KR20070083387A - New Purification Method of Human Growth Hormone - Google Patents

Abstract

The present invention utilizes a mixture of a phenyl column, an eluent, an aqueous buffer and an organic solvent, and then desalting and lyophilizing, resulting in the cleavage position present on the macromolecules of human growth hormone and presenting it as impurities A commercial method for purifying human growth hormone obtained by DNA recombination techniques using hydrophobic interaction chromatography, which efficiently separates a truncated molecule from a target human growth hormone, furthermore, elution of the truncated molecule of human growth hormone It relates to a method that occurs before elution of the target human growth hormone to obtain hGH with a purity of at least 99.5%.

Description

A novel method of purifying human growth hormone {A NOVEL PROCESS FOR PURIFICATION OF HUMAN GROWTH HORMONE}

The present invention is a method for purifying human growth hormone (hGH) on a commercial scale using hydrophobic interaction chromatography that effectively separates the clipped hormone moiety formed during the production of growth hormone by DNA recombination technology. It is about.

Human growth hormone (hGH) is a pituitary derived protein with a number of important biological functions, including protein synthesis, cell proliferation and metabolism. hGH is a polypeptide of about 22 kDa with 191 amino acid residues. Recombinant human growth hormone (hGH) has been expressed in E. coli in the form of both intracellular and secreted proteins. Thereafter, a series of chromatographic and / or non-chromatographic methods are used to obtain the purified protein. It is known that the region between 140-150 amino acid residues of human growth hormone is sensitive to a number of proteases. This produces a protein that is truncated by proteolysis, whose physical properties are indistinguishable from that of an intact molecule. The variant form of hGH can appear in the purification process and its removal is a major challenge in producing therapeutic grade growth hormone proteins. In order to use hGH for therapeutic purposes it is necessary to remove the truncated molecule.

The prior art differs from the present invention in many respects, such as process parameters, equipment used, priority of target moiety separation, the use of multiple phases, the use of solvents, and the form of isolated impurities. . A few prior art documents disclose techniques that are more useful as analytical techniques than industrial separation methods.

US4332717 discloses the use of hydrophobic interaction chromatography for purifying human growth hormone. The patent purifies hGH extracted from human pituitary gland and is not related to recombinant hGH or its purification. Thus, the impurities generated in the method differ from those provided by recombinant hormones. In addition to using different media and different columns, the method uses different pressure conditions, different temperature conditions and different binding and elution buffers. Dissolution gradients are also different. The document also discloses the use of blue sepharose or agarose for separation and does not suggest the use of an organic and aqueous mixture, which is one of the essential properties of the present invention for elution.

The following references relate to the purification of growth hormone obtained by recombinant DNA technology. In the literature, a small number of patents are known, such as US4861868, which discloses a method for producing recombinant porcine growth hormone; US4705848, US4694073, US4731440, US5064943, US4975529, US5023323, US5109117, and US6410694 treat solubilization and maturation methods as essential properties, but they are outside the scope of the present invention.

US Patent 6022858 relates to the formulation of hGH, which hGH is pretreated with Zn and optionally lysine or calcium ions, then benzyl alcohol is added and the pH is adjusted to 2-9.

US Patent 5734024 discloses a method for determining the biological activity of recombinant hGH and is excellent for analytical purposes.

U. S. Patent 5182369 discloses a process wherein the operation is performed at a pH of 6.5 or below, and two-stage precipitation is an essential characteristic. However, process conditions for introducing acidic pH are disadvantageous because they can lead to aggregation or acid hydrolysis of proteins.

U. S. Patent 6451347 discloses a method for purifying hGH, in which complex formation with metal ions such as Zn ions is carried out, which is not a characteristic of the present invention. The patent discloses variants resulting from degradation of hGH but not truncated moieties.

U.S. Patent 6451987 emphasizes the use of cation exchangers for the purification of peptides containing hGH, but does not mention the separation of truncated hormone moieties from intact hGH molecules.

