JP4146728B2 - Method for purifying molecules using unbranched alkyl-terminated diols - Google Patents

Description

  The present invention relates to a method for purifying molecules using unbranched alkyl-terminated diols. In particular, the method of the present invention is performed on a reverse phase liquid chromatography column using a buffer containing 1,5-pentanediol, 1,6-hexanediol or 1,7-heptanediol, in particular molecules, Useful for purifying proteins and peptides.

  Proteins and peptides play a vital role in metabolism, gene expression, signal transduction, cellular and extracellular structures, and are essential for the survival and / or reproduction of any organism. Many proteins and peptides are useful in therapeutic and / or diagnostic applications and are particularly useful when available in pure form. Impurities can interfere with achieving therapeutic and / or diagnostic purposes and endanger the patient's health.

  Protein purification and peptide purification are particularly important challenges, especially when large amounts of material are required for therapeutic or diagnostic purposes. There is a strong need for a method that can easily and quickly provide important proteins and peptides in a pure form and in a high yield regardless of the scale.

  Proteins and peptides are often created by recombinant DNA technology. This is because large amounts of foreign material can be expressed in bacteria or other host cells. As is well known in the art, recombinant DNA technology involves injecting a host cell with DNA encoding the protein and allowing the cell to grow under conditions that favor expression of the heterologous protein. (See, for example, US Pat. No. 4,565,785, US Pat. No. 4,673,641, US Pat. No. 4,738,921).

  Human growth hormone (hGH) and hGH antagonists (ie, growth hormone antagonists (GHAs)) are examples of recombinantly produced proteins that are available in several therapeutic areas. Human growth hormone treats hypophyseal dwarfism and all pathologies caused by reduced levels of hGH production, whether the condition is due to genetic defects, damage or pituitary resection It is used for. Human growth hormone can also speed up the recovery of burn victims and other hospitalized patients. On the other hand, GHA has been used for the treatment of acromegaly, a form of giantism caused by overexpression of hGH (Kopick et al., US Pat. Nos. 5,681,809, 5,958,879, 5,350). , 836). In addition, GHA could be used to prevent retinopathy in diabetic patients and to treat cancer patients with tumors that overexpress receptors that bind growth hormone (Clark et al., 1996, International Publication WO 97). / 11178).

  Reversed phase liquid chromatography (“RP-LC”) and reversed phase high performance liquid chromatography (“RP-HPLC”) are widely used to purify molecules such as peptides and proteins produced by synthetic or recombinant methods. used. The RP-LC and RP-HPLC methods are capable of efficiently separating closely related impurities and are used to purify many diverse molecules (Lee et al., “Preparative HPLC” 8th Biotechnology Symposium, Part 1, 593-610 (1988)). Furthermore, RP-LC and RP-HPLC have been used successfully for the purification of molecules, especially proteins, on an industrial scale (Olsen et al., 1994, J. Chromatog. A, 675, 101). page).

  Typical eluents for RP-LC and RP-HPLC are ethanol, isopropanol, methanol and acetonitrile. Acetonitrile has been used for the purification of proteins such as insulin (Kroeff et al., 1989, J. Chromatography, 461, 45). However, all of these solvents are highly flammable and toxic. When used in a large amount, they become a problem that cannot be ignored for safety. In addition, acetonitrile often exhibits a denaturing effect when used as an eluent for reverse phase chromatography. The disposal of toxic organic solvents, especially on a manufacturing scale, requires considerable costs.

  Accordingly, in the art, RP-LC and RP using non-flammable solvents that are less toxic, cheaper and less denatured than typical solvents used as eluents in RP-LC and RP-HPLC. There is a need for a method that selectively separates molecules such as peptides, polypeptides (eg, hGH and GHA) and organic compounds from impurities by HPLC.

  The present invention provides a method for purifying molecules by RP-LC and RP-HPLC using an unbranched alkyl-terminated diol as the eluting solvent. Specifically, the present invention relates to molecules, particularly proteins and peptides, by reverse phase liquid chromatography columns using a buffer containing 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol. Is to be purified. These non-flammable terminal alkyl diols are less toxic, less expensive and less denaturing than typical solvents used as eluents in RP-LC and RP-HPLC.

  In one embodiment, the present invention provides a method for purifying molecules from a mixture. First, the mixture is loaded onto a reverse phase liquid chromatography column. The molecules are then eluted from the column with a buffer containing one diol that is 1,5-pentanediol, 1,6-hexanediol, or 1,7-heptanediol. In a preferred embodiment, the diol is 1,6-hexanediol. In another embodiment, the column is a high pressure reverse phase liquid chromatography column.

  The pH of the buffer is preferably between about 2.0 and about 12.0, more preferably between about 7.0 and about 11.0, and most preferably between about 6.0 and about 8.0. The concentration of 1,6-hexanedioe in the buffer is preferably between about 0% and about 80%, more preferably between about 0% and about 50%.

  In one embodiment, the molecule is a polypeptide, preferably hGH or GHA. In a preferred embodiment, the polypeptide is GHA. In another preferred embodiment, the polypeptide is human growth hormone. In other embodiments, the molecule is a peptide, preferably α-MSH, enkephalin, somatostatin or somatotropin.

  In one embodiment, the column is a high performance liquid chromatography column. In other embodiments, the column is a preparative column. The diameter of the column is preferably between about 5 cm and about 2.0 m, more preferably between about 10 cm and about 100 cm.

