Optimised method of purifying recombinant protein of growth factor

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to method of obtaining purified recombinant GDF-5-like protein. Method includes destruction of E.coli cells by homogeniser of high pressure at pressure of destruction from 800 to 900 bar. Inclusion bodies are separated from destructed cells E.coli by centrifugation. Separated inclusion bodies are processed with denaturate buffer, which contains carbamide and L- arginine, for solubilisation. Solubilised monomer of recombinant GDF-5-like protein is purified by centrifugation and chromatography.

EFFECT: invention makes it possible to obtain purified recombinant GDF-5-like protein with high output.

7 cl, 10 dwg, 2 tbl, 5 ex

 

This invention relates to an improved method for the effective production in prokaryotes and purification of recombinant proteins growth factor. More specifically, the invention relates to modifications of the method, leading to a higher yield of protein, to a higher purity of the product and to improved industrial applicability of the above method.

Growth factors and Differentiation (GDF) are homodimeric cytokines, promoting cell proliferation/differentiation and tissue regeneration. Among all factors of growth/differentiation is most useful in a wide range of medical applications is a Factor of Growth/Differentiation 5 (GDF-5). Previously most successfully applied osteogenic properties of GDF-5, for example, when support for the local treatment of bone fractures. GDF-6 and GDF-7 are the most related to GDF-5 protein with similar biological functions and extremely high amino acid homology. Group GDF-5/-6/-7 conservative among mammals, but has no orthologues in invertebrates (Ducy and Karsenty 2000, Kidney Int. 57, 2207-2214).

In vivo, the representatives of this protein family is initially synthesized in the form of large protein precursors that are subsequently subjected to proteolytic degradation consisting of basic amino acid residues cluster at a distance V-140 amino acid residues from the C-end of the protein; thus, from the N-terminal predomina separated C-terminal Mature bioactive protein fragments. All Mature polypeptides are structurally similar and contain conservative bioactive domains containing six or seven canonical cysteines. Disulfide bridges between the amino acid residues create typical of that protein family of three-dimensional motif of the "cystine knot".

Previously it had been the expression of Mature GDF-5 in prokaryotic cells-hosts (see, e.g., Biochem. Biophys. Res. Commun., 204, pp.646-652, 1994). However, these proteins are difficult to obtain in pure form. When their expression in E. coli in large quantities, and the corresponding protein often consists of Monomeric and inactive protein with a molecular mass of 14 kDa, which accumulates in inclusion bodies. With the aim of obtaining bioactive dimeric growth factor (28 kDa), Monomeric protein of Taurus inclusion should be solubilisation, peeled and renaturierung with the formation of glycosilated with the typical structure of the cysteine site. This procedure is often called the "refolding".

Because of the extremely low solubility in aqueous solutions at pH values from 4 to 9, as well as other rare protein properties, methods of purification and refolding produced in prokaryotes GDF-5-like proteins need to include a few selected stages. For example, POSCO is ICU after refolding GDF-5-like proteins, usually adsorbed chromatographic media, it becomes apparent that the purification of the desired protein in large quantities cannot be carried out in accordance with standard protocols purification using aqueous chromatographic components. After refolding of the protein are applicable primary methods of treatment on the basis of organic solvents (such as, ortofena chromatography).

A recently developed method of obtaining and purification of recombinant GDF-5-like proteins disclosed in WO 96/33215. The method is based on the clearing before refolding Monomeric protein and contains the following essential steps:

1. Growing bacterial culture, the destruction of cells and the release of Taurus enable,

2. Processing of denaturing agents to obtain solubilizing monomer,

3. Separation by ion-exchange chromatography,

4. Sulfonation (phase sulfonation is not mandatory),

5. Separation by gel-filtration,

6. Refolding,

7. Division isoelectric precipitation,

8. Division obetovannoi chromatography.

Although the above-described method, in General, is applicable in this case, the first two stages of processing are some of the challenges that affect both the yield and purity of the target protein. Achievable output GDF-5-like protein considerable is lower than theoretically expected, mainly due to the partial degradation due to unusual turbidity/viscosity of the solution obtained by solubilization of protein from Taurus inclusion. Thus, it is obvious that these parameters and conditions of the method should be improved.

The objectives of this invention are to overcome the above problems and to optimize the yield and purity of recombinant GDF5-like proteins.

These objectives are solved by developing disclosed further improved methods for obtaining recombinant GDF5-like proteins in E. coli.

The most commonly used terms should be defined and shown to a detailed description of the invention:

Used herein, the term "domain cystine knot"means well-known, conservative, rich in cysteine amino acid stretch present in the Mature part of the protein superfamily of TGF-beta, such as human GDF-5, and generates three-dimensional protein structure, known as cystine knot. In this domain is important to the proper location relative to each other cysteine residues and, in order to preserve the biological activity is allowed only minor changes. It was demonstrated that the domain cystine knot itself is sufficient for biological activity of the protein (Schreuder et al. (2005), Biochem Biophys es Commun. 329, 1076-86). Consensus sequences for domains cystine knot is well known in this field. By definition, a specific protein domain here cystine knot begins with the first cysteine residue that is involved in cystine node of the corresponding protein, and ends at amino acid residue, following after the last cysteine involved in cystine node of the corresponding protein. For example, the domain cystine knot protein precursor of human GDF-5 (SEQ ID NO:1) consists of amino acid residues 400-501 (see also figure 1).