U.S. Patent 6437101 discloses a technique in which an aqueous two-phase extraction is introduced without the use of chaotropic agents. The method is cumbersome and therefore not easy to perform.

Patents referred to in this document by patent number are considered to be incorporated by reference, so far as the body of the patent is concerned.

Even today, the real problem with this purification is the separation of the truncated molecule, which is the result derived from a mega target molecule. The truncated moieties remain agglomerated together due to certain other bonds present in the molecule.

Non-patent prior art documents include the following three published articles on the use of hydrophobic interaction chromatography to separate hGH variants comprising truncated variants.

Pavlu and Gellerfors (Bioseparation (1993), 3: 257-265) discloses hydrophobic interaction chromatography of the recombinant human growth hormone Genotropin.

Gellerfors et al., Acta Pediatr Scand (Suppl) (1990), 370: 93-100 disclose the isolation and identification of growth hormone variants by high performance liquid chromatography techniques.

Wu et al, J. Chromatogr. (1990), 500: 595-606 disclose the application of high efficiency hydrophobic interaction chromatography for the characterization of DNA-derived human growth hormone.

The techniques disclosed in all of the above references use a surfactant Brij (polyoxyethylene 23 lauryl ether) as the mobile phase B, the peak corresponding to the truncated molecule appears after the main peak. . The papers disclose the use of acetonitrile of 0.5% in mobile phase A and 5% in mobile phase B with 0.075% breeze. Breeze 35 was used to improve product recovery from 70% to 99%. Detergent Breeze 35 helps improve recovery by reducing protein-matrix interactions. Interestingly, the peak of the target molecule exceeds the peak of the variant. These papers essentially disclose HPLC techniques and are better used for analytical purposes than for industrial production purposes. The main drawback of the above methods is that using detergents such as breezes is not preferred on an industrial scale because it can be difficult to remove them from solutions containing proteins.

The present invention utilizes organic solvents in much higher proportions and does not use such breezes. In addition, it is noted that the peak corresponding to the variant exceeds the peak of the target molecule.

The prior art shows the separation between truncated and intact molecules at analytical level loading of approximately 50-150 μg semi-purified protein per 3.3 ml column volume. In the present invention, separation is achieved at loading of 1 mg semi-tablet protein per ml column volume.

A limitation of this prior art is the use of Breeze 35, a nonionic surfactant, to improve process recovery at the assay level. The presence of such nonionic surfactants may be undesirable at the preparation level because it is difficult to remove the surfactant that remains bound to the protein molecule. The claimed method uses surfactants to improve process recovery by using organic solvents in greater amounts in eluant that can be easily removed by tangential flow filtration or gel filtration chromatography. Overcome the need to use

Purpose of the Invention

It is a main object of the present invention to provide a method for purifying human growth hormone which can effectively separate the truncated molecule from the target molecule.

Another object of the present invention is to provide a method for purifying human growth hormone which effectively removes the truncated molecule first to better control the production of the target molecule.

Another object of the present invention is to describe a method for purifying human growth hormone that can be used for multiple purposes such as analytical methods for hGH, isolation of hGH and industrial purification of hGH.

Another object of the present invention is to provide a purification method that can be effectively performed in the pH range of 8.0 to 9.0.

Another object of the present invention is to develop a method for purifying human growth hormone that does not use detergents or surfactants at all.

Summary of the Invention

The present invention relates to a novel method of purifying hGH obtained by recombinant technology using hydrophobic interaction chromatography. The present invention also provides polymer hydrophobic beads and eluents, which are solid supports, to separate target hGH molecules from impurities that are present as impurities and finally desalted and lyophilized by gel filtration to obtain purified hGH. It relates to using a mixture of an aqueous buffer and an organic solvent.

The features of the present invention will become more apparent from the following detailed description of the subject matter of the present invention and the description of the preferred embodiments.

