Methods for purifying recombinant proteins

Abstract

The present invention provides a method for purifying recombinant proteins, particularly recombinant human albumin, comprising the steps of: (a) adding aminoguanidine and a medium- to long-chain fatty acid to a sample containing the recombinant protein; and (b) subjecting the resulting sample to chromatography with a chromatography buffer containing aminoguanidine.

Description

[Technical field]

[0001] The present invention relates to a method for purifying recombinant proteins, particularly recombinant human albumin, and more particularly to a method for efficiently purifying recombinant proteins from a sample containing the recombinant proteins. [Background technology]

[0002] The structure of human albumin is a single-chain nonglycosylated protein with a heart-shaped structure, 585 amino acids, 17 pairs of disulfide bonds, one free thiol group, and a molecular weight of 66,438 daltons. The half-life of human albumin in the human body is 19-21 days. The heart-shaped structure of human albumin consists of three main domains and six subdomains wrapped with 17 disulfide bonds, which are loosely held together by van der Waals forces. As can be seen from its crystal structure, the disulfide bridges provide rigidity to the helical globular structure while providing sufficient flexibility to allow the protein to undergo conformational changes in response to changes in the surrounding medium.

[0003] Human albumin is generally produced by extraction, separation and purification from human serum, and is collectively referred to as human serum albumin. Human albumin derived from human blood is subject to quantitative restrictions on plasma sources, viral contamination from plasma donors, and individual antibody and protein differences, which poses a great risk in clinical use. Therefore, the instructions for use of human serum albumin in many countries all include, for example, an explanation of virus safety, which states that "standard measures taken to prevent infection from the use of human blood or plasma products include the selection of blood donors, screening of single donated blood or plasma pools for special infection marks, and the use of effective manufacturing processes for virus inactivation / removal. Nevertheless, when using medicinal products produced from blood or plasma, the possibility of infection by infectious pathogens cannot be excluded. This includes unknown or newly emerging viruses and other pathogens." Therefore, using recombinant gene methods is the optimal route to effectively obtain albumin free of viral contamination.

[0004] Currently, the most common method for mass production of recombinant human albumin by microbial expression is mainly yeast expression system, with Saccharomyces cerevisiae and Pichia yeast being the most mature. However, since human albumin belongs to the category of large-volume injections, the dose per injection can reach 5-30 g. Therefore, the overall host protein residues per injection and the ELISA test results for contaminants during the production process must be less than 1 ng / ml (200 mg / ml-rHA). Regardless of the method used to produce recombinant human albumin, its immunogenicity further includes post-translational modifications of the protein, such as glycosylation, oxidation, multimerization and aggregation. Therefore, efficient and specific purification is a key element in the process of obtaining recombinant human albumin with high purity.

[0005] Conventional purification processes use existing purification media, and the process is complicated, and generally cannot obtain ultra-high purity recombinant human albumin. Chinese Patent Application No. 1127299 discloses a protease in which yeast activity is inactivated by heating both the fermentation liquid and the bacteria, and in the first step of concentration and purification using Sepharose-Streamline-SP, about 20-30% or more of dimers appear, which need to be depolymerized by heating and reducing, then the 45KDa albumin fragment is removed by HIC hydrophobic chromatography, and then the pigment is removed by Sepharose-DEAE chromatography. In this application, the fermentation liquid and the bacteria are heated at 58-65°C or higher to inactivate the protease, so that crosslinking of heat shock proteins, recombinant human albumin, and host proteins occurs in the yeast, making the subsequent chromatography purification difficult. In addition, the separation and purification and operation efficiency of Sepharose-Streamline-SP chromatography, in which the bacteria and the fermentation liquid flow together through a fluidized bed, are both low.

[0006] Chinese Patent Applications Nos. 101768206, 1854155 and 1496993 disclose the use of highly salt tolerant Sepharose HSL type cationic medium to concentrate and capture recombinant human albumin in the fermentation broth, followed by the use of Sepharose phenyl HIC medium to remove albumin fragments, and further replacing Sepharose DEAE with Sepharose aminobutyl anion exchange chromatography medium to improve the yield.

