Chromatographic purification of at least one enzyme selected from the group consisting of 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 at least one hydrophobic interaction chromatography as a method step, 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 invention further relates to the use of the enzymes thus purified for pharmaceutical, cosmetic, and / or biochemical purposes.

Description

[Technical Field]

[0001] 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 at least one hydrophobic interaction chromatography step, wherein the stationary phase in the hydrophobic interaction chromatography comprises a material selected from the group consisting of polypropylene glycol (PPG) and butyl sepharose. The present invention further relates to the use of the enzymes thus purified for pharmaceutical, cosmetic, and / or biochemical purposes. [Background technology]

[0002] Clostridia are Gram-positive, obligately anaerobic, spore-forming bacteria belonging to the family Clostridiaceae. They are widespread and ubiquitous, especially in soil and the digestive tract of higher organisms.

[0003] When grown on or in an appropriate nutrient medium, Clostridium histolyticum secretes a complex mixture of enzymes containing collagenase, various other proteases, and low-molecular-weight components. Depending on the substrate being converted, collagenases are subdivided into type I and type II (EC 3.4.24.3), briefly also called collagenase I and collagenase II, and have molecular weights of 115–125 kDa. Another component of the enzyme mixture secreted by Clostridium histolyticum is the SH protease clostripain (EC 3.4.22.8), which exists as a heterodimer and has a molecular weight of approximately 59 kDa. Clostripain specifically cleaves target proteins at arginine residues. Additionally, a nonspecific neutral protease with a molecular weight of approximately 34 kDa is secreted by Clostridium histolyticum.

[0004] In this way, all proteases can be easily separated from the cells, since they are secreted into the culture medium by the bacteria and, in their natural environment, have the role of degrading tissue or fibrous collagen, making the released peptides and amino acids available to the bacteria as a source of nutrients.

[0005] One of the commercial applications of collagenases is their use as a biochemical for the in vitro isolation of cells from tissue media. In some cases, this cell isolation requires, in addition to collagenase, other proteases, such as neutral protease and / or clostripain. However, optimal results can only be achieved if the proteases are present in specific ratios depending on the intended cell isolation. To achieve optimal results, the two collagenase types must also be present in specific ratios depending on the application.

[0006] Another area of ​​application for proteases derived from Clostridium histolyticum is their use as biological agents for the production of medicines, including, among others, the treatment of fibrous bands in the palms and fingers in Dupuytren's disease, but also for various other diseases. Another pharmaceutical application of these proteases is their use in ointments for wound healing; for example, collagenase can be used for the enzymatic treatment of skin ulcers. In addition to collagenase, the corresponding active ingredient also contains neutral protease and clostripain. The usual manufacturing process for this active ingredient for wound treatment only involves desalting, concentrating, and drying the cell-free culture supernatant. The enzymes are not separated in this process. Therefore, the ratio of the enzymes relative to each other is determined by fermentation and cannot be affected by the current process.

[0007] Because Clostridium histolyticum secretes a complex mixture of enzymes into the culture supernatant, it is necessary to isolate the desired target enzyme and therefore purify it from the culture supernatant. A method for purifying enzymes from the culture supernatant of Clostridium histolyticum is known from DE 101 34 347 A1. However, a multi-step purification process using only chromatographic materials based on styrene / divinylbenzene or ceramic hydroxyapatite is provided. To purify the enzyme, a first chromatographic step is required, which uses a column packed with ceramic hydroxyapatite. This is followed by a second chromatographic step consisting of anion exchange chromatography using a column matrix based on styrene / divinylbenzene. This is followed by a third chromatographic step, this time in the context of cation exchange chromatography, also with a column matrix based on styrene / divinylbenzene. Therefore, a total of three chromatographic steps are required in this method, which makes the purification of the enzyme from the culture supernatant very costly and time-consuming.

[0008] Another method for purifying collagenase from a liquid culture of Clostridium histolyticum bacteria has been described (see Patent Document 1). The first step performed is protein precipitation with ammonium sulfate. This is followed by a second step, hydrophobic interaction chromatography, and a third purification step involving anion exchange chromatography. This method again involves a series of several different method steps that are material- and time-consuming, and has the result that only purified collagenase types I and II are obtained, but no purified fractions of neutral protease and clostripain are obtained.

[0009] Therefore, the described purification processes are strongly focused on two collagenases (type I and type II). To achieve the required collagenase purity, at least three purification steps are required in each case. The purification of neutral proteases and clostripain is not described in these methods. With regard to the separation of collagenase I and collagenase II, the known methods require an additional, separate chromatography step that must be carried out specifically for this purpose. The materials used for this purpose in known methods are usually anion exchangers. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2011 / 0070622 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention is therefore based on the objective of avoiding the disadvantages of the prior art. Preferably, an economical method should be provided for purifying at least one enzyme selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain from a mixture of substances. In particular, such a method should be provided for purifying at least one enzyme selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain from the culture supernatant of Clostridium histolyticum. Furthermore, material- and time-saving purification and separation of the enzyme should be provided. It is also an object of the present invention to provide a method by which various enzymes can be separated from each other so that they can subsequently be mixed in a predetermined ratio. Separation of the enzymes from each other should preferably be carried out with high yield and at the required quality. A further object is to reduce the number of steps required for the purification of enzymes from the culture supernatant of Clostridium histolyticum. In particular, it is an object of the present invention to reduce the number of chromatographic steps for enzyme purification and / or separation. [Means for solving the problem]

