The method of purification of recombinant interleukin-8 person

 

(57) Abstract:

The method of purification of recombinant interleukin-8 person obtained in various ways, includes lysis of the original cells, the combined ultrafiltration and chromatography followed by dialysis or ultrafiltration. The source cell may be a cell eukaryotic systems expression or transformed mammalian cells. For each stage of purification of selected special conditions of its implementation. As a result of implementation of the method are interleukin-8 with high yield and high purity. 32 C.p. f-crystals, 2 ill., 3 table.

The present invention relates to a method of purification of recombinant interleukin-8 person, obtained in different ways.

Methods purification of recombinant interleukin-8 person already known. Various research groups have developed different methods of cleaning. Cleaning varies according to the selected chromatographic stages were described the following methods: Mono S with subsequent HPLC (liquid chromatography high resolution) with reversed phase (Lindley, I.; Aschauer, N.; Seifert, J.-M.; Lam, C.; Brunowski, W.; Kownatzki, E.; Thelen, M.; Peveri, P. ; Dewald, B.; von Tscharner, V.; Waltz, A.; Baggiolini, M. (1988): Synthesis and Express and recombinant neutrophil-activating factor. Proceedings of the National Academy of Science, USA, 85), chromatography on heparin-Sepharose nesuderinamas on DEAE Sephacel fractions followed by chromatography on a column of Sephacryl S-200 column (CM-3SW (Matsushima, K.; Oppenheimer, J. J. (1988): the Interleukin and MCAF: novel cytokines associated with inflammation induced by IL-1 and TNF. Cytokine 1), chromatography on CM-Sepharose CL-6B followed by chromatography on Toyopearl HW-55 (Furuta, R.; Yamagishi, J.; Kotani, H.; Sakamoto, F.; Fukui, T.; Matsui, Y.; Sohmura, Y.; Yamada, M.; Yoshimura, T.; Larsen, C. G.; Oppenheim, J. J. ; Matsushima, K. (1989): Production and characterization of recombinant human factor chemotaxis of neutrophils. Journal of Biochemistry, Vol. 106, N 3) and adsorption using batch procedures (with shaking) with silicic acid and subsequent chromatography on heparin-Sepharose CL-6B and Ultragel ACA 54 (Van Damme, J.; Van Beeumen, J.; Conings, R.; Decock, B.; Billau, A. (1989): Purification of peptide chemotaxis of granulocytes/interleukin-8 identifies the heterogeneity of the N-terminal sequence similar to that of thromboglobulin, European Journal of Biochemistry 181). Subsequent processing for the production of interleukin-8 in accordance with the GMP conditions on a commercial scale is unknown.

The disadvantages of the known methods are low yield and lack of cleanliness of interleukin-8 with accompanying high materialistic purposes, that is, the inability to convert them to production methods that meet the requirements of GMP.

Currently established method of purification of recombinant interleukin-8, which overcome the disadvantages of the known methods.

The method of purification of interleukin-8 of the present invention differs in that

a) first, the cells are lysed in buffered solution and

b) the resulting lysate is treated by removing components with different molecular weight by means of repeated ultrafiltration with cross current, so that

i) at the first stage filter interleukin-8 is separated from the larger associated components and

ii) the second stage filter is separated from smaller components,

C) buffered the filtrate obtained in stage b) of the method, is subjected to high purification using cation exchange chromatography, in which the surface of the molecule interleukin-8 with a high positive charge is used in such a form that the pH value of the application and elution is chosen high enough interleukin-8 was still connected, and then

d) buffered solution of the eluate of interleukin-8 from stage C) of the method zamanay in stage d) of the method, lyophilizer.

Lysis of cells in accordance with stage a) of the method is only needed in the case of intracellular educated enzymatic proteins (for example, using lysozyme), chemical (e.g., using organic solvents) and other physical methods of cell lysis (e.g., using ultrasonic treatment), which are also possible. For technical lysis of the cells is generally preferable physical methods (Schwedes and Bunge, 1988 Mechanische Zellaufschlubverfahren, Jahrbuch der Biotechnologie, VCH Verlagsgesellschaft mhB, Weinheim, Germany). Of the physical methods, not counting the ball mill can be especially marked homogenization at high pressure as a reasonable and well-documented method of lysis of the cells.

