CHROMATOGRAPHIC PURIFICATION OF AT LEAST ONE ENZYME SELECTED FROM A GROUP INCLUDING COLLAGENASE TYPE I, COLLAGENASE TYPE II, NEUTRAL PROTEASE AND CLOSTRIPAIN

Abstract

The present invention relates to a method for purifying at least one enzyme selected from the group consisting of collagenase type I, collagenase type II, neutral protease and clostripain from a mixture of substances, comprising as a method step at least one hydrophobic interaction chromatography, characterized in that, in the hydrophobic interaction chromatography, the stationary phase comprises a material selected from the group consisting of polypropylene glycol and butyl sepharose. The present invention further relates to the use of an enzyme thus purified for pharmaceutical, cosmetic and/or biochemical purposes.

Claims

1-20. (canceled)

21. A method for the purification of at least one enzyme, selected from the group consisting of collagenase type I, collagenase type II, neutral protease and clostripain, from a mixture of substances, the method comprising: subjecting the at least one enzyme to at least one hydrophobic interaction chromatography having a stationary phase that comprises a material selected from the group consisting of polypropylene glycol and butyl sepharose, and at least one aqueous solution as a mobile phase, the at least one aqueous solution comprising at least one salt selected from the group consisting of ammonium sulfate and potassium chloride.

22. The method according to claim 21, wherein the stationary phase comprises butyl sepharose and wherein at least one aqueous solution comprising ammonium sulfate is used as a mobile phase.

23. The method according to claim 22, wherein the stationary phase comprises butyl sepharose and wherein the at least one enzyme is eluted from the stationary phase with at least one aqueous solution comprising ammonium sulfate.

24. The method according to claim 21, wherein the method does not comprise a step of protein precipitation with ammonium sulfate.

25. The method according to claim 21, wherein the mixture of substances comprises at least two enzymes selected from the group consisting of collagenase type I, collagenase type II, neutral protease and clostripain.

26. The method according to claim 21, wherein the mixture of substances is a culture supernatant of Clostridium histolyticum.

27. The method according to claim 21, wherein the at least one aqueous solution has a molar concentration of ammonium sulfate of up to 1.5 mol/l.

28. The method according to claim 26, wherein the at least one aqueous solution has a molar concentration of ammonium sulfate in the range from 0.3 to 1.5 mol/l.

29. The method according to claim 21, wherein a step elution, a gradient elution or a combination of these two is carried out in the hydrophobic interaction chromatography.

30. The method according to claim 21, wherein a step elution is carried out in the hydrophobic interaction chromatography and at least three elution steps are carried out.

31. The method according to claim 30, wherein the two collagenases, neutral protease and clostripain are separated from one another, so that collagenase type I and collagenase type II are mixed in a common fraction and neutral protease and clostripain are separated from one another in two further fractions separate from the fraction comprising the two collagenases.

32. The method according to claim 30, wherein collagenase type I, collagenase type II, neutral protease and clostripain are separated from one another.

33. The method according to claim 21, wherein a gradient elution or a combination of gradient and step elution is carried out in the hydrophobic interaction chromatography and at least three elution steps are carried out.

34. The method according to claim 33, wherein the two collagenases, neutral protease and clostripain are separated from each other, so that collagenase type I and collagenase type II are mixed in a common fraction and neutral protease and clostripain are separated from each other and in two further fractions separate from the fraction comprising the two collagenases.

35. The method according to claim 33, characterized in that collagenase type I, collagenase type II, neutral protease and clostripain are separated from one another.

36. An enzyme obtained according to the method of claim 21.

37. A composition comprising at least one enzyme of claim 36.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1: Chromatogram of the purification and separation of the concentrate of the culture supernatant on polypropylene glycol (PPG-600M) showing the fractions containing clostripain (F2-F5), collagenases (F6) and neutral protease (F7).

[0037] FIG. 2: MonoQ chromatogram of the collagenase value fraction (equivalent to fraction F6 in FIG. 1) after loading the polypropylene glycol (PPG-600M) column in HIC chromatography with different amounts (measured in volume-specific PZ activity according to Wunsch) of the concentrate of the culture supernatant.

[0038] FIG. 3: SDS-PAGE of the collagenase value fraction after separation on polypropylene glycol (PPG-600M) (equivalent to fraction F6 in FIG. 1). M: marker proteins; K: sample application concentrate (before purification/separation); Kol: Collagenase value fraction (F6).

