed samples was then performed ( FIG.  26    and  FIG.  27   ). 
     The results from these gels indicated that the levels of clostripain, even in the very low concentrated post-dialyzed samples (lanes 6 and 7 of  FIGS.  26  and  27   ) were extremely low. This is more evident in the case of strain 013 compared to strain 004. 
     Thus the invention relates to collagenase compositions which are free of clostripain, such as those produced by the fermentation processes described herein. 
     Measurement of Clostripain Activity: 
     In order to investigate further the role of clostripain an enzymatic assay was set up to measure the clostripain activity of post dialyzed samples. The following method was used: 
     Enzymatic Assay of Clostripain: 
     
       
         
         
             
             
         
       
     
     Conditions: T=25° C., pH=7.6 m A 253 nm , Light path=1 cm 
     Method: Continuous Spectrophotometric Rate Determination 
     Unit definition: One unit will hydrolyze 1.0 μmole of BAEE per minute at pH 7.6 at 25° C. in the presence of 2.5 mM DTT. 
     Analysis of Post Dialysed Samples for Clostripain Activity: 
     The clostripain activity assay was used to analyze the post-dialyzed samples from the fermentations with the TSB/Proteose (PBFT58) and the vegetable based fed-batch fermentation (PBFT57). Table 8 summarizes the results. 
     The results demonstrate that there was very low clostripain activity in the case of TSB/Proteose fermentations. In contrast the clostripain activity in the case of the fed-batch PBFT58 was very high. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Enzymatic activities of post-dialyzed samples 
               
            
           
           
               
               
               
               
            
               
                   
                 PBFT57 
                 PBFT58c 
                 PBFT58d 
               
               
                   
                 (Animal-free, 
                 (Animal-derived,  
                 (Animal-derived,  
               
               
                   
                 strain 004) 
                 strain 004) 
                 strain 013) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Clostripain 
                 56.4 * 
                 1.0 * 
                 0.1 * 
               
               
                 activity (U/mL) 
                   
                   
                   
               
               
                 Specific clostripain 
                 205.5 
                 5.5 
                 0.7 
               
               
                 activity (U per mg 
                   
                   
                   
               
               
                 total collagenase) 
               
               
                   
               
               
                 * Clostripain activity determined in the post precipitated/dialyzed sample 
               
            
           
         
       
     
     Investigation of Alternative Peptones 
     Screening Experiments in Shake Flask: 
     The experimental procedure used is described in  FIG.  28   . The media recipes are detailed in Table 9, whereas a list of the peptones used is shown in Table 10. A control shake flask was also conducted, containing phytone peptone. In all cases, 50 g/L of yeast extract and 100 g/L of each peptone were used in an effort to mimic the concentrations of these components at the harvest point of the developed fed-batch fermentation (see Table 4). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Composition of media used in shake  
               
               
                 flask experiment. All media were filter sterilised. 
               
               
                 Vegetable media 
               
            
           
           
               
               
            
               
                 Component 
                 Concentration 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Alternative Peptone 
                 100 
                 g/L 
               
               
                 Yeast extract 
                 50 
                 g/L 
               
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
               
               
                 NaCl 
                 2.5 
                 g/L 
               
               
                 Magnesium 
                 0.08 
                 g/L 
               
               
                 Vitamin solution 
                 10  
                 mL/L 
               
               
                 Glucose 
                 0.9 
                 g/L 
               
               
                   
               
            
           
         
       
     
     The shake flasks were incubated for 18 hours. The cultures were analysed for OD 600 nm  and viable cell counts. The cultures were filtered and the supernatants analysed by SDS PAGE. The results from the OD 600 nm  measurements and viable cell counts are summarized in Table 10. 
     Most of the vegetable peptones resulted in higher net OD values compared to the phytone peptone. However, the OD values did not correlate to the viable cell counts. This could be partly attributed to the variability of the viable cell count method or to the fact that the cells had already started to lyse before the pre-selected harvest point (18 hours). 
     Interestingly, the SDS-PAGE gel indicated that there was no expression of collagenase (gel not shown) in all the flasks, including that of the control (phytone peptone). A possible reason for this could be the fact that the concentrations of the phytone peptone and yeast extract used were very high and as a result the repressed the expression of collagenases. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Results from 1 st  screening experiment 
               
            
           
           
               
               
               
            
               
                   
                 Net OD 600 nm  after  
                   
               
               
                 Type of peptone 
                 18 h growth 
                 CFU/mL 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Phytone peptone (control) 
                 2.65 
                 1.2 × 10 8   
               
               
                 Proteose peptone (vegetable) 
                 2.58 
                 7.4 × 10 8   
               
               
                 Tryptone (vegetable) 
                 3.05 
                 4.8 × 10 8   
               
               
                 Vegetable extract 
                 3.22 
                 1.0 × 10 9   
               
               
                 Vegetable extract 1 
                 3.11 
                 9.6 × 10 9   
               
               
                 Vegetable extract 2 
                 3.05 
                 7.8 × 10 9   
               
               
                 Vegetable hydrolysate 2 
                 3.01 
                 8.4 × 10 9   
               
               
                   
               
            
           
         
       
     
     Fed-Batch Fermentations Using Alternative Peptones—DCFT27a,b 
     Based on information from the previous shake flasks experiments that no expression of collagenases was observed, it was decided to evaluate the alternative peptones using the developed fed-batch strategy. 
     Two fed-batch fermentations were conducted, DCFT27a (vegetable extract 2) and DCFT27b (vegetable hydrolyzate 2). In both fermentations the fed-batch strategy that was developed for the media containing phytone peptone was used. Table 11 describes the media recipes, whereas  FIG.  29    the strategy used. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Media recipe for fed-batch fermentations DCFT27a and DCFT27b 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 DCFT27a,b 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Concentrations  
               
               
                 Component 
                 Batch phase 
                 Feed 
                 at harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Alternative 
                 60 
                 g/L 
                 151.4 
                 g/L 
                 100  
                 g/L 
               
               
                 Peptone 
                   
                   
                   
                   
                   
                   
               
               
                 Yeast extract 
                 20 
                 g/L 
                 127.1 
                 g/L 
                 50  
                 g/L 
               
               
                 Gineose 
                 7.5  
                 g/L 
                 17.8 
                 g/L 
                 10  
                 g/L 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Filtered  
                   
                   
               
               
                   
                   
                   
                 sterilized 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
                   
                   
                   
                   
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
                   
                   
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
                   
                   
                   
                   
               
               
                 NaCl 
                 2.5 
                 g/L 
                   
                   
                   
                   
               
               
                 Magnesium 
                 0.080 
                 g/L 
                   
                   
                   
                   
               
               
                 Vitamin solution 
                 10 
                 mL/L 
                   
                   
                   
                   
               
               
                 Volume 
                 3.6  
                 L 
                 1.4 
                 L 
                 ~5  
                 L 
               
               
                   
               
            
           
         
       
     
     Growth Curves: 
     The growth curves (Net OD 600 nm  vs. time) for DCFT27a and DCFT27b are depicted in  FIG.  30   . In both fermentations, the cells grew to a slightly higher OD 600 nm . compared to the media containing phytone peptone (fermentation PBFT57,  FIG.  16   ). This was in accordance with the viable cell counts (approximately 2×10 9  CFU/mL for DCFT27a,b compared to 1.5×10 9  CFU/mL for PBFT57). 
     SDS Page Gels: 
     As with the shake flask experiments the SDS PAGE analysis indicated that there was no expression of collagenases in both DCFT27a and DCFT27b (gels not shown). 
     This could be attributed to the fact that the media, which consist of high amounts of peptone, supports the expression collagenases when phytone peptone is used, but is too rich when an alternative peptone is used and thus represses the expression of any metabolite, including collagenase and clostripain. It seems that the cells experience luxurious growth conditions in the media containing the alternative peptones and do not need to produce any proteases. 
     Batch Fermentations Using Alternative Peptones—PBFT59a,b,c 
     The results from DCFT27a and DCFT27b fed-batch fermentations, led to further work to investigate three additional alternative peptones, however using lower concentrations than previously used. 
     Three 5 L batch fermentations were conducted, PBFT59a (vegetable tryptone), PBFT59b (vegetable extract) and PBFT59c (vegetable extract no. 1). The fermentations were harvested after 18 hours. 
     All peptones were used at concentrations of 50 g/L in an effort to mimic the concentration of the proteose peptone in the animal media (Proteose/Peptone) and the concentration of phytone peptone that was used previously. The media recipe is shown in Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Media recipe and fermentation strategy  
               
               
                 for 5 L fermentations PBFT59a,b,c 
               
            
           
           
               
               
            
               
                 Component 
                 Concentration 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Alternative Peptone 
                 50 
                 g/L 
               
               
                 Yeast extract 
                 8.5 
                 g/L 
               
               
                 Glucose 
                 5 
                 g/L 
               
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
               
               
                 NaCl 
                 2.5 
                 g/L 
               
               
                 Magnesium 
                 0.08 
                 g/L 
               
               
                 Vitamin solution 
                 10 
                 mL/L 
               
               
                 Volume 
                 5 
                 L 
               
               
                   
               
            
           
         
       
     
     Growth Curves: 
     The growth curves obtained from PBFT59a,b,c fermentations are depicted in  FIG.  31   . In all cases the cells grew to a lower OD 600 nm  (between 1.8 and 2.8) compared to the DCFT27 fed-batch fermentations. This was also in accordance with the viable cell counts (between 0.7×10 9  CFU/mL to 1.2×10 9  CFU/mL for PBFT59a,b,c compared to 2×10 9  CFU/mL for DCFT27a,b). In the media containing tryptone the cells demonstrated the slowest growth rate and achieved the lowest cell density after 18 hours. 
     SDS Page Gels: 
     As for the shake flask experiment and the DCFT27a,b fed-batch fermentations no collagenase expressions was seen in the SDS PAGE gels (gels not shown). 
     These results show that the alternative peptones, although they support the cell growth, they do not allow the expression of collagenases. As suggested before this could be due to the fact that these peptones are very rich in nutrients, e.g., free amino acids, small peptides. 
     4 th  Set of Fed-Batch Fermentations—DCFT27d 
     As the results from the experiments using the alternative vegetable peptones were not successful the next aim of this work was to investigate the possibility of decreasing the levels of clostripain in the developed fed-batch fermentation using the phytone peptone media. As described previously, the clostripain was probably causing the degradation of collagenases during the purification process. 
     A fed-batch fermentation was carried out using the standard phytone peptone media supplemented with three amino acids, i.e., glutamine, tryptophan and asparagine. This fermentation was performed as the concentrations of these particular amino acids were lower in the phytone peptone compared to the animal TSB/Proteose media, based on the amino acid composition of these components, provided by the manufacturers. 
     The aim here was to investigate whether addition of these amino acids could reduce any nutrient limitation that may be a contributing factor for the expression of clostripain. The media recipe is shown in Table 13. The fermentation strategy used was the standard fed-batch strategy used for DCFT26 and PBFT57 fermentations (see  FIG.  6   ). 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Media recipe for fed-batch fermentation DCFT27d 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 DCFT27a,b 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Batch 
                   
                   
                 Concentrations at 
               
            
           
           
               
               
               
               
            
               
                 Component 
                 phase 
                 Feed 
                 harvest point 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Phytone Peptone 
                 80 
                 g/L 
                 151.4 
                 g/L 
                 100 
                 g/L 
               
               
                 Yeast extract 
                 20 
                 g/L 
                 127.1 
                 g/L 
                 50 
                 g/L 
               
               
                 Glucose 
                 7.5 
                 g/L 
                 17.8 
                 g/L 
                 10 
                 g/L 
               
               
                 Amino acids 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Glutamine 
                 2.8 
                 g/L 
                 Filtered 
                   
                   
               
               
                 Tryptophan 
                 0.35 
                 g/L 
                 sterilised 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Asparginine 
                 0.18 
                 g/L 
                   
                   
                   
                   
               
               
                 KH 2 PO 4   
                 1.92 
                 g/L 
                   
                   
                   
                   
               
               
                 K 2 HPO 4   
                 1.25 
                 g/L 
                   
                   
                   
                   
               
               
                 Na 2 HPO 4   
                 3.5 
                 g/L 
                   
                   
                   
                   
               
               
                 NaCl 
                 2.5 
                 g/L 
                   
                   
                   
                   
               
               
                 Magnesium 
                 0.08 
                 g/L 
                   
                   
                   
                   
               
               
                 Vitamin solution 
                 10 
                 mL/L 
                   
                   
                   
                   
               
               
                 Volume 
                 3.6 
                 L  
                 1.4 
                 L 
                 ~5 
                 L 
               
               
                   
               
            
           
         
       
     
     Growth Curve: 
     The growth curve obtained from DCFT27d fermentation is depicted in  FIG.  32   . The growth profile obtained was very similar to that obtained for the standard fed-batch fermentation in the absence of amino acids (DCFT26b and PBFT57) shown previously. SDS PAGE gel. 
       FIG.  33   a    shows the SDS PAGE gel of the supernatant samples taken throughout the fermentation. The level of collagenases is similar to that seen for the standard fed-batch fermentation (see  FIG.  10    for SDS PAGE gel from DCFT26b). Although clostripain is still present in the fermentation, it did seem that its level was lower than that in DCFT26b. 
     In order to investigate this further, the clostripain activity of the post-dialysed harvest point sample was estimated using the clostripain activity assay. In addition, the clostripain activity of the post-dialysed harvest point sample taken from the 20 L lyophilization batch was also estimated. Since this particular batch was purified without showing significant collagenase degradation, knowledge of its clostripain activity would be informative. Table 14 summarizes the enzymatic activities of the post-dialyzed samples. It also includes the enzymatic activities for the standard fed-batch fermentation PBFT57 and the animal TSB/Proteose peptone presented in Table 8, for comparative purposes. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Enzymatic activities of post-dialyzed samples 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 DCFT27d 
                   
                   
               
               
                   
                 PBFT57 
                 Fed-batch 
                   
                 PBFT58c 
               
               
                   
                 Standard fed- 
                 plus amino 
                 20 L Lyo 
                 Animal TSB/ 
               
               
                   
                 batch 
                 acids 
                 batch 
                 Proteose 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Clostripain 
                 56.4 
                 15.2 
                 16.6 
                 1.0 
               
               
                 activity (U/mL) 
                   
                   
                   
                   
               
               
                 Specific 
                   
                   
                   
                   
               
               
                 clostripain 
                   
                   
                   
                   
               
               
                 activity (U per 
                 205.8 
                 55.3 
                 184.4 
                 5.5 
               
               
                 mg total 
                   
                   
                   
                   
               
               
                 collagenase) 
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     The results from DCFT27d indicate that the addition of the amino acids reduces the activity of clostripain produced by the strain. The ratio of clostripain to collagenase is approximately four fold lower in the amino acid supplemented fermentation compared to the control fed-batch fermentation. The ratio of clostripain to collagenase in the animal-derived fermentation was ten fold lower than the amino acid supplemented fed-batch fermentation. It is possible that the reduction of clostripain activity may result in significant reduction on the degradation of collagenases during purification. 
     A series of 5 L fermentations were conducted to assess several fed-batch fermentation strategies. The strategies were assessed based on their yield of collagenase, quantity of contaminants and scalability. Based on these results and optimum fed-batch strategy was identified that resulted in a productivity of total collagenases of approximately 280 mg/L. the fermentation strategy was modified by slightly increasing the batch media concentration and reducing the fed-batch media concentration to improve its scalability. This change to the fermentation strategy had no effect on the productivity or levels of contaminants. 
     The second objective was to optimize the primary recovery step of the collagenases. Optimization of this step involved improvement in the yield of the process step or a reduction in the quantity of contaminants recovered or an increase in scalability. A range of ammonium sulphate concentrations from 100 to 520 g/L were assessed. The effect of lowering the pH to 6.0 and oxygenating the media were also assessed. All ammonium sulphate concentrations below 400 g/L showed very low recoveries of collagenases. No difference in the recovery of collagenase or clostripain was observed in any of the ammonium sulphate concentrations between 400 and 520 g/L. The pellet from the 400 g/L precipitation was the easiest to re-suspend and this concentration was therefore defined as the optimum level. 
     A benchmarking experiment was carried out in order to determine and compare the growth and production of collagenases and clostripain in an animal-derived media with  C. histolyticum  strains 013 and 004. The animal-derived media recipe was taken from the Process I fermentation media, utilizing TSB and protease peptone. This experiment also allowed a comparison of strain 004 grown in animal and non-animal media. The results from SDS-PAGE analysis showed that much lower quantities of clostripain from  C. histolyticum  grown in the animal-derived media. These results were confirmed using an enzymatic assay for clostripain activity. The assay demonstrated a significant reduction in the activity of clostripain in fermentations using the animal-derived media. When the two strains were compared 004 showed a higher clostripain activity than 013. 
     Selections of alternative nitrogen sources were assessed for their ability to replace the Phytone peptone in the fed-batch fermentation strategy. These peptones were Vegetable Extract No. 2 (Sigma, 49869) and Vegetable Hydrolysate No. 2 (Sigma, 07436). The  C. histolyticum  grew extremely well on the vegetable peptones reaching optical densities (600 nm) of 4 to 5 units. SDS-PAGE analysis of these fermentations showed no expression of either collagenase or clostripain. Due to the luxuriant cell growth observed on these peptones it was thought that the concentration of complex nitrogen source was too high resulting in an inhibition of protease expression. A second set of fermentations was therefore carried out using the alternative peptones at 50 g/L in a batch strategy. Vegetable Tryptone (Sigma, 16922) Vegetable Extract (Sigma, 05138) and Vegetable Extract No. 1 (Sigma, 04316) were used as alternative peptones for these experiments. When the fermentations were analyzed by SDS-PAGE no expression of collagenase or clostripain was seen. A fed-batch fermentation using Phytone peptone was supplemented with three amino acids, glutamine, tryptophan and asparagine. These amino acids were identified as being present in lower amounts in the non-animal media. The growth profile of the fermentation was very similar to that of the fed-batch fermentation without amino acid supplementation. SDS-PAGE analysis showed a similar yield of collagenases but a slightly lower level of clostripain. The clostripain assay showed reduced activity in the amino supplemented when compared to the control fed-batch fermentation. The reduction in clostripain activity whilst still significant was not as great as the difference between animal and non-animal media. 
     Materials and Methods: 
     Inoculum Media for Fermentations Using Vegetable Media 
     Throughout this development work the following recipes for the inoculum media were used. 
     
       
         
           
               
            
               
                   
               
               
                 Inoculum media-Vegetable 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Concentration 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Vegetable Peptone 
                 50 
                 g/L 
               
               
                   
                 Yeast extract 
                 8.5 
                 g/L 
               
               
                   
                 Glucose 
                 0.9 
                 g/L 
               
               
                   
                 KH 2 PO 4   
                 1.92 
                 g/L 
               
               
                   
                 K 2 HPO 4   
                 1.25 
                 g/L 
               
               
                   
                 Na 2 HPO 4   
                 3.5 
                 g/L 
               
               
                   
                 NaCl 
                 2.5 
                 g/L 
               
               
                   
                 Magnesium 
                 0.08 
                 g/L 
               
               
                   
                 Vitamin solution 
                 10  
                 mL/L 
               
               
                   
                   
               
               
                   
                 The media was filter sterilized 
               
            
           
         
       
     
     Inoculation Procedure 
     A vial from the internal cell bank was thawed and 0.025 mL was used to inoculate 5 mL of the inoculum media in a 30 mL universal. The 5 mL culture was incubated at 37° C. in an anaerobic jar in the presence of anaerobic gas generators. After approximately 13 to 15 hours of incubation, 4 mL of the culture was used to inoculate 200 mL of the inoculum media in a 500 mL flask. As previously the flask was placed in an anaerobic jar in the presence of anaerobic gas generators. After approximately 13 to 15 hours of incubation at 37° C. and 75 rpm, the whole content of the flask was used to inoculate the fermenter. 
     The pH and the temperature of the fermenters were controlled at 7.0 and 37° C., respectively. The nitrogen flow rate was set at 1 L/min (˜0.2 vvm) and the stirrer speed at 100 rpm. The fermenter was sampled at regular time intervals for OD 600 nm  measurements and viable cell counts. Samples were filtered through a 0.22 μm filter. The filtrates were stored at −20° C. and were frozen at −20° C. for SDS PAGE analysis.  FIG.  33   b    depicts a schematic diagram the inoculation procedure. 
     A preferred recipe for the fed-batch fermentation is set forth below. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Component 
                 Quantity 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Batch Phase 
                   
                   
               
               
                   
                 KH 2 PO 4   
                 2.91 
                 g/L 
               
               
                   
                 K 2 HPO 4   
                 1.89 
                 g/L 
               
               
                   
                 Na 2 HPO 4   
                 5.30 
                 g/L 
               
               
                   
                 NaCl 
                 3.79 
                 g/L 
               
               
                   
                 Phytone 
                 64.45 
                 g/L 
               
               
                   
                 Bacto Yeast Extract 
                 21.80 
                 g/L 
               
               
                   
                 MgSO 4   
                 0.12 
                 g/L 
               
               
                   
                 FeSO 4  × 7H 2 O 
                 18.18 
                 mg/L 
               
               
                   
                 Riboflavin 
                 0.758 
                 mg/L 
               
               
                   
                 Niacin 
                 1.52 
                 mg/L 
               
               
                   
                 Calcium Pantothenate 
                 1.52 
                 mg/L 
               
               
                   
                 Pimelic acid 
                 1.52 
                 mg/L 
               
               
                   
                 Pyridoxine 
                 1.52 
                 mg/L 
               
               
                   
                 Thiamine 
                 1.52 
                 mg/L 
               
               
                   
                 Volume for 5 L fermentation 
                 3.3 
                 L 
               
               
                   
                   
               
               
                   
                 Fed-batch Phase 
                   
                   
               
               
                   
                 Glucose 
                 17.86 
                 g/L 
               
               
                   
                 Phytone 
                 151.43 
                 g/L 
               
               
                   
                 Bacto Yeast Extract 
                 127.14 
                 g/L 
               
               
                   
                 Volume for 5 L fermentation 
                 1.4 
                 L 
               
               
                   
                   
               
            
           
         
       
     
     It is also desirable to scale-up the fermentation process further without detracting from the quality or yields of the collagenase products, thus the invention further relates to an approximately 200 liter fed batch process as described in the flow chart in  FIG.  33     c.    
     Viable Cell Counting Method 
     Samples taken from the shake flasks were diluted by a factor of 10 −4  to 10 −7  and plated out onto TB agar plates. Plates were incubated at 37° C. for approximately 48 hours in a Genbox Jar. An Anaerobic Gas Generator Pack was used in order to create anaerobic conditions within the Jar. The number of colonies was then counted. 
     Ammonium Sulphate Precipitation: 
     Materials: Sorvall Evolution Centrifuge 
     Chemicals: Ammonium Sulphate, GPR Grade (BDH) 
     Supernatant samples (100 mL to 500 mL) were filtered through a 0.22 μm filter. Depending on the experiment various amount of ammonium sulphate were added (from 15% to 80% saturation). The solution was mixed slowly in a magnetic stirrer for approximately 15 minutes, until all the ammonium sulphate had dissolved. It was then held without mixing for ˜3.5 hours at +2-8° C. following the hold step, significant amount of precipitate was formed. The solution was then centrifuged at 7,200×g 20 minutes at 4° C. The supernatant was decanted and the pellet stored at −20° C. 
     Dialysis 
     Materials: 10 kDa MWCO SnakeSkin Dialysis Tubing (68100, Pierce) 
     Magnetic Stirrer 
     Chemicals: Potassium Dihydrogen Orthophosphate AnalaR (BDH) 
     Water for Injection (WFI) 
     The pellets obtained from a 100 mL ammonium sulphate sample were re-suspended in 3.3 mL of WFI. The re-constituted pellet was transferred into a pre-wetted 10 kDA MWCO SnakeSkin dialysis tubing and dialyzed against 100 mM of K 2 HPO 4  (pH 6.7) for ˜12 to 16 hours at 2-8° C. The WFI was then changed and dialysis continued for 2 to 4 hours. The dialyzed material was recovered and the volume determined. The post-dialyzed sample was stored at −20° C. 
     SDS-PAGE Analysis (8% Tris-Glycine Gels) 
     Materials: Xcell SureLock Mini-Cell 
     Chemicals: 
     SDS-PAGE Standards High Molecular Weight (161-0303, Bio Rad) 
     Novex 8% Tris-Glycine gels, 1.5 mm, 10 well (EC6018Box, Invitrogen)
 
Novex 8% Tris-Glycine gels, 1.5 mm, 15 well (EC60185Box, Invitrogen)
 
     Novex Tris-Glycine Running Buffer (10×) (LC2675, Invitrogen) 
     Novex Tris-Glycine Sample Buffer (2×) (LC2676, Invitrogen) 
     NuPAGE Sample Reducing Agent (10×) (NP0009, Invitrogen) 
     Collodial Blue Staining kit (LC6025, Invitrogen) 
     Ethylenediaminetetra-acetic acid disodium salt Analar R (BDH) 
     Samples were prepared for reducing SDS PAGE by adding 10 μl of sample to 10 μl sample Buffer (2×), 2.5 μl reducing agent (10×) and 2 μl of 0.1M EDTA (to achieve final concentration of 10 mM). The high molecular weight (BMW) marker was prepared by adding 10 μl of concentrated stock to 80 μl reducing agent (10×), 310 μl WFI and 400 μl sample buffer (2×). The diluted HMW standard was then heated at 95° C. for 5 minutes before aliquoting and storage at −20° C. for use in subsequent gels. Samples (150) containing collagenases were run directly (i.e. with no prior heat treatment) on 8% Tris-Glycine gels using Tris-Glycine running buffer at 130V for ˜1 hour 50 mins. After electrophoresis, the gels were stained with colloidal blue stain reagent as per the manufacturer&#39;s instructions. 
     Purification Process 
     Method Summary for 5 L Process of Purification: 
     
         
         Step 1. Ammonium sulphate precipitation of culture media supernatant (secreted protein)
       Reconstitution and dialysis into 0.1M potassium phosphate, 0.1M arginine pH6.7   
     
         Step 2. Hydroxyapatite chromatography (in presence of 200 μM leupeptin) Elute with 0-100% gradient of 0.264M potassium phosphate pH6.7 over 4 CV Pool 2 late-eluting peaks where A 280 &gt;A 260 , load straight onto TMAE 
         Step 3. Fractogel TMAE ion exchange (in presence of 200 μM leupeptin) Nucleic acid removal (a Pall MUSTANG Q filter can also be used) Collect and pool unbound flowthrough 
         Step 4. Dialysis into 10 mM Tris pH8.0 
         Step 5. Q Sepharose HP ion exchange (in presence of 200 μM leupeptin)
       Separates AUXI from AUXII   Elute with 0-40% gradient of 10 mM Tris, 3 mM CaCl 2 , 350 mM NaCl pH8.0 over 20 CV   2 peaks collected: Peak 1=AUXII
           Peak 2=AUXI   
           Arginine added to 0.1M to AUXI and AUXII containing fractions   
     
         Step 6. AUXI and AUXII pools concentrated by pressurized stirred-cell 
         Step 7. Superdex 75 Gel Filtration
       Removal of clostripain and gelatinase from AUXI and AUXII   AUXI and AUXII run individually on separate columns   Samples loaded at 5% CV   Buffer: 10 mM Tris, 3 mM CaCl 2 , 150 mM NaCl, 0.1M Arginine pH8   
     
         Step 8. The AUXI and AUXII are pooled and concentrated individually, diafiltered into water and then pooled to for the final drug product.
 