Various methods of lysis of the cells mentioned above, can be applied in the purification method of the invention.

For stage a) of the method of the invention preferably choose homogenization at high pressure. Lysis of the cells is achieved at a pressure of from 2000 to 15000 psi (0.137-1.034 108n/m2), to be applied for from 1 to 6 cycles, preferably at a pressure of from 5000 to 7000 psi (0.344-0.482 108n/m2) used within 3 to 5 cycles, most preferably at a pressure of 6000 psi is x, for example, as cells of E. coli.

The exception of components with different molecular weight by means of repeated ultrafiltration with a cross-current in accordance with the method of stage b) can in principle be reduced to a filter module filters 1 and 3 kDa NMWL (nominal molecular weight) (Cheryan, M. (1986): guidelines for ultrafiltration, Technomic Publishing Company, Inc., Lancaster, Pennsylvania, USA). This filtering is a physical method of separation (Vlauck, W. R. A.; Muller, N. A. (1994): Grundoperationen chemischer Verfahrenstechnik, Deutscher Verlag fur Grundstoff-industrie, Leipzig, Federal Republic of Germany) and is often used in post-processing to separate the insoluble part. Accordingly, ultrafiltration cross-talk is often used in subsequent processing in the separation of whole cells and damaged cells, as well as in the separation of inclusion bodies. Examples of separation of soluble accompanying substances, especially accompanying proteins described for ultrafiltration in laboratory scale (Cheryan, M. (1986): guidelines for ultrafiltration, Technomic Publishing Company, Inc., Lancaster, Pennsylvania, USA). Industrial application of ultrafiltration with cross-current to separate soluble accompanying substances is unknown, but it is not obvious.

On stesa applies sequential organization of two stages of ultrafiltration with cross-current, with different boundary separation. This organization is selected so that at the first stage filter interleukin-8 were separated from the larger associated components, and the second stage of filtration is concentrated and separated from smaller components. To achieve the most clear separation of interleukin-8 and related components suitable for different approaches. When the first ultrafiltration with cross flow by choosing the appropriate operating parameters it is possible to get interleukin-8 in the filtrate of a membrane having nominally lower border of separation than the limit, the corresponding molecular weight of interleukin-8. Of the operating parameters are crucial type of membrane and the boundary separating, defining the fundamental possibility of the passage of molecules interleukin through the membrane. In the method of the present invention, the boundary separating membrane should be lower than the molecular weight of interleukin-8. By changing the operating parameters of the boundary separation increases, the membrane becomes permeable to interleukin-8.

Preferably, ultrafiltration cross-current to the first filter phase i) was conducted at Membranula pressure, for example, by increasing the circulating volume or speed reduction filtering, adding additives such as organic solvents, for example, propanol, butanol, etc., salts such as sodium chloride, ammonium sulfate and so on, chaotropic agents, such as urea, guanidine salt, and so on, surfactants such as sodium dodecyl sulphate (SDS), Triton, tween, and so on, or by changing the pH. The advantage of the process of the present invention is the ability to control the transfer of the main part of the set of molecules through the membrane by changing the operating parameters. Thus, before you raise the operating parameters of the filtrate obtained without a given molecule can be discarded, so that the following process some of the associated components will be already separated. In this case, a controlled transfer of the bulk of interleukin-8 through the membrane can be achieved by increasing the circulating volume.

When the second ultrafiltration with cross flow boundary separation, lower than the molecular weight of Il-8 may be obtained by selecting appropriate operating parameters. In General, we apply the same features that already the exhaust gas would be separated from smaller associated components by selecting a membrane, having sufficiently low boundary separation, i.e. by choosing the appropriate operating parameters.

Ultrafiltration with cross-current to the second filtration stage ii) stage b) of the method is carried out with the boundary from 0.1 to 1.5 kDa, preferably with boundary 1 kDa.

In stage C) of the method due to the properties of the buffer, providing a high total positive charge of interleukin-8, a cleaning stage with high resolution using cation exchange chromatography is carried out so that the pH of the deposition and elution is selected to be sufficiently high to interleukin-8 was still connected. Elution is achieved by increasing the concentration of sodium chloride in the field of pH 8 to 10, preferably at pH 9.5. The advantages of these terms chromatography is the high capacity of the chromatographic material, because very few of polluting compounds (related proteins and endotoxins) still retain the ability to bind, high purity of the eluate and high output.