[0007] Chinese patent applications 1810834, 1550504 and 1880334 disclose the use of Sepharose SP FF to capture and concentrate recombinant human albumin, and then affinity chromatography with Delta Blue column to adsorb albumin and remove 45KDa albumin fragments and yeast host proteins, but the blue column has the disadvantages of blue dye ligand shedding and safety, and the process design of affinity chromatography to separate recombinant human albumin reduces the efficiency of purification. The subsequent step of S-200HR molecular sieve also has low purification efficiency.

[0008] Although the above-disclosed patents can effectively obtain high-purity recombinant human albumin, a large amount of polymer is generated during the capture and concentration process in the initial stage of purification, which requires depolymerization again, and the depolymerization process may result in recombinant human albumin containing immunogenicity, such as mismatches.

[0009] Thus, there is a continuing need in the art for methods to efficiently purify recombinant proteins, particularly recombinant human albumin, that reduce polymer formation and inhibit interactions of the recombinant protein with host proteins, pigments and sugar substances. Summary of the Invention

[0010] According to one embodiment of the present invention, (a) adding aminoguanidine and a medium-chain fatty acid to a sample containing a recombinant protein; (b) subjecting the obtained sample to chromatography using a chromatography buffer containing aminoguanidine.

[0011] In one embodiment of the present invention, the sample containing the recombinant protein is a fermentation supernatant, preferably obtained by using common techniques such as centrifugation, solid-liquid separation, heat inactivation, hollow fiber ultrafiltration and / or depth filtration for clarification and separation.

[0012] In one embodiment of the present invention, solid-liquid separation is performed using a centrifuge to quickly separate the fermentation bacteria and the supernatant, thereby maintaining the homogeneity of the fermentation liquid, and after performing solid-liquid separation on the fermentation liquid, the fermentation supernatant is heated at 55°C to 68°C under the condition of a heat stabilizer such as sodium octanoate to perform solid-liquid separation again, and then a clarification treatment is performed using a flat membrane cassette or hollow fiber membrane of 300 to 500 KDa, or a hollow fiber with a membrane pore size of 0.1 to 2 μm. The clarification treatment may be performed once each before and after inactivation by heating.

[0013] In another embodiment of the present invention, the concentration of the aminoguanidine in step (a) is 2 to 100 mmol / g (recombinant protein), preferably 3 to 80 mmol / g (recombinant protein).

[0014] In one embodiment of the present invention, the medium-chain fatty acid is selected from one or more of octanoic acid, capric acid, myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:4), and salts thereof. In one embodiment, the concentration of the medium-chain fatty acid is 2-300 mmol / g (recombinant protein). Preferably, the concentration of the medium-chain fatty acid is 6-150 mmol / g (recombinant protein).

[0015] In one embodiment of the invention, the chromatography comprises cation exchange chromatography and hydrophobic chromatography.

[0016] In other embodiments, the recombinant protein may be G-CSF, GLP-1, interferons, growth hormones, interleukins, analogs thereof, and fusion proteins of the above proteins with albumin.

[0017] Unlike the prior art, the inventors of the present application add aminoguanidine and medium- and long-chain fatty acids to a sample containing recombinant human albumin, and then perform cation chromatography and hydrophobic chromatography (performed with a chromatography buffer containing aminoguanidine), which can achieve the following effects: First, aminoguanidine can prevent and reduce the phenomenon that traditional cation exchange media is prone to form dimers, multimers and heteromers, and medium- and long-chain fatty acids, as active and strong ligands, suppress the interaction of albumin with most host proteins, pigments and saccharide substances. Therefore, the polymers, heteromers, pigments and host proteins commonly present in cation exchange chromatography are greatly reduced by the embodiment of the present invention. Second, the presence of aminoguanidine and medium- and long-chain fatty acids suppresses the inter-disulfide bond polymerization between recombinant albumin fragments with a molecular weight of 45 KDa, thereby exposing the hydrophobic regions of many unfoldable protein fragments, which allows the small molecular fragments of recombinant human albumin and large amounts of hydrophobic impurities to be more thoroughly removed in hydrophobic chromatography, and yeast pigments with strong hydrophobic structures to be removed.

[0018] In one embodiment of the present invention, the concentration of aminoguanidine in the chromatography equilibration solution, washing solution, or elution buffer in the cation exchange chromatography is 1 to 200 mmol / L, and preferably 1 to 150 mmol / L.