Column details:
 
       
    
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Column specifications for 5 L process 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Volume 
                   
                 Bed height 
                   
                 Plates/ 
               
               
                 Media 
                 (mL) 
                 Column 
                 (cm) 
                 Asymmetry 
                 meter 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 HA 
                 300 
                 XK50/30 
                 15 
                 1.85 
                 9227 
               
               
                 Fractogel 
                 58 
                 XK26/40 
                 10 
                 1.02 
                 5368 
               
               
                 TMAE  
                   
                   
                   
                   
                   
               
               
                 Q 
                 100 
                 XK50/20 
                 5 
                 1.35 
                 19,367 
               
               
                 Sepharose 
                   
                   
                   
                   
                   
               
               
                 Superdex 
                 880 
                 XK50/60 
                 45 
                 1.24 
                 18,644 
               
               
                 75-1 
                   
                   
                   
                   
                   
               
               
                 Superdex 
                 880 
                 XK-50/60 
                 45 
                 1.85 
                 13,576 
               
               
                 75-2 
                   
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     Column Packing 
     
         
         
           
             Columns were packed as manufacturer&#39;s instructions where possible 
             TMAE column—no issues were encountered 
             Q Sepharose and Superdex 75—difficulties were encountered in packing to correct pressure due to the size of the column. However, the columns could be run at the recommended pressure 
             HA—packed as a 50% slurry and run at 10 mL/min.
 
Yields/Recoveries from 5 L Process:
 
           
         
       
    
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Purification from AS ppt to Q-Sepharose IEX 
               
               
                 Chromatography step yields in    bold     
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Protein 
                   
                   
                 Total 
                 Step 
               
               
                 Process 
                 Concentration 
                   
                 Volume 
                 Protein 
                 Yield 
               
               
                 Step 
                 (mg/mL) 
                 Method 
                 (g) 
                 (mg) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Post AS ppt 
                 1.12 
                 Bradford 
                 346.45 
                 388.02 
                 — 
               
               
                 and dialysis 
                   
                   
                   
                   
                   
               
               
                 Pre HA 
                 1.08 
                 Bradford 
                 359.85 
                 388.64 
                 — 
               
               
                 (post-leupeptin 
                   
                   
                   
                   
                   
               
               
                 addition) 
                   
                   
                   
                   
                   
               
               
                 Post HA 
                 0.51 
                 Bradford 
                 646.85 
                 329.89 
                 
                   
                     84.88 
                   
                 
               
               
                 Pre-TMAE 
                 0.51 
                 Bradford 
                 646.85 
                 329.89 
                 — 
               
               
                 Post-TMAE 
                 0.51 
                 UV 
                 647.2 
                 330.07 
                 
                   
                     100.05 
                   
                 
               
               
                 Post dialysis 
                 0.404 
                 UV 
                 715.0 
                 288.86 
                  87.51 
               
               
                 Pre-IEX 
                 0.388 
                 UV 
                 744.0 
                 288.67 
                 — 
               
               
                 Post IEX 
                 0.454 
                 UV 
                 188.1 
                 85.40 
                 
                     
                   
                     29.58 
                   
                 
               
               
                 
                   
                     ABC I 
                   
                 
                   
                   
                   
                   
                   
               
               
                 (peak 2) 
                   
                   
                   
                   
                   
               
               
                 Post IEX 
                 0.536 
                 UV 
                 220.7 
                 118.29 
                 
                     
                   
                     40.98 
                   
                 
               
               
                 
                   
                     ABC II 
                   
                 
                   
                   
                   
                   
                   
               
               
                 (peak 1) 
                   
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Purification from Q-Sepharose IEX to post Superdex 75 GPC. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Protein 
                   
                   
                 Total 
                 Step 
               
               
                 Process 
                 Concentration 
                   
                 Volume 
                 Protein 
                 Yield 
               
               
                 Step 
                 (mg/mL) 
                 Method 
                 (g) 
                 (mg) 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Pre-stirred 
                 0.454 
                 UV 
                 188.1 
                 85.40 
                 — 
               
               
                 cell 
                   
                   
                   
                   
                   
               
               
                 ABC I 
                   
                   
                   
                   
                   
               
               
                 Post-stirred 
                 1.901 
                 UV 
                 41.6 
                 79.08 
                  92.6 
               
               
                 cell 
                   
                   
                   
                   
                   
               
               
                 ABC I 
                   
                   
                   
                   
                   
               
               
                 Pre GPC 
                 1.901 
                 UV 
                 40.6 
                 77.18 
                 — 
               
               
                 ABC I 
                   
                   
                   
                   
                   
               
               
                 Post GPC 
                 
                   1.12 
                 
                 UV 
                 
                   60.0 
                 
                 
                   67.2 
                 
                 
                     
                   
                     87.07 
                   
                 
               
               
                 ABC I 
                   
                   
                   
                   
                   
               
               
                 Pre-stirred 
                 0.536 
                 UV 
                 220.7 
                 118.29 
                 — 
               
               
                 cell 
                   
                   
                   
                   
                   
               
               
                 ABC II 
                   
                   
                   
                   
                   
               
               
                 Post-stirred 
                 2.76 
                 UV 
                 45.5 
                 125.58 
                 106.16 
               
               
                 cell 
                   
                   
                   
                   
                   
               
               
                 ABC II 
                   
                   
                   
                   
                   
               
               
                 Pre GPC 
                 2.46 
                 UV 
                 44.0 
                 108.24 
                 — 
               
               
                 ABC II 
                   
                   
                   
                   
                   
               
               
                 
                   Post GPC 
                 
                 
                   1.192 
                 
                 UV 
                 
                   59.3 
                 
                 
                   70.68 
                 
                 
                     
                   
                     65.3 
                   
                 
               
               
                 ABC II 
               
               
                   
               
            
           
         
       
     
     Yields from a 5 L process are approximately 60-75 mg each of ABCI and ABCII 
     For the scale up, depend on fermentation, yields of 250-300 mg for 20 L and 2500-3000 mg for 200 L could be expected. 
     Individual Chromatography Steps of 5 L Scale Process: 
     Hydroxyapatite Chromatography 
     
         
         Column size: 2×300 mL in XK50/30 (15 cm bed height each) 
         Buffer A: 0.1M potassium phosphate, 200 μM leupeptin, pH6.7 
         Buffer B: 0.264M potassium phosphate, 200 μM leupeptin, pH6.7 
         Sample: ˜350 mL (in 0.1M potassium phosphate, 0.1M Arginine pH6.7) loaded at &lt;1.0 mg/mL media* 
         Flow rate: 9.8 mL/min 
         Elution: 0-100% B over 4 CV 
       
    
       FIG.  34    shows a chromatogram after hydroxyapatite with a loading of 1.0 mg/L media, wherein a considerable loss of resolution and target degradation occurs. 
     Fractogel TMAE Anion Exchange 
     
         
         Column size: 58 mL in XK26/60 (10 cm bed height) 
         Buffer A: 10 mM potassium Phosphate, 0.2M NaCl, 200 μM leupeptin, pH6.7 
         Buffer B: 10 mM potassium Phosphate, 2M NaCl, pH6.7 
         Sample: ˜650 mL @ 0.5 mg/mL (in Potassium Phosphate pH6.7, straight from HA column) loaded at ˜5.5 mg/mL media 
         Flow rate: 8.8 mL/min 
         Elution: (100% B to elute nucleic acid) 
       
    
       FIG.  35    illustrates a chromatogram after Fractogel TMAE anion exchange. The unbound fraction pooled to give ˜650 mL at 0.5 mg/mL. Dialysed into 10 mM Tris at pH8. 
       FIG.  36    shows a SDS-PAGE gel of Pre HA, Post HA and Post TMAE material from 5 L scale process. The gel is stained with Colloidal blue. 
     Q Sepharose HP Anion Exchange with Original Elution Gradient
     Column size: 100 mL in XK50/20 (5.0 cm bed height)   Buffer A: 10 mM Tris, 3 mM CaCl 2 , 200 μM leupeptin, pH8.0   Buffer B: 10 mM Tris, 3 mM CaCl 2 , 360 mM NaCl, 200 μM leupeptin, pH8.0   Sample: —650 mL at 0.5 mg/mL (in 10 mM Tris pH8.0+200 μM leupeptin) loaded at ˜3.0 mg/mL media   Flow rate: 18.0 mL/min   Elution: 0-40% B over 20 CV   

       FIG.  37    illustrates a chromatogram after Q Sepharose HP anion exchange with original elution gradient. Arginine is added to 0.1M to ABCI and ABCII containing fraction. Peak1 fraction (ABCII) pooled to give ˜220 mL at 0.55 mg/mL which was concentrated by stirred-cell to give ˜45 mL at 2.8 mg/mL. Peak 2 fractions (ABCI, excluding gelatinase shoulder) pooled to give ˜190 mL at 0.45 mg/mL, which was concentrated by stirred-cell to give ˜42 mL at 2 mg/mL. 
       FIG.  38    shows a SDS-PAGE gel of Q Sepharose IEX chromatography of post TMAE material run in the presence of leupeptin for Peak 1 (ABCII). The gel is stained with Colloidal blue. 
       FIG.  39    shows a SDS-PAGE gel of Q Sepharose IEX chromatography of post TMAE material run in the presence of leupeptin for Peak 2 (ABCI). The gel is stained with Colloidal blue. 
     Q Sepharose HP Anion Exchange with Modified Gradient 
     Small scale test of NaCl addition to Buffer A and using a steeper/faster gradient. Sample was from a 1/3 5 L process, post TMAE, previously froze (−20° C.).
     Column size: 1 mL   Buffer A: 10 mM Tris, 30 mM NaCl, 3 mM CaCl 2 , 200 μM leupeptin, pH8.0   Buffer B: 10 mM Tris, 3 mM CaCl 2 , 360 mM NaCl, 200 μM leupeptin, pH8.0   Sample: 3 mg post TMAE, post dialysis into 10 mM Tris, 30 mM NaCl, 200 μM leupeptin, pH8.0. Loaded at 3.0 mg/mL media   Gradient: 0-25% B over 2CV, 25% B for 2CV, 25-40% B over 7.5CV   

       FIG.  40    illustrates a chromatogram after Q Sepharose HP anion exchange with modified elution gradient. Good separation of ABCI and ABCII is observed. The second part of the gradient can be made steeper to sharpen ABCI peak. Improvement of the peak can also be made using 5 mL CV loaded at 3 and 10 mg/mL media. 
     Superdex 75 Gel Permeation Chromatography of ABCII (Peak 1 from IEX)
     Column size: 880 mL in XK50/60 (54 cm bed height)   Buffer: 10 mM Tris, 3 mM CaCl 2 , 150 mM NaCl, 0.1M arginine, pH8.0   Sample: ˜44 mL (5% CV) at 2.5 mg/mL (in 10 mM Tris, 3 mM CaCl 2 , ˜60 mM NaCl, 0.1M arginine, pH8.0   Flow rate: 8.8 mL/min   

       FIG.  41    illustrates a chromatogram after superdex 75 gel permeation chromatography of ABCII (Peak 1 from IEX). Peak pooled to give ˜60 mL ABC II at 1.2 mg/mL. 
       FIG.  42    shows a SDS-PAGE gel of superdex 75 gel permeation chromatography of concentrated ABC II run in the presence of arginine. The gel is stained with Colloidal blue. 
     Superdex 75 Gel Permeation Chromatography of ABCI (Peak 2 from IEX)
     Column size: 880 mL in XK50/60 (54 cm bed height)   Buffer: 10 mM Tris, 3 mM CaCl 2 , 150 mM NaCl, 0.1M arginine, pH8.0   Sample: ˜42 mL (5% CV) at 2.0 mg/mL (in 10 mM Tris, 3 mM CaCl 2 ), ˜60 mM NaCl, 0.1M arginine, pH8.0   Flow rate: 8.8 mL/min   

       FIG.  43    illustrates a chromatogram after superdex 75 gel permeation chromatography of ABCI (Peak 2 from IEX). Peak pooled to give ˜60 mL ABC I at 1.1 mg/mL. 
       FIG.  44    shows a SDS-PAGE gel of superdex 75 gel permeation chromatography of concentrated ABC I run in the presence of arginine. The gel is stained with Colloidal blue. 
     Scale Up Column Sizing 
       
     
       
         
           
               
               
               
             
               
                 TABLE 18 
               
               
                   
               
             
            
               
                   
                 Hydroxyapatite 
                 Fractogel TMAE 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Process 
                 Media 
                 Column 
                 Bed 
                 Media 
                 Column 
                 Bed 
               
               
                 Scale 
                 volume 
                 type 
                 height 
                 volume 
                 type 
                 height 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1/3 
                 220 
                 mL 
                 XK50/30 
                 ~11 cm 
                 18 
                 mL 
                 XK16/20 
                 ~9 
                 cm 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 5 L 
                 2 ×  
                 XK50/30 
                 ~15 cm 
                 54 
                 mL 
                 XK26/40 
                 ~10 
                 cm 
               
               
                   
                 300 mL 
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 L 
                 2.4 L 
                 * 
                 * 
                 216 
                 mL 
                 XK50/20 
                 ~11 
                 cm 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 (at 1 
                   
                   
                   
                   
                   
                   
               
               
                   
                 mg/mL 
                   
                   
                   
                   
                   
                   
               
               
                   
                 load) 
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 200 L 
                 24 
                 L 
                 * 
                 * 
                 2.2 
                 L 
                 * 
                 * 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Q Sepharose HP 
                 Superdex 75 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Process 
                 Media 
                 Column 
                 Bed 
                 Media 
                 Column 
                 Bed 
               
               
                 Scale 
                 volume 
                 type 
                 height 
                 volume 
                 type 
                 height 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1/3 
                 65 
                 mL 
                 XK26/20 
                 ~12 cm 
                 300 
                 mL 
                 XK26/70 
                 ~57 cm 
               
               
                 5 L 
                 100 
                 mL 
                 XK50/20 
                  ~5 cm 
                 880 
                 mL 
                 XK50/60 
                 ~45 cm 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 400 mL 
                   
                   
                   
                   
                   
                   
               
               
                 20 L 
                 (at 3  
                 * 
                 * 
                 4 
                 L 
                 * 
                 * 
               
               
                   
                 mg/mL 
                   
                   
                   
                   
                   
                   
               
               
                   
                 load) 
                   
                   
                   
                   
                   
                   
               
               
                 200 L 
                 4 L 
                 * 
                 * 
                 40 
                 L 
                 * 
                 * 
               
               
                   
                 (at 
                   
                   
                   
                   
                   
                   
               
               
                   
                 3 mg/mL 
                   
                   
                   
                   
                   
                   
               
               
                   
                 load) 
               
               
                   
               
               
                 * Column type and resulting bed height to be further optimized. 
               
               
                 Media volumes are linear scale up from 5 L scale. 
               
            
           
         
       
     
     In yet other embodiments of the invention, the dialysis steps of the purification process described above can be substituted with ultrafiltration/diafiltration (UF/DF) operations using dialysis and stirred cells will be replaced by TFF, tangential flow filtration. The TMAE step discussed above is optional. 
     The invention includes the collagenase products that are produced by (or can be produced by) the above purification processes. Such collagenase products possess exceptional high degrees of purity and retained enzymatic activity. For example, the compositions are free of clostripain (e.g., possess negligible or undetectable levels of clostripain). 
     Optimization of the Manufacturing Process: 
     In order to support clinical studies and provide a commercial-scale process, optimization of the manufacturing process earlier developed was completed. The process changes are described briefly below, and are outlined in Table 19. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Summary of Process Changes between BTC (Process 1) and Auxilium Supplies 
               
               
                 (Process 2 and 3) 
               
            
           
           
               
               
               
               
               
            
               
                 Stage 
                   
                 Process 1 
                 Process 2 
                 Process 3 
               
               
                   
               
               
                 Fementation and 
                   
                   
                   
                   
               
               
                 Primary Recovery 
                   
                   
                   
                   
               
               
                   
                 Cell line 
                 013 and 004 
                 004 
                 004 
               
               
                   
                 Cell line storage form 
                 Lyophilized 
                 Frozen liquid culture 
                 Frozen liquid culture 
               
               
                   
                 Cell bank medium 
                 Bovine-derived 
                 Non-animal derived 
                 Non-animal derived 
               
               
                   
                 Seed medium 
                 Bovine-derived 
                 Non-animal derived 
                 Proteose peptone (porine-derived) 
               
               
                   
                 Seed scale up strategy 
                 1 WCB vial 
                 2 WCB vials  
                 1 WCB vials  
               
               
                   
                   
                 →1 × 500 mL boils 
                 → 2 × 30 mL → 
                 → 3 × 25 mL → 
               
               
                   
                   
                 →45 L fermentor 
                 2 × 500 mL flasks → 
                 4 × 200 mL flasks →  
               
               
                   
                   
                   
                 1 × 20 L fermentor 
                 1 × 20 L fermentor  
               
               
                   
                   
                   
                   
                 → 200 L fermentor 
               
               
                   
                 Production medium 
                 Bovine-derived 
                 Non animal-derived 
                 Proteose peptone (porcine-derived) 
               
               
                   
                 Production medium  
                 Autoclaved 
                 Sartoclear maxicap and 
                 In situ media sterilization and 0.2 
               
               
                   
                 sterilization 
                   
                 0.2 μm liters 
                 micron filtration 
               
               
                   
                 Production strategy 
                 Batch 
                 Fed-batch 
                 Batch 
               
               
                   
                 Production scale 
                 45 L 
                 20 L 
                 200 L 
               
               
                   
                 Harvest method 
                 10 μm and 1 μm  
                 Millipore Millistak HC  
                 Millipore Millistak HC Pod filter 
               
               
                   
                   
                 filter train 
                 Pod 
                   
               
               
                   
                 Ammonium Sulfate 
                 95% saturation 
                 60% saturation 
                 Capture proteins using Phenyl 
               
               
                   
                 precipitation (AS ppt) 
                   
                   
                 Sepharose FF Low Substitution 
               
               
                   
                   
                   
                   
                 chromatography 
               
               
                   
                 Resuspended AS ppt  
                 Dialysis 
                 Dialysis 
                 N/A 
               
               
                   
                 buffer exchange 
                   
                   
                   
               
               
                   
                 Temperature control 
                 None 
                 2-8° C. solutions 
                 2-8° C. solutions 
               
               
                 Purification 
                   
                   
                   
                   
               
               
                   
                 New 
                 NA 
                 Mustang  O filter   
                 Mustang  Q filter   
               
               
                   
                 chromatography/filitration 
                   
                   
                   
               
               
                   
                 step 
                   
                   
                   
               
               
                   
                 New chromatography step 
                 NA 
                 Superdex 75 GPC 
                 Elimination of hydroxyapatite (HA) 
               
               
                   
                   
                   
                   
                 and GPC 
               
               
                   
                 HA and QQ HP buffer  
                 minus leupeptin 
                 200 μM leupeptin 
                 200 μM leupeptin 
               
               
                   
                 systems 
                   
                   
                   
               
               
                   
                 Temperature control 
                 None 
                 2-8° C. buffers and  
                 2-8° C. buffers and column packings 
               
               
                   
                   
                   
                 column packings 
                   
               
               
                   
                 Buffer exchange Pre-Q HP 
                 Dialysis 
                 Dialysis 
                 TFF 
               
               
                   
                 Buffer exchange Post-Q HP 
                 Dialysis 
                 Dialysis 
                 NA 
               
               
                   
                 Concentrate/diafilter into final 
                 Dialysis 
                 Dialysis 
                 TFF 
               
               
                   
                 formulation 
                   
                   
                   
               
               
                   
                 Scale-Up all steps for 200 L 
                 NA 
                 NA 
                 4.5 times 
               
               
                   
                 fermentation 
                   
                   
                   
               
               
                 Formulation 
                 Formulation of Drug 
                 DS in WFI dilute 
                 DS in dilute  
                 DS in 10 mM Tris, 60 mM Sucrose, 
               
               
                   
                 Substrance (DS) 
                 w/lactose 
                 w/lactose 
                 pH 8.0 
               
               
                   
               
            
           
         
       
     
     Fermentation Optimization 
     Removal of the bovine-derived raw materials from the original cell bank and fermentation process was carried out. Strain 004 of  Clostridium histolyticum  was propagated for use as the master cell bank based on passage viability required for scale-up. The specifications and analytical results for the master cell bank are captured in Table 20. In order to increase biomass and production of collagenase, a fed-batch fermentation strategy was developed utilizing animal-free raw materials in growth medium at a 20 Liter fermentation scale. Further fermentation scale-up to 200 Liter was observed to require the use of a porcine-derived media component (i.e., Proteose Peptone #3, infra) to assure consistent cell growth, collagenase expression, and an improved impurity profile. Subsequent changes were made to increase the yield and purity of collagenase over the downstream process. These changes include the addition of new separation and filtration strategies, as well as scale-up of the production equipment to support the 200 Liter batch fermentation scale.  FIG.  45    depicts a flow chart of the fermentation process 3. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Analytical Specifications and Test Results for Master Cell Bank 
               
            
           
           
               
               
               
            
               
                 Test 
                 Specification 
                 Result 
               
               
                   
               
               
                 Identity 
                 Expected profile,  
                 Expected profile, 99.9% ID 
               
               
                   
                 &gt;95% ID 
                   
               
               
                 Viable Count 
                 ≥1 × 10 6  cfu/mL on 
                 1.3 × 10 7  cfu/mL on  
               
               
                   
                 TB agar 
                 TB agar 
               
               
                 Purity Test 
                 No extraneous organisms 
                 No extraneous organisms 
               
               
                   
                 observed 
                 observed 
               
               
                 Colony Morphology  
                 Irregular shape, 1-2 mm 
                 Irregular, flat elevation, 
               
               
                 (anaerobic at 37° C.) 
                 in size, grey to white 
                 undulate margin 1-2 mm 
               
               
                   
                 color 
                 diameter (48 hr), grey, white 
               
               
                   
                   
                 color 
               
               
                 Gram Stain 
                 Gram positive rods 
                 Gram positive rods 
               
               
                 Phenol Red Dextrose  
                 Negative 
                 Negative 
               
               
                 fermentation 
                   
                   
               
               
                 Hydrogen Sulfide production 
                 Positive 
                 Positive 
               
               
                 Gelatinase Test 
                 Positive 
                 Positive 
               
               
                 Spore Test 
                 Negative 
                 Negative on all media 
               
               
                 Growth in cooked meat media 
                 Positive 
                 Positive 
               