  Preferably, the column is packed with a polymer resin. In one preferred embodiment, the polymer resin is styrene divinylbenzene (ie, styrenic). In other preferred embodiments, the polymer resin is methacrylate or acrylic.

  In one embodiment, the mixture is loaded onto the column at a rate of about 10.0 g molecules / packed bed volume (L). In other embodiments, after fractionation with RP-LC, the molecule is further purified.

  The present invention relates to a method for purifying molecules using linear alkyl-terminated diols. Details for carrying out the present invention are described below.

Definitions As used herein, “molecule” refers to a peptide, polypeptide or non-peptide compound.

  As used herein, “peptide” refers to molecules in which up to about 30 amino acids, including natural and unnatural amino acids that are L-form or D-form isomers, and derivatives and / or analogues thereof are covalently linked. Point to. Examples of peptides include molecules such as enkephalin, somatostatin and α-MSH. Other peptides that can be purified using the methods of the present invention include, but are not limited to, those bioactive peptides listed in the catalog available from Bachem Biosciences Limited (Philadelphia, PA). .

  As used herein, “polypeptide” refers to peptides containing more than 30 amino acids and includes both endogenous and exogenous (ie, heterologous) polypeptides. Examples of proteins include, but are not limited to, human growth hormone and growth hormone antagonists.

  As used herein, “non-peptidized compound” refers to an inorganic or organic compound having a molecular weight of about 200-800 daltons. The compound is preferably an organic compound. Particularly preferred organic compounds are antibiotics such as vancomycin, cephalosporin and penicillin.

  As used herein, “growth hormone antagonist (“ GHA ”)” refers to an antagonist of human growth hormone. GHA has a growth inhibitory action, that is, a human growth hormone antagonistic action. GHA can be used to reduce human growth hormone activity in patients suffering from diabetes, diabetic retinopathy, diabetic nephropathy, growth hormone tumors, acromegaly or giantosis.

  As used herein, “buffer” refers to a solution that prevents pH change by an acid-base conjugate component. Suitable free acids to form the buffer include citric acid, acetic acid, phosphoric acid, maleic acid, malonic acid, phthalic acid, salicylic acid, fumaric acid, dimethylmalonic acid, mandelic acid, malic acid, formic acid, tartaric acid, Itaconic acid, lactic acid, barbituric acid, ascorbic acid, 2,2-dimethylsuccinic acid, succinic acid, benzoic acid, propionic acid and the like are included. Suitable free bases for forming the buffer include Tris, triethylamine, imidazole, brucine, tricine, glycinamide, histidine, ethanolamine, glycine, ethylamine, dimethylamine and the like. Those skilled in the art will appreciate that buffers can be prepared using various other acids and bases. Preferred buffer pH is generally about 2.0 to about 12.0.

Source of molecules The molecules can be prepared by any technique known in the art. For example, a protein or peptide may be a prokaryotic culture secreting a wild-type or heterologous protein or peptide, a prokaryotic lysis, a prokaryotic lysis expressing a heterologous protein or peptide, a eukaryotic lysis, a heterologous protein or peptide Can be obtained by lysis of eukaryotic organisms expressing E. coli, growth of eukaryotic organisms secreting soluble proteins or peptides, total synthesis, and the like. Organic or inorganic molecules can be prepared using synthetic methods and procedures known to those skilled in the art. Molecules are commercially available from known chemical suppliers (eg, Alfa Aesar (Wardhill, Mass.), Merck KgaA (Darmstadt, Germany)).

  Proteins or peptides are obtained by prokaryotic cell lysis. Several methods can be used to lyse bacterial cells, such as bead grinding, osmotic shock, freeze disruption, and enzyme treatment. Preferably, the bacterial cells are lysed using a high-pressure homogenizer such as a microfluidizer. Alternatively, a protein or peptide can be obtained by culturing a microorganism that secretes a wild-type or heterologous protein or peptide. Wild-type prokaryotic cells or prokaryotic cells expressing heterologous proteins can be grown under various conditions known to those skilled in the art. Inoculum growth methods, inoculum media are known to those skilled in the art, and specific methods are described in the art. Preferred media, time, temperature, and pH for culturing microorganisms are also known in the art. Thus, for example, the cells can be grown in a medium suitable for the growth of the cells, such as a minimal medium or a complete medium (ie, a rich medium).

  Heterologous proteins can be produced by recombinant DNA techniques, advantageously using techniques well known in the art for gene expression. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. For example, Sambrook et al., “Molecular Cloning” 1-3 (1989) and its periodic revisions, Cold spring Harbor Laboratory Press (Cold Spring Harbor, NY), and “Current Protocols” edited by Ausubel et al. See the technology described in 1989, Green Publishing Associates, Inc, and John Wiley & Sons, Inc (New York). DNA and RNA encoding any heterologous protein can be chemically synthesized using, for example, a synthesizer. For example, Gait, M. et al. J. et al. See the technique described in the edit “Oligonucleotide Synthesis”, 1984, GIRL Press, Oxford.