[0039] FIG. 4: SDS-PAGE of the neutral protease value fraction after separation on polypropylene glycol (PPG-600M) (equivalent to fraction F7 in FIG. 1). M: marker proteins; K: sample application concentrate (before purification/separation); NPf: Neutral protease value fraction (F7) after chromatography; NPe: Neutral protease after concentration of F7 to lyophilized final product.

[0040] FIG. 5: Chromatogram of the purification and separation of the concentrate of the culture supernatant on butyl sepharose (Butyl Sepharose HP) showing the—compared to FIG. 1—additional separation of collagenase I and collagenase II.

[0041] FIG. 6: MonoQ chromatogram for comparative analysis of the respective collagenase value fractions after purification and separation of the cell-free, concentrated culture supernatant by HIC chromatography. A: Collagenase value fraction after separation on polypropylene glycol (PPG-600M) (procedure variant 1); B: Collagenase type II value fraction after separation on butyl sepharose (Butyl Sepharose HP) (procedure variant 2); C: Collagenase type I value fraction after separation on butyl sepharose (Butyl Sepharose HP) (procedure variant 2).

[0042] FIG. 7: SDS-PAGE for comparison of a currently commercially available collagenase (Ka) purified via several chromatographic steps with the collagenase (Kn) obtained in one chromatographic step according to method variant 2 of the invention. M: Marker proteins.

DETAILED DESCRIPTION

[0043] The first sub-step in the hydrophobic interaction chromatography consists of applying the mixture of substances to be separated to the chromatographic column, preferably with the aid of a so-called application buffer. The application buffer is usually the specifically modified, highly saline aqueous matrix in which the mixture of substances dissolved therein accumulates for purification and separation, in the present method according to the invention, in particular in the context of the processing from the culture supernatant of a culture of the bacterium Clostridium histolyticum. In the extremely hydrophilic medium of the application buffer, very strong hydrophobic interactions of the proteins with the stationary phase occur, so that most proteins are almost completely fixed to the stationary phase.

[0044] As a second sub-step of the hydrophobic interaction chromatography—and thus before the elution of the value fractions with the target proteins, in the present method according to the invention with the enzymes collagenase I, collagenase II, neutral protease and/or clostripain—at least one so-called washing step is preferably carried out. In this washing step, the chromatography column including the proteins bound to the stationary phase is washed, wherein any impurities which are rather polar or hydrophilic and therefore not bound to the stationary phase, and which are often of low molecular weight, are washed out and thus separated from the target proteins. An aqueous buffer solution also containing a high salt content is usually used as the so-called wash buffer.

[0045] The composition of the application buffer and the wash buffer can differ. However, one and the same buffer solution can also be used both as an application buffer and as a wash buffer.

[0046] In the hydrophobic interaction chromatography, the washing step(s) are followed by the elution steps as further sub-steps. In particular with regard to the present method according to the invention, these are those sub-steps in which the value fractions with the target proteins are eluted from the column by successive adjustment of the composition of the mobile phase (elution buffer), in the case of hydrophobic interaction chromatography in particular by reducing the salt content of the mobile phase. This elution can be gradually carried out isocratically, i.e. in steps with constant composition of the mobile phase (step elution), as a gradient, i.e. with a targeted continuously changing composition of the mobile phase (gradient elution), or as a combination of step and gradient elution.

[0047] In certain constellations of hydrophobic interaction chromatography, one of the target proteins may show less strong hydrophobic interactions with the stationary phase than the remaining target proteins of the substance mixture and is therefore not bound to the stationary phase with the same intensity already in the application and wash buffer. In these cases, therefore, the wash buffer can under certain circumstances simultaneously serve as the first elution buffer, so that the same sub-step of the hydrophobic interaction chromatography represents in parallel both the wash step and the first elution step. In this case, the rather polar or hydrophilic, often low-molecular impurities are first washed out in the same sub-step and then the first value fraction is eluted with the less strongly bound target protein. In such constellations, the volume of the first elution buffer used—irrespective of whether it also functions as a wash buffer—can control the quality of separation of the target protein in question from the remaining target proteins of the mixture. In this case, the amount of mobile phase used is often expressed as the column volume (CV).

