Improved method for producing collagenase

Abstract

The present invention relates to the field of collagenase production and collagenase products, in particular to increasing the reproducibility, purity and stability of collagenase I and collagenase II compositions wherein said compositions have a purity of at least 95% in area as measured by reversed-phase high pressure liquid chromatography (RP-HPLC) and are essentially free of neutral proteases.

Description

[0001] This application is a divisional application of application number 201880032230.2 filed on March 28, 2018, and the invention name is “Improved method for producing collagenase”.

[0002] Related Applications

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 477,846, filed on March 28, 2017, the entire contents of which are incorporated herein by reference to the fullest extent permitted by law. Technical Field

[0004] The present invention relates to the field of collagenase production and collagenase products, and in particular to improving the reproducibility, purity and stability of collagenase I and collagenase II compositions, wherein the purity of the compositions is at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC) and is essentially free of neutral proteases. Background Art

[0005] A process for making high purity (at least 95% pure) collagenase from a fermentation product of Clostridium histolyticum has been previously described in U.S. Pat. No. 7,811,560. Collagenase I and II are enzymes that break down collagen, which is the most abundant structural protein in mammals. These enzymes are used to treat a variety of collagen-mediated diseases, such as Dupuytren's contracture, Peyronie's disease, lipomas, and adhesive capsulitis. U.S. Pat. Nos. 6,086,872 and 5,589,171 disclose the use of collagenase preparations to treat Dupuytren's disease. U.S. Pat. No. 6,022,539 discloses the use of collagenase preparations to treat Peyronie's disease. U.S. Pat. Nos. 6,958,150 and 7,842,673 disclose the use of collagenase for the treatment of lipomas. U.S. Patent Application Publication No. 2006 / 020448A1 discloses the use of collagenase to treat adhesive capsulitis. U.S. Patent Application Publication Nos. 2014 / 0335072 and 2016 / 0279046 disclose the use of collagenase for treating cellulite.

[0006] The main source of collagenase is the fermentation product of Clostridium histolyticum. Injectable formulations containing C. histolyticum collagenase I and collagenase II are marketed under the trade name is sold and approved by the U.S. Food and Drug Administration for the treatment of Dupuyt's contracture and Peyronie's disease. In Europe and other countries, the injectable formulation is known as The amino acid sequences of collagenase I and collagenase II are encoded by colG and colH genes, respectively. The amino acid sequence of colG is described in GenBank Acc. No. D87215 and Matsushita et al. (1999), Journal of Bacteriology 181 (3): 923-933, and the amino acid sequence of colH is described in GenBank Acc. No. D29981 and Yoshihara et al. (1994), Journal of Bacteriology 176 (21): 6489-6496. Collagenase AUX I has a single polypeptide chain consisting of approximately 1000 amino acids, with a molecular weight of about 113 kDa. Collagenase AUX II also has a single polypeptide chain consisting of approximately 1000 amino acids, with a molecular weight of about 112 kDa.

[0007] Collagenase I and collagenase II are metalloproteinases and require tightly bound zinc and loosely bound calcium for their activity (Eddie L. Angleton and HE Van Wart, Biochemistry 1988, 27, 7406-7412). Both collagenase I and collagenase II have broad specificity for all types of collagen (Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260p 2771-2776). These collagenases digest collagen by hydrolyzing the triple helical region of collagen under physiological conditions (Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260p 2771-2776). Even though each collagenase displays a different specificity (i.e., each has a different preferred amino sequence for cleavage), together they have synergistic activity on collagen (Mandl, I, (1964), Biochemistry, 3: p. 1737-1741; Vos-Scheperkeuter, GH, (1997), Cell Transplantation, 6: p. 403-412).

[0008] Collagenase for treatment can be obtained from a variety of sources including mammals, fungi and bacteria. A common source of crude collagenase comes from bacterial fermentation processes, particularly fermentation of Clostridium histolyticum. Any of several chromatographic techniques can be used to purify the crude collagenase obtained from Clostridium histolyticum. However, in addition to collagenase, many other enzymes are secreted in the fermentation broth, including a variety of toxins.

[0009] As previously described in U.S. 2015 / 0010532, genome sequencing and analysis were performed for secreted toxins from Clostridium histolyticum strain 004 to study the function of these toxins in the fermentation of producing collagenase. The toxins studied were alpha toxin (lethal factor), beta toxin (type I and type II collagenase), gamma toxin (clostripain), delta toxin (neutral protease) and epsilon toxin (oxygen-unstable hemolysin). According to genome sequence analysis, only collagenase and clostripain were considered to be functional toxins of strain 004. Other toxins were considered to be non-functional based on the key amino acid sequence differences between Clostridium histolyticum protein and the corresponding model protein.

[0010] Delta toxin, also known as neutral protease (NP), is a metalloprotease from Clostridium histolyticum. Neutral protease is a member of the M4 family of metalloproteases and according to the MEROPS peptidase database, neutral proteases share structural similarities with the M4 family, including common sequence motifs, substrate specificity, and cofactor requirements. Thermolysin is the most studied and best understood enzyme in the M4 family. Relevant to this study, thermolysin from Bacillus thermoproteolyticus has been previously cloned and sequenced and the information has been deposited in GenBank under accession number CAA54291. Thermolysin is a zinc metalloprotease with a mature enzyme molecular weight of 34.6 kDa. Thermolysin and all members of the M4 peptidase family consist of a signal peptide, a prosequence, and a mature sequence.

[0011] When a cell produces thermolysin, it starts as an inactive enzyme (zymogen) because the preprotein sequence preceding the mature enzyme sequence inhibits thermolysin. The presequence represents two-thirds of the size of the secreted preenzyme, while the mature enzyme represents one-third. The presequence in thermolysin is autocatalytically cleaved, resulting in the mature enzyme being activated in its destination environment. The secretion strategy of thermolysin is shared with neutral proteases and is the basis for the conclusion that Clostridium histolyticum neutral protease also starts as an inactive protease. Similarly, the mature form of Clostridium histolyticum neutral protease has a molecular weight similar to that of thermolysin, as described in Herber (U.S. 2015 / 0010532) and in Maeda et al., Cloning the neutral protease in Clostridium histolyticum, determining its substrate specificity and designing specific substrates, Appl. Microbiology. Biotechnology, 99: 10489-99 (2015).

[0012] The similarity of thermolysin in its synthesis, secretion, activation, and substrate specificity makes thermolysin a model for the neutral protease of Clostridium histolyticum strain 004. Although these properties between thermolysin and neutral protease are comparable based on shared structural features, sequence differences do exist between the two enzymes. Relevant to the present invention, previous gene fractionation analysis and homology comparison of delta toxin with thermolysin in Herber (U.S. 2015 / 0010532) suggests that the neutral protease may be secreted into the growth medium but is inactive due to divergent consensus sequences in the autocatalytic site.

[0013] The performance characteristics of thermophilins (and all neutral proteases) are that they can act on a wide range of proteins. The presence of nonspecific proteases in pharmaceuticals, especially those injected into the body, is problematic because they undesirably degrade non-targeted enzymes in cells and tissues in the body - even if present in trace amounts.

[0014] Previous processes used to make collagenase sometimes resulted in atypical collagenase degradation. For efficient and effective commercial production of collagenase, reproducible production of high purity collagenase free of toxins is required. Summary of the invention

[0015] The present disclosure provides an improved method for making collagenase, which removes detectable amounts of neutral proteases from collagenase I and II products. The present disclosure provides a collagenase product that is less prone to degradation and purer than the collagenase compositions used in previous medicine. In one embodiment, an investigation was conducted to identify the reasons for the degradation of atypical collagenases I and II from Clostridium histolyticum in a manufacturing process. It has been found that the neutral protease from Clostridium histolyticum is present in the fermentation product of Clostridium histolyticum in its active, mature form. In addition, the neutral protease is highly active on collagenase I and collagenase II, and may cause significant degradation of the two collagenases. Its continued presence in the purification process may also degrade the two collagenases.

[0016] Neutral proteases previously thought to be inactive unexpectedly become more active in the presence of elevated amounts of one or more metals, such as nickel and zinc. This metal-mediated activity can produce elevated levels of collagenase product fragments in the collagenase manufacturing process in the presence of parts per million levels of such metals, thereby causing purity failures in at least the ion exchange (IEX) chromatography step of the process.

[0017] The present invention also provides methods for isolating the neutral protease from the manufacturing process, methods for removing it from the process, and methods for determining its presence throughout the manufacturing process. In addition, the present invention provides improved reproducibility and stability of highly purified collagenases, provides collagenase I and II products that are essentially free of neutral proteases (i.e., below the detectable limit), and provides safer and purer collagenase compositions that are essentially free of neutral proteases.

[0018] By controlling metals in fermentation and purification processes, the repeatability of manufacturing can be improved. Surprisingly, it was found that the presence or absence of certain metals during the purification of collagenase I and II from Clostridium histolyticum affects the purity of the collagenase product. In one embodiment, as measured by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), about 80ppm or higher zinc levels, or about 1ppm or higher nickel levels cause collagenase I product and collagenase II product impurities to be at an increased level, thereby causing the impurity levels in the drug product to be unacceptable.

[0019] In another embodiment, when the zinc level is higher than about 2.75ppm and less than about 80ppm, the collagenase is not degraded into fragments. And, in at least one step of the purification process, when the nickel content is higher than about 0.4ppm and lower than about 1ppm, the collagenase is not degraded into fragments. In addition, the control of the drug purification process to maintain the zinc level at about 3ppm or lower, or to maintain the nickel level at about 0.5ppm or lower, improves the reliability and repeatability of the manufacture of the high-purity collagenase product. Compared with the previous process, operating the collagenase manufacturing process with trace amounts of metallic zinc and nickel (or other metals) will not cause the collagenase degradation level to increase, thereby significantly improving reliability and repeatability.

[0020] In certain embodiments, when nickel or zinc is present in the buffer of the hydrophobic interaction chromatography (HIC) step of the collagenase manufacturing process, the neutral protease actively degrades collagenase I and II into smaller fragments and produces low purity (less than 95% purity as measured by SDS-PAGE) collagenase products. In some embodiments, nickel (in an amount between about 0.4ppm and about 1ppm) and zinc (in an amount between about 2.8ppm and 84ppm) do not amplify the specific activity of the neutral protease in the HIC and tangential flow filtration (TFF) unit operations, and do not cause a significant increase in product fragment impurities in the IEX unit operation. In one embodiment, controlling the nickel or zinc level in the collagenase manufacturing process is important for obtaining high purity collagenase I and II products.

[0021] Neutral proteases are eliminated or at least reduced from the manufacturing process of collagenase so that the collagenase composition is essentially free of neutral proteases, the safety of the product is improved, and the degradation of the collagenase in the product by neutral proteases is prevented. The collagenase composition is improved by adding at least one neutral protease elimination step in the manufacturing process. Neutral proteases produced by the manufacture of the collagenase product can be reduced or eliminated by one or more of the following elimination steps: (a) excluding fractions having a collagenase I or II content of less than about 90% and any single impurity content of more than about 7% as measured by SDS PAGE gel or HPLC; (b) excluding fractions having a detectable level of neutral proteases as measured by SDS PAGE gel or zymography; or (c) excluding any fractions calculated to contain an excessive level of neutral proteases. Each step can be used alone or in combination.

[0022] More specifically, the elimination step may include the exclusion of fractions, which are collected after collagenase passes through a separation column or filter (or other separation media), and before merging collagenase I or collagenase II fractions, comprise the neutral protease of the detectable level tested by SDS-PAGE or zymogram analysis. In certain embodiments, even if the fraction comprises a large amount of collagenase, it may be excluded. Elimination may also require exclusion or discarding any collagenase I or II fraction, as measured by SDS PAGE gel or HPLC, the collagenase I or II content of these fractions being lower than about 90% and the content of any single impurity being higher than about 7%. The elimination step may include using the collagenase II of the estimated amount produced in the process to control which collagenase I fractions are merged for collagenase I products. Any of these steps can be used for the manufacturing process of collagenase to produce collagenase I and II products essentially free of neutral proteases.

[0023] One embodiment of neutral protease elimination enables process control of the purity of ion exchange chromatography (IEX) fractions, wherein each fraction is analyzed for purity and maximum amount of impurities, and each fraction has to meet the criteria in order for the fraction to be forward processed (acceptable for pooling, drug product, and use as a pharmaceutical formulation). In this embodiment, the positional nature of the neutral protease elution at the end of the collagenase I peak of the IEX step provides an effective mechanism for removing neutral proteases by automatically rejecting fractions that show detectable limits of neutral proteases by SDS-PAGE, SDS-PAGE with densitometry, or zymography (with or without densitometry) analysis. In another embodiment, the peak fractions of the collagenase I fraction collected from the IEX elution are forward processed based on the fractions that meet the desired purity and impurity criteria, and only the collagenase I peak fractions are pooled until the approximate amount of collagenase I pooled matches the approximate amount of collagenase II produced by the process. This control strategy eliminated the other end fractions of the collagenase I peak, rendering the pooled collagenase I fractions essentially free of neutral proteases.

[0024] The present invention further provides a method for preparing a pharmaceutical formulation according to the present disclosure, and a method for treating a patient suffering from a collagen-mediated disease using a collagenase composition of the present invention. For example, a method for treating a collagen-mediated disease by administering an effective amount of collagenase, collagenase I, collagenase II, or a combination thereof is contemplated. In one embodiment, the present disclosure relates to a method for treating a collagen-mediated disease (e.g., cellulite) in a patient in need thereof, wherein the method includes the step of administering an effective amount of a collagenase composition to the patient, wherein the collagenase composition comprises a collagenase I product and a collagenase II product, which are combined in a mass ratio of collagenase I to collagenase II of about 0.6:1.4 and a mass ratio of collagenase I to collagenase II of about 1.4:0.6, and wherein the composition is essentially free of neutral proteases. In addition, the present disclosure provides a more stable and safer pharmaceutical formulation.

[0025] According to the following description, drawings, examples and claims, other embodiments of the compositions and methods of the present invention, etc. will be apparent. It will be understood from the foregoing and following description that each feature described herein and each combination of two or more such features are included within the scope of the present disclosure, as long as the features included in the combination are not mutually inconsistent. In addition, any feature or combination of features can be explicitly excluded from any embodiment or aspect. Additional aspects and embodiments are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.

[0026] References to color drawings

[0027] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The foregoing features of the embodiments will be more readily understood by reference to the following detailed description and accompanying drawings, in which:

[0029] Figure 1 is an example of a graph showing the linearity of the AUX-I / AUX-II band response. It is a graph of trace amounts of collagenase in the microgram range of 1.125 micrograms to 1.875 micrograms relative to a known reference standard, and R 2 The value is greater than 98%.

[0030] Figure 2 : is an example of an annotated image report generated by Bio-Rad Quantity One software. Lane 1 contains molecular weight standards with known molecular weights. Lane 2 contains the approximate AUX intermediate reference standard. The remaining lanes contain samples of each IEX fraction. The main product band is the highest band in each lane, and each additional lower band represents an impurity.

[0031] Figure 3 For the AUX-I 33 kD impurity, the frequency of occurrence of the fraction impurities and the mean and range of the relative percentage amounts of the fraction impurities are shown.

[0032] Figure 4 For the AUX-I 45 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0033] Figure 5 For the AUX-I 55 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0034] Figure 6 For the AUX-I 80 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0035] Figure 7 For the AUX-I 90 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0036] Figure 8 For the AUX-I 96 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0037] Fig. 9 For the AUX-II 25 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0038] Fig.10 For the AUX-II 38 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0039] Fig.11 For the AUX-II 50 kD impurity, the frequency of occurrence of the fraction impurities and the mean and range of the relative percentage amounts of the fraction impurities are shown.

[0040] Fig.12 For the AUX-II 60 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0041] Fig.13 For the AUX-II 80 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0042] Fig.14 For the AUX-II 90 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0043] Fig.15 For the AUX-II 92 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0044] Fig.16 For the AUX-II 96 kD impurity, the frequency of occurrence of the fraction impurity and the mean and range of the relative percentage amounts of the fraction impurity are shown.

[0045] Fig.17 Typical grams of protein per IEX fraction are shown.

[0046] Fig.18 Typical estimates of grams of the 90 kD impurity for each IEX fraction are shown.

[0047] Fig.19 The estimated grams of 90 kD impurity relative to grams of protein for each AUX peak is shown.

[0048] Fig. 20 A control chart for the AUX-1 impurity is shown.

[0049] Fig.21A and 21BA control chart for AUX-II impurities is shown.

[0050] Fig. 22 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-II fraction after the IEX column during a control run from a zinc doping study (DEV-25C). The AUX-II peak from the IEX column was collected in six (6) fractions. The gel shows typical purity of the AUX-II fractions and a typical pattern of product related impurities.

[0051] Lane 1: Molecular weight marker

[0052] Lane 2: Collagenase II

[0053] Lane 3: Fraction #1 - 94.9% purity (1.5 μg per lane)

[0054] Lane 4: Fraction #2 - 98.1% purity (1.5 μg per lane)

[0055] Lane 5: Fraction #3 - 98.4% purity (1.5 μg per lane)

[0056] Lane 6: Fraction #4 - 97.3% purity (1.5 μg per lane)

[0057] Lane 7: Fraction #5 - 96.3% purity (1.5 μg per lane)

[0058] Lane 8: Fraction #6 - 89.9% purity (1.5 μg per lane)

[0059] Lane 9: Blank

[0060] Lane 10: Collagenase I

[0061] Lane 11: Fraction #16 of collagenase I peak (1.5 μg per lane)

[0062] Fig.23 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-I fraction after the IEX column during a control run of a zinc doping study (run DEV-25C). The AUX-I peak from the IEX column was collected in ten (10) fractions. The gel shows typical purity of the AUX-I fraction and a typical pattern of product related impurities.

[0063] Lane 1: Molecular weight marker

[0064] Lane 2: Collagenase I

[0065] Lane 3: Fraction #7 - 87.7% purity

[0066] Lane 4: Fraction #8 - 86.5% purity

[0067] Lane 5: Fraction #9 - 92.0% purity

[0068] Lane 6: Fraction #10 - 95.8% purity

[0069] Lane 7: Fraction #11 - 97.8% purity

[0070] Lane 8: Fraction #12 - 97.1% purity

[0071] Lane 9: Fraction #13 - 97.0% purity

[0072] Lane 10: Fraction #14 - 96.8% purity

[0073] Lane 11: Fraction #15 - 96.5% purity

[0074] Fig.24 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-II fractions after an IEX column spiked to 84 ppm (run DEV-25B) based on a zinc spike study. The AUX-II peak from the IEX column was collected in eight (8) fractions. The gel shows a degradation pattern due to the presence of zinc, with the number and intensity of impurities increasing and the purity of the AUX-II fractions decreasing. None of the AUX-II fractions in this gel met the combined purity criteria.

