Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF

Definitions

• the present invention relates to a method for purifying a factor for stimulating granulocyte colonies (denoted G-CSF for "Granulocyte Colony Stimulating Factor") by chromatography using a hydroxyapatite chromatography stage.
• European patent application EP 243153 describes a process for the purification of human G-CSF from bladder carcinoma cell lines HBT5637;
• European patent application EP215126 describes the purification of recombinant human G-CSF produced in E. coli.
• the methods described above correspond to multiple stages of purification in which the initial concentration of the starting biological preparations is generally obtained by the conventional methods of ultrafiltration or of precipitation by salt, followed by liquid chromatography in reverse phase (noted RP -HPLC) successive which have the known drawback of leading to significant yield losses, for example because the protein is denatured by organic solvents.
• RP -HPLC liquid chromatography in reverse phase
• 5,055,555 describes a selective and simplified method for purifying recombinant human G-CSF produced in yeast on a larger scale, by precipitation with NaCl preceded by concentration by chromatography on a column of heat exchanger. cations (S Sepharose® or Mono S®), but whose yield and purity obtained are not mentioned.
• Phenyl Sepharose® CL-6B (Pharmacia) has been described by N A. Nicola et al., Journal of Biological Chemistry, Vol. 258, p. 9017-9023, 1983 for the purification of G-CSF produced naturally by murine leukemia cells. After preliminary concentration of the medium on hollow fiber and "salting out” chromatography, the G-CSF was directly fixed on the Phenyl Sepharose® column, then eluted using a decreasing salt gradient, then a linear gradient of ethylene glycol.
• One of the objects of the present invention is to provide a process which allows isolate and purify G-CSFs, on a large scale and with high yields, by a stage of chromatography on hydroxyapatite from biological samples previously concentrated and enriched using hydrophobic interaction chromatography.
• the method of the invention can be used for example as the first stage of purification of a G-CSF in a multistep process for preparing a G-CSF having a purity allowing clinical use.
• the subject of the invention is a method for purifying G-CSF from a biological sample comprising the stages of a) reducing the volume of the biological sample containing G-CSF by hydrophobic interaction chromatography to obtain a concentrated fraction , desalted and enriched, b) pass the concentrated fraction over hydroxyapatite in conditions where the G-CSF is weakly bound to obtain a concentrated, desalted and enriched fraction containing the G-CSF and c) collecting the G-CSF.
• the above process makes it possible to purify G-CSF under non-denaturing conditions and to isolate the biologically active G-CSF.
• the G-CSF purified according to the process of the invention can be any known G-CSF having a biological and pharmaceutical interest.
• G-CSF is included a G-CSF produced constitutively by cells, for example by cell lines established from tumor cells as described by Watson et al., J. Immunol. , Flight. 137, p.
• a G-CSF produced by activation of the G-CSF gene (noted GA-GCSF for "Gene Activation-GCSF) in human cells as described in international patent application WO 95/31560 or a G-CSF produced by recombinant DNA technology by host cells
• the host cells can be eukaryotic cells such as mammalian cells, for example monkey COS cells, hamster CHO cells or C127 cells of mice or such as yeasts, for example S. cerevisiae or prokaryotic cells, for example E. coll.
• G-CSF examples include recombinant G-CSF.
• EP217404 which describes a G-CSF produced in C127 cells or in CHO cells
• US Patent 5,055,555 which describes a G-CSF produced by S. cerevisiae
• WO 87/01132 which describes a G-CSF produced in COS cells as well as 'a G-CSF produced in E.
• the process makes it possible to purify both glycosylated or non-glycosylated G-CSFs.
• the biological sample from which the method of the invention makes it possible to purify a G-CSF comprises the biological fluids of cell cultures such as cell lysates, inclusion bodies or culture supernatants when the G-CSF is excreted.
• the biological sample used for the purification of G-CSF was preferably previously separated from the cells or debris cells by methods known to those skilled in the art, for example by filtration, centrifugation or ultrafiltration.
• Hydrophobic interaction chromatography means chromatography on a substance separation support on the basis of their differences in interaction with hydrophobic groups attached to a matrix without ionic groups.
• the hydrophobic group may be an aliphatic ligand, for example a butyl or octyl group or an aromatic ligand, for example a phenyl group or a phenylbutylamine group and the matrix is generally a gel, for example agarose such as Sepharose® .
• the supports used are marketed products. In all the methods of using hydrophobic interaction chromatography, the proteins are fixed to the hydrophobic gel in the presence of high salt concentrations.