  A variety of host-expression vector systems may be utilized to express the protein. Expression systems that can be used for the purposes of the present invention include phagemid DNA containing a nucleotide sequence encoding a desired protein, a microorganism such as a bacterium (eg, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA Or a cosmid DNA expression vector, a yeast gene-injected with a recombinant yeast expression vector containing a nucleotide sequence encoding the protein of interest (eg, Sacchromyces, Pichia), a recombinant virus containing a nucleotide sequence encoding the protein of interest Insect cell systems infected with expression vectors (eg, baculovirus), recombinant viral expression vectors (eg, cauliflower mosaic virus CaMV, tag) containing nucleotide sequences encoding the protein of interest Promoters derived from genomes of plant cells or mammalian cells infected with recombinant mosaic expression vectors (eg, Ti plasmid) or mammalian cells infected with a comosa mosaic virus TMV) or mammalian viruses Mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3, U937) containing a recombinant expression construct containing a promoter derived from (eg, adenovirus late promoter, vaccinia virus 7.5K promoter) .

In eukaryotic systems, several selection systems are available, such as herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11, 223), hypoxanthine-guanine phosphoribosyltransferase. (Szybalska et al., 1962, Proc. Natl. Acad. Sci., USA 48, 2026) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell, 22: 817), respectively, tk ⁻ , Hprt ⁻ , aprt ⁻ cells. Antimetabolite resistance is also used as a basis for selection for the following genes: That is, dhfr (Wigler et al., 1980, Proc. Natl. Sci., USA 77, 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci., USA 78, 1527, which confers resistance to methotrexate. Page), gpt which imparts resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78, 2072), neo (Colberre-Garapin et al.) Which imparts resistance to aminoglycoside G-418, 1981, J. Mol. Biol. 150, 1) and hygro (Santerre et al., 1984, Gene 30, 147) conferring resistance to hygromycin.

  In bacterial systems, several expression vectors can be selected as described above. Bacteria suitable for the practice of the present invention are gram positive and gram negative bacteria. In a preferred embodiment, the soluble protein fluid is obtained by expression of a heterologous protein in Escherichia coli (“E. coli”) followed by cell lysis. Proteins can be expressed in prokaryotes using expression systems known to those skilled in the biotechnology art. Expression systems useful in the practice of the present invention are described in US Pat. Nos. 5,795,745, 5,714,346, 5,637,495, 5,496,713, 5,334,531, 4,634. No. 6,677, No. 4,604,359, No. 4,601,980.

  Prokaryotes can be grown under a variety of conditions known to those skilled in the art. In one embodiment of the invention, the cells are grown in a medium suitable for the growth of the cells, such as a minimal medium or a complete (ie, rich) medium.

Purification of molecules using terminal alkyl diols As a first step of the invention, it is necessary to load a mixture containing the molecules onto a reverse phase liquid chromatography column. The mixture can include several biological components such as carbohydrates, lipids, proteins, nucleic acids and the like. Mixtures can also include isomers closely related to the molecule, such as positional isomers, geometric isomers, stereoisomers, and the like.

  The molecule may be a peptide, polypeptide or organic molecule (eg antibiotic). Preferred peptide molecules are enkephalin, somatostatin, somatropin, and α-MSH. Other molecules that can be purified using the methods of the present invention include, but are not limited to, Bachem Biosciences Inc. Those listed in the list of bioactive peptides available from (Philadelphia, Pennsylvania). Thus, atrial natriuretic peptide, basal fibroblast inhibitory peptide, bradykinin, corticotropin inhibitory peptide, etc. are examples of molecules that can be purified using the method of the present invention.

  Preferred protein molecules are GHA and hGH. Other molecules that can be purified using the methods of the present invention include, but are not limited to, blood clotting factors, factor VIII, relaxin, insulin-like growth factor, interferon, tPA, antibodies, viral vaccine surface antigens such as porcine, bovine Or animal growth factor derived from sheep, insulin, erythropoietin, granulocyte-macrophage colony stimulating factor, IGF-2, interleukin and other cytokines, soluble receptor, soluble selectin, heregulin, vascular endothelial growth factor, epidermal growth factor ("EGF"), EGF-like growth factor, epidermal transforming growth factor, keratinocyte growth factor, tumor necrosis factor transforming growth factor, thrombopoietin and the like. Preferred organic molecules include antibiotics such as vancomycin, cephalosporin C, penicillins G and V. Other organic molecules that can be purified using the methods of the present invention include, but are not limited to, polyene macrolides, terpenes, alkaloids, carbohydrates, polyketides, and the like.

  The column may be a low pressure, medium pressure or high pressure column, the latter column being packed with a packing having a particle diameter of less than about 20 μm. A low pressure column is preferred for purification on a preparative scale. The column is packed with a filler having a particle diameter of preferably about 1 μm to about 300 μm, more preferably about 15 μm to about 120 μm, and most preferably about 35 μm to about 120 μm. The column packing preferably has a pore size of about 0 to about 1000, more preferably about 150 to about 300. Thus, non-porous (ie non-porous resin beads) and porous fillers can be used to pack RP-LC columns.