In the applicable conditions in stage d) of the method of replacing the buffer solution of the eluate interleukin-8 is achieved preferably by dialysis.

Use the, the use of prokaryotic expression systems such as E. coli cells, eukaryotic expression systems such as yeast cells, especially Pichia pastoris systems, insect cells, especially Baculovirus-infected and Baculovirus-transfected insect cells or permanent systems of insect cells using mammalian cells, and transformed mammalian cells such as CHO and BHK, and instead of cells can be applied to the milk of transgenic animals, such as cows and goats.

Methods cloning and expression of genes in different expression systems known to experts in the field of science and can be derived from the relevant literature.

Ultrafiltration with cross current at the first stage (i) filter stage b) of the method may be carried out in two steps.

Depending on the design of the ultrafiltration system with cross-talk, especially from the surface area of the filter and the type of membrane surface area of the filter can range from 500 to 10000 cm2for production scale. The resulting transmembrane pressure

PTRANSmembRUNNth= (Pint- Pouter)/2 - Pthe filtrate< / BR>
can the total volume and filtration rate thus varies in the range from 1 to 10 l/min and from 10 to 20 ml/min This applies to both first and second phases of the first stage filter.

Preferably by ultrafiltration with cross current circulating volume ranged from 1 to 4 l/min, the filtration rate of 5 to 8 ml/min and a transmembrane pressure of from 1 to 20 psi (0.0689 to 1.378 105n/m2in the first phase and the circulating amount of from 3 to 6 l/min, a filtration rate of 10 to 15 ml/min and a transmembrane pressure of from 20 to 50 psi (1.378 to 3.44 105n/m2in the second phase.

Especially preferably by ultrafiltration circulating volume ranged from 2 to 3 l/min, the filtration rate of 6.3 ml/min and a transmembrane pressure of 10 psi (0.689 105n/m2in the first phase, and circulating volume from 4 to 5 l/min, the filtration rate from 12.7 ml/min and a transmembrane pressure of 35 psi (2.4 10 n/m2in the second phase.

Also especially preferably by ultrafiltration circulating volume ranged from 2 to 3 l/min, the filtration rate of 6.3 ml/min and a transmembrane pressure of 10 psi (0.689 105n/m2in the first phase, and circulating volume from 4 to 5 l/min, the filtration rate from 18.7 ml/min and a transmembrane pressure of 35 psi (2.4 105n/m2in the second phase.

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When ultrafiltration with cross current at the second stage (ii) filtering stage b) of the method depending on the type of filter surface area filter can range from 500 to 10000 cm2. The resulting transmembrane pressure

PTRANSmembreTES= (Pint- Pouter)/2 - Pthe filtrate< / BR>
can respectively be in the range from 5 to 150 psi (0.344 to 10.34 105n/m2). The operating parameters of the circulating volume and filtration rate thus will vary in the range from 0.5 to 10 l/min and from 1 to 20 ml/min

Preferably by ultrafiltration with a cross-current in accordance with the second phase ii) filtering stage b) of the method of circulating volume ranged from 0.5 to 3 l/min, the filtration rate of 4 to 6 ml/min and a transmembrane pressure of from 8 to 12 psi (0.552 to 0.827 105n/m2).

Especially preferably by ultrafiltration with a cross-current in accordance with step ii) filtering stage b) of the method of circulating volume ranged from 1 to 2 l/min, the filtration rate of 5 ml/min and a transmembrane pressure of 10 psi (0.689 105n/m2). The surface of the filter in this case is preferably 700 cm2
a) can first be treated with urea to achieve a 8 M solution of the latter, then

b) the solution in the urea may be subjected to double homogenization at high pressure to solubilize nerastvorimogo interleukin-8 and, finally,

(C) thus obtained product may be further purified in accordance with the stages C), d) and e) of the process.

pH 8M urea solution that is specified in a) and b), preferably communicated to the optimal value of 9.5.

Homogenization at high pressure, performed in b) is carried out at a pressure of from 2000 to 10000 psi (0.138-0.690 108n/m2), to be applied for from 1 to 6 cycles, preferably at a pressure of from 4000 to 8000 psi (0.276-0.551 108n/m2) applied for 1 to 3 cycles, and particularly preferably at a pressure of 6000 psi (0.414 108n/m2) applied within 2 cycles.