[0019] In one embodiment of the present invention, the pH of the chromatography equilibration solution and washing solution in the cation exchange chromatography is between 4.0 and 6.0, preferably between 4.0 and 5.5, and the pH of the elution solution is between 7.0 and 9.5, preferably between 7.0 and 8.5.

[0020] In one embodiment of the present invention, the conductivity of the chromatography equilibration solution and washing solution in cation exchange chromatography is 15 ms / cm or less, preferably 10 ms / cm or less, and the conductivity of the elution solution is 30 ms / cm or less, preferably 25 ms / cm or less.

[0021] In one embodiment of the present invention, the chromatographic equilibration solution and washing solution in the cation exchange chromatography are phosphate buffer, acetate buffer or Tris buffer, preferably phosphate buffer or acetate buffer.

[0022] In one embodiment of the present invention, the medium substrate in the cation exchange chromatography is a polyacrylate substrate, a polystyrene-divinylbenzene substrate, an agarose substrate, or a modified cellulose substrate.

[0023] In one embodiment of the invention, the cation exchange chromatography medium is coupled with a hydrophobic cationic ligand, including the highly salt tolerant Sepharose Capto MMC.

[0024] In one embodiment of the present invention, the medium in the cation exchange chromatography is selected from the Uni-SP system, the UniGel-SP system, the NanoGel-SP system, the MonoMix-HC SP system, the MonoMix-MC SP system, the agarose Sepharose system, or the Bestarose system.

[0025] In one embodiment of the present invention, the medium in the hydrophobic chromatography is a hydrophilically modified agarose, polyacrylate, polystyrene-divinylbenzene based microsphere to which a phenyl or butyl based hydrophobic ligand is coupled, or the medium in the hydrophobic chromatography is a hydrophilic polymethacrylate based microsphere to which a phenyl or butyl based hydrophobic ligand is coupled.

[0026] In one embodiment of the invention, the medium in the hydrophobic chromatography is selected from the UniHR Phenyl system, the NanoHR Phenyl system, the UniHR Butyl system, the NanoHR Butyl system, the MonoMix-MC Butyl system, the MonoMix-MC Phenyl system, the agarose Sepharose system or the Bestarose system.

[0027] For cation exchange and hydrophobic chromatography, separation media are preferably selected in which cationic or hydrophobic ligands are coupled to hydrophilically surface-modified polyacrylate, polystyrene-divinylbenzene or polymethacrylate substrate microspheres.

[0028] The hydrophilically modified polyacrylate polymer microsphere-based separation media used in the present invention may further be, for example, polymethacrylate-based hydrophilically modified ion exchange, hydrophobic interaction and affinity chromatography filler products of the Fractogel® type manufactured by Merck Millipore, including, but not limited to, cation exchange media Fractogel® EMD SO3-(S) Resin system, Fractogel® SO3-(strong CEX) system, Fractogel® SE Hicap(strong CEX) system or Eshmuno® S(strong CEX) system.

[0029] The present invention includes, but is not limited to, cationic and hydrophobic separation media that are hydrophilically modified after synthesis of the same base medium.

[0030] In the present invention, the hydrophilically modified polymethacrylate-based separation medium of the Fractogel® type manufactured by Merck Millipore as described above and the hydrophilically modified polyacrylate or polystyrene-divinylbenzene-based microsphere coupling and separation medium manufactured by Suzhou Nanomicrotech Co., Ltd. or Sepax Technologies, Inc. are preferably selected, and the hydrophilically modified polyacrylate or polystyrene-divinylbenzene-based microsphere coupling and separation medium manufactured by Suzhou Nanomicrotech Co., Ltd. or Sepax Technologies, Inc. are most preferably selected.

[0031] The buffer exchange and concentration processes during each chromatography step of the present invention may be carried out using devices and machines such as hollow fiber membranes and flat membrane cassettes with separation pore sizes ranging from 1 KDa to 30 KDa in molecular weight, including, but not limited to, sequential use and cross-use.

[0032] For buffer exchange during the chromatography steps of the present invention, the buffer exchange process may be carried out using Sephadex G25 or Superdex G75.