  Column resins include, but are not limited to, polystyrene divinylbenzene resin (eg, CG-300, CG-1000 or CG-161), acrylate resin or methacrylate resin (eg, CG-71-C or CG-71-M), Silica-based resins (eg, silica with C1-C18 alkyl groups, phenyl derivatives, aminoalkyl or aminophenyl compounds), agarose-based resins (eg, agarose with C1-C18 alkyl groups or phenyl derivatives) Polymer resins such as sepharose-based resins (eg, sepharose with C1-C18 alkyl groups or phenyl derivatives), sucrose-based resins (eg, sucrose with C1-C18 alkyl groups or phenyl derivatives) Including Is any suitable hydrophobic material. Those skilled in the art will appreciate that many other hydrophobic adsorbents are known in the art and can be used in the present invention. Column resins can be obtained from, for example, Bayer Corporation (Morris Township, NJ), Serva (Heidelberg, Germany), Alltech Associates, Inc. (Deerfield, Ill.) TosoHaas (Montgomery, Pennsylvania), Pharmacia (Piscataway, NJ), The Separation Group Inc. (Hesperia, CA), Waters Corporation (Milford, MA), Bio-Rad laboratories (Hercules, CA) and Polymer Labs (available from Church Stretton, Shropshire, UK) The type of resin used to purify the molecule can be readily determined by one skilled in the art and depends on factors such as the nature of the molecule, the required purity, and the amount of purified molecule required.

  The column can be an analytical column or a preparative column. The amount of molecules loaded on the column is generally from about 0.01 g to about 70.0 g, preferably from about 0.02 g to about 40.0 g, more preferably from about 0.05 g to about 0, per liter of packed bed. 30.0 g, more preferably about 1.0 g to about 25.0 g, most preferably about 3.0 g to about 15.0 g. In a preferred embodiment, the amount of molecules loaded on the column is about 10 grams per liter of packed bed. In general, the lower limit of the load is an amount capable of detecting the substance eluted from the column packed bed, and the upper limit of the load is the saturation capacity of the resin. The above amounts are only general guidelines, and the loading is determined by experimental factors such as the nature of the molecule, the flow rate, and the type of resin.

  In a preferred embodiment, the column is a preparative column, i.e. a column that runs on a preparative scale and / or loads for preparative. The column diameter is preferably about 5 cm to about 2 m, more preferably about 10 cm to about 100 cm.

  The column length must be in an appropriate ratio to the column diameter. In general, this determination is made empirically and can be easily made by one skilled in the art. Factors that may be important in determining the relationship between column diameter and column length include: bead size, elution method (eg isocratic vs. gradient elution), resin type (eg ion exchange column Short, size exclusion columns are longer, i.e. both columns are different in length due to different separation methods), the desired processing time and so on. Ideally, the optimum column length for a certain diameter is the length that allows the resin to efficiently separate impurities from the molecule. Usually, after determining the column length for each purification process, scaling up requires increasing the flow rate in proportion to the diameter, with the column length remaining fixed.

  In general, the column size is determined by the required amount of purified molecules. Thus, for example, columns with a diameter of 2 or 3 meters are used, especially when purifying large quantities of material.

  The solvent for loading may be any solvent, but a buffered aqueous solution containing a peptide or protein is particularly preferred for large-scale purification. In some cases (ie, purification of GHA), the loading solvent can include a large amount (about 10-20% w / v) of polyethylene glycol.

  The column flow rate can vary between about 30 cm / hr to about 300 cm / hr. The flow rate is preferably about 60 cm / hour when the column is loaded with molecules and about 120 cm / hour when the molecules are eluted from the column. It should be noted that the flow rate depends on the speed of the mobile phase, the bead size, the retention efficiency, and the pressure limit of the resin and equipment. The gradient gradient is preferably about 0.1% to about 5% (w / w) diol / column volume (CV), more preferably about 1.25% to about 5% (w / w) diol / CV, Most preferred is about 2.5% (w / w) diol / CV.

  As the next step of the invention, the molecules are eluted from the column using a buffer containing a diol such as 1,5-pentanediol, 1,6-hexanediol or 1,7-heptanediol. Usually, various components in the mixture are fractionated by the column during elution. The pH of the buffer is preferably from about 2.0 to about 12.0, more preferably from about 7.0 to about 11.0, and most preferably from about 6.0 to about 8.0. Those skilled in the art will appreciate that acidic chromatography requires a pH of about 2.0 to about 6.0. Similarly, neutral chromatography requires a pH of about 6.0 to about 8.0, and basic chromatography requires a pH of about 8.0 to about 12.0. In practicing the present invention, any suitable buffer that maintains the pH in the desired range for purification (eg, phosphate, acetate, Tris-HCL, triethylamine, trifluoroacetic acid, citrate, etc.) Can be used. One skilled in the art will be able to select an appropriate concentration of buffer.

  The concentration of diol used for elution varies depending on factors such as the type of molecule to be purified, the type of resin used, and the flow rate. However, one of ordinary skill in the art can readily determine the concentration of diol required to elute a molecule from the column without undue experimentation.

  The eluting solvent is preferably a terminal alkyl diol, more preferably 1,5-pentanediol, 1,6-hexanediol or 1,7-heptanediol, most preferably 1,6-hexanediol. In general, the maximum concentration of eluting solvent in a buffer is determined by the solubility limit of both the molecule and the diol. The concentration of 1.6-hexanediol in the buffer is preferably about 0% to 80%, more preferably about 0% to about 50%. The concentration of 1,5-pentanediol in the buffer is preferably about 0% to 80%, more preferably about 0% to about 70%. The preferred elution temperature is generally about 25 ° C., but may be lower or higher.