Detailed description of the invention

In accordance with this object, the present invention discloses a purification technique for hGH. Escherichia coli cells comprising the recombinant human growth hormone (hGH) gene were grown in a 1 L shake flask under standard conditions. After 8-10 h induction, cells were harvested by centrifugation at 4-8 ° C. for 15 min. The supernatant was discarded and the cell pellet was suspended in lysis buffer containing 20-50 mM Tris, pH 7.0-9.0 and 100-500 mM NaCl. Cells were disrupted for 30 minutes using ultrasound. The temperature during crushing was maintained at 4-8 ° C. by placing the sample on ice.

Crude lysate was clarified by centrifugation at 16,000 rpm for 1 hour at 4 ° C. After centrifugation, the pellet was discarded and imidazole was added to the clear supernatant to a final concentration of 20-40 mM. It was loaded on a 10 ml chelating Sepharose bead column filled with NiSO ₄ . The column was equilibrated at a flow rate of 5-20 ml / min with a buffer containing 20-50 mM Tris pH 7.0-9.0, 100-500 mM NaCl, 20-40 mM imidazole. Unbound material was washed using the equilibration buffer and after absorbance at 280 fell to baseline, it included 20-50 mM Tris pH 7.0-9.0, 100-500 mM NaCl, 200-500 mM imidazole. Elution of the bound protein was performed using an elution buffer. Protein concentration in the eluate was measured to a concentration of 5-10 mg / ml and maintained at 4-10 ° C. for enzymatic digestion. Digestion was performed for 15-24 hours and stopped at the final concentration of 0.2-0.4 M by addition of 2 M K ₂ HPO ₄ , pH 7-9.0 solution. Digested samples were phenyl Sepharose FF column equilibrated at a flow rate of 5-10 mL / min with a buffer containing 20-50 mM Tris, 0.2-0.4 MK ₂ HPO ₄ , pH 7.0-9.0 (column volume = 15 mL). Loaded onto the bed. Bound protein eluted with water and peak fractions were collected. This was loaded on a Q Sepharose FF column (column volume = 10 mL) equilibrated with 20-50 mM Tris pH 7.0-9.0. Elution was carried out using 15-30 column volumes of 0% to 30% (v / v) buffer "A" linear gradient with 0.5-2 M NaCl. The main peak at 280 nm containing human growth hormone was collected and 2 MK ₂ HPO ₄ solution was added to obtain a final concentration of 0.2-0.6 M.

The samples were analyzed by varying pH, equilibration buffer and eluent conditions using hydrophobic interaction chromatography.

According to the present invention there is provided a method for purifying human growth hormone from a truncated moiety of hGH molecules using hydrophobic interaction chromatography techniques, the method comprising

(a) loading a sample into a column in the presence of a high concentration of inorganic salts;

(b) equilibrating the column loaded with the sample with an aqueous buffer;

(c) eluting the column of step (b) with a linear gradient of the aqueous buffer and organic solvent mixture;

(d) collecting and binding the eluted fraction corresponding to the hGH peak and concentrating the bound fraction;

(e) desalting and lyophilizing by filtering the concentrated fraction of step (d) on a Sephacryl S-200 gel equilibrated with a solution of disodium hydrogen phosphate at pH 6.0-9.0; And

(f) obtaining purified human growth hormone.

The method uses semi-purified human growth hormone as a sample for purification.

The column used is a hydrophobic resin and is a crosslinked polystyrene divinyl benzene polymer with hydrophobic ligands selected from the group consisting of ether, isopropyl, butyl, octyl and phenyl.

The hydrophobic ligand is preferably a phenyl group.

The inorganic salt is selected from the group consisting of ammonium sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate or sodium chloride, preferably disodium hydrogen phosphate, most preferably dipotassium hydrogen phosphate.

The concentration of the inorganic salts used is in the range of 0.2-0.6 M, more preferably 0.3-0.4 M.

The buffer used to equilibrate the column is a mixture of aqueous disodium phosphate and dipotassium hydrogen phosphate. The pH of the equilibration buffer is in the range of 6.0 to 9.0, preferably in the range of 8.0 to 9.0.

The buffer used for protein elution is a mixture of disodium hydrogen phosphate or Tris buffer with organic solvent.

The pH of the elution buffer is preferably in the range between 8.0 and 9.0.