• the process of the invention comprises a hydrophobic interaction chromatography characterized by a binding of the protein with low conductivity or with low salt content, for example ammonium sulphate or NaCl, and reduces the volume of the initial biological sample while eliminating salts as well as a high percentage of contaminating proteins.
• the process of the invention therefore makes it possible to obtain a concentrated fraction, desalted and enriched in
• the invention particularly relates to the above process in which the collected G-CSF has a purity of at least 90%.
• Another subject of the invention is also the above method in which the biological sample is a cell culture supernatant as well as the method in which the G-CSF is a human G-CSF (denoted hG-CSF).
• a further subject of the invention is also the above method, in which the volume reduction stage comprises bringing the biological sample into contact on a chromatography support by hydrophobic interaction of the phenyl type under conditions allowing the fixation of the G -CSF, then its elution.
• the invention more particularly relates to the above process in which the phenyl-type support is a Phenyl Sepharose®.
• the subject of the invention is especially the above process in which the fixation on Phenyl Sepharose® is carried out in a buffer having an ionic strength of between 0 and 60 mSi and the elution is carried out by reduction of the ionic strength or of the salt concentration in the fixing buffer.
• the invention also especially relates to the above process in which the fixation on Phenyl Sepharose® is carried out in a buffer containing NaCl at a concentration of between 0.1 and 1 M.
• a more specific subject of the invention is the above process in which the fixation on Phenyl Sepharose® is carried out in a buffer containing NaCl at a concentration of between 0.1 and 0.5 M and the elution is carried out by 'water.
• a further subject of the invention is also the process of the above invention in which the step of passage over hydroxyapatite is carried out in a buffer with an ionic strength of between 2 and 30 mSi and at a pH between 5.5 and 7 5.
• the subject of the invention is more particularly the above method in which the buffer comprises phosphate at a concentration between 1 and 10 iru.
• the invention also more particularly relates to the above process in which the buffer is a 1 mM phosphate buffer and the pH is between 6.0 and 7.5.
• the invention also relates to a method for purifying G-CSF which can be included in a multistage process for purifying G-CSF from a biological sample comprising the steps of a) reducing the volume of the biological sample containing G -CSF by hydrophobic interaction chromatography to obtain a concentrated, desalted and enriched fraction, b) passing the concentrated fraction over hydroxyapatite under conditions where the G-CSF is weakly bound to obtain a concentrated, desalinated and enriched fraction containing the G-CSF and c) collect the G-CSF.
• the invention particularly relates to the above method in which the multistep method further comprises one or more stages of chromatography chosen from the group consisting of ion exchange chromatography, gel filtration, reverse phase or affinity.
• the invention also relates to a method for removing contaminating proteins from a solution containing G-CSF and contaminating proteins comprising: a) passing the solution over hydroxyapatite by which the contaminating proteins are attached to the hydroxyapatite and G-CSF is weakly bound and b) elution of G-CSF.
• the invention particularly relates to the above method in which the elution of G-CSF is carried out by simple washing with the fixing buffer.
• the contaminating proteins present in the solutions containing G-CSF were for example added in the cell culture media.
• the added proteins can be, for example, serum, such as beef serum or fetal calf serum, for example partially purified serum proteins, such as albumin or transferrin or mixtures thereof.
• the method of the invention makes it possible to remove these contaminating proteins by passing the solution containing G-CSF over hydroxyapatite during which the undesirable proteins are strongly fixed on the support and retained during the elution of G-CSF.
• the invention also relates particularly to the above method in which the solution containing G-CSF is prepared by hydrophobic interaction chromatography of a biological sample containing G-CSF.
• the hydrophobic interaction chromatography is carried out on a phenyl-type support, for example on Phenyl Sepharose® as is illustrated below in the experimental part.
• the method of the invention advantageously makes it possible to remove these contaminating proteins during the first stage of purification of G-CSF from a biological sample.
• Analytical methods 1. Determination of G-CSF by HPLC
• G-CSF is eluted at a concentration of approximately 65% acetonitrile.
• the G-CSF concentration is measured against a G-CSF standard.
• An assessment of purity is measured by the ratio of the area of the G-CSF peak to the area of all the peaks other than the injection peak.
• the G-CSF concentration is measured using the ELISA kit from R&D System Inc and the protocol recommended by the supplier.