[0033] The inventors of the present application have unexpectedly discovered that in the above chromatography, preferably polyacrylate, polymethacrylate or polystyrene-divinylbenzene microspheres having a diameter of 10 μm to 150 μm are used, which are modified by hydrophilic coating, and then grafted with cationic ion exchange media such as Uni-SP series, UniGel-SP or NanoGel-SP series, MonoMix-HC SP or MonoMix-MC SP series, or hydrophobic ligands such as UniHR Phenyl series, NanoHR Phenyl series, UniHR Butyl series and NanoHR Butyl series, MonoMix-MC Butyl series or MonoMix-MC Phenyl series for hydrophobic chromatography, and such separation and purification media retain to a certain degree the hydrophobicity of the polyacrylate, polymethacrylate or polystyrene-divinylbenzene itself or the coating, thereby making it easier to remove dyes and host proteins in the chromatography of recombinant human albumin containing aminoguanidine and medium- to long-chain fatty acids.

[0034] In one embodiment of the present invention, the pH in the hydrophobic chromatography is between 6.0 and 8.5, and preferably between 6.5 and 8.0.

[0035] In one embodiment of the present invention, the conductivity in the hydrophobic chromatography is 30 ms / cm or less, preferably 25 ms / cm or less.

[0036] In one embodiment of the present invention, the concentration of aminoguanidine in the loading solution in the hydrophobic chromatography is 1 to 100 mmol / g (recombinant protein).

[0037] In one embodiment of the invention, the aminoguanidine is in the form of its salt, preferably its hydrochloride salt.

[0038] The method of the present invention is mainly applied to the early stage of the purification process of fermentation supernatant expressed in yeast, which improves the purification accuracy during the concentration process of the fermentation liquid, and helps to improve the efficiency, increase the yield, and reduce the cost of the later purification.

[0039] The methods of the present invention may also be applied to other processes for recombinant human albumin, including, but not limited to, purification processes, intermediate purification processes.

[0040] The hydrophilically modified polyacrylate, polymethacrylate or polystyrene-divinylbenzene polymeric microspheres used in another specific embodiment of the present invention have a pore size between 300 Å and 3000 Å and a diameter between 10 μm and 150 μm.

[0041] In another specific embodiment of the present invention, microspheres between 10 μm and 150 μm made of polyacrylate or polystyrene-divinylbenzene polymer belong to high strength polymer materials and are packed so that the column bed is between 100 mm and 800 mm, preferably between 250 mm and 600 mm.

[0042] The polyacrylate or polystyrene-divinylbenzene polymer microspheres used in other specific embodiments of the present invention have uniform diameter distribution, relatively low back pressure, and are amenable to separation and purification by continuous flow chromatography.

[0043] The chromatography conditions of the present invention can be routinely adjusted and modified according to the general instruction manual.

[0044] During the chromatography of the present invention, some other commonly used steps such as dialysis, ultrafiltration and low temperature inactivation can be carried out without affecting the practical effect of the present invention.

[0045] For purification of recombinant human albumin, optionally, after the above chromatography is finally completed, 100KDa and / or 30KDa and / or 10KDa flat membrane cassettes can be used to retain high molecular aggregates, remove small molecular substances, continue purification, exchange buffer, and concentrate to a recombinant human albumin stock solution with a concentration of more than 20%. Note that host cells expressing recombinant human proteins include, but are not limited to, yeast, Saccharomyces cerevisiae including Saccharomyces cerevisiae, Kluyreromyces cerevisiae, Polymorpha cerevisiae, and Pichia cerevisiae.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification will control, and the following describes preferred methods and materials, although any methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention. The materials, methods, and examples disclosed herein are illustrative and not limiting.

[0047] As used herein, the term "recombinant human albumin" may also be referred to as "recombinant human serum albumin," "recombinant human serum albumin," "rHA," and / or "rHSA." The term "human serum albumin" refers to human albumin extracted from human serum, and may also be referred to as "human serum albumin," "HSA," "HA," and / or "pdHSA." [Brief description of the drawings]