               
                 Growth in thioglycollate media 
                 Growth as finger-like 
                 Growth as finger-like 
               
               
                   
                 projection 
                 projection 
               
               
                 Motility Test (MIO media) 
                 Non-motile 
                 Non-motile 
               
               
                 Bacteriophage 
                 None detected 
                 No confirmed evidence of 
               
               
                   
                   
                 phage 
               
               
                   
               
            
           
         
       
     
     Primary Recovery and Purification Optimization 
     Further development to optimize the primary recovery and downstream purification process is being undertaken. Substitution of the ammonium sulfate precipitation with phenyl sepharose fast flow low sub column chromatography to capture the collagenases has been implemented to improve yields, eliminate the use of bulk ammonium sulphate and improve aseptic processing. 
     With regards to purification, the Pall MUSTANG Q filter has been implemented for residual DNA and impurity clearance to further enhance yields and simplify the production process train and validation requirements. The Quaternary Amine Sepharose high Performance (Q HP) operating parameters have been optimized to eliminate the Gel Permeation Chromatography (GPC) step. In addition to the process changes cited above, the drug substance formulation has been modified to include 10 mM Tris, 60 mM Sucrose, pH 8.0, improving both product solubility and drug substance and drug product stability. 
     The optimization process took place in two stages. The initial process (Process 2) utilizes an animal-free medium for all cell banking and fermentation stages with the fed-batch fermentation performed at the 20 Liter scale. The downstream process has been adapted from Process 1 to include MUSTANG Q filtration for residual DNA removal and Superdex 75 GPC for additional host cell contaminant clearance. Leupeptin has also been added to the chromatography buffer systems to prevent proteolytic degradation. Process 2 material has been bridged analytically with Process 1 material (Table 21A), and was tested in a side-by-side pre-clinical study outlined herein. 
     Process 2 material has been proposed for use in early stage of Phase 3 clinical program, The specifications for Process 2 intermediates and drug substance are detailed in Tables 22 and 23 respectively. Further process, formulation and lyophilization development provided an optimized manufacturing process (Process 3). These changes include the addition of new separation and filtration strategies, as well as scale-up of the production equipment to support the 200 Liter batch fermentation scale as outlined in Table 19.  FIG.  46    depicts a flow chart of the purification for process 3. Declaration of dose: The initial in vitro potency assay was a bovine collagenase assay and did not differentiate collagenase types I and II. This assay was utilized for the material used in the open label, DUPY101 and DUPY 202 clinical studies only, with the 0.58 mg dose typically resulting in a potency of 10,000 Units. Analysis of Process 1 material utilizing the current separate in vitro potency assays for type I collagenase and 43,000 to 69,000 Units/dose (0.58 mg dose) for type II collagenase. Analysis of Process 2 material utilizing the current in vitro potency assays has confirmed that similar relative potency values compared to Process 1 material are typically achieved. 
     Demonstration of analytical comparability between Process 1 and Process 2: In order to support the changes between Process 1 and Process 2, comparability data have been submitted in the form of release testing and analytical characterization. These data are presented in Table 21. 
     Comparison of the intermediates, described as AUX-I and AUX-II, and drug substance from the previous process (Process 1; Reference) with a process of the invention (Process 2). This analytical comparison shows that material manufactured from Process 2 is comparable to that made with Process 1 (Table 21). In particular, the identity, potency and purity between these materials are comparable. 
     The purity level of Process 2 intermediates is shown in  FIG.  47   , a reduced SDS-PAGE Coomasie stained gel, The gel shows a single band for each intermediate with no other minor bands evident. AUX-I has an apparent MW of 115 kDa and compares with the reference (ABC I), while AUX-II has an apparent MW of 110 kDa and compares with the reference (ABC II).  FIG.  48    shows a reduced SDS-PAGE Coomasie stained gel depicting drug substance. As with intermediates, drug substance manufactured by Process 2 compares with the reference (Process 1). A silver stained SDS-PAGE gel is depicted in  FIG.  49    further substantiating the high purity level of the Process 2 drug substance. In summary, the release testing and analytical characterization for the intermediates (AUX-I and AUX-II) and drug substance manufactured using Process 2 clearly demonstrated comparability with Process 1 (Reference) materials. Additionally, further release testing was performed on Process 2 material and is listed in Table 21B. In conclusion, the direct analytical comparison between Process 1 and Process 2 materials (Table 21), and the further intermediate and release testing (Table 22) indicate that Process 2 material is suitable for use in the human studies. Table 23 and 24 further list the analytical specifications resulting from Process 2 manufacturing process. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Analytical comparability between (Process 1) and Auxilium (Process 2) 
               
               
                 intermediates and drug substance. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Intermediate 
                 Intermediate 
                 Drug 
                 Drug Substance 
               
               
                 Test 
                 AUX-I 
                 AUX-II 
                 Substance 
                 Specification 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Identity by SDS- 
                 Conforms to 
                 Conforms to 
                 Conforms 
                 Major 
                 Major 
               
               
                 PAGE 
                 reference (see 
                 reference (see 
                 to 
                 collagenase 
                 collagenase 
               
               
                   
                 attached) 
                 attached) 
                 reference 
                 band 
                 band 
               
               
                   
                   
                   
                 (see 
                 between 100- 
                 between 107- 
               
               
                   
                   
                   
                 attached) 
                 115 kDa; 
                 110 kDa; 
               
               
                   
                   
                   
                   
                 no minor 
                 no minor 
               
               
                   
                   
                   
                   
                 bands 
                 bands 
               
            
           
           
               
               
               
               
               
            
               
                 Rat Tail Tendon 
                 2310  
                 — 
                 2866 
                 1700-3500 units/mg 
               
            
           
           
               
               
               
               
               
               
            
               
                 Collagen Assay for 
                 units/mg 
                   
                 units/mg 
                   
                   
               
               
                 Potency (AUX-I) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Process 1 Reference 
                 2704  
                 — 
                 2018 
                 * 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 units/mg 
                   
                 units/mg 
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Potency for Class II 
                 — 
                 179704 
                 50955 
                 43000-69000 units/mg 
               
            
           
           
               
               
               
               
               
               
            
               
                 Collagenases 
                   
                 units/mg 
                 units/mg 
                   
                   
               
               
                 (AUX-II) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Process 1 Reference 
                 — 
                 174045 
                 58491 
                 * 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 units/mg 
                 units/mg 
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Analysis of proteins 
                 100% main 
                 100% main 
                 100% main 
                 ≥99% main peak; 
               
               
                 using the Agilent 1100 
                 peak; 0% 
                 peak; 0% 
                 peak; 0% 
                 ≤1% aggregates by area 
               
               
                 HPLC System (Purity 
                 aggregates 
                 aggregates 
                 aggregates 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 and aggregation by 
                   
                   
                   
                   
                   
               
               
                 size exclusion 
                   
                   
                   
                   
                   
               
               
                 chromatography) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Process 1 Reference 
                 87% main 
                 90% main 
                 Intermediates 
                 * 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 peak; 13% 
                 peak; 10% 
                 used** 
                   
                   
               
               
                   
                 aggregates 
                 agnregates 
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Analysis of proteins 
                 99% AUX-I; 
                 100% AUX-II 
                 100% 
                 2 major peaks (AUX I &amp; 
               
               
                 using the Agilent 1100 
                 1% AUX-II 
                   
                 AUX-I and 
                 AUX II), combined ≥97% 
               
               
                 HPLC System 
                   
                   
                 AUX-II 
                 by area; Retention times of 
               
               
                 (Identity and purity by 
                   
                   
                   
                 AUX-I and AUX-II within 
               
               
                 reverse phase liquid 
                   
                   
                   
                 5% of reference 
               
            
           
           
               
               
               
               
               
               
            
               
                 chromatography) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Process 1 Reference 
                 89.4% ABC-I; 
                 93% ABC-II; 
                 Intermediates 
                 * 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 5.4% ABC-II; 
                 0.5% ABC-I; 
                 used** 
                   
                   
               
               
                   
                 5.2% other 
                 6.5% other 
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Analysis of proteins 
                 &lt;1% 
                 &lt;1% 
                 &lt;1% 
                 &lt;1% by area 
               
            
           
           
               
               
               
               
               
               
            
               
                 using the Agilent 1100 
                   
                   
                   
                   
                   
               
               
                 HPLC System 
                   
                   
                   
                   
                   
               
               
                 (Gelatinase by anion 
                   
                   
                   
                   
                   
               
               
                 exchange 
                   
                   
                   
                   
                   
               
               
                 chromatography) 
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Process 1 Reference 
                 &lt;1% 
                 &lt;1% 
                 &lt;1% 
                 * 
               
               
                 Peptide Mapping by 
                 Peak pattern 
                 N/A 
                 Peak 
                 Conforms to reference 
               
            
           
           
               
               
               
               
               
               
            
               
                 Tryptic Digest and 
                 conforms to 
                   
                 pattern 
                   
                   
               
               
                 Reverse Phase HPLC 
                 Reference 
                   
                 conforms 
                   
                   
               
               
                   
                   
                   
                 to 
                   
                   
               
               
                   
                   
                   
                 Reference 
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 N- &amp; C-terminal 
                 Sequence 
                 Sequence 
                 Not 
                 Conforms is to reference 
               
               
                 sequencing 
                 identical to 
                 identical to 
                 required 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Reference*** 
                 Reference 
                   
                   
                   
               
               
                   
               
               
                 *Process 1 preliminary specifications not included here 
               
               
                 **Drug Substance not available for these tests, limited supplies on hand 
               
               
                 ***N-terminal sequencing completed for AUX-I (identical to reference), but further development required for the AUX-II as N-terminus appears to be blocked. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Analytical results for Process 2 intermediates and drug substance 
               
            
           
           
               
               
               
               
            
               
                   
                 Intermediate 
                 Intermediate  
                   
               
               
                 Test 
                 AUX-I 
                 AUX-II 
                 Drug Substance 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 pH of Solution 
                 Not required 
                 Not required 
                 6.8 
               
               
                 Protein Concentration  
                 Not required 
                 1.54 mg/mL 
                 Not required 
               
               
                 by Bradford Assay 
                   
                   
                   
               
               
                 Total Protein by 
                 1.36 mg/mL 
                 1.39 mg/mL 
                 1.41 mg/mL 
               
               
                 Absorbance 
                   
                   
                   
               
               
                 Spectrophotometry 
                   
                   
                   
               
               
                 Residual Host Protein 
                 Not required 
                 Not required 
                 Band pattern  
               
               
                   
                   
                   
                 similar 
               
               
                   
                   
                   
                 to Reference 
               
               
                 Residual Host DNA 
                 Residual  
                 Not required 
                 2.9 ng/mL* 
               
               
                   
                 Host DNA 
                   
                   
               
               
                 Endotoin 
                 Not required 
                 Not required 
                 8.7 EU/mg 
               
               
                 Residual Leupeptin 
                 Not required 
                 Not required 
                 &lt;1 μg/mL 
               
               
                   
               
               
                 *Result is at the LOQ of the previous residual DNA method 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Analytical Specifications for Process 2 AUX-I and AUX-II Intermediates 
               
            
           
           
               
               
            
               
                   
                 Specification 
               
            
           
           
               
               
               
            
               
                 Test 
                 AUX-I 
                 AUX-II 
               
               
                   
               
               
                 Appearance 
                 Clear colorless and  
                 Clear colorless and  
               
               
                   
                 free from  
                 free from  
               
               
                   
                 particulate matter 
                 particulate matter 
               
               
                 *Endotoxin 
                 &lt;10 EU/mL  
                 &lt;10 EU/mL  
               
               
                 Identity (and purity) by SDS- 
                 Major band between  
                 Major band between  
               
               
                 PAGE (Reduced conditions, 
                 110-115 kDa, and  
                 107-110 kDa, and  
               
               
                 Coomasie and silver stained) 
                 no minor bands 
                 no minor bands 
               
               
                 *Total Protein by  
                 1.0-1.5 mg/mL 
                 1.0-1.5 mg/mL 
               
               
                 Absorbance Spectroscopy 
                   
                   
               
               
                 SRC assay (AUX-I) 
                 1900-3300 units/mg 
                 Not applicable 
               
               
                 GPA assay (AUX-II) 
                 Not applicable 
                 4300-6400 units/mg 
               
               
                 Analysis of Proteins using 
                 ≥99% main peak;  
                 ≥99% main peak;  
               
               
                 the Agilent 1100 HPLC 
                 ~1% aggregates by  
                 ≤1% aggregates by  
               
               
                 System (Aggregation by 
                 area 
                 area 
               
               
                 size exclusion  
                   
                   
               
               
                 chromatography) 
                   
                   
               
               
                 *Analysis of Proteins using  
                 ≥97% by area  
                 ≥97% by area  
               
               
                 the Agilent 1100 HPLC  
                   
                   
               
               
                 System (Purity by reverse  
                   
                   
               
               
                 phase liquid chromatography) 
                   
                   
               
               
                 Analysis of Proteins using the 
                 &lt;1% by area  
                 &lt;1% by area  
               
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Residual gelatinase by anion 
                   
                   
               
               
                 exchange chromatography) 
                   
                   
               
               
                 Analysis of Proteins using the 
                 &lt;1% by area  
                 &lt;1% by area  
               
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Residual clostripain by 
                   
                   
               
               
                 reverse phase liquid 
                   
                   
               
               
                 chromatography) 
                   
                   
               
               
                 Identity by Peptide Mapping 
                 Conforms to  
                 Conforms to  
               
               
                   
                 reference 
                 reference 
               
               
                 Bioburden 
                 &lt;100 CFU/mL  
                 &lt;100 CFU/mL 
               
               
                   
               
               
                 *Tests required for provisional release of intermediates for further manufacturing 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Analytical Specifications for Process 2 Drug Substance 
               
            
           
           
               
               
            
               
                   
                 Specification 
               
            
           
           
               
               
               
            
               
                 Test 
                 AUX-I 
                 AUX-II 
               
               
                   
               
            
           
           
               
               
            
               
                 Appearance 
                 Clear colorless and essentially free from 
               
               
                   
                 particulate matter 
               
               
                 Potentiometric Measure  
                 6.0 to 7.0  
               
               
                 of pH of Solution 
                   
               
               
                 Endotoxin 
                 &lt;10 EU/mL  
               
            
           
           
               
               
               
            
               
                 Identity (and purity) by 
                 Major collagenase  
                 Major collagenase  
               
               
                 SDS-PAGE (Reduced 
                 band between 100-  
                 band between 107-  
               
               
                 conditions, Coomasie and 
                 115 kDa; no minor  
                 110 kDa; no minor  
               
               
                 silver stained) 
                 bonds 
                 bands 
               
            
           
           
               
               
            
               
                 *Total Protein by  
                 1.1-1.5 mg/mL  
               
               
                 Absorbance Spectroscopy 
                   
               
            
           
           
               
               
               
            
               
                 *SRC assay (AUX-I) 
                 1700-3500 units/mg 
                 NA 
               
               
                 *GPA assay (AUX-II) 
                 NA 
                 43000-69000  
               
               
                   
                   
                 units/mg 
               
            
           
           
               
               
            
               
                 Residual host cell protein 
                 &lt;10 ppm 
               
               
                 Residual host cell DNA 
                 &lt;10 pg/dose 
               
               
                 Analysis of Proteins using  
                 ≥99% main peak; ≤1% aggregates by area  
               
               
                 the Agilent 1100 HPLC  
                   
               
               
                 System (Aggregation by  
                   
               
               
                 size exclusion  
                   
               
               
                 chromatography) 
                   
               
               
                 *Analysis of Proteins using 
                 2 major peaks (AUX I &amp; AUX II), combined 
               
               
                 the Agilent 1100 HPLC 
                 ≥97% by area; Retention times of AUX-I  
               
               
                 System (Identity and purity 
                 and AUX-II within 5% of AA4500 reference 
               
               
                 by reverse phase liquid 
                   
               
               
                 chromatography) 
                   
               
               
                 Analysis of Proteins using 
                 &lt;1% by area 
               
               
                 the Agilent 1100 HPLC 
                   
               
               
                 System (Residual  
                   
               
               
                 clostripain by reverse phase 
                   
               
               
                 liquid chromatography 
                   
               
               
                 Analysis of Proteins using 
                 &lt;1% by area 
               
               
                 the Agilent 1100 HPLC 
                   
               
               
                 System (Residual  
                   
               
               
                 gelatinase by anion  
                   
               
               
                 exchange  
                   
               
               
                 chromatography) 
                   
               
               
                 Residual leupeptin by 
                 &lt;1% by area 
               
               
                 reverse phase 
                   
               
               
                 chromatography 
                   
               
               
                 *Bio burden 
                 &lt;1 CFU/mL 
               
               
                   
               
               
                 *Tests required for provisional release of Drug Substance for further manufacturing 
               
            
           
         
       
     
     DETAILED EXPERIMENTAL FOR PROCESS 3 
     Process 3 Fermentation: 
     The fermentation process using Phytone peptone employed during Process 2 had shown significant variability during both supplies for DSP development and GMP manufacture. 
     During previous work an animal derived Proteose Peptone had been shown to support the growth of  C. histolyticum  very well. The animal derived Proteose Peptone culture produced significantly less clostripain than observed during Process 2 and expressed AUXI and AUXII at a 1:1 ratio. As a result a regulatory acceptable animal derived peptone, Proteose Peptone #3 from Becton Dickinson (PP3), was evaluated in 5 L fermenters. Initial comparison to the existing Phytone based process (Process 2) showed that using the PP3 at 50 g/L generated a high biomass concentration with a rapid exponential growth rate. The fermentation resulted in a higher product yield of &gt;350 mg/L total collagenase opposed to ˜230 mg/L from Process 2 (by semi quantitative SDS-PAGE analysis). Further fermentations using PP3 demonstrated that significantly less clostripain was produced using the animal derived fermentation medium. The first three fermentations (using one batch of PP3) demonstrated very consistent growth profiles. When the product was analysed by SDS-PAGE the yield and purity of collagenase was found to be very reproducible between the three fermentations. 
     To supply DSP with material for process development several fermentations were conducted using PP3. For this supply material three different batches of PP3 were used. It was noted that when two of these batches were used the growth profiles of the cultivations were not consistent with previous PP3 fermentations and demonstrated variability in the growth profile between fermentations. A small scale investigation showed that batch to batch variability in the PP3 caused this variation. The small scale study also demonstrated that an increase in the PP3 concentration to 100 g/L would prevent this variation. 
     Two 5 L fermentations were conducted with 100 g/L PP3 using two batches of the peptone, one that resulted in the typical growth profile and one which did not (as demonstrated during the small scale experiment). The experiment showed that the increase in concentration ensured that the two fermentations with different batches of PP3 were reproducible. The growth profiles were highly similar and the product was expressed at a similar yield and purity. 
     The optimized fermentation process utilizing 100 g/L PP3 was finally scaled to 200 L. The 200 L growth profile was very similar to that seen at 5 L scale. SDS-PAGE analysis of the fermentation filtrate showed a high yield from the 200 L fermentation, —320 mg/L total collagenase (by quantitative densitometry analysis). The purity of the collagenase product (post fermentation) was similar at both 5 L and 200 L scale. 20 L of the 200 L fermentation filtrate was processed by the DSP group to represent a partial scale-up for the downstream process (infra). 
     The Proteose Peptone #3 fermentation process (Process 3) generated collagenase with a higher yield and with less clostripain than the existing Phytone process. At 100 g/L PP3 was shown to yield  C. histolyticum  cultivations with reproducible growth curves despite using various batches of PP3. Both the yield and purity of collagenase were also shown to be reproducible when using various lots of PP3. 
     Evaluation of Proteose Peptone #3 as a Raw Material for Production of Collagenase from  Clostridium histolyticum.    
     Due to the variability observed in fermentations utilizing Phytone peptone as a complex nitrogen source the suitability of Proteose Peptone #3 (Becton Dickinson, 212230) (PP3) was evaluated in 5 L fermentations. A simple batch strategy with 50 g/L PP3 was used. The exact medium composition can be found in the materials and methods section. 
       FIG.  51    compares the growth curve of the 50 g/L PP3 (a lower concentration than the Phytone concentration in Process 2) fermentation to the Phytone fed-batch fermentation. The PP3 cultivation demonstrates a very rapid specific growth rate during exponential growth before entering stationary phase approximately 8 hours after inoculation. The PP3 fermentation reached a maximum optical density (600 nm) of 4.7 units. The culture was left for a further 12 hours in stationary phase to monitor product formation/degradation. 
       FIG.  52    shows SDS-PAGE semi-quantitative analysis of the concentration of the collagenase products from the 20 hour point of the PP3 cultivation.  FIG.  53    shows the same analysis for the Phytone fed-batch process. It can be observed that the PP3 fermentation generates more product than the Phytone based process (an increase from 230 mg/L to 360 mg/L total collagenase, based on the semi-quantitative analysis in  FIGS.  52  and  53   ). The PP3 culture also expressed AUXI and AUXII at a 1:1 ratio, whereas the Process 2 produced the two proteins at a 1:1:6 ratio. 
     Reproducibility of Proteose Peptone #3 Batch Fermentation. 
     The reproducibility of the PP3 batch process was further examined using lot #5354796 of Proteose Peptone #3. All three runs illustrated in  FIG.  54    demonstrate consistent growth profiles with a maximal optical density (600 nm) of approximately 4.5 obtained after 8 hours. 
     Semi-quantitative SDS-PAGE analysis of the harvest points of the fermentation showed that yield of total collagenase to be ˜350-400 mg/L. 
     The harvest point of the fermentation was also evaluated during this study. The fermentations were harvested at 8, 11 and 20 hours.  FIGS.  55  and  56    show SDS-PAGE analysis of the time course of PP3 fermentation GCFT05d (harvested at 11 hours). The gel depicted in  FIG.  55    has been stained with colloidal blue and the gel in  FIG.  56    has been silver stained. A third higher molecular weight band can be observed above the two collagenase bands on the gels in  FIGS.  55  and  56   . It is thought that this band corresponds to an AUXI precursor protein reported in the literature. The precursor band is present during the exponential growth phase. At the end of exponential growth the precursor band decreases in intensity and is not present after 11 hours (in GCFT05d). The main lower molecular weight contaminants can be seen on the silver stained gel at approximately 90, 60, 55, 45 and 40 kDa. It must be noted that these contaminants are present at a low level and are only clearly detected on the silver stained gel. The optimal harvest point for the fermentation was determined to be ˜11 hours at this stage of development.  FIG.  57    shows SDS-PAGE analysis of samples from the time course of a standard Phytone fed-batch fermentation. A 40 kDa contaminant can be observed on the gel in  FIG.  57   . This 40 kDa contaminant band from the Phytone fed-batch process was identified as the protease clostripain. By comparing the gels in  FIGS.  55  and  57    it is possible to determine that the quantity of clostripain produced using the PP3 fermentation process is significantly lower than the Phytone based fermentation. 
     Generation of Supply Material for Downstream Process Development 
     To support downstream process (DSP) development several fermentations were conducted using 50 g/L PP3. During these fermentations two different lots of PP3 were used (U.S. Pat. Nos. 5,332,398 and 5,325,635).  FIG.  58    depicts the growth curves of these fermentations (shown in diamond) compared to a fermentation (shown in square) using lot #5354796 (GCFT05d). The fermentations with the new batches of PP3 display highly varied growth profiles. Although the initial growth rates of the cultures are all very similar, the point at which they enter stationary phase and therefore the maximum biomass concentrations differ considerably. The optical densities (600 nm) in the inoculum cultures showed very little variation (OD600 of 5 mL stage; 2.9-3.6 units, OD600 of 200 mL stage; 4.5-5.9 units) and no reduction from previous inocula using PP3 lot #5354796. The variation and reduced optical density (600 nm) only manifested itself in the final (fermentation) stage of the cultivation. This suggests that reason for the variation was a nutrient limitation in the PP3 and the quantity of the limiting nutrient varied between batches of PP3. 
     Although these fermentations were successfully used for DSP development and SDS-PAGE analysis showed that there was not a huge variation in the quantity of collagenase produced (350-400 mg/L total collagenase based on semi-quantitative SDS-PAGE analysis, data not shown) it was decided that it was still critical to investigate the reason for the variation. The variation in the growth profile would make it very difficult to predict a harvest point of the fermentation. There were also concerns that nutrient limitation may induce expression of other proteases as seen with the Phytone fed-batch process and specifically the protease, clostripain. 
     Investigation into the Variation Between Batches of Proteose Peptone #3. 
     Initial work with PP3 had demonstrated a highly robust process with a higher product yield and lower levels of the protease clostripain. When new batches of PP3 were employed it was observed that the process robustness decreased significantly with highly variable growth profiles. A shake flask experiment was conducted to directly compare the three batches of PP3 used so far (lots 5354796, 5325635 and 5332398). The experiment replicated the two stage inoculum process from the 5 L process but replaced the final fermentation phase with another 200 mL culture. Having this third stage was critical, as the variation was only observed in the final fermentation stage of the process in previous experiments. The optical densities (600 nm) of the cultures were measured at each transfer stage and the cultures were used to inoculate the next stage. Media was prepared using the three batches of PP3 at 50 g/L. One of the two batches that had resulted in lower biomass concentrations of  C. histolyticum  during 5 L experiments (lot #5332398) was also prepared at 100 g/L. 
       FIG.  59    shows the results from the small scale experiment. It can be observed that lot 5325635 and 5332398 showed reduced optical densities (600 nm) in the third stage of approximately 2.5 units, these were deemed to be “poor” batches of PP3. Lot 5354796 maintains an optical density (600 nm) of 5 units in the third stage of cultivation, this was deemed to be a “good” batch of PP3. Interestingly when the concentration of a “poor” batch of PP3 (5332398) was increased to 100 g/L the same optical density (600 nm) was achieved in the second and third stage of the cultivation. This data does support the theory that the deviations in growth profiles are caused by variation in the quantity of a limiting nutrient between batches of PP3. It was not possible to identify this nutrient by analytical testing of the batches of PP3. 
     Evaluation of Proteose Peptone #3 at 100 g/L in 5 L Fermentation 
     The results of the small scale study demonstrated that increasing the concentration of PP3 from 50 to 100 g/L removed the issue of batch to batch variability. This process change was tested at 5 L scale using a “good” and “poor” batch of PP3 (lot 5354796 and 5325635, respectively) as determined during the small scale investigation into PP3 variability.  FIG.  60    shows the growth profiles of the two fermentations. The two cultures show identical specific growth rates during the exponential phase. The fermentation enter stationary phase and reach very similar maximal optical densities (600 nm) of approximately 6.5 units. This data demonstrates that increasing the concentration of PP3 alleviates the issue of batch to batch variability of the PP3. Due to the higher biomass concentration achieved and longer exponential phase in the fermentation harvest point was extended to 12 hours. 
       FIGS.  61  and  62    show SDS-PAGE analysis of the two fermentations utilising 100 g/L PP3. The gels demonstrate consistent expression of collagenase in both fermentations. The samples from both fermentations appear to contain similar levels of contaminant described in  FIG.  56   , although PBFT70d appears contain slightly more of the 40 kDa band (clostripain). It is possible that these small differences are due to staining or loading differences. Again the quantity of clostripain produced using the PP3 process is significantly lower than the Phytone process. The precursor band appears to persist longer into the time course of the fermentation. It was recommended that future fermentations at 100 g/L should be extended to a 14 hour harvest. 
     The presence of the precursor band highlights the importance of the harvest point definition and its qualification during process validation. 
       FIG.  63    displays data from densitometry analysis of the gel in  FIG.  61   . The chart compares product and precursor formation (densitometry peak area) to cell growth (OD600). Product formation appears to be consistent with cell growth and the rate of production decreases as the cultivation enters stationary phase. The precursor band decreases in intensity as exponential growth ends but is still present at the harvest point of the fermentation. 
     Scale-Up of 100 g/L Proteose Peptone #3 Fermentation to 200 L. 
     Following the increase in the PP3 concentration to 100 g/L the process was scaled to 200 L. To generate the required quantity of inoculum for the 200 L vessel a third inoculum stage was introduced using a 15 L working volume fermenter. 3×200 mL cultures were used to inoculate the 15 L fermenter and following 12 hours of growth 8 L of the 15 L were inoculated into the 200 L vessel.  FIG.  64    compares the growth curve of the 200 L fermentation to the two 5 L fermentation using 100 g/L PP3. As recommended the growth profile was extended to 14 hours to ensure that the precursor band had completely disappeared before processing began. The growth profile of the 200 L fermentation is very similar to the fermentation at 5 L scale, demonstrating successful scale up of the cultivation. 
       FIG.  65    shows SDS-PAGE analysis of the time course of the 200 L fermentation. The gel shows product formation during the course of the fermentation. The material at the 14 hour harvest point contains no detectable pre-cursor and very low levels of contaminants. The product generated from the 200 L fermentation appears very similar to that produced from the 5 L process, indicating that the increased generation number of the 200 L process has not had a detrimental effect.  FIG.  66    displays data from densitometry analysis of the gel in  FIG.  64   . The chart compares product and precursor formation (densitometry peak area) to cell growth (OD 600 ). Product formation appears to be consistent with cell growth and the rate of production decreases as the cultivation enters stationary phase. The precursor band decreases in intensity as exponential growth ends. The precursor band decreases in intensity more rapidly in the 200 L fermentation than the 5 L cultivation, PBFT70c ( FIG.  63   ).  FIG.  67    shows SDS-PAGE analysis using a 4-12% Bis-Tris gel on the 200 L fermentation time course. The approximate molecular weights of the detected contaminants are annotated on the gel. 
     The harvest process (clarification by filtration) developed for Process 2 was evaluated during the 200 L scale up fermentation. The cell culture was successfully clarified using the existing process with no blockage of the filter train. The harvest process is described in the materials and methods section. 20 L of filtrate from the 200 L fermentation was processed by DSP to demonstrate a partial scale up of the downstream Process 3 (infra). 
     Quantification of Product Yield by Densitometry Analysis 
     A more accurate and quantifiable method was required to determine product concentration during the upstream process step than the semi-quantitative SDS-PAGE analysis ( FIGS.  62  and  63   ). The fermentation filtrate has a high quantity of pigment and peptides from the growth medium that makes standard protein quantification techniques such as UV and the Bradford assay unusable. The semi-quantitative analysis carried out previously was modified and updated by carrying out densitometry analysis of the Coomassie stained gels. The method involved loading a range of quantities (0.2-1.2 μg/lane) of mixed AUXI and AUXII reference material and dilutions of the sample to be quantified onto a Tris Glycine gel. The scanned image was then analysed and the peak area for estimated for the standards and the samples. A standard curve was then constructed (total collagenase) and used to quantify the amount of total collagenase in the sample dilutions.  FIG.  68    shows an example of a collagenase standard curve and highlights the linearity of the quantification method within the anticipated range of the samples. The Tris Glycine gels did not completely resolve AUXI and AUXII therefore the total collagenase was quantified rather than attempting to separately quantitate the two proteins. 
     The quantity of collagenase was analysed for PBFT70c, PBFT70d and the 200 L scale-up fermentations. The quantity was found to be ˜280-350 mg/L total collagenase for all three fermentations. 
     Materials and Methods 
     Media Preparation: 
     IL Media Preparation 
     The phosphates for the inoculum preparation (table 25) were autoclaved in a IL bottle at 121° C. for 20 minutes. The bulk media (table 26) was initially heated in a microwave to 60° C. to fully dissolve components before autoclaving in a 1 L bottle at 121° C. for 20 minutes. The PSA 1 (table 27) was filtered through a 0.2 μm Sartopore 2 150 cm 2  filter into a 250 mL sterile bottle. The 300 mL autoclaved phosphates, 600 mL autoclaved bulk media and 100 mL sterile filtered PSA 1 were pooled before aliquoting into 30 mL gamma irradiated universals (8×5 mL) and 500 mL Erlenmeyer flasks (4×200 mL). 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 Phosphate composition for inoculum preparation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
               
