WO2022120547A1 - 纯化重组蛋白的方法 - Google Patents

纯化重组蛋白的方法 Download PDF

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WO2022120547A1
WO2022120547A1 PCT/CN2020/134385 CN2020134385W WO2022120547A1 WO 2022120547 A1 WO2022120547 A1 WO 2022120547A1 CN 2020134385 W CN2020134385 W CN 2020134385W WO 2022120547 A1 WO2022120547 A1 WO 2022120547A1
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series
chromatography
medium
hydrophobic
acid
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PCT/CN2020/134385
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English (en)
French (fr)
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项炜
岳志蕾
赵洪志
邵聪伟
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通化安睿特生物制药股份有限公司
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Priority to US18/026,831 priority Critical patent/US20230331772A1/en
Priority to EP20964494.7A priority patent/EP4169938A4/en
Priority to CA3200066A priority patent/CA3200066A1/en
Priority to CN202080087052.0A priority patent/CN114885608A/zh
Priority to JP2023518533A priority patent/JP2024501601A/ja
Priority to PCT/CN2020/134385 priority patent/WO2022120547A1/zh
Priority to KR1020237011047A priority patent/KR20230060524A/ko
Publication of WO2022120547A1 publication Critical patent/WO2022120547A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • human albumin The conventional production method of human albumin is to extract, separate and purify from human serum, which is collectively referred to as human serum albumin.
  • Human albumin derived from human blood is affected by the quantitative limitation of plasma sources, viral contamination of plasma donors, and individual antibody and protein differences, and there will be greater risks in clinical use.
  • virus safety claims are included in the human albumin instructions for use in many countries, for example: "Standard measures to prevent infection from the use of human blood or plasma products include donor selection, screening of single blood donations Or the screening of special infectious markers in plasma pools and the adoption of effective production steps for inactivating/removing viruses. Even so, when selecting medicinal products prepared from blood or plasma, the possibility of being infected by infectious pathogenic agents cannot be ruled out. This including unknown or emerging viruses and other pathogens". Therefore, the use of genetic recombination is the best way to effectively obtain albumin without virus contamination.
  • human albumin is a large-volume injection, the dose per injection can reach 5-30 g. Therefore, it must be required that the total host protein residue of each injection dose and the contaminant ELISA test result of the production process should not be greater than 1ng/ml (200mg/ml-rHA).
  • the fermentation broth and bacterial cells are heated above 58-65°C to inactivate protease, resulting in cross-linking of heat shock protein, recombinant human albumin and host protein in yeast, which brings difficulties to subsequent chromatographic purification .
  • the separation, purification and operation efficiency of the Sepharose-Streamline-SP chromatography in which the bacteria and the fermentation broth co-flow through the fluidized bed are very low.
  • Chinese patent applications CN1810834 and CN1550504 and CN1880334 disclose the use of Sepharose SP FF to capture and enrich recombinant human albumin, and then use Delta blue column affinity chromatography to adsorb albumin to remove 45KDa albumin fragments and yeast host proteins.
  • the disadvantages are the ligand shedding and safety of the blue dye, and the process design of the separation of recombinant human albumin by affinity chromatography leads to a decrease in the efficiency of purification.
  • the molecular sieve of S-200HR used subsequently is also a step with low purification efficiency.
  • a method for purifying recombinant protein, especially recombinant human albumin comprising:
  • the recombinant protein-containing sample is a fermentation supernatant.
  • the clear supernatant is obtained from the fermentation broth by conventional techniques of centrifugation, solid-liquid separation, heat inactivation, hollow fiber ultrafiltration or/and depth filtration clarification separation.
  • a centrifuge is used for solid-liquid separation, which can quickly separate the fermentation cells from the supernatant to maintain the consistency of the fermentation liquid; after the solid-liquid separation of the fermentation liquid, the Under the condition of heat stabilizer, the fermentation supernatant is heated between 55 °C ⁇ 68 °C, and solid-liquid separation is carried out again; The hollow fiber is clarified.
  • the clarification treatment can be treated once before and after heat inactivation.