[0048] Accordingly, in the present method according to the invention, it is possible to control the content of clostripain in the corresponding eluted value fractions (elution fractions) via the volume of the first elution buffer (clostripain elution buffer) used, since clostripain shows less strong hydrophobic interactions with the stationary phase than the remaining target proteins of the mixture of substances (collagenase I, collagenase II and neutral protease). Thus, a large volume of the first elution buffer results in clostripain eluting completely or nearly completely in a separate, pure clostripain fraction. If, on the other hand, a smaller volume of the first elution buffer is used, the quantitative ratios of clostripain in the respective elution fractions shift. This then leads to the fact that not all or almost all of the clostripain is eluted in a separate fraction, but that the eluted clostripain is distributed both to a separate, pure clostripain fraction and to the respective subsequent collagenase fraction. Thus, the amount of the first elution buffer can be used to select whether clostripain should be eluted almost completely in a separate, pure clostripain fraction or whether part of the clostripain should be eluted as a component of the respective subsequent collagenase fraction and thus also occur together with collagenase(s) in one fraction. In the latter case, the choice of suitable salt concentrations of the mobile phase during the elution of the collagenases can control whether the second part of the clostripain accumulates together with both collagenases (type I and type II) in a common elution fraction (column material polypropylene glycol or butyl sepharose without additional separation of the two collagenase types) or together only with collagenase II in a common elution fraction (column material butyl sepharose with additional separation of the two collagenase types). The necessary volume of the mobile phase (of the first elution buffer) leading to an almost complete elution of the clostripain in a separate, pure clostripain fraction and thus to an almost complete separation of the clostripain from the collagenases and the neutral protease depends on the specific combination of different factors (stationary phase, mobile phase, dimensions of the column, etc.). However, preferred for separating the clostripain according to the invention is a method characterized in that the chromatography column is eluted with at least 10 column volumes, more preferably with at least 12 column volumes, and most preferably with at least 15 column volumes, of the first elution buffer. With such volumes of clostripain elution buffer, the clostripain is usually eluted completely or almost completely from the column.

[0049] The present method allows to obtain collagenase I, collagenase II and mixtures of these two types of collagenases in a purity of at least 80%, preferably of at least 90%, wherein the purity of the collagenases is determined by analytical anion-exchange chromatography (e.g. using a GE Healthcare MonoQ column with tris buffer as mobile phase at room temperature). A method according to the invention in which collagenase I, collagenase II or mixtures of these collagenases are obtained in a purity (determined as indicated above) of at least 80%, preferably at least 90%, is therefore preferred. Neutral protease can be obtained by the method according to the invention in a purity of at least 70%, preferably of at least 80%, wherein the purity of the neutral protease is determined by SDS-PAGE (according to Laemmli U. K.; Nature 227: 680-685, 1970). Also preferred, therefore, is a method according to the invention in which neutral protease is obtained in a purity (determined as indicated above) of at least 70%, preferably of at least 80%. Clostripain can be obtained by the method according to the invention in a purity of at least 60%, preferably of at least 70%, wherein the purity of the clostripain is determined by SDS-PAGE (according to Laemmli U. K.; Nature 227: 680-685, 1970). Also preferred, therefore, is a method according to the invention in which clostripain is obtained in a purity (determined as indicated above) of at least 60%, preferably of at least 70%. All of these purity values are purity levels that satisfy the requirements when these enzymes are used for most biochemical and medical purposes. Most preferred is a method according to the invention in which collagenase I, collagenase II or mixtures of these collagenases are obtained in a purity of at least 80% (determined as indicated above), neutral protease in a purity of at least 70% (determined as indicated above) and clostripain in a purity of at least 60% (determined as indicated above). Most preferred is a method according to the invention in which collagenase I, collagenase II or mixtures of these collagenases are obtained in a purity of at least 90% (determined as indicated above), neutral protease in a purity of at least 80% (determined as indicated above) and clostripain in a purity of at least 70% (determined as indicated above).

[0050] These enzymes are also obtainable in this purity in a one-step method.

[0051] Furthermore, a process according to the invention is preferred, which is characterized in that a linear flow rate of 100 to 300 cm/h, in particular of 150 to 250 cm/h, is used in the hydrophobic interaction chromatography. These flow rates result in optimal purification and separation of the enzymes.