  The preferred operating pressure depends on whether the column design is for high pressure or low pressure. The high pressure column preferably operates from about 300 psi to about 5,000 psi, more preferably from about 500 psi to about 2,000 psi. The low pressure column preferably operates from about 0 psi to about 500 psi, more preferably from about 0 psi to about 50 psi.

  The conditions for purifying GHA include a RP-LC column having a diameter of about 45 cm to about 200 cm, a resin based on methacrylic acid ester (for example, CG71) or styrene (for example, CG300), and a buffer having a neutral pH. Preferably, the liquid is filled and loaded with about 1.0 g to about 10 g of GHA per liter of packed bed. GHA is eluted from the column using about 0-50% (v / v) 1,6-hexanediol dissolved in 50 mM TrisHCL solution. The column length is preferably about 10 cm to about 20 cm. The operating pressure is preferably from about 0 psi to about 500 psi, more preferably from about 0 psi to about 50 psi.

Post-treatment of molecules purified by unbranched alkyl-terminated diols The molecules can be further processed, for example to obtain higher purity molecules. The required purity depends on the potential use of the molecule. In some cases, it is possible to make the method of the present invention the final step of purification by simply replacing or concentrating the buffer prior to use for the intended application. Also, in some cases, it may be necessary to purify after purification by the method of the present invention and before use in the intended application.

  If necessary, any purification method known to those skilled in the art for further purification can be used. Such purification techniques are directed to proteins such as Walker, J. et al. M.M. Ed., Humana Press (Clifton, NJ), 1988, “New Protein Technologies: Methods in Molecular Biology” and Scopes, R .; K. Written by Springer-Verlag (New York, NY), published in 1987, “Protein Purification; Principles and Practice third edition”. In general, but not limited to, ammonium sulfate precipitation, centrifugation, ion exchange chromatography, affinity chromatography, gel filtration, reverse phase chromatography (and its HPLC or FPLC type), recrystallization, and adsorption chromatography (ie, The molecules can be further purified using techniques including (on silica or alumina).

  Hereinafter, the present invention will be further described with reference to examples, but the scope of the present invention is not limited thereto.

Experiments with BAKER BOND BUTYL A BAKER BOND BUTYL experiment was performed using an AKTA column (Amersham Pharmacia Biotech, Piscataway, NJ). GHA was eluted with a step gradient and a linear gradient. The gradient duration and gradient gradient were adjusted according to the experimental results. The eluate was monitored by UV absorbance at 280 nm. The buffer system is Buffer A which is 15 mM triethylamine phosphate ([TEAP]) (pH 7.2) and buffer which is 15 mM TEAP (pH 7.2) containing 60% to 70% 1,6-hexanediol. It consists of liquid B.

  The elution profile of GHA from a large pore butyl column (Mallinckrodt Baker Inc., Phillsburg, NJ) using 1,6-hexanediol as the eluent is shown in FIG. The results of SDS-PAGE analysis of the eluent are shown in FIG. 2 (lanes 2 to 4). Lane 4 corresponding to fraction 29 clearly contains GHA. Lanes 2 and 3 (fractions 8 and 22 respectively) contain some GHA but are much less than lane 4. Fraction 29 corresponds to the peak of the absorption peak at 280 nm. FIG. 3 shows the results of C-4 RP-HPLC analysis of the pooled fractions, with GHA peaks and succinylated GHA peaks appearing at 12.6 minutes and 13.4 minutes, respectively. The recovery rate of GHA was about 34%.

Small-scale experiment using methacrylate column CG71-M A 15 mM TEAP solution (pH 7.2) was flowed at a flow rate of 2 ml / min, and a 10 mm × 10 cm (8 ml) Amberchrom CG71-M column (TosoHaas, Montgomeryville, Pa.) Was used. Equilibrated. 27 ml of GHA solution (2.6 mg / ml, 50 mM Tris, pH 7.2, dissolved in 200 mM NaCl) was loaded onto the equilibrated CG71-M column and then washed with equilibration buffer. The concentration was initially 32.5%, then 49% and then 65% hexanediol in 15 mM TEAP solution (pH 7.2) and eluted stepwise from the column. The eluent was monitored by absorbance at 280 nm and the collected fractions were analyzed by SDS-PAGE and RP-HPLC.

  FIG. 4 shows the results of chromatography obtained with an 8.0 ml AmberchromCG71-M column. GHA was eluted with 98% recovery by a step gradient method of 32.5% hexanediol TEAP solution (pH 7.2). The results of SDS-PAGE analysis of the eluted protein are shown in FIG. As can be seen from FIG. 5, the eluted protein in lane 10 contains some impurities but is mainly GHA. Fractions 10 to 14 were pooled and analyzed for protein content by C-4 RP-HPLC. The results are shown in FIG. The volume of the pooled solution is 20 ml, the concentration is 3.43 mg / ml, and the total GHA is 68.6 mg. The amount of GHA loaded on the column is about 69 mg.

PH experiment using CG71-C AmberchromCG71-C (TosoHaas, Montgomeryville, Pa.) One column (5 mm x 10 mm, 2.0 ml) was equilibrated with a 15 mM TEAP solution (pH 7.2) and at the same time AmberchromCG71-C Another column (5 mm × 10 mm, 2.0 ml) was equilibrated with 0.1% trifluoroacetic acid solution (TFA). In experiments at low pH, Buffer A was 0.1% TFA solution and Buffer B was 80% 1,6-hexanediol 0.1% TFA solution. In the experiment at neutral pH, buffer A was a 15 mM TEAP solution (pH 7.2), and buffer B was a 65% 1,6-hexanediol 15 mM TEAP (pH 7.2) solution. Each column was loaded with a total of 15 mg of GHA obtained by the method of Hayenga et al. US patent application Ser. No. 09 / 307,549. A linear gradient from 0% to 100% of buffer B was performed until the column volume exceeded 20 times. The eluate was monitored by absorbance at 280 nm. Fractions were collected and analyzed by RP-HPLC and SDS-PAGE.