[0075] Lane 1: Molecular weight marker

[0076] Lane 2: Collagenase II

[0077] Lane 3: Fraction #1 - 66.4% purity (1.5 μg per lane)

[0078] Lane 4: Fraction #2 - 75.5% purity (1.5 μg per lane)

[0079] Lane 5: Fraction #3 - 79.5% purity (1.5 μg per lane)

[0080] Lane 6: Fraction #4 - 76.7% purity (1.5 μg per lane)

[0081] Lane 7: Fraction #5 - 72.1% purity (1.5 μg per lane)

[0082] Lane 8: Fraction #6 - 63.9% purity (1.5 μg per lane)

[0083] Lane 9: Fraction #7 - 52.9% purity (1.5 μg per lane)

[0084] Lane 10: Fraction #8 - 45.0% purity (1.5 μg per lane)

[0085] Fig.25 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-I fractions after an IEX column spiked to 84 ppm (run DEV-25B) based on zinc spike studies. The AUX-I peak from the IEX column was collected in ten (10) fractions. The gel shows a degradation pattern due to the presence of zinc, with an increase in the number and intensity of impurities and a decrease in the purity of the AUX-I fractions. None of the AUX-I fractions in this gel met the combined purity criteria.

[0086] Lane 1: Molecular weight marker

[0087] Lane 2: Collagenase I

[0088] Lane 3: Fraction #9 - 54.9% purity (1.5 μg per lane)

[0089] Lane 4: Fraction #10-5 0.9% purity (1.5 μg per lane)

[0090] Lane 5: Fraction #11 - 56.2% purity (1.5 μg per lane)

[0091] Lane 6: Fraction #12 - 65.2% purity (1.5 μg per lane)

[0092] Lane 7: Fraction #13 - 70.4% purity (1.5 μg per lane)

[0093] Lane 8: Fraction #14 - 70.0% purity (1.5 μg per lane)

[0094] Lane 9: Fraction #15 - 67.5% purity (1.5 μg per lane)

[0095] Lane 10: Fraction #16 - 62.4% purity (1.5 μg per lane)

[0096] Lane 11: Fraction #17 - 58.8% purity (1.5 μg per lane)

[0097] Lane 12: Fraction #18 - 52.6% purity (1.5 μg per lane)

[0098] Fig.26is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-II fraction after an IEX column from a control run for a nickel doping study (run DEV-25A). The AUX-II peak from the IEX column was collected in six (6) fractions. The gel shows typical purity for the AUX-I fraction and a typical pattern of product related impurities.

[0099] Lane 1: Molecular weight marker

[0100] Lane 2: Collagenase II

[0101] Lane 3: Fraction #1 - 95.9% purity (1.5 μg per lane)

[0102] Lane 4: Fraction #2 - 97.1% purity (1.5 μg per lane)

[0103] Lane 5: Fraction #3 - 99.1% purity (1.5 μg per lane)

[0104] Lane 6: Fraction #4 - 98.2% purity (1.5 μg per lane)

[0105] Lane 7: Fraction #5 - 96.9% purity (1.5 μg per lane)

[0106] Lane 8: Fraction #6 - 92.3% purity (1.5 μg per lane)

[0107] Lane 9: Blank

[0108] Lane 10: Collagenase I

[0109] Lane 11: Fraction #16 of collagenase I peak (1.5 μg per lane)

[0110] Fig. 27 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-I fraction after the IEX column from a control run for a nickel doping study (run DEV-25A). The AUX-I peak from the IEX column was collected in ten (10) fractions. The gel shows typical purity of the AUX-II fraction and a typical pattern of product related impurities.

[0111] Lane 1: Molecular weight marker

[0112] Lane 2: Collagenase I

[0113] Lane 3: Fraction #7 - 85.6% purity (1.5 μg per lane)

[0114] Lane 4: Fraction #8 - 85.2% purity (1.5 μg per lane)

[0115] Lane 5: Fraction #9 - 90.7% purity (1.5 μg per lane)

[0116] Lane 6: Fraction #10 - 95.5% purity (1.5 μg per lane)

[0117] Lane 7: Fraction #11 - 96.6% purity (1.5 μg per lane)

[0118] Lane 8: Fraction #12 - 97.0% purity (1.5 μg per lane)

[0119] Lane 9: Fraction #13 - 96.6% purity (1.5 μg per lane)

[0120] Lane 10: Fraction #14 - 96.8% purity (1.5 μg per lane)

[0121] Lane 11: Fraction #15 - 96.8% purity (1.5 μg per lane)

[0122] Fig.28 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-II fractions after an IEX column spiked to 1.2 ppm (run DEV-25D) based on a nickel spike study. The AUX-II peak from the IEX column was collected in seven (7) fractions. The gel shows a degradation pattern caused by the presence of nickel, with an increase in the number and intensity of impurities and a decrease in the purity of the AUX-II fractions. In this gel, none of the AUX-II fractions met the combined purity criteria.

[0123] Lane 1: Molecular weight marker

[0124] Lane 2: Collagenase II

[0125] Lane 3: Fraction #1 - 74.8% purity (1.5 μg per lane)

[0126] Lane 4: Fraction #2 - 81.8% purity (1.5 μg per lane)

[0127] Lane 5: Fraction #3 - 84.6% purity (1.5 μg per lane)

[0128] Lane 6: Fraction #4 - 85.6% purity (1.5 μg per lane)

[0129] Lane 7: Fraction #5 - 83.0% purity (1.5 μg per lane)

[0130] Lane 8: Fraction #6 - 72.3% purity (1.5 μg per lane)

[0131] Lane 9: Fraction #7 - 64.9% purity (1.5 μg per lane)

[0132] Lane 10: Blank

[0133] Lane 11: Collagenase I

[0134] Lane 12: Fraction #8 of collagenase I peak - 62.7% purity (1.5 μg per lane)

[0135] Fig.29 is a SDS-PAGE Coomassie stained gel showing impurities in the AUX-I fractions after an IEX column spiked to 1.2 ppm (run DEV-25D) based on a nickel spike study. The AUX-I peak from the IEX column was collected in ten (10) fractions. The gel shows a degradation pattern caused by the presence of nickel, with an increase in the number and intensity of impurities and a decrease in the purity of the AUX-I fractions. In this gel, none of the AUX-I fractions met the combined purity criteria.

[0136] Lane 1: Molecular weight marker

[0137] Lane 2: Collagenase I

[0138] Lane 3: Fraction #9 - 60.3% purity (1.5 μg per lane)

[0139] Lane 4: Fraction #10 - 67.6% purity (1.5 μg per lane)

[0140] Lane 5: Fraction #11 - 81.6% purity (1.5 μg per lane)

[0141] Lane 6: Fraction #12 - 84.7% purity (1.5 μg per lane)

[0142] Lane 7: Fraction #13 - 84.1% purity (1.5 μg per lane)

[0143] Lane 8: Fraction #14 - 82.6% purity (1.5 μg per lane)

[0144] Lane 9: Fraction #15 - 82.0% purity (1.5 μg per lane)

[0145] Lane 10: Fraction #16 - 72.4% purity (1.5 μg per lane)

[0146] Lane 11: Fraction #17 - 68.6% purity (1.5 μg per lane)

[0147] Fig.30It is a Q-Sepharose column high performance (Q-HP) chromatogram and SDS-PAGE with Coomassie stained gel image. The combined fractions of AUX I and AUX II loaded and fused are shown. The Q HP chromatogram represents the neutral protease from the wastewater stream of the HIC chromatography step purified using an IEX column. The gel image describes the fractions collected in the IEX loading and purification process. The neutral protease eluted at the end of the gradient and was observed in fraction 3. The N-termini of the impurities were determined by Edman degradation and liquid chromatography-mass spectrometry (LC-MS) using purified product fragments. Thermolysin was used as a model protease to determine whether the cleavage end matched the thermolysin cleavage point in the protein analysis program peptide cleavage (website: http: / / expasy.org). In all cases, the cleavage end point was hit by thermolysin cleavage. The purified neutral protease was sequenced by Edman degradation and LC-MS to identify the neutral protease N-terminus of the current process. This is also used for degradation studies to confirm that the neutral protease isolated by the process is functional (active).

[0148] Fig.31 is the deduced genomic sequence of the neutral protease (delta-toxin, CHL 2576) of Clostridium histolyticum (CCH). The blue highlight indicates the N-terminus of the mature neutral protease from Clostridium histolyticum strain 004, which was determined by Edman degradation assay and highlighted in blue. The green highlight indicates the portion of the mature neutral protease from Clostridium histolyticum detected by Lys-C / trypsin LC-mass spectrometry.

[0149] Fig.32 The nomenclature of Schechter and Berger protease subsites is described.

[0150] Fig.33 yes r Sequence alignment of npr A and collagenase Clostridium histolyticum (abbreviated CCH) neutral protease (CLH 2576) showing complete homology between the two enzymes.

[0151] Fig.34 12% Tris-Glycine in the process product stream of Clostridium histolyticum (CCH) Casein zymography PAGE images: Mustang-Q filtrate (MQF), HIC load, HIC eluate, and AUS-II pool from batch 100808. The gel used was 12% Tris-Glycine Zymography casein gel. The manufacturing process that generated batch 100808 produced high levels of impurities and used an older process control method. In the image, neutral protease activity is observed in the MQF, HIC load, and HIC eluate, but not in the AUX-II pool. The low signal observed in the HIC eluate sample suggests that the major clearance of the neutral protease occurs at the HIC step.

[0152] Lane 1: + control (thermolysin), 1 ng

[0153] Lane 2: Molecular weight reference

[0154] Lane 3: Blank

[0155] Lane 4: MQF Dev-25A, 2 μg

[0156] Lane 5: Blank

[0157] Lane 6: HIC loading Dev-13, 3 μg

[0158] Lane 7: Blank

[0159] Lane 8: Blank

[0160] Lane 9: HIC eluted Dev-25A, 4 μg

[0161] Lane 10: Blank

[0162] Lane 11: Aux II pool 1000808, 10 μg

[0163] Lane 12: Blank

[0164] Fig.35 12% Tris-Glycine in the process product stream of Clostridium histolyticum (CCH) Casein zymography PAGE images: first tangential flow filter (TFF-1) concentrate, AUX-1 pool, AUX-1 intermediate, AUX-II intermediate, drug product from a variety of species affected by HIC buffer with high zinc and nickel content. The gel used was 12% Tris-Glycine Zymography casein gel. In this case, neutral proteases were observed in the TFF-1 concentrate, at low levels in AUX-I and the drug product, but not in AUX-II. These data confirm that neutral proteases are separated from the product throughout the IEX step and eluted with AUX-1.

[0165] Lane 1: + control (thermolysin), 1 ng

[0166] Lane 2: Molecular weight reference

[0167] Lane 3: Blank

[0168] Lane 4: TFF-1 concentrate 10000808, 10 μg

[0169] Lane 5: Blank

[0170] Lane 6: AUX-I pool 10000808, 10 μg

[0171] Lane 7: Blank

[0172] Lane 8: AUX-I intermediate 10000808, 10 μg

[0173] Lane 9: Blank

[0174] Lane 10: AUX-II intermediate 10000808, 10 μg

[0175] Lane 11: Blank

[0176] Lane 12: Drug substance 10000808, 10 μg

[0177] Fig.36 12% Tris-Glycine from the collagenase Clostridium histolyticum (CCH) IEX fraction retained from the AUX-II fraction of batch 1000414 Casein zymography PAGE image. The gel used was 12% Tris-glycine Zymography casein gel. These fractions are from manufacturing batches produced before active neutral proteases were found in the process. No neutral proteases were detected in any of the AUX-II fractions.

[0178] Lane 1: + control (thermolysin), 1 ng

[0179] Lane 2: Molecular weight reference

[0180] Lane 3: AUX-II protein 1 1000414, 6.4 μg

[0181] Lane 4: AUX-II protein 2 1000414, 14.4 μg

[0182] Lane 5: AUX-II protein 3 1000414, 10 μg

[0183] Lane 6: AUX-II protein 4 1000414, 10 μg

[0184] Lane 7: AUX-II protein 5 1000414, 10 μg

[0185] Lane 8: AUX-II protein 6 1000414, 11.2 μg

[0186] Lane 9: AUX-II protein 7 1000414, 6.2 μg

[0187] Lane 10: AUX-II protein 8 1000414, 5.8 μg

[0188] Fig.37 12% Tris-Glycine from the collagenase Clostridium histolyticum (CCH) IEX fraction retained from the AUX-I fraction of batch 1000414 Casein zymography PAGE image. The gel used was 12% Tris-glycine Zymography casein gel. These fractions are from manufacturing batches produced before active neutral proteases were found in the process. Neutral proteases were not detected in any of the AUX-1 fractions using this method.

[0189] Lane 1: Molecular weight reference

[0190] Lane 2: AUX-I protein 9 1000414, 8.8 μg

[0191] Lane 3: AUX-I protein 10 1000414, 10 μg

[0192] Lane 4: AUX-I protein 11 1000414, 10 μg

[0193] Lane 5: AUX-I protein 12 1000414, 10 μg

[0194] Lane 6: AUX-I protein 13 1000414, 10 μg

[0195] Lane 7: AUX-I protein 14 1000414, 10 μg

[0196] Lane 8: AUX-I protein 15 1000414, 10 μg

[0197] Lane 9: AUX-I protein 16 1000414, 14 μg

[0198] Lane 10: AUX-I Fxn 17 1000414, 10 μg

[0199] Fig.38 12% Tris-Glycine from the collagenase Clostridium histolyticum (CCH) IEX fraction retained from batch 1000414 AUX-II fractions 1, 7 and AUX-1 fractions 8, 17 Casein zymography PAGE image. The gel used was 12% Tris-glycine Zymography casein gel. The manufacturing process that produced batch 1000414 resulted in low impurity levels and used older process control methods. On this gel, the fractions were run at a higher concentration than the original gel ( Fig.16 and 17 ). Neutral protease was detected only in the last fraction of the AUX-1 peak (fraction 17), which confirmed the positional nature of the elution at the end of the peak.

[0200] Lane 1: + control (thermolysin), 1 ng

[0201] Lane 2: Molecular weight reference

[0202] Lane 3: AUX-II protein 1 1000414 Tris-Glycine, 10 μg

[0203] Lane 4: AUX-II protein 1 1000414LDS, 10 μg

[0204] Lane 5: AUX-II protein 7 1000414 Tris-Glycine, 9.3 μg

[0205] Lane 6: AUX-II protein 7 1000414LDS, 9.3 μg

[0206] Lane 7: AUX-I protein 8 1000414 Tris-Glycine, 6.5 μg

[0207] Lane 8: AUX-I protein 8 1000414LDS, 6.5 μg

[0208] Lane 9: AUX-I protein 17 1000414 Tris-Glycine, 11 μg

[0209] Lane 10: AUX-I protein 17 1000414LDS, 11 μg

[0210] Tris-glycine and LDS are two buffer systems used for SDS-PAGE. This gel was run to evaluate whether one system is better than the other. No differences were recorded.

[0211] Fig.39 12% Tris-Glycine from the collagenase Clostridium histolyticum (CCH) IEX fraction retained from batch 001138 AUX-I tail fractions 17, 18, 19 Casein zymography PAGE image. The gel used was 12% Tris-glycine Zymography casein gel. This gel depicts the fractions that were rejected at the end of the AUX-1 peak. Each of the three rejected fractions contained detectable neutral proteases. Fractions 17 and 18 passed the in-process pooling criteria but were rejected based on a 1:1 yield target pooling strategy. This demonstrates process control to remove residual neutral proteases from both AUX-1 and the drug product.

[0212] Lane 1: TFF-1 concentrate Dev-25A, 34 μg

[0213] Lane 2: Molecular weight reference

[0214] Lane 3: Blank

[0215] Lane 4: AUX-1 protein 7Dev-25A, 4.5 μg

[0216] Lane 5: Blank

[0217] Lane 6: AUX-I protein 17Dev-25A, 5 μg

[0218] Lane 7: Blank

[0219] Lane 8: AUX-I protein 17 001138, 14.8 μg

[0220] Lane 9: Blank

[0221] Lane 10: AUX-I protein 18 001138, 10.3 μg

[0222] Lane 11: Blank

[0223] Lane 12: AUX-I protein 19 001138, 8.1 μg

[0224] Fig.40A -B is collagenase Clostridium histolyticum (CCH) in 12% Tris-glycine, manufacturing batch 0010987 Casein zymography SDS-PAGE gel image. The gel used was 12% Tris-Glycine Zymography casein gel. This gel depicts the fractions that were rejected at the end of the AUX-1 peak. Each of the three rejected fractions contained detectable neutral proteases. Fractions 17 and 18 passed the in-process pooling criteria but were rejected based on the 1:1 yield target pooling strategy. This demonstrates process control to remove residual neutral proteases from AUX-1 and the drug product. Fig.40A The purification status of AUX-1 throughout the process is shown. Fig.40B The purification status of AUX-II throughout the process is shown.

[0225] Fig.40A

[0226] Lane 1: Positive control (TL)

[0227] Lane 2: Molecular weight reference

[0228] Lane 3: Empty

[0229] Lane 4: Mustang Q filtrate, 2.1 μg load Lane 5: Empty

[0230] Lane 6: HIC eluate, 4.9 μg load

[0231] Lane 7: Empty

[0232] Lane 8: AUX-I pool, 10 μg loading

[0233] Lane 9: Empty

[0234] Lane 10: AUX-I intermediate, 10 μg loading

[0235] Lane 11: Empty

[0236] Lane 12: Analysis blank

[0237] Fig.40B

[0238] Lane 1: Positive control (TL)

[0239] Lane 2: Molecular weight reference

[0240] Lane 3: Empty

[0241] Lane 4: TFF-1 concentrate, 10 μg load

[0242] Lane 5: Empty

[0243] Lane 6: AUX-II pool, 10 μg load

[0244] Lane 7: Empty

[0245] Lane 8: AUX-II intermediate, 10 μg loading

[0246] Lane 9: Empty

[0247] Lane 10: Raw drug substance (drug product), 10 μg load Lane 11: Empty

[0248] Lane 11: Analysis blank

[0249] Fig.41 The collection of collagenase I fractions and collagenase II are depicted and their positive processing based on purity criteria.

[0250] Fig.42 The percent purity of the AUX-1 product relative to the total protein amount in each AUX elution peak is provided.

[0251] Fig.43 The percent purity of the AUX-II product relative to the total protein amount in each AUX elution peak is provided.

[0252] Fig.44 A control chart for the AUX-I 90 kDa impurity is presented. Fig.42 The decrease in percent purity level of the AUX-1 elution peak seen in Figure 3 is primarily due to increased levels of the AUX-1 90 kDa impurity.

[0253] Fig.45 A control chart for the AUX-II 96 kDa impurity is presented. Fig.43 The decrease in percent purity level of the AUX-II elution peak seen in Figure 3 is primarily due to increased levels of the AUX-II 96 kDa impurity.

[0254] Figures 46A-46D Estimated product peak percentages for various AUX peaks and bands are provided. These figures provide data for statistical analysis for the evaluation of impurities in the IEX fractions after implementation of the corrective and preventive actions associated with deviation 3797. DETAILED DESCRIPTION

[0255] Various aspects and embodiments will now be fully described herein. However, these aspects and embodiments may be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided to make this disclosure more thorough and complete and to fully convey the scope of the subject matter to those skilled in the art. All publications, patents, and patent applications cited herein, whether supra or infra, are incorporated herein by reference in their entirety.

[0256] I. Definitions

[0257] Unless otherwise defined, all terms and phrases used herein include the meanings that the terms and phrases have acquired in the art, unless the context in which the term or phrase is used clearly indicates a contrary meaning or a contrary meaning is readily apparent therein. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods and materials are now described.