[0052] Preferably, the method according to the invention does not comprise gel filtration steps and/or protein precipitation steps. Gel filtration is a very time-consuming method, which thus entails high costs and increases the risk of self-digestion of the enzymes to be separated. Protein precipitation steps, such as ammonium sulfate precipitation, can lead to undesirable structural changes in the proteins. Also, protein precipitation does not allow enzymes to be obtained with the high purity provided by the present method.

[0053] Preferably, at least one enzyme purified with the method according to the invention is used for pharmaceutical and/or biochemical purposes. Pharmaceutical purposes include any use of one or more of said enzyme(s) as an active pharmaceutical ingredient, wherein the use is not subject to any restriction as to indication, dosage form/preparation or mode of application. Furthermore, the enzyme(s) may be used for biochemical purposes, such as in vitro cell isolation.

[0054] Also preferably, at least one enzyme purified with the method according to the invention is used for cosmetic purposes. The use of the enzyme or enzymes thus also extends to the use for purely cosmetic purposes, i.e., a cosmetic use as distinguished from a therapeutic purpose.

[0055] The invention also includes an enzyme itself which is obtained by the method according to the invention. In addition, the invention also includes compositions comprising at least one enzyme obtained by the method according to the invention. The present invention further relates to the use of an enzyme purified with the method according to the invention for pharmaceutical, cosmetic and/or biochemical purposes.

[0056] After cultivation of clostridium histolyticum carried out in a suitable fermentation medium, the cells and other non-soluble components are separated from the culture supernatant, for example by centrifugation and/or filtration. The culture supernatant, which contains the enzymes collagenase I, collagenase II, neutral protease and clostripain, can be concentrated in the usual way before purification and separation of these proteins by hydrophobic interaction chromatography.

[0057] Hydrophobic interaction chromatography (HIC) is then performed to purify and separate the enzymes from the culture supernatant. For this purpose, the cell-free and, if necessary, suitably concentrated culture supernatant is applied, for example, to a chromatography column filled with polypropylene glycol (PPG) and/or butyl sepharose, wherein the enzymes—among other components—bind to the column material. After washing out unbound molecules, the elution of the initially bound components, including the target proteins, is carried out in a buffered system in the pH range of 6.0 to 9.5 at a linear flow rate of 100 to 300 cm/h by means of step elution, gradient elution, or a combination of these two, wherein the salt content is successively reduced. The elution of the proteins is preferably carried out in three or more elution steps. Here, three value fractions can be obtained, wherein one fraction contains the collagenases (type I and type II) together, another fraction contains neutral protease, and the third fraction contains clostripain (separation on PPG or butyl sepharose). In the case of chromatography on butyl sepharose, an additional separation of the two collagenases (type I and type II) from each other is possible. This separation is then preferably performed with a combination of step and gradient elution in at least three elution steps or with a four-step elution. Accordingly, four value fractions are obtained, where—as before—one fraction contains neutral protease and another fraction contains clostripain. However, the collagenases are now obtained separately in a third fraction containing collagenase I and a separate fourth fraction containing collagenase II.

[0058] The individual elution fractions can now be desalted and/or concentrated by means of conventional methods, such as a tangential flow filtration (TFF) process. Subsequently, the resulting material can be lyophilized by means of likewise customary methods, e.g. freeze-drying. If necessary, further purification steps can also follow.

[0059] A flow diagram of the entire process is shown in scheme 1 below. Depending on the variant used, this results in three or four different end products which are then available for the desired further use or further processing, in particular also a specific and defined mixture of several of these end products.

##STR00001##

[0060] Depending on the stationary phase and mobile phase used (type and amount of salts in the mobile phase), this method can therefore achieve, for example, the following:

[0061] By using PPG as column material and ammonium sulfate (0.3-1.5 mol/l, in particular 0.5-1.0 mol/l) as salt, a separation of collagenases, neutral protease and clostripain is achieved, wherein the two types of collagenases (type I and type II) are present mixed in a common fraction, while neutral protease and clostripain are present in two further fractions separate from each other and separate from the collagenases. The use of butyl sepharose as column material with potassium chloride (1.0-3.0 mol/l, especially 1.5-2.5 mol/l) as salt allows an additional separation of the collagenases into collagenase I and collagenase II. The same is achieved when butyl sepharose is used as column material with ammonium sulfate (0.3-1.5 mol/l, especially 0.5-1.0 mol/l) as salt.