[0012] According to the present invention, the above-described object is achieved by a method for purifying at least one enzyme selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain from a mixture of substances, comprising at least one hydrophobic interaction chromatography (HIC) method step, 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. In all embodiments of the present invention, it is further preferred that the stationary phase consists of polypropylene glycol or butyl sepharose. [Brief explanation of the drawings]

[0013] [Figure 1]Chromatograms for the purification and separation of concentrates of culture supernatant on polypropylene glycol (PPG-600M) showing fractions containing clostripain (F2-F5), collagenase (F6), and neutral protease (F7). [Figure 2] This is a MonoQ chromatogram of the collagenase value fraction (corresponding to fraction F6 in Figure 1) after loading various amounts of culture supernatant concentrate (measured by volume-specific PZ activity according to Wunsch) onto a polypropylene glycol (PPG-600M) column in HIC chromatography. [Figure 3] SDS-PAGE of the collagenase-rich fraction (corresponding to fraction F6 in Figure 1) after separation on polypropylene glycol (PPG-600M). M: marker proteins; K: sample application concentrate (before purification / separation); K1: collagenase-rich fraction (F6). [Figure 4] SDS-PAGE of the neutral protease-rich fraction (corresponding to fraction F7 in Figure 1) after separation on polypropylene glycol (PPG-600M). M: marker protein; K: sample application concentrate (before purification / separation); NPf: neutral protease-rich fraction (F7) after chromatography; NPe: neutral protease after concentration of F7 into the lyophilized final product. [Figure 5] FIG. 2 is a chromatogram for the purification and separation of the concentrate of culture supernatant on butyl sepharose (butyl sepharose HP), showing additional separation of collagenase I and collagenase II compared to FIG. 1. [Figure 6]MonoQ chromatograms for comparative analysis of collagenase-rich fractions after purification and separation of cell-free concentrated culture supernatants by HIC chromatography: A: Collagenase-rich fraction after separation on polypropylene glycol (PPG-600M) (Procedure Variant 1); B: Collagenase type II-rich fraction after separation on butyl-Sepharose (Butyl-Sepharose HP) (Procedure Variant 2); C: Collagenase type I-rich fraction after separation on butyl-Sepharose (Butyl-Sepharose HP) (Procedure Variant 2). [Figure 7] 1 is a diagram of an SDS-PAGE for the comparison of currently commercially available collagenases (Ka) purified through several chromatographic steps with collagenases (Kn) obtained in one chromatographic step according to method variant 2 of the invention. M: marker proteins. DETAILED DESCRIPTION OF THE INVENTION

[0014] The separation principle of hydrophobic interaction chromatography is based on the interaction of non-polar surface regions of proteins with a hydrophobic stationary phase. These hydrophobic interactions are enhanced by increasing the salt concentration in the solution serving as the mobile phase. This leads to the partial removal of the hydrate shell and thus the exposure of the hydrophobic regions of the protein. These hydrophobic surface regions of the protein now in turn interact with the hydrophobic residues of the stationary phase.

[0015] In hydrophobic interaction chromatography, the stationary phase, i.e., the column material, is usually composed of a polymer that has been chemically modified, i.e., hydrophobized, with specifically selected nonpolar functional groups. Here, the selectivity and capacity of the stationary phase depend on the selectivity and density of the functional groups used. In the present invention, the use of polypropylene glycol (PPG) or butyl sepharose as the column material has proven particularly advantageous. For example, commercially available PPG-600M from Tosoh Bioscience LLC can be used as the polypropylene glycol column material. When butyl sepharose is used as the column material, commercially available butyl sepharose High Performance (abbreviated as butyl sepharose HP) and butyl sepharose Fast Flow (abbreviated as butyl sepharose FF) from GE Healthcare can be used. Butyl sepharose HP is particularly preferred.

[0016] Butyl Sepharose (also called "butyl agarose" or "cross-linked butyl agarose") is a cross-linked agarose in which some of the hydrogen atoms of the OH groups have been replaced by 3-n-butoxy-2-hydroxypropyl residues (-CH2-CHOH-CH2-O-CH2-CH2-CH3), thus etherifying the associated OH groups. The degree of cross-linking is preferably 1-7%, particularly preferably 2-6%. Butyl Sepharose is preferably present in the form of spherical particles with an average particle size in the range of 20-150 μm, particularly preferably 30-100 μm. For the separation of collagenase I and collagenase II from each other, an average particle size of 70 μm or less is preferred. An average particle size of 50 μm or less is particularly preferred for this purpose. The number of 3-n-butoxy-2-hydroxypropyl residues is preferably 10 to 200 μmol, particularly preferably 20 to 100 μmol, and most preferably 30 to 70 μmol per milliliter of medium.

[0017] Thanks to the method according to the present invention, purified enzymes can be obtained with high purity. The enzymes are very stable with respect to hydrophobic interactions with the stationary phase polypropylene glycol and butyl-Sepharose, as well as with respect to the buffer conditions of the mobile phase required for this purpose, and are virtually undegraded. A particular advantage is that there is virtually no denaturation of the enzymes to be purified, so that the enzymes are obtained in their native form and retain their biological activity. Therefore, the purified enzymes contain no or only very small amounts of denatured enzymes or their parts. This is highly advantageous, since denatured enzymes are often very difficult to separate from the corresponding native enzymes, since they often have similar chromatographic retention times. Therefore, thanks to the method according to the present invention, purified enzymes can be obtained with particularly good yields and high purity. Therefore, in the method according to the present invention, the proteins to be purified essentially retain their native form, and therefore their enzymatic activity. Therefore, the proteins can be subsequently used for their further use, such as in pharmaceutical or biochemical compositions, where retention of enzymatic activity is essential. This is also of great importance with regard to the reproducible quality of the active pharmaceutical ingredient and the pharmaceutical preparations made therefrom.