                   
                 KH 2 PO 
                 1.92 g 
               
               
                   
                 K 2 HPO 
                 1.25 g 
               
               
                   
                 Na 2 HPO2 
                  3.5 g 
               
               
                   
                 NaCl 
                  2.5 g 
               
               
                   
                 Deionised Water 
                 Up to 300 mL 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 Bulk medium composition for inoculum preparation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
               
                   
                 Proteose Peptone # 3 
                 50 g of 100 g* 
               
               
                   
                 Yeast Extract 
                 8.5 g 
               
               
                   
                 Deionised Water 
                 Up to 600 mL 
               
               
                   
                   
               
               
                   
                 *Medium recipe includes PP3 at 50 and 100 g/L. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 PSA I Magnesium/Glucose composition for inoculum preparation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MgSO 4  × 7H 2 O 
                 0.08 
                 g 
               
               
                 Glucose 
                 5 
                 g 
               
               
                 Vitamin solution 
                 10 
                 mL 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 100 mL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 Vitamin solution for inoculum preparation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 FeSO4 × 7H 2 O  
                 1.2 
                 g 
               
               
                   
                 Riboflavin 
                 50 
                 mg 
               
               
                   
                 Niacin 
                 100 
                 mg 
               
               
                   
                 Calcium Pantothenate 
                 100 
                 mg 
               
               
                   
                 Pimelic acid 
                 100 
                 mg 
               
               
                   
                 Pyridoxine 
                 100 
                 mg 
               
               
                   
                 Thiamine 
                 100 
                 mg 
               
            
           
           
               
               
               
            
               
                   
                 Deionised Water 
                 Up to 1 litre 
               
               
                   
                   
               
            
           
         
       
     
     5 L Media Preparation 
     The phosphate solution for the 5 L scale (table 29) was autoclaved in a 1 L bottle at 121° C. for 20 minutes. The bulk medium (table 30) was added directly to the 5 L vessel and autoclaved at 121° C. for 20 minutes. The PSA 1 (table 31) was filtered through a 0.2 μm Sartopore 2 150 cm 2  filter into a 500 mL sterile bottle. The 250 mL phosphate solution and 200 mL PSA 1 was separately pumped into the 5 L vessel on completion of autoclaving and cooling of the vessel. 
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Phosphate composition for 5 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 KH 2 PO 4   
                 9.22 
                 g 
               
               
                 K 2 HPO 4   
                 6 
                 g 
               
               
                 Na 2 HPO 4   
                 16.8 
                 g 
               
               
                 NaCl 
                 12 
                 g 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 250 mL/278.35 g 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Bulk medium composition for 5 L fermentation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
               
                   
                 Proteose Peptone #3 
                 240 g or 480 g* 
               
               
                   
                 Bacto Yeast Extract 
                 40.8 g 
               
               
                   
                 Deionised Water 
                 Up to 4.35 L 
               
               
                   
                   
               
               
                   
                 *Medium recipe includes PP3 at 50 and 100 g/L. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 PSA I Magnesium/Glucose composition for 5 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MgSO 4  × 7H 2 O 
                 0.38 
                 g 
               
               
                 Glucose 
                 24 
                 g 
               
               
                 Vitamin solution 
                 48 
                 mL 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 200 mL/200 g 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Vitamin solution for 5 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 FeSO4 × 7H2O  
                 1.2 
                 g 
               
               
                 Riboflavin 
                 50 
                 mg 
               
               
                 Niacin 
                 100 
                 mg 
               
               
                 Calcium Pantothenate 
                 100 
                 mg 
               
               
                 Pimelic acid 
                 100 
                 mg 
               
               
                 Pyridoxine 
                 100 
                 mg 
               
               
                 Thiamine 
                 100 
                 mg 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 1 litre 
               
               
                   
               
            
           
         
       
     
     15 L Media Preparation 
     The phosphate solution (table 33) was filtered through a 0.2 μm Sartopore 2 300 cm 2  filter into a sterile 2 L bottle. The bulk medium (table 34) was added directly to the 20 L vessel prior to Stream-In-Place (SIP) sterilisation of the vessel. The PSA 1 (table 35) was filtered through a 0.2 μm Sartopore 2 300 cm 2  filter into a 1 L sterile bottle. The 750 mL phosphates and 600 mL PSA 1 were separately pumped into the 20 L vessel on completion of SIP and cooling of the vessel. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Phosphate composition for 15 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 KH 2 PO 4   
                 27.66 
                 g 
               
               
                 K 2 HPO 4   
                 18 
                 g 
               
               
                 Na 2 HPO 4   
                 50.4 
                 g 
               
               
                 NaCl 
                 36 
                 g 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 750 mL/835.05 g 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Bulk medium composition for 15 L fermentation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Proteose Peptone #3 
                 1.44 
                 kg 
               
               
                   
                 Bacto Yeast Extract 
                 122.4 
                 g 
               
            
           
           
               
               
               
            
               
                   
                 Deionised Water 
                 Up to 13.05 L 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 PSA I Magnesium/Glucose composition for 15 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MgSO 4  × 7H 2 O  
                 1.14 
                 g 
               
               
                 Glucose 
                 72 
                 g 
               
               
                 Vitamins solution 
                 144 
                 mL 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 600 mL/600 g 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Vitamin solution for 15 L fermentation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 FeSO4 × 7H2O  
                 1.2 
                 g 
               
               
                   
                 Riboflavin 
                 50 
                 mg 
               
               
                   
                 Niacin 
                 100 
                 mg 
               
               
                   
                 Calcium Pantothenate 
                 100 
                 mg 
               
               
                   
                 Pimelic acid 
                 100 
                 mg 
               
               
                   
                 Pyridoxine 
                 100 
                 mg 
               
               
                   
                 Thiamine 
                 100 
                 mg 
               
            
           
           
               
               
               
            
               
                   
                 Deionised Water 
                 Up to 1 litre 
               
               
                   
                   
               
            
           
         
       
     
     200 L Media Preparation 
     The phosphate solution (table 37) was filtered through a 0.2 μm Sartopore 2 300 cm 2  filter into a Gammasart Biosystem SA10 10 L bag. The bulk media (table 38) was added directly to the 200 L vessel prior to SIP sterilisation of the vessel. The PSA 1 solution (table 39) was filtered through a 0.2 μm Sartopore 2 300 cm 2  filter into a Gammasart Biosystem SA10 10 L bag. The 10 L phosphates and 8 L PSA 1 were separately pumped into the 200 L vessel on completion of SIP and cooling of the vessel. 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Phosphate composition for 200 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 4 × Fermenters 
               
               
                   
               
            
           
           
               
               
               
            
               
                 KH 2 PO 4   
                 368.8 
                 g 
               
               
                 K 2 HPO 4   
                 240 
                 g 
               
               
                 Na 2 HPO 4   
                 672 
                 g 
               
               
                 NaCl 
                 480 
                 g 
               
            
           
           
               
               
            
               
                 Deionised Water 
                 Up to 10 L/11.134 kg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 38 
               
             
            
               
                   
               
               
                 Bulk medium composition for 200 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
               
                 Proteose Peptone #3 
                  19.2 kg 
               
               
                 Bacto Yeast Extract 
                 1.632 kg 
               
               
                 Deionised Water 
                 Up to 174 L 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 PSA 1 Magnesium/Glucose composition for 200 L fermentation 
               
            
           
           
               
               
            
               
                 Component 
                 Quantity Required 
               
               
                   
               
               
                 MgSO 4  × 7H 2 O 
                 15.2 g 
               
               
                 Glucose 
                  960 g 
               
               
                 Vitamins solution 
                 1.92 L 
               
               
                 Deionised Water 
                 Up to 8 L/8 kg 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 Vitamin solution for 200 L fermentation 
               
            
           
           
               
               
               
            
               
                   
                 Component 
                 Quantity Required 
               
               
                   
                   
               
               
                   
                 FeSO4 × 7H2O 
                 2.4 g  
               
               
                   
                 Riboflavin 
                 100 mg 
               
               
                   
                 Niacin 
                 200 mg 
               
               
                   
                 Calcium Pantothenate 
                 200 mg 
               
               
                   
                 Pimelic acid 
                 200 mg 
               
               
                   
                 Pyridoxine 
                 200 mg 
               
               
                   
                 Thiamine 
                 200 mg 
               
               
                   
                 Deionised Wirer 
                 Up to 2 L/2 kg 
               
               
                   
                   
               
            
           
         
       
     
     Fermentation 
       FIG.  69    illustrates overviews of the process flows for Phytone and PP3 fermentation processes at 5 and 200 L scale. 
     5 L Scale Fermentation 
     A vial of WCB (2005#1019D) was thawed and 50 μL aliquots were used to binoculate 8×5 mL of inoculum media in a 30 mL gamma irradiated universals. The 5 mL cultures were incubated at 37° C. in an anaerobic jar in the presence of 3 anaerobic gas packs. After approximately 12 hours of incubation (OD600 3.0-4.0) 2×5 mL cultures were selected and used to inoculate 2×200 mL inoculum media in 500 mL Erlenmeyer flasks. The two flasks were placed together in an anaerobic jar with 3 gas packs and were incubated at 37° C. in a shaking incubator (70 rpm) for 12 hours. After 12 hours of incubation (OD600 6.0-7.0) each 200 mL inoculum was used to inoculate 5 L vessel. 
     The working volume of the 5/7 L vessels FT Applikon vessels was 5 L of which 4% (v/v) was inoculum from the 200 mL stage. The agitation rate was set at 100 rpm. The pH, dO2 and temperature were controlled at 7.00 units, 0% of saturation and 37° C. respectively. The pH was controlled with additions of either HCl (5M) of NaOH (5M). The dO2 concentration was maintained at 0% by continuous sparing of nitrogen, with a flowrate of 1 L/min. Samples were taken during the fermentation and filtered through 0.2 μm filters before storing at −20° C. for analytical purposes. The fermentations began to enter stationary phase at an OD600 of 6.0-7.0. After 12 hours the fermenter was cooled to 10-20° C. before commencing harvest recovery. 
     200 L Scale Fermentation 
     A vial of the WCB (2005#1019D) was thawed and 50 μL aliquots were used to inoculate 8×5 mL of inoculum media in 30 mL gamma irradiated universals. The 5 mL cultures were incubated at 37° C. in an anaerobic jar in the presence of 3 anaerobic gas packs. After approximately 12 hours of incubation (OD600 3.0-4.0), 4×5 mL cultures were selected and used to inoculate 4×200 mL inoculum media in 500 mL Erlenmeyer flasks. Two flasks were placed together in anaerobic gas jars with 3 gas packs and left to incubate at 37° C. in a shaking incubator (70 rpm) for 12 hours. After 12 hours of incubation (OD600 6.0-7.0) three of the four flasks were pooled together and used to inoculate the 20 L vessel. 
     The working volume of the 20 L vessels was 15 L of which 4% (v/v) was inoculum from the 200 mL stage. The agitation rate was set at 100 rpm. The pH, dO2 and temperature were set at 7.00 units, 0% and 37° C. respectively. The pH was controlled with additions of either HCl (5M) of NaOH (5M). The dO2 concentration was maintained at 0% by continuous headspace sparging of nitrogen, with a flowrate of 20 L/min. 
     After 12 hours of growth in the 20 L vessel (OD600 6.0-7.0), 8 L of culture were used to inoculate the 200 L vessel. The running conditions were identical to the 20 L scale. The final optical density (600 nm) at harvest was 6.0-7.0. After 14 hours the fermenter was cooled to 10-20° C. before commencing harvest recovery. 
     Harvest 
     5 L Harvest 
     The 5 L cultures were pumped with a flow rate of 5 L/h through a Millistak+10″ Opticap depth filter (Millipore, KCOHC10FF1) and 0.2 μm Sartopore 2 300 cm 2  filter into sterile 250 mL bio-containers. The processed material was either stored at −20° C. or stored at 4° C. overnight before processing by DSP. 
     200 L Harvest 
     The 200 L harvest was performed using a filtration harvest train. The culture was pumped with a flow rate of 200 L/h through a Milistak+ (MCOHC10FS1) disposable depth filter with a filtration area of 4×1 m2 followed by two 0.2 μm Express Opticap XL filters, 2×0.49 m2 (Millipore, KHGES10TT1). The process time for primary clarification was 1 hour. An additional 10 min was allowed at the end of the harvest to retrieve residual product held up in the filters. The clarified supernatant was collected in a 200 L Stedim Palletank with the filtrate weight recorded 20 L of filtrate was passed through a MUSTANG Q high affinity DNA column with a flowrate ˜6 L/min and collected into two sterile 20 L stedim bags, prior to storage at 4° C. overnight. 
     Analysis 
     Optical Density Measurements 
     The spectrophotometer was blanked using PBS at wavelength 600 nm. Fermentation samples were diluted by factors of 10, 20 or 100 (dependent on cell density) using PBS. 1 mL of each diluted sample was transferred into a 1 mL cuvette; the top was sealed and inverted 5 times before recording triplicate optical density readings at a wavelength of 600 nm. 
     Tri-Glycine Gels 
     Fermentation samples were filtered through 0.2 μm filters before preparing them for SDS-PAGE analysis. 10 μl of filtered sample was added to 10 μl sample buffer (2×), 2.5 μl reducing agent (10×) and 2 μl of 0.1M EDTA (to achieve final concentration of 10 mM). The high molecular weight (HMW) marker was prepared by adding 10 μl of concentrated stock to 80 μl reducing agent (10×), 3100 WF1 and 400 μl sample buffer (2×). The diluted HMW standard was heated to 95° C. for 5 minutes before aliquoting and storage at −20° C. for use in subsequent gels. 15 μL of fermentation sample and 10 μL of HMW marker were run on 8% Tris-Glycine gel using pre-cooled (4° C.) Tris-Glycine running buffer at 130V, 400 mA and 100 W for ˜1 hour and 50 minutes. After electrophoresis, the gels were immersed in 100 mL colloidal blue stain reagent (55 mL WFI, 20 mL methanol, 5 mL stainer A, 20 mL stainer B) and left to stain for 5 h on an orbital shaker at 60 rpm. Gels were de-stained with 200 mL WFI. The gel was left in WFI for 15-20 h until excess stain was removed after which the gel was scanned and dried according to the manufactures instructions. 
     Bis-Tris Gels 
     The fermentation samples were prepared for SDS-PAGE analysis by adding 10 μl of 0.2 μm filtered sample to 4 μl sample buffer (4×), 1.5 μl reducing agent (10×) and 1.7 μl of 0.1M EDTA (to achieve final concentration of 10 mM). 15 μL of fermentation sample and 10 μL of Mark 12 marker were run on a 4-12% Bis-Tris gel and run using MES running buffer at 200V, 400 mA and 100 W for ˜40 mins. After electrophoresis, the gels were immersed in a 100 mL fixing solution (40 mL dH2O, 50 mL methanol, 10 mL acetic acid) for 10 minutes before replacing with a 95 mL staining solution (55 mL dH2O, 20 mL methanol, 20 mL stainer A) for a further 10 minutes. 5 mL of stainer B was added to the staining solution and the gels were left to stain for 5 h on an orbital shaker at 60 rpm before de-staining with 200 mL WFI. The gel was left in WFI for 15-20 h until excess stain was removed after which the gel was scanned and dried according to the manufactures instructions. 
     Process 3 Purification: 
     The first 20 L scale run-through of a newly developed process (Process 3) for the purification of collagenases from  Clostridium histolyticum , which was modified from Process 2 performed to GMP at 20 L scale. Significant process changes were introduced in the development of Process 3 in order to make the purification more robust and more amendable to scale up and subsequent process validation. One significant factor in facilitating this process change was in the choice of fermentation component. Process 2 had been based on the requirement to maintain a phytone based fermentation media whereas for process 3 proteose peptone No. 3 was use. The process run-through is split into the key steps of the down stream purification and the collagenases AUXI and AUXII. These include the treatment of the fermentation filtrate using a MUSTANG Q capsule, hydrophobic interaction chromatography, tangential flow filtration step 1 (denoted TFF1), anion exchange chromatography and tangential flow filtration step 2 (denoted TFF2). AUX1 and AUXII co-purify in the initial steps of the purification and are only separated during the anion exchange chromatography step (performed using QSepharose HP media). AUXI and AUXII are then processed separately and formulated. The intermediates are then mixed in a 1:1 ratio (based on protein content determined by UV) and filtered to form the drug substance. In developing process 3, key steps associated with process 2 were removed. Notably the ammonium sulphate precipitation step, two chromatography steps (hydroxyapaptite and gel permeation chromatography) and all −20° C. hold steps were eliminated. The use of un-scalable steps such as stirred cells and dialysis were also removed and replaced with tangential flow filtration (TFF). The issue of product instability, which was evident in process 2 (and eliminated the use of TFF), was not apparent in the 20 L scale run of process 3. The contaminant profile associated with process 3 was however different to process 2 in which clostripain and gelatinase had been major components. Most notable a 40 kDa, 55 kDa and two 90 kDa contaminants (one co-purifying with AUXI and the other with AUXII) were detected by SDS-PAGE. As a result of these new contaminants, some of the QC assays (such as RPHPLC and SEC-HPLC) were of limited use since they did not resolve all process 3 impurities. The inability to utilize established QC assays for in-process purity determination, resulted in the need to define a method for establishing which material for the QSepharose column was suitable for further purification. This was required since the contaminants were not clearly resolved from the AUXI and AUXII products on the QSepharose column and it was therefore necessary to collect eluted material in discrete fractions, which could be analyzed retrospectively. Analysis was performed by SDS-PAGE and the pooling decision for the 20 L run-through was based on experience of the relative staining intensity of impurity to product using a standardized 1 μg load. 
     Retrospective densitometry analysis of SDS-PAGE enabled the pooling criteria to be described based on relative percent product purity. Further densitometry analysis using material from the 200 L demonstration run enabled a standardized method to be established as well as an approximation of assay variation. this led to an agreed procedure for the poling of in process fractions to be implemented in the first GMP campaign. 
     In addition to the process description, preliminary work describing a buffer stability and in-process sample stability study is presented along with initial characterization of some of the impurities associated with Process 3. 
     Process 3 differed from process 2 in three main areas. Firstly, the ammonium sulphate precipitation step and hydroxyapatite chromatography steps were removed, secondly, the gel permeation chromatography (GPC) step was eliminated and thirdly, all buffer exchange steps were performed by tangential flow filtration. the precipitation step was replaced by the use of hydrophobic interaction chromatography (HIC) at the client&#39;s recommendation. Development of this step resulted in the successful implementation of HIC for (i) product capture (thereby serving as a concentration step), and (ii) some protein and pigment contaminant removal. The HIC step was also subsequently shown to reduce levels of dsDNA. As a result of the process development program, the introduction of HIC and inclusion of a MUSTANG Q filter step removed the need for both the ammonium sulphate precipitation step and the hydroxyapatite chromatography step. The overall effect was to simplify the up front capture of product and to remove a potential hold step associated with Process 2. This latter point had significance in that previously the fermentation could be assessed prior to down stream purification since the pellets resulting from the precipitation step could be held at −20° C. prior to processing. 
     Following the HIC step, product was buffer exchanged using tangential flow filtration (TFF). This was performed using 30 kDa molecular weight cut off (MWCO) membranes and replaced the dialysis procedure used to Process 2. Aggregate contamination, which when present was detected as AUXII-derived, appeared to be removed during the anion exchange chromatography step (IEX). As a result, the GPC step was eliminated since both AUXI and AUXII intermediates were within specification for aggregates following IEX. Finally, the final concentration and formulation of the AUXI and AUXII intermediates was performed using TFF instead of the previous method of utilizing stirred cells. 
     Overall, Process 3 represented a simpler process that was more amenable to scale up and validation than Process 2. In addition, the reduction in consumable cost was apparent by the elimination of the need for hydroxyapaptite and gel permeation media and by the reduced number of steps requiring leupeptin. An overview of publication scheme for Process 3 is given in  FIG.  46   . 
     Non-GMP Demonstration Run at 20 L Scale 
     Process 3 was performed at 20 L scale in the process development laboratories in order to demonstrate if material of suitable quality could be generated using this modified process at 20 L scale. A key requirement for processing was the ability to limit potential protease activity by performing steps chilled wherever possible and by the inclusion of the cysteine protease inhibitor leupeptin at key stages in the procedure. A full 20 L of fermentation filtrate was processed since the feedstock was generated from 200 L fermentation PP3. Details of the fermentation and subsequent harvest and filtration are documented in a separate report. 
     MUSTANG Q Filter Treatment of Fermentation Filtrate 
     Following 0.2 μm filtration, approximately 22 L of fermentation supernatant was loaded onto a MUSTANG Q chromatography capsule as describe previously. Some visible pigment contamination (green/brown) appeared to be removed by the MUSTANG Q capsule during the filtration of the first 10 L since the contents of the first 10 L Stedim bag appeared visibly less pigmented than the second. The ability of the MUSTANG Q capsule to remove dsDNA was monitored across this step by pico green analysis of pre and post MUSTANG Q filter samples (Table 41). In process analysis indicated that unlike previous data generated at small-scale, bulk nucleic acid removal was not evident at the MUSTANG Q filter step. The robustness and application of this step therefore requires further investigation. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 41 
               