Interleukin-8, purified using the method of the present invention may be used for research, therapeutic and diagnostic purposes.

In particular, the process of the invention for the purification of interleukin-8 is a very economical process, which I, calculated on the basis of the soluble portion is approximately 50%. This is a very high output compared to other industrial purification methods to obtain expressed in bacterial recombinant proteins in accordance with GMP and more than satisfactory from the point of view of economy. Accordingly, there is a distinct advantage compared to conventional processes in which to achieve the same result requires several chromatographic stages using the liter scale. Theoretically, under the conditions of traditional processes using at least 4 chromatographic stage with DMF materials and average output stage 80%, you can set that the output will be 0.84= 0.41 or 41%. When this was not taken into account starting losses associated with the removal of cellular debris and DNA, and losses due to change of buffer. For a good industrial development that meets the conditions of GMP, the acceptable yield of protein expressed in bacteria, comprising from 10 to 20%.

With regard to material costs included in production, first of all it is possible to make some General assessments, in accordance with which the receiving buttersworth cost occurs because expensive chromatographic material. When classical methods of this type are several chromatographic columns liter size to achieve the same output high-purity protein, as in the case of the present process. One of the major achievements of the developed process is that the allocation of purified protein required only one chromatographic stage, which in contrast to classical liter sizes characterized milliliters. Thus, in comparison with the classical treatment processes, there is a significant cost reduction.

In Fig. 1 shows the dependence of the total concentration of protein from the number of cycles. All mathematical models are reflected in the respective formulas and parameters.

In Fig. 2 shows the total number of interleukin-8 in the filtrate III depending on the time.

The following examples illustrate the purification process of the invention is not limited presents working examples.

Working examples

1. Cloning and fermentation of interleukin-8

Part of the gene interleukin-8 person encoding the proteins of 72 amino acids, was inserted into plasmid pMS119EH(L). The strain E. coli TG1 transformed received Victoriei were as follows:

from the flowing culture of E. coli TG1 (pIL8/tac), stored at -70oC in glycerol, selected 20 μl, and incubated for 48 h at 37oC in culture on agar in L medium (10 g/l tryptophan (Difco), 5 g/l yeast extract (Difco), 10 g/l NaCl) and 200 mg/l ampicillin. The colony was then washed with 5 ml of L medium. 100 μl of this culture was used for inoculation of 500 ml bottle for shaking with 100 ml of L medium. This first pre-culture was incubated for 16 h at 37oC at a frequency of shaking 180 Rev/min to 10 ml of this pre-culture was used for inoculation of the second pre-culture in a 2-liter bottle to shake containing 1000 ml of L medium. Incubation was carried out for 48 h under the conditions mentioned above. 1000 ml of this second pre-culture was used for inoculation of 300-liter fermenter containing 200 l L environment. The fermenter was stirred at 150 rpm at 28oC and aeronavali at 5 m3/h After 4 h of fermentation induced expression of interleukin-8 by the addition of 0.25 mmol/l IPTG, and the fermentation was continued for further 8 hours, the Bacterial cells were then killed by the addition of octanol to the concentration of 0.1%. After adding octanol batch was stirred for 2 m is eparatory at 6600 x g and 200 l/h and concentrated from 200 liters to 7 liters Sediment bacterial cells were obtained from these 7 l using centrifugation.

2. Purification of interleukin-8

2.1. Lysis of the cells by homogenization at high pressure (stage a) of the method)

423.35 g cell mass (wet weight) resuspendable 50 mm NaCl, 20 mm TRIS, pH 8.0 to obtain a final volume 1730 ml and literally using homogenization at high pressure, and lysis resuspending cell mass was carried out for 4 cycles at a pressure of 6,000 psi (0.414 108n/m2). The presence of lysis of the cells was analyzed by determining the total protein, SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate, SDS page with LTOs) and enzyme immunoassay. The dependence of the concentration of total protein from the number of cycles of homogenization at high pressure is shown in Fig. 1. Looking at the concentration of total soluble protein, it can be seen that the lysis of the cells is a first order reaction. The curve of concentration of total protein demonstrates a marked decrease in slope with increasing number of cycles. This indicates adequate lysis of the cells, preferably in 4 cycles. Moreover, it is possible to find an empirical correction for the description of cell lysis. The concentration of internal after the 4th cycle. When the page with LTOs best effect separation of individual bands was observed with increasing number of cycles, which probably can be attributed to DNA fragmentation. The total amount of soluble protein, defined by the ICA method (method of coloring Kumasi), as well 6643.2 mg. per share of interleukin-8 from this number accounts for careful evaluation to 4.4%.