[0018] The method according to the present invention not only allows the purification of the aforementioned enzymes, collagenase I, collagenase II, neutral protease, and clostripain, from impurities, but also allows the separation of these enzymes from one another. Therefore, a method according to the present invention is also preferred, characterized in that the mixture of substances contains at least two enzymes selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain. A method according to the present invention is also preferred, characterized in that the mixture of substances contains at least two enzymes, at least one enzyme selected from the group consisting of collagenase I and collagenase II, and at least one enzyme selected from the group consisting of neutral protease and clostripain. A method according to the present invention is particularly preferred, characterized in that the mixture of substances contains at least three enzymes selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain. However, a method according to the invention is very particularly preferred, characterized in that the mixture of substances comprises collagenase I, collagenase II, neutral protease and clostripain enzymes.

[0019] The present invention provides a material- and time-saving, thus cost-effective, method for producing and isolating enzymes in high yield and with the required quality. This has the advantage that the composition of active ingredients containing one or more of these enzymes can be varied as desired by selectively mixing the respective enzymes. This improves the quality of the active ingredients and leads to new active ingredients with defined compositions and better efficacy.

[0020] Collagenase I, collagenase II, neutral protease, and clostripain enzymes are expressed and secreted by Clostridium histolyticum bacteria. Because Clostridium histolyticum secretes a complex mixture of enzymes into the culture supernatant, these desired target enzymes must be obtained by purifying and separating them from one another. Therefore, the method according to the present invention is most preferred, characterized in that the mixture of substances is obtained from the culture supernatant of a Clostridium histolyticum bacterial culture. Equally preferred is the method according to the present invention, characterized in that the mixture of substances is obtained from the culture supernatant of a Clostridium histolyticum bacterial culture and contains at least one enzyme selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain. Most preferably, the mixture contains at least two of the enzymes selected from the group consisting of collagenase I, collagenase II, neutral protease, and clostripain, and most preferably at least three of the enzymes. Most preferably, the mixture of substances contains all four enzymes.

[0021] The mixture of substances can be obtained, for example, from the culture supernatant of a culture of Clostridium histolyticum bacteria by separating the insoluble components, for example by centrifugation or by filtration, to remove cells, cell debris, and other components that are necessarily insoluble. Further, for example, desalting, rebuffering, and / or concentration, or other method steps typically used in the preparation of culture supernatants, can be carried out.

[0022] Thanks to the method according to the invention it is possible to purify both single and several target proteins from mixtures of substances, in particular from culture supernatants.

[0023] Surprisingly, it has been shown that the method according to the present invention allows the desired enzyme to be obtained from the culture supernatant in ready-to-use purity by only a single chromatographic step. Preferably, the method according to the present invention does not include any other chromatographic steps other than hydrophobic interaction chromatography with polypropylene glycol or butyl sepharose as the stationary phase. Particularly preferably, the method according to the present invention includes only a single method step in which hydrophobic interaction chromatography is carried out using a stationary phase comprising a material selected from the group consisting of polypropylene glycol and butyl sepharose, and no further chromatographic steps are carried out. This embodiment is referred to below as a "one-step process." However, if necessary, further purification steps can follow or precede the chromatography.

[0024] Using the method according to the present invention, it is possible to purify and separate the above-mentioned enzymes, collagenase I, collagenase II, neutral protease, and clostripain, from each other in a single chromatographic step. The developed method can provide the required quality of enzymes in a one-step process, thereby significantly reducing production costs. Compared to established methods, firstly, significantly less material and time are required to carry out the method, and secondly, the yield is much higher.

[0025] In addition, one-step purification and separation also offers clear advantages in terms of the stability of various proteases when purifying enzymes from a mixture of substances, especially from culture supernatants.In fact, the longer a mixture of enzymes that are all proteases remains in the same mixture of substances, especially in the same aqueous solution, the higher the risk and actual degree of mutual proteolysis, and therefore the irreversible destabilization and inactivation of the enzymes.This means that the sooner the proteases are separated as completely as possible from each other, the higher the expected yield, achievable purity, and stability of the individual enzymes purified.In particular, storage stability increases.This is also of great importance for the reproducible quality of active pharmaceutical ingredients and pharmaceutical preparations made therefrom.

[0026] The method according to the present invention is preferably characterized in that in the hydrophobic interaction chromatography, at least one aqueous solution containing at least one salt selected from the group consisting of ammonium sulfate and potassium chloride is used as the mobile phase.When such a mobile phase is used, the method can be carried out particularly effectively, and the advantages described herein become particularly clear.In particular, the use of these mobile phases allows the four enzymes to be clearly separated from each other.

[0027] A particularly preferred method of the present invention is characterized in that the stationary phase comprises polypropylene glycol and at least one aqueous solution containing ammonium sulfate is used as the mobile phase for hydrophobic interaction chromatography. This embodiment allows the four enzymes to be obtained in three separate fractions, with one fraction containing only a mixture of collagenases, i.e., collagenase I and collagenase II, another fraction containing only a neutral protease, and a third fraction containing only clostripain. This is advantageous for many applications because the collagenase mixture can be used without the need for precise mixing ratios. At the same time, this embodiment of the method of the present invention can be carried out in a simple and material-saving manner. In this context, it is further preferred that the stationary phase consists of polypropylene glycol. Therefore, a method of the present invention is also preferred in which the purified enzymes are obtained as a mixture of collagenase I and collagenase II in one fraction and as the neutral protease and clostripain separately in two additional fractions.