               
                   
                   
               
               
                   
                 Sample description 
                 Result ng/ml 
               
               
                   
                   
               
             
            
               
                   
                 Fermentation filtrate 
                 230.65  
               
               
                   
                 Post Mustang Q 
                 216.53  
               
               
                   
                 Post HIC 
                  1.02 
               
               
                   
                 Post TFF 
                  6.34 
               
               
                   
                 Post IEX Aux I 
                  2.33 
               
               
                   
                 Post IEX Aux II 
                  3.41 
               
               
                   
                   
               
            
           
         
       
     
     Hydrophobic Interaction Chromatography (HIC) 
     The use of HIC served three functions in the purification. Firstly, the product was reduced in volume since condition were identified in which collagenases bound to the resin. Secondly, some pigment and protein contaminant was removed at this stage and thirdly, pico green analysis from this run indicated reduction of dsDNA. The HIC step was performed using supernatant processed directly from the fermentation (after MUSTANG Q treatment) and, as a result a hold step, (evident in Process 2 as the ammonium sulphate pellet) was no longer present for Process 3. 
     In order to provide conditions for collagenases to bind in the HIC column, product (20 L) from the MUSTANG Q filter step was diluted with a 3M-ammonium sulphate solution to a final concentration of 1M. After filtration, product was loaded onto the column and eluted using a 2-step isocratic elution. 
     The protein concentration of the HIC load material was difficult to determine accurately and was estimated in two ways. Firstly, a Bradford assay was performed on the material prior to ammonium sulphate addition. This was performed with undiluted material in order to standardize the contribution from pigment present in the fermentation media, which was known to interfere with the assay. Secondly, the estimate was based on volume of fermentation media loaded per mL of column resin. The column load was estimated to be 5.9 mg of total protein/mL resin by Bradford assay or alternatively ˜13 mL of fermentation media per mL resin. An estimate of the total amount of target protein eluted from the column was determined as 3.4 g using UV (see Table 42). Assuming that the total protein present in the HIC load was 9 g (Bradford assay), this equated to a 38% recovery. This value was only regarded as a relative measure, however, due to the inaccuracy of the assay for the samples containing fermentation media components. 
     An alternative method for estimating the HIC load concentration was determined using densitometry although it was recognized that this estimation would give a collagenase content rather than estimate of total protein (which could vary between fermentations). Using this approach, the total collagenases were estimated as 360 mg/L with an approximate ratio of AUXI to AUXII estimated as 40:60. Using this data, the total collagenase expected in the HIC load would be 7.2 g giving a step yield of 47%. 
     The chromatogram resulting from the HIC step is shown in  FIG.  70   . Visible pigment was apparent in the flow-through as well as bound to the column. After washing the column with equilibration buffer to remove the flow-through contamination, peak 1 was eluted using an intermediate concentration ammonium sulphate solution (0.3M). 
     This peak was shown to contain protein contaminants although some AUXII was also eluted at this stage ( FIG.  71   ). This loss in product was expected and had been noted previously. In order to minimize the amount of product lost, without compromising purity, the elution volume for peak 1 removal was set at 5 column volumes. Peak 2, containing the majority of the product, was then eluted using buffer with no ammonium sulphate. Peak 2 was collected as a single pool with the chromatography method programmed so that collection began after % of a column volume of elution buffer had been applied to the column. 
     Collection was then terminated after a total of 4 column volumes had been collected. In order to minimize potential proteolysis in the product at this stage in the process, leupeptin was added to the post HIC elute and the material held at 2-8° C. The hold time for the post HIC eluate was of 2 day duration. 
     Tangential Flow Filtration 1 (TFF1) 
     TFF using 30 kDa membranes was introduced following the HIC in order to reduce the volume of product (5-fold) and to exchange the buffer into conditions suitable for binding to the anion exchange column. Of particular importance was the sufficient reduction in ammonium sulphate such that the conductivity of the 1EX load sample was &lt;1.8 mS. The diafiltration buffer was chilled and leupeptin added prior to use to reduce the likelihood of proteolysis. No loss in protein was estimated over the course of this step (&gt;100% recovery) although this may reflect the inaccuracy in protein concentration estimation at this stage in the process due to the presence of pigment in the pre TFF1 material. Approximately 97.5% of the total protein (3325 mg) was recovered in the retentate with an additional 204.8 mg recovered in the first membrane rinse (infra). Filtration of the total protein from the combined retentate and rinse was performed at the end of the TFF step prior to holding the material overnight at 2-8° C. SDS-PAGE analysis indicated no significant differences were detected before and after the TFF step ( FIG.  71   ). 
     Q-Sepharose Chromatography 
     The Q-Sepharose column was loaded at a maximum capacity of 5 mg total protein per mL resin. As a result, not all of the available material from the TFF step was utilized in this step (see Table 421). The Q-Sepharose column resolved AUXI and AUXII collagenases as expected ( FIG.  72   ). The start of AUXII elution began at approximately 13.6% B (where buffer A=10 mM Tris, 0.2 mM leupeptin pH 8 and buffer B=Buffer A+360 mM NaCl) which equated to a post column conductivity of 5.7 mS. Fractions (100 mL) were collected throughout the elution of AUXII until the absorbance value dropped to 25% of the peak height (550 mAU). A small peak was eluted at approximately 8 mS (20.3% B) following AUXII elution. In-process analysis of this peak from previous small-scale experiments indicated this to be AUXII derived aggregate material. The start of AUXI elution was at approximately 27% B (which equated to 10.4 mS). As before, 100 mL fractions were collected until the absorbance dropped to the required 25% value (190 mAU). 
     Each AUXI and AUXII fraction collected was analyzed by SDS-PAGE and subjected to densitometry ( FIGS.  73 - 76   ). Densitometry was performed retrospectively, so the decision on fraction pooling was based on experience of the levels of contaminant visible by Colloidal blue staining. In consultation with Auxilium, fractions 6-12 were pooled for the AUXII product and fractions 19-26 pooled for AUXI. The step yields and protein concentrations present in the material pooled from the Q-Sepharose run are included in table 42. 
     SDS-PAGE analysis of the post IEX AUXI and AUXII products from the 20 L demonstration run ( FIGS.  77  and  78   ) showed few contaminants visible by SDSPAGE. In addition, the contaminants detected were in accordance with previous small-scale experiments although there were noted differences in the resolution of the contaminants, which appeared to be more defined (i.e. separate peaks or shoulders) in the small-scale model. These contaminants were also different to those identified for Process 2 in which clostripain and gelatinase had been major components. As a result, the QC protocols developed for Process 2 were not optimized for the detection of the new contaminants associated with Process 3. 
     Retrospective densitometry of the pooled material estimated the purity at 95.1% for AUXI and 99.4% for AUXII. Currently, however the purity specification of 97% is specified by RP-HPLC and no final product specification has been established using densitometry. 
     Concentration and Buffer Exchange of AUXI and AUXII 
     The separated AUXI and AUXII products from the Q Sepharose column were processed separately by TFF using a 30 kDa membrane. This step was required to: (i) remove/reduce leupeptin in the final product; (ii) formulate the intermediates into the correct buffer (10 mM Tris, 60 mM sucrose pH 8); and (iii) to achieve the required target protein concentration of 0.9-1.1 mg/mL. A total of 799 mg (˜683 mL at 1.17 mg/mL) of AUXII and 860 mg (796 mL at 1.08 mg/mL) of AUXII was concentrated to a target concentration of 1.75 mg/mL. This theoretical concentration was based on the calculated reduction in volume required assuming no loss of product during the concentration step. Diafiltration was then performed into the required formulation buffer, the membranes washed with the minimum volume of the TFF system (−250 mL) and the full amount combined with the concentrate to achieve the required target concentration of 0.9-1.1 mg/mL) were available after filtration. In both cases, the majority of product was recovered in the retentate and was estimated as 95.4% (762 mg) for AUXII and 83.1% (715 mg) for AUXI. The additional material provided by the membrane rinse was estimated as 153 mg and 89.6 mg for AUXII and AUXI respectively. 
     Mixing of Intermediates to Drug Substance 
     Approximately 200 mg of each intermediate was combined to give 400 mg of the drug substance. This was then filtered and approximately 26 mg provided to QC for testing. The QC results for AUXI, AUXII intermediates and the drug substance are provided in Table 43. All tests on the drug substance and AUXII intermediate passed the required specification. The test for potency of the intermediate AUXI however, was not within the specified range although all other tests passed. With the exception of the AUXI potency result, these data indicated that Process 3 was capable of generating material of the required specification when purified at the 20 L scale. 
     As well as QC testing, material from the 20 L demonstration run was utilized for method validation at KBI BioPharma, Inc. At the client&#39;s request, 200 mg of drug substance was shipped on dry ice to KBI for drug substance and drug product methods validation. The latter testing was performed after lyophilisation of the drug substance at KBI. In addition, 25 mg of each intermediate was supplied to KBI for validation of analytical methods. 
     The individual step yields for the 20 L demonstration run are given in table 42. An extrapolation of the data in which all the available material had been loaded onto the Q-Sepharose column indicated that the maximum total amount of available drug substance from this process run-through was 1.6 g (assuming no loss of material through retains). This equates to an approximate overall process yield of 17.8% based on the initial estimate of 9 g (using the Bradford assay) for the amount of total protein available to load onto the HIC column. With the limitation on the load for the Q-Sepharose column, a maximum of 1.4 g of drug substance was available from the current run-through if all the available intermediate had been mixed to form the drug substance. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 42 
               
               
                   
               
               
                   
                 Protein conc. 
                 Amount 
                 Total protein 
                   
               
               
                 Process step 
                 (mg/mL) 
                 (weight/volume) 
                 (mg) 
                 Step yield 
               
               
                   
               
             
            
               
                 Fermentation 
                 — 
                 200 L 
                 — 
                 — 
               
               
                 Pre Mustang Q 
                 — 
                  22 L 
                   
                   
               
               
                 Post Mustang Q 
                 0.45 
                  22 L 
                 9000 
                 — 
               
               
                   
                 (Bradford) 
                   
                   
                   
               
               
                 HIC load 
                   0.30* 
                  30 L 
                 9000 
                 100 
               
               
                   
                 (theoretical) 
                   
                   
                   
               
               
                 HIC peak 2 
                 0.56 
                 6101.7 mL 
                 3416.95 
                 38 
               
               
                 (after leupeptin) 
                   
                   
                   
                   
               
               
                 Pre-TFF1 
                 0.54 
                 6317.7 mL 
                 3411.6 
                 100 
               
               
                 Post TFF1 
                 2.55 
                 1378.5 g  
                 3515.2 
                 &gt;100% 
               
               
                 (with wash 1 
                   
                   
                   
                   
               
               
                 and post 
                   
                   
                   
                   
               
               
                 filtration) 
                   
                   
                   
                   
               
               
                 Q load 
                 2.55 
                 1216 mL 
                 3100.8 
                 100 
               
               
                 (5 mg/mL resin) 
                   
                   
                   
                   
               
               
                 Q AUXII pool 
                 1.17 
                  682.8 mL 
                 798.88 
                 25.8 
               
               
                 Q AUXI pool 
                 1.08 
                  796.4 mL 
                 860.11 
                 27.7 
               
               
                 Pre TFF2 
                 1.17 
                 682.80 mL  
                 798.88 
                 100 
               
               
                 AUXII 
                   
                   
                   
                   
               
               
                 Intemtediate 
                 1.03 
                 795.6 g  
                 819.47 
                 &gt;100 
               
               
                 AUXII 
                   
                   
                   
                   
               
               
                 (post filtration) 
                   
                   
                   
                   
               
               
                 Pre TFF2 
                 1.08 
                 796.40 mL 
                 860.11 
                 100 
               
               
                 AUXI 
                   
                   
                   
                   
               
               
                 Intermediate 
                 1.09 
                 731.2 g  
                 797.01 
                 92.7 
               
               
                 AUXI 
                   
                   
                   
                   
               
               
                 (post filtration) 
               
               
                   
               
               
                 *calculated based on dilution factor after ammonium sulphate addition 
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 43 
               
               
                   
               
               
                   
                   
                 Drug substance 
                 Intermediate AUXI 
                 Intermediate AUXII 
               
               
                 Test 
                 Method 
                 (AXS2006A0754H) 
                 (AXS2006A0745H) 
                 (AXS2006A0737H) 
               
               
                   
               
             
            
               
                 Appearance 
                 QC SOP 
                 Clear, colourless with 2-3 
                 Clear, colourless and free 
                 Clear, colourless with 2-3 
               
               
                 of solution 
                 001 
                 small exogenous fibres 
                 from Particulate matter 
                 small exogenous fibres 
               
               
                   
                   
                 1 mm in length 
                 (AK/1573/121) 
                 1 mm in length 
               
               
                   
                   
                 (AK/1573/121) 
                   
                 (AK/1573/121) 
               
               
                 Potentiometric 
                 QCSOP 
                 7.6 
                 Not required 
                 Not required 
               
               
                 measurement of pH 
                 002 
                 (FR/1598/098) 
                   
                   
               
               
                 Endotoxin 
                 QCSIO 
                 &lt;0.5 EU/mL 
                 &lt;0.5 EU/mL 
                 0.136 EU/mL 
               
               
                 Determination 
                 018 
                 (AS/1597/128) 
                 (AS/1597/128) 
                 (AS/1597/128) 
               
               
                 Identity by SDS-PAGE 
                 QC SOP 
                 Major collagenase bands 
                 Major collagenase bands 
                 Major collagenase bands 
               
               
                 Coomassie Stained 
                 103 
                 between 107 and 110 kDa 
                 between 110 and 115 kDa 
                 between 107 and 110 kDa 
               
               
                   
                   
                 and 110 and 115 kDa 
                 (AS/1597/133) 
                 (AS/1597/133) 
               
               
                   
                   
                 (AS/1597/133) 
                   
                   
               
               
                 Total Protein by 
                 QC SOP 
                 1.04 mg/mL 
                 1.11 mg/mL 
                 0.90 mg/mL 
               
               
                 Absorbance 
                 144 
                 (AS/1597/106) 
                 (AS/1597/106) 
                 (AS/1597/106) 
               
               
                 Spectrophotometry 
                   
                   
                   
                   
               
               
                 Rat Tail Tendon 
                 QC SOP 
                 2326 
                 1483 
                 Not required 
               
               
                 Collagen Assay for 
                 105 
                 (1700-3500) 
                 (1900-3300) 
                   
               
               
                 Potency 
                   
                 (AS/1597/124) 
                 (AS/1597/124) 
                   
               
               
                   
                   
                   
                 (OOS/Keele/2006/0038) 
                   
               
               
                 Reference 
                   
                 2097 
                 2097 
                   
               
               
                   
                   
                 (2014-3440) 
                 (2014-3440) 
                   
               
               
                 Potency for Class II 
                 QC SOP 
                 57677 GPA unit/mg 
                 Not required 
                 119552 GPA unit/mg 
               
               
                 Collagenases 
                 106 
                 (50000-90000) 
                   
                 (79000-170000) 
               
               
                   
                   
                 (AS/1597/111) 
                   
                 (AS/1597/111) 
               
               
                 Reference 
                   
                 69523 GPA units/mg 
                   
                 69523 GPA units/mg 
               
               
                   
                   
                 (58000-95000) 
                   
                 (58000-95000) 
               
               
                 Host Cell Protein Assay 
                 QCSOP 
                 &lt;LOD 
                 Not required 
                 Not required 
               
               
                   
                 107 
                 (FR/1589/108) 
                   
                   
               
               
                 Host Cell DNA Assay 
                 External 
                 New Lab 
                 Not required 
                 Not required 
               
               
                 Analysis of proteins 
                 QC SOP 
                 100% main peaks 
                 100% main peak 
                 100% main peak 
               
               
                 using the Agilent 1100 
                 109 
                 (47.70% AUX-I, 
                 0% aggregates 
                 0% aggregates 
               
               
                 HPLC System 
                   
                 52.30% AUX-II) 
                 (AK/1573/122) 
                 (AK/1573/122) 
               
               
                 (Identity and purity by 
                   
                 (AK/1573/122) 
                   
                   
               
               
                 size exclusion 
                   
                   
                   
                   
               
               
                 chromatography) 
                   
                   
                   
                   
               
               
                 Reference 
                   
                 AUX-I 100% 
                 AUX-I 100% 
                 AUX-II 100% 
               
               
                   
                   
                 AUX-II 100% 
                   
                   
               
               
                 Analysis of proteins 
                 QC SOP 
                 48.89% AUX-I 
                 99.14% AUX-I 
                 0.39% AUX-I 
               
               
                 using the Agilent 1100 
                 109 
                 50.99% AUX-II 
                 0.37% AUX-II 
                 99.35% AUX-II 
               
               
                 HPLC System 
                   
                 0.12% Others 
                 0.49% Others 
                 0.26% Others 
               
               
                 (Identity and purity by 
                   
                 (AK/1573/125) 
                 (AK/1573/125) 
                 (AK/1573/125) 
               
               
                 reverse phase liquid 
                   
                   
                   
                   
               
               
                 chromatography) 
                   
                   
                   
                   
               
               
                 Reference 
                   
                 AUX-I 93.92% 
                 AUX-I 93.92% 
                 AUX-II 100% 
               
               
                   
                   
                 AUX-II 4.96% 
                 AUX-II 4.96% 
                   
               
               
                   
                   
                 Others 1.13% 
                 Others 1.13% 
                   
               
               
                   
                   
                 AUX-II 100% 
                   
                   
               
               
                 Analysis of proteins 
                 QC SOP 
                 0.3% Gelatinase 
                 0.4% Gelatinase 
                 0% Gelatinase 
               
               
                 using the Agilent 1100 
                 109 
                 (AK/1573/131) 
                 (AK/1573/131) 
                 (AK/1573/131) 
               
               
                 HPLC System 
                   
                   
                   
                   
               
               
                 (Gelatinase by  
                   
                   
                   
                   
               
               
                 anion exchange 
                   
                   
                   
                   
               
               
                 chromatography) 
                   
                   
                   
                   
               
               
                 Reference 
                   
                 AUX-I 0% Gelatinase 
                 AUX-I 0% Gelatinase 
                 AUX-I 0% Gelatinase 
               
               
                   
                   
                 AUX-II 0% Gelatinase 
                 AUX-II 0% Gelatinase 
                 AUX-II 0% Gelatinase 
               
               
                 Peptide mapping by 
                 QC SOP 
                 Not required 
                 Tuesday 
                 Tuesday 
               
               
                 Tryptic Digest and 
                 110 
                   
                   
                   
               
               
                 Reverse Phase HPLC 
                   
                   
                   
                   
               
               
                 Residual Leupeptin 
                 QCSOP 
                 Not detected 
                 Not required 
                 Not required 
               
               
                   
                 141 
                 &lt;0.5% w/w 
                   
                   
               
               
                   
                   
                 (AK1573/136) 
                   
                   
               
               
                 Bioburden 
                 QCSOP 
                 0 cfu/5 mL 
                 0 cfu/5 mL 
                 0 cfu/5 mL 
               
               
                   
                 223 
                 (JM/1505/115) 
                 (JM/1505/114) 
                 (JM/1505/112) 
               
               
                   
               
            
           
         
       
     
     Sample Stability Study 
     During the 20 L demonstration run, samples were taken at key process points. As the demonstration run was performed as a continuous process (with no hold steps) an attempt was made to assess the stability of in-process material during the hold times anticipated for GMP batches. The extended run duration expected for GMP was recognized due to the requirement to obtain equipment clearance data between process steps. In-process material was held at 2-8° C. for approximately the duration expected for the GMP manufacture. In addition samples were held for an extended time representing twice that expected for the GMP campaign. A description of the samples taken, along with the respective hold times is given in table 44. The processing times for the 20 L demonstration run are represented in table 45. All samples were submitted to QC for SDS-Page, RP-HPLC, SEC-HPLC and UV analysis ( FIGS.  79 - 83   ). 
     Overall, the results showed no detectable deterioration in the product over the first hold point with respect to purity (as determined by RP-HPLC), degradation (as detected by 8% Tris-Glycine SDS-PAGE) and aggregation (as determined by SECHPLC). Some of the assays, however, were recognized to be limiting since low molecular mass components would not be detected by 8% SDS-Page and RPHPLC assay had not been developed to detected the 40 kDa, 55 kDa and 90 kDa contaminants associated with Process 3. Some assays were also less relevant for crude samples such as the UV and SEC-HPLC in the fermentation samples. Despite these limitations, the only detected changes in product profile was identified for the second hold point (day 12) for the AUXII in-process sample taken from the Q-Sepharose column. This showed an increase in aggregate level between day 5 and day 12 although this increase was only from 0 to 0.62%. 
     A second stability study was performed on the in-process retains which were taken at the point of manufacture during the 20 L demonstration and stored at −20° C. In this study, samples were thawed and incubated at room temperature and at 37° C. and monitored by 4-12% SDS-Page analysis to allow the full molecular mass range of contaminants to be evaluated ( FIGS.  84 - 88   ). These data demonstrated that the samples prior to Q-Sepharose anion exchange were vulnerable to degradation. Following separation of the collagenases AUXI and AUXII (by the Q-Sepharose column), the samples appeared to be relatively stable and looked comparable to the time zero samples by SDS-PAGE. 
     Taken together, both studies indicate that providing the temperature is maintained between 2-8° C., in-process material is not expected to deteriorate during processing over the hold times investigated. This gives a level of confidence that the use of leupeptin and temperature control is sufficient to restrict levels of product degradation during processing over the durations anticipated in GMP. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 44 
               
               
                   
               
               
                   
                 Duration held 
                   
                   
                   
               
               
                   
                 at 2-8° C. 
                   