  • the concentration of aminoguanidine in the above step (a) is 2-100 mmol/g (recombinant protein), preferably the concentration of aminoguanidine is 3-80 mmol/g (recombinant protein).
  • the medium and long chain fatty acids are selected from caprylic acid, capric acid, myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), one or more of linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (C20:4) and salts thereof.
  • the concentration of the medium and long chain fatty acids is 2-300 mmol/g (recombinant protein).
  • the concentration of the medium and long chain fatty acids is 6-150 mmol/g (recombinant protein).
  • the chromatography includes cation exchange chromatography and hydrophobic chromatography.
  • the recombinant protein can also be G-CSF, GLP-1, interferon, growth hormone, interleukin and analogs thereof, as well as fusion proteins of the above proteins and albumin.
  • aminoguanidine and medium and long-chain fatty acids were added to a sample containing recombinant human albumin, followed by cation chromatography and hydrophobic chromatography (optionally with a layer containing aminoguanidine). analysis buffer), which can have several unexpected effects.
  • aminoguanidine can prevent and reduce the phenomenon that cation exchange media are prone to produce dimers, multimers and heteromers in the past; medium and long-chain fatty acids act as active strong ligands to inhibit most host proteins, pigments and carbohydrates. Interaction of substances with albumin.
  • embodiments of the present invention result in greatly reduced aggregates, heteromers, pigments and host proteins commonly found in cation exchange chromatography.
  • the presence of aminoguanidine and medium and long-chain fatty acids inhibits the mutual disulfide bond polymerization between the 45KDa molecular weight recombinant albumin fragments, so as to expose more hydrophobic regions of the protein fragments that cannot be folded, and make the hydrophobic chromatography more thorough. Remove small molecular fragments and a large number of hydrophobic impurities of recombinant human albumin, and remove yeast pigment with strong hydrophobic structure.
  • the concentration of aminoguanidine in the chromatographic equilibrium solution, washing solution or elution buffer of the cation exchange chromatography is 1-200 mmol/L, preferably the chromatographic equilibrium solution, washing solution or washing solution
  • the concentration of aminoguanidine in the debuffering solution is 1-150 mmol/L.
  • the pH of the chromatographic equilibrium solution and the washing solution of the cation exchange chromatography is between 4.0-6.0, preferably between 4.0-5.5; the pH of the eluent is between 7.0-9.5 between, preferably between pH 7.0-8.5.
  • the chromatographic equilibrium solution and washing solution of the cation exchange chromatography are phosphoric acid or acetic acid or Tris buffer solution, preferably phosphoric acid or acetate buffer solution.
  • the medium of the cation exchange chromatography is coupled with a hydrophobic cationic ligand, and the hydrophobic cationic ligand comprises a highly salt-tolerant Sepharose Capto MMC.
  • the medium of the cation exchange chromatography is selected from Uni-SP series, UniGel-SP series, NanoGel-SP series, MonoMix-HC SP series, MonoMix-MC SP series, Sepharose of agarose series or Bestarose series.
  • the medium of the hydrophobic chromatography is selected from UniHR Phenyl series, NanoHR Phenyl series, UniHR Butyl series, NanoHR Butyl series, MonoMix-MC Butyl series, MonoMix-MC Phenyl series, Sepharose of agarose series or Bestarose series.
  • the present invention includes, but is not limited to, hydrophilically modified cationic and hydrophobic separation media synthesized from the same substrate media.
  • the replacement buffer and concentration process between the various chromatographic steps of the present invention can be implemented by using devices and equipment such as hollow fiber membranes and flat membrane packages with separation pore sizes between 1KDa and 30KDa, including but not limited to the sequence of use and cross use.
  • the replacement buffer between the chromatographic steps of the present invention can also be treated with Sephadex G25 or Superdex G75.
  • polyacrylate or polymethacrylate or polystyrene-divinylbenzene microspheres with a diameter of 10 ⁇ m to 150 ⁇ m are preferably used.