[0028] Particularly preferred are methods according to the present invention, characterized in that in hydrophobic interaction chromatography, the stationary phase comprises butyl-Sepharose and at least one aqueous solution containing at least one salt selected from the group consisting of ammonium sulfate and potassium chloride is used as the mobile phase. Very particularly preferred are methods according to the present invention, characterized in that in hydrophobic interaction chromatography, the stationary phase comprises butyl-Sepharose and at least one aqueous solution containing ammonium sulfate is used as the mobile phase. Most preferred are methods according to the present invention, characterized in that in hydrophobic interaction chromatography, the stationary phase comprises butyl-Sepharose and at least one aqueous solution containing potassium chloride is used as the mobile phase. These embodiments allow the four enzymes collagenase I, collagenase II, neutral protease, and clostripain to be obtained separately from each other in four distinct fractions. For these embodiments, the use of a stationary phase comprising butyl-Sepharose is particularly preferred. Most preferably, the stationary phase consists of butyl-Sepharose.

[0029] Thus, by using this method, it is possible to both separate collagenase, neutral protease, and clostripain from each other in addition to purifying the enzymes, and to separate two different types of collagenase (type I and type II) from each other in addition to separating neutral protease and clostripain, all in a one-step process.

[0030] The concentrations of ammonium sulfate and potassium chloride in the mobile phase affect the hydrophobic interaction and therefore the binding properties to the respective stationary phases. Those skilled in the art can easily determine the amounts of these salts required for purification and separation by preliminary experiments. A preferred method according to the present invention is characterized in that at least one mobile phase is used for hydrophobic interaction chromatography, with a molar concentration of ammonium sulfate in the range of 0.3 to 1.5 mol / L, preferably in the range of 0.5 to 1.0 mol / L. A preferred method according to the present invention is also characterized in that at least one mobile phase is used for hydrophobic interaction chromatography, with a molar concentration of potassium chloride in the range of 1.0 to 3.0 mol / L, preferably in the range of 1.5 to 2.5 mol / L.

[0031] The mobile phase used in the method according to the present invention may also contain additional components typically used in mobile phases, such as additional salts. Examples of such salts are sodium chloride and calcium chloride. To the extent that the mobile phase contains sodium chloride, it preferably contains sodium chloride in a molar concentration of 0.2 to 5 mol / L. If the mobile phase contains calcium chloride, it preferably contains calcium chloride in a molar concentration of up to 50 mmol / L, particularly preferably up to 15 mmol / L. Furthermore, the mobile phase may contain tris(hydroxymethyl)-aminomethane (Tris). To the extent that the mobile phase contains Tris, it preferably contains Tris in a molar concentration of up to 60 mmol / L, particularly preferably up to 30 mmol / L. In addition, the mobile phase may contain organic solvents. These are preferably fairly polar solvents. Alcohols, especially isopropanol, and / or polyols are particularly preferred. Among polyols, glycols are most preferred, and among these, glycol and propylene glycol are again most preferred. To the extent that the mobile phase contains an organic solvent, it preferably contains at most 50% by weight, preferably at most 40% by weight, particularly preferably at most 30% by weight of the organic solvent. The pH of the mobile phase is preferably in the range of pH 6.0 to 9.5.

[0032] The mixture of substances to be separated is preferably applied to the stationary phase using a so-called application buffer. The composition and properties of any application buffer used are the same as for the mobile phase.

[0033] The hydrophobic interaction chromatography is preferably carried out by step elution, gradient elution, or a combination of these two. Therefore, the method according to the present invention is particularly preferred, characterized in that the hydrophobic interaction chromatography is carried out by step elution, gradient elution, or a combination of these two. Step elution is particularly preferred for use on a production scale.

[0034] Further preferred is a method according to the present invention, characterized in that step elution, gradient elution, or a combination of the two is performed during hydrophobic interaction chromatography, and at least three elution steps are performed. Such a method is particularly well suited for separating collagenase I, collagenase II, or a mixture of collagenases from neutral proteases and clostripain. Particularly preferred is a method according to the present invention, characterized in that at least three elution steps are performed during hydrophobic interaction chromatography, and two collagenases, neutral proteases, and clostripain are separated from each other. Thus, the two collagenase types (type I and type II) are present in a common fraction, mixed therein, while the neutral proteases and clostripain are present in two additional fractions, separate from each other and from the collagenases. Particularly preferred is a method according to the present invention, characterized in that at least three elution steps are performed during hydrophobic interaction chromatography, and collagenase I, collagenase II, neutral proteases, and clostripain are separated from each other.

[0035] Also preferred is a method according to the present invention, characterized in that in hydrophobic interaction chromatography, at least three elution steps are carried out in the form of step elution, gradient elution, or a combination of these two, to separate two collagenases (type I and type II), a neutral protease, and clostripain from one another, or to separate collagenase I, collagenase II, a neutral protease, and clostripain from one another. In the latter case, the additional separation of the two collagenases is preferably carried out by an additional elution step, preferably by a linear gradient or in the form of an elution step. Thus, elution with at least three elution steps results in the purification and separation of collagenase, neutral protease, and clostripain in at least three useful fractions.