                 Storage, 
                   
               
               
                 Sample 
                 before freezing 
                 Volume 
                 Retain 
                 Container 
               
               
                   
               
             
            
               
                 Fermentation Filtrate 
                 Day 4  
                 1 × 2 mL 
                 −70° C. 
                 Bag 
               
               
                   
                 Day 5  
                 1 × 2 mL 
                   
                   
               
               
                 Post Mustang Q 
                 Day 4  
                 1 × 2 mL 
                 −70° C. 
                 Bag 
               
               
                 Post HIC 
                 Day 3  
                 1 × 2 mL 
                 −70° C. 
                 Bag 
               
               
                   
                 Day 6  
                 1 × 2 mL 
                   
                   
               
               
                 Post TFF 
                 Day 2  
                 1 × 2 mL 
                 −70° C. 
                 Bag 
               
               
                   
                 Day 4  
                 1 × 2 mL 
                   
                   
               
               
                 Post IEX AUX I 
                 Day 5  
                 2 × 1 mL 
                 −70° C. 
                 Biotainer 
               
               
                   
                 Day 12 
                 2 × 1 mL 
                   
                   
               
               
                 Post IEX AUX II 
                 Day 5  
                 2 × 1 mL 
                 −70° C. 
                 Biotainer 
               
               
                   
                 Day 12 
                 2 × 1 mL 
                   
                   
               
               
                 AUX I Intermediate 
                 Day 6  
                 2 × 1 mL 
                 −70° C. 
                 Biotainer 
               
               
                   
                 Day 12 
                 2 × 1 mL 
                   
                   
               
               
                 AUX II Intermediate 
                 Day 6  
                 2 × 1 mL 
                 −70° C. 
                 Biotainer 
               
               
                   
                 Day 12 
                 2 × 1 mL 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 45 
               
               
                   
                   
               
               
                   
                   
                 20 L 
               
               
                   
                 Process step 
                 demonstration 
               
               
                   
                   
               
             
            
               
                   
                 Fermentation harvest 
                 Day 1  
               
               
                   
                 Mustang Q 
                 Day 1  
               
               
                   
                 HIC 
                 Day 2  
               
               
                   
                 TFF1 
                 Day 5  
               
               
                   
                 IEX (Q-Sepharose) 
                 Day 6  
               
               
                   
                 TFF2 (AUXI) 
                 Day 7  
               
               
                   
                 TFF2 (AUXII) 
                 Day 9  
               
               
                   
                 DS mixing 
                 Day 12 
               
               
                   
                   
               
            
           
         
       
     
     Buffer Stability Study 
     Buffer samples illustrated in table 46 were reserved from the 20 L demonstration and retested after storage at 2-8° C. The pH, conductivity, temperature and appearance of the buffers were noted at the time of completion and after 12-13 day&#39;s storage. The results of this study are given in table 47. Small differences were observed in the values for pH and conductivity but this may be due to differences in temperature between the original buffers and the tested retains. In particular, the HIC buffers showed the largest variation in conductivity and temperature. As a result, future studies on buffer stability should include specification of an accepted temperature range for recording all parameters. In all cases, the buffer retains were clear in appearance at time zero and after the required hold time. 
     
       
         
           
               
               
             
               
                 TABLE 46 
               
               
                   
               
               
                 BUFFER 
                 CONSTITUENTS 
               
               
                   
               
             
            
               
                 HIC A 
                 10 mM Tris, 1.0 M Ammonium Sulphate, pH 8.0 
               
               
                 HIC A2 
                 10 mM Tris, 0.3 M Ammonium Sulphate, pH 8.0 
               
               
                 HIC B 
                 10 mM Tris, pH 8.0 
               
               
                 DIAFILTRATION 
                 10 mM Tris, 200 μm Leupeptin, pH 8.0 
               
               
                 IEX A 
                 10 mM Tris, 3 mM CaCl 2 , 200 μm Leupeptin, pH 8.0 
               
               
                 IEX B 
                 10 mM Tris, 3 mM CaCl 2 , 200 μm Leupeptin, 360 mM NaCl pH 8.0 
               
               
                 IEX SCRUB 
                 10 mM Tris, 3 mM CaCl 2 , 1.5 M NaCl pH 8.0 
               
               
                 FORMULATION 
                 10 mM Tris, 60 mM Sucrose pH 8.0 
               
               
                 3.0 M AMMONIUM  
                 10 mM Tris, 3.0 M Ammonium Sulphate pH 8.0 
               
               
                 SULPHATE STOCK 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 47 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Repeat 
                   
               
               
                   
                   
                   
                   
                   
                 pH and 
                 testing of pH 
                   
               
               
                   
                   
                   
                 Number 
                 Original 
                 conductivity 
                 and cond.  
                   
               
               
                   
                 Date 
                 Date of 
                 of days 
                 buffer pH 
                 of retain 
                 Of retain 
                 Buffer 
               
               
                 Buffer 
                 prepared 
                 testing 
                 elapsed 
                 and cond. 
                 samples 
                 samples 
                 appearance 
               
               
                   
               
             
            
               
                 HIC A 
                 2 May 
                 15 May 
                 13 days 
                 pH 7.98 
                 pH 8.10 
                 pH 7.89 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 127.6 mS 
                 137.5 mS 
                 134.9 mS 
                   
               
               
                   
                   
                   
                   
                 @17.8° C. 
                 @15.8° C. 
                 @22.0° C. 
                   
               
               
                 HIC A2 
                 2 May 
                 15 May 
                 13 days 
                 pH 8.03 
                 pH 8.05 
                 pH 7.85 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 32.7 mS 
                 49.7 mS 
                 49.6 mS 
                   
               
               
                   
                   
                   
                   
                 @18.5° C. 
                 @ 15.1° C. 
                 @22.0° C. 
                   
               
               
                 HIC B 
                 2 May 
                 15 May 
                 13 days 
                 pH 8.06 
                 pH 7.78 
                 pH 7.81 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 0.699 mS 
                 0.879 mS 
                 0.842 mS 
                   
               
               
                   
                   
                   
                   
                 @19.4° C. 
                 @18.2° C. 
                 @22.1° C. 
                   
               
               
                 Diafiltration 
                 2 May 
                 15 May 
                 13 days 
                 pH 8.05 
                 pH 7.70 
                 N/A 
                 Clear 
               
               
                 buffer 
                 2006 
                 2006 
                   
                 0.668 mS 
                 0.793 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @19.2° C. 
                 @18.2° C. 
                   
                   
               
               
                 IEX A 
                 3 May 
                 15 May 
                 14 days 
                 pH 8.00 
                 pH 7.69 
                 N/A 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 1.585 mS 
                 1.646 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @20.3° C. 
                 @18.2° C. 
                   
                   
               
               
                 IEX B 
                 3 May 
                 15 May 
                 14 days 
                 pH 8.00 
                 pH 7.87 
                 N/A 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 34.6 mS 
                 37.6 mS 
                   
                   
               
               
                   
                   
                   
                   
                 1@9.4° C. 
                 @18.8° C. 
                   
                   
               
               
                 IEX Scrub 
                 3 May 
                 15 May 
                 14 days 
                 pH 8.00 
                 pH 7.95 
                 N/A 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 105.2 mS 
                 122.9 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @10.0° C. 
                 @18.3° C. 
                   
                   
               
               
                 Formulation 
                 3 May 
                 15 May 
                 14 days 
                 pH 7.97 
                 pH 7.89 
                 N/A 
                 Clear 
               
               
                   
                 2006 
                 2006 
                   
                 0.735 mS 
                 0.987 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @18.5° C. 
                 @19.1° C. 
                   
                   
               
               
                 3.0M 
                 3 May 
                 15 May 
                 14 days 
                 pH 7.95 
                 pH 7.96 
                 N/A 
                 Clear 
               
               
                 AS Stock 
                 2006 
                 2006 
                   
                 251.0 mS 
                 235 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @16.5° C. 
                 @19.9° C. 
                   
                   
               
               
                 3.0M 
                 4 May 
                 15 May 
                 15 days 
                 pH 8.00 
                 pH 7.97 
                   
                 Clear 
               
               
                 AS Stock 
                 2006 
                 2006 
                   
                 224.0 mS 
                 254 mS 
                   
                   
               
               
                   
                   
                   
                   
                 @14.6° C. 
                 @20.4° C. 
               
               
                   
               
            
           
         
       
     
     Contaminant Identification by N-Terminal Sequencing 
     Three main impurities were detected for Process 3 by SDS-PAGE analysis. These appeared to be co eluted with the AUXI and AUXII collagenases and were only resolved by fractionation of the peaks eluted from the Q-Sepharose column. The contaminants were assigned by their apparent molecular mass on SDS-PAGE as 40 kDa, 55 kDa and 90 kDa contaminants. Fractions with elevated levels of a particular contaminant were submitted for N-terminal sequencing after excision of the band from SDS-PAGE. 
     Sequence analysis was successful for both the 55 kDa and 40 kDa contaminant isolated from the 20 L demonstration run. The N-terminus of the 55 kDa contaminant band associated with AUXI (Lanes 1-5;  FIG.  89   ) was shown to match a region of the Col G sequence for collagenase AUXI whereas the 40 kDa contaminant band from AUXII (Lanes 6-10;  FIG.  89   ) was identical to a region of the Col H sequence for collagenase AUXII. A previous attempt was made to sequence the 90 kDa band associated with both the AUXI and AUXII products ( FIG.  90   ). Sequencing of the 90 kDa contaminant associated with the AUXI product was successful in that identity was correlated with the N-terminus of the AUXI sequence. In contrast, it was not possible to obtain a complete sequence for the 90 kDA contaminant associated with AUXII, which suggested that the two 90 kDa contaminants were different products. 
     The main contaminants associated with Process 3 appeared to be product related and were either identified as N-terminally cleaved products of AUXI (55 kDa) and AUXII (40 kDa) or a C-terminally cleaved product of AUXI (90 kDa). As these contaminants were different to those identified in Process 2, the QC assays utilized for the specification of the intermediates and drug substance did not resolve the new contaminants as the assay development had originated around Process 2. In particular, the standard purity assay (RP-HPLC) could not be used to detect levels of the 40 kDa and 55 kDa contaminants. 
     Densitometry Analysis 
     20 L Demonstration Run 
     The 40 kDa, 55 kDa and 90 kDa contaminants associated with Process 3 were identified and resolved by SDS-PAGE. These contaminants were clearly detected in fractions eluted from the Q-Sepharose column and appeared to elute at the leading and trailing edges of the peak profile (See  FIGS.  72 - 76   ). The decision for which fractions were included or excluded for further purification was based on experience of the relative intensity of staining for contaminants and product on Colloidal blue stained gels. In order to make this a less subjective estimation, densitometry was utilized to determine specific pooling criteria for fractions following the Q-Sepharose step. Densitometry was used in preference to the current QC assay for purity (RP-HPLC) since this assay could not resolve the new contaminants associated with Process 3. 
     Densitometry Data from Post Q Fractions from the 20 L Demonstration Run 
     The densitometry values from 2 separate analyses of the post-IEX fractions were averaged and are shown in table 48. Fractions 1-12 and the last 25% (tail) of peak 1 contain AUXII and the associated contaminating proteins of 40, 75 and 90 kDa. Fractions 13-27 and the last 25% (tail) of peak 2 contain AUXI and the associated contaminants of 55 and 90 kDa. The pools of the fractions selected, based on SDS-PAGE without densitometric analysis, are highlighted. 
                         TABLE 48                  Fraction   Relative percentage (5) of band intensity                                 number   AUXII   48   75   90                                          1   55.9   44.1   0   0        2   57.8   42.2   0   0        3   68.0   32.0   0   0        4   83.2   18.8   0   0        5   93.3   5.7   0   0        6   96.3   1.7   0   0        7   100   0   0   0        8   100   0   0   0        9   100   0   0   0       10   98.1   0   0.7   1.2       11   94.2   0   1.9   3.9       12   94.3   0   2.2   3.5       tail   92.3   0   3.5   4.1           AUXI   55   90           13   60.8   12.       7.2           14   77.8   15.3   6.9           15   75.7   19.1   5.7           16   74.5   20.8   4.7           17   77.8   17.5   4.5           18   82.0   13.7   4.3           19   87.8   0.7   2.8           20   93.1   3.7   3.7           21   94.1   3.0   2.9           22   94.4   2.8   4.8           23        .7   0.9    4.               24   95.8   0.1   4.2           25   93.6   0.1   6.2           26   91.5   0.0   0.4           27   90.0   0.1   3.8           tail   88.5   0.5   10.8                    
Densitometry Summary Documents from Post Q Fractions from the 200 L Demonstration Run
 
     Summary of Densitometry Analysis 
     Post IEX fractions from the 200 L engineering run have been analyzed multiple times to establish a pooling criteria that can be documented in the IEX BMR for the GMP campaign. This pooling criterion is based on the assumption that: (i) the quality of material generated from the engineering run is appropriate for the GMP material; and (ii) the approximation of the densitometry method is acceptable. If the aim is to generate material of higher quality in the GMP campaign, the specification for pooling criteria will need to be revised. 
     Specification for Pooling from the IEX 
     In total, the samples from the 200 L engineering run have been analyzed 6 times (2 operators and 3 repeats of each gel) and the average data presented in table 49. The fractions that were pooled for the engineering run are highlighted in red. 
     From this analysis, the following pooling criteria can be established: 
     (i) Any fraction of purity greater than or equal to 88.5% can be pooled.
 
(ii) Any fraction with a single impurity greater or equal to 10% cannot be pooled.
 
(iii) Fractions to be pooled must be from consecutive fractions.
 
(iv) The calculated theoretical purity of the pool should be:
 
Greater than or equal to 93% in theoretical purity for AUXI;
 
Greater than or equal to 96% theoretical purity for AUXII.
 
     This last point was based on the estimates from the 200 L engineering run in which the total protein in available fractions was estimated (although one limitation was that not all fractions were present for UV analysis for the AUXI). The data from this analysis is presented in table 50. 
     **NOTE: from these criteria, fraction 7 for AUXII peak would now be excluded. 
     Assay Variation 
     From the data of the post IEX fractions from the 200 L engineering run, the following level of accuracy has been estimated: 
     (i) for the product (AUXI and AUXII) the % CV had been calculated as 2.1 (AUXI) and 2.3% (AUXII). Therefore the purity specification of 88.6% for pooling could be in the range 86.3-90.9%.
 
(ii) for the impurities, the % CV is much greater and the range has been estimated as 18.5%-33.7% depending on the impurity. Consequently, the purity specification of excluding fractions with a single impurity of no greater than 10% could be for fractions with an actual impurity range of 6.63-13.37%. Therefore the value for the purity of the product (and not the impurities) is the most reliable value for pooling specification.
 
     Estimated Purity of Final Material by Densitometry 
     Densitometry analysis of the final material (DS and intermediates) for the 200 L engineering run has also been determined by densitometry and is as follows: 
     AUXI=96.0% (3.1% of 90 kDa contaminant) 
     AUXII=98.7% (1.2% of 90 kDa contaminant) 
     DS=97.6% (2.1% of 90 kDa contaminant) 
     (Note: This is the range determined for a single SDS-PAGE analysed 3 times by 3 different operators.) 
     Standardisation of the Method 
     Over the course of the repeat analysis, the densitometry method has been standardized to minimize error between operators and variation between gels and will be documented in an SOP. Most notably:
         (i) The standard loading of total protein in each lane of the gel will be 1 μg.   (ii) A maximum of 16 fractions will be selected for analysis from each of the product peaks (AUXI and AUXII). This will limit the number of gels for densitometry analysis to 4.   (iii) The 16 fractions selected will start at the last fraction to be collected for each peak and work forward consecutively. This is to ensure accuracy in the figure calculated for the average purity (since all fractions to be pooled are likely to be included).       

     
       
         
           
               
             
               
                 TABLE 49 
               
               
                   
               
               
                 Average relative quantities of product and impurities in  
               
               
                 the post IEX fractions from the 200 L Engineering 
               
               
                 run, as determined by densitometry analysis. 
               
               
                 The fractions pooled are highlighted in red. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Quantity 
                 Relative quantity (%) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Fraction # 
                 loaded (μg) 
                 Product 
                 40 
                 90 
                 80 
                 55 
               
               
                   
               
               
                 AUXI (peak 2) 
                   
                   
                   
                   
                   
                   
               
               
                 3 
                 1 
                 88.91 
                 0 
                 7.43 
                 0 
                 3.66 
               
               
                 4 
                 1 
                 85.81 
                 0 
                 7.28 
                 0 
                 6.91 
               
               
                 5 
                 1 
                 84.57 
                 0 
                 6.76 
                 0 
                 8.65 
               
               
                 6 
                 1 
                 80.41 
                 0 
                 6.96 
                 0 
                 12.53 
               
               
                 8 
                 1 
                 80.00 
                 0 
                 5.46 
                 0 
                 14.54 
               
               
                 9 
                 1 
                 80.61 
                 0 
                 5.53 
                 0 
                 13.86 
               
               
                 10 
                 1 
                 88.53 
                 0 
                 4.20 
                 0 
                 7.27 
               
               
                 11 
                 1 
                 90.43 
                 0 
                 4.19 
                 0 
                 5.39 
               
               
                 13 
                 1 
                 94.68 
                 0 
                 4.36 
                 0 
                 0.97 
               
               
                 14 
                 1 
                 94.10 
                 0 
                 4.94 
                 0 
                 0.96 
               
               
                 15 
                 1 
                 93.93 
                 0 
                 5.18 
                 0 
                 0.89 
               
               
                 16 
                 1 
                 93.57 
                 0 
                 5.83 
                 0 
                 0.50 
               
               
                 18 
                 1 
                 92.03 
                 0 
                 7.97 
                 0 
                 0 
               
               
                 19 
                 1 
                 91.40 
                 0 
                 8.60 
                 0 
                 0 
               
               
                 20 
                 1 
                 90.30 
                 0 
                 9.70 
                 0 
                 0 
               
               
                 21 
                 1 
                 90.14 
                 0 
                 9.86 
                 0 
                 0 
               
               
                   
               
               
                 AUXII (peak 1)  
                   
                   
                   
                   
                   
                   
               
               
                 3 
                 1 
                 58.48 
                 21.55 
                 9.97 
                 0 
                 0 
               
               
                 4 
                 1 
                 55.51 
                 36.80 
                 7.69 
                 0 
                 0 
               
               
                 5 
                 1 
                 55.90 
                 36.73 
                 4.96 
                 2.41 
                 0 
               
               
                 6 
                 1 
                 67.59 
                 25.09 
                 4.53 
                 2.79 
                 0 
               
               
                 7 
                 1 
                 50.70 
                 12.00 
                 4.41 
                 2.55 
                 0 
               
               
                 8 
                 1 
                 87.61 
                 5.18 
                 4.58 
                 2.62 
                 0 
               
               
                 9 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 10 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 11 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 12 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 13 
                 1 
                 95.59 
                 0 
                 2.92 
                 1.79 
                 0 
               
               
                 14 
                 1 
                 93.56 
                 0 
                 4.12 
                 2.31 
                 0 
               
               
                 15 
                 1 
                 91.90 
                 0 
                 5.05 
                 3.06 
                 0 
               
               
                 16 
                 1 
                 91.45 
                 0 
                 4.68 
                 3.67 
                 0 
               
               
                 17 
                 1 
                 89.95 
                 0 
                 5.57 
                 4.48 
                 0 
               
               
                 18 
                 1 
                 87.85 
                 0 
                 7.03 
                 5.11 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 50 
               
               
                   
               
               
                 Theoretical relative amounts of product and impurities 
               
               
                 in the post IEX pools from the 200 L Engineering run, 
               
               
                 as determined by densitometry analysis. The fractions 
               
               
                 pooled are highlighted in red. The theoretical average  
               
               
                 product purity is calculated as 92.3% and 95.8% for the 
               
               
                 AUXI and AUXII intermediates respectively. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Product 
                   
               
               
                   
                 Total protein 
                 (AUXI) 
                 Quantity of all 
               
               
                 AUXI 
                 qauntity (mg) 
                 quantity (mg) 
                 Impurities (mg) 
               
               
                   
               
               
                 AUXI 
                 954.09 
                 844.67 
                 109.42 
               
               
                   
                 1290.61 
                 1167.07 
                 123.55 
               
               
                   
                 1524.29 
                 1443.16 
                 81.13 
               
               
                   
                 1507.35 
                 1418.46 
                 85.89 
               
               
                   
                 1339.47 
                 1258.18 
                 81.29 
               
               
                   
                 1256.06 
                 1175.32 
                 80.74 
               
               
                   
                 1063.30 
                 979.57 
                 84.73 
               
               
                   
                 972.75 
                 890.05 
                 83.70 
               
               
                   
                 774.62 
                 699.46 
                 78.15 
               
               
                   
                 588.06 
                 530.06 
                 58.00 
               
               
                 Total 
                 11270.60 
                 10404.00 
                 866.60 
               
               
                 % 
                   
                 92.31 
                 7.69 
               
               
                   
               
               
                 AUXI 
                 200.20 
                 161.56 
                 38.64 
               
               
                   
                 323.14 
                 283.11 
                 40.02 
               
               
                   
                 533.02 
                 533.02 
                 0.00 
               
               
                   
                 882.09 
                 882.09 
                 0.00 
               
               
                   
                 1226.58 
                 1226.58 
                 0.00 
               
               
                   
                 1508.94 
                 1508.94 
                 0.00 
               
               
                   
                 1206.73 
                 1153.47 
                 53.27 
               
               
                   
                 943.46 
                 882.75 
                 60.73 
               
               
                   
                 684.41 
                 628.99 
                 55.43 
               
               
                   
                 537.35 
                 491.40 
                 45.96 
               
               
                   
                 312.80 
                 281.35 
                 31.46 
               
               
                   
                 348.53 
                 306.19 
                 42.34 
               
               
                 Total 
                 8707.28 
                 8339.45 
                 367.82 
               
               
                 % 
                   
                 95.78 
                 4.22 
               
               
                   
               
            
           
         
       
     
     GMP Pooling Criteria for Post Q-Sepharose Fractions 
     Detail to be Specified in the Ion Exchange BMR 
     A. The following pooling criteria is to be specified for fractions from both the AUXI and AUXII peaks which have been analyzed by densitometry: 
     (i) A maximum of 16 fractions will be selected for analysis from each of the product peaks (AUXI and AUXII). This will limit the number of gels for densitometry analysis to 4. 
     (ii) Any fraction of purity greater than or equal to 90.00% (reported to 2 decimal places) can be pooled. 
     (iii) Any fraction with a single impurity greater than or equal to 9.00% (to 2 dp) cannot be pooled. 
     (iv) Fractions to be pooled must be from consecutive fractions. 
     B. The following pooling criteria is to be specified for fractions from the AUXII peak which have been analyzed by SEP-HPLC. 
     (i) The maximum number of samples to be submitted for SEC-HPLC is 10 and must be from the last fraction collected for this peak and consecutive fractions forward. 
     (ii) Any fraction with greater than or equal to 2.00% (to  2   dp ) aggregate cannot be pooled. 
     Details to be Recorded for Information Only. 
     A. The estimated theoretical purity of the pool should be calculated for information only and is expected to be: 
     Greater than or equal to 93.00% theoretical purity for AUXI; 
     Greater than or equal to 97.00% theoretical purity for AUXII; 
     B. The minimum quantity of protein in each pool should be noted to establish if criteria for excluding fractions with less that 0.5 g could be used in the future. 
     C. Fractions for the AUXI peak will be submitted for RP-HPLC but will be analyzed retrospectively and for information only. These data will NOT be considered as a part of the pooling criteria. 
     Expected Impact on Pooling 
     The following has been calculated from the average data set presented in table 51 to reflect the effect on yield and fraction selection following the new pooling criteria: 
     
       
         
           
               
               
               
             
               
                 TABLE 51 
               
               
                   
               
               
                   
                 AUXI 
                 AUXII 
               
               
                   
               
             
            
               
                 Fractions pooled from the 200 L  
                 #10-21 
                 #7-18 
               
               
                 engineering run 
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 Total protein determined for the post IEX  
                 13.18 
                 g 
                 8.37 
                 g 
               
               
                 pools from the 200 L engineering run 
                   
                   
                   
                   
               
            
           
           
               
               
               
            
               
                 Fraction which would be pooled following 
                 #11-21 
                 #9-16 
               
            
           
           
               
               
               
               
               
            
               
                 GMP criteria 
                   
                   
                   
                   
               
               
                 Estimated reduction in total protein due to 
                 0.95 
                 g 
                 1.18 
                 g 
               
               
                 fractions excluded by the GMP criteria 
                   
                   
                   
                   
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 52 
               
               
                   
               
               
                 Average relative quantities of product and impurities in  
               
               
                 the post IEX fractions from the 200 L Engineering run, 
               
               
                 as determined by densitometry analysis. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Quantity 
                 Relative quantity (%) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Fraction # 
                 loaded (μg) 
                 Product 
                 40 
                 90 
                 80 
                 55 
               
               
                   
               
               
                 AUXI (peak 2) 
                   
                   
                   
                   
                   
                   
               
               
                 3 
                 1 
                 96.91 
                 0 
                 7.43 
                 0 
                 3.66 
               
               
                 4 
                 1 
                 85.81 
                 0 
                 7.28 
                 0 
                 6.91 
               