[0258] Unless otherwise specified, the use of each numerical value is expressed as an approximation, just as if these values ​​were preceded by the word "about" or "approximately". Similarly, unless otherwise explicitly stated, the numerical values ​​within each range specified in this application are expressed as approximations, just as the minimum and maximum values ​​within the range are all preceded by the word "about" or "approximately". In this way, changes above and below the range can be used to obtain results substantially the same as the values ​​within the range. As used herein, the terms "about" and "approximately" should have their simple and ordinary meanings to those of ordinary skill in the art who are most closely related to or associated with the disclosed subject matter or those of ordinary skill in the art who are related to the range or element in question when referring to numerical values. The amount extended from the strict numerical boundaries depends on many factors. For example, some factors that can be considered include the criticality of the element and / or the effect that a change in a given amount will have on the performance of the claimed subject matter, as well as other considerations known to those skilled in the art. As used herein, the use of different numbers of significant figures for different numerical values ​​does not mean how the use of the word "about" or "approximately" will expand a specific numerical value or range. Therefore, generally, "about" or "approximately" widens the numerical value. Likewise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values, as well as extensions of the range provided by use of the terms "about" or "approximately." Therefore, recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0259] As used herein, "collagenase" refers to one or more proteins including collagenase activity, such as collagenase I and / or collagenase II, that are shown in a standard collagenase assay. The collagenase composition can include at least one of two microbial collagenases, sometimes referred to as AUX I and AUX II. The terms "collagenase I", "ABC I", "AUX I", "AUX-1", "collagenase AUX I" and "collagenase ABC I" refer to the same enzyme with the same amino acid sequence and can be used interchangeably. Similarly, the terms "collagenase II", "ABC II", "AUX 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. In some aspects, the collagenase is separated and purified from the culture supernatant of Clostridium histolyticum by chromatography. In other aspects, the collagenase is recombinantly separated and purified, and / or derived from a variety of sources, including mammals, fungi and bacterial sources. Both collagenases are specific proteases and have the same EC number (EC 3.4.24.3). In other aspects, the collagenase composition comprises collagenase I or collagenase II. In other embodiments, the collagenase composition comprises both collagenase I and collagenase II.

[0260] As used herein, "collagen-mediated condition" refers to any disease or disorder involving collagen. Examples of collagen-mediated conditions that can be treated by the compositions and methods described herein include, but are not limited to: Dupuyt's disease; Peyronie's disease; frozen shoulder (adhesive capsulitis), keloids; hypertrophic scars; depressed scars such as those caused by inflammatory acne; postoperative adhesions; acne vulgaris; lipomas, and disfiguring conditions such as wrinkles, cellulite, and hypertrophic fibrosis.

[0261] The phrase "collagenase I product" refers to collagenase I that has been purified from a fermentation, wherein the eluate of the collagenase I in a purification step has been collected in fractions, and any number of these fractions have been pooled together.

[0262] The phrase "collagenase II product" refers to collagenase II that has been purified from a fermentation, wherein the eluate of the collagenase II in a purification step has been collected in fractions, and any number of these fractions have been pooled together.

[0263] The phrase "derived from" Clostridium histolyticum refers to collagenases that originate from Clostridium histolyticum. It includes collagenases obtained from fermentations of Clostridium histolyticum and fermentations of other organisms using recombinant collagenases.

[0264] It will be understood that the terms "drug substance," "drug product," or "collagenase composition" are used interchangeably to refer to collagenase I, collagenase II, or a combination of both collagenase I and collagenase II prior to any optional lyophilization.

[0265] As used herein, "substantially free of neutral proteases" means below the detectable limit of the particular detection assay employed (eg, SDS PAGE, zymography, HPLC, etc.).

[0266] As used herein, "forward processing" refers to highly purified collagenase I or II fractions that are used for pooling to produce the final collagenase I drug substance or collagenase II drug substance.

[0267] As used herein, "highly purified" or "high purity" or "high purity" collagenase I or II, or a combination of collagenases I and II, refers to a purity of at least 95% by area, or a purity of at least 96% by area, or a purity of at least 97% by area, a purity of at least 98% by area, a purity of at least 99% by area, a purity of about 100% by area, as determined by reverse phase HPLC (RP-HPLC). For further clarification, this includes all values ​​for purity determined by RP-HPLC that are between at least 95% and about 100% by area.

[0268] As used herein, "the largest impurity" refers to the collagenase fragment with the highest presence in a given step (e.g., after IEX), as measured by SDS-PAGE gel with or without densitometry, or by another acceptable method. Collagenase fragments can be found throughout the purification process and can be of any size, but the most common are collagenase I fragments with a molecular weight of about 33 kDa, 45 kDa, 55 kDa, 80 kDa, or 90 kDa, and collagenase II fragments of about 25 kDa, 38 kDa, 50 kDa, 60 kDa, 80 kDa, 90 kDa, 92 kDa, 92 kDa, or 96 kDa.

[0269] As used herein, "neutral protease elimination" or "neutral protease elimination step" refers to the removal or rejection of the fraction collected after the collagenase filtrate passes through a separation column or filter during manufacturing.

[0270] "Optional" or "optionally" means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component or circumstance occurs and instances where it does not.

[0271] As used herein, the term "pharmaceutically acceptable carrier" or "excipient" refers to a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. Some examples of materials that can be used 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 carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, flavoring agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0272] As used herein, "purified fraction" means that the undegraded collagenase I has a purity of at least about 90%, as measured by SDS-PAGE gel densitometry, and the maximum amount of degraded collagenase I or II impurities does not exceed 7.5%. For collagenase II fractions from the collagenase manufacturing process step, the qualification level is "purified fractions", as measured by SDS-PAGE gel densitometry, the undegraded collagenase II has a purity of at least about 90%, and the maximum amount of degraded collagenase I or II impurities does not exceed 7.5%. For example, as measured by SDS-PAGE gel densitometry, the purified collagenase I fraction can have at least about 90% or about 91.2% undegraded AUX-I, and, as measured by SDS-PAGE gel densitometry, the maximum amount of degraded AUX-I or AUX-II impurities does not exceed about 5% or about 5.8%. . As a second example, in order for an AUX-II fraction to qualify as a purified fraction, the fraction can have at least about 90% or about 91.2% purity of undegraded AUX-II as measured by SDS-PAGE gel densitometry, and no more than about 5% or about 5.8% purity of the most degraded AUX-I or AUX-II impurity as measured by SDS-PAGE gel densitometry.

[0273] The terms "subject" or "patient" are used interchangeably herein to refer to a human or other mammal.

[0274] II. Introduction

[0275] Collagenase can be produced by a variety of fermentation methods known to those skilled in the art. The crude collagenase obtained from Clostridium histolyticum has previously been purified by a variety of methods, including dye ligand affinity chromatography, heparin affinity chromatography, ammonium sulfate precipitation, hydroxyapatite chromatography, exclusion chromatography, ion exchange chromatography, and metal chelate chromatography. Crude and partially purified collagenase is commercially available from many sources, including Sigma Aldrich (SIAL-Millipore) and Advance Biofactures, Inc., Linbrook, New York. Fermentation and purification methods for the crude collagenase obtained from Clostridium histolyticum have also been described in U.S. Patent No. 7,811,560.

[0276] As described in detail below, the present invention provides an improved method for making collagenase products by: (a) controlling the metal content during purification; (b) reducing or eliminating neutral proteases from the final collagenase product, such as collagenase I and collagenase II drug substances. The present invention has general applicability to the manufacture of collagenases and the use of collagenases, regardless of the source. It is not intended to be limited to collagenases I and II derived from Clostridium histolyticum.

[0277] III. Improved production of collagenase

[0278] In a general aspect of the method of the present invention for making a highly purified collagenase (e.g., collagenase I and II drug substance), the method comprises the following steps: (1) fermenting bacteria that secrete at least collagenase and neutral protease into a liquid fermentation medium; (2) purifying the collagenase from the medium (optionally including treatment with a high salt composition); (3) separating collagenase I and collagenase II from each other; (4) reducing or eliminating neutral protease from one or both of the collagenase I and collagenase II fractions so that they are essentially free of neutral protease; and (5) producing a highly purified collagenase drug substance after removal of neutral protease. Optionally, a pharmaceutically acceptable excipient is added to this drug substance to form a pharmaceutical formulation as described elsewhere herein.

[0279] A. Fermentation to produce collagenases such as collagenase I and collagenase II

[0280] The present disclosure encompasses ferments of any bacteria that secrete collagenase into a fermentation medium. Examples of such ferments include those from Actinomycetes madura, Actinobacillus actinomycetemcomitans, Bacillus cereus, Clostridium histolyticum, Clostridium perfringens, Streptococcus mutans, Staphylococcus, Vibrio alginolyticus, and Vibrio vulnificus. This list is not exhaustive, and other bacterial organisms that secrete collagenase known to those skilled in the art may be used. See, e.g., U.S. Patent No. 7,811,560. Proteins secreted by Clostridium histolyticum include type I and type II collagenases, as well as various toxins (e.g., neutral proteases, clostripain, aerolysin-like hemolysins, and oxygen-labile hemolysins).

[0281] Fermentation medium can be any medium into which collagenase is secreted. Fermentation medium can include carbon source, nitrogen source, salt, micronutrients and water. Carbon source is not limited, but can include sugar or other carbohydrates. Nitrogen source is not limited, but can include meal, such as soy flour, or can include extract, such as yeast extract, or can include pre-digested polypeptide, such as peptone or tryptone. Peptone can be derived from animals, such as cattle-derived medium or pig-derived medium. Peptone can be derived from a source different from an animal, such as derived from vegetables, or derived from other plants.

[0282] B. Purification of Collagenase I and Collagenase II

[0283] Common purification techniques include, but are not limited to, filters, chromatography, and precipitation. Certain aspects of the purification of collagenases I and II from Clostridium histolyticum can be performed as known in the art. See, e.g., U.S. Patent No. 7,811,560.

[0284] The collagenase purification process may further include at least one of the following steps: (1) controlling the concentration of zinc and / or nickel in at least the first anion exchange step, the HIC step, and the first buffer exchange step; (2) discarding AUX-I from the subsequent fractions obtained from any anion exchange step; (3) after separating collagenase I from collagenase II, combining only the peak fractions of collagenase I until the amount of collagenase I combined from the peak fractions is approximately equal to the amount of collagenase II obtained from the manufacturing process, and (4) setting purity standards for the combination step of collagenase I, collagenase II, or both collagenase I and collagenase II. The use of these steps produces a collagenase I product that is essentially free of neutral proteases. Similarly, the collagenase II product is essentially free of neutral proteases.

[0285] Filtering collagenase and toxins from the culture medium can be accomplished in a variety of ways, such as by using depth filters, centrifugation, microfiltration, tangential flow filtration, diatomaceous earth filter beds, and vacuum filtration. In one embodiment, filtration is performed using a depth filter followed by a 0.2 micron sterilizing grade filter.

[0286] In one embodiment, the purification process may include harvesting collagenase from fermented Clostridium histolyticum (e.g., porcine-derived culture medium such as peptone) through a depth filter (e.g., Millipore Millistak HCPOD), followed by anion exchange filtration (e.g., Mustang Q filter), followed by filtration (e.g., 0.45μ filter), and conditioning the filtrate by exposing the filtrate to a high salt composition (e.g., ammonium sulfate) at a concentration of about 0.8M-1.2M, followed by a hydrophobic interaction chromatography column (e.g., phenyl SFF low-sub), followed by buffer exchange (e.g., tangential flow filtration or dialysis), followed by ion exchange chromatography (e.g., using a Q chromatography HP anion exchange column), followed by buffer exchange (e.g., tangential flow filtration or dialysis). Further, leupeptin may be added to the hydrophobic interaction chromatography step and maintained during the ion exchange step.

[0287] High salt compositions allow separation of proteins based on solubility. Salts of the Hofmeister series (easily soluble series) can be used. In certain embodiments, the composition is a solution selected from the group consisting of ammonium sulfate, sodium chloride, potassium chloride, sodium sulfate, or a mixture thereof. High salt compositions can be used at various concentrations. For example, ammonium sulfate can be used at a concentration ranging from about 0.8 M to about 1.2 M, or from about 0.9 M to about 1.1 M, or about 1 M.

[0288] Advantageously, the high salt composition contains no metal contaminants, or has a low metal content. In some embodiments, the high salt composition meets one or more of the following specifications:

[0289]

[0290] In other embodiments, the high salt composition may have zinc present in an amount less than about 85ppm, or 80ppm, or 75ppm, or 70ppm, or 65ppm, or 60ppm, or 55ppm, or 50ppm, or 45ppm, or 40ppm, or 35ppm, or 30ppm, or 25ppm, or 20ppm, or 15ppm, or 10ppm, or 9ppm, or 8ppm, or 7ppm, or 6ppm, or 5ppm, or 4ppm, or 3ppm, or 2ppm, or 1ppm. Nickel may be present in the high salt composition in an amount less than about 1.5ppm, or 1.4ppm, or 1.3ppm, or 1.2ppm, or 1.1ppm, or 1.0ppm, or 0.9ppm, or 0.8ppm, or 0.7ppm, or 0.6ppm, or 0.5ppm, or 0.4ppm, or 0.3ppm, or 0.2ppm, or 0.1ppm.

[0291] The metal content of the raw materials can be controlled by purchasing ultrapure raw materials from qualified suppliers. In one aspect, the nickel concentration is less than about 1.2 ppm, or the zinc concentration is less than about 84.4 ppm, or the nickel concentration is less than about 1.2 ppm and the zinc concentration is less than about 84.4 ppm. In other aspects, the nickel concentration, the zinc concentration, or the nickel and zinc concentrations are all less than about 0.1 ppm.

[0292] In one embodiment, collagenase I and collagenase II from Clostridium histolyticum are isolated and purified, and the purification process controls metal levels. In a further embodiment, collagenase I and collagenase II from Clostridium histolyticum are isolated and purified, and the purification process controls the levels of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, lead, or a combination thereof.

[0293] In another embodiment, the present invention is directed to a method for isolating and purifying collagenase I and collagenase II from Clostridium histolyticum, and the purification process controls nickel and / or zinc levels.

[0294] In one embodiment of the invention, the nickel concentration during the manufacturing process of proenzyme I and / or II and the formation of the drug product is less than about 100 ppm, or less than about 75 ppm, or less than about 50 ppm, or less than about 40 ppm, or less than about 30 ppm, or less than about 20 ppm, or less than about 10 ppm, or less than about 5 ppm, or less than about 3.0 ppm, or less than about 2.9 ppm, or less than about 2.8 ppm, or less than about 2.7 ppm, or less than about 2.6 ppm, or less than about 2.5 ppm, or less than about 2.4 ppm, or less than about 2.3 ppm, or less than about 2.2 ppm, or less than about 2.1 ppm, or less than about 2.0 ppm, or less than about 1.9 ppm, or less than about 1.8 ppm, or less than about 1.7 ppm, or less than about 1.6 ppm, or less than about 1.5 ppm, or less than about 1.4 ppm ppm, or less than about 1.3 ppm, or less than about 1.2 ppm, or less than about 1.1 ppm, or less than about 1.0 ppm, or less than about 0.9 ppm, or less than about 0.8 ppm, or less than about 0.7 ppm, or less than about 0.6 ppm, or less than about 0.5 ppm, or less than about 0.4 ppm, or less than about 0.3 ppm, or less than about 0.2 ppm, or less than about 0.1 ppm, or less than about 0.09 ppm, or less than about 0.08 ppm, or less than about 0.07 ppm, or less than about 0.06 ppm, or less than about 0.05 ppm, or less than about 0.04 ppm, or less than about 0.03 ppm, or less than about 0.02 ppm, or less than about 0.01 ppm.

[0295] In another embodiment of the invention, during the manufacturing process of collagenase I and / or II and the formation of the drug product, the zinc concentration is less than about 1000 ppm, or less than about 950 ppm, or less than about 900 ppm, or less than about 850 ppm, or less than about 800 ppm, or less than about 750 ppm, or less than about 700 ppm, or less than about 650 ppm, or less than about 600 ppm, or less than about 550 ppm, or less than about 500 ppm, or less than about 450 ppm, or less than about 400 ppm, or less than about 350 ppm, or less than about 300 ppm, or less than about 250 ppm, or less than about 200 ppm, or less than about 150 ppm, or less than about 125 ppm, or less than about 100 ppm, or less than about 95 ppm, or less than about 90 ppm, or less than about 85 ppm, or less than about 84 ppm, or less than about 80 ppm, or less than about or less than about 75 ppm, or less than about 70 ppm, or less than about 65 ppm, or less than about 60 ppm, or less than about 55 ppm, or less than about 50 ppm, or less than about 45 ppm, or less than about 40 ppm, or less than about 35 ppm, or less than about 30 ppm, or less than about 25 ppm, or less than about 20 ppm, or less than about 15 ppm, or less than about 10 ppm, or less than about 9 ppm, or less than about 8 ppm, or less than about 7 ppm, or less than about 6 ppm, or less than about 5 ppm, or less than about 4 ppm, or less than about 3 ppm, or less than about 2 ppm, or less than about 1 ppm, or less than about 0.9 ppm, or less than about 0.8 ppm, or less than about 0.7 ppm, or less than about 0.6 ppm, or less than about 0.5 ppm, or less than about 0.4 ppm, or less than about 0.3 ppm, or less than about 0.2 ppm, or less than about 0.1 ppm.

[0296] C. Separation of Collagenase I and Collagenase II

[0297] Collagenase I and II can be separated by any technique known to those skilled in the art. For example, collagenase I and II can be separated from each other using ion exchange chromatography. One type of ion exchange chromatography can be an anion exchange column, such as a Q-agarose column.

[0298] D. Reduce or eliminate neutral proteases

[0299] In general, if multiple collagenases are present, the step of eliminating neutral proteases occurs after the collagenases are separated from each other. During the elution step, ion exchange chromatography is used to separate the collagenases. Collagenase elution generally occurs in peaks, with collagenase I in one peak and collagenase II in a second peak. Each peak can be collected as a fraction for each elution peak time.

[0300] Neutral proteases from collagenase I and collagenase II products can be reduced or eliminated by one or more of the following elimination steps: (a) excluding any fractions having a collagenase I or II content of less than about 90% and any single impurity content greater than about 5% as determined by SDS PAGE gel or HPLC; (b) excluding fractions having detectable levels of neutral proteases as measured by SDS PAGE gel or zymography; (c) excluding any fractions calculated to contain excessive levels of neutral proteases. Each is described below and can be used alone or in combination.

[0301] The scale of the fraction is not limited, but exemplary industrial scale fractions may be less than about 200 ml, or 300 ml, or 400 ml, or 500 ml, or 600 ml, or 700 ml, or 800 ml, or 900 ml, or 1000 ml, or 1100 ml, or 1200 ml, or 1300 ml, or 1400 ml, or 1500 ml, or 1600 ml, or 1700 ml, or 1800 ml, or more.