2.2. The exception molecular weight using repeated ultrafiltration with cross flow (stage b) of the method)

Full homogenate cell lysate from stage a) of the method was subjected to ultrafiltration with cross flow) using a membrane having a split line 3 kDa. The obtained filtrate was then processed using the same technique with boundary separating 1 kDa. Ultrafiltration with boundary separating 3 kDa were performed in two stages, by performing filtering using a 3 kDa NMWL (nominal molecular weight) of the modified PES membrane with a surface area of the filter 2100 cm2. Selected applicable conditions were as follows:

buffer for filtration: 5 mm EDTA, 50 mm NaCl, 20 mm TRIS, pH 9.5,

the initial phase of the circulating volume: 2-3 l/min,

the filtration rate of 6.3 ml/min,

transmembrane pressure of 10 psi (0.68910
filtration rate 12.7 ml/min,

transmembrane pressure 35 psi (2.4105n/m2).

Received 8620 ml of filtrate I and 1110 ml withholding material I. Determination of total protein using the ICA showed that of the original 6643.2 mg total soluble protein in the cell lysate, 2508.6 mg could still be detected in withholding material I and 2293.3 mg in the leachate I. Analysis of soluble interleukin-8 after analytical HPLC using a C4 column with reversed phase showed that its concentration in the retained material I and the filtrate I is below the lower limit of determination, less than 0.005 mg/ml for Further analysis using ELISA method showed interleukin-8 in withholding material 1 at a concentration of 0.002 mg/ml (2.2 mg interleukin-8).

The filtrate I was further processed by filtering at the border division 1 kDa. 1 kDa ultrafiltration with cross flow was performed using a 1 kDa NMWL (nominal molecular weight) of the modified PES membrane with a surface area of the filter 700 cm2. Selected applicable conditions were as follows:

circulating volume: 1-2 l/min,

filtration rate 5.0 ml/mi is about 600 ml of withholding material II with the formation 8020 ml of the filtrate II. Analysis of the retained material II using SDS page with LTOs found intense band in the area corresponding to the molecular weight of interleukin-8. In the field of molecular weight lower than that of interleukin-8, it was possible to detect two substantially weaker bands. A rough estimate of the purity of interleukin-8 determined on the basis of total protein according to the profile of elution from HPLC with reversed phase amounted to a value of 25%. Sequencing by Edman N-Terminus of interleukin-8 in withholding material II confirmed a similar purity. Further analysis is conducted to determine the concentration using ELISA revealed the concentration of interleukin-8 in withholding material II, equal to 0.31 mg/ml (186 mg of interleukin-8), and the filtrate II concentration of interleukin-8 was below 0.0001 mg/ml (< 0.8 mg of interleukin-8). After one pre-processing the detection level was 63.2% and a purity of > 25%.

2.3. Cleaning with high resolution using cation-exchange chromatography (stage C) of the method)

45-ml column of CM Sepharose (HC column FPLC system) inflicted 600 ml withholding material II, which accounted for 90% of its capacity. The sequence of stages chromatography 45-ml column of CM Sepharose prestolee 90%; related proteins when this is no longer detected. As for the eluate of interleukin-8, obtained by stepwise elution was obtained 173.4 mg of interleukin-8 by the results of analytical C4-HPLC with reversed phase. Concentration of the eluate was 2.1 mg/ml, which is a relatively high value, and related proteins could not be detected. Because endotoxins and DNA are also usually separated during chromatography on a weak cation-exchanger, the eluate of interleukin-8 were investigated on purity (using SDS page with LTOs with the overloading of the gel) and the content of endotoxins (LAL test). Related proteins also were not found after reloading the page with LTOs at colouring protein, Kumasi. Removal of endotoxin, as it turned out, was very effective. The concentration of endotoxin was less than 1.5 PG/ág Il-8, which was below the limit of detection. The eluate was air-conditioned and subjected to control as a final product.