• the samples are analyzed on ready-to-use polyacrylamide gels (Novex) containing a gradient of 10 to 20% of polyacrylamide and silver staining using the Silver staining kit from Biorad for a deposit of 50 ng to l ⁇ g from G-CSF.
• FIG. 1 is a chromatogram showing the fractionation on phenyl Sepharose of a supernatant of cells expressing GA-GCSF and containing 0.1 M NaCl. The arbitrary units represent respectively the conductivity and the optical density (OD) of the effluent from the column expressed as a percentage.
• FIG. 2 is a chromatogram showing the fractionation on phenyl Sepharose of a supernatant of cells expressing GA-GCSF and containing 0.5 M NaCl. The arbitrary units have the same meaning as in FIG. 1.
• FIG. 3 is a chromatogram showing the fractionation of GA-GCSF on MacroPrep® ceramic Type I hydroxyapatite after phenyl Sepharose. Arbitrary units have the same meaning as in Figure 1.
• FIG. 4 is a chromatogram of RP-HPLC analytical of GA-GCSF after phenyl Sepharose and hydroxyapatite Type I.
• FIG. 5 is a chromatogram showing the fractionation of GA-GCSF on hydroxyapatite MacroPrep® ceramic Type II after phenyl Sepharose.
• Arbitrary units have the same meaning as in Figure 1.
• FIG. 6 is an analytical RP-HPLC chromatogram of GA-GCSF after phenyl Sepharose and hydroxyapatite Type II.
• FIG. 7 represents the SDS-PAGE analysis of the purification of GA-GCSF successively in a filtered culture supernatant (well 3), an eluate of phenyl Sepharose (well 4), a hydroxyapatite eluate (well 5), an SP Sepharose eluate (well 6), a UF concentrate (well 7), a filtration gel eluate in PBS buffer (well 9), a filtration gel eluate in acetate buffer pH 5.5 (well 11) with standard molecular weight markers (well 1).
• the band corresponding to the apparent PM of the GA-GCSF is indicated by an arrow.
• Example 1 Concentration of a biological sample of G-CSF by chromatography on phenyl Sepharose.
• the starting material is the centrifugal supernatant of a culture broth of human cell lines expressing a human GA-GCSF obtained according to international patent application WO95 / 31560 in an Endotronics® hollow fiber bioreactor in DMEM / F12 (Hyclone) containing 0.9% fetal calf serum. After centrifugation, the supernatant was stored at -20 ° C before use.
• the thawed supernatant was chromatographed on phenyl Sepharose after addition of NaCl q.s.p. 0.1 M and filtration on a 0.22 ⁇ m Millipore membrane, at a temperature of around 15 to 20 ° C.
• the column is then washed at a flow rate of 4 ml / min with 220 ml of 0.05 M NaCl solution, collecting the column effluent in 40 ml fractions.
• the column is then eluted at the same flow rate with 150 ml of Milli-Q demineralized water, collecting the column effluent in fractions of 2 ml.
• the column is finally regenerated by washing at the same rate with an 8 M urea solution.
• the total proteins in the column effluent are detected by absorption at 280 nm and the salt concentration is monitored using a conductivity meter.
• the presence in the column effluent of a first peak of protein eluted by water, then of a second peak of protein eluted by washing with urea is shown in FIG. 1.
• the fractions collected during the elution with water were analyzed for their G-CSF content by analytical RP-HPLC chromatography and by ELISA using the conditions described above.
• the fractions containing the combined G-CSF (40 ml) contain 29.3 mg of GA-GCSF titrated by HPLC corresponding to a yield of 56% and a purity of 58%.
• the GA-GCSF phenyl Sepharose solution thus obtained has a conductivity of 0.161 mS. cm -1 .
• Example 2 Chromatography on phenyl Sepharose, then on hydroxyapatite as the first stage of purification of G-CSF.
• the starting material is the supernatant of a culture broth of human cell lines expressing a human GA-GCSF obtained as in Example 1 but using a 5-liter bioreactor instead of an Endotronics® bioreactor.
• the thawed supernatant was chromatographed on phenyl Sepharose after addition of 0.5 M NaCl qsp and filtration through a 0.45 ⁇ m Millipore membrane.
• a Pharmacia XK16 column packed with 50 ml of Phenyl Sepharose® Fast Flow High Substitution (Pharmacia) stored under 25% ethanol then washed with Milli-Q demineralized water before use, the concentration by chromatography on phenyl Sepharose was performed as follows:
• 1640 ml of salted and filtered supernatant obtained above (conductivity 56.3 mS.cm "1 ) are applied to the column at the flow rate of 4 ml / min and the column effluent is collected in 400 ml fractions.