               
                 5 
                 1 
                 54.57 
                 0 
                 6.78 
                 0 
                 8.65 
               
               
                 6 
                 1 
                 50.41 
                 0 
                 6.96 
                 0 
                 12.63 
               
               
                 8 
                 1 
                 80.00 
                 0 
                 5.46 
                 0 
                 14.54 
               
               
                 9 
                 1 
                 60.61 
                 0 
                 5.53 
                 0 
                 13.86 
               
               
                 10 
                 1 
                 89.53 
                 0 
                 4.20 
                 0 
                 7.27 
               
               
                 11 
                 1 
                 90.43 
                 0 
                 4.19 
                 0 
                 5.30 
               
               
                 13 
                 1 
                 94.68 
                 0 
                 4.35 
                 0 
                 0.97 
               
               
                 14 
                 1 
                 94.16 
                 0 
                 4.94 
                 0 
                 0.96 
               
               
                 15 
                 1 
                 93.83 
                 0 
                 5.18 
                 0 
                 0.69 
               
               
                 16 
                 1 
                 93.67 
                 0 
                 5.83 
                 0 
                 0.60 
               
               
                 18 
                 1 
                 92.03 
                 0 
                 7.97 
                 0 
                 0 
               
               
                 19 
                 1 
                 91.40 
                 0 
                 8.00 
                 0 
                 0 
               
               
                 20 
                 1 
                 90.30 
                 0 
                 9.70 
                 0 
                 0 
               
               
                 21 
                 1 
                 90.14 
                 0 
                 9.86 
                 0 
                 0 
               
               
                   
               
               
                 AUXII (peak 1) 
                   
                   
                   
                   
                   
                   
               
               
                 3 
                 1 
                 68.48 
                 21.55 
                 9.97 
                 0 
                 0 
               
               
                 4 
                 1 
                 55.51 
                 36.8 
                 7.69 
                 0 
                 0 
               
               
                 5 
                 1 
                 55.90 
                 36.73 
                 4.96 
                 2.41 
                 0 
               
               
                 6 
                 1 
                 67.59 
                 25.09 
                 4.53 
                 2.79 
                 0 
               
               
                 7 
                 1 
                 80.70 
                 12.33 
                 4.41 
                 2.55 
                 0 
               
               
                 8 
                 1 
                 87.61 
                 5.18 
                 4.58 
                 2.62 
                 0 
               
               
                 9 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 10 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 11 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 12 
                 1 
                 100 
                 0 
                 0 
                 0 
                 0 
               
               
                 13 
                 1 
                 95.59 
                 0 
                 2.92 
                 1.49 
                 0 
               
               
                 14 
                 1 
                 93.56 
                 0 
                 4.12 
                 2.31 
                 0 
               
               
                 15 
                 1 
                 91.90 
                 0 
                 5.05 
                 3.05 
                 0 
               
               
                 16 
                 1 
                 91.46 
                 0 
                 4.85 
                 3.67 
                 0 
               
               
                 17 
                 1 
                 89.95 
                 0 
                 5.57 
                 4.48 
                 0 
               
               
                 18 
                 1 
                 87.85 
                 0 
                 7.03 
                 5.11 
                 0 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 53 
               
               
                   
               
               
                 Theoretical relative amounts of product and impurities 
               
               
                 in the post IEX pools from the 200 L Engineering run, 
               
               
                 as determined by densitometry analysis, the fractions 
               
               
                 pooled are highlighted in red. the theoretical average 
               
               
                 product purity is calculated as 92.3% and 95.8% for the 
               
               
                 AUXI and AUXII intermediates respectively. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Product 
                   
               
               
                   
                 Total protein 
                 (AUXI) 
                 Quantity of all 
               
               
                 AUXI 
                 quantity (mg) 
                 quantity (mg) 
                 impurities (mg) 
               
               
                   
               
               
                 #10 
                 954.09 
                 844.67 
                 109.42 
               
               
                 #11 
                 1290.61 
                 1167.07 
                 123.55 
               
               
                 #13 
                 1524.29 
                 1443.16 
                 81.13 
               
               
                 #14 
                 1507.35 
                 1418.46 
                 88.89 
               
               
                 #15 
                 1339.47 
                 1258.18 
                 81.29 
               
               
                 #16 
                 1256.06 
                 1175.32 
                 80.74 
               
               
                 #18 
                 1063.3 
                 978.57 
                 84.73 
               
               
                 #19 
                 972.75 
                 889.05 
                 83.7 
               
               
                 #20 
                 774.62 
                 699.46 
                 75.15 
               
               
                 #21 
                 588.06 
                 530.06 
                 58 
               
               
                 Total 
                 11270.6 
                 10404 
                 866.6 
               
               
                 % 
                   
                 92.31 
                 7.69 
               
               
                   
               
               
                   
                   
                 Product 
                   
               
               
                   
                 Total protein 
                 (AUXII) 
                 Quantity of all 
               
               
                 AUXII 
                 quantity (mg) 
                 quantity (mg) 
                 Impurities (mg) 
               
               
                   
               
               
                 #7  
                 200.20 
                 161.56 
                 38.64 
               
               
                 #8  
                 323.14 
                 283.11 
                 40.02 
               
               
                 #9  
                 533.02 
                 533.02 
                 0 
               
               
                 #10 
                 882.09 
                 882.09 
                 0 
               
               
                 #11 
                 1226.58 
                 1226.58 
                 0 
               
               
                 #12 
                 1508.94 
                 1508.94 
                 0 
               
               
                 #13 
                 1206.73 
                 1153.47 
                 53.27 
               
               
                 #14 
                 943.48 
                 882.75 
                 60.73 
               
               
                 #15 
                 584.41 
                 628.99 
                 55.43 
               
               
                 #16 
                 537.35 
                 491.4 
                 45.95 
               
               
                 #17 
                 312.8 
                 281.35 
                 31.45 
               
               
                 #18 
                 348.53 
                 306.19 
                 42.34 
               
               
                 Total 
                 8707.28 
                 8339.45 
                 367.82 
               
               
                 % 
                   
                 95.78 
                 4.22 
               
               
                   
               
            
           
         
       
     
     A comparison of 2 data sets (i.e. the same in-process samples run on different gels by different operators) allowed the following retrospective pooling criteria to be noted for the average data set although one additional fraction (fraction 27 from AUXI) would be included from those actually pooled in the 20 L run: 
     AUXI 
     Pool all fractions with a purity of ≥87% but which do not have a single impurity of ≥10%. 
     AUXII 
     Pool all fractions with a purity of ≥94% but which do not have a single impurity of ≥4%. 
     QC data from the analysis of the final material from the 20 L demonstration run showed that the AUXII intermediate was 99.4% pure, the AUXI intermediate was 99.1% pure and the drug substance was 99.9% collagenase by RP-HPLC. Therefore, the criteria specified for the pooling process would be expected to result in material that passes the release specifications for the final material. 200 L demonstration run 
     The criteria established for the 20 L demonstration run previously mentioned was different to that implemented for the 200 L engineering run. In this case, pooling was specified for both the AUXI and AUXII products as fractions with a purity of ≥86.5% but which did not have a single impurity contaminant of ≥10%. AUXII samples with an impurity level ≥2% detected by SEC-HPLC for were also excluded. The resulting AUXI/AUXII intermediates and drug substance were also analyzed by densitometry, using a standardized method, and shown to have the following estimated purity based on analysis of a single gel 3 times (3 different operators): AUXI=96.0% (3.1% of 90 kDA contaminant); AUXII−98.7% (1.2% of 90 kDa contaminant); DS=97.6% (2.1% of 90 kDa contaminant). 
     In addition the QC determined purity of the intermediates and drug substance was show to pass specification by the RP-HPLC assay (AUXI=98.2%; AUXII=98.1%; drug substance=99.4%). Consequently, the pooling criteria followed for the 200 L engineering run was successful in delivering product of suitable purity based on the current available analytical methods. 
     Materials and Methods 
     MUSTANG Q Chromatography (20 L scale run) 
     Equipment: 
     MUSTANG Q Chromatography Capsule, 60 mL (CL3MSTGQP1, Pall) 
     Conductivity and pH Meter 4330 (Jenway) 
     Chemicals: 
     Sodium chloride (USP grade, Merck)
 
Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)
 
Tris (hydroxymethyl) methylamine (USP grade, Merck)
 
Ammonium sulphate (Extra Pure, Merck)
 
     Hyclone Water for Injection—Quality Water (WFI-QW) 
     A 60 mL bed volume MUSTANG Q chromatography capsule was sanitized with 1M NaOH at a flow rate of 30 mL·min for 30 minutes. The capsule was then preconditioned for the same time and flow-rate using 1M NaCl. the capsule was equilibrated with 2 L of MUSTANG Q Equilibration buffer (10 mM Tris, 1M ammonium sulphate, pH 8), at a flow rate of 60 mL/min. The outlet flow was checked to ensure pH was Supernatant (22 L) from 200 L fermentation PP3 (which had been 0.2 μm filtered) was loaded onto the capsule at a flow rate of 540 mL/min (approximately 40 min. duration). The maximum recommended operating flow rate for the capsule was 600 mL/min. the filtered material was stored in 2×10 L Stedim bags at 2-8° C. overnight. 
     Hydrophobic interaction chromatography (20 L scale run) 
     Equipment: 
     AKTA Pilot installed with Unicorn V 5.01 software (GE Healthcare)
 
Vantage S130 column (cross sectional area 125 cm2, Millipore)
 
     Conductivity and pH Meter 4330 (Jenway) 
     Sartopore 2 0+0.45 μm filter capsule (Sartorius) 
     Medical Refrigeration Unit MP150 (Electrolux) 
     Chemicals: 
     Phenyl Sepharose 6 FF low sub (GE Healthcare) 
     Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)
 
Sodium Chloride (USP grade, Merck)
 
Tris(hydroxymethyl)methylamine (USP grade, Merck)
 
Ammonium sulphate (Extra Pure, Merck)
 
     Leupeptin (MP Biomedicals, Inc) 
     Hyclone Water for Injection—Quality Water (WFI-QW) 
     HIC column packing 
     2400 mL of Phenyl Sepharose 6 FF Low Sub (Lot #312089) slurry was settled for 3 hours and the ethanol removed and replaced with 1800 mL WFI. The media was reslurried (50%), settled and washed once with WFI and twice with 1800 mL 200 mM NaCl, with settling overnight between washes. The media was reslurried with 1800 mL 200 mM NaCl, poured into the column and allowed to settled for 1 h. The adaptor was brought down to ˜1 cm above the resin bed (removing all air bubbles) and the media packed in 200 mM NaCl at a flow rate of 400 mL·min (192 cm/hr) for 10 mins. This packing flow rate was utilized as equivalent to the maximum operating flow rate for the K-prime system available in GMP. The adaptor was brought down to the top of the bed and the column packed at 192 cm/hr for 10 mins before screwing the adaptor into the top of the bed and the column packed at 192 cm/hr for a further 10 mins, during which no compression of the resin was observed. the pack test was carried out using the AKTA Pilot method: HIC 1500 mL Pack Test. For this, the column was equilibrated with 1 column volume (CV) of 200 mM NaCl in WFI and pack tested with 15 mL (1% CV) of 1M NaCl in WFI at a flow rate of 313 mL/min (150 cm/hr). The column was flushed with 2CV WFI and stored with 2CV 10 mM NaOH. The packed column had an asymmetry of 1.2, a plate count of 2659 plates/meter, a CV of 1525 mL and a bed height of 12.2 cm. 
     Column Sanitisation and Equilibration 
     The Phenyl Sepharose 6 FF (low sub) column was sanitized with 0.5M NaOH for 60 minutes, washed with 2 column volumes (CV) WFI and equilibrated with 5CV 10 mM Tris, pH 8 (HIC Buffer B) followed by 5CV 10 mM Tris, 1.0M ammonium sulphate, pH 8 (HIC Buffer A). 
     Preparation of the HIC Load 
     13.48 kg (11.05 L) of 3.0M ammonium sulphate, 10 mM Tris, pH 8 was added to 22.1 kg fermentation filtrate after MUSTANG Q filter treatment (section 3.1). the filtrate was mixed for 5 minutes before filtering through a 0.05 m2 filter capsule (0.8+0.45 μm). The filtered material (denoted the HIC load material) was stored on ice (approximately 30 minutes duration) until use. 
     HIC Column Run 
     The HIS run was performed at a constant linear flow rate of 150 cm/hour using chilled buffers maintained at 2-8° C. 30 L feedstock (equivalent to 20 L post MUSTANG Q filtrate) was loaded onto the 1525 mL Phenyl Sepharose 6FF (low sub) column previously equilibrated with 2CV 10 mM Tris, 1.0M ammonium sulphate, pH8 (HIC Buffer A2). Unbound material was washed off the column with 10CV HIC buffer A. the column was then washed with 5 CV 10 mM Tris, pH 8 (HIC Buffer B). The first 0.67 CV (1 L) of the elution buffer was discarded and a post-HIC pool of 4CV was collected, Leupeptin was added (126.4 mL) to the post-HIC pool (6191.3 g) to a final concentration of 200 μM from a stock solution of 10 mM leupeptin, 10 mM Tris, pH 8. the mixed solution (6.3 kg) was stored at 2-8° C. for 2 days before further processing by tangential flow filtration. 
     Tangential Flow Filtration step 1 (TFF1 20 L scale run) 
     Equipment: 
     ProFlux M12 TFF system (Millipore)
 
Conductivity and pH meter 4330 (Jenways)
 
Sartopore 2 0.8+0.45 μm filter capsule (Sartorius)
 
Pellicon 2 “Mini” Filter 0.1 mz 30 kDa MWCO PES membranes (Millipore)
 
     Medical Refrigeration Unit MP150 (Electrolux) 
     Materials/Chemicals 
     Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)
 
Tris(hydroxymethyl)methylamine (USP grade, Merck)
 
     Leupeptin (MP Biomedicals, Inc.) 
     Hyclone Water For Injection Quality Water (WFI-QW) 
     System Step Up 
     The ProFlux M12 TFF system was set up according to the manufacturer&#39;s instructions with two Pellicon 2 “Mini” Filter 30 kDa MWCO PES membranes, sanitised with 0.5M NaOH for 60 minutes and stored in 0.1M NaOH until use. The system was drained and flushed with 14 L WFI and the normal water permeability (NWP) measured as 23 L/m2/hr/psi at 25° C. at a trans-membrane pressure (TMP) of 15 sig (inlet pressure of 20 psig and outlet pressure of 10 psig). The system was flushed with 0.5 L 10 mM Tris, pH 8 (diafiltration buffer) and equilibrated with 1 L of the same buffer for 10 minutes. The conductivity of pH of the permeate was determined and checked against that of the diafiltration buffer to ensure the membranes were equilibrated prior to use. 
     Concentration and Diafiltration 
     The concentration and diafiltration steps were performed with chilled diafiltration buffer (10 mM Tris, pH 8) containing 200 μM leupeptin. the TFF system was flushed with 1 L chilled buffer just before use. 2 L of the post-HIC material (6.3 L total volume) was pumped into the TFF system reservoir and recirculated for 0 minutes without back-pressure to condition the membrane. the level sensor on the reservoir was set to 1.2 L and the post-HIC material concentrated at a TMP of 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig) until all the material had entered the system. the permeate was collected and stored at 2-8° C. for analysis. The inlet tubing was connected to the diafiltration buffer and diafiltration of the material was performed at a TMP of 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig) for approximately 8.5 turnover volumes (TOV), maintaining the volume of material in the reservoir at 1.2 L. The conductivity and pH of the permeate was determined after 5, 7 and 8.5 TOV and checked against the diafiltration buffer. The retentate was drained from the system and stored at 2-8° C., 250 mL diafiltration buffer was pumped into the reservoir, recirculated around the system for 10 minutes without backpressure to rinse the system, drained, the rinse repeated and both rinses were stored separately at 2-8° C. The protein concentration of the retentate and rinses were determined (by UV) and the first rinse (204.8 g weight) added to the retentate (1231.4 g weight). this post TFF1 material (1.4 kg) was then filtered through a Sartopore 2.0+0.45 μm filter capsule and stored at 2-8° C. overnight until further processing by Q Sepharose ion exchange chromatography. 
     Ion Exchange Chromatography (20 L Scale Run) 
     Equipment: 
     AKTA Pilot installed with Unicorn 5.01 software (GE Healthcare) 
     Conductivity and pH Meter 4330 (Jenway) 
     Vantage S90 Column (cross sectional area 62 cm 2 , Millipore) 
     Medical Refrigeration Unit MP150 (Electrolux) 
     Chemicals: 
     Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)
 
Sodium chloride (USP grade, Merck)
 
Tris(hydroxymethyl)methylamine (USP grade, Merck)
 
Calcium chloride 2-hydrate (USP grade, Merck)
 
     Leupeptin (MP Biomedicals, Inc.) 
     Q Sepharose HP (GE Healthcare) 
     Hyclone Water For Injection Quality Water (WFI-QW) 
     Column Packing and Preparation 
     A Vantage S90 column was packed using an AKTA Pilot chromatography system with Q Sepharose HP media in WFI to give a packed column with a 10 cm bed height, therefore a column volume (CV) of 620 mL. The packing was performed in accordance to the manufacturers instruction but with the pressure limit of the Vantage column imposed (0.3 MPa) which equated to a packing flow rate of 210 cm/hr and pressure limit of 0.28 MPa. After packing, the column was equilibrated with 2CV of 0.2M NaCl and pack tested with 1% CV (6.2 mL) 1M NaCl at a flow rate of 100 cm/hr (103 mL/min). The packed column had an asymmetry of 1.6 and a plate count of 12605 plates/meter, which was within specification for the media (asymmetry between 0.8 and 1.8, with a plate count &gt;10,000). The column was stored in 10 mM NaOH until required. 
     Prior to use, the Q Sepharose column was washed with 1.5 column volumes (CV) of WFI to remove the storage buffer, sanitised with 0.5M NaOH for 60 mins at 40 cm/hr before flushing again with 1.5CV WFI. The column was then changed and equilibrated in accordance to the manufacturers instructions with 2CV 10 mM Tris, 3 mM calcium chloride, 8 pH followed by 2CV 10 mM Tris, 3 mM CaCl 2 , 360 mM NaCl, pH 8 and finally 5CV 10 mM Tris, 3 mM CaCl 2 , pH 8. 
     Column Run 
     Immediately prior to the sample being loaded onto the column, the column was reequilibrated with chilled 10 mM Tris, 3 mM CaCl 2 , 200 μm leupeptin pH 8 (IEX Buffer A). 1216 mL of chilled post TFF 1 material at a concentration of 2.55 mg/mL (determined by UV) was loaded onto the column at a flow rate of 100 cm/hr (103 mL/min). This equated to a column load of 5 mg total protein per mL of media. Following loading of the product, the column was washed with 3 column volumes (CV) of IEX Buffer A and the protein eluted with 10 mM Tris, 3 mM CaCl 2 , 200 μm leupeptin pH 8 (IEX Buffer B) with a gradient of 0-40% elution buffer (A to B), over 20CV at a flow rate of 70.2 ml/min (68 cm/hour). Elution was monitored at 280 nm and 260 nm and 100 mL fractions collected across the two product peaks containing AUX II and AUX I. Fraction collection was started from the breakthrough of the peak and continued until 25% of the peak height on the trailing edge. A total of 12 fractions were collected across until 25% of the peak height on the trailing edge. A total of 12 fractions were collected across the AUX II peak and 15 fractions across the AUX I peak. The Q Sepharose HP chromatography was carried out at a standard laboratory temperature of 18-23° C., although the buffers used were pre-chilled. Fractions were stored at 2-8° C. until a result was obtained from the SDSPAGE analysis. Fractions 6 to 12 (peak 1) were pooled as AUX II collagenase with the volume determined as 683 g (after sampling) and the concentration by UV analysis measures as 1.17 mg/mL. Fractions 19 to 26 (peak 2) were pooled as AUX I collagenase with the volume determined as 796 g (after sampling) and the concentration by UV analysis measures as 1.08 mg/mL. 
     Tangential Flow Filtration Step 2 (TFF2 20 L Scale Run) 
     Equipment: 
     ProFlux M12 TFF system (Millipore)
 
Conductivity and pH meter 4330 (Jenways)
 
Pellicon 2 “Mini” Filter 0.1 mz 30 kDa MWCO PES membranes (Millipore)
 
90 mm Filter Unit (1 L) 0.2μ PES membrane (Nalgene)
 
     Medical Refrigeration Unit MP150 (Electrolux) 
     Materials/Chemicals 
     Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)
 
Tris(hydroxymethyl)methylamine (USP grade, Merck)
 
Sucrose (BP grade, Merck)
 
     Leupeptin (MP Biomedicals, Inc.) 
     Hyclone Water For Injection Quality Water (WFI-QW) 
     Frensius Kabi Water for Injection (WFI) 
     System Set Up 
     The ProFlux M12 TFF system was set up according to the manufacturer&#39;s instructions with one Pellicon 2 “Mini” Filter 30 kDa MWCO PES membrane, sanitized with 0.5M NaOH for 60 minutes and stored in 0.1M NaOH until use. The system was drained and flushed with 14 L WFI and the normal water permeability (NWP) measured as 19.5 L/mz/hr/psi for the membrane used for AUXI and as 14.5 L/mz/hr/psi at 25° C. for the membrane used for AUXII at 25° C. and at a trans-membrane pressure (TMP) of 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig). The system was flushed with 0.5 L 10 mM Tris, 60 mM sucrose, pH8 (formulation buffer), and equilibrated with 1 L of the same buffer for 10 minutes. The conductivity and pH of the permeate was determined and checked against that of the formulation buffer. 
     Concentration and Formulation 
     The concentration and diafiltration steps were performed separately on each of the post IEX pools of AUXI and AUXII. All steps were performed using chilled formulation buffer (10 mM Tris, 60 mM sucrose, pH 8) maintained at 2-8° C. The TFF system was flushed with 1 L chilled buffer just before use. The post-IEX pool (683 g weight of AUXII and 796 g weight of AUXI) was pumped into the TFF system reservoir and recirculated at 10% pump speed for 10 minutes without backpressure to condition the membrane. The level sensor on the reservoir was set to approximately 400 mL and the AUXI or AUXII pool concentrated at a TMP of 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig) until the volume in the reservoir had been reduced to approximately 360-390 mL (this assumed a system hold up volume of 100 mL). The target volume reduction was based on achieving a theoretical concentration of 1.75 mg/mL for the product assuming no loss in protein during the concentration operation. The permeate was collected and stored at 2-8° C. for analysis. For the diafiltration operation the inlet tubing was connected to the formulation buffer and diafiltration performed at a TMP of 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig). Approximately 12 turnover volumes (TOV) were performed for AUXII and 8.5 TOV&#39;s for AUXI, maintaining the volume of material in the reservoir at ˜400 mL. The conductivity and pH of the permeate was determined after 12 TOV for AUXII and after 6, 7, and 8.5 TOVs for AUXI and checked against that of the formulation buffer. The retentate was drained from the system and stored at 2-8° C. 250 mL formulation buffer was used to wash residual product from the membranes by re-circulated around the system for 10 minutes (without backpressure). After draining the rinse solution, a second wash was performed and both rinse 1 and rinse 2 were stored at 2-8° C. After UV protein content determination of the retentate and rinses, the first rinse was added to the retentate, mixed and a UV protein concentration of the mix determined. For AUXII, 122 g of the second rinse was also added to the retentate plus rinse 1 to give a theoretical AUXII concentration of 1.1 mg/mL. For AUXI, 94 g of the second rinse was added to the material to give a theoretical AUXI concentration of 1.1 mg/mL. Both the AUXI and AUXII material were filtered through a 1 L Nalgene 0.2 μm filter unit in a Class II hood and the post filtered protein concentration determined. The AUXI and AUXII intermediates were stored at 2-8° C. 
     Protein Concentration Determination 
     Absorbance 
     Equipment: 
     DU800 Spectrophotometer (Beckham) 
     In process samples were analyzed by UV spectrophotometry by performing a UV scan of samples between 220 and 330 nm. The appropriate buffer was used as a blank and a scan of the buffer blank performed before scanning the samples. If necessary, samples were diluted with the same buffer to ensure the A280&lt;1.0 AU. Protein concentrations (mg/mL) were determined according to the Beer-Lambert law, c=A/b −ε , where A is the absorbance (A 280 -A 330 ), b is the pathlength (1.0 cm) and ε is the extinction coefficient of the protein. Extinction coefficients of 1.48 mg- 1 cm- 1 mL for AUXI, 1.576 mg- 1 cm- 1 mL for AUXII and 1 428 mg- 1 cm- 1 mL for an AUXI/AUXII mix were used. 
     Bradford Assay 
     Materials 
     Lyophilised BSA (hydrated to 1.4 mg/mL) 
     Chemicals: 
     Protein Assay Dye Reagent Concentrate (500-0006, Bio-Rad) 
     A BSA standard curve was prepared by diluting the BSA with water, to known concentrations. The Bio-Rad protein assay dye reagent was prepared by diluting one part concentrate with four parts water. Test samples were prepared by diluting with water. 50 μL of test sample either neat or diluted was added to a cuvette and 2.5 mL diluted regent added. Samples were prepared in duplicate. The samples were incubated for 10 minutes before reading the OD. The standard curve of OD 595 nm  vs. protein concentration was obtained by measuring the OD 595 nm  of BSA solutions of known concentration. The test samples were then assayed and the protein concentration determined from the standard protein assay curve. Samples from the post MUSTANG Q step were always analyzed without dilution in order to standardize the contribution from the pigment. In this case, 50 μL of the undiluted post MUSTANG Q material was utilized in the assay. 
     SDS-PAGE Analysis 
     Equipment: 
     Xcell SureLock Mini-Cell Electrophoresis System (Invitrogen) 
     Electrophoresis Power Supply EPS 601, (Amersham Pharmacia Biotech) 
     Rocky shaker platform, (Scientific Laboratory Supplies) 
     Chemicals: 
     SDS-PAGE Standards High Molecular Weight (161-0303, Bio Rad) 
     Mark12 Unstained Standard (LC5677, Invitrogen) 
     Novex 8% Tris-Glycine gels, 1.5 mm, 10 well (EC6018BOX, Invitrogen)
 