2.4. Dialysis (stage d) of the method)

The eluate interleukin-8 were dialyzed against PBS and brought to a concentration of 1.00 mg/ml Dialysis was performed with a membrane having a boundary separating 3.5 kDa. The eluate interleukin-8 were dialyzed for 1, 2.5 and 20 h, each time against 5 l of free pie from the following dialysis the concentration of brought up to 1.00 mg/ml of interleukin-8 by using free of pyrogen PBS buffer and tested using analysis of protein Pierce BCA, using as protein comparison BSA (bovine serum albumin).

2.5. Lyophilization (stage e) of the method)

After creating a concentration of 1.00 mg/ml of interleukin-8 in PBS were lyophilization. For this purpose, the solution carried in suitable small container in the form of aliquot 1.00 ml (1.00 mg of interleukin-8 on capacity) and subjected to deep freezing at -70oC. After 12 h at -70oC lyophilization was performed within 36 hours the vessels of the samples were sealed and kept at -70oC. Randomly selected sample was subjected to control in the quality of the final product.

3. The output of the purification method

The output of the purification method of the invention is approximately 50%. This unexpectedly high value for bacterial expressed protein. Table 2 summarizes the results of the analysis of the purification method:

Optimization experiments with double ultrafiltration with cross flow) showed that the yield of 63.2% can be increased to more than 80%. Accordingly, an optimized cleaning process can provide outputs up to 65%

4. The production properties of the way

To control the product to perform tests on a clean final product in relation to identity, concentration, purity, GMP production for use in humans and were as follows:

4.1. Identification

Sequencing by Edman (1950) the N-end.

The required sequence of interleukin-8 can be unequivocally confirmed.

The Western blot turns with poly - and monoclonal antibodies against interleukin-8.

The Western blot turns showed a direct linear relationship between signal intensity and concentration of purified applied drug.

4.2. Concentration

Analysis of protein Pierce BCA

Determination of concentration of total protein analysis protein Pierce BCA using as protein comparison BSA gives a 1.00 mg/ml

4.3. Purity

HPLC with reversed phase

In the test with HPLC with reversed phase for a foreign protein using a C4 column associated protein could not be detected at 214 nm.

SDS page with LTOs with color silver

SDS page with LTOs with the color silver is also found only one lane of interleukin-8. Other bands could not be detected.

SDS page with LTOs with Coomassie blue staining and by measuring the intensity of the bands

Analysis of SDS page with LTOs with Coomassie blue staining and subsequent measurement of the intensity of the band gave a purity of more than 99% pure product.

4.4. Endotoxins

LAL test

LAL test show the ideological activity

The reaction of chemotaxis of neutrophils

The value of the ED50measured by the reaction of chemotaxis of neutrophils, was 0.84 mmol/l

5. Solubilization of interleukin-8, the remaining cell debris

Fragments of cells, which were stored at -70oC, literally using homogenization at high pressure and were processed using filtering with boundary separating 3 kDa (see the second stage of purification of interleukin-8), was thawed on ice, was heated to room temperature and was dissolved by stirring with a crystalline urea to obtain the final concentration of the last 8 M. the pH was 9.5. Then conducted twice homogenization at high pressure using a homogenizer, and the suspension in 8 M urea were subjected to two cycles at a pressure of 6000 psi (0.414 108n/m2). Some stages were analyzed using immunoassay test for interleukin-8. Table 3 schematically shows the individual stages of solubilization of interleukin-8 on an industrial scale and their results.

After the last stage of the process of industrial solubilization was obtained 228.0 mg of interleukin-8. It was found that in the process of production reactions adding m is the situation, on the other hand, gave only a weak increase in the concentration of interleukin-8.

Subsequent tests showed that a higher value of output obtained when longer incubation solution of 8 M urea at pH 9.5. In laboratory scale treatment of urea for 24, 36 and 48 h under the same conditions could give two to three times the output.

After this treatment the solution of 8 M urea brought deionized H2O up to 4 M last (system Millipore Q) (dilution 1:1) and again subjected to filtration with a boundary separating 3 kDa, and the resulting filtrate III was further subjected to filtration with a boundary separating 1 kDa in accordance with the procedure described above. Filtering withholding material I have performed in two stages. In the first stage, a solution of 4 M urea concentrated and in the second phase was filtered with 5 mm EDTA, 50 mm NaCl, 20 mm TRIS, pH 9.5.