[0036] Also preferred are methods according to the invention in which gradient elution or a combination of gradient and step elution is performed during hydrophobic interaction chromatography, characterized in that at least three elution steps are performed. Particularly preferred are methods according to the invention in which two collagenases, a neutral protease, and clostripain are separated from one another, such that collagenase type I and collagenase type II are present mixed in a common fraction, and the neutral protease and clostripain are present in two additional fractions separate from one another and from the collagenase. Particularly preferred are methods according to the invention in which collagenase type I, collagenase type II, the neutral protease, and clostripain are separated from one another.

[0037] The first substep in hydrophobic interaction chromatography consists of applying the mixture of substances to be separated to the chromatography column, preferably using a so-called application buffer. The application buffer is usually a specially modified highly saline aqueous matrix in which the mixture of dissolved substances accumulates for purification and separation in the method according to the invention, especially in the context of processing from the culture supernatant of a Clostridium histolyticum bacterial culture. In the extremely hydrophilic medium of the application buffer, very strong hydrophobic interactions occur between the proteins and the stationary phase, which results in almost complete fixation of most proteins to the stationary phase.

[0038] As a second substep of hydrophobic interaction chromatography, at least one so-called washing step is preferably carried out before elution of the useful fraction containing the target protein, which in the method according to the present invention contains collagenase I, collagenase II, neutral protease, and / or clostripain enzymes. In this washing step, the chromatography column containing the protein bound to the stationary phase is washed, and any impurities that are fairly polar or hydrophilic and therefore not bound to the stationary phase, and often have a low molecular weight, are washed away and thus separated from the target protein. Aqueous buffer solutions containing a high salt content are also used as so-called washing buffers.

[0039] The composition of the application buffer and the wash buffer can be different, however, one and the same buffer solution can also be used as both the application buffer and the wash buffer.

[0040] In hydrophobic interaction chromatography, the washing step is followed by an elution step as a further substep. In particular with regard to the method according to the invention, this is a substep in which the useful fractions carrying the target protein are eluted from the column by successively adjusting 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 carried out isocratically, i.e., in steps with a constant composition of the mobile phase (step elution), as a gradient, i.e., with a continuously changing target composition of the mobile phase (gradient elution), or gradually as a combination of step and gradient elution.

[0041] In certain classes of hydrophobic interaction chromatography, one of the target proteins may exhibit a weaker hydrophobic interaction with the stationary phase than the remaining target proteins in the mixture, and therefore may not already be bound to the stationary phase with the same strength in the application and wash buffers. Therefore, in these cases, the wash buffer can, under certain circumstances, simultaneously function as the first elution buffer, thereby allowing the same substep of hydrophobic interaction chromatography to represent both the wash step and the first elution step in parallel. In this case, fairly polar or hydrophilic impurities, often small molecules, are washed away first in the same substep, and then the first useful fraction is eluted with the less strongly bound target protein. In such classes, the volume of the first elution buffer used can control the quality of separation of the target protein in question from the remaining target proteins in the mixture, regardless of whether it also functions as a wash buffer. In this case, the amount of mobile phase used is often expressed as a column volume (CV).

[0042] Therefore, in the method according to the present invention, since clostripain exhibits a weaker hydrophobic interaction with the stationary phase than the remaining target proteins (collagenase I, collagenase II, and neutral protease) of the substance mixture, the content of clostripain in the corresponding eluted useful fractions (elution fractions) can be controlled by the volume of the first elution buffer (clostripain elution buffer) used. Thus, a larger volume of the first elution buffer results in complete or almost complete clostripain elution in a separate pure clostripain fraction. On the other hand, if a smaller volume of the first elution buffer is used, the ratio of the amount of clostripain in each elution fraction will shift. This then leads to the fact that not all or almost all of the clostripain is eluted in a separate fraction, but the eluted clostripain is distributed both in the separate pure clostripain fraction and in each subsequent collagenase fraction. Therefore, the amount of the first elution buffer can be used to select whether clostripain should be almost completely eluted into a separate, pure clostripain fraction, or whether a portion of clostripain should be eluted as a component of each subsequent collagenase fraction and therefore also present in one fraction together with collagenase. In the latter case, selection of an appropriate salt concentration of the mobile phase during collagenase elution can control whether the second portion of clostripain accumulates in a common elution fraction with both collagenases (type I and type II) (column material: polypropylene glycol or butyl-Sepharose, without additional separation of the two collagenase types) or in a common elution fraction with collagenase II only (column material: butyl-Sepharose, with additional separation of the two collagenase types). The required volume of mobile phase (of the first elution buffer) that leads to almost complete elution of clostripain in a separate pure clostripain fraction, and therefore almost complete separation of clostripain from collagenase and neutral protease, depends on the specific combination of various factors (stationary phase, mobile phase, column dimensions, etc.).However, preferred for separating clostripain according to the present 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, such that clostripain is usually completely or almost completely eluted from the column.