[0302] The amount of collagenase in each fraction is not limited, but exemplary industrial-scale fractions may include a protein concentration of between about 0.1 g / L to about 5 g / L or more, such as about 0.2 g / L, or 0.5 g / L, or 0.8 g / L, or 1.1 g / L, or 1.4 g / L, or 1.7 g / L, or 2.0 g / L, or 2.3 g / L, or 2.6 g / L, or 2.9 g / L, or 3.2 g / L, or 3.5 g / L, or 3.8 g / L, or 4.1 g / L, or 4.4 g / L, or 4.7 g / L, or 5.0 g / L, or 6 g / L, or 7 g / L, or 8 g / L, or 9 g / L, or 10 g / L, or more.

[0303] The percent purity of the collagenase of each fraction can be determined using any method known to those skilled in the art. An exemplary method is to measure purity using SDS-PAGE or SDS-PAGE and densitometry. Densitometry software can be any software that converts the SDS-PAGE band on the gel into an intensity quantity. An example of such software is "Quantity-@". The SDS-PAGE gel can have any number of lanes, and the common number of lanes includes 10 lanes or 15 lanes. The sample well size of each lane may be different; the common sample well size is between 15 and 50 microliters. Generally, any amount of protein suitable for the sample well can be loaded, but the typical amount sufficient to distinguish the target product is any amount of about 0.5 to about 5 micrograms of protein.

[0304] It is also possible to determine whether there is a neutral protease in the fraction. Any method known to those skilled in the art for determining the presence of a neutral protease in the fraction can be used. Exemplary methods include SDS-PAGE as just described or SDS-PAGE with densitometry. Other exemplary methods include casein zymography or casein zymography with densitometry. Casein zymography is a SDS-PAGE gel, usually embedded with bovine casein in the gel, to which neutral proteases can act, but collagenase cannot. Therefore, the band in the zymography gel containing neutral proteases is displayed as a different color from the band containing collagenase or other impurities. Typically, the limit of detection (LOD) of the zymography assay related to the present disclosure using densitometry software is about 0.5 nanograms of protein, intuitively about 0.2-0.3 nanograms. However, less amount may be detected depending on the exact method used.

[0305] Generally, when used for impurity detection, a higher amount of protein is loaded in the sample wells than for distinguishing the main target product. Impurities can be detected using the same protein amount as the main target product, but it may be necessary to load at least about 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 12 or 15 or 20 or more times of loading in the sample wells to distinguish or detect the main target product. This is especially true as the target product purity increases. Generally, the amount of protein loaded in the sample wells to detect the neutral protease associated with the disclosure is between about 1 microgram to about 100 micrograms, such as about 5 micrograms, 8 micrograms, or 10 micrograms, or 12 micrograms, or 15 micrograms, or 20 micrograms, or 30 micrograms, or 50 micrograms, or 75 micrograms or more.

[0306] In certain aspects, the present disclosure provides three different ways to reduce or eliminate neutral proteases:

[0307] 1. Elimination strategy 1

[0308] According to this strategy, fractions that do not meet the selected purity specification are set aside and excluded from further forward processing. Each fraction is assayed using SDS-PAGE or SDS-PAGE with densitometry, and each band in the gel lane corresponds to the percentage of total protein loaded in the wells above the lane. These bands may correspond to collagenase I or collagenase II, or may correspond to impurities in the form of fragments of collagenase or other proteins (such as neutral proteases). An acceptable level of collagenase I or collagenase II for the combined steps used to produce a collagenase I product or a collagenase II product, respectively, is at least 80% by area as measured by SDS-PAGE.

[0309] In other embodiments, an acceptable level of collagenase I for the combined steps to produce a collagenase I product is at least about 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 88.5%, or 89.0%, or 89.5%, or 90%, or 90.5%, or 91%, or 91.5%, or 92%, or 92.5%, or 93%, or 93.5%, or 94%, or 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 100% by area as measured by SDS-PAGE.

[0310] In one embodiment, an acceptable level of collagenase II for the combined steps to produce a collagenase II product is at least about 81%, or 82%, or 83%, or 84%, or 85%, or 86%, or 87%, or 88%, or 88.5%, or 89.0%, or 89.5%, or 90%, or 90.5%, or 91%, or 91.5%, or 92%, or 92.5%, or 93%, or 93.5%, or 94%, or 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 100% by area as measured by SDS-PAGE.

[0311] In one embodiment, the acceptable level of the largest impurity of the collagenase I drug substance (after purification and separation from collagenase II, but before mixing with collagenase II) is less than about 20%, or 19%, or 18%, or 17%, or 16%, or 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.75%, or 0.5%, or 0.25%, or 0.1% (w / w) of the collagenase I drug substance as determined by SDS-PAGE. The amount of the largest impurity can also be measured, for example, by SDS-PAGE with densitometry.

[0312] In another embodiment, the acceptable level of the largest impurity of the collagenase II drug substance (after purification and separation from collagenase I, but before mixing with collagenase I) is less than about 20%, or 19%, or 18%, or 17%, or 16%, or 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.75%, or 0.5%, or 0.25%, or 0.1% (w / w) of the collagenase I drug substance as determined by SDS-PAGE. The amount of the largest impurity can also be measured, for example, by SDS-PAGE with densitometry.

[0313] In addition, the acceptable level of the maximum amount of impurity of the mixed collagenase I and collagenase II drug substance is less than about 5%, or 4%, or 3%, or 2%, or 1%, or 0.75%, or 0.5%, or 0.25%, or 0.1% (w / w) of the drug substance as measured by SDS-PAGE. The amount of the maximum amount of impurity can also be measured, such as by SDS-PAGE with densitometry.

[0314] In addition, in certain embodiments, once the selected fractions are combined into the collagenase I or collagenase II product, the total purity of the combined fractions is at least about 95%, or 96%, or 97%, or 98%, or 99%, or 100% as determined by RP-HPLC. In other embodiments, the total purity of the combined fractions is between about 80% and 100% as measured by RP-HPLC.

[0315] 2. Elimination strategy 2

[0316] According to this strategy, if the fraction has a neutral protease of detectable level measured by, for example, SDS-PAGE, SDS-PAGE with densitometry, casein zymography, casein zymography with densitometry or its combination, then the fraction is excluded from further processing (forward processing). Determine the concentration of protein in each fraction by any method known to those skilled in the art. Each sample loading hole of gel can load any number of proteins. In some embodiments, the amount of the protein loaded is between about 0.1 microgram and about 100 micrograms, such as about 90,80,70,60,50,40,30,25,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2 or 1 microgram. Any fraction containing the neutral protease of detectable level on gel is excluded from merging.

[0317] 3. Elimination strategy 3

[0318] According to this strategy, collagenase I fractions are pooled based on the position of the fractions relative to the elution peak and the estimated amount of collagenase II that is pooled. In other words, the elution peak of collagenase II serves as an identifier for which collagenase I fractions can be pooled.

[0319] The fraction of collagenase I through merging can be from any fraction of collagenase I elution peak.When multiple fractions are merged, these fractions can be continuous fractions or discontinuous fractions.In some embodiments, merge fractions start from the maximum elution peak of collagenase I fraction, and move to the tail evenly on both sides of the peak.In other embodiments, merge fractions start from the first fraction that meets the specified purity standard for collagenase I purity or maximum impurity or both, and then move towards the maximum collagenase I elution peak.In some embodiments, collect fractions from any fraction (measured by any technology known to those skilled in the art) that does not contain a detectable level of neutral protease, and move to the starting point of the collagenase I elution peak.

[0320] The amount of collagenase I combined may be different from the estimated amount of collagenase II (total collagenase II elution peak, the amount of collagenase II combined, or other). In some embodiments, the estimated amount of collagenase II can be about two times the estimated amount of collagenase I, or about one and a half times the estimated amount of collagenase I, or about three times the estimated amount of collagenase I. Any particular ratio works, such as a ratio of collagenase I to collagenase II of about 1:1.2, or a ratio of collagenase I to collagenase II of about 1:1.1, or 1:1.3, or 1:1.4, or more.

[0321] For example, the strategy includes: (a) calculating the approximate amount of eluted collagenase II contained in the fractions passing the above purity requirement, and (b) discarding tail fractions from the collagenase I elution peak that exceed the approximate amount of collagenase II to produce an approximately 1:1 mass ratio of collagenase I to the amount of collagenase II calculated in (a), wherein the pooling of collagenase I fractions begins with approximately the first passing fraction of the tail of the collagenase I elution peak and moves toward the final collagenase I fraction. The collagenase composition produced in this way is essentially free of neutral proteases.

[0322] In another embodiment, the present disclosure relates to a process for making collagenase I and collagenase II derived from Clostridium histolyticum, the process comprising the steps of: (a) separating collagenase I from collagenase II using chromatography, (b) collecting elution fractions from step (a) for collagenase I and collagenase II, respectively, (c) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total amount of protein in the fraction as measured by SDS-PAGE analyzed by densitometry, and any single impurity such as greater than about 5.8% of the total amount of protein in the fraction as measured by SDS-PAGE analyzed by densitometry, and (d) calculating the approximate amount of eluted collagenase II contained in the fraction as required by the purity of (c), and discarding tail fractions from the collagenase I elution peak that exceed the approximate amount of collagenase II to produce an approximately 1:1 mass ratio of collagenase I to the amount of collagenase II calculated in (d), wherein discarding the tail collagenase I fraction begins at about the tail of the collagenase I elution peak and moves toward the largest peak. The collagenase composition produced in this manner is substantially free of neutral proteases.

[0323] E. Finalization of drug product and formulation after neutral protease removal

[0324] The collagenase I drug substance, after the neutral protease elimination step and before it is mixed with collagenase II, may have less than about 50 ng of neutral protease per mg of collagenase I drug substance as measured by casein zymography with or without densitometry. Alternatively, the neutral protease per mg of collagenase I drug substance may be less than about 40 ng, or 30 ng, or 20 ng, or 10 ng, or 5 ng, or 1 ng, or less as measured by casein zymography with or without densitometry.

[0325] In another aspect, the collagenase II drug substance, after the neutral protease elimination step, and prior to mixing with collagenase I, may have less than about 50 ng of neutral protease per mg of collagenase II drug substance, as measured by casein zymography with or without densitometry. Alternatively, the neutral protease per mg of collagenase II drug substance may be less than about 40 ng, or 30 ng, or 20 ng, or 10 ng, or 5 ng, or 1 ng, or less, as measured by casein zymography with or without densitometry.

[0326] In addition, the neutral protease in the final mixed collagenase I and collagenase II drug substance per mg of collagenase I and II drug substance can be less than about 50 nanograms, as measured by casein zymography with or without densitometry. Alternatively, the neutral protease in the final mixed collagenase I and collagenase II drug substance can be less than about 40 nanograms, or 35 nanograms, or 30 nanograms, or 25 nanograms, or 20 nanograms, or 15 nanograms, or 10 nanograms, or 5 nanograms, or 4 nanograms, or 3 nanograms, or 2 nanograms, or 1 nanogram, or less, as measured by casein zymography with or without densitometry.

[0327] In other embodiments, the collagenase drug substance has less than about 1000 ng, or 750 ng, or 500 ng, or 250 ng, or 100 ng, or 75 ng, or 50 ng, or 40 ng, or 35 ng, or 30 ng, or 25 ng, or 20 ng, or 15 ng, or 10 ng, or 5 ng, or 4 ng, or 3 ng, or 2 ng, or 1 ng, or less neutral protease per mg of collagenase as measured by casein zymography with or without densitometry.

[0328] In one example, the amount of neutral protease present is determined by comparing the amount of neutral protease measured in the sample wells with about 5 micrograms to about 20 micrograms of collagenase. For example, the sample for the zymography test method is prepared with 0.5 micrograms of AUX-1 per microliter of sample and loaded onto the SDS-PAGE gel with 20 microliters. This produces 10 micrograms of AUX-1 loading on the gel. The detection limit of the zymography test method is 0.5 nanograms of neutral protease. Therefore, the detection limit is 0.5 nanograms of NP per 10 micrograms of AUX-I.

[0329]

[0330] In another embodiment, the present disclosure relates to a collagenase composition derived from Clostridium histolyticum, wherein the collagenase composition comprises a collagenase I product and a collagenase II product, wherein the purity of the collagenase product is at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC), and wherein the collagenase I product is essentially free of neutral proteases.

[0331] In some embodiments, the collagenase composition or pharmaceutical formulation has a mass ratio of collagenase I to collagenase II of approximately 1 to 1. However, other mass ratios of collagenase I to collagenase II are also envisioned. The mass ratio of collagenase I to collagenase II can be in any range of about 1:10 to about 10:1. Preferably, the mass ratio of collagenase I to collagenase II is between about 1:3 to about 3:1. More preferably, the mass ratio of collagenase I to collagenase II is between about 0.9:1 to about 1:1.4. For example, the mass ratio of collagenase I to collagenase II can be about 1:1, or 1:1.5, or 1.5:1, or 2.4:1, 1:2.1, or 1:1.3, or 1:1.4, or 1.4:1, or 1.3:1. In one aspect, the mass ratio of collagenase I to collagenase II is about 1:1. In one embodiment, the collagenase concentrate has an extinction coefficient of 1.528.

[0332] In addition, the present invention provides a collagenase composition comprising collagenase I and collagenase II derived from Clostridium histolyticum, wherein collagenase I and collagenase II have a mass ratio of collagenase I to collagenase II of about 0.9:1 to about 1:1.4 (for example, about 1:1), wherein the purity of the collagenase composition as measured by RP-HPLC is at least 90% by area, or the purity as measured by RP-HPLC is at least about 95% by area, or the purity as measured by RP-HPLC is at least 97% by area, or the purity as measured by RP-HPLC is at least 98% by area, or the purity as measured by RP-HPLC is at least 99% by area.

[0333] The pharmaceutical formulation of the present invention comprises a therapeutically effective amount of the collagenase composition of the present invention, which is formulated together with one or more pharmaceutically acceptable carriers or excipients.

[0334] In another embodiment, the concentration of zinc and / or nickel is controlled in the manufacturing process of collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, and the formation of the drug product results in a process reproducibility of about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% for a collagenase product, or a collagenase I product, or a collagenase II product, having a purity by area of ​​at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% as measured by RP-HPLC (reverse phase high pressure liquid chromatography).

[0335] In one embodiment, the present disclosure relates to a method for isolating and purifying collagenase I and collagenase II from Clostridium histolyticum, wherein nickel levels and zinc levels are controlled in the collagenase purification process, thereby resulting in improved process reproducibility of collagenase I or collagenase II with a purity of greater than about 95%.

[0336] In one example, the purification process includes harvesting collagenase from fermented Clostridium histolyticum (such as from porcine derived culture medium (preferably, protease peptone) medium) through a filter (e.g., Millipore Millistak HC POD), followed by adjusting the Mustang Q filtrate to 1 M using an ammonium sulfate concentrate (e.g., 60% saturated ammonium sulfate) to promote binding of the collagenase to a HIC chromatography column, and then resuspending or dissolving the precipitated collagenase in ammonium sulfate, followed by anion exchange chromatography (e.g., using a Mustang Q column), followed by filtration (e.g., a 0.45μ filter), followed by buffer exchange (e.g., dialysis or tangential flow filtration), followed by running the filtrate on a hydrophobic interaction chromatography column (e.g., Q-agarose), followed by the addition of leupeptin, followed by buffer exchange.

[0337] In another example, the purification process includes passing through a depth filter (e.g., Millipore Millistak HC POD) from fermented Clostridium histolyticum (e.g., fermented in a plant-derived medium), followed by addition of salt (preferably ammonium sulfate) to a concentration of about 0.8 M to about 1.2 M (e.g., to about 1 M ammonium sulfate), followed by use of a hydrophobic interaction chromatography column (e.g., phenyl sepharose), followed by buffer exchange (e.g., using TFF and removing ammonium sulfate), followed by anion exchange chromatography (e.g., using a Q-sepharose column), followed by collection of fractions of the filtrate produced by the anion exchange chromatography for AUX-I and AUX-II, followed by combining the fractions of AUX-I together, and combining the fractions of AUX-II together. Leupeptin can be added to the purification process in the hydrophobic interaction chromatography step, the first buffer exchange step, and the second anion exchange chromatography step.

[0338] One embodiment of a purification process according to the present invention includes harvesting collagenase from fermented Clostridium histolyticum through a filter (e.g., Millipore Millistak HC POD), followed by filtration through at least one 0.2 micron filter, followed by anion exchange chromatography (such as Mustang Q filtration), wherein the effluent is adjusted to an ammonium sulfate concentration of about 0.8 M to about 1.2 M, followed by use of a hydrophobic interaction (HIC) column (e.g., phenyl agarose), followed by buffer exchange (e.g., using TFF and removing ammonium sulfate), followed by anion exchange chromatography (e.g., Q-agarose), followed by collection of fractions of AUX-I and AUX-II eluates generated by anion exchange chromatography for AUX-I and AUX-II, respectively, followed by combining the fractions of AUX-I together, and combining the fractions of AUX-II together, followed by buffer exchange to a pharmaceutical formulation buffer (e.g., 10 mM Tris, 60 mM sucrose at pH 8 using tangential flow filtration). Leupeptin is optionally added to the purification process in the hydrophobic interaction chromatography step, the first buffer exchange step and the second anion exchange chromatography step.

[0339] In certain embodiments of the invention, the collagenase purification process comprises the following steps: a) filtering the crude harvest using ion exchange chromatography; preferably using an anion exchange capsule filter (such as MUSTANG Q); b) adding ammonium sulfate; preferably to a final concentration of about 1 M; c) filtering the crude harvest; preferably, through a 0.45 micron filter; d) treating the filtrate by hydrophobic interaction chromatography (HIC); preferably, a phenyl sepharose 6FF (low sub) column; e) adding leupeptin to the eluate; preferably, the final concentration of the eluted product after HIC is about 0.2 mM; f) removing ammonium sulfate and maintaining leupeptin; preferably, using buffer exchange by tangential flow filtration (TFF); g) filtering the mixture of step (f); preferably, through a 0.45 micron filter; h) separating collagenase I and collagenase II using ion exchange chromatography; preferably, using a Q-Sepharose High Performance (Q HP) column; i) performing a neutral protease elimination step. Optionally, additional steps may be performed, including j) performing buffer exchange of collagenase I and II, respectively, after step (i); preferably preparing TFF concentrates and formulations of collagenase I and collagenase II, respectively, wherein TFF is a tangential flow filter with a 10 and / or 30K MWCO (molecular weight cut-off) PES or RC-polyethersulfone or regenerated cellulose filter membrane (TFF provides a means of retaining and concentrating selected proteins and exchanging proteins from one buffer solution to another); and k) preferably filtering the buffer-exchanged collagenases I and II, respectively, through a 0.2 micron filtration system.

[0340] One embodiment of the invention includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is controlled in the process such that the concentration of nickel is less than about 0.2 ppm in the manufacturing process, and / or the concentration of zinc is less than about 1 ppm in the manufacturing process, and these metal concentrations produce a process reproducibility of greater than about 90%, and achieve a purity of greater than about 95% by area as measured by RP-HPLC for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II. Another embodiment of the invention includes monitoring the levels of zinc and nickel in the manufacturing process of collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of nickel is maintained at less than about 0.2 ppm in the manufacturing process, and / or the concentration of zinc is maintained at less than about 1 ppm in the manufacturing process, such that these metal concentrations produce a process reproducibility of greater than about 95% for a collagenase composition having a purity of greater than about 95%.