The organic solvent used for protein elution is selected from the group consisting of C ₁ to C ₄ alcohols, acetonitrile and mixtures thereof.

The organic solvent in the elution mixture is in the range between 40-70% v / v, more preferably 40% -50% v / v.

The temperature for chromatographic separation is preferably in the range of 20-30 ° C., more preferably 22-24 ° C.

The growth hormone peak eluted in the hydrophobic interaction chromatography step was concentrated and further desalted on a Sephacryl S-200 gel filtration column equilibrated with 2-10 mM disodium hydrogen phosphate at pH 7.0-9.0. The desalted fractions were collected and lyophilized to obtain purified hGH.

It is known that 0.5 to 5% of acetonitrile cannot separate the moieties cut from recombinant hGH (Gellerfors P et al., Acta Pediatr Scand (Supple) (1990) 370, 93-100, growth by high efficiency liquid chromatography technique. Isolation and identification of hormonal variants). In the present invention, it has been found that by using acetonitrile in the range of 50% ± 10%, unwanted truncated molecules are first separated, and in turn, the target molecules can be effectively separated at a purity of 99.5% or more. pH also plays an important role in the separation efficiency. When the experiment was performed, it was observed that when the pH was acidic it was not effectively separated, whereas as the pH was increased above neutral it was more effectively separated. It was observed that a pH in the range of 8-9 yielded an optimal tablet, i.e. a product of the desired quality (FIGS. 1 and 2). After a series of experiments, it was concluded that pH plays an important role in achieving separation of the truncated hGH and intact hGH molecules using Resource phenyl chromatography. Separation is most effective at pH 8-9. Dropping the pH below 8.0 reduces the separation (Figure 3) and at pH 6.0 both molecules elute with a single peak. In all chromatograms, the left peak corresponds to truncated hGH and the right peak corresponds to intact hGH.

Various other techniques we have attempted to separate the truncated molecule from intact hGH are those mentioned below:

1.Ion-exchange with both polymer and Sepharose beads-using an aqueous buffer as well as a mixture of aqueous-organic solvents and detergents

2. Hydrophobic Interaction Chromatography with Sepharose Beads with Different Hydrophobicity

3. Gel Filtration Chromatography Using Native and Reduction-Modified Conditions

A brief description of the items of conditions used for purification and the results are shown below.

Experimental details

Ion-Exchange Chromatography: Ion exchange chromatography with source 15 Q beads was attempted to separate intact molecules from hGH molecules that were cut using a sodium chloride gradient for elution (FIG. 27).

Observation: Separation of truncated molecules from intact hGH was not observed in the chromatography method in any fraction analyzed by SDS-PAGE gel.

Gel Filtration Chromatography-

Column: Superdex 75 HR 10/30, Amersham

Buffer: 20 mM Tris, pH 8.0 / 5% Glycerol / 150 mM NaCl

Sample: denatured in hGH-6 M urea with truncated molecules and reduced to 50 mM DTT for 2 hours at room temperature

Detection: 280 nm

Fraction: 1 ml

System used: FPLC at 0.5 ml / min flow rate

Assay: 10 μl of each fraction analyzed on SDS-PAGE followed by silver staining (FIG. 28)

Observation: Separation of truncated molecules from intact hGH was not observed in the chromatography method in any fraction analyzed by SDS-PAGE gel.

Sepharose Bead  Interaction Chromatography of Phase (FIG. 29)-

Column: High Trap 1 ml column of Phenyl Sepharose FF (high sub) / Butyl Sepharose FF / Phenyl Sepharose HP / Octyl Sepharose FF

Buffer A: 20 mM sodium phosphate, pH 7.0 / 0.5 m (NH ₄ ) ₂ SO ₄

Buffer B: 20 mM sodium phosphate, pH 7.0

Sample: hGH with truncated molecules

Detection: 280 nm

Fraction: 1 ml

System used: AKTA Explorer at 1 ml / min flow rate

Observation: No truncated molecular separation from intact hGH was observed in this chromatography method.