[0043] This method allows obtaining collagenase I, collagenase II, and mixtures of these two types of collagenase with a purity of at least 80%, preferably at least 90%, as determined by analytical anion exchange chromatography (e.g., at room temperature using a GE Healthcare MonoQ column with Tris buffer as the mobile phase). Therefore, methods according to the present invention are preferred in which collagenase I, collagenase II, or mixtures of these collagenases are obtained with a purity of at least 80%, preferably at least 90%, as determined as described above. Neutral proteases can be obtained by the methods according to the present invention with a purity of at least 70%, preferably at least 80%, as determined by SDS-PAGE (according to Laemmli, UK; Nature 227:680-685, 1970). Therefore, methods according to the present invention are also preferred, in which the neutral protease is obtained with a purity of at least 70%, preferably at least 80% (determined as set forth above). Clostripain can be obtained by methods according to the present invention with a purity of at least 60%, preferably at least 70%, as determined by SDS-PAGE (according to Laemmli, UK; Nature 227:680-685, 1970). Therefore, methods according to the present invention are also preferred, in which the clostripain is obtained with a purity of at least 60%, preferably at least 70% (determined as set forth above). All of these purity values ​​are purity levels that meet the requirements for the use of these enzymes for most biochemical and medical purposes. Most preferred is a method according to the invention in which collagenase I, collagenase II, or a mixture of these collagenases is obtained with a purity of at least 80% (determined as set forth above), neutral protease with a purity of at least 70% (determined as set forth above), and clostripain with a purity of at least 60% (determined as set forth above).Most preferred is a method according to the invention in which collagenase I, collagenase II, or a mixture of these collagenases is obtained with a purity of at least 90% (determined as set forth above), neutral protease with a purity of at least 80% (determined as set forth above), and clostripain with a purity of at least 70% (determined as set forth above).

[0044] These enzymes can also be obtained in this purity using a one-step method.

[0045] Furthermore, a process according to the invention is preferred, characterized in that a linear flow rate of 100 to 300 cm / h, in particular 150 to 250 cm / h, is used in the hydrophobic interaction chromatography, as these flow rates result in optimal purification and separation of the enzyme.

[0046] Preferably, the method according to the present invention does not include a gel filtration step and / or a protein precipitation step. Gel filtration is a very time-consuming method, which therefore entails high costs and increases the risk of autolysis of the enzyme to be separated. Protein precipitation steps, such as ammonium sulfate precipitation, can lead to undesirable structural changes of the protein. Furthermore, protein precipitation does not allow the enzyme to be obtained with the high purity provided by the present method.

[0047] Preferably, at least one enzyme purified using the method according to the present invention is used for pharmaceutical and / or biochemical purposes. Pharmaceutical purposes include any use of one or more of said enzymes as active pharmaceutical ingredients, without any restrictions regarding indication, dosage form / preparation, or mode of application. Furthermore, said enzymes may be used for biochemical purposes, such as in vitro cell isolation.

[0048] Also preferably, the at least one enzyme purified using the method according to the invention is used for cosmetic purposes. Thus, the use of the enzyme(s) also extends to purely cosmetic uses, i.e. cosmetic uses as distinguished from therapeutic uses.

[0049] The present invention also includes the enzymes themselves obtained by the methods according to the present invention. In addition, the present invention also includes compositions comprising at least one enzyme obtained by the methods according to the present invention. The present invention further relates to the use of enzymes purified using the methods according to the present invention for pharmaceutical, cosmetic, and / or biochemical purposes.

[0050] Description of the Preferred Method According to the Invention After culturing Clostridium histolyticum in a suitable fermentation medium, cells and other insoluble components are separated from the culture supernatant, for example, by centrifugation and / or filtration. The culture supernatant containing collagenase I, collagenase II, neutral protease, and clostripain enzymes can be concentrated in the usual manner before purification and separation of these proteins by hydrophobic interaction chromatography.

[0051] Hydrophobic interaction chromatography (HIC) is then performed to purify and separate the enzyme from the culture supernatant. For this purpose, the cell-free, if necessary appropriately concentrated, culture supernatant is applied to a chromatography column packed with, for example, polypropylene glycol (PPG) and / or butyl-Sepharose, where the enzyme, among other components, binds to the column material. After washing away unbound molecules, elution of initially bound components, including the target protein, is carried out by step elution, gradient elution, or a combination of the two, at a linear flow rate of 100-300 cm / h in a buffered system with a pH range of 6.0-9.5, with the salt content being successively reduced. Protein elution is preferably carried out in three or more elution steps. Here, three useful fractions can be obtained: one containing collagenase (type I and type II) together, another containing neutral protease, and a third containing clostripain (separation on PPG or butyl-Sepharose). Chromatography on butyl-Sepharose allows for the additional separation of the two collagenases (type I and type II) from each other. This separation is then preferably carried out using a combination of step and gradient elution in at least three elution steps, or using a four-step elution. Thus, four useful fractions are obtained, one containing neutral protease and the other containing clostripain, as before. However, here, the collagenases are separated into a third fraction containing collagenase I and a separate fourth fraction containing collagenase II.

[0052] Here, the individual eluted fractions can be desalted and / or concentrated by conventional methods, such as a tangential flow filtration (TFF) step. The resulting material can then be lyophilized by similarly conventional methods, such as freeze-drying. Further purification steps can also follow, if necessary.

[0053] A flow diagram of the entire process is shown below in Scheme 1. Depending on the variant used, this leads to three or four different end products, which are then available for further use or further processing as desired, especially specific and defined mixtures of some of these end products.

[0054] [ka]

[0055] Thus, depending on the stationary and mobile phases used (type and amount of salt in the mobile phase), this method can achieve, for example:

[0056] By using PPG as the column material and ammonium sulfate (0.3-1.5 mol / L, especially 0.5-1.0 mol / L) as the salt, separation of collagenase, neutral protease, and clostripain is achieved; the two types of collagenase (type I and type II) are present mixed in a common fraction, while the neutral protease and clostripain are present in two additional fractions separate from each other and from collagenase. The use of butyl-Sepharose as the column material in combination with potassium chloride (1.0-3.0 mol / L, especially 1.5-2.5 mol / L) as the salt allows for additional separation of collagenase into collagenase I and collagenase II. The same is achieved when butyl-Sepharose is used as the column material in combination with ammonium sulfate (0.3-1.5 mol / L, especially 0.5-1.0 mol / L) as the salt. [Example]

[0057] All mobile phases, application buffers, washing buffers, and elution buffers used in the examples are aqueous solutions. The complete composition of the mobile phases used is given below in each case.