  • graft cationic and hydrophobic ligands such as Uni-SP series, UniGel-SP or NanoGel-SP series, MonoMix-HC SP or MonoMix-MC SP series of cation exchange media; UniHR of hydrophobic chromatography Phenyl series, NanoHR Phenyl series, UniHR Butyl series and NanoHR Butyl series, MonoMix-MC Butyl series or MonoMix-MC Phenyl series, such separation and purification media retain polyacrylate or polymethacrylate or polystyrene-divinyl A certain amount of hydrophobicity of the base benzene itself or the coating makes it easier to remove pigments and host proteins in chromatography with recombinant human albumin containing aminoguanidine and medium and long chain fatty acids.
  • the pH of the hydrophobic chromatography is between 6.0-8.5, preferably the pH is between 6.5-8.0.
  • the conductivity of the hydrophobic chromatography is not higher than 30 ms/cm, preferably not higher than 25 ms/cm.
  • the concentration of aminoguanidine in the loading solution of the hydrophobic chromatography is 1-100 mmol/g recombinant protein.
  • the aminoguanidine is in the form of its salt, preferably its hydrochloride.
  • the method of the invention is mainly applied in the early stage of the yeast expression fermentation supernatant purification process, which is beneficial to improve the purification precision in the process of enriching the fermentation liquid, so as to improve the efficiency and increase the yield in the later purification and purification, Reduce the cost.
  • Some specific embodiments of the present invention use polyacrylate or polystyrene-divinylbenzene polymer with microspheres between 10 ⁇ m and 150 ⁇ m, which belong to high-strength polymer material column bed packing between 100 mm and 800 mm. It is preferably between 250mm and 600mm.
  • the polyacrylate or polystyrene-divinylbenzene polymer microspheres have uniform diameter distribution and relatively low back pressure, and are separated and purified by continuous flow chromatography.
  • chromatographic conditions described in the present invention can be adjusted and modified according to general instruction manuals.
  • the steps of dialysis, ultrafiltration, pasteurization and the like may be performed by other conventional methods between the chromatography described in the present invention, which does not affect the implementation effect of the present invention.
  • a membrane bag of 100KDa and/or 30KDa and/or 10KDa can be used to intercept macromolecular aggregates and remove small molecular substances for continued purification,
  • the buffer was exchanged and concentrated to a stock solution of recombinant human albumin with a concentration greater than 20%.
  • host cells expressing recombinant human proteins include, but are not limited to, yeast, Saccharomyces cerevisiae including Saccharomyces spp., Kluyveromyces spp., Hansenula spp. and Pichia spp.
  • human albumin may also be referred to as “recombinant human serum albumin” and/or “recombinant human serum albumin” and/or “rHA” and/or “rHSA”.
  • human serum albumin refers to human albumin extracted from human serum, which may also be referred to as “human serum albumin” and/or “HSA” and/or “HA” and/or “pdHSA”.
  • Figure 1 shows the production of aggregates in the first step of enrichment and purification using the purification process without aminoguanidine.
  • Figure 2 shows that addition of aminoguanidine can prevent and reduce the production of dimers, multimers and heteromers according to a method according to one embodiment of the present invention.
  • Fig. 3 shows the hydrophobic chromatography collection liquid HPLC-C4 detection pattern without the addition of medium and long chain fatty acids
  • Figure 4 shows the HPLC-C4 detection pattern of the hydrophobic chromatography collection liquid containing medium and long chain fatty acids, indicating that medium and long chain fatty acids act as active strong ligands to inhibit most host proteins, pigments and carbohydrates and albumin. Interaction.
  • Figures 5 and 6 show the reduced and non-reduced profiles of anti-HCP-Western Blotting purified by two purification processes without and with aminoguanidine and medium and long chain fatty acids, respectively, according to the method of Example 4.
  • Lanes 1-6 are cation exchange collection solution-1, cation exchange collection solution-3, cation exchange collection solution-4, cation exchange collection solution-5, cation exchange collection solution-6 and cation exchange collection solution-7 (sample loading).
  • the volume is 0.14ul), wherein the number label is the sub-batch number of the purification process, and the samples are reserved for other experiments during the -2 batches of experiments; -1/-3/-4 are samples produced by the production process without aminoguanidine, -4 /-5/-6 Samples of aminoguanidine and medium and long chain fatty acid production process added according to the present invention.