The table below shows the chromatographic separation conditions for the semi-purified hGH in connection with each figure.

Drawing number Chromatography Buffer A Buffer B Sample history One Column: Resources PHE (1.0 mL) Amersham 20 mM Sodium Phosphate /0.4 M K ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH  9.0 / 50% Acetonitrile Sample: 1.0 mg detection 280 nm  2 Column: Resources PHE (1.0 mL) Amersham 20 mM Sodium Phosphate /0.4 M K ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH  9.0 / 40% Acetonitrile Sample volume: 1.0 mL detection 280 nm  3 Column: Resources PHE (1.0 mL) Amersham 20 mM Sodium Phosphate /0.4 M K ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 30% acetonitrile Sample volume: 1.0 mL detection 280 nm  4 Column: Resources PHE (1.0 mL) Amersham 20 mM Sodium Phosphate /0.4 M K ₂ HPO ₄ , pH 8.0 20 mM sodium phosphate, pH  8.0 / 50% Acetonitrile Sample: 1 mg detection 280 nm  5 Column: Resources PHE (1.0 mL) Amersham 20 mM Sodium Phosphate /0.4 M K ₂ HPO ₄ , pH 7.0 20 mM sodium phosphate, pH  7.0 / 50% Acetonitrile Sample: 1.0 mg detection 280 nm

Drawing number Chromatography Buffer A Buffer B Sample history 6 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 6.0 20 mM sodium phosphate, pH 6.0 / 50% acetonitrile Sample: 1.0 mg detection 280 nm 7 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 50% methanol Sample: 1.0 mg detection 280 nm 8 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 40% methanol Sample: 1.0 mg detection 280 nm 9 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 30% methanol Sample: 1.0 mg detection 280 nm 10 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 8.0 20 mM sodium phosphate, pH 8.0 / 50% methanol Sample: 1 mg detection 280 nm 11 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 7.0 20 mM sodium phosphate, pH 7.0 / 50% methanol Sample: 1 mg detection 280 nm 12 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 6.0 20 mM sodium phosphate, pH 6.0 / 50% methanol Sample: 1 mg detection 280 nm 13 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 50% isopropanol Sample: 1 mg detection 280 nm 14 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 40% isopropanol Sample: 1 mg detection 280 nm 15 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 30% isopropanol Sample: 1 mg detection 280 nm 16 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 8.0 20 mM sodium phosphate, pH 8.0 / 50% isopropanol Sample: 1 mg detection 280 nm 17 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 7.0 20 mM sodium phosphate, pH 7.0 / 50% isopropanol Sample: 1 mg detection 280 nm 18 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 6.0 20 mM sodium phosphate, pH 6.0 / 50% isopropanol Sample: 1 mg ion exchange purified sample detection 280 nm 19 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 25% acetonitrile / 25% methanol Sample: 1 mg detection 280 nm 20 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 25% acetonitrile / 25% isopropanol Sample: 1 mg detection 280 nm