  The results are shown in FIGS. 7 and 8 and show that GHA can be eluted from a methacrylate-based column with 1,6-hexanediol. The peak of the elution profile has a shoulder, and it is clear from the profile that other proteins and impurities closely related thereto are fractionated. The elution profile in the neutral pH experiment has a narrower elution volume range compared to the profile at low pH. SDS-PAGE analysis of experiments at low pH (FIG. 9, gel 1, lanes 5-9 and gel 2, lanes 2-4) and SDS-PAGE analysis of experiments at neutral pH (FIG. 9, gel 2, lanes) 5-9) revealed that these fractions contained GHA, which could be confirmed by C-4 RP-HPLC analysis. The pooled amounts of fractions were 15 ml and 33 ml in the pH 7.2 and low pH experiments, respectively. The recovery rate of each column exceeds 96%, indicating that the recovered amount of GHA is about 14.5 mg.

Screening of methacrylate resins (CG71-M and CG71-C) and styrene divinylbenzene resin (CG300-M), respectively, Amberchrom CG71-M (TosoHaas, Montgomeryville, PA), Amberchrom CG71-C and Amberchrom CG300-M Three columns packed with Montgomeryville, PA were equilibrated with 15 mM TEAP solution (pH 7.2). 40 ml of dilute upper phase solution (prepared by two-phase extraction using the method of Hayenga et al. Of US Patent Application No. 09 / 307,549) containing about 0.5 mg / ml GHA in each column Loaded. The load on the column is 10 mg / ml. After loading, each column was washed with a 15 mM TEAP solution (pH 7.2). GHA was eluted from the column by a linear gradient method in which the hexanediol concentration in a 15 mM TEAP solution (pH 7.2) was changed from 0% to 65% (the amount of solvent used was about 20 times the column volume). The eluate was monitored by absorbance at 280 nm and the appropriate fractions were collected. Fractions were analyzed by C-4 RP-HPLC and SDS-PAGE.

  US Pat. Application No. 09 / 307,549 is a reverse phase resin based on styrene divinylbenzene that has been used to purify GHA from dilute upper phase liquid obtained using the method of Hayenga et al. It was decided to adopt the method of the present invention instead of Amberchrom CG1000. Acetonitrile was commonly used to elute GHA from Amberchrom CG1000, but due to its handling requirements and costly disposal, the cost of the process was rather high.

  Therefore, the gradient method using 1,6-hexanediol detailed above was used to elute the Amberchrom CG71-C, Amberchrom CG71-M methacrylate resin, and Amberchrom CG300-M styrene divinylbenzene resin loaded as described above. I did it. The elution profiles of each resin are shown in FIGS. From each chromatogram, it can be seen that protein can be eluted from the respective resin by 1,6-hexanediol. FIG. 13 shows the results of SDS-PAGE analysis of pooled eluents from CG71-C and CG71-M. Figures 15 and 16 are C-4 RP-HPLC chromatograms of pooled eluents from CG71-C and CG71-M, respectively.

  In the experiment of Amberchrom CG71-C, fractions 11 to 18 were pooled into one fraction (24 ml) (GHA recovery was 84%). Since unrecovered material was found in the removed eluent, the retention capacity of this resin against GHA appears to be about 8 mg / ml. In the experiment of Amberchrom CG71-M, fractions 10 to 16 were pooled (21 ml) (recovery rate 103%). The load on this column is 10 mg / ml with respect to GHA. In the Amberchrom CG71-M experiment, no GHA was detected in the removed eluent. The data is summarized in Table 1 below.

  From the Amberchrom CG300-M experiment and the resulting C-4 RP-HPLC and SDS-PAGE analysis results (FIGS. 17 and 14, respectively), GHA is eluted to some extent from this column by 1,6-hexanediol. I understand that. However, when looking at the profile (FIG. 11), the elution band broadens and continues until after the end of the gradient. Therefore, under these conditions, GHA cannot be completely eluted from the resin with 50% 1,6-hexanediol.

Scale-up using a step gradient In the preliminary experiments described in Example 4, good results were obtained with Amberchrom CG71-M resin, so this resin was selected for scale-up experiments. A 50 mM TrisHCL solution (pH 7.2) was passed through a 235 ml (5 cm × 12 cm) AmberchromCG71-M column for equilibration. 4 liters of a 2-fold diluted upper phase solution obtained using the method of Hayenga et al. Of US patent application Ser. No. 09 / 307,549 was loaded onto the column at a flow rate of 20 ml / min and 50 mM TrisHCL (pH 7.2). Elution was carried out by a step gradient method using 50% 1,6-hexanediol. The column was monitored by absorbance at 280 nm, divided into fractions and analyzed by C-4 RP-HPLC and SDS-PAGE.