[0048] [Figure 1] The figure shows the formation of mers when the first step of concentration and purification is carried out using a purification process that does not contain aminoguanidine. [Diagram 2] FIG. 1 shows a method according to one embodiment of the present invention whereby the addition of aminoguanidine can prevent and reduce the formation of dimers, multimers and heteromers. [Diagram 3]3 shows the HPLC-C4 detection spectrum of the liquid obtained by hydrophobic chromatography when no medium- to long-chain fatty acids were added. [Figure 4] The HPLC-C4 detection spectrum of the liquid obtained by hydrophobic chromatography when it contains medium- to long-chain fatty acids is shown, indicating that medium- to long-chain fatty acids act as active and strong ligands and inhibit the interaction of albumin with most host proteins, pigments, and sugar substances. [Diagram 5] 1 shows the reduction spectrum of anti-HCP Western blotting when purified using a purification process containing / not containing aminoguanidine and medium- to long-chain fatty acids according to the method of Example 4. [Figure 6] The figure shows the non-reduced spectrum of anti-HCP-Western blotting when purified by the purification process containing / not containing aminoguanidine and medium-long chain fatty acid according to the method of Example 4. Lanes 1 to 6 are solution-1 obtained by cation exchange, solution-3 obtained by cation exchange, solution-4 obtained by cation exchange, solution-5 obtained by cation exchange, solution-6 obtained by cation exchange, and solution-7 obtained by cation exchange, respectively (the volume of the loading solution is 0.14ul in each case), in which the numerals indicate the sub-batch numbers in the purification process, and the samples in the experimental process of the -2 batch were left to carry out other experiments, -1 / -3 / -4 are samples produced by a manufacturing process that does not contain aminoguanidine, and -4 / -5 / -6 are samples produced by a manufacturing process that adds aminoguanidine and medium-long chain fatty acid according to the present invention. [Figure 7]This is a non-reducing electrophoresis SDS-PAGE spectrum obtained by comparative detection of two types of purification processes for a liquid obtained by the same cation exchange chromatography according to the method of Example 4. In this experiment, a purification process containing aminoguanidine and medium-chain fatty acids and a purification process not containing aminoguanidine and medium-chain fatty acids were respectively performed on a liquid obtained by the same batch of cation exchange chromatography to purify proteins and compare the production status of monomers. -1 represents a sample purified by a purification production process without adding aminoguanidine and medium-chain fatty acids, and 2 represents a sample purified by a purification production process adding aminoguanidine and medium-chain fatty acids according to the present invention. The loading solution status of lanes 1 to 10 is as follows. 1 Liquid obtained by cation exchange chromatography 0.2ul 2 Loading flow-through solution-1 in hydrophobic chromatography 5ul 3 Target solution obtained by hydrophobic chromatography-1 0.2ul 4 Washing solution-1 in hydrophobic chromatography 1ul 5 Cleaning solution-1 in hydrophobic chromatography 5ul 6 Loading flow-through solution 1#sample-2 in hydrophobic chromatography 5ul 7 Loading flow-through solution 2#sample-2 in hydrophobic chromatography 0.5ul 8 Target solution obtained by hydrophobic chromatography-2 0.2ul 9 Washing solution-2 in hydrophobic chromatography 2ul 10 Cleaning solution-2 in hydrophobic chromatography 2ul DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The fermentation of the present invention is carried out in equipment with a scale of 10L, 20L, 3000L and 100000L, and purification is carried out at a scale with a column diameter of 10cm, 45cm and 120cm, respectively, and is highly scalable. This embodiment includes, but is not limited to, the above scales. The following examples are provided to further understand the features and advantages of the present invention with reference to the drawings, and are not intended to limit other contents to be interpreted from the present invention in any manner. EXAMPLES

[0050] Fermentation and solid-liquid separation

[0051] According to the method of Chinese Patent No. 102190722, Pichia yeast was constructed and fermented with optimized medium and culture parameters, and fermented for 300 hours to obtain a fermentation liquid of 12 g / L recombinant human albumin. The cells in the fermentation liquid were centrifuged to obtain a supernatant, and a heat stabilizer (sodium octanoate) was added to a final concentration of 20 mM, aminoguanidine to a final concentration of 30 mM, cysteine ​​to a final concentration of 10 mM, and N-acetyltryptophan to a final concentration of 5 mM, and the mixture was heated at 64 °C for 60 minutes to inactivate the protease. The mixture was filtered through a 0.22 μm hollow fiber membrane to clarify it, washed with water for injection, and then adjusted to a pH of 4.0-4.5 with acetic acid.