Drawing number Chromatography Buffer A Buffer B Sample history 21 Column: Rixophenyl (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM sodium phosphate, pH 9.0 / 25% isopropanol / 25% methanol Sample: 1 mg detection 280 nm 22 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 8.0 20 mM sodium phosphate, pH 8.0 / 50% acetonitrile Sample: GH, IEX fraction, sample volume: 1.0 mL, detection 280 nm 23 Column: Resources PHE (1.0 mL) Amersham 20 mM Tris / 0.4 MK ₂ HPO ₄ , pH 9.0 20 mM Tris, pH 9.0 / 50% acetonitrile Sample: 1.0 mg detection 280 nm 24 Column: Sephacryl S-200 HR (Amersham) 100 ml bed volume 20 mM sodium phosphate, pH 8.0 Detection 280 nm 25 Column: Resources PHE (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 8.0 20 M Na-P, pH 8.0 Sample: 1 mg detection 280 nm 26 Column: Phenyl Sepharose FF (1.0 mL) Amersham 20 mM sodium phosphate / 0.4 MK ₂ HPO ₄ , pH 8.0 20 M Na-P / 50% Acetonitrile, pH 8.0 Sample: 1 mg detection 280 nm 27 Column: Sauce 15 Q, Amersham 10 mM NH ₄ Cl / 10 mM Tris, pH 8.0 / 3% EtOH 20 mM NH ₄ Cl, pH 8.0 /7.5% EtOH / 500 mM NaCl Sample: Cu-IDA purified hGH detection with truncated molecules 280 nm Fraction: 1 ml System used: FPLC analysis at 1 ml / min flow rate: 10 μl of each fraction was analyzed on SDS-PAGE gel and then stained silver 28 Column: Superdex 75 HR 10/30, Amersham 20 mM Tris, pH 8.0 / 5% Glycerol / 150 mM NaCl Sample: Denature in hGH-6 M urea with truncated molecules and reduce for 2 hours at room temperature with 50 mM DTT 280 nm Fraction: 1 ml System used: FPLC analysis at 0.5 ml / min flow rate: 10 μl of each fraction of SDS Analyzed on -PAGE gel and then stained 29 Column: High Trap 1 ml column of Phenyl Sepharose FF (high sub) / Butyl Sepharose FF / Phenyl Sepharose HP / Octyl Sepharose FF 20 mM sodium phosphate, pH 7.0 / 0.5 m (NH ₄ ) ₂ SO ₄ 20 mM sodium phosphate, pH 7.0 Sample: hGH detection with truncated molecules 280 nm Fraction: 1 ml System used: AKTA Explorer at 1 ml / min flow rate

The following examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The invention has been described in terms of specific embodiments, and variations and equivalents thereof will be apparent to those skilled in the art and are intended to be included within the scope of the invention.

Example 1

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecules eluted before the intact GH molecules (FIG. 1). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 2

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 40% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 2). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 3

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 30% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules were not isolated from intact molecules (FIG. 3).

Example 4

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 8.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 8.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecules eluted before the intact GH molecules (FIG. 4). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 5

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 7.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 7.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE (FIG. 5) (purity:> 80%, yield: 35%).

Example 6

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 6.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 6.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules were not isolated from intact molecules (FIG. 6).

Example 7

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 50% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 7). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 8

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 40% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 8). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 9

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 30% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules were not isolated from intact molecules (FIG. 9) (purity:> 95%, yield: 45%).

Example 10

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 8.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 8.0 / 50% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 10). High purity human growth hormone was obtained (purity:> 98%, yield: 85%).

Example 11

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 7.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 7.0 / 50% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 11) (purity was not obtained).

Example 12

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 6.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 6.0 / 50% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecules eluted before the intact GH molecules (FIG. 12) (purity was not obtained).

Example 13

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 50% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 13). High purity human growth hormone was obtained (purity:> 99%, yield: 85%).

Example 14

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 40% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecule eluted before the intact GH molecule (FIG. 14). High purity human growth hormone was obtained (purity:> 98%, yield: 80%).

Example 15

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 30% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 15) (purity:> 95%, yield: 60%).

Example 16

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 8.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 8.0 / 50% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecule eluted before the intact GH molecule (FIG. 16) (purity was not obtained).

Example 17

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 7.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 7.0 / 50% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecule eluted before the intact GH molecule (FIG. 17) (purity was not obtained).

Example 18

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 6.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 6.0 / 50% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. The truncated hGH molecules eluted before the intact GH molecules (FIG. 18) (purity was not obtained).

Example 19

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. The bound protein was eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 25% acetonitrile / 25% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 19). High purity human growth hormone was obtained (purity:> 99%, yield: 80%).

Example 20

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 25% acetonitrile / 25% isopropanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 20). High purity human growth hormone was obtained (purity:> 99%, yield: 85%).

Example 21

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 25% isopropanol / 25% methanol. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 21). High purity human growth hormone was obtained (purity:> 99%, yield: 85%).