  The chromatogram for the 235 ml AmberchromCG71-M experiment is shown in FIG. The elution profile has a single peak, which is consistent with the elution step with 50% 1,6-hexanediol. The column load in this experiment was 8.3 mg / ml (about 2000 mg per 235 ml resin). No GHA was observed in the C-4 RP-HPLC chromatogram (Figure 19a) of the excluded eluent. However, the GHA recovery rate was only 67%. Fractions 6-12 constitute the main peak and account for 47% of the eluate. The rest is in fractions 1-5 and fractions 13-20. According to the C-4 RP-HPLC chromatogram (Figure 19b) of the pooled fractions, the GHA was almost pure. In subsequent experiments, an excellent GHA recovery rate, usually 80% to 95% recovery rate, is obtained by the linear gradient method of 0% -50% 1,6-hexanediol 50 mM TrisHCL solution (pH 7.2). It has been found.

Large Scale GHA Production A 100 L (100 cm × 13 cm) Amberchrom CG71-M column was equilibrated by flowing 3 column volumes of 50 mM Tris solution (pH 7.2) at a rate of 150 cm / min. The diluted upper phase liquid (about 1000 L) obtained by the method of Hayenga et al. In US Patent Application No. 09 / 307,549 was loaded onto the column at a flow rate of 7.9 L / min. The column was loaded with about 10 g of GHA per liter of resin. Next, the column was washed by flowing a column volume of 50 mM Tris solution (pH 7.2) at 60 cm / hour and a volume of 50 mM Tris solution (pH 7.2) at 120 cm / hour. GHA was eluted using a gradient method with a 1,6-hexanediol concentration of 0% to 50% using an eluent of 20 column volumes. Buffer A is a 50 mM Tris solution (pH 7.2) (10 times the column volume), and Buffer B is a 50 mM Tris solution (pH 7.2) having a concentration of 1,6-hexanediol of 50% (w / w). ) (10 times the column volume). The elution of GHA was monitored by absorbance at 280 nm.

  Two main peaks elute in the gradient, but each fractioned separately. In general, the first peak elutes second to third counting in column volume units. The main peak containing GHA elutes at the sixth to twelfth (about 6 volumes) counting in column volume units, and becomes about 600 L when pooled. Both peaks were analyzed by C-4 RP-HPLC to determine the concentration of GHA. Usually, the GHA recovery was about 80% to 95% at the main peak. FIG. 20 is a typical chromatogram in 100 L scale purification using CG71-M resin. Table 2 below shows the recoveries of five different GHA purification experiments using the same resin.

  At the end of each purification experiment, the column is divided into two column volumes of 0% to 100% isopropanol gradient, one column volume of isopropanol, 100% to 0% isopanol gradient, and subsequent columns. It was regenerated with 1 volume of water and 1.5 column volumes of 0.5N sodium hydroxide solution. The column was contacted with 0.5N NaOH for about 12-18 hours. The column was then washed with twice the column volume of distilled water. The column can be stored in 20% ethanol and 0.01 N NaOH.

Experiments with other proteins and peptides Five proteins or peptides were selected in order to investigate the general applicability of this method. Synthetic methionine enkephalin (Try-Gly-Gly-Phe-Met, Bachem Biosciences Inc (Philadelphia, Pennsylvania), 0.5 mg / ml), synthetic α-MSH (acetyl-ACTH, Bachem Biosciences, Inc. (Philpen, Inc., Phila, Pennsylvania) 0.5 mg / ml), synthetic somatotropin (Bachem Biosciences Inc (Philadelphia, PA), 0.5 mg / ml), somatostatin (isolated from porcine pituitary, 0.5 mg / ml), and recombinant hGH (Bachem Biosciences Inc (Philadelphia, Pennsylvania) 0.5 mg / ml) was separately added to each 1 ml (5 mm × 5 cm) Amberchrom CG71-M column was loaded at a rate of about 1 mg / ml resin. AKTA apparatus (ie, Amersham Pharmacia Biotech, Piscataway, FPLC apparatus, NJ) with 50 mM TrisHCL (pH 7.5) buffer A, and 50 mM TrisHCL (pH 7.5) with 50% 1,6-hexanediol concentration. The buffer B was ready for use. Elution from each column was performed by a linear gradient method in which the ratio of the buffer B was 0% to 100% and the column volume (CV) was 20 times or more. Fractions were collected and analyzed by C-4 HPLC and SDS-PAGE.

  The elution profile of enkephalin eluted from CG71-M by the gradient method using 1,6-hexanediol is shown in FIG. Enkephalin elutes when the solvent entering the column contains about 31% buffer B.

  A chromatogram depicting the elution of α-MSH is shown in FIG. Protein elutes when the solvent entering the column contains approximately 54% buffer B. FIG. 23 (a) is a control, and FIG. 23 (b) is a C-4 RP-HPLC analysis result of the eluate from CG-71.

  Somatotropin begins to elute when the solvent flowing into the CG71-M column contains approximately 80% buffer B, but is completely removed even after flowing 10 times the column volume of an eluent with a hexanediol concentration of 50%. Did not elute (see Figure 24). According to SDS-PAGE analysis, it can be seen that there is some fractionation within the peak (FIG. 25). C-4 RP-HPLC did indeed show the presence of other chemical species, but not quantitatively as suggested by the results of SDS-PAGE analysis. It is considered that different somatotropin species in the sample are fractionated by the CG71-M column.