  • Fig. 7 shows the non-reducing electrophoresis SDS-PAGE pattern obtained by the comparative detection of the two purification processes using the same cation exchange chromatography collection solution according to the method of Example 4.
  • the same batch of cation exchange chromatography collected liquid was used to purify the protein correspondingly by two purification processes without aminoguanidine and medium and long chain fatty acids and with aminoguanidine and medium and long chain fatty acids, and the production of aggregates was compared;- 1 is identified as the sample purified by the purification production process without adding aminoguanidine and medium and long chain fatty acids, and 2 the sample purified by the production process of adding aminoguanidine and medium and long chain fatty acids according to the present invention.
  • the loading conditions for lanes 1-10 are as follows:
  • the fermentation scale of the present invention was carried out on 10L, 20L, 3,000L and 10,0000L equipment, and the purification scale was carried out on a column diameter of 10cm, 45cm, and 120cm, respectively, and the linear scale was good.
  • This embodiment includes, but is not limited to, the above scale.
  • the strain of Pichia pastoris was constructed and the optimized medium and culture parameters were fermented. After 300 hours of fermentation, a fermentation broth of 12g/L recombinant human albumin was obtained. The fermentation broth was centrifuged to separate the bacterial cells, the supernatant was harvested, and a heating stabilizer (sodium caprylate 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 were added. Acid to a final concentration of 5 mM and heating at 64°C for 60 min to inactivate the protease. After filtration and clarification through a 0.22 ⁇ m hollow fiber membrane and washing with injection water, the pH was adjusted to between 4.0 and 4.5 with acetic acid.
  • Example 1 The eluate collected in Example 1 was directly loaded onto an equilibrated hydrophobic chromatography column (UniHR Phenyl-80L, column bed height 400mm), and the equilibration solution was: 50mM PB+160mM NaCl pH 7.8+10mM aminoguanidine, using The column was washed with the equilibration solution, the recombinant human albumin fraction samples were collected, and the medium was thoroughly cleaned and regenerated with 0.01M NaOH and water.
  • Example 2 The protein solution collected in Example 2 was replaced with a 30KDa and/or 10KDa membrane package, and stabilizers such as aminoguanidine were removed, and the purification was continued; replaced and concentrated into a recombinant human albumin stock solution with a concentration greater than 20%.