• column is then eluted at the same flow rate of 4 ml / min with 240 ml of 0.5M NaCl, collecting the column effluent in 40 ml fractions
• the column is then eluted at the same flow rate with 150 ml of Milli demineralized water -Q by collecting the column effluent in fractions of 2 ml.
• the column is finally regenerated by washing at the same rate with an 8M urea solution.
• Example 2 The total proteins in the column effluent and the salt concentration are detected as in Example 1. The presence in the column effluent of a first peak of protein eluted by washing with water, then a second peak of protein eluted by washing with urea is shown in FIG. 2.
• the fractions collected during elution with water were analyzed for their G-CSF content by analytical RP-HPLC chromatography and by ELISA.
• the fractions containing the combined G-CSF (50 ml) contain 45.1 mg of GA-GCSF titrated by HPLC corresponding to a yield of 90% with a purity of 61%.
• the GA-GCSF phenyl Sepharose solution thus obtained has a conductivity of 4.22 mS.cm-1 which allows it to be used as it is in the next stage of chromatography on hydroxyapatite.
• Example 3 Chromatography on phenyl Sepharose, then on hydroxyapatite as the first stage of purification of G-CSF.
• 24 ml of the GA-GCSF solution of phenyl Sepharose obtained in Example 2 were chromatographed on a hydroxyapatite column according to the conditions described in Example 2, but using the hydroxyapatite Macro-Prep® Ceramic Type II (Biorad) instead of Type I.
• the starting material is a supernatant of a culture broth of human cell lines expressing a GA-GCSF obtained according to Example 2 but using a 100-liter bioreactor and the medium without calf serum.
• SDS-PAGE analysis of the filtered culture supernatant thus obtained is shown in Figure 7 (well 3).
• the fractions corresponding to the peak of protein eluted by water were analyzed for their G-CSF content by analytical RP-HPLC chromatography and by ELISA.
• the combined G-CSF fractions (400 ml) contain 357 mg of GA-CSF titrated by HPLC corresponding to a yield of 67.9% with a purity of 20.6%.
• the GA-GCSF phenyl Sepharose solution thus obtained has a conductivity of 2 S. cm "1 at 20 ° C.
• the phenyl Sepharose solution was also analyzed by SDS-PAGE (FIG. 7, well 4). After stabilization by adding Pefabloc (0.2 mg / ml) and benzamidine (ImM), the solution was immediately used for the next stage of chromatography on hydroxyapatite.
• the fractions collected were analyzed by analytical RP-HPLC and by ELISA.
• the combined fractions (400 ml) contain 331 mg of GA-GCSF titrated by HPLC corresponding to a chromatography yield of 92.5% with a purity greater than 90% estimated by HPLC.
• the example illustrates the stages of subsequent purification of G-CSF which can be used after passing over hydroxyapatite in a multistep purification process.
• a chromatography stage on a cation exchanger then a gel filtration chromatography stage were carried out as follows: 1 ) chromatography on cation exchanger.
• 390 ml of GA-GCSF hydroxyapatite solution obtained in Example 4 are applied to the column of SP Sepharose at a flow rate of 13.2 ml / min.
• the column is then washed at the same rate with 414 ml of 20 mM buffer, pH 5.3, then with 1 liter of elution buffer corresponding to an NaCl gradient, varying from 0 to 250 mM in 5 column volumes (850 ml) of 20 mM buffer, pH 5.3 over 52 minutes, collecting the column effluent in fractions of 13.2 ml.
• the elution of a protein peak is observed.
• the fractions collected were analyzed for their G-CSF content by analytical RP-HPLC chromatography and by ELISA.
• the combined fractions (237 ml) contain 255 mg of GA-GCSF titrated by HPLC corresponding to a yield of 78.8% and a purity of 98.7%.
• the SP Sepharose solution thus obtained was also analyzed by SDS-PAGE ( Figure 7, well 6). 2) gel filtration chromatography.
• the UF concentrate was then subjected to a gel filtration chromatography stage as follows:
• FIG. 7 shows the analysis by SDS-PAGE of the filtration gel solution obtained respectively in the PBS buffer (well 9) and in the acetate buffer, pH 5.5 (well 11).

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• 1999
  • 1999-07-08 FR FR9908831A patent/FR2796071B1/fr not_active Expired - Fee Related
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