[0058] Example 1 A culture of Clostridium histolyticum was grown in liquid culture using an appropriate nutrient medium according to standard methods to the desired cell density. After cell separation by standard methods such as centrifugation and / or filtration, hydrophobic interaction chromatography according to the method of the present invention was performed. For this purpose, a chromatography column (bed height approximately 20 cm) packed with polypropylene glycol (PPG-600M, Tosoh Bioscience LLC) was equilibrated with a mobile phase (aqueous solution, 0.85 mol / L ammonium sulfate, 20 mmol / L Tris, 7 mmol / L CaCl2, 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. Elution of the target protein was carried out at a linear flow rate of 250 cm / h in three elution steps. The first useful fraction was obtained by isocratic elution using the aforementioned mobile phase and contained clostripain. The second fraction was obtained by isocratic elution with a different mobile phase (aqueous solution, 0.2 mol / L ammonium sulfate, 20 mmol / L Tris, 7 mmol / L CaCl, pH 7.5) and contained 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 / L Tris, 7 mmol / L CaCl, pH 7.5) and contained neutral proteases.

[0059] Figure 1 shows a chromatogram of the above-described purification and separation of cell-free concentrated culture supernatant by hydrophobic interaction chromatography with PPG-600M as the column material. The figure shows the three different product fractions with corresponding elution ranges. Clostripain elutes in subfractions F2 to F5, collagenase 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.

[0060] Figure 2 shows the analysis of the collagenase-informative fraction (corresponding to fraction F6 in Figure 1) after separation on PPG-600M using a MonoQ chromatogram. The quality obtained using various column loads in HIC chromatography is shown (loads measured by volume-specific PZ activity according to Wunsch E., Heidrich H.-G.; Z. Physiol. Chem. 333:149-151, 1963). For this purpose, samples of each collagenase-informative fraction were analyzed using a MonoQ column (column: MonoQ 5 / 20 GL, GE Healthcare; application buffer: aqueous solution, 10 mmol / L Tris, 2 mmol / L CaCl2, pH 7.5; elution buffer: aqueous solution, 10 mmol / L Tris, 2 mmol / L CaCl2, 1 mol / L NaCl, pH 7.5; gradient elution). Thus, both the purity and content of collagenase types, as well as the ratio of collagenases to each other, can be determined each time. As can be seen from the chromatograms, the obtained collagenase fractions contain only very few impurities in addition to the two collagenase types, regardless of the PPG column load. Therefore, the method is reproducible and robust. Values ​​of >90% were determined for the purity of the collagenase fractions (see Figures 2 and 3).

[0061] Because there is no meaningful activity assay currently available for collagenase I, the quality and quantity of collagenase was assessed using MonoQ analysis. As shown in Figure 2, no significant degradation products could be observed in the MonoQ analysis of the collagenase I fraction, and therefore the enzyme corresponds to the quality of commercially available products.

[0062] Figure 3 shows an SDS-PAGE of the collagenase-rich fraction (corresponding to fraction F6 in Figure 1) after separation with PPG-600M. This was performed according to the UK Laemmli protocol using 14% Tris-glycine gels (Anamed) and stained with Coomassie R-250 (Invitrogen) (Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4; Nature 227:680-685, 1970). The left lane, labeled M, plots marker proteins (Novex Mark 12, Invitrogen). The middle lane, labeled K, is the cell-free, concentrated culture supernatant before purification and separation. The right lane (K1) plots collagenase-rich fraction F6 from Figure 1, which contains collagenase I and collagenase II. Thus, the figure shows a direct comparison of collagenase purity before and after purification and separation on a PPG column. Although the collagenase protein was purified and separated using only one chromatography column, the purity of the collagenase beneficial fraction is >90%.

[0063] Figure 4 shows the corresponding SDS-PAGE of the neutral protease-rich fraction (corresponding to fraction F7 in Figure 1) after separation on the PPG-600M column. Marker proteins were again applied to the left lane labeled M. The center lane labeled K again represents the cell-free, concentrated culture supernatant before purification and separation. To one of the two right lanes labeled NPf, the neutral protease-rich fraction F7 from Figure 1 was applied immediately after purification and separation on the PPG column. One of the two right lanes labeled NPe shows the neutral protease end product previously obtained from F7 by desalting, concentration, and lyophilization, redissolved for comparative analysis. Figure 4 shows that the neutral protease also exhibits a high purity of well over 80% after purification and separation on the PPG column. This is significantly higher than the purity of neutral protease products currently available on the market.

[0064] Table 1 shows the relative yields of the enzyme activity after purification and isolation of the enzyme from the cell-free concentrated culture supernatant by hydrophobic interaction chromatography on PPG-600M as the column material. Values ​​of >90% were determined several times for the corresponding yield of collagenase II.