  • Example 1 and Example 2 of the present invention Purify, through the comparison of purification effect, liquid phase detection comparison ( Figure 1, Figure 2, Figure 3, Figure 4), electrophoresis detection comparison ( Figure 5, Figure 6, Figure 7), sugar detection results (Table 1) comparison, The difference is obvious.
  • HPLC detection method The main detection method of chromatography 1 is HPLC-SEC detection method/HPLC-C4 detection method:
  • Detection method Referring to the 2015 edition of the Chinese Pharmacopoeia, the three-part general rule 3121 Human albumin multimer determination method, establish a corresponding detection method to measure the recombinant human albumin solution in the polymer (including multimer and dimer);
  • Electrophoresis detection method The main detection method of hydrophobic chromatography is SDS-PAGE electrophoresis:

Abstract

一种纯化重组蛋白，特别是重组人白蛋白的方法，包括：(a)将氨基胍和中长链脂肪酸加入含有重组蛋白的样品中；和(b)对所得样品进行层析，所述层析任选用含有氨基胍的层析缓冲液进行。

Description

纯化重组蛋白的方法 技术领域
本发明涉及重组蛋白的纯化方法。更具体而言，本发明涉及从含有重组蛋白，特别是重组人白蛋白的样品中高效纯化重组蛋白的方法。
背景技术
人白蛋白的结构是一个心型结构的单链非糖基化蛋白，有585个氨基酸，17对二硫键，一个自由巯基，分子量为66438道尔顿。人白蛋白在人体中的半衰期为19-21天。人白蛋白的心形结构由三个主要结构域和六个被17个二硫键包裹的亚结构域组成，它们通过范德华力松散地结合在一起。从其晶体结构可以看出，二硫化物桥赋予螺旋状的球状结构刚度，但提供足够的灵活性，使蛋白质能够根据周围介质的变化进行构象变化。
人白蛋白常规的生产方法是从人血清中提取分离纯化出来，统称为人血清白蛋白。人血来源的人白蛋白受到血浆来源的数量限制和血浆供者的病毒污染以及个体抗体和蛋白差异的影响，其在临床使用中将存在较大的风险。因此，在很多国家的人血白蛋白使用说明书中都有病毒安全性声明，例如：“为预防因使用人血或血浆制品导致的感染所采取的标准措施包括供血者选择、单次供血的筛选或血浆池特殊感染标记的筛选及灭活/去除病毒的有效生产步骤的采用。即便如此，当选用由血液或血浆制备的医药产品时，被传染性致病因子感染的可能性不能排除。这包括了未知的或新出现的病毒和其它病原体”。因此，采用基因重组方法是有效获取无病毒污染的白蛋白的最好途径。
目前，最常用的可实现规模化生产的基因重组微生物表达人白 蛋白的方式，主要是酵母表达系统，其中以酿酒酵母和毕赤酵母最为成熟。然而，由于人白蛋白属于大容量注射剂，每次注射的剂量可达到5-30g。所以，必须要求每次注射剂量的总宿主蛋白残留和生产过程的污染物ELISA测试结果不得大于1ng/ml(200mg/ml-rHA)。不管采用何种方式进行重组人白蛋白的生产，其免疫原性还包含蛋白质的翻译后修饰，例如糖基化、氧化、多聚和聚集等等。因此，高效特异性纯化是获得高纯度重组人白蛋白的工艺关键要素。
以往的纯化工艺是采用现有的纯化介质，其工艺复杂，通常不能获得超高纯度重组人白蛋白。中国专利申请CN1127299公开了发酵液与菌体共同加热灭活酵母活性的蛋白酶，采用其Sepharose-Streamline-SP第一步的富集纯化中出现约20-30％以上的二聚体，需要加热还原解聚，然后用HIC疏水层析去除45KDa的白蛋白碎片，再用Sepharose-DEAE层析法去除色素。在该申请中，发酵液与菌体在大于58-65℃以上加热灭活蛋白酶，导致酵母菌出现热激蛋白和重组人白蛋白以及宿主蛋白交联，为其后的层析纯化带来难度。同时，菌体与发酵液共同流穿流化床的Sepharose-Streamline-SP层析的分离纯化和运行效率都很低。
中国专利申请CN101768206和CN1854155以及CN1496993中公开了采用Sepharose HSL型高耐盐的阳离子介质富集捕获发酵液中的重组人白蛋白，然后采用Sepharose phenyl的HIC介质去除白蛋白碎片，再用Sepharose aminobutyl阴离子交换层析介质替代Sepharose DEAE以提高收率。
中国专利申请CN1810834和CN1550504以及CN1880334中公开了采用Sepharose SP FF捕获富集重组人白蛋白，然后采用Delta蓝柱亲和层析吸附白蛋白以去除45KDa的白蛋白碎片和酵母的宿主蛋白，蓝柱的缺点是蓝色染料的配基脱落和安全性，且以亲和层析分离重组人白蛋白的工艺设计导致纯化的效率降低。其后采用的S-200HR的分子筛也是纯化效率低的步骤。
以上三个系列公开的专利可以有效的得到纯度较高的重组人白蛋白，但纯化前期的捕获富集过程中聚合体的产生量较大，需要重新解聚，该解聚工艺有可能出现错配等含免疫原性的重组人白蛋白。
因此，本领域对于高效纯化重组蛋白，特别是重组人白蛋白的方法存在持续需求，使其减少聚合体的产生并抑制宿主蛋白、色素和糖类物质与重组蛋白的相互作用。
发明内容
在本发明的一个实施方案中，提供了一种纯化重组蛋白，特别是重组人白蛋白的方法，包括：
(a)将氨基胍和中长链脂肪酸加入含有重组蛋