[0062] FIG. 1: Chromatogram of the purification and separation of the concentrate of the culture supernatant on polypropylene glycol (PPG-600M) showing the fractions containing clostripain (F2-F5), collagenases (F6) and neutral protease (F7).

[0063] FIG. 2: MonoQ chromatogram of the collagenase value fraction (equivalent to fraction F6 in FIG. 1) after loading the polypropylene glycol (PPG-600M) column in HIC chromatography with different amounts (measured in volume-specific PZ activity according to Wunsch) of the concentrate of the culture supernatant.

[0064] FIG. 3: SDS-PAGE of the collagenase value fraction after separation on polypropylene glycol (PPG-600M) (equivalent to fraction F6 in FIG. 1). [0065] M: marker proteins; K: sample application concentrate (before purification/separation); Kol: Collagenase value fraction (F6).

[0066] FIG. 4: SDS-PAGE of the neutral protease value fraction after separation on polypropylene glycol (PPG-600M) (equivalent to fraction F7 in FIG. 1). [0067] M: marker proteins; K: sample application concentrate (before purification/separation); NPf: Neutral protease value fraction (F7) after chromatography; NPe: Neutral protease after concentration of F7 to lyophilized final product.

[0068] FIG. 5: Chromatogram of the purification and separation of the concentrate of the culture supernatant on butyl sepharose (Butyl Sepharose HP) showing the—compared to FIG. 1—additional separation of collagenase I and collagenase II.

[0069] FIG. 6: MonoQ chromatogram for comparative analysis of the respective collagenase value fractions after purification and separation of the cell-free, concentrated culture supernatant by HIC chromatography. [0070] A: Collagenase value fraction after separation on polypropylene glycol (PPG-600M) (procedure variant 1); B: Collagenase type II value fraction after separation on butyl sepharose (Butyl Sepharose HP) (procedure variant 2); C: Collagenase type I value fraction after separation on butyl sepharose (Butyl Sepharose HP) (procedure variant 2).

[0071] FIG. 7: SDS-PAGE for comparison of a currently commercially available collagenase (Ka) purified via several chromatographic steps with the collagenase (Kn) obtained in one chromatographic step according to method variant 2 of the invention. [0072] M: Marker proteins

EXAMPLES OF EMBODIMENTS

[0073] All mobile phases, application buffer, wash buffer and elution buffer used in the examples, are aqueous solutions. The complete ingredients of the mobile phases used are given below in each case.

Example 1

[0074] A culture of Clostridium histolyticum was cultured to the desired cell density in liquid culture using a suitable nutrient medium according to standard methods. After separation of the cells by standard methods, such as centrifugation and/or filtration, the hydrophobic interaction chromatography of the method according to the invention was performed. For this purpose, a chromatography column (bed height about 20 cm) filled with polypropylene glycol (PPG-600M, Tosoh Bioscience LLC) was equilibrated by means of a mobile phase (aqueous solution, 0.85 mol/l ammonium sulfate, 20 mmol/l tris, 7 mmol/l CaCl.sub.2, pH 7.5). After loading the cell-free concentrated culture supernatant, the column was washed with 10 column volumes (CV) of the same mobile phase. The elution of the target proteins was carried out at a linear flow rate of 250 cm/h in three elution steps. The first value fraction was obtained by isocratic elution with the aforementioned mobile phase and contained the clostripain. The second fraction was obtained by isocratic elution with another mobile phase (aqueous solution, 0.2 mol/l ammonium sulfate, 20 mmol/l tris, 7 mmol/l CaCl.sub.2, pH 7.5) and contained the collagenases (collagenase I and collagenase II). The third fraction was obtained by isocratic elution with a third mobile phase (aqueous solution, 12% (m/m) propylene glycol, 20 mmol tris, 7 mmol CaCl.sub.2, pH 7.5) and contained the neutral protease.

[0075] FIG. 1 shows a chromatogram of the above-mentioned purification and separation of the cell-free concentrated culture supernatant by means of hydrophobic interaction chromatography on PPG-600M as column material. The figure shows the value fractions of the three different products with the corresponding elution ranges. Clostripain elutes in subfractions F2 to F5, the collagenases in fraction F6, and neutral protease in fraction F7. The collagenase fraction (F6) contains both collagenases (type I and type II) in approximately equal proportions.