[0052] The column was packed with UniGel SP, the column bed height was 400 mm, the chromatography column was equilibrated with an equilibration solution of 50 mM HAc + 50 mM NaCl + 10 mM aminoguanidine with a pH of 4.1, the clarified and separated fermentation broth (containing sodium octanoate with 30 mmol / g albumin) was loaded, the chromatography column was washed with the equilibration solution, and the target protein was eluted with an elution solution of 50 mM PB + 170 mM NaCl + 10 mM aminoguanidine with a pH of 8.3. After the elution was completed, the medium was thoroughly washed and regenerated with a solution of 1 M NaCl + 0.5 M NaOH and water. EXAMPLES

[0053] Hydrophobic Chromatography

[0054] The eluate obtained in Example 1 was directly loaded onto an equilibrated hydrophobic chromatography column (UniHR Phenyl-80L, column bed height 400 mm), the equilibration solution was 50 mM PB+160 mM NaCl+10 mM aminoguanidine with a pH of 7.8, the chromatography column was washed with the equilibration solution to obtain a sample containing recombinant human albumin components, and the medium was thoroughly washed and regenerated with 0.01 M NaOH and water. EXAMPLES

[0055] Removal of aminoguanidine by ultrafiltration

[0056] The protein solution obtained in Example 2 was subjected to solution exchange using a 30 KDa and / or 10 KDa flat membrane cassette to remove stabilizers such as aminoguanidine, and was then purified and exchanged to concentrate the recombinant human albumin stock solution at a concentration of more than 20%. EXAMPLES

[0057] Comparison of the purification process of the present invention with a purification process that does not contain aminoguanidine and medium-long chain fatty acids

[0058] The fermentation liquid produced by the same fermentation process was purified under purification conditions without adding aminoguanidine and medium- to long-chain fatty acids, and also under conditions with the addition of aminoguanidine and sodium oleate according to the methods of Examples 1 and 2 of the present invention. Comparisons of the purification effects, liquid phase detection (Figures 1, 2, 3, and 4), electrophoretic detection (Figures 5, 6, and 7), and sugar detection results (Table 1) showed clear differences.

[0059] [Table 1] EXAMPLES

[0060] Methods for detecting quality control items in protein purification processes

[0061] 1. HPLC assay: The main detection method of chromatography was HPLC-SEC assay / HPLC-C4 assay.

[0062] Detection method: Based on the method for determining the polymers in human serum albumin in the 2015 edition of the Chinese Pharmacopoeia, Part 3, General Principle 3121, a corresponding detection method was established to determine the monomers (including polymers and dimers) in recombinant human albumin solution; Detection method: Referring to the high performance liquid chromatography in Part 3 General Principle 0512 of the 2015 edition of the Chinese Pharmacopoeia, the corresponding detection method was established to measure the pigment-binding protein and some sugar-binding protein in recombinant human albumin solution.

[0063] 2. Electrophoretic detection method: The main detection method for hydrophobic chromatography was SDS-PAGE electrophoresis.

[0064] Detection method: Sample detection was performed according to the Chinese Pharmacopoeia 2015 Edition, Part 4 (General Rule 0541, Method 5, SDS-polyacrylamide gel electrophoresis method). The detection of relevant samples was performed by referring to Part 4 of the 2015 edition of the Chinese Pharmacopoeia (Immunoblotting method in General Rule 3401). 3. Detection of sugar content by PAS method:

[0065] The test sample solution was added with hydrochloric acid solution to make the solution pH acidic, sodium periodate was added, mixed thoroughly, and the cis-ethylene glycol group of the polysaccharide in the test sample was oxidized to aldehyde at room temperature, the reaction was stopped in an ice bath, and ice zinc sulfate and sodium hydroxide were added to precipitate the protein. After centrifugation at 8000 rpm for 15 minutes, the supernatant was aspirated and placed in a 96-well plate, and a freshly prepared mixture of acetylacetonate and ammonium acetate was added and incubated at 37°C for 1 hour. The sugar content was calculated by colorimetry at a wavelength of 405 nm using an automatic microplate reader.

[0066] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will occur to those skilled in the art, and it is therefore to be understood that the appended claims are intended to cover all such modifications and changes that are within the true spirit and scope of the invention.