[0341] The present disclosure further includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is monitored and / or controlled during the process such that the concentration of nickel is less than about 0.2 ppm during the manufacturing process and / or the concentration of zinc is less than about 1 ppm during the manufacturing process, and these metal concentrations produce a process repeatability of greater than about 90%, resulting in collagenase, collagenase I, or collagenase II, or both collagenase I and collagenase II, having a purity greater than about 95%. Another embodiment of the invention includes monitoring the levels of zinc and nickel in a manufacturing process for collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of nickel in the manufacturing process is maintained at less than about 0.2 ppm, and / or the concentration of zinc in the manufacturing process is maintained at less than about 1 ppm, such that these metal concentrations produce a process reproducibility of greater than about 95%, and a purity of greater than about 95% by area, as measured by RP-HPLC for collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II.

[0342] Another embodiment of the invention includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is monitored and / or controlled during the process such that the concentration of nickel is less than about 0.5 ppm during the manufacturing process, and / or the concentration of zinc is less than about 10 ppm during the manufacturing process, and these metal concentrations result in a process reproducibility greater than about 90%, such as achieving a purity of greater than about 95% by area for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II as measured by RP-HPLC. Yet another embodiment of the invention includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is monitored and / or controlled during the process such that the concentration of nickel is less than about 0.5 ppm during the manufacturing process and / or the concentration of zinc is less than about 10 ppm during the manufacturing process, and these metal concentrations result in a process reproducibility greater than about 95%, such as achieving a purity of greater than about 95% by area for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II as measured by RP-HPLC.

[0343] Yet another embodiment of the invention includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is controlled during the process such that the concentration of nickel is less than about 0.5 ppm during the preparation process and / or the concentration of zinc is less than about 10 ppm during the manufacturing process, and these metal concentrations result in a process reproducibility greater than about 95%, such as achieving a purity of the collagenase composition greater than about 95% by area as measured by RP-HPLC.

[0344] Another embodiment of the invention includes a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is controlled in the process such that the concentration of nickel in the manufacturing process is less than about 0.5 ppm and / or the concentration of zinc in the manufacturing process is less than about 10 ppm, and these metal concentrations produce a process reproducibility greater than about 95%, such as achieving a purity of greater than about 95% by area for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II as measured by RP-HPLC.

[0345] Further disclosed is a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is controlled in the process such that the concentration of nickel in the manufacturing process is less than about 0.5 ppm and / or the concentration of zinc in the manufacturing process is less than about 10 ppm, and these metal concentrations result in a process repeatability greater than about 90%, such as achieving a purity of greater than about 97% by area for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II as measured by RP-HPLC.

[0346] The present disclosure further contemplates a method for manufacturing collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II, wherein the concentration of zinc and / or nickel is controlled in the process such that the concentration of nickel in the manufacturing process is less than about 0.5 ppm and / or the concentration of zinc in the manufacturing process is less than about 10 ppm, and these metal concentrations result in a process repeatability greater than about 95%, such as achieving a purity of greater than about 97% by area for the collagenase, collagenase I, collagenase II, or both collagenase I and collagenase II as measured by RP-HPLC.

[0347] In another embodiment, the present disclosure relates to a process for producing collagenase I and collagenase II derived from Clostridium histolyticum, the process comprising the steps of: (a) separating collagenase I from collagenase II using chromatography, (b) separately collecting elution fractions of collagenase I and collagenase II produced in step (a), (c) analyzing each fraction for neutral protease using SDS-PAGE, SDS-PAGE and densitometry, or zymography, (d) discarding fractions containing collagenase I or collagenase II as measured by SDS-PAGE with densitometry, and (e) discarding the fractions containing collagenase I or collagenase II. The invention relates to a method for producing a collagenase composition comprising: combining only the fractions that pass through collagenase I for the collagenase I product and combining only the fractions that pass through collagenase II for the collagenase II product, wherein the purity of the combined collagenase I product or the collagenase II product is at least 95% as measured by RP-HPLC. The collagenase composition produced by this method is essentially free of neutral proteases.

[0348] In another embodiment, the present disclosure relates to a process for producing collagenase I and collagenase II derived from Clostridium histolyticum, the process comprising the steps of: (a) separating collagenase I from collagenase II using chromatography, (b) separately collecting elution fractions of collagenase I and collagenase II produced in step (a), (c) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total amount of protein as measured by SDS-PAGE with densitometry analysis, and any single impurity greater than about 1% of the total amount of protein as measured by SDS-PAGE with densitometry analysis. The total amount of protein in the fractions measured by SDS-PAGE with densitometry analysis is about 5.8%, and (d) calculating the approximate amount of eluted collagenase II contained in the fractions as required by the purity of (c), and discarding the tail fractions from the collagenase I elution peak that exceed the approximate amount of collagenase I to produce an approximately 1:1 mass ratio of collagenase I to the amount of collagenase II calculated in (d), wherein the combined collagenase I fractions start from about the first pass fraction at the tail of the collagenase I elution peak and move toward the final collagenase I fraction. The collagenase composition produced in this way is essentially free of neutral proteases.

[0349] The present disclosure relates to a process for producing collagenase I and collagenase II derived from Clostridium histolyticum, the process comprising the steps of: (a) separating collagenase I from collagenase II using chromatography, (b) separately collecting eluted fractions of collagenase I and collagenase II produced in step (a), (c) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total amount of protein as measured by SDS-PAGE with densitometry, (d) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total amount of protein as measured by SDS-PAGE with densitometry, and (e) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total amount of protein as measured by SDS-PAGE with densitometry. any single impurity is greater than about 5.8% of the total amount of protein in the fraction as measured by SDS-PAGE, and (d) calculating the approximate amount of eluted collagenase II contained in the fractions meeting the purity requirement of (c), and discarding tail fractions from the collagenase I elution peak that exceed the approximate amount of collagenase I to produce a mass ratio of collagenase I to the amount of collagenase II calculated in (d) of approximately 1:1, wherein discarding the tail collagenase I fraction starts from near the tail of the collagenase I elution peak and moves toward the maximum peak. The collagenase composition produced by this method is essentially free of neutral proteases.

[0350] The present disclosure relates to a process for producing collagenase I and collagenase II derived from Clostridium histolyticum, wherein (a) neutral proteases are substantially eliminated from the collagenase I product during the manufacturing process, and (b) the concentration of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, lead, or a combination thereof, is controlled during the manufacturing process.

[0351] In another embodiment, the present disclosure relates to a method for purifying collagenase I and collagenase II, comprising: (a) filtering the fermentation broth from Clostridium histolyticum through an anion exchange filter; (b) adding ammonium sulfate to the filtrate of step (a); (c) passing the filtrate of step (b) through a hydrophobic interaction chromatography column; (d) adding leupeptin to the eluate of step (c); (e) removing ammonium sulfate from the mixture of step (d); (f) filtering the mixture of step (e); and (g) separating collagenase I and II in the mixture of step (f) using ion exchange chromatography, wherein the concentration of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, lead, or a combination thereof is controlled during the purification process.

[0352] In a further embodiment, the present disclosure relates to a method for purifying collagenase I and collagenase II, comprising: (a) filtering the fermentation broth from Clostridium histolyticum through an anion exchange filter; (b) adding ammonium sulfate to the filtrate of step (a); (c) passing the filtrate of step (b) through a hydrophobic interaction chromatography column; (d) adding leupeptin to the eluate of step (c); (e) removing ammonium sulfate from the mixture of step (d); (f) filtering the mixture of step (e); and (g) separating collagenase I and II in the mixture of step (f) using ion exchange chromatography, wherein the nickel level or zinc level is controlled during the purification process.

[0353] The present disclosure also relates to a method for purifying collagenase I and collagenase II, which comprises: (a) filtering the fermentation broth from Clostridium histolyticum through an anion exchange filter; (b) adding ammonium sulfate in the filtrate step of step (a); (c) passing the filtrate of step (b) through a hydrophobic interaction chromatography column; (d) adding leupeptin to the eluate of step (c); (e) removing ammonium sulfate from the mixture of step (d); (f) filtering the mixture of step (e); and (g) separating collagenase I and II in the mixture of step (f) by ion exchange chromatography, and (h) removing neutral protease from the collagenase I or collagenase II produced by the elution of step (g).

[0354] In addition, the present invention relates to a method for purifying collagenase I and collagenase II, which comprises: (a) filtering the fermentation broth from Clostridium histolyticum through an anion exchange filter; (b) adding ammonium sulfate to the filtrate of step (a); (c) subjecting the filtrate of step (b) to a hydrophobic interaction chromatography column; (d) adding leupeptin to the eluate of step (c); (e) removing ammonium sulfate from the mixture of step (d); (f) filtering the mixture of step (e); and (g) separating collagenase I and II in the mixture of step (f) using ion exchange chromatography, wherein the nickel level and the zinc level are controlled in the collagenase purification process.

[0355] In another embodiment, the present disclosure relates to a process for producing a drug product consisting of isolated and purified collagenase I and collagenase II derived from Clostridium histolyticum, wherein the mass ratio of collagenase I to collagenase II is about 1:1, and the purity of the drug product is at least 95% by area as measured by reverse phase high performance liquid chromatography, comprising the following steps: (a) fermenting Clostridium histolyticum; (b) harvesting a crude fermentation product comprising collagenase I and collagenase II; (c) purifying collagenase I and collagenase II from the crude harvest by filtration and column chromatography, comprising the following steps: (i) filtering the purified collagenase I and collagenase II from Clostridium histolyticum through an anion exchange filter; fermentation broth; (ii) adding ammonium sulfate to the filtrate of step (i); (iii) passing the filtrate of step (ii) through a hydrophobic interaction chromatography column; (iv) adding leupeptin to the eluate of step (iii); (v) removing ammonium sulfate from the mixture of step (iv); (vi) filtering the mixture of step (v); and (vii) separating collagenase I and collagenase II in the mixture of step (vi) by ion exchange chromatography; and (d) combining collagenase I and collagenase II purified by step (c) in a ratio of about 1:1, wherein the concentration of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, lead or a combination thereof is controlled in the manufacturing process.

[0356] Further, the present disclosure relates to a process for producing a drug product, which consists of isolated and purified collagenase I and collagenase II derived from Clostridium histolyticum, wherein the mass ratio of collagenase I to collagenase II is about 1:1, and the purity of the drug product is at least 95% by area as measured by reversed-phase high performance liquid chromatography, which comprises the following steps: (a) fermenting Clostridium histolyticum; (b) harvesting a crude fermentation product containing collagenase I and collagenase II; (c) purifying collagenase I and collagenase II from the crude harvest by filtration and column chromatography, which comprises the following steps: (i) separating the collagenase I and collagenase II from Clostridium histolyticum through an anion exchange filter; and (ii) separating the collagenase I and collagenase II from the crude harvest by filtration and column chromatography. (ii) filtering the fermentation broth from step (i); (iii) passing the filtrate from step (ii) through a hydrophobic interaction chromatography column; (iv) adding leupeptin to the eluate from step (iii); (v) removing ammonium sulfate from the mixture from step (iv); (vi) filtering the mixture from step (v); and (vii) separating collagenase I and collagenase II in the mixture from step (vi) by ion exchange chromatography; and (d) combining collagenase I and collagenase II purified from step (c) in a ratio of about 1:1, wherein the nickel level or zinc level, or the levels of zinc and nickel, are controlled in the manufacturing process.

[0357] The present disclosure relates to a process for producing a drug product, which consists of isolated and purified collagenase I and collagenase II derived from Clostridium histolyticum, wherein the mass ratio of collagenase I to collagenase II is about 1:1, and the purity of the drug product is at least 95% by area as measured by reverse phase high performance liquid chromatography, which comprises the following steps: (a) fermenting Clostridium histolyticum; (b) harvesting a crude fermentation broth containing collagenase I and collagenase II; (c) purifying collagenase I and collagenase II from the crude harvest by filtration and column chromatography, which comprises the following steps: (i) filtering the fermentation broth from Clostridium histolyticum through an anion exchange filter; (ii) adding ammonium sulfate to the filtrate of step (i); (iii) passing the filtrate of step (ii) through a hydrophobic interaction chromatography column; (iv) adding leupeptin to the eluate of step (iii); (v) removing ammonium sulfate from the mixture of step (iv); and (vi) ) filtering the mixture of step (v); and (vii) separating collagenase I and II in the mixture of step (vi) by ion exchange chromatography; wherein the separation step further comprises (a) collecting the eluted fractions of collagenase I and collagenase II produced in (vii), respectively, (b) analyzing the fractions for each neutral protease using SDS-PAGE or zymography, (c) discarding any fractions in which collagenase I or collagenase II is less than about 91.8% of the total protein in the fraction as measured by SDS-PAGE with densitometry analysis, any single impurity greater than about 5.8% of the total protein in the fraction as measured by SDS-PAGE with densitometry analysis, or a detectable level of neutral protease as tested in (b); and (d) combining only the fractions that pass through collagenase I for the collagenase I product, and combining only the fractions that pass through collagenase II for the collagenase II product. The collagenase composition produced by this method is essentially free of neutral protease.

[0358] IV. Examples

[0359] The following examples are included to demonstrate certain embodiments of the present disclosure. However, based on the present disclosure, it should be understood by those skilled in the art that modifications can be made in the specific embodiments disclosed and similar results can be obtained without departing from the spirit and scope of the present invention. Therefore, all contents set forth should be interpreted as illustrative rather than restrictive.

[0360] Example 1 - Classification and trending of impurities in IEX fractions when analyzed by optimized SDS-PAGE method

[0361] As shown in Table 1, IEX fractions were tested from 13 different batches of collagenase I and collagenase II prepared from Clostridium histolyticum. In each batch, each collagenase fraction collected from the IEX eluate was run on SDS-PAGE gel electrophoresis and analyzed using a Bio-Rad densitometer and corresponding optical density analysis software. For each fraction of each batch, the percentage purity of undegraded collagenase I or II and the percentage of each visible impurity (collagenase fragment or other) were determined.

[0362] Table 1: Batches of fractions impurity classification used to determine the optimal SDS-PAGE method

[0363]

[0364]

[0365] Using the molecular weight values ​​reported by the software and the vertical position of the impurities compared to the vertical position of the known molecular weight bands in the molecular weight marker lanes, the impurities were divided into four main categories (90 kD, 80 kD, 55 kD and 40 kD). Impurities that do not belong to a category are classified as "others". Compared with using only the current SDS-PAGE method, the SDS-PAGE method using gel densitometry can resolve more impurity bands.

[0366] A. Quantification of major and minor bands in collagenase C. histolytica ion exchange chromatography fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry

[0367] The method uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 4-12% NuPAGE gel (MES buffer) and colloidal Coomassie staining to determine the purity and impurities of collagenase Clostridium histolyticum ion exchange chromatography fractions. The method uses the optical density measurement analysis of the gel to allow the relative quantification of the major band (percentage of AUX-1, AUX-II or both) and minor bands of product fragments or other impurities (percentage of minor bands).

[0368] Typically, the amount of collagenase loaded into the wells of an SDS-PAGE gel is linearly related to the intensity of the bands seen using densitometry software. For example, the linearity of the response to the collagenase band can be observed for 1.123-1.875 μg of collagenase (75%-125% of target), R 2 Values ​​> 0.98 of collagenase on micrograms of a known reference standard are demonstrated in the graph.

[0369] A reference standard should be used for the sample being analyzed. For example, an AUX-1 intermediate reference standard can be used to analyze the AUX-1 elution fraction from the IEX step. The amount of reference standard and sample required to form a gel final sample (C2 = 0.15 mg protein / mL) is calculated as follows: C1V1 = C2V2

[0370] C1 = initial concentration of the sample to be tested, in mg / mL

[0371] V1 = sample volume required at initial concentration, in microliters

[0372] C2 = 1 mg / mL (final sample preparation concentration)

[0373] V2 = final sample preparation volume in microliters

[0374] B. Classification of impurities

[0375] i. Impurity classification method

[0376] Fraction impurities can be determined based on analysis of gels produced by analyzing fraction samples using SDS-PAGE with densitometry. Each gel is placed on a densitometer and the gel image is scanned into the densitometer software (e.g., Bio-Rad Quantity One Software) using a densitometer (e.g., Bio-Rad Densitometer). The analyst ensures that the software correctly identifies the gel lanes and appropriate bands. The software generates an annotated image report, such as Figure 2 Representative images shown in .

[0377] exist Figure 2 In the ELISA, lane 1 contains molecular weight standards with bands of known molecular weight. Lane 2 contains the appropriate AUX intermediate reference standards. The remaining lanes contain samples from each IEX fraction. The main product band is the highest band in each lane, and each additional lower band represents an impurity.

[0378] Trend analysis of impurities observed in the IEX fractions can provide important process performance information. Impurities should be consistently classified based on molecular weight from batch to batch for successful trend analysis. Although densitometry software may report the apparent molecular weight of each impurity, the categories based on the reported apparent molecular weight have some normal variation in the gel image, and this variation combined with the close proximity of the bands can result in ambiguous and overlapping classification ranges. Instead, the recommended molecular weight categories for AUX-I and AUX-II impurities can be based on a visual comparison of the vertical (y-axis) position of the fraction impurities relative to the vertical position of the bands in the known molecular weight lanes.

[0379] A total of 128 AUX-I fractions and 99 AUX-II fractions were analyzed. Impurities that appeared in three or more of the 13 batches were assigned to a category. A description of each category is provided below.

[0380] ii. Impurity classification results

[0381] AUX-I

[0382] The impurity categories of AUX-1 fraction include: 33kD, 45kD, 55kD, 80kD, 90kD and 96kD. Figures 3 to 8 A description of each impurity class is provided. Each figure includes:

[0383] • Group 1: The percentage frequency with which a given impurity was observed in each fraction of the 13 batches included in the evaluation.

[0384] - Second set: Mean values ​​(points) and ranges (error bars) of the relative amounts (reported as percentages) of a given impurity according to fraction number.

[0385] • Group 3: Description of the intuitive vertical position of the bands relative to the molecular weight standards.

[0386] • Group 4: The apparent molecular weight range of a given band is reported by the software.

[0387] AUX-II

[0388] The impurity categories of AUX-II fractions include: 25kD, 38kD, 50kD, 60kD, 80kD, 90kD, 92kD and 96kD. Figures 9 to 16 A description of each impurity is provided. The set of figures is the same as the description of AUX-1.

[0389] Among the 877 impurity bands analyzed, 3 bands did not fit into the above categories and were classified as “others.” Table 2 lists the details of these impurities.

[0390] Table 2: Impurities classified as “Others”

[0391]

[0392] C. Impurity Trend

[0393] i. Impurity Trend Method

[0394] After the impurities have been classified, they can be trended to monitor process performance. Fraction impurity trending can be complex because there are multiple impurities, multiple IEX fractions at different protein concentrations, and the results are recorded as relative amounts. However, as described below, trending can be simplified by recording the impurity fractions as a percentage of the impurity of interest relative to the total protein for each AUX peak.

[0395] The amount of protein in each IEX fraction can be calculated by multiplying the fraction volume by the fraction protein concentration by the UV A280 The resulting value is the grams of protein in each fraction. Fig.17 A typical plot of grams of protein in each fraction is provided.