  Somatostatin elutes when the solvent entering the CG71-M column contains approximately 66% buffer B (FIG. 26). C-4 RP-HPLC analysis confirmed the presence of somatostatin in the peak fraction.

  Human growth hormone elutes when the solvent flowing into the CG71-M column contains about 60% buffer B (FIG. 27). Human growth hormone has a single peak and is considered homogeneous. According to the SDS-PAGE analysis, there are other components (FIG. 28). On the C-4 HPLC chromatogram, a shoulder occupying 5% of the total integrated area was observed at the tailing end of the main peak.

  The scope of the present invention is not limited by the illustrated embodiments, and the illustrated embodiments are intended to illustrate aspects of the present invention, and any functionally equivalent aspect is intended to Included in the range. Moreover, various modifications in addition to those disclosed herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

  All publications cited herein are hereby incorporated by reference in their entirety.

It is a figure which shows the elution profile of GHA eluted from the WP-butyl column by the gradient method using 0%-60% 1, 6- hexanediol 50 mM TrisHCL solution (pH 7.2). It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the elution profile of GHA eluted from the CG71-M RP-LC column by the step gradient method using 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the elution profile of GHA eluted from the CG71-CRP-LC column by the gradient method using a 1, 6- hexanediol 15 mM triethylamine phosphate solution. It is a figure which shows the elution profile of GHA eluted from the CG71-CRP-LC column by the gradient method using a 1, 6- hexanediol 0.1% trifluoroacetic acid solution. It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. 7 and FIG. It is a figure which shows the elution profile of GHA eluted from the CG71-C column by the gradient method using a 0-65% 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the elution profile of GHA eluted from the CG71-M column by the gradient method using a 0-65% 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the elution profile of GHA which eluted from the CG300-M column by the gradient method using a 0-65% 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. 10 and FIG. It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the elution profile of GHA which eluted from the 240 mLCG71-M column by the step gradient method using a 50% 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the C-4 RP-HPLC analysis result of the fraction excluded by the column experiment of FIG. It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the elution profile of GHA eluted from the 100L CG71-M column by the gradient method using a 0%-50% 1, 6- hexanediol 15 mM triethylamine phosphate solution (pH 7.2). It is a figure which shows the elution profile of the enkephalin eluted from the CG71-M column by the gradient method using a 0% -50% 1, 6- hexanediol 50 mM Tris solution (pH 7.5). It is a figure which shows the elution profile of (alpha) -MSH eluted from the CG71-M column by the gradient method using 0%-50% 1, 6- hexanediol 50 mM Tris solution (pH 7.5). It is a figure which shows the C-4 RP-HPLC analysis result of an alpha-MSH solution (1.0 mg / mL). It is a figure which shows the C-4 RP-HPLC analysis result of the eluent of FIG. It is a figure which shows the elution profile of the somatotropin eluted from the CG71-M column by the gradient method using 0%-50% 1, 6- hexanediol 50 mM Tris solution (pH7.5). It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG. It is a figure which shows the elution profile of the somatostatin eluted from the CG71-M column by the gradient method using 0-50% 1, 6- hexanediol 50 mM Tris pH7.5. It is a figure which shows the elution profile of hGH which eluted from the CG71-M column by the gradient method using 0%-50% 1, 6- hexanediol 50 mM Tris solution (pH 7.5). It is a figure which shows the SDS-PAGE analysis result of the eluent of FIG.

Claims (22)

  Loading the mixture onto a reverse phase liquid chromatography column and using a buffer containing a diol selected from the group consisting of 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol Eluting the molecule from the column and purifying the molecule from the mixture.   The method of claim 1, wherein the molecule is a polypeptide.   3. The method of claim 2, wherein the molecule is selected from the group consisting of human growth hormone and growth hormone antagonist.   The method of claim 1, wherein the molecule is a peptide. 5. The method of claim 4 , wherein the peptide is selected from the group consisting of [alpha] -MSH, enkephalin, somatostatin and somatotropin.   The process of claim 1, wherein the diol is 1,6-hexanediol.   The method of claim 1, wherein the column is a high performance liquid chromatography column. The method of claim 1, wherein the column is a preparative column.   The method of claim 1, wherein the column has a diameter of about 5 cm to about 2.0 m.   The method of claim 9, wherein the column has a diameter of about 10 cm to about 100 cm.   The method of claim 1, wherein the column comprises a polymer resin.   The method of claim 11, wherein the polymer resin is styrene divinylbenzene.   The method of claim 11, wherein the polymer resin is methacrylate or acrylic. 12. The method of claim 11, wherein the column is loaded with the mixture at a rate of about 1.0 g to about 25.0 g molecules per liter packed bed volume.   4. The method of claim 3, wherein the polypeptide is a growth hormone antagonist.   4. The method of claim 3, wherein the polypeptide is human growth hormone.   The method of claim 1, wherein the pH of the buffer is from about 2.0 to about 12.   The method of claim 17, wherein the pH of the buffer is from about 7.0 to about 11.0.   The method of claim 18, wherein the pH of the buffer is from about 6.0 to about 8.0.   8. The method of claim 7, wherein the concentration of 1,6-hexanediol in the buffer is from about 0% to about 80%.   21. The method of claim 20, wherein the concentration of 1,6-hexanediol in the buffer is from about 0% to about 50%.   The method of claim 1, further comprising further purification of the molecule.