[0076] FIG. 2 shows the analysis of the collagenase value fraction after separation on PPG-600M (equivalent to fraction F6 in FIG. 1) using MonoQ chromatograms. Shown is the quality obtained with different column loadings in HIC chromatography (loading measured in volume-specific PZ activity according to Wunsch E., Heidrich H.-G.; Z. Physiol. Chem. 333: 149-151, 1963). For this purpose, a sample of the respective collagenase value fraction is analyzed using a MonoQ column (column: MonoQ 5/20 GL, GE Healthcare; application buffer: aqueous solution, 10 mmol/l tris, 2 mmol/l CaCl.sub.2, pH 7.5; elution buffer: aqueous solution, 10 mmol/l tris, 2 mmol/l CaCl.sub.2, 1 mol/l NaCl, pH 7.5; gradient elution). Thus, both the purity and the content of the collagenase types as well as their ratio to each other can be determined in each case. As can be seen from the chromatograms, the collagenase fraction obtained contains only minor impurities in addition to the two collagenase types, irrespective of the loading of the PPG column. The method is thus reproducible and robust. A value of >90% was determined for the purity of the collagenase fraction (see FIGS. 2 and 3).

[0077] Since no meaningful activity assay is currently available for collagenase I, its quality and quantity were evaluated using MonoQ analytics. As shown in FIG. 2, no significant degradation products can be detected in the MonoQ analytics of the collagenase I fraction, and the enzyme thus corresponds to the quality of the products available on the market.

[0078] FIG. 3 shows an SDS-PAGE of the collagenase value fraction after separation on PPG-600M (equivalent to fraction F6 in FIG. 1). This was carried out according to the protocol of U. K. Laemmli using a 14% tris glycine gel (Anamed) and stained with Coomassie (Coomassie R-250, Invitrogen) (Laemmli U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4; Nature 227: 680-685, 1970). In the left lane, labeled M, marker proteins (Novex Mark12, Invitrogen) are plotted. The middle lane, labeled K, is the cell-free, concentrated culture supernatant before purification and separation. In the right lane (Kol), the collagenase value fraction F6 from FIG. 1, containing collagenase I and collagenase II, was plotted. The figure thus shows a direct comparison of the purity of the collagenases before and after purification and separation on the PPG column. Although the collagenase proteins were purified and separated using only one chromatography column, the purity of the collagenase value fraction is >90%.

[0079] FIG. 4 shows a corresponding SDS-PAGE of the neutral protease value fraction after separation on PPG-600M (equivalent to fraction F7 in FIG. 1). Marker proteins were again applied to the left lane, labeled M. The middle lane, labeled K, is again the cell-free, concentrated culture supernatant before purification and separation. In the one of the two right lanes labeled NPf, the neutral protease value fraction F7 from FIG. 1 was applied directly after purification and separation on the PPG column. One of the two right lanes, labeled NPe, shows the neutral protease end product dissolved again for comparative analytics, which had previously been obtained from F7 by desalting, concentration and lyophilization (freeze-drying). It can be seen from FIG. 4 that the neutral protease also exhibits a high purity of well >80% after purification and separation on the PPG column. This is significantly higher than the purity of the neutral protease products currently available on the market.

[0080] Table 1 shows the respective relative yield of enzymatic activity after purification and separation of the enzymes from the cell-free, concentrated culture supernatant by means of hydrophobic interaction chromatography on PPG-600M as column material. Values of >90% were determined several times for the corresponding yield of collagenase II.

TABLE-US-00001 TABLE 1 Relative yield of enzymatic activity after purification and separation of enzymes (results from several experiments). Collagenase Clostripain Neutral type II (PZ).sup.a (BAEE).sup.b protease Sample (%) (%) (%) Application 100 100 100 Flow (waste) — — — Value fraction clostripain — 40-80.sup.d — Value fraction collagenase 65-95  0-40.sup.d — Value fraction — — 60-100 neutral protease .sup.adetermined according to Wünsch E., Heidrich H.-G.; Z. Physiol. Chem. 333, 149-151, 1963 .sup.bdetermined according to Mitchell W. M., Harrington W. F.; Methods Enzymol. 19: 635-642, 1970 c: determined according to Moore S., Stein W. H.; J. Biol. Chem. 176: 367-388, 1948 .sup.dA portion is lost in the washing step with the waste fraction.