[0396] Using the described SDS-PAGE and densitometry, IEX fractions were generated as a percentage of the total protein detected in each SDS-PAGE gel fraction lane. The same amount of protein (1.5 μg protein / lane) was loaded into each fraction lane; therefore, the grams of each impurity in each fraction can be estimated by converting the impurity percentage to a decimal (dividing by 100) and then multiplying by the grams of protein in each fraction. For example, Fig.18 A typical plot for the 90 kD impurity typically observed in AUX-I and AUX-II fractions is shown, expressed in grams.

[0397] The grams of protein for each AUXI, AUXI or impurity can be calculated based on the fraction. Alternatively, the grams of protein for each fraction and the target impurity can be summed by the AUX peak. Fig.19 A visual representation of this mathematical operation is provided.

[0398] The percentage of the target impurity relative to the amount of protein in each AUX peak can then be calculated by dividing the grams of the target impurity by the grams of the total AUX peak protein and then multiplying the result by 100 to convert the resulting value to a percentage. The value is expressed as a percentage. The equation for deriving the final value expressed as a percentage of the target impurity relative to the total protein in each AUX peak is summarized below:

[0399]

[0400] in:

[0401] [protein] i,j is the protein concentration of the ith fraction containing the jth impurity

[0402] V i,j is the volume in liters of the ith fraction containing the jth impurity

[0403] Impurity value i,jis the impurity percentage of the jth impurity in the ith fraction

[0404] [protein] i Through UV A280 Protein concentration of the ith fraction

[0405] V i is the volume of the ith fraction in liters

[0406] L is one liter

[0407] The above equation can be used to trend the amount of each impurity detected to assess process performance. The above equation can also be easily modified to represent the relative amount of the target impurity only in the fraction being forward processed.

[0408] Trending can be performed using a variety of methods. One method is to use a Shewhart control chart for individual values. An individual Shewhart control chart is a statistical tool used to distinguish between results due to normal variation (within the control chart limits) and results due to abnormal variation (outside the control limits). The control limits are calculated using the moving range of two consecutive observations to assess the variability of the process. The green horizontal line in the control chart represents the average result. The red horizontal lines represent the calculated upper and lower control limits, calculated by the following formula:

[0409] in is the average value, is the moving range of the average value.

[0410] Limits formed using the Shewhart control chart for individual values ​​are not intended to be indicative of product quality. Therefore, results outside the control range will not automatically indicate a quality impact and need to be evaluated holistically in the context of all available data.

[0411] ii. Impurity trending results

[0412] Individual Shewhart control charts were used to trend the impurities in the batches listed in Table 1. Fig. 20 The AUX-I impurities are trended in Fig.21A and 21B AUX-II impurities were trended in Table 3. Batches were introduced in phases by manufacturing site. A different x-axis scale was used for each control chart. Table 3 lists the recommended impurity categories based on the evaluation presented in this report.

[0413] Table 3: Common IEX fraction impurity categories determined using the optimized SDS-PAGE method

[0414] AUX-I degradation products (MW) AUX-II degradation products (MW) About 33 kDa About 25 kDa About 45 kDa About 38 kDa About 55 kDa About 50 kDa About 80 kDa About 60 kDa About 90 kDa About 80 kDa About 96 kDa About 90 kDa - About 92 kDa - About 96 kDa other other

[0415] Trending of impurities in IEX fractions can provide important information about process performance. It is recommended to simplify impurity trending by recording impurities as a percentage of the impurity of interest relative to total protein in each AUX peak.

[0416] Example 2 - Metals in a Purification Process

[0417] A. Abnormal impurity levels in the manufacture of collagenase I and II products

[0418] The purity of collagenase I and II products is greatly reduced in normal manufacturing process. An analysis was carried out, which included the identity of the impurities seen after the ion exchange chromatography (IEX) step of determining the manufacturing process. The molecular weight (MW) of collagenase I product is about 113kDa, and the MW of collagenase II product is about 112kDa. From the IEX step, it is obvious that during the conventional manufacturing collagenase, high levels of impurities have occurred inexplicably. On the optimized SDS-PAGE gel, these high levels of impurities appear at 40kDa, 50kDa, 55kDa, 80kDa, 90kDa and 96kDa.

[0419] B. Potential causes of increased impurity products

[0420] The cause of the high level of impurities was then studied. Clostridium histolyticum produces two proteases, clostripain and neutral protease, in addition to collagenase (AUX-I and AUX-II). Both proteases require metal cofactors for activity. Calcium is a cofactor for clostripain and is required for activity. Calcium is also a cofactor for neutral protease, but is only required for structural stability. If calcium is removed from the binding site of neutral protease, the enzyme will be degraded by autocatalysis. Zinc is a cofactor for neutral protease and is required for neutral protease activity.

[0421] During the manufacturing process, significant product degradation was first detected in the manufacturing process stream after the hydrophobic interaction chromatography (HIC) unit operation. The HIC process buffer raw materials studied included: approximately 37% ammonium sulfate (AS), Tris-Base (tromethamine or "Tris"), and concentrated hydrochloric acid (HCl). The study included determining the metal components in the raw materials. Ion coupled plasma mass spectrometry (ICP-MS) was selected to measure metal content because of its high sensitivity and the wide range of metals that can be detected and quantified. ICP-MS has a sensitivity of parts per billion (ppb) and is used to measure thirty-four (34) metal ions, including calcium and zinc.

[0422] Trace metals tested by ICP-MS for each of the above raw materials are listed below, with respect to levels of calcium, nickel, and zinc. These specific metal levels were found at significantly higher concentrations in some of the ammonium sulfate batches tested (Tables 4-6) compared to the Tris batches (Table 7) and HCl batches (Table 8). Further testing ruled out Tris-Base and concentrated HCl based on the low levels of zinc, calcium, and nickel metals detected and the lower concentrations used in the process buffers.

[0423] i. Ammonium sulfate test:

[0424] Table 4: ICP-MS trace metal data for ammonium sulfate batches

[0425]

[0426] Calcium was ruled out as a cause of the impurity because the ammonium sulfate batches that produced low levels of the impurity (less than or equal to about 5% by area, as measured by SDS-PAGE) had higher calcium levels than the ammonium sulfate batches that produced high levels of the impurity (greater than 5% by area, as measured by SDS-PAGE). These data rule out calcium contamination as a potential cause of the observed degradation of collagenase I and II to impurities during the manufacturing process. The ammonium sulfate batches tested produced high levels of impurities in collagenase manufacturing, with increased levels of zinc and nickel. These data suggest a possible link between nickel contamination and the observed product degradation. The impact of zinc on the manufacturing process is unclear from these data, as both batches contained fairly high levels of nickel. The potential impact on zinc and nickel ion levels is explored in the spike-in studies discussed further below.

[0427] Historical batches of ammonium sulfate were also investigated.

[0428] Table 5: ICP-MS trace metal data for historical batches of ammonium sulfate

[0429]

[0430] Table 6: EMD Millipore ICP-MS trace metal data for ACS, NF grade ammonium sulfate

[0431]

[0432] The calcium levels of five historical ammonium sulfate batches (Table 5) range from 240 to 870 ppb. Once again, any elevated calcium levels did not produce high levels of impurities in the manufacturing process abnormalities. The zinc content ranged from about 130 to 2980 ppb. The nickel content of the historical batches ranged from about 140 to 180 ppb. These data were compared with the nickel content of two ammonium sulfate batches that produced high-level product impurities of about 1150 ppb and about 1020 ppb, respectively, and the tolerance to nickel contamination was low. Although the relative difference of nickel between ammonium sulfate batches was six to eight times, this difference was much lower than other metals, such as zinc, which varied more than ten times within the range of the historical ammonium sulfate batches. Similarly, the low zinc content of Avantor ammonium sulfate batch 99428 (250 ppb) showed that the high-level impurities seen in the manufacturing process were highly sensitive to nickel. In short, the study showed that the product of the purification process was sensitive to both nickel and zinc levels in the process, but the tolerance to zinc in the process was higher than the tolerance to nickel.

[0433] Calcium levels in the EMD-Millipore ammonium sulfate batches (Table 6) were lower than the deviation-related ammonium sulfate batches tested and comparable to calcium levels in historical ammonium sulfate batches (Table 5). Zinc ion levels in the EMD-Millipore ammonium sulfate batches were generally lower than the Avantor batches tested (Table 3). Nickel ion levels were below the limit of quantitation (LOQ) of 100 ppb for the four ammonium sulfate development batches tested by EMD-Millipore.

[0434] ii.Tris test:

[0435] The two batches of Tris-Base represent a raw material that results in high levels of impurities used in commercial production prior to the manufacturing process and in the manufacturing process (Avantor batch 61712), and a development material used for laboratory-scale investigations (Fisher batch 126928). As shown in Table 7, the calcium and nickel levels of Tris-Base were comparable between batches. The significant difference between the two batches was the zinc content; the zinc content of the development material was below the LOQ 100 ppb. Tris is underestimated as a raw material that contributes to collagenase degradation because the nickel and zinc levels present in the manufacturing process that produce high and low levels of impurities are very similar.

[0436] Table 7: ICP-MS trace metal data for Tris-Base

[0437]

[0438] iii.HCl test:

[0439] Both HCl batches were raw materials used during the manufacturing process, which produced high-level impurities. Table 8 shows that the calcium level was comparable to the level detected in the AS batch, while the zinc and nickel contents were below the LOQ (100 ppb). HCl was underestimated as a raw material that contributes to collagenase degradation because the nickel and zinc levels present in the manufacturing process, which produced high-level impurities and low-level impurities, were very similar and below the detection limit.

[0440] Table 8: ICP-MS trace metal data for concentrated HCl

[0441]

[0442] C. Zinc and Nickel Incorporation Studies

[0443] Laboratory scale purification studies were performed using two different ammonium sulfate batches known to be essentially free of zinc or nickel (Fisher batch 1476214A, Table 4 and EMD batch AM0556316, Table 5) to determine whether the presence of relative concentrations of zinc or nickel detected in the deviation-related ammonium sulfate batches resulted in product degradation observed in the manufacturing process that produced high levels of impurities. Early investigations using ammonium sulfate Fisher batch 1476214A at laboratory scale produced a typical number of passed AUX-I and AUX-II fractions, wherein nearly all of the AUX-I and AUX-II fractions had an AUX-I / AUX-II undegraded purity of at least 91.2% as measured by SDS-PAGE gel densitometry, and the maximum amount of degraded AUX-I or AUX-II impurity did not exceed 5.8% as measured by SDS-PAGE gel densitometry. Testing of the EMD ammonium sulfate batch showed that the levels of zinc and nickel were below the LOQ (<100 ppb).

[0444] Using these ammonium sulfate batches for the study, a control run consisting of a qualified HIC and IEX small-scale model was performed, which was used as the thawed starting material, which was a previously frozen MustangQ filtrate (MQF) from a manufacturing process that produced high levels of impurities. The MQF was processed through the HIC, TFF and IEX steps. Both the HIC and IEX steps were loaded at the midpoint of the effective load range of the chromatography steps. All unit operations were performed under refrigerated conditions, with the chromatography cabinet set to 10°C in the HIC step and 4°C in the IEX step. The IEX fractions were analyzed by SDS-PAGE with densitometry. The fractions passed for merging required a purity of 91.2% or higher, undegraded collagenase I or II, and a maximum amount of degraded collagenase I or II impurities of 5.8% or less.

[0445] The zinc doping study used ammonium sulfate from Fisher lot 1476214A. This study preceded the doping study, which used ammonium sulfate from EMD lot AM0556316. Both lots of ammonium sulfate were determined to have zinc and nickel levels below 100 ppb.

[0446] In the zinc doping study, ammonium sulfate from Fisher lot 1476214A was spiked with ZnCl2 to raise the zinc concentration in the HIC buffer to about 84 ppm. The results of the IEX fractions of the material treated with the zinc doping buffer were compared to the IEX fractions of the material treated without adding additional zinc to the treatment buffer. For the nickel doping study, ammonium sulfate from EMD lot AM0556316 was spiked with about 1.2 ppm NiCl2. Each of these studies is discussed separately below.

[0447] i. Zinc doping study:

[0448] The control run DEV-25C produced typical results as determined by SDS-PAGE analysis of IEX fractions. DEV-25B, which was performed using a zinc-doped buffer, resulted in significant degradation of AUX-I and AUX-II.

[0449] IEX SDS-PAGE image of control run DEV-25C is shown in Fig. 22 and 23 The densitometry data for these figures are tabulated in Tables 9 and 10 below. The AUX-II peak consists of six fractions, of which fractions 1 to 5 meet the pooling criteria. The last fraction does not pass the pooling criteria, which is consistent with typical process performance (ref. Fig. 22 and Table 9). The AUX-I peak consisted of ten fractions (fractions 7-16), of which fractions 9 to 15 met the merging criteria. In order for the AUX-I fraction to meet the merging criteria, the purity of the fraction was required to be at least 91.2% of the undegraded AUX-I, and the purity of the largest degraded AUX-I or AUX-II impurity did not exceed 5.8%. Similarly, in order for the AUX-II fraction to meet the merging criteria, the purity of the fraction was required to be at least 91.2% of the undegraded AUX-I, and the purity of the largest degraded AUX-I or AUX-II impurity did not exceed 5.8%. Under these special requirements, the first two AUX-I fractions (the seventh and eighth) and the last AUX-I fraction (the sixteenth) did not pass the merging criteria (reference Fig.23 and Table 10). Since the fractions represent the peaks of AUX-I or AUX-II being purified, a bell-shaped purity curve across the fractions will typically appear.

[0450] Table 9: Optical density measurement results of DEV-25C AUX-II fractions

[0451]

[0452] Table 10: Density determination results of DEV-25C AUX-I fractions

[0453]

[0454]

[0455] In contrast, SDS-PAGE / densitometry analysis of IEX fractions generated from run DEV-25B spiked with 84 ppm zinc contained collagenase impurity fragments well above the maximum allowed limit of <5.8% (Maximum Impurity %). None of the AUX-I or AUX-II fractions met the pooling criteria ( Fig.24 and 25 , Tables 11 and 12). This study demonstrated that zinc contamination in the HIC buffer reached levels found in ammonium sulfate from Avantor lot 70339, which promoted collagenase degradation similar to that observed in the manufacturing process and produced high levels of impurities.

[0456] Table 11: SDS-PAGE densitometry results of DEV-25B AUX-II fractions

[0457]

[0458] Table 12: SDS-PAGE densitometry results of DEV-25B AUX-I fractions

[0459]

[0460] ii. Nickel doping research:

[0461] IEX SDS-PAGE image of control run DEV-25A is shown in Fig.26 and 27 The optical density data for these figures are tabulated in Tables 13 and 14 below. The AUX-II peak consists of six fractions. All fractions met the pooling criteria (see Fig.26 and Table 13). The AUX-I peak consisted of ten fractions, of which fractions 9 to 15 met the criteria for merging. The first three AUX-I fractions (fractions 7-9) and the last AUX-I fraction (fraction 16) did not meet the criteria for merging (ref. Fig. 27 and Table 14). Likewise, since the fractions represent the AUX-I or AUX-II peak being purified, a bell-shaped purity curve across the fractions will generally appear.

[0462] Table 13: SDS-PAGE densitometry results of DEV-25A AUX-II fractions

[0463]

[0464] Table 14: SDS-PAGE densitometry results of DEV-25A AUX-I fractions

[0465]

[0466] In contrast, in Run 4 with nickel doping, the HIC buffer contained 1.2 ppm of nickel chloride. The presence of 1.2 ppm of nickel in the HIC buffer formed product fragments in the AUX-I and AUX-II fractions comparable to those in the manufacturing process that produced high levels of impurities. No AUX-I or AUX-II fractions met the pooling criteria ( Fig.28 and 29 , Tables 15 and 16). These results indicate that 1.2 ppm nickel contamination in the ammonium sulfate batch promoted collagenase degradation, which is consistent with the high levels of impurities seen from an aberrant manufacturing process.

[0467] Table 15: SDS-PAGE density determination results of DEV 25D AUX-II fractions

[0468]

[0469]

[0470] Table 16: SDS-PAGE densitometry results of DEV 25D AUX-I fractions

[0471]

[0472] D. Isolation and identification of neutral proteases from HIC CIP washes

[0473] Prior to the present invention, impurities in the form of collagenase I or II fragments were considered to be the result of clostripain degradation, which is a known host cell contaminant in the collagenase purification process. Clostripain is considered to be split into fragments by collagenase I and II, and it is known that they are present and active in Clostridium histolyticum fermentation and collagenase purification process. Previous homology modeling and genome sequence analysis in Herber (US2015 / 0010532) have predicted that neutral protease (another lyase) can be secreted during fermentation, but due to the significant mutation of the autocatalytic region between the C-terminal of the presequence region and the N-terminal of the mutant protein, the neutral protease is non-functional.

[0474] In conventional manufacturing processes, the increased level of impurities requires further investigation. This investigation determines that impurities are fragments of collagenase I (AUX-I) and collagenase II (AUX-II) produced from the neutral protease of Clostridium histolyticum. The amino acid sequences of AUX-I and AUX-II have been used in the polypeptide cutting program on the bioinformatics resource portal ExPASy.org to form a theoretical protein cleavage site list using clostripain and thermolysin splitting rules. Thermolysin is selected as the model protease of the neutral protease of Clostridium histolyticum because it is the full type of the M4 family. Inquiries to the cleavage sites produced by clostripain and thermolysin in AUX-I and AUX-II sequences have produced the positive matching of most of the terminal cleavage sites in the characteristic conventional product fragments contained in the IEX fractions (40kDa, 50kDa, 55kDa, 80kDa, 90kDa and 96kDa). The characteristics of the main product fragments and their respective lytic enzymes are summarized in Table 17 below:

[0475] Table 17. Matching of IEX fragment signature data and cleavage endpoints using clostripain and thermolysin cleavage rules

[0476]

[0477] A complete list of IEX fraction impurity categories from IEX fractions is described in Example 1 in Table 3. Impurities may occur at any of the molecular weights (MW) recorded in Table 3; however, the most common impurities occur at 45 kDa, 55 kDa, 80 kDa, and 90 kDa for AUX-I and at 38 kDa, 50 kDa, 60 kDa, 80 kDa, and 90 kDa for AUX-II. Occasionally, impurities may occur at a different MW than specified in Table 3 (referred to as "Other" in Table 3).

[0478] To produce a purified neutral protease from Clostridium histolyticum, a method was developed to purify the neutral protease from the clean-in-place (CIP) waste stream of a hydrophobic interaction chromatography (HIC) step. Two-liter fractions of the HIC CIP water wash waste were manually collected in the peak. The fraction containing the largest peak was concentrated tenfold by volume before buffer exchange with six diavolumes of 10 mM Tris, 3 mM CaCl2, pH 8.0, 5°C. This operation was performed using a single 0.1 m 2 The column was washed with 5kDa theoretical molecular weight cutoff tangential flow filter. The concentrated, buffer-exchanged wash was loaded onto a Q agarose high performance (Q HP) column, which was balanced with 10mM Tris, 3mM CaCl2 at pH 8.0. The column was washed after loading the equilibration buffer before a 25 column volume gradient of 0-50% buffer B (10mMTris, 3mM CaCl2, 360mM NaCl, pH 8.0). Two fused peak clusters were eluted from the column and collected by automatic fractionation. SDS-PAGE analysis using Coomassie staining showed that within the second peak cluster, the eluted 34kDa protein had no other protein species within the detection limit ( Fig.30 , gel lane 3). The peak maximum sample buffer was exchanged into 20 mM potassium phosphate and stored frozen at -70°C.