Example 22

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a resource phenyl column (purchased from Amersham Bioscience) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 9.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE according to the method of Laemlli. The gel was run for 45 min at 25 mA and then stained to visualize protein bands (FIG. 22). The truncated hGH molecules eluted before the intact GH molecules and separated as shown in the gel photograph.

Example 23

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Tris / 0.4 MK ₂ HPO ₄ , pH 9.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Tris, pH 9.0 / 50% acetonitrile. 1 ml fractions were collected and analyzed by SDS-PAGE. Truncated hGH molecules eluted before intact GH molecules (FIG. 23). High purity human growth hormone was obtained (purity:> 99.5%, yield: 85%).

Example 24

Resource PHE Purified Protein Fraction of 9 mM Hydrogen Phosphate It was loaded into a 100 ml bed volume of Sephacryl S-200 HR column equilibrated with disodium buffer , pH 8.0. The column was eluted at 0.4 ml / min with the same buffer. Protein peaks detected at 280 nm were collected and lyophilized to obtain purified hGH (> 99.5%) with a yield of at least 95% (FIG. 24).

Example 25

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml resource phenyl column (30 × 6.4 mm) equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 8.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ , pH 8.0. 1 ml fractions were collected and analyzed by SDS-PAGE (FIG. 25). No separation of the truncated molecule and the intact hGH molecule is obtained.

Example 26

To the ion-exchange purified hGH fraction, K ₂ HPO ₄ is added to a final concentration of 0.4 M. It was injected onto a 1 ml phenyl Sepharose FF column equilibrated with 20 mM Na ₂ HPO ₄ /0.4 MK ₂ HPO ₄ , pH 8.0. Bound proteins were eluted with a 20 ml linear gradient of 20 mM Na ₂ HPO ₄ /50% acetonitrile, pH 8.0. 1 ml fractions were collected and analyzed by SDS-PAGE (FIG. 26). No separation of the truncated molecule and the intact hGH molecule is obtained.

Claims (11)

(a) loading a sample into a column in the presence of an inorganic salt having a concentration in the range of 0.2-0.5 M; (b) equilibrating the column of step (a) with an aqueous buffer having a pH in the range of 6.0-9.0; (c) eluting the equilibrated column of step (b) with a linear gradient of aqueous buffer and organic solvent in the range of 40-70% v / v; (d) collecting and binding the eluted fraction corresponding to the hGH peak and concentrating the bound fraction; (e) desalting and lyophilizing by filtering the concentrated fraction of step (d) on a Sephacryl S-200 gel equilibrated with disodium hydrogen phosphate solution at pH 6.0-9.0; And (f) purifying human growth hormone from a truncated moiety of hGH molecules using hydrophobic interaction chromatography techniques, comprising obtaining purified human growth hormone. The method of claim 1, The sample used in step (a) is a semi-purified human growth hormone method. The method of claim 1, The column used in step (a) is a hydrophobic resin comprising a crosslinked polystyrene divinyl benzene polymer attached with a hydrophobic ligand selected from the group consisting of ether, isopropyl, butyl, octyl and phenyl. The method of claim 3, Wherein said hydrophobic ligand is preferably phenyl. The method of claim 1, The inorganic salt used in step (a) is selected from the group consisting of ammonium sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate or sodium chloride, preferably disodium hydrogen phosphate, most preferably dipotassium hydrogen phosphate. . The method of claim 1, In step (b), the aqueous buffer used to equilibrate the column is a mixture of aqueous disodium phosphate and dipotassium hydrogen phosphate. The method of claim 1, The aqueous buffer of step (c) is disodium hydrogen phosphate or tris buffer. The method of claim 1, The organic solvent used in step (c) is selected from the group consisting of C ₁ to C ₄ alcohols, acetonitrile and / or mixtures thereof. The method of claim 1, The linear gradient range of step (c) is preferably 40-50%. The method of claim 1, The pH of the aqueous buffer and organic solvent of step (c) is preferably in the range of 6.0-9.0. The method of claim 1, The temperature for the chromatographic separation is preferably in the range 20-30 ° C., more preferably 22-24 ° C.

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