[0081] A special feature of the developed process is that the distribution of clostripain between a separate, pure clostripain fraction (comprising subfractions F2 to F5 in FIG. 1) and the collagenase fraction (fraction F6 in FIG. 1) can be controlled by how much column volume (CV) of the first elution buffer (clostripain elution buffer) is used to elute the chromatography column. A respective volume starting at about 12 CV leads to up to about 80% yield of total clostripain in the separate, pure clostripain fraction with corresponding losses with the waste fraction in the wash step and only very low clostripain content in the collagenase fraction. In contrast, a respective volume of approx. 4 CV, however, leads to a distribution of the total clostripain of approx. 40% in the separate, pure clostripain fraction and approx. 40% in the subsequent collagenase fraction, again with corresponding losses with the waste fraction in the washing step (see tables 1 and 2).

TABLE-US-00002 TABLE 2 Dependence of the amount of clostripain in the collagenase value fraction on the volume of the first elution buffer (clostripain elution buffer). Volume clostripain Clostripain activity in the elution buffer collagenase value fraction (CV) (%) 4 approx. 40 7 approx. 20 10 <5 12 <1

Example 2

[0082] A culture of clostridium histolyticum was cultured to the desired cell density in liquid culture using a suitable nutrient medium according to standard methods. After separation of the cells by standard methods, such as centrifugation and/or filtration, the hydrophobic interaction chromatography of the method according to the invention was carried out. For this purpose, a chromatography column (bed height 20 cm) filled with butyl sepharose (Butyl Sepharose High Performance, abbreviated as Butyl Sepharose HP, GE Healthcare) was equilibrated by means of a mobile phase (aqueous solution, 2 mol/l KCl, 20 mmol/l tris, 7 mmol/l CaCl.sub.2, pH 9). After application of the cell-free concentrated culture supernatant, the column was washed with 4 column volumes (CV) of the same mobile phase and eluted isocratically. The elution of the target proteins was carried out at a linear flow rate of 250 cm/h. The subsequent second elution step was carried out as a gradient over 20 CV with linearly decreasing salt concentration starting from the aforementioned mobile phase to the target buffer (aqueous solution, no KCl (0 mol/l), 20 mmol/l tris, 7 mmol/l CaCl.sub.2, pH 9). The first value fraction hereby obtained contained the clostripain. The second fraction contained the collagenase II. The third fraction contained the collagenase I. The fourth fraction was obtained by a third elution step by isocratic elution with 5 CV of a further mobile phase (aqueous solution, 25% (m/m) propylene glycol, 20 mmol tris, 7 mmol CaCl.sub.2, pH 9) and contained the neutral protease.

[0083] FIG. 5 shows a chromatogram of the above-described purification and separation of the cell-free, concentrated culture supernatant by hydrophobic interaction chromatography on butyl sepharose HP as column material. Shown is—in comparison to FIG. 1—the additional separation into collagenase I and collagenase II, wherein the first main peak in this respect corresponds to collagenase II and the second main peak in this respect corresponds to collagenase I.

[0084] FIG. 6 shows the comparative MonoQ analysis of the respective collagenase value fractions after purification and separation of the cell-free, concentrated culture supernatant by means of different variants of the hydrophobic interaction chromatography. A shows the collagenase value fraction after separation on PPG-600M as column material (method variant 1). B represents the collagenase type II value fraction after separation on butyl sepharose HP as column material (method variant 2). C shows the collagenase type I value fraction after separation on butyl sepharose HP as column material (method variant 2). Thus, a quality of purity and, if necessary, separation is achieved in one method step, which requires three or more method steps in the known methods.

[0085] FIG. 7 shows an SDS-PAGE after silver staining (Argent Quick Silver Staining Kit, Anamed). On the left lane, labeled M, the marker proteins were again applied. Next to it, for direct comparison, a currently commercially available “classical” collagenase purified over several chromatographic steps (middle lane, labeled Ka) and a collagenase obtained by a variant of the one-step purification method according to the invention (right lane, labeled Kn) are shown. It can be seen from the figure that the purity of the collagenases obtained by the one-step method according to the invention is at least as high as that of the “classical” collagenase currently available on the market. Due to the one-step method, the yield of ≥80% is also significantly higher than the yield of the established methods.