[0479] The wastewater stream, the water wash step of the HIC Cleaning in Place (CIP) procedure, is produced by flushing the HIC column with water for injection (WFI) after the HIC unit operation is completed by flushing the column with 0.5M NaOH before starting CIP. The water wash causes the neutral protease to elute from the HIC column. The neutral protease was successfully isolated from the wastewater stream and the presence of the enzyme was confirmed in the collagenase manufacturing process, although also in the wastewater stream of the HIC process. The isolated neutral protease has an apparent molecular weight of 34 kDa.

[0480] Due to the proline residue at the potential cleavage site P2' position, the 80kDa fragment is the only characteristic fragment that does not meet the protease cleavage rule using the polypeptide cleavage program. However, by mixing the neutral protease of AUX-1 and process separation or the neutral protease of Clostridium histolyticum (from Serva) of commercial version, the 80kDa fragment is produced again. Further, the evaluation of the cleavage site of the 80kDa fragment using the MEROPS database shows that thermolysin can split the Thr-693-Gly694 peptide bond to set up the 80kDa fragment from AUX-1. Therefore, all five AUX-1 and AUX-III product fragments that typically occur in the manufacture of collagenase products are the proteolytic cleavage products of neutral protease. Under manufacturing conditions, 50kDa and 96kDa impurities can also be the cleavage products of clostripain degradation.

[0481] The uncharacterized 96 kDa fragment present in the AUX-II fraction theoretically matches multiple cleavage sites at the N- and C-termini of clostripain and neutral proteases. The 96 kDa fragment is produced again by exposing AUX-II to neutral proteases. This in silico analysis and neutral protease digestion studies show that neutral proteases are reliable enzymes for product fragment formation in Clostridium histolyticum fermentation and / or purification.

[0482] As described above, the isolated neutral protease produced by the collagenase manufacturing process has an apparent molecular weight of 34 kDa. This indicates that the neutral protease prosequence has been cleaved by autoactivation or another host enzyme and suggests that the neutral protease is active in the C. histolytica manufacturing process.

[0483] Next, studies were conducted to determine whether the prosequence cleaves itself to produce an active and mature neutral protease, even though it is predicted not to do so. Identification of the N-terminus of the neutral protease will provide key data to assess the autocatalytic ability of the proenzyme by comparing the consensus sequence requirements of thermolysin with the sequences flanking the N-terminus of the neutral protease of Clostridium histolyticum. The purified neutral protease was subsequently subjected to N-terminal characterization, and residue Gln220 was identified as the N-terminus of the enzyme. This N-terminus matches the N-terminus of the neutral protease of Clostridium histolyticum recently published by Maeda et al. in 2015.

[0484] In addition, it is found that neutral protease rather than clostripain is a proteolytic enzyme that produces all conventional collagenase fragments observed during the collagenase manufacturing process. As determined by SDS-PAGE analysis, the product fragments produced using the isolated neutral protease from HIC CIP washing in AUX-I / AUX-II digestion studies are consistent with the apparent molecular weight of conventional fragments. These digestion studies have used the neutral protease isolated from the manufacturing process as described above.

[0485] E. Identification of the N-terminus of Clostridium histolyticum neutral protease

[0486] The N-terminal identification methods used were orthogonal, namely Edman degradation sequencing and LC-MS / MS identification of the N-terminus following Lys-C / trypsin digestion. Samples used for both tests were prepared from the same purification run and represented different fractions of the neutral protease elution peak. Edman degradation analysis identified the following five residues in the neutral protease N-terminal region: Gln220-Ala224. LC-MS / MS digestion analysis identified a larger stretch of the neutral protease N-terminal region, with two peptides detected in the analysis: Gln220Ala-Arg227 and Gly228-Lys236. The identified amino acids were Fig.31 is highlighted.

[0487] The N-terminus of the identified neutral protease is used to examine the hexapeptide consensus sequence region of the proenzyme. The consensus sequence corresponds to the three residues at the C-terminus of the presequence of thermolysin, combined with the N-terminus and the following two residues of the mature enzyme. For thermolysin, the consensus sequence is Val230LysSerIleTherGly236. Ile233 is the N-terminus of thermolysin. The Schechter & Berger protease subsite nomenclature will be used to describe the consensus sequence (reference Fig.32 The autocatalytic activity of thermolysin requires amino acid features of a nonpolar residue at the P3 position (Gly, Ala, Ile, Leu or Val), a polar residue or proline (Ser, His, Glu, P) at the P1 position, and a nonpolar residue at the P1' position. Inspection of the consensus sequence of thermolysin shows that it meets all three requirements.

[0488] Using the identified N-terminus of Clostridium histolyticum neutral protease (Gln220), the theoretical consensus sequence is Lys217SerCysGlnAlaThr222. This hexapeptide region does not have any subsite features for autocatalysis, except that Cys219 is a polar residue. However, it is not listed as a suitable residue in the rule. Based on the literature, it is unclear whether the structural characteristics of cysteine ​​exclude it from being a suitable residue in the P1 site, or researchers have not yet studied it. According to the autocatalytic sequence rule of thermolysin, Clostridium histolyticum neutral protease should not have autocatalytic ability.

[0489] The complete gene product sequence (secretory sequence, pro-sequence and mature sequence) of the neutral protease of Clostridium histolyticum ATTC 19401 was recently published by Maeda et al. in 2015. The gene product was cloned and expressed to allow the study of the neutral protease substrate specificity, thereby producing active neutral protease. Recombinant neutral protease ( r nprA) and the neutral protease gene product sequence of Clostridium histolyticum yielded 100% homology (ref. Fig.33 ). Thus, it was determined that the neutral protease in the collagenase Clostridium histolyticum (CCH) manufacturing process has autocatalytic ability, is secreted as an inactive proenzyme, which self-cleaves the prosequence in the growth medium to become a functional mature enzyme, and is a typical feature of CCH fermentation.

[0490] F. Investigating the Presence of Neutral Proteases in the Manufacturing Process

[0491] An investigation was conducted to determine whether the active neutral protease was unique to the abnormal production process run or a regular feature of the process. The collagenase fragment pattern in the manufacturing process was analyzed. In the abnormal manufacturing process run, the degradation pattern of the AUX-I and AUX-II IEX fractions was consistent with the typical fragment relative molecular weight in the typical manufacturing process run, but the level was much higher. The cleavage site of each characteristic product fragment for the cleavage rules of clostripain and neutral protease was evaluated, which indicated that the neutral protease was producing product fragments identified as impurities. SDS-PAGE analysis was performed on the entire HIC unit operation (including the CIP wastewater stream) to determine whether there was a protein band of approximately 34kDa in both process streams. The water wash portion of the CIP method contained a band consistent with the apparent molecular weight by SDS-PAGE evaluation. In the analytical spiking study, the HIC water wash sample was also shown to degrade AUX-I and AUX-II (similar to the pattern observed in the abnormal manufacturing process run).

[0492] In addition to these steps in the collagenase manufacturing process, residual neutral proteases were found in the HIC eluate and TFF-1 concentrate product streams. Investigation of IEX fractions, AUX-I and AUX-II IEX pools, AUX-I intermediates, AUX-II intermediates, and drug product indicated that the neutral proteases were sequestered into the tail AUX-I fractions of the IEX unit operation, at a position where they co-eluted with the tail of the AUX-I peak. As these fractions did not meet the in-process purity limits as analyzed by SDS-PAGE / densitometry, these fractions could be removed from the product stream.

[0493] Example 3 - Process Control

[0494] Neutral protease activity was found in process samples of Mustang Q filtrate, HIC eluate, and tangential flow filtration (TFF) concentrate from three manufacturing process runs. IEX AUX-I fractions from the conventional manufacturing process were tested in the neutral protease zymography activity assay, and the protease showed activity in the tail fractions of these samples. These data suggest that residual neutral protease co-eluted from the HIC column is sequestered in the tail AUX-I fraction eluted from the IEX column. Most of the tail fraction will not pass the purity in-process limit tested by SDS-PAGE densitometry fractions and therefore will not be combined to generate AUX-I intermediates. However, other process controls have been implemented that ensure that fractions containing small amounts of neutral protease are not forward processed. Additional procedures are used to exclude additional AUX-I tail fractions from the forward process. The apparent positional nature of the neutral protease elution indicated by the zymography activity analysis suggests that these procedures act to eliminate neutral proteases from the product stream prior to formulation of the drug product. These process controls are discussed below.

[0495] Zymography is an enzyme activity assay based on electrophoresis, which utilizes the copolymerization substrate (casein) in the SDS-PAGE gel and the renaturation ability of some enzymes after removing SDS from the gel. After removing SDS, the enzyme through renaturation is allowed to be incubated for a period of time to degrade the copolymerized casein substrate. Activity is detected by Coomassie staining, in which the casein region corresponding to the apparent molecular weight of the enzyme shows as a white region that does not absorb the dye. The remainder of the gel is compounded with the casein contained in the gel by Coomassie dye and dyed blue. Casein is a bovine milk protein, which is susceptible to the proteolytic attack of Clostridium histolyticum neutral protease (rather than Clostridium histolyticum clostripain or collagenase). Therefore, this activity assay based on PAGE provides the ability to screen and manufacture the product stream to have active neutral protease.

[0496] The assay shows neutral protease signals in the Mustang Q filtrate, HIC eluate, TFF-1 concentrate, and the final IEX AUX-I tail fraction. Neutral proteases are primarily removed in the HIC step, where they remain firmly bound to the column and are eluted in the water wash of the column cleaning process. Trace amounts of neutral proteases are co-purified with collagenase from the HIC column and are bound to the IEX column. In the final AUX-I fraction, this trace amount of neutral protease begins to desorb from the column at the end of product collection. After separation of collagenase I from collagenase II and collection of the respective eluates in fractions of the eluate peak, neutral proteases can be removed by performing at least one of the following elimination steps: (1) incorporating improved standard limits for fractions of collagenase I, collagenase II, or both collagenase I and collagenase II that can be pooled, (2) incorporating an improved fraction pooling strategy that excludes fractions containing detectable levels of neutral proteases as tested by SDS-PAGE or zymography electrophoresis, and (3) incorporating a pooling strategy that incorporates approximately 1:1 (equal amounts) of AUX-I and AUX-II purified fractions. Zymography data and IEX process control will now be discussed.

[0497] A. Improved pooling - collect only fractions free of neutral proteases for casein zymography

[0498] Samples of the bench-scale process of Mustang Q filtrate, HIC eluate, TFF-1 concentrate, AUX-I and AUX-II intermediates, and drug product were tested by zymography electrophoresis. Fig.34 and 35The samples were from two different runs (Dev-13 and Dev 25A). The zymography images were scanned by densitometry and annotated to highlight the neutral protease bands as some sample types exhibited low neutral protease signals. To aid in the visualization of the bands, a reverse image of the gel was used to generate a typical Coomassie gel image (proteolytic bands stained blue). Neutral proteases were detected in early process samples such as MQF, HIC load, and HIC eluate (ref. Fig.34 ). In both the MQF and HIC load samples, the neutral protease band is easily observed at approximately 34 kDa apparent molecular weight. Note that for all three sample types, the total protein load increased from 2 μg (MQF), 3 μg (HIC load), and 4 μg (HIC eluate), but the HIC eluate neutral protease response decreased significantly. The neutral protease band in the HIC eluate sample was faint enough to be highlighted using the "Select Band" feature of the densitometry software. This demonstrates significant removal of the neutral protease throughout the HIC step.

[0499] A higher total loading mass of 10 μg of total protein was used for the TFF-1 concentrate, AUX-I and AUX-II pools, intermediates, and drug product to amplify potential neutral protease signals in these samples. The TFF-1 concentrate (Dev-25A) sample contained sufficient neutral protease concentration to produce a diffuse band of approximately 34 kDa. The AUX-1 pool, AUX-1 intermediates, and drug product contained detectable neutral protease, which was highlighted by the densitometry software and can be observed in lanes 6, 8, and 12 (ref. Fig.35 A 34 kDa band was visible in the gel. However, the fidelity of the resulting image was not sufficient to clearly see the band. The AUX-II intermediate contained neither detectable neutral protease nor its predecessor, the AUX-II pool ( Fig.34 ). These data indicate that the neutral protease is dissociated from the product during the IEX step and co-elutes in the AUX-I peak, which is evident in the AUX-I pool and the AUX-I intermediate. These data also indicate that any fractions containing detectable levels of the neutral protease as measured by zymography should be rejected and not included in the pooling step.

[0500] IEX fractions of the AUX-I and AUX-II elution peaks from a typical manufacturing process were run through a casein zymography assay to determine if neutral proteases were detectable in the fractions and if they were contained in specific locations. Fig.36 and 37 Gel images from the assay are included. None of the AUX-II fractions contained neutral proteases at concentrations detectable by this method. Selected samples were run at higher concentrations to exclude the presence of neutral proteases in the IEX fractions (ref. Fig.38 ).

[0501] A low neutral protease signal was detected in the last AUX-I fraction (17) of this typical manufacturing process. The location of the neutral protease elution from the IEX column is Fig.38 (lanes 9, 10) and 39 (lanes 8, 10, and 12). This detection again shows that residual neutral proteases (not removed in the HIC or TFF-1 steps) are partially desorbed from the IEX column at the end of the AUX-I peak collection. The positional nature of the neutral protease contamination in the tail of the AUX-I peak of this manufacturing process, as well as the inclusion of the last AUX-I fraction in the AUX-I pool of the old manufacturing process, suggests the presence of neutral proteases detected in AUX-I intermediates and drug products in the AUX-I pool of the manufacturing process, as shown in Figure 2. Fig.35 Thus, pooling fractions that do not contain detectable levels of neutral proteases as measured by casein zymography assay can produce a collagenase composition that is essentially free of neutral proteases.

[0502] The present invention may also include an automatic exclusion feature. Prior to the present invention, these AUX-I fractions were artificially excluded during the mixing of the AUX-I fractions passing through to produce the AUX-I pool. The automatic exclusion of at least the last two AUX-I fractions prevents residual neutral proteases from entering the AUX-I intermediates, as it would normally co-elute in these fractions.

[0503] As described in the following sections, implementation of an improved IEX pooling strategy can eliminate this tail-end contamination during pooling of AUX-I and AUX-II fractions.

[0504] B. Improved pooling - pooling fractions that meet the lowest purity limit as measured by SDS-PAGE

[0505] During the gradient elution with AUX-II and the gradient elution with AUX-I, collagenase, AUX-I and AUX-II are eluted from the IEX column in the form of discrete peaks. The peak is collected by manual fractionation, which is about one liter of aliquots. The purity of these fractions is tested by SDS-PAGE with densitometry. Continuous AUX-I or AUX-II fractions can be used for merging to generate collagenase I product or collagenase II product. In the past, the fractions of AUX-I or AUX-II were merged to generate collagenase I product or collagenase II product, wherein as measured by SDS-PAGE with densitometry, the purity of these fractions was at least 88.5% by area, and, as measured by SDS-PAGE with densitometry, no single impurity was greater than 10% by area. The fractions that fail to reach these restrictions will not be forward processed, and therefore can be eliminated from the product stream.

[0506] Improving the criteria for passing through fractions not only improves the overall purity of the collagenase composition, but also serves to further eliminate neutral proteases from the AUX-1 tail. Increasing the limit for passing through fractions can remove neutral proteases when the AUX-I and AUX-II fractions have an AUX-I or AUX-II purity of at least 91.2% by area as measured by SDS-PAGE with densitometry, and the highest single impurity is no greater than 5.8% by area as measured by SDS-PAGE with densitometry.

[0507] As part of the investigation, a planned deviation was made to the pool AUX-I and AUX-II fractions based on historical batch data. AUX-I fractions 12-16 and AUX-II fractions 2-5 were pooled and processed to produce the drug substance (sometimes referred to as the drug substance or BDS). Release testing of the drug product obtained upper out of specification (OOS) purity by SDS-PAGE and out of trend (OOT) purity by reverse phase HPLC (RP-HPLC), confirming the lower purity of the IEX fraction. This planned deviation resulted in Fig.35 Data shown in Figure 2. The neutral protease detected in the AUX-I pool, AUX-I intermediates, and drug product generated from the manufacturing process was contributed by the tail AUX-1 fraction (16) that was pooled according to the planned deviation. The detection limit of the zymography analysis was approximately 0.5 ng of neutral protease. It can be seen on the actual casein zymography gel itself. Fig.35 The band was only below the 0.5 ng limit to approximately 0.2-0.3 ng of neutral protease using a 10 mg loading amount of the collagenase composition.

[0508] C. Improved merging - matching AUX-II outputs to minimize excess AUX-I outputs

[0509] The present invention may also include evaluating a theoretical gram amount of AUX-II, which is produced by combining a passed AUX-II fraction and a passed AUX-I fraction to produce a pool having a mass ratio of AUX-I to AUX-II of about 1:1 in the combining step. For example, the amount of AUX-I combined may be 1.3 times the grams of AUX-II. After manufacturing the drug substance (collagenase composition), any excess AUX-I or AUX-II can be disposed of. This typically results in one or more tail AUX-I fractions that meet the initial process limits being excluded from the AUX-I pool. This implementation is an auxiliary process control for eliminating neutral proteases in the manufacturing process.

[0510] For example, the peak fractions of the collagenase I fractions collected from the IEX eluate are forward processed according to the fractions that meet the required purity and impurity standards, and only the collagenase I peak fractions are merged until the approximate amount of the combined collagenase I matches the approximate amount of the collagenase II produced by the process. First, after the AUX-II fractions (excluding the final tail fractions) are collected, the theoretical total AUX-II amount of the process can be calculated. Secondly, the theoretical amount of AUX-I in each fraction is determined. Then, starting from the first AUX-I fraction, as measured by SDS-PAG with densitometry, the purity of the first AUX-I fraction is at least 91.2% by area, and as measured by SDS-PAG with densitometry, there is no single impurity greater than about 5.8%, and the continuous fractions are merged until the number of AUX-I merged is about 1.3 times the total amount calculated for AUX-II. The collagenase composition produced in this way is essentially free of neutral proteases.

[0511] This control strategy eliminated the other end fractions of the collagenase I peak, rendering the pooled collagenase I fractions essentially free of neutral proteases.

[0512] Example 4 - Neutral protease produced by the cleaning process

[0513] The small-scale spike-in studies discussed above demonstrated that amplification of intrinsic neutral protease activity due to nickel or zinc resulted in high levels of impurities in the IEX fraction. Importantly, the neutral protease clearance study found detectable neutral protease activity in only one of the three HIC eluates (batch 0011338) included in the study, while all three batches had detectable neutral protease activity in the TFF-1 concentrate samples. Trace levels present in typical HIC eluates and TFF-1 concentrates do not promote elevated product fragments in the IEX fraction, but manufacturing processes that produce high levels of impurities and small-scale spike-in study processes increase the fragments present in the HIC eluate, TFF-1 concentrate, and IEX fractions. These results indicate that nickel and zinc ions amplify the specific activity of neutral proteases in the HIC and TFF-1 unit operations, resulting in increased product fragment impurities in the IEX unit operation.