Buffer for filtration: 5 mm EDTA, 50 mm NaCl, 20 mm TRIS, pH 9.5,

the initial phase of the circulating volume: 2-3 l/min,

the filtration rate of 6.3 ml/min,

transmembrane pressure of 10 psi (0.689 105n/m2).

After two changes of volume of the filtering options increased.

second phase: circulating volume: 4-5 l/min,

speed Filina-8 in the filtrate III was analyzed using ELISA method. The results are shown in Fig. 2. The filtering process described by sigmoidal curve, and the area of the curve is determined primarily by the increase in operating parameters at the stage of filtration. Consequently, there is a picture of the filtering process, characteristic of first order reactions.

The total number solubilizing interleukin-8 in the filtrate III, which could be separated from the withholding of material I for 18 h using a filter with boundary separating 3 kDa, was 232.0 mg with a concentration of 0.015 mg/ml of the Filtrate III was further subjected to filtration with a boundary separating 1 kDa. After concentration 7815 ml of the filtrate III to 1250 ml withholding material IV, by analysis with C4-HPLC with reversed phase detected concentration of interleukin-8 0.087 mg/ml (109.0 mg of interleukin-8). This corresponds to a yield of over 90%. The concentration of interleukin-8 in the filtrate IV was less than 0.005 mg/ml and was below the limit of detection. You can see that the total yield of the interleukin-8 can be almost doubled by using this phase solubilization.

1. The method of purification of interleukin-8, characterized in that a) the cells are lysed in a buffer solution, (b) the resulting lysate to exclude molecular weight ACC is tdelay interleukin-8 from the accompanying components with molecular weight greater than interleukin-8, ii) at the second stage filter is separated from components with molecular weight less than that of interleukin-8, C) buffered the filtrate obtained in stage b), is subjected to high purification using cation exchange chromatography, in which the positively charged surface molecules interleukin-8 is used in the form, allowing you to select the pH of the deposition and elution, sufficient to interleukin-8 remained strongly connected, then d) buffered solution of the eluate of interleukin-8 from stage C) is subjected to gel filtration, the dialysis or ultrafiltration, and then (e) the solution of interleukin-8, obtained in stage d) lyophilizer.

2. The method according to p. 1, characterized in that during ultrafiltration with cross current at the first stage (i) filter stage b) of the method, the boundary separating membrane is lower than the molecular weight of interleukin-8.

3. The method according to p. 2, characterized in that the ultrafiltration cross-current in the first stage (i) filtering is performed at the boundary of division 3 kDa.

4. The method according to p. 1, characterized in that during ultrafiltration with cross current at the second stage (ii) filtering stage b) how the boundary richeldis fact, what ultrafiltration with cross current at the second stage (ii) filtering stage b) of the method is carried out at the boundary separating 0.1 to 1.5 kDa.

6. The method according to p. 5, characterized in that the ultrafiltration cross-current in the second phase ii) filtering stage b) of the method is carried out at the border division 1 kDa.

7. The method according to any of paragraphs.1 - 6, characterized in that stage a) of the method of lysis of the cells is achieved by homogenization under high pressure.

8. The method according to any of paragraphs.1 - 6, characterized in that stage a) of the method of lysis of the cells is achieved enzymatically or chemically.

9. The method according to any of paragraphs.1 to 7, characterized in that the lysis of cells at the stage a) of the method is achieved at a pressure of from 2000 to 15,000 psi (0,137 to 1,034108n/m2) applied for 1 to 6 cycles.

10. The method according to p. 9, characterized in that the lysis of cells at the stage a) of the method is achieved at a pressure of 5,000 to 7,000 psi (0,344 to 0,482108n/m2) applied for 3 to 5 cycles.

11. The method according to p. 9 or 10, characterized in that the lysis of the cells is achieved at a pressure of 6000 psi (0,413108n/m2) administered for 4 cycles.

12. The method according to any of paragraphs.1 - 11, characterized in that cell, p is tives such as those the cells subjected to lysis, are E. coli cells.