[0065] [Table 1]

[0066] A unique feature of the developed process is that the distribution of clostripain between the separate pure clostripain fractions (including subfractions F2 to F5 in Figure 1) and the collagenase fraction (fraction F6 in Figure 1) can be controlled by the number of column volumes (CV) of the first elution buffer (clostripain elution buffer) used to elute the chromatography column. Starting with approximately 12 CV, each volume leads to a yield of up to approximately 80% of total clostripain in the separate pure clostripain fraction, with corresponding losses to the waste fraction in the wash steps and only negligible clostripain content in the collagenase fraction. In contrast, however, each volume of approximately 4 CV leads to a distribution of approximately 40% of total clostripain in the separate pure clostripain fraction and approximately 40% in the subsequent collagenase fraction, again with corresponding losses to the waste fraction in the wash steps (see Tables 1 and 2).

[0067] [Table 2]

[0068] Example 2 A culture of Clostridium histolyticum was grown in liquid culture using an appropriate nutrient medium according to standard methods to the desired cell density. After cell separation by standard methods such as centrifugation and / or filtration, hydrophobic interaction chromatography according to the method of the present invention was performed. For this purpose, a chromatography column (20 cm bed height) packed with butyl Sepharose (Butyl Sepharose High Performance, abbreviated as Butyl Sepharose HP, GE Healthcare) was equilibrated with a mobile phase (aqueous solution, 2 mol / L KCl, 20 mmol / L Tris, 7 mmol / L CaCl2, 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. Elution of the target protein was performed at a linear flow rate of 250 cm / h. The subsequent second elution step was performed as a gradient over 20 CV with a 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, pH 9). The first useful fraction thus obtained contained clostripain. The second fraction contained collagenase II. The third fraction contained collagenase I. The fourth fraction was obtained by a third elution step by isocratic elution with 5 CV of additional mobile phase (aqueous solution, 25% (m / m) propylene glycol, 20 mmol Tris, 7 mmol CaCl, pH 9) and contained neutral protease.

[0069] Figure 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 the column material. Compared to Figure 1, an additional separation into collagenase I and collagenase II is shown, with the first main peak corresponding to collagenase II and the second main peak corresponding to collagenase I.

[0070] 6 shows a comparative MonoQ analysis of the collagenase-rich fraction after purification and separation of the cell-free concentrated culture supernatant by different variants of hydrophobic interaction chromatography. A shows the collagenase-rich fraction after separation on PPG-600M as column material (method variant 1). B shows the collagenase type II-rich fraction after separation on Butyl Sepharose HP as column material (method variant 2). C shows the collagenase type I-rich fraction after separation on Butyl Sepharose HP as column material (method variant 2). Thus, the quality of purity and, if necessary, separation are achieved in one method step, which requires three or more method steps in known methods.

[0071] Figure 7 shows an SDS-PAGE after silver staining (Argent Quick Silver Staining Kit, Anamed). Marker proteins were again applied in the left lane labeled M. Next to it, for direct comparison, are shown a currently commercially available "classical" collagenase purified through several chromatographic steps (middle lane labeled Ka) and a collagenase obtained by a variant of the one-step purification method according to the present invention (right lane labeled Kn). It can be seen from the figure that the purity of the collagenase obtained by the one-step method according to the present invention is at least as high as that of the "classical" collagenase currently available on the market. Thanks to the one-step method, the yield of ≥ 80% is also significantly higher than that of established methods.

Claims

1. 1. 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 at least one hydrophobic interaction chromatography as a method step, In the hydrophobic interaction chromatography, the stationary phase comprises cross-linked butyl agarose, and in the hydrophobic interaction chromatography, at least one aqueous solution containing at least one salt selected from the group consisting of ammonium sulfate and potassium chloride is used as the mobile phase.

2. 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 at least one hydrophobic interaction chromatography as a method step, In the hydrophobic interaction chromatography, the stationary phase comprises polypropylene glycol, and in the hydrophobic interaction chromatography, at least one aqueous solution containing at least one salt selected from the group consisting of ammonium sulfate and potassium chloride is used as the mobile phase.

3. 3. The method according to claim 1 or claim 2, wherein at least one aqueous solution containing ammonium sulfate is used as the mobile phase.

4. 4. The method of claim 1, 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.

5. 5. The method according to claim 1, wherein the mixture of substances is a culture supernatant of Clostridium histolyticum.

6. 6. The method according to claim 1, wherein in the hydrophobic interaction chromatography at least one mobile phase is used having a molar concentration of ammonium sulfate in the range of 0.3 to 1.5 mol / l.

7. 7. The method of claim 1, wherein the hydrophobic interaction chromatography is performed by step elution, gradient elution, or a combination of the two.

8. 8. The method according to claim 1, wherein step elution is performed in the hydrophobic interaction chromatography.

9. 9. The method of claim 8, wherein at least three elution steps are performed.

10. 10. The method of claim 9, wherein the two collagenases, neutral protease, and clostripain are separated from one another, such that collagenase type I and collagenase type II are present mixed in a common fraction, and neutral protease and clostripain are present in two further fractions separate from one another and from the collagenase.

11. 10. The method of claim 9, wherein collagenase type I, collagenase type II, neutral protease, and clostripain are separated from each other.

12. 2. The method of claim 1, wherein the cross-linked butyl agarose is present in the form of spherical particles having an average particle size of 20 to 150 μm.

13. The method described in claim 2, wherein the stationary phase is chemically modified with the polypropylene glycol.

14. 13. The method according to claim 1 or 12, wherein the cross-linked butyl agarose is a cross-linked agarose having 3-n-butoxy-2-hydroxypropyl residues, and the degree of cross-linking of the cross-linked agarose is 1 to 7%.

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