Used here, the term "GDF-5-like protein" means any naturally or artificially derived protein that contains a domain cystine knot, homologous amino acid residues, at least 60% of the 102 AK domain cystine knot of human GDF-5 (amino acids 400-501 sequence SEQ ID NO:1). This term includes proteins with similar biophysical properties belonging to the group of proteins GDF-5, GDF-6 and GDF-7 vertebrates or mammals, as well as their recombinant variants, since the latter demonstrate the above-mentioned percentage homology domain cystine knot of human GDF-5. Equal to 60 % of the limit value is well suited for separating members of a group of proteins GDF-5/-6/-7, as well as their variants from other proteins is in such as GDF and other BMP proteins. The comparison consists of 102 AK domains cystine knot of human GDF-5 and human GDF-7 (see figure 2) revealed a high degree of amino acid homology between these proteins. Human GDF-6 matches in 87 amino acid residues (85%), and human GDF-7 - 83 (81%) of amino acid residues from the domain cystine knot of human GDF-5. The corresponding domains of molecules GDF-5/-6/-7 from other vertebrates and mammals, certain at the moment, also show a high percentage of homology average of at least 75% (from 79% to 99%) when compared with human GDF-5. In contrast, GDF and BMP not belonging to the subgroup of GDF-5/-6/-7, show a much lower degree of homology, less than 60% (see figure 3).

The determination of the respective positions of amino acids in the corresponding amino acid sequences, and the calculation of the percent homology between them can be easily implemented using well-known algorithms for alignment, and by using these algorithms computer programs. For example, amino acid homology in this patent application (e.g., figure 2) was calculated for the aligned sequences, using freely-available program ClustalX (Version 1.81) left with the default settings, and the subsequent counting of identical residues manually. Led the kami default pairwise (slow-exact alignment are: the penalty for opening deletions: 10; the penalty for continued deletions: 0,10; matrix comparison of amino acids: gonnet on 250. The program ClustalX described in detail in the work Thompson.J.D., Gibson.T.J., Plewniak.F., Jeanmougin.F. and Higgins.D.G. (1997):

The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.

Nucleic Acids Research 24:4876-4882.

ClustalX is ported to Windows program multiple sequence alignment ClustalW, i.e. available from various sources, for example, is available by anonymous FTP addresses ftp-igbmc.u-strasbg.fr, ftp.emblheidelberg.de, ftp.ebi.ac.uk or available by download from the following web pages: http://www-igbmc.u-strasbg.fr/Biolnfo/. The program ClustalW algorithm described in detail in the work of Thompson, J. D., Higgins, D. G. and Gibson, T.J. (1994):

CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.

Nucleic Acids Research 22:4673-4680.

Used herein, the terms "variant", refers to the following polypetides:

a) to biologically active fragments of the protein,

b) to the biologically active protein structures that contain additional sequence beyond the original protein sequence,

C) to any combination of a) and b).

The terms "buffer for dilution or buffer to solubilize Taurus inclusion mean solutions which are used to solubilize Taurus on and denaturation of these proteins Taurus enabled.

The term "biological activity" on the mean activity of therapeutic compounds including, for example, GDF-5-like protein, measured by conventional in vitro analysis of alkaline phosphatase, for example, as described in example 5 or Takuwa et al. (1989), Am. J. Physiol. 257, E797-E803. Cell lines ATDC-5 or MCHT 1/26 suitable for use in this analysis of alkaline phosphatase.

Detailed description of the invention

Method for the production of recombinant GDF-5-like proteins and particularly recombinant human GDF-5 consists of the following primary stages: the stage of culturing E. coli; the collection phase of the biomass; the stage of cell disruption; stage collection/cleaning Taurus on and the stage of dissolution Taurus inclusion in denaturing conditions. Then denatured protein at later stages is subjected to purification and refolding as, for example, described in WO 96/33215.

The above-mentioned stage of cell disruption is usually done using high-pressure homogenizer. Then, usually, Taurus inclusion (TV) is collected by centrifugation and (optionally) several times washed. Thorough dissolution (solubilization) of protein from Taurus activate before the subsequent steps of purification is achieved by forming a suspension in the buffer to solubilize containing a high amount of urea denatured.

It is noteworthy that the solution to solubilize containing monomer and denatured protein from Taurus inclusion, which is extremely turbid and viscous, even after the preliminary stages of filtration or centrifugation. At the same time, is the time-dependent fragmentation Monomeric GDF-5 (see figure 5), a process that ultimately leads to the destruction/change of weight of a significant proportion of the monomers GDF-5. Less than 1.5 hours weight Mature Monomeric protein in solution for dissolution is significantly reduced from the original 14 kDa to about 10 kDa. This junk and fast degradation, apparently, is dependent on amino acid sequence/conformation of the protein and occurs exclusively on the stage solubilization Taurus inclusion. The process is time-dependent degradation especially interferes with protein production in large industrial scale as production periods due to increased volume is usually increased, which consequently leads to a significant reduction of the yield and purity of the GDF-5-like protein obtained at the end of the whole procedure cleanup.

In order to overcome the described problems, the inventors have conducted numerous studies using different approaches, which led, eventually, to a modification of the method of purification. These experiments included: variations of the procedure of destruction of cells; experiments on the inactivation of proteases in order to combat potential animations.com/proteolytic pollution; correction concentrations the critical value the components of the buffer to solubilize and/or washing; and the addition of various chemical compounds in the buffer to solubilize.

Taking into account the fact that different approaches to testing and inactivation expected, contributing to the observed degradation of proteins, proteolytic activity failed (see example 3: Chemical inhibition and thermal inactivation), the inventors have found that reducing fragmentation of proteins and higher output/purity of the protein can, however, be achieved by introducing either individually or (preferably) together, two important, related to the way of modifications. These modifications are specific embodiments of the disclosed invention and are to 1) adaptation of procedures for destruction of cells and 2) optimization of the composition of the buffer to solubilize.

They further illustrated in more detail:

1) Modification of cell disruption by homogenization at high pressure

It was found that an unusually high turbidity and the viscosity of the solution to solubilize (containing solubilization bullock enable) are detrimental to subsequent stages of treatment, GDF-5-like proteins and should be avoided. Taking into account the fact that no filtration, no additional steps of centrifugation before solubilization Taurus enable, can not solve the problem, it has been unexpectedly discovered is, that its solution can be found by highly selective modifications made to the destruction of the cell pressure. Taking into account the fact that the pressure normally varies within a wide range (for example, from 100 to 2000 bar) without dramatic consequences for solubilization Taurus inclusion, it is extremely important to limit the pressure in a narrow range during cleaning GDF-5-like proteins. More precisely, if the destruction of the applied pressure between 800-900 bar, it is much more transparent solution solubilizing Taurus on and increases the product yield after the first part of the method of purification of GDF-5-like proteins. In addition, the ratio of recombinant GDF-5 to the total protein was significantly improved at high pressure to fracture. Because the best point total processing time becomes smaller and, therefore, the time-dependent fragmentation is reduced. Pressure fracture above and below this range are harmful and lead to a significant reduction of the yield of protein (see, for example, Fig.6).

2) Modification of the composition of the buffer to solubilize

The following modifications of the components of the buffer to solubilize covered by this invention:

Urea/Supplement with L-arginine.

Although the deleterious effect of urea on the stability of the primary structure of growth factors and differentiation has not been previously op the San for a given level of technology, the inventors, it was found that the fragmentation of the GDF-5-like proteins does not occur if the urea is completely removed from all contact with cells of the inclusion fluids (e.g., buffers for washing and solubilization). However, removing the denaturing agent from the buffer to solubilize impossible because of the need to maintain the desired effect denaturation. Unfortunately, replacement of urea to guanidine hydrochloride as an alternative denaturing agent is not recommended for use in the equipment of industrial production due to the corrosive properties of guanidine salts (which in some cases can lead to the reduction of the economically viable resource of pipes and tanks). In addition, the guanidine hydrochloride is very expensive and can increase associated with the way costs.

Therefore, the inventors searched for an alternative way to resolve the above-mentioned related proteases, the collapse of GDF-5. Through careful experiments, it was found that the fragmentation of the GDF-5-like proteins can be resolved in the buffer to solubilize containing urea, if the mentioned solutions as a protective additive to Supplement L-arginine in a certain concentration.

In example 3,/7 and 8 demonstrated that the addition of L-arginine containing urea solutions at ensait or eliminates degradation of GDF-5, depending on the concentration. Degradation can be reduced by approximately 50 percent with buffers containing at least 100 mm L-arginine, and may be stopped when using buffers for dilution, containing 500 mm (or more) of L-arginine. Even low concentrations of L-arginine (for example, 1 mm L-arginine in the buffer A4 of example 3) demonstrate detected, inhibiting fragmentation effect.

The use of L-arginine has several advantages when used as an additional ingredient in urea-containing buffer to solubilize Taurus inclusion with GDF-5-like proteins. First, L-arginine is a relatively inexpensive chemical and economic efficiency of the method of purification protein persists despite the addition of this substance. Secondly, the combination of urea and L-arginine significantly less corrosion than denaturing solution with guanidine hydrochloride. Third, L-arginine is more environmentally friendly chemical compound compared with the salts of guanidine, which require special disposal. This advantage makes the invention particularly useful for industrial installations consisting of metal devices. Moreover, L-arginine can easily be removed in the cleaning process using a simple phase diafiltration, for example, directly after solubili the purpose Taurus inclusion. This is particularly important because the proposed addition of L-arginine to the buffer to solubilize interfere with the subsequent binding of GDF-5-like proteins on ionoobmennoi chromatographic column (see example 4). Holding diafiltration and ion-exchange chromatography simplified if after solubilization Taurus inclusion in order to remove high molecular weight contaminants such as cell debris, apply (if necessary) additional cleaning steps (e.g., centrifugation, three-dimensional filtering and/or sterilizing filtration). Possible parameters of pore sizes for three-dimensional filtering is 0.1 to 0.7 μm, for sterilizing filtration, for example, 0.22 μm.

Thus, in accordance with the preferred embodiment of the invention and in order to prevent the protein fragmentation/degradation, the buffer to solubilize Taurus inclusion with GDF-5-like proteins must contain L-arginine. The preferred concentration of this additive is in the range from 100 to 1000 mm L-arginine in the buffer to solubilize according to the invention. The most preferred concentration is equal to 400-500 mm L-arginine. However, it is also possible to use higher concentrations of L-arginine (for example, up to 2000 mm), which can be useful in the case of extremely long periods of incubation/processing.

Buffer to solubilize from which briteney characteristic of the content of urea as a denaturing agent at a concentration of from 2 to 10 M Preferably, if the concentration of urea is in the range from 4 M to 8 M, Most preferred is a buffer to solubilize containing 6 M urea.

Other parts of the invention are further modifications of the above-mentioned buffer to solubilize having less strong, but, nevertheless, a significant impact on the performance of the method.

pH:

In accordance with the method of purification of recombinant GDF-5 disclosed in WO 1996/033215, pH 8.3, described as suitable for buffer to solubilize GDF-5-like proteins. However, the currently detected (see also example 3 /7)that the use of buffers to solubilize higher pH values in the range of from 9.0 to 11.0 reduces degradation and increases the amount of total protein obtained in the purification process. This fact can be explained by the profile of pH-dependent solubility of GDF-5, which is shown in Fig.9. Its low solubility at pH of 8.3, but significantly increased at higher pH values. Thus, the pH in the range between 9.0 and 11.0 is also useful for buffers to solubilize according to the invention.

The chelators:

It also can be customized concentration of chelators in the buffer to solubilize. The chelators are used to secure bind metals such as mercury, arsenic and lead. Nai is more commonly used synthetic chelator is EDTA, used in the solubilization buffers according to the invention (for example, in the form of Na2EDTA or Na3EDTA). According to the experiments described in example 3, it is advantageous to increase the concentration of chelators was originally described from 1 mmol/liter (see WO/1996/033215) to 5-100 mmol/l, preferably up to 5-50 mmol/L.

The most preferred buffer to solubilize contain the following components: 20 mm Tris-HCl

6 M urea

64 mm DTT

500 mm L-arginine

5 mm Na3EDTA

Major modifications of the method according to the invention shown in figure 10. It should be noted, in the form of precautions that the proposed treatment scheme represents a preferred embodiment of the invention, but the invention is in no way limited to this order or the number of processing steps (especially steps 5 through 9 to figure 10). Single stages can be omitted, replaced or supplemented with other cleaning methods, provided that, if the entire cleaning procedure includes the following primary steps: 1. bacterial cell culture (preferred bacterial host is E. coli, particularly preferred strains owners are W3110 and D1210), 2. the destruction of the cells 3. the release of Taurus on and 4. solubilization Taurus enabled.

The open invention was confirmed by the example of recombinant GDF-5 in the quality of the ve test substance. However, due to unusually high sequence homology (see figure 2) cleaning methods can also be used to clean other GDF-5-like proteins. The term "GDF-5 protein" includes functionally similar proteins belonging to the group of proteins of vertebrates consisting of GDF-5, GDF-6 and GDF-7, as well as their recombinant variants. A common feature of all GDF-5-like proteins is the presence of biologically active domain cystine knot with amino acid homology equal to at least 60% of 102 amino acids of domain cystine knot of human GDF-5, and which is sufficient for the biological function of the protein. As can be seen in figure 3, is equal to 60% preferred limit value separates the group members, GDF-5/-6/-7 from more distant GDF proteins and BMP. Particularly preferred proteins demonstrate the amino acid homology is equal to at least 75%, 80%, or 90% of the 102 AK domain cystine knot of human GDF-5.

Non-limiting examples of GDF-5-like proteins in vertebrates and mammals are precursors and Mature proteins of human GDF-5 (disclosed as MR in WO 95/04819 and as a human GDF-5 in Them et al. 1994, Btochem. Biophys Res. Commun. 204, 646-652), recombinant human GDF-5/MR (WO 96/33215), MR Arg (WO 97/06254); high molecular weight human MP52s (WO 97/04095), CDMP-1 (WO 96/14335), mouse (Mus musculus) GDF-5 (US 5,801,014), rabbit (Oryctolagus cuniculus) GDF-5 (Sanyal et al 2000, Mol Biotechnol. 16, 203-210), chicken (Gallus gallus) GDF-5 (NCBI accession no. NP_989669), GDF-5 clawed frogs (Xenopus laevis)(NCBI accession no. AAT99303), Monomeric GDF-5 (WO 01/11041 and WO 99/61611), human GDF-6/BMP-13 (US 5,658,882), mouse GDF-6 (NCBI accession no NP_038554), GDF-6/CDMP-2 (WO96/14335), human GDF-7/BMP-12 (US 5,658,882), mouse GDF-7 (NCBI accession no AAP97721), GDF-7/CDMP-3 (WO96/143335). The invention also covered GDF-5-like proteins having additional mutations, such as substitutions, insertions and deletions, provided that these additional mutations do not disrupt the fully biological activity of the protein. Some of the preferred options are the mutant GDF-5-like proteins with improved biological activity. In which, for example, one or more residues normally present in the protein is the precursor of human GDF-5 (see figure 1) are substituted by other amino acids: arginine at position 438 predecessor of human GDF-5 is replaced by glycine, alanine, valine, leucine, isoleucine, methionine or asparagine; and/or serine 439 is replaced with aspartic acid, glutamic acid, glycine, leucine, or isoleucine; and/or asparagine 445 is replaced by a serine or a threonine. In another high-level mutant, methionine 453 and/or methionine 456 is replaced by alanine, valine or isoleucine. Also of special interest mutants, in which the leucine 441 is replaced by Proline.

The biological activity of GDF-5-p is such proteins can be easily determined using recognized test systems. The most suitable and preferred is generally accepted in vitro test known as the analysis of alkaline phosphatase (Takuwa et al. 1989, Am. J. Physiol. 257, E797-E803), which is also described in example 5. GDF-5-like proteins show increased activity of alkaline phosphatase, for example, ROB-C26 cells (Yamaguchi et al. 1991, Calcif. Tissue Int. 49, 221-225) as described in WO 95/04819 in embryonic ATDC5 cells (Riken Gene Bank, ROB 0565), in murine stromal cells MNT-1/26 and HPDL cells, as shown in the work of Nakamura et al. 2003, J Periodontal Res. 38, 597-605.

The following limitiruyuschie examples together with figures and protocols sequences are intended to further illustrate the invention.

Sequence:

SEQ ID NO:1 is the protein sequence of the precursor of human GDF-5.

SEQ ID NO:2 is the DNA sequence of the precursor of human GDF-5.

SEQ ID NO:3 is the protein sequence of the Mature human GDF-5, consisting of 120 amino acid residues. Recombinante-derived proteins may also consist of 119 amino acid residues, and to begin, therefore, with the second amino acid (Proline) sequence SEQ ID NO:3.

SEQ ID NO:4 is the protein sequence of 120 amino acid residues of Monomeric Mature human GDF-5. A protein may consist of 119 amino acids, and to begin, therefore, with the second the second amino acid (Proline) sequence SEQ ID NO:4.

Figures

Figure 1 shows additional features of the protein precursor of human GDF-5 in accordance with the sequence SEQ ID NO:1:

amino acid residues 001-381 Pres prodomain (bold)

amino acid residues 001-027 - signal peptide (underlined bold font)

amino acid residues 382-501 - a fragment of the Mature protein

amino acid residues 400-501 domain cystine site (underlined font).

Figure 2 shows a comparison of the 102 amino acids of domain cystine knot of human GDF-5 (SEQ ID NO:1), human GDF-6 (sequence 2 from patent US 5,658,882) and human GDF-7 (sequence 26 from patent US 5,658,882). Amino acid residues that are identical in all three molecules is selected.

Figure 3 shows a table homology sequences of domains cystine knot several known proteins BMP and GDF domain cystine knot of human GDF-5.

Figure 4 shows a map of plasmid for expression of recombinant Mature human GDF-5 as described in example 1 and (in more detail) in WO 1996/033215.

Figure 5 shows the LTO-PAG electrophoresis showing the time-dependent fragmentation of recombinant Mature GDF-5 during solubilization buffer to solubilize (8 M urea, 20 mm Tris, 10 mm DTT, 1 mm Na2EDTA, pH 8.3). Monomeric GDF-5 is reduced from 14 kDa to 10 kDa (fragm the t). Fragmentation is almost complete after three hours solubilization.

Figure 6 shows the LTO-PAG electrophoresis showing the effect of modification of the pressure cell disruption on the fragmentation of the protein, the yield and purity in accordance with example 2. In this group of experiments, the bursting pressure is equal to 560 bar (top picture) is compared with the bursting pressure equal to 850 bar (bottom picture). Higher pressure equals 860 bar, leads to a significant reduction in the fragmentation of proteins and to higher yield/purity of the protein.

7 and 8 shows the LTO-PAG electrophoresis showing the effects of different buffers to solubilize the fragmentation Monomeric GDF-5, dissolved in the buffer to solubilize. The compositions of the buffers shown in example 3.

Figure 9 shows the profile of the dependence of the solubility of the Mature GDF-5 from pH.

Figure 10 shows a modification of the method of obtaining GDF-5 in accordance with the invention.

Information confirming the possibility of carrying out the invention

Example 1. Production and purification of recombinant GDF-5

(1) Constructing expressing vector and transformation of E. coli

System construction of plasmid vectors to obtain the Mature recombinant human GDF-5 (amino acid residues 1 to 119 of the sequence Seq ID No. 3) and the transformation strain of the host E. coi W3110 (W3110M) was performed, as described in example 1 of WO 1996/033215.

(2) Culturing in E. coli.

E. coli expressing the protein of the invention, precultural in a modified environment SOC (20 g/l of Bacteriophora, 5 g/l Backdragging extract, 0.5 g/l NaCl, 2,03 g/l MgCl2×6H2O, 3.6 g/l glucose). 100 ml of bacterial suspension was inoculable 5 litres medium for producing (5 g/l of Bacteriophora, 4.3 g/l Citric acid, 4,675 g/l K2HPO4, 1,275 g/l KH2PO4, 0,865 g/l NaCl, 100 mg/l FeSO4×7H2O, 1 mg/l CuSO4×5H2O, 0.5 mg/l MnSO4·×nH2O, 2 mg/l CaCl2×2H2O, 0,225 mg/l Na2B4O7×10H2O, 0.1 mg/l (NH4)6Mo7O24×4H2O, 2.25 mg/l ZnSO4×7H2O, 6 mg/l CoCl2×6H2O, 2.2 g/l MgSO4×7H2O 5.0 mg/l Thiamine HCl, 3 g/l glucose), which was cultured in a 10-liter fermentor with aeration-stirring, and then, upon reaching the early logarithmic growth phase (OD 550=5.0)was added isopropyl-beta-D-thio galactopyranoside to a final concentration of 1 mm and continued cultivation to achieve the OD 550=150. The cultivation temperature was maintained at 32°C, and pH was maintained with the help of ammonia at the level of 7.15. In order to prevent the reduction of dissolved oxygen concentration, stirring was carried out at high speed, in order to keep the level to which ncentratio dissolved oxygen equal to 50% of its content in the atmosphere. Continued cultivation with the addition of 50% glucose solution up to a level of 0.2% to obtain a high density of cells, indicating a sharp increase in the concentration of dissolved oxygen.

(3) Preparation of Taurus inclusion of E. coli

The culture fluid obtained by the method described above was ofcentrifugal to collect cells, which then resuspendable in 25 mm Tris-HCl containing 10 mm ethylenediaminetetraacetic acid (pH 7.3). Cells were destroyed by the high-pressure homogenizer and centrifuged again to collect containing calf enable precipitate.

(4) Washing and solubilization Taurus inclusion of E. coli

After 3 times washing (for example, using 1% Triton X-100) bullock include E. coli was centrifuged at 3000 g for 30 minutes at 4°C, and then the resulting precipitate was solubilizers ultrasound in the buffer to solubilize (20 mm Tris-HCl buffer, 8 M urea, 10 mm DTT, and 1 mm Na2EDTA, pH 8,3). In connection with the observed partial degradation of the protein GDF-5 of Taurus inclusion in containing urea buffers (see figure 5) were also tested various additional buffer to solubilize described in example 3.

(5) Preparation of monomers

The solubilized solution was centrifuged at 20000 g for 30 minutes at 4°C and the resulting supernatant was collected. The obtained supernatant was applied to a column of SP-Sepharose FF (harmacia AB), equilibrated with buffer containing 20 mm Tris-HCl, pH 8.3, 6 M urea and 1 mm EDTA, and then, after washing with the same solution, the search was suirable the same solution containing 0.5 M NaCl. Protein in the eluate was sulfurously by adding Na2SO3and Na2S4O6to the final concentrations respectively 111 mm and 13 mm, and incubated at 4°C and for 15 hours. Sulfonated solution was subjected to gel filtration on a column of Sephacryl S-200 HR (Pharmacia AB), equilibrated with buffer containing 20 mm Tris-HCl, pH 8.3, 6 M urea, 0.2 M NaCl, and 1 mm EDTA to obtain purified, sulfonated monomers of the protein of the invention.

(6) the Refolding

The solution from sulphonated monomers were added to nine volumes of buffer containing 50 mm sodium salt of glycine, pH of 9.8, 0.2 M NaCl, 16 mm CHAPS, 5 mm EDTA, 2 mm GSH (reduced form of glutathione) and 1 mm GSSG (oxidized form of glutathione) with stirring and then were incubated 24 hours at 4°C for oxidation and refolding of the protein of the invention.

(7) Preparation of homodimeric

The solution for refolding was diluted in the same volume of purified water and then adding 6 N NaCl adjust the pH to about 7.4 and subjected to isoelectric precipitation. The precipitate, collected by centrifugation at 3000 g for 20 minutes, was solubilizers in a solution of 30% acetonitrile containing 0.1% TFA. The solution was diluted with the same amount the purified water and was applied to a column RESOURCE RPC (Pharmacia AB) to obetovannoi HPLC pre-equilibrated with 25% acetonitrile, containing 0.05% TFA and then suirable linear gradient of 25-45% acetonitrile containing 0.05% TFA. The eluate was monitored by absorption at 280 nm. Fractions purified homodimeric protein was collected and liofilizirovanny using a SpeedVac Concentrator (Servant Co.). Optionally, the purified protein was subjected to end-stage ultra/diafiltration.

Example 2. Option I - modification of the pressure cell disruption

There have been several experiments with different pressure cell disruption, in order to evaluate the effect of cell disruption to release/degradation of protein purity and filterability, Biomass after each cultivation resuspendable in the buffer for homogenization (25 mm Tris, 10 mm Na2EDTA, pH 7.3), homogenized and stirred for 30 to 60 minutes using a magnetic stirrer. Next, the suspension of biomass destroyed three times in the high-pressure homogenizer at different pressures destruction. Received bullock enable washed with wash buffer (20 mm Tris, 5 mm Na2EDTA pH 8,3) and kept at <-70°C. After thawing overnight at +4°C, bullock inclusion was dissolved in pre-cooled buffer to solubilize containing 6 M urea and 0.5 M L-arginine, homogenized and again mixed with a magnetic stirrer for 30 to 60 minutes. After this, the solution Taurus enable centrifuged to use the e for 30 minutes at 10°C, when dynamic load 10000 g (=7500 rpm). The supernatant decantation to separate Taurus inclusion from the insoluble components were filtered through volume filter (CUNO Zeta Plus BC0030A90ZA08A). Then, the filtrate was filtered again through a sterilizing filter (Nalgene Bottle Top Filter 0.2 µm). The sterile filtrate was concentrated and definitavely against cation exchange buffer A (6 M urea, 20 mm Tris, 1 mm Na2EDTA, 50 mm NaCl, 10 mm DTT, pH 8,3) before loading them into the cation exchange column. The test samples obtained at different stages, were analyzed using known analytical test methods, such as LTO-SDS page electrophoresis, the color of the dye Coomassie-Brilliant-Blue, and based on ELISA methods for the determination of proteins of E. coli.

The results of this study (see Fig.6) show that significant improvement in the primary cleaning method can be achieved if the destruction of the cells is carried out at a pressure fracture between 800 and 900 bar. Thus, we obtain the bullock include improved quality, which leads to a higher ratio of recombinant GDF-5 to the total protein (for example, 57% at 850 bar, against 35% at 560 bar), and reduction of proteins of E. coli in the final product (for example, <30 µg/mg at 850 bar, against >50 μg/mg at 560 bar). These improvements are also useful for filterability. The required area of the filter DL the production scale can be reduced (for example, from a theoretical 2.6 m2at 560 bar, to <1 m2at 850 bar) on a large scale. This leads to a reduction in the time method, reduced fragmentation of the protein and reducing associated costs in obtaining recombinant GDF-5.

Example 3. Option II - Solubilization Taurus inclusion

In order to prevent degradation of GDF-5 and similar proteins, the standard phase solubilization Taurus enable such as that described in example 1, modified in various aspects. Efforts were focused on experiments to identify/inhibition potential proteolytic activity, as well as on amendments to the composition buffer for solubilization as described in example 1 (e.g., pH, urea, Na2EDTA and DTT, guanidine HCl, amino acids such as L-arginine).

(3.1) experiments on the inhibition of proteases

(3.1.1) Chemical inhibition

In this series of experiments used a cocktail protease inhibitors. In the subgroup of Taurus include additional resuspendable for 20 minutes in 25% HCl (pH 2,7) for the inactivation of proteases associated with the outer cell wall. After 3 times washing, 8 g of Taurus include with recombinant human GDF-5 was dissolved in 50 ml of a standard buffer to solubilize containing 8 M urea. 2 tablets containing a mixture of protease inhibitors (Roche Diagnostics Protease Inhibitor Cocktail Tablets at. No. 11 697 498 001), was added and thoroughly mixed with a solution Taurus inclusion. After 1.5 h and 3 h of incubation at room temperature, samples were centrifuged and analyzed. Recombinant GDF-5 was found in a significantly degraded in all groups, demonstrating that chemical inhibition of protein degradation using HCl and protease inhibitors are not effective.

(3.1.2) Thermal inactivation

After 3 times washing 15 g Taurus inclusion with recombinant human GDF-5 was dissolved in 100 ml of buffer containing 10 mm Na2EDTA, 25 mm Tris (pH 7.3). Heat inactivation was performed using incubation at 65°C for various periods of time (from 20 min to 2 hours). Then, the samples were subjected to standard phase solubilization described in example 1. Results: despite thermal inactivation of protease, recombinant GDF-5 was degraded in all these samples.

(2) Correction of compositions of buffers to solubilize.

Attempts to reduce fragmentation of GDF-5-like proteins by modification of the buffer used to solubilize were successful. Some of the tested buffers to solubilize are listed below:

Buffers with urea:

Standard: 8 M urea, 20 mm Tris, 10 mm DTT 1 mm Na2EDTA, pH 8,3

The buffer U1: 8 M urea, 20 mm Tris, 64 mm DTT, 50 mm Na2EDTA, pH 8,3

The buffer U2: 6 M urea, 20 mm Tris 64 mm DTT, 50 mm Na2EDTA, pH 8,3

The buffer U3: 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8,3

Buffer U4: 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA 50 mm NaCl, pH 8,3

The buffer U5: 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 9,5

Buffers with L-arginine:

Buffer A1: 100 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8,3

Buffer A2: 30 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, rn,3

Buffer A3: 10 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8,3

Buffer A4: 1 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8,3

The A5 buffer: 200 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8,3

Buffer A6: 100 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 9,5 Buffer A7: 500 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 9.5 Buffer A8: 500 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8.3 Buffer A9: 300 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8.3

Buffer a10: 400 mm arginine, 6 M urea, 20 mm Tris, 64 mm DTT, 5 mm Na2EDTA, pH 8.3

To verify degradation, 0.1 g Taurus inclusion with GDF-5 was mixed with 0.9 ml of buffer to solubilize. Degradation was checked after 4-5 hours of incubation Taurus include dissolved in buffer to solubilize. The results were analyzed using LTO-PAG electrophoresis and subsequent staining Coomassie Brilliant Blue.

Results: Among others, were achieved following negative resultsthe tests on degradation:

- Na2EDTA: increasing the concentration from 1 to 5-50 mm leads to a slight decrease in the degradation

- pH: change from 8.3 to higher values (between 9.0 and 11.0 in) reduce degradation and increase in total protein. For example, an increase in pH from 8.3 to 9.5 in the buffer for solubilization (see, for example, buffers A7 and A8 7) improved and the total amount of protein and the degree of degradation. Even if bullock include dissolved in buffers with a small amount of L-arginine, after 5 hours of incubation at room temperature, at elevated pH of these buffers will still contain recombinant GDF-5 (see, for example, the buffer A6 7).

- DTT: Ineffective changes

- Amino acids, especially L-arginine: the Following initial results were obtained with buffers to solubilize containing from 0 to 100 mm L-arginine (pH 8.3):

used bufferthe incubation perioddegradation of recombinant GDF-5
Buffer without arginineincubation 0 halmost complete
incubation for 4 hours at room temperaturealmost complete
Buffer A1incubation 0 habout 50%
incubation for 4 hours at room temperatureabout 50%
Buffer A3 (30 mm L-Arg, pH 8,3)incubation 0 habout 50%
incubation for 4 hours at room temperaturealmost complete
Buffer A2(10 mm L-Arg, pH 8,3)incubation 0 habout 50%
incubation for 4 hours at room temperaturealmost complete
Buffer A4 (1 mm L-Apr, pH 8,3)incubation 0 hThe degradation is less than in the control sample
incubation for 4 hours at room temperaturealmost complete

In subsequent experiments used the increased concentration of L-arginine. The incubation time was increased to 5 hours. In this experiment tested the effect of (a) high concentration of L-arginine in the buffer for dilution and b) shift the pH to more alkaline conditions in the money is adalu recombinant GDF-5. Were the results:

used buffertotal protein, mg/mlrecombinant GDF-5, mg/mlthe ratio of the rivers. GDF-5 (REC. GDF-5/total protein)Degradation of rivers. GDF-5
Buffer without Arg (pH 8,3)of 5.92rivers. GDF-5 was not detected1)rivers. GDF-5 was not detected1)almost full2)
Buffer without Arg (pH 9,5)7,79rivers. GDF-5 was not detected1)rivers. GDF-5 was not detected1)almost full2)
Buffer A6 with 100 mm Arg (pH 9.3 in)there is a 10.033.3133%almost no
The A5 buffer with 200 mm Arg (pH 8,3)a 7.621,6422%about 50%2)
Buffer A9 with 300 mm Arg (pH 8,3)to 7.59334 44%almost no2)
Buffer a10 with 400 mm Arg (pH 8,3)7,293,5749%almost no2)
Buffer A7 with 500 mm Arg (pH 9,5)11,125,1747%almost no2)
Buffer A8 with 500 mm Arg (pH 8,3)7,684,7662%almost no2)
1)Values outside the calibration
2)The level of degradation was visually estimated using LTO-PAG electrophoresis

In accordance with the quantitative assessment, the level of degradation clearly decreases with increase in the concentration of arginine in the buffer for solubilization (table above and Fig.7 and 8). However, there remains some level of degradation of recombinant GDF-5 when using a 400 mm arginine (in the buffer for dilution) a10. Almost no (degraded) recombinant GDF-5 was not found in the granules Taurus inclusion, if judged visually on LTO-PAA is electrophoresis and quantification. Thus, the solubility of recombinant GDF-5 containing arginine buffers to solubilize good. The proportion of recombinant GDF-5 increased with increasing concentration of L-arginine in the buffer to solubilize. Best share recombinant GDF-5, which constitutes 62%, can be achieved by using arginine-containing buffer for dissolving A8 (500 mm L-arginine). The concentration is at least 500 mm L-arginine in the buffer to solubilize Taurus inclusion is considered to be optimal for obtaining the recombinant GDF-5 and similar proteins.

Example 4. The effect of L-arginine on ion-exchange chromatography

The objective of this experiment was to test the influence of the content of L-arginine in the buffer to solubilize Taurus enable the subsequent protein purification by ion exchange chromatography.

Various samples Taurus enable obtained by cultivation, after solubilization was applied to a cation exchange column consisting of chromatographic media SP Sepharose FF Packed in a column XK 16/20 (CV=28 ml). The tested buffers contained (among other described components) 8 M urea without L-arginine (standard buffer for solubilization) or 6 M urea, 500 mm L-arginine (modified buffer to solubilize).

Taurus inclusion was obtained by destruction of producing GDF-5 cells of E. coli using the homogenizer is a high pressure (three cycles, 850 bar) and then twice with washing. 10,37 g the Taurus inclusion was dissolved in 100 ml of modified buffer to solubilize (buffer with 6 M urea containing 0.5 M arginine). 80 ml of a solution with calves enable remained after centrifugation, dead-end filtering and sterilizing filtration Taurus inclusion. 40 ml of the filtered solution with calves inclusion was applied undiluted on a cation exchange column (approximately 172,4 mg total protein). Analyzed the content of total protein and recombinant GDF-5 in the eluate, hillshade and fractions both runs cation-exchange chromatography.

Results: due to the changed conductivity of the modified buffer to solubilize (18 mS/cm instead of 5 mS/cm standard buffer for solubilization), linking with the carrier cation-exchange column in the modified buffer is not full. With modified buffer to solubilize possibly only partial binding with the carrier cation-exchange column (protein yield 10% instead of 60%). Therefore, the necessary additional steps to replace the buffer (for example, diafiltrate through a 5 kDa cellulose membrane) prior to cation exchange chromatography.

Example 5. Testing of biological activity of alkaline phosphatase

The biological activity of GDF-5-like proteins and their colloidal compositions can be readily determined using recognized test systems. The most convenient and preferred is a regular analysis of alkaline phosphatase (Takuwa et al. 1989, Am. J. Physiol. 257, E797-E803). In this in vitro test system, the biological activity of GDF-5-like growth factors measured after cocultivation different concentrations of osteogenic protein/chondrogenesis cells. GDF-5 and similar proteins with osteo/chondrogianni potential to increase the expression of alkaline phosphatase in such cells, for example, in cells ATDC-5, ROB-C26 or MSNT-1/26. Alkaline phosphatase activity in these cell lysates determined by colorimetric analysis. The reaction is based on the hydrolysis of p-Nitrophenylphosphate (PNPP) and turning it into p-NITROPHENOL, which becomes visible under alkaline conditions, in the form of a yellow p-Nitrophenylamino. The goal was to measure activity of the tested LMP-compositions and compared with the activity of alkaline phosphatase caused by known concentrations of the reference GDF-5.

The standardized analysis of alkaline phosphatase, 1×104cells cell lines ATDC-5, MSNT 1/26 incubated overnight in 96-meadow tablet in culture medium (alpha-MEM, Penicillin/Streptomycin, 2 mm L-glutamine, 10% FCS) at 37°C, 5% CO2in a saturated water atmosphere). The next day cells were stimulated GDF-5-like proteins or compositions within 72 hours with the indicated concentrations of ligands. The cell is subsequently washed with phosphate-saline buffer solution. Lysis of cells was performed in 100 μl of alkaline buffer for lysis 1 (0.1 M glycine, pH of 9.6, 1% NP-40, 1 mm MgCl2, 1 mm ZnCl2) for 1 hour at room temperature. Then added 100 μl of alkaline buffer for lysis 2 (0.1 M glycine, pH of 9.6, 1 mm MgCl2I mm ZnCl2+2 mg/ml PNPP). The plates were incubated at 37°C, 5% CO2in a saturated water atmosphere. Then the reaction of the alkaline phosphatase was stopped using 100 μl of a solution of 30 g/l NaOH and finally, the optical density was measured using an automatic tablet reader at a wavelength of 405 them with subtraction of blank sample.

1. A method of obtaining a purified recombinant GDF-5-like protein, which includes the stage of destruction of recombinant bacterial cells of E. coli expressing GDF-5-like protein, and solubilization Taurus enable to obtain solubilizing monomer GDF-5-like protein, in which:
a) destroy these cells E. coli using high-pressure homogenizer at a pressure of destruction from 800 to 900 bar;
b) allocate bullock inclusion of the destroyed E.coli cells by centrifugation;
c) process selected bullock include denaturing buffer to solubilize containing urea and L-arginine; and
d) clean the solubilized monomer recombinant GDF-5-like protein by centrifugation and chromatography.

2. The method according to claim 1, in cat the rum buffer to solubilize contains 4-8 M urea and 400-500 mm arginine, preferably 6 M urea, 500 mm arginine.

3. The method according to claim 1, wherein the buffer to solubilize contains a chelator at a concentration of from 5 to 100 mm.

4. The method according to claim 1, wherein the buffer to solubilize has a pH between 9.0 and 11.0 in.

5. The method according to claim 1, additionally including the removal of high molecular weight impurities directly after step solubilization Taurus inclusion by implementing one or more methods selected from the group consisting of centrifugation, three-dimensional filtering and sterilizing filtration.

6. The method according to claim 1, further comprising removing using diafiltration mentioned L-arginine from a solution containing the solubilized protein Taurus enabled.

7. The method according to claim 1, wherein the recombinant bacterial cells of E. coli expressing GDF-5-like protein in inclusion bodies, selected from E. coli D1210, and E. coli W3110.



 

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