Claims

1. (a) adding aminoguanidine and a medium-chain fatty acid to a sample containing a recombinant protein; (b) subjecting the obtained sample to chromatography with a chromatography buffer containing aminoguanidine; (c) removing aminoguanidine by ultrafiltration; having In the step (a), the fermentation liquid containing the recombinant protein is subjected to solid-liquid separation to obtain a supernatant containing the recombinant protein, aminoguanidine, medium-long chain fatty acid, cysteine ​​and N-acetyltryptophan are added to the obtained supernatant, and the mixture is heated at 55°C to 68°C for 60 minutes to inactivate proteases, and the liquid after the inactivation treatment is filtered. In the step (b), the liquid obtained after the filtration in the step (a) is subjected to cation exchange chromatography containing aminoguanidine as a chromatography smoothing solution, a washing solution and an elution buffer, and the cation exchange chromatography is followed by hydrophobic chromatography containing guanidine as a chromatography equilibration solution; In the step (c), the protein solution is subjected to solution exchange by ultrafiltration using a 30 KDa and / or 10 KDa membrane to remove guanidine; In the step (a), The recombinant protein is a protein selected from the group consisting of recombinant human albumin, G-CSF, GLP-1, interferon, growth hormone, interleukin, or a fusion protein of any of them with albumin; the medium-long chain fatty acid is selected from one or more of octanoic acid, capric acid, myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:4), and salts thereof; In the step (a), the concentration of the aminoguanidine is 2-100 mmol / g (recombinant protein), the concentration of the medium-long chain fatty acid is 2-300 mmol / g (recombinant protein), the concentration of cysteine ​​is 10 mmol / g (recombinant protein), and the concentration of N-acetyltryptophan is 5 mmol / g (recombinant protein); In the step (b), The medium in the cation exchange chromatography is a hydrophilically modified polyacrylate substrate or a polystyrene-divinylbenzene substrate, and a hydrophobic cationic ligand including highly salt-tolerant Sepharose Capto MMC is coupled to the medium in the cation exchange chromatography; the concentration of aminoguanidine in the chromatography equilibration solution, the washing solution, or the elution buffer in the cation exchange chromatography is 1 to 200 mmol / L; the pH of the chromatography equilibration solution and the washing solution in the cation exchange chromatography is between 4.0 and 6.0, and the pH of the elution solution is between 7.0 and 9.5; The medium in the hydrophobic chromatography is a medium in which a phenyl or butyl-based hydrophobic ligand is coupled to a hydrophilically modified agarose, polyacrylate, or polystyrene-divinylbenzene base microsphere, or a medium in which a phenyl or butyl-based hydrophobic ligand is coupled to a hydrophilic polymethacrylate base, The pH of the chromatography equilibration solution in the hydrophobic chromatography is between 6.5 and 8.0, and the concentration of aminoguanidine in the loading solution in the hydrophobic chromatography is between 1 and 100 mmol / g (recombinant protein); Methods for purifying recombinant proteins.

2. The concentration of the medium-long chain fatty acid is 6 to 150 mmol / g (recombinant protein); The method of claim 1.

3. The pH of the chromatography equilibration solution and washing solution in the cation exchange chromatography is between 4.0 and 5.5, and the pH of the elution solution is between 7.0 and 8.

5. The method of claim 1.

4. The conductivity of the chromatography equilibration solution and the washing solution in the cation exchange chromatography is 15 ms / cm or less, and the conductivity of the elution solution is 30 ms / cm or less. The method of claim 1.

5. The conductivity of the chromatography equilibration solution and the washing solution in the cation exchange chromatography is 10 ms / cm or less, and the conductivity of the elution solution is 25 ms / cm or less. The method of claim 1.

6. The chromatography equilibration solution and washing solution in the cation exchange chromatography are phosphate buffer or acetate buffer. The method of claim 1.

7. The medium in the cation exchange chromatography is selected from the group consisting of Uni-SP system, UniGel-SP system, NanoGel-SP system, MonoMix-HC SP system, MonoMix-MC SP system, agarose Sepharose system, and Bestarose system; The method of claim 1.

8. The medium in the hydrophobic chromatography is selected from the group consisting of UniHR Phenyl system, NanoHR Phenyl system, UniHR Butyl system, NanoHR Butyl system, MonoMix-MC Butyl system, MonoMix-MC Phenyl system, agarose Sepharose system, and Bestarose system; The method of claim 1.

9. The conductivity in the hydrophobic chromatography is 25 ms / cm or less. The method of claim 1.

10. The aminoguanidine is its hydrochloride salt. The method according to any one of claims 1 to 9.

11. The sample containing the recombinant protein is a fermentation supernatant. The method according to any one of claims 1 to 10.

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