[0514] A neutral protease clearance study was conducted to confirm that neutral proteases were effectively eliminated from the product stream of the collagenase manufacturing process prior to creating the drug product. This study was performed on three historical drug product batches by testing product stream retain samples from downstream processes using two recently developed neutral protease activity-based assays: (1) an AUX-I pooling step that rejects the AUX I elution fractions from the ion exchange step, which contain detectable levels of neutral proteases as measured by SDS-PAGE or zymography, and (2) a pooling of the largest peak fractions from AUX I based on an approximately 1:1 mass ratio of AUX II pooled from the ion exchange step. The product streams tested in this study are listed below in Table 19. The assays used to detect neutral protease activity in the manufacturing product streams were a fluorescence-based ELISA plate assay and an SDS-PAGE zymography assay. The data for each of these assays are discussed below.

[0515] Table 19: Product streams tested in neutral protease clearance studies

[0516]

[0517]

[0518] A. Neutral protease activity assay using fluorescent ELISA plate

[0519] The sample types listed in Table 19 were tested using a semi-quantitative fluorescence activity assay. Since the purity of the only commercial source of neutral protease was not suitable for use, the assay utilized commercially prepared thermolysin (TL) of a standard curve. The limit of quantification (LOQ) for the assay was 0.0625 AU / mL (0.01866 AU / mg TL). Only the Mustang Q filtrate contained quantifiable levels of neutral protease activity. All sample types further downstream were below the lowest limit of quantification for the assay. Data from three manufacturing batches tested are provided below in Table 20.

[0520] Table 20: Neutral protease activity of the C. histolytica product stream measured by fluorescent enzyme labeling assay

[0521] sample Batch 0009749 Batch 0010987 Batch 0011338 Mustang Q Filtrate 0.77AU / mg 1.13AU / mg 0.96AU / mg HIC eluent <LOQ <LOQ <LOQ TFF-1 Concentrate <LOQ <LOQ <LOQ AUX-I Pool <LOQ <LOQ <LOQ AUX-II Pool <LOQ <LOQ <LOQ AUX-I Intermediate <LOQ <LOQ <LOQ AUX-II Intermediate <LOQ <LOQ <LOQ API <LOQ <LOQ <LOQ

[0522] B. Enzymography

[0523] All Mustang Q filtrate samples, a batch of HIC eluate (batch 0011338) and all TFF-1 concentrate samples tested positive for neutral protease bands due to zymography assays for neutral protease activity. None of the sample types downstream of the TFF-1 concentrate showed neutral protease activity. Data from C. histolytica batches 0009749, 0010987 and 0011338 are given in Table 21 below. The zymography gel images for batch 0010987 are shown in Figure 40. These images are Coomassie reverse stain representations, which provide improved contrast to enable low amplitude signal detection, such as neutral protease bands.

[0524] Table 21: Qualitative activity results of neutral protease zymography assay

[0525]

[0526]

[0527] ND = Not Detected; TQ = Optical Density (OD) x mm and is a measure of the area under the lane tracer curve using the densitometry software described in this method.

[0528] *Total protein load on gel and trace amount (area under the curve) in the “Quantity One” software shown in brackets.

[0529] Neutral protease clearance studies indicated that neutral proteases were primarily removed during the HIC chromatography step as evidenced by the lack of detectable activity following testing of the Mustang Q filtrate samples in ELISA plate analysis and low levels of activity in one HIC eluate sample (Batch 0011338) as assessed by zymography. Zymography analysis showed low neutral protease activity in all three TFF-1 concentrate samples, confirming that the neutral protease was partially desorbed from the HIC column during the elution step, while the majority of the enzyme remained bound to the column. The lack of neutral protease activity in the two HIC eluate samples with low activity detected in the respective TFF-1 concentrates was due to the six-fold concentration that occurred prior to the initiation of diafiltration in this unit operation. Residual neutral protease present in the TFF-1 concentrate was removed from the product stream during the IEX unit operation, with the neutral protease partially co-eluting with the AUX-I tail fraction (see Fig.39 Purity process limits for the IEX fractions and process controls (automatic rejection of the last two AUX-I fractions to target equal intermediate mass) exclude the tail AUX-I fraction from the AUX-I pool, thereby removing residual neutral proteases from the manufacturing process prior to generating the AUX-I intermediates and drug product.

[0530] Example 5—Effect of Removing Trace Neutral Proteases

[0531] As shown in Example 4, less A was incorporated at the tail end of the collagenase peak. 280 The collagenase peak fraction also had most, if not all, trace amounts of neutral proteases removed, rendering the collagenase I product produced by pooling fractions of AUX I essentially free of neutral proteases. After the IEX purification step for each collagenase, the pooled elution fractions were quantitatively evaluated to compare the effects of the improved manufacturing process with the previous one.

[0532] Trend analysis of the relative purity and impurity levels observed in IEX fractions provides important process performance information. One such approach is described in Example 1. In this example, collagenase I and collagenase II were tested from the following batches:

[0533] Table 22: Batches used to compare purity and impurities of collagenase I and collagenase II fractions

[0534]

[0535] For each manufacturing run listed in Table 22, the product purity and percent impurities of the AUX I and AUX II elution fractions were measured as described in Example 1. The percent impurities in each fraction were given both on a per fraction basis and on a total impurity basis.

[0536] The percentage of purity and the percentage of each impurity for the atypical ammonium sulfate batch effect relative to the amount of total protein in each AUX I or AUX II elution peak are plotted using the computer program Individual Control Chart. The Individual Control Chart is a statistical tool used to distinguish between results due to routine variation (within the control chart limits) and those due to abnormal variation (outside the control chart range). The control limits are calculated using the moving range of two consecutive observations to estimate the process variability. The green horizontal line in the control chart represents the average result. The red horizontal lines represent the upper and lower limits of the control chart, calculated as X ± 2.66 x mR, where X is the mean and mR is the mean moving range.

[0537] exist Fig.42 and Fig.43 The percent purity of the AUX-I and AUX-II products relative to the amount of total protein in each AUX elution peak is provided in . Fig.42 and 43 The decrease in the percentage purity level of the AUX-I and AUX-II elution peaks seen in Figure 1 is mainly due to the increase in the levels of the AUX-I 90 kDa and AUX-II 96 kDa impurities, respectively. Fig.44 and Fig.45 shown.

[0538] exist Fig.46A and 46B The raw data for the peak percentages of AUX I and AUX II products during a typical manufacturing process are shown in Figures 46B-46D The corresponding raw data of the major impurity percentages for AUX I and AUX II are shown in Figure 2. The major impurities are 90 kDa and 96 kDa degradation products generated by collagenase I and collagenase II due to the presence of neutral proteases in the upstream manufacturing process.

[0539] like Figures 46A-46D As shown, the raw data of collagenase I purified using the previous treatment method has an average purity of 94.5% + / - 1.0%. The raw data of collagenase I purified using the improved processing method has an improved average purity of 95.8% + / - 1.0%. By using the improved manufacturing method, the statistically significant improvement of AUX I merged was 0.3%, which further proved that the collagenase I product essentially free of neutral proteases was generated. As expected, the purity of collagenase II (AUX-II) remained unchanged because neutral proteases would not be eluted from the ion exchange column with AUX II. The continued presence of 90kDa and 96kDa collagenase impurity peaks in the purified product is not surprising, because even with the improved process, even in the earlier purification steps in the improved process, neutral proteases are still retained.

[0540] The embodiments of the present invention described above are exemplary only. Many variations and modifications are apparent to those skilled in the art. All of these variations and modifications are intended to fall within the scope of the present invention as defined by any of the appended claims.

Claims

1. A pharmaceutical product comprising isolated and purified collagenase, wherein the pharmaceutical product is essentially free of neutral proteases.

2. The pharmaceutical product of claim 1, wherein the product comprises less than about 100 nanograms of neutral protease per milligram of the product.

3. The pharmaceutical product of claim 1, wherein the product comprises less than about 75 nanograms of neutral protease per milligram of the product.

4. The pharmaceutical product of claim 1, wherein the product comprises less than about 50 nanograms of neutral protease per milligram of the product.

5. The pharmaceutical product of claim 1, wherein the product comprises less than about 25 nanograms of neutral protease per milligram of the product.

6. The pharmaceutical product according to claim 1, wherein the collagenase comprises collagenase I and collagenase II derived from Bacillus histolyticus.

7. The drug product according to claim 1, wherein the collagenase is at least one of collagenase I and collagenase II.

8. The pharmaceutical product according to claim 7, wherein the collagenase I and collagenase II are present in a ratio of approximately 1:

1.

9. An isolated and purified collagenase I obtained or derived from Clostridium histolyticum, wherein the collagenase I is substantially free of neutral proteases.

10. The collagenase I according to claim 9, wherein the collagenase I comprises an undetectable amount of neutral protease.

11. The collagenase I of claim 9, wherein it comprises less than about 1000 nanograms of neutral protease per milligram of collagenase I.

12. The collagenase I of claim 9, wherein it comprises less than about 500 nanograms of neutral protease per milligram of collagenase I.

13. A method for the isolation and purification of collagenase I and collagenase II obtained or derived from Clostridium histolyticum comprising controlling metal levels during the purification of collagenase I and collagenase II.

14. The method of claim 13, wherein the process is exposed to low levels of metals selected from the group consisting of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, and lead.

15. The method of claim 14, wherein the nickel is present in an amount less than about 2.0 ppm.

16. The method of claim 14, wherein the nickel is present in an amount less than about 1.2 ppm.

17. The method of claim 14, wherein the nickel is present in an amount less than about 1.0 ppm.

18. The method of claim 14, wherein the nickel is present in an amount less than about 0.5 ppm.

19. The method of claim 14, wherein the nickel is present in an amount selected from the group consisting of less than about 0.9 ppm, less than about 0.8 ppm, less than about 0.7 ppm, less than about 0.6 ppm, less than about 0.5 ppm, less than about 0.4 ppm, less than about 0.3 ppm, less than about 0.2 ppm, less than about 0.1 ppm, less than about 0.09 ppm, less than about 0.08 ppm, less than about 0.07 ppm, less than about 0.06 ppm, less than about 0.05 ppm, less than about 0.04 ppm, less than about 0.03 ppm, less than about 0.02 ppm, or less than about 0.01 ppm.

20. The method of claim 14, wherein the zinc is present in an amount less than about 80 ppm.

21. The method of claim 14, wherein the zinc is present in an amount less than about 50 ppm.

22. The method of claim 14, wherein the zinc is present in an amount less than about 25 ppm.

23. The method of claim 14, wherein the zinc is present in an amount less than about 10 ppm.

24. The method of claim 14, wherein the zinc is present in an amount less than about 5 ppm.

25. The method of claim 14, wherein the zinc is present in an amount less than about 3 ppm.

26. The method of claim 14, wherein the zinc is present in an amount less than about 1 ppm.

27. A method of preparing isolated and purified collagenase comprising preparing the collagenase in a process in which metal content is controlled and limited, and wherein the process comprises an elimination step to separate a plurality of highly pure collagenase fractions from neutral proteases.

28. The method of claim 27, wherein the collagenase is collagenase I and the depletion step isolates a collagenase I fraction.

29. The method of claim 27, wherein the collagenase is collagenase II and the depleting step isolates a fraction of collagenase II.

30. The method of claim 27, wherein the collagenase comprises collagenase I and collagenase II, and wherein the high purity fractions of collagenase I and collagenase II are mixed, and wherein the resulting mixture has less than 50 nanograms of neutral protease per milligram of mixture.

31. The method of claim 27, wherein the process is limited to low levels of metals selected from the group consisting of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, and lead.

32. The method of claim 31 , wherein the metal is nickel and is present in an amount less than about 2.0 ppm.

33. The method of claim 31 , wherein the metal is nickel and is present in an amount less than about 1.2 ppm.

34. The method of claim 31 , wherein the metal is nickel and is present in an amount less than about 1.0 ppm.

35. The method of claim 31 , wherein the metal is nickel and is present in an amount less than about 0.5 ppm.

36. The method of claim 31 , wherein the metal is nickel and is present in an amount selected from the group consisting of less than about 0.9 ppm, less than about 0.8 ppm, less than about 0.7 ppm, less than about 0.6 ppm, less than about 0.5 ppm, less than about 0.4 ppm, less than about 0.3 ppm, less than about 0.2 ppm, less than about 0.1 ppm, less than about 0.09 ppm, less than about 0.08 ppm, less than about 0.07 ppm, less than about 0.06 ppm, less than about 0.05 ppm, less than about 0.04 ppm, less than about 0.03 ppm, less than about 0.02 ppm, or less than about 0.01 ppm.

37. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 80 ppm.

38. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 50 ppm.

39. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 25 ppm.

40. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 10 ppm.

41. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 5 ppm.

42. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 3 ppm.

43. The method of claim 31 , wherein the metal is zinc and is present in an amount less than about 1 ppm.

44. The method of claim 31, wherein the depleting step comprises testing each fraction on an SDS-PAGE gel and combining only the fractions that have no detectable amount of neutral protease on the gel.

45. A method for preparing isolated and purified collagenase I obtained or derived from Clostridium histolyticum, comprising (a) isolating a plurality of highly pure collagenase I fractions from a neutral protease, and (b) combining the highly pure collagenase I fractions into a collagenase I drug substance.

46. ​​The method of claim 45, wherein the separating step comprises testing each fraction for the presence of a neutral protease using zymography, and the combining step combines only fractions containing collagenase I that have no detectable amount of neutral protease on the zymography gel.

47. The method of claim 45, wherein the collagenase I drug substance is substantially free of neutral proteases.

48. The method of claim 45, wherein the collagenase I drug substance comprises less than 100 nanograms of neutral protease per milligram of drug substance.

49. The method of claim 45, wherein the collagenase I drug substance comprises less than 75 nanograms of neutral protease per milligram of drug substance.

50. The method of claim 45, wherein the collagenase I drug substance comprises less than 50 nanograms of neutral protease per milligram of drug substance.

51. The method of claim 45, wherein the collagenase I drug substance comprises less than 25 nanograms of neutral protease per milligram of drug substance.

52. The method of claim 45, wherein the collagenase I drug substance comprises less than 20 nanograms of neutral protease per milligram of drug substance.

53. The method of claim 45, wherein the collagenase I drug substance comprises less than 10 nanograms of neutral protease per milligram of drug substance.

54. A process for producing isolated and purified collagenase I obtained or derived from Clostridium histolyticum comprising the following steps: a. Fermented Clostridium histolyticum; b. harvesting the crude fermentate containing collagenase I and collagenase II; c. Purifying collagenase I and collagenase II, comprising the following steps: i. filtering the crude fermentate through an anion exchange filter; ii. the filtrate produced by precipitation step (c)(i), the filtrate having ammonium sulfate comprising less than about 80 ppm zinc and less than about 1.2 ppm nickel; iii. resuspending the precipitated product produced in step (c) (ii); iv. passing the resuspension produced in step (c) (iii) through a hydrophobic interaction chromatography column; v. separating the collagenase I and collagenase II from the filtrate of step (c) (iv) using ion exchange chromatography; and vi. collecting the filtrate from the fractions of step (c) (v); d. estimating the total amount of collagenase II collected in each fraction produced in step (c)(vi); e. estimating the amount of collagenase I collected in each fraction produced in step (c)(vi); and f. Pooling fractions comprising the peak of collagenase I in said fractions toward the tail of said collagenase I in said fractions until said pooled amount of collagenase I is approximately the same as said amount of collagenase II estimated in step (d).

55. The process of claim 54, wherein the ammonium sulfate comprises less than about 80 ppm zinc and less than about 1.2 ppm nickel.

56. A process for producing isolated and purified collagenase I obtained or derived from Clostridium histolyticum comprising the following steps: a. Fermented Clostridium histolyticum; b. harvesting the crude fermentate containing collagenase I and collagenase II; c. Purifying collagenase I and collagenase II, comprising the following steps: i. filtering the crude fermentate through an anion exchange filter; ii. the filtrate produced in the precipitation step (i), the filtrate having ammonium sulfate containing essentially no transition metals; iii. resuspending the precipitated product produced in step (ii); iv. passing the resuspension produced in step (iii) through a hydrophobic interaction chromatography column; v. separating the collagenase I and collagenase II from the filtrate produced in step (iv) using ion exchange chromatography; and vi. collecting the filtrate in the fractions of step (v); d. separating multiple high-purity collagenase I fractions from neutral proteases; and e. Combine the high purity fractions of collagenase I.

57. The process of claim 17, wherein the separation step (d) comprises testing each fraction on an SDS-PAGE gel and the pooling step (e) pools only fractions containing collagenase I that have no detectable amount of neutral protease on the gel.

58. A process for preparing a drug product consisting of separated and purified collagenase I and collagenase II, obtained or derived from Clostridium histolyticum, respectively, wherein the collagenase I and collagenase II have a mass ratio of about 1:1 and the purity of the drug product as measured by reverse phase high performance liquid chromatography is at least 95% by area, comprising the following steps: a. Fermented Clostridium histolyticum; b. harvesting the crude fermentate containing collagenase I and collagenase II; c. Purifying collagenase I and collagenase II from the crude harvest by filtration and column chromatography, which comprises treating the collagenase I and collagenase II with an ammonium sulfate solution having a zinc level of less than about 80 ppm and a nickel level of less than about 1.2 ppm.

59. The method of claim 58, wherein the process is limited to low levels of metals selected from the group consisting of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium and lead.

60. The method of claim 59, wherein the nickel is present in an amount less than about 2.0 ppm.

61. The method of claim 59, wherein the nickel is present in an amount less than about 1.2 ppm.

62. The method of claim 59, wherein the nickel is present in an amount less than about 1.0 ppm.

63. The method of claim 59, wherein the nickel is present in an amount less than about 0.5 ppm.

64. A method according to claim 59, wherein the nickel is present in an amount selected from the group consisting of: less than about 0.9 ppm, less than about 0.8 ppm, less than about 0.7 ppm, less than about 0.6 ppm, less than about 0.5 ppm, less than about 0.4 ppm, less than about 0.3 ppm, less than about 0.2 ppm, less than about 0.1 ppm, less than about 0.09 ppm, less than about 0.08 ppm, less than about 0.07 ppm, less than about 0.06 ppm, less than about 0.05 ppm, less than about 0.04 ppm, less than about 0.03 ppm, less than about 0.02 ppm or less than about 0.01 ppm.

65. The method of claim 59, wherein the zinc is present in an amount less than about 80 ppm.

66. The method of claim 59, wherein the zinc is present in an amount less than about 50 ppm.

67. The method of claim 59, wherein the zinc is present in an amount less than about 25 ppm.

68. The method of claim 59, wherein the zinc is present in an amount less than about 10 ppm.

69. The method of claim 59, wherein the zinc is present in an amount less than about 5 ppm.

70. The method of claim 59, wherein the zinc is present in an amount less than about 3 ppm.

71. The method of claim 59, wherein the zinc is present in an amount less than about 1 ppm.

72. A method for producing a neutral protease obtained from Clostridium histolyticum, comprising the steps of: a. Fermented Clostridium histolyticum; b. harvesting the crude fermentation product containing neutral protease; as well as c. Purifying the neutral protease using an anion exchange filter and hydrophobic interaction chromatography to obtain the neutral protease.

73. The method of claim 72, further comprising the step of adding ammonium sulfate prior to step (c).

74. The method of claim 73, wherein the ammonium sulfate is from about 0.8M to about 1.2M.

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