NuPAGE Novex 4-12% Bis-Tris gels, 1.0 mm, 12 well (NP0322BOX, Invitrogen)
 
     Novex Tris-Glycine SDS Running Buffer (10×) (LC2675, Invitrogen) 
     NuPAGE MES SDS Running Buffer (20×) (NP0002, Invitrogen) 
     Novex Tris-Glycine SDS Sample Buffer (2×) (LC2676, Invitrogen) 
     NuPAGE LDS Sample Buffer (4×) (NP00007, Invitrogen) 
     NuPAGE Sample Reducing Agent (10×) (NP0009, Invitrogen) 
     Colloidal Blue Staining kit (LC6025, Invitrogen) 
     Ethylenediaminetetra-acetic acid disodium salt AnalaR R (BDH) 
     Tris-Glycine Gels 
     Samples were prepared for reducing SDS-PAGE by adding 12 μl of sample to 20 μl sample Buffer (2×), 4 μl reducing agent (10×) and 4 μl of 0.1M EDTA (to achieve final concentration of 10 mM). The high molecular weight (HMW) marker was prepared by adding 10 μl of concentrated stock to 80 μl reducing agent (10×), 310 μl WFI and 400 μl sample buffer (2×). The diluted HMW standard was then heated at 95° C. for 5 minutes before aliquoting and storage at −20° C. for use in subsequent gels. Samples (20 μl load volume) containing collagenases were run directly (i.e. with no prior heat treatment) on 8% Tris-Glycine gels using Tris-Glycine running buffer at 130V for ˜2 hours. After electrophoresis, the gels were stained with colloidal blue stain reagent as per the manufacturer&#39;s instructions. 
     Bis-Tris Gels 
     Samples were prepared for reducing SDS-PAGE by adding 16.50 of sample to 7.5 μl sample buffer (4×), 3 μl reducing agent (10×) and 3 μl of 0.1M EDTA (to achieve final concentration of 10 mM). MARK 12 marker loaded neat GOO). Samples (15 μl load volume) containing collagenases were run directly (i.e. with no prior heat treatment) on 4-12% Bis-Tris gels using either MES running buffer at 200V for ˜40 mins. After electrophoresis, the gels were stained with either colloidal blue stain reagent as per the manufacturer&#39;s instructions or silver stained using a standard procedure (GE Healthcare). 
     Densitometry analysis of post-IEX fractions 
     Equipment: 
     Xcell SureLock Mini-Cell Electrophoresis System (Invitrogen) 
     Electrophoresis Power Supply EPS 601, (Amersham Pharmacia Biotech) 
     Rocky shaker platform, (Scientific Laboratory Supplies) Flatbed scanner (Hewlett Packard) 
     Materials/Chemicals: 
     NuPAGE Novex 4-12% Bis-Tris gels, 1.0 mm, 12 well (NP0322BOX, Invitrogen) 
     NuPAGE MES SDS Running Buffer (20×) (NP0002, Invitrogen) 
     NuPAGE LDS Sample Buffer (4×) (NP00007, Invitrogen) 
     NuPAGE Sample Reducing Agent (10×) (NP0009, Invitrogen) 
     Mark12 Unstained Standard (LC5677, Invitrogen) 
     Colloidal Blue Staining kit (LC6025, Invitrogen) 
     Ethylenediaminetetra-acetic acid disodium salt (EDTA) (AnalaR, BDH)
 
Purified water
 
     Reducing SDS-PAGE 
     The post-IEX samples were run on 4-12% Bis-Tris gels using MES running buffer at 1 μg/lane loading. Samples were prepared by adding 20 μL of diluted post-IEX material to 8 μL Sample Buffer (4×), 3 μL Reducing Agent (10×) and 3.4 μL of 0.1M EDTA. 15 μL of each sample was loaded into the well directly after mixing (i.e. with no heat treatment) and run at 200V for 40 mins. After electrophoresis, the gels were stained with Colloidal Blue stain reagent according to the manufacturers instructions but with a fixed staining duration to reduce staining variation (10 minute fix, 5 hours stain, 15-20 hours destain with purified water). 
     Gel Scanning and Densitometry 
     Gels were placed between 2 sheets of acetate ensuring removal of all air bubbles, scanned on a flat-bed scanner at 600 dpi resolution and the image cropped, resized and colour corrected with HP Image zone software. The image was converted to an 8-bit greyscale TIFF image with Alpha EaseFC software and the protein bands were analysed using QuantityOne gel documentation software (BioRad). After background substitution, the intensity peak areas of selected bands were converted to relative percentage values of product (AUXI or AUXII) and impurity(s) in each lane. 
     Buffer Stability 
     Equipment: 
     Peristaltic Pump (Watson Marlow) 
     125 ml PETG biotainers (Cellon)
 
Watson Marlow Tubing for peristaltic pump
 
     Conductivity and pH Meter 4330 (Jenway) 
     Sartopore 2 300 (0.45/0.2 μm) filter capsule (Sartorius) 
     Buffers for the 20 L demonstration run were filtered after preparation through a 0.45/0.2 μm filter capsule into 10 or 20 L Stedim bags for storage at 2-8° C. prior to use. When the majority of the buffer had been filtered, approximately 75 mls of the remaining buffer was collected into pre-labelled 125 ml PETG biotainers and stored at 2-8° C. The pH, conductivity, temperature and date of buffer preparation were recorded. On completion of the 20 L demonstration run, the buffer samples were retrieved from cold storage and retested for pH, conductivity, and appearance. The temperature of the buffer at the time of testing was also recorded. 
     Preparation of samples for N-terminal sequencing analysis 
     Equipment: 
     Electrophoresis Power Supply EPS 601, (Amersham Pharmacia Biotech) 
     Xcell SureLock Mini-Cell Electrophoresis System, (Invitrogen) 
     Rocky shaker platform, (Scientific Laboratory Supplies) 
     Chemicals: 
     Novex 8% Tris-Glycine gels, 1.5 mm, 10 well, (Invitrogen) 
     High Molecular Wight Marker, (BioRad) 
     NuPAGE Sample Reducing Agent (10×), (Invitrogen) 
     Novex Tris-Glycine SDS Running Buffer (10×), (Invitrogen) 
     Novex Tris-Glycine SDS Sample Buffer (2×), (Invitrogen) 
     Colloidal Blue Staining kit, (Invitrogen)
 
Ethylenediaminetetra-acetic acid disodium salt (EDTA) (AnalaR, BDH)
 
     Methanol, AnalaR (BDH) 
     Acetic Acid, AnalaR (BDH) 
     Water for injection (WFI) 
     Purified Water 
     Samples for N-terminal sequencing were prepared and separated on 8% Tris-Glycine gels as outlined previously. Samples identified as enriched for the 40 kDa contaminant (fraction 2 from the post IEX AUXII peak, CTL2006#0610H;) and 55 kDa contaminant (fraction 16 from the post IEX AUXI peak, CTL2006#061111) were each loaded in 5 lanes of the gel to provide enough material for sequencing ( FIG.  89   ). Post JEX fractions from a previous 20 L fermentation (20 L PP3), which were enriched for the 90 kDa contaminants associated with both AUXI (fraction B7 R2, CTL2006#0581P) and AUXII (fraction D1, CTL200#0582P) were also loaded in multiple lanes ( FIG.  90   ). After electrophoresis, the gels were stained with colloidal blue stain reagent according to the manufacturers instructions and the contaminant bands excised and submitted to Alta Bioscience (Birmingham University, UK) for N-terminal sequencing. The 90 kDa AUXI associated contaminant (CTL200#0612H) from the 20 L demonstration run was also submitted for sequencing but no data was obtained. 
     Summary of the Manufacturing of Process 3 
     Fermentation 
     The Phytone fed-batch fermentation process (Process 2) for production of collagenase from  Clostridium histolyticum  has been shown to be highly variable due to batch-to-batch variability in the Phytone peptone. For this reason Proteose Peptone #3 (PP3) was evaluated in 5 L fermentations. The evaluation demonstrated that when one specific batch of PP3 was used at 50 g/L the fermentation process was robust and reproducible. However when other batches of PP3 were employed at 50 g/L large variations were seen in the growth profiles of the cultivations. The maximum biomass concentration the various batches of PP3 would support were assessed in a small scale evaluation. These batches were deemed “good” or “poor” based on their ability to support high or low biomass concentrations of  C. histolyticum  respectively. When two fermentations were carried out at 5 L scale with “poor” and “good” batches of PP3 at 100 g/L both demonstrated highly similar growth profiles and product yields. This experiment showed that increasing the concentration of PP3 to 100 g/L alleviated the problem associated to batch to batch variation in the peptone. 
     A scale up fermentation was carried out at 200 L. The fermentation used the optimized concentration of PP3 (100 g/L). The fermentation was successful and replicated both the growth profile and the product yield/quality observed at the 5 L scale. The harvest process (clarification by filtration) developed for Process 2 was evaluated during the 200 L scale up fermentation. The cell culture was successfully clarified using the existing process with no blockage of the filtration train. 
     The quantification of collagenase concentration in crude fermentation samples was improved using densitometry analysis of Coomassie stained Tris Glycine gels. A standard curve of mixed AUXI and AUXII was loaded with dilutions of fermentation samples. The relationship between collagenase concentration and densitometry peak area was shown to be linear within the range of the sample dilutions. The concentrations of collagenase in the samples were then extrapolated using their peak area and the standard curve. This method estimated the yield of collagenase to be 280-350 mg/L from the 100 g/L PP3 process at 5 and 200 L scale. 
     The optimized PP3 fermentation process generated a higher biomass concentration (OD600 7 units) and increased the product yield (280-350 mg/L total collagenase, by quantitative densitometry) when compared to the Phytone fed-batch process. The ratio of AUXI:AUXII was closer to 1 compared to that observed during evaluation of Process 2. In summary the PP3 process increased the product yield, purity (post-fermentation) and reproducibility of the fermentation. 
     Purification 
     Process 3 was developed in an accelerated time frame in order to improve the processes previously developed at Cobra (Process 2) and run at 20 L scale in GMP. Major improvements to the process were made in order to simplify the purification procedure, facilitate robustness as well as make the process more amenable to scale up to 200 L. These improvements were also considered key to assisting process validation. 
     Process 3 was performed using material from a 200 L fermentation of  Clostridium histolyticum  in which a full 20 L of fermentation was purified. Material was processed directly from the fermentation and no hold steps were implemented. Following filtration, product was passed through a MUSTANG Q filter since small-scale experiments demonstrated reduction of dsDNA (as detected by pico green analysis) using this procedure. Analysis of in-process samples from the 20 L demonstration run however, showed no reduction in dsDNA suggesting that the robustness and application of this step required further investigation. A comparison of the parameters used for the 20 L run-through and previous small-scale experiments demonstrated dsDNA removal when the capsule was oversized by a factor of 1000 (based on the DNA binding capabilities of 15-25 mg DNA/mL capsule described by the manufacturer). In comparison, the capsule used in the 20 L run-through was oversized by a factor of approximately 177-296. Material from the MUSTANG Q capsule was held overnight at 2-8° C. An off-line stability study on sample material taken at this stage in the process indicated that maintaining a low temperature was key to the product stability at this point in the process since samples incubated at RT and 37° C. were susceptible to degradation as indicated by SDS-PAGE analysis. 
     Product from the MUSTANG Q capsule was prepared for hydrophobic interaction chromatography (HIC) by the addition and mixing of an ammonium sulphate solution (3M) to achieve a final concentration of 1M. This provided conditions suitable for collagenase binding to Phenyl Sepharose FF (low sub) media. A proportion of protein contaminants and pigment were then eluted from the HIC column using a step elution of 0.3M ammonium sulphate followed by collagenase product elution with a solution containing no ammonium sulphate. Criteria for collection of the product peak were established as a fixed volume of 4 column volumes (although this was later extended to 5 column volumes for the 200 L scale demonstration run). Leupeptin was then added immediately following elution and the material held for a period of 2 days at 2-8° C. 
     The yield from this step was difficult to determine accurately due to the complex nature of the feedstock. The process step yield was estimated as (i) 38% based on Bradford assay of the load and UV of the eluted material or (ii) 47% based on collagenase content in the load estimated by densitometry and UV of the eluted material. Alternatively, 0.17 g of total protein was eluted from the HIC column for the equivalent of every 1 L of fermentation filtrate applied. 
     The post HIC pool was prepared for Q-Sepharose purification by concentration (5-fold) and buffer exchange using tangential flow filtration (TFF1) using 2×0.1 m2 30 kDa membranes. No loss was detected over this step and the reported increase in protein recovered may reflect the inaccuracy of UV at this point in the process. Inaccuracy could be attributed to pigment contamination or the use of the extinction coefficient for collagenases, which will be less accurate for material earlier in the purification when a complex of proteins are likely to be present. The TFF step was completed by a product filtration step before holding the material at 2-8° C. over night. 
     As with Process 2, the Q-Sepharose column was a key purification step in Process 3 and resulted in the separation of the AUXI and AUXII collagenases. The contaminants associated with process 3, however, were different to those in process 2 and appeared to closely co-purify with the AUXI and AUXII products. It was possible however, to remove the contaminants from the products by fractionation of the product peaks since the contaminants appeared to elute at either the leading or tail edges of both peaks. The contaminants were denoted by their relative molecular mass on reducing SDS-PAGE. Those associated with the AUXII product (the first peak eluted from the Q-Sepharose column) were identified as (i) 40 kDa (associated with the leading edge of the peak) and (ii) 75 kDa and 90 kDa (associated with the trailing edge of the peak). N-terminal amino acid sequencing indicated that the 40 kDa was AUXII related since the sequence matched identity with a region of the Col H sequence. In comparison, no identity could be confirmed for the 90 kDa contaminant due to issues of low signal. Contaminants associated with AUXI product (the second peak eluted form the Q-Sepharose column) were (i) 55 kDa (associated with the leading edge of the peak) and (ii) 90 kDa (associated with the trailing edge of the peak). N-terminal sequencing showed both the 55 kDa and 90 kDa contaminants to be identified as AUXI-related where the 55 kDa contaminant showed sequence identity with a mid region in the Col G sequence and the 90 kDa showed identical N-terminal match to AUXI. Consequently, the major impurities identified at this stage in the process were all product related and either identified as internal cleavage products of AUXI (55 kDa) and AUXII (40 kDa) or a C-terminally cleaved product of AUXI (90 kDa). 
     Following the Q-Sepharose column, a key process step was in the decision as to which fractions should go forward for further purification. For the 20 L demonstration run this criteria was based on the relative staining intensities of contaminants to product when analyzed by 4-12% SDS-PAGE and stained with Colloidal Blue stain. The decision was subjective and based on the collective experience of the process development group as well as requests from the client. In order to establish defined criteria that was described the pooling procedure, densitometry was performed on SDSPAGE. From this, the pooling was described as including fractions that were ≥87% pure (with no single impurity ≥10%) for AUXI and ≥94% pure (with no single impurity ≥4%) for AUXII. This resulted in a step yield based on UV estimation of 27.7% and 25.8% for AUXI and AUXII respectively. Further refinement and standardization of the densitometry method was achieved from data acquired from the 200 L scale demonstration run which resulted in definition of modified criteria for the subsequent GMP run. 
     Fractions containing AUXI or AUXII product from the Q-Sepharose column were formulated separately by TFF (denoted TFF2) using 1×0.1 m2 30 kDa membrane for each collagenase. The formulation buffer of 10 mM Tris, 60 mM sucrose pH 8, had been established by KBI BioPharma Inc. Product was filtered following the TFF2 step and the overall step yields for TFF and filtration estimated as 97.5% for AUXI and 92.2% for AUXII. At this stage samples were referred to as intermediates and were retained at 2-8° C. for QC analysis and prior to mixing of the drug substance. A retrospective stability study indicated the intermediates were stable over a period of at least 5 days at 2-8° C. as determined by SDS-PAGE, UV, RP-HPLC and SEC-HPLC analysis. The only detected deterioration in intermediates was identified in the AUXII intermediate after a 12 day hold in which aggregate levels increased from 0 to 0.62%. 
     The AUXI and AUXII intermediates were mixed in equal ratio (as determined by UV) to generate the drug substance before performing a final product filtration. Only 400 mg of drug substance was prepared of which 200 mg was shipped to KBI BioPharma Inc. along with 25 mg of each intermediate. The overall process yield was estimated for the 20 L demonstration run in which all available material from the 20 L of fermentation feedstock had been processed and assuming all material had been mixed as a drug substance. This gave a predicted yield of 1.6 g drug substance for the 20 L scale purification. This equated to a process recovery of 17.8% based on then assumption that the initial estimate of 9 g (using the Bradford assay) for the amount of total protein available to load onto the HIC column was accurate. Alternatively, if the total available protein was related to the collagenase content in the HIC load (as estimated by densitometry) the overall process yield was calculated as 22%. 
     In addition to the process run-through, some preliminary studies were preformed on sample and buffer retains taken from the process to access stability. These data indicated that for the product, low temperature was a key factor in controlling degradation and samples taken early in the purification (prior to the Q-Sepharose column) were more susceptible to proteolysis. A product hold study showed however, that the combination of leupeptin and temperature control (2-8° C.) was successful in maintaining the product quality over the time courses anticipated for the GMP process. 
     Tables 54 and 55 detailed the analytical specifications AUX-I and AUX-II intermediates and also for Drug Substance for Process 3. 
     
       
         
           
               
             
               
                 TABLE 54 
               
             
            
               
                   
               
               
                 Analytical Specifications for Process 3 AUX-I and AUX-II Intermediates 
               
            
           
           
               
               
            
               
                   
                 Specification 
               
            
           
           
               
               
               
            
               
                 Test 
                 AUX-I 
                 AUX-II 
               
               
                   
               
               
                 Appearance 
                 Clear colorless and free 
                 Clear colorless and free 
               
               
                   
                 from particulate matter 
                 from particulate matter 
               
               
                 *Endotoxin 
                 ≤10 EU/mL 
                 &lt;10 EU/mL 
               
               
                 Identity (and purity) by SDS- 
                 Major hand between 
                 Major hand between 
               
               
                 PAGE (Reduced conditions, 
                 98-188 kDa, and  
                 98-188 kDa, and  
               
               
                 Coomasie and silver stained) 
                 no minor bands 
                 no minor bands 
               
               
                 *Total Protein by Absorbance 
                 0.8-1.2 mg/mL 
                 0.8-1.2 mg/mL 
               
               
                 Spectroscopy 
                   
                   
               
               
                 SRC assay (AUX-I) 
                 12 000-21 000 fSRC 
                 Not applicable 
               
               
                   
                 units/mg 
                   
               
               
                 GPA assay (AUX-II) 
                 Not applicable 
                 370 000-680 000 fGPA 
               
               
                   
                   
                 units/mg 
               
               
                 Analysis of Proteins using 
                 ≥98% main peak 
                 ≥98% main peak 
               
               
                 the Agilent 1100 HPLC 
                   
                   
               
               
                 System (Aggregation by 
                   
                   
               
               
                 size exclusion chromatography) 
                   
                   
               
               
                 *Analysis of Proteins using the 
                 ≥97% by area 
                 ≥97% by area 
               
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Purity by reverse phase liquid 
                   
                   
               
               
                 chromatography) 
                   
                   
               
               
                 Analysis of Proteins using the 
                 ≤1% by area 
                 ≤1% by area 
               
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Residual gelatinase by anion 
                   
                   
               
               
                 exchange chromatography) 
                   
                   
               
               
                 Analysis of Proteins using the 
                 ≤1% by area 
                 ≤1% by area 
               
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Residual clostripain by 
                   
                   
               
               
                 reverse phase liquid 
                   
                   
               
               
                 Chromatography) 
                   
                   
               
               
                 Identity by Peptide Mapping 
                 Conforms to reference 
                 Conforms to reference 
               
               
                 Bioburden 
                 ≤CFU/mL 
                 ≤CFU/mL 
               
               
                   
               
               
                 *Tests required for provisional release of intermediates for further manufacturing 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 55 
               
             
            
               
                   
               
               
                 Analytical Specifications for Process 3 Drug Substance 
               
            
           
           
               
               
            
               
                   
                 Specificatioin 
               
            
           
           
               
               
               
            
               
                 Test 
                 AUX-I 
                 AUX-II 
               
               
                   
               
            
           
           
               
               
            
               
                 Appearance 
                 Clear colorless and essentially free 
               
               
                   
                 from particulate matter 
               
               
                 Potentiometric Measure of 
                 7.5 to 8.5 
               
               
                 pH of Solution 
                   
               
               
                 Endotoxin 
                 &lt;10 EU/mL 
               
            
           
           
               
               
               
            
               
                 Identity (and purity) by 
                 Major collagenase band 
                 Major collagenase band 
               
               
                 SDS-PAGE (Reduced 
                 between 98-188 kDa 
                 between 97-200 kDa; 
               
               
                 conditions Coomasi and 
                 MW markers 
                 MW markers; major bands 
               
               
                 silver stained) 
                   
                 comparable to reference 
               
               
                   
                   
                 standard 
               
            
           
           
               
               
            
               
                 *Total Protein by 
                 0.8-1.2 mg/mL 
               
            
           
           
               
               
               
            
               
                 Absorbance Spectroscopy 
                   
                   
               
               
                 *SRC assay (AUX-I) 
                 13 000-23 000 fSRC 
                 NA 
               
               
                   
                 units/mg 
                   
               
               
                 *GPA assay (AUX-II) 
                 NA 
                 200 000-380 000 fGPA 
               
               
                   
                   
                 units/mg 
               
            
           
           
               
               
            
               
                 Residual host cell protein 
                 Comparable to reference standard; no individual 
               
               
                   
                 impurity band exhibiting greater intensity than 1% 
               
               
                   
                 BSA intensity marker 
               
               
                 Residual host cell DNA 
                 ≤10 pg/dose 
               
               
                 Analysis of Proteins using the 
                 ≥98% main peak; ≤2% aggregates by area 
               
            
           
           
               
               
               
            
               
                 Agilent 1100 HPLC System 
                   
                   
               
               
                 (Aggregation by size 
                   
                   
               
               
                 exclusion chromatography) 
                   
                   
               
            
           
           
               
               
            
               
                 *Analysis of Proteins using 
                 2 major peaks (AUXI &amp; AUXII), 
               
               
                 the Agilent 1100 HPLC 
                 combined ≥97% by area; Retention times 
               
               
                 System (Identity and purity 
                 of AUX-I and AUX-II within 5% of reference 
               
            
           
           
               
               
               
            
               
                 by reverse phase liquid 
                   
                   
               
               
                 chromatography) 
                   
                   
               
            
           
           
               
               
            
               
                 Analysis of Proteins using 
                 ≤1% by area 
               
            
           
           
               
               
               
            
               
                 the Agilent 1100 HPLC 
                   
                   
               
               
                 System (Residual clostripain 
                   
                   
               
               
                 by reverse phase liquid 
                   
                   
               
               
                 chromatography 
                   
                   
               
            
           
           
               
               
            
               
                 Analysis of Proteins using 
                 ≤1% by area 
               
            
           
           
               
               
               
            
               
                 the Agilent 1100 HPLC 
                   
                   
               
               
                 System (Residual gelatinase 
                   
                   
               
               
                 by anion exchange 
                   
                   
               
               
                 chromatography) 
                   
                   
               
            
           
           
               
               
            
               
                 Residual leupeptin by 
                 ≤1 ug/mg w/w 
               
            
           
           
               
               
               
            
               
                 reverse phase 
                   
                   
               
               
                 chromatography 
                   
                   
               
            
           
           
               
               
            
               
                 *Bioburden 
                 &lt;1 cfu/mL 
               
               
                   
               
               
                 *Tests required for provisional release of Drug Substance for further manufacturing. 
               
            
           
         
       
     
     The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.