14. The method according to any of paragraphs.1 - 11, characterized in that the cells are subjected to lysis, represent cells eukaryotic expression systems.

15. The method according to p. 14, characterized in that the cells are subjected to lysis, are yeast cells, insect cells and mammalian cells.

16. The method according to p. 15, wherein the yeast cells originate from Pichia pastoris and insect cells come from Baculovirus-transfected insect cells.

17. The method according to p. 15, wherein the transformed mammalian cells are Cho and KSS.

18. The method according to p. 14, characterized in that instead of the cells may also be applicable to the milk of transgenic animals, such as cows and goats.

19. The method according to any of paragraphs.1 to 18, characterized in that stage a) of the method, the pH value of the solution for application and elution is 8 - 10.

20. The method according to p. 19, characterized in that the pH of the solution for application and elution is 9.5.

21. The method according to any of paragraphs.1 to 20, characterized in that the ultrafiltration cross-current in the first stage (i) filter stage b) sponem current applied circulating volume 1 - 10 l/min, the speed of filtration of 1 to 20 ml/min and a transmembrane pressure of 1 to 150 psi (0,0689 to 10,34105n/m2as the first phase and the second phase.

23. The method according to PP.21 or 22, characterized in that during ultrafiltration with cross current in the first phase applied circulating volume 1 - 4 l/min, the filtration rate of 5 - 8 ml/min and a transmembrane pressure of 1 to 20 psi (0,0689 to 1,378105n/m2), and the second phase applied circulating volume 3 - 6 l/min, a filtration rate of 10 to 15 ml/min and a transmembrane pressure of 20 to 50 psi (1,378 to 3,44105n/m2).

24. The method according to p. 23, characterized in that during ultrafiltration in the first phase used a recirculating volume of 2 to 3 l/min, the filtration rate from 6.3 ml/min and a transmembrane pressure of 10 psi (0,689105n/m2), and the second phase used a recirculating volume of 4 to 5 l/min, the filtration rate of 12.7 ml/min and a transmembrane pressure of 35 psi (2,4105n/m2).

25. The method according to p. 22, characterized in that during ultrafiltration in the first phase used a recirculating volume of 2 to 3 l/min, the filtration rate from 6.3 ml/min and a transmembrane pressure of 10 psi (0,689105n/m2), and the second phase used a recirculating volume of 4 to 5 l/min, speed Phi is .1 - 25, characterized in that during ultrafiltration with cross current at the second stage (ii) filtering stage b) of the method is used in the circulating volume of 0.5 - 10 l/min, the speed of filtration of 1 to 20 ml/min and a transmembrane pressure of 5 to 150 psi (0,345 to 10,34105n/m2).

27. The method according to p. 26, characterized in that during ultrafiltration with cross current at the second stage (ii) filtering stage b) of the method is used in the circulating volume of 0.5 to 3 l/min, the filtration rate of 4 to 6 ml/min and a transmembrane pressure to 8 - 12 psi (0,552 to 0,827105n/m2).

28. The method according to p. 26 or 27, characterized in that during ultrafiltration with cross current applied circulating volume 1 - 2 l/min, the filtration rate of 5 ml/min and a transmembrane pressure of 10 psi (0,689105n/m2).

29. The method according to any of paragraphs. 1 to 28, characterized in that the cellular debris, obtained at the first stage (i) filter stage b) method (a) is treated with urea to the solution concentration of urea 8 M, then b) the urea solution is subjected to double homogenization at high pressure to solubilize nerastvorimogo interleukin-8, and finally (C) thus obtained product is subjected to further purification in accordance whom paragraphs (a) and (b), has an optimum pH of 9.5.

31. The method according to p. 29 or 30, characterized in that the homogenization at high pressure, performed in subparagraph (b), performed at a pressure of 2000 to 10000 psi (was 0.138-0,690108n/m2) applied for 1 to 6 cycles.

32. The method according to p. 31, characterized in that the homogenization at high pressure, performed in subparagraph (b), performed at a pressure of 4000 to 8000 psi (0,276-0,551108n/m2) applied for 1 to 3 cycles.

33. The method according to p. 31 or 32, characterized in that the homogenization at high pressure, performed in subparagraph (b), performed at a pressure of 6000 psi (0,414108n/m2) applied within 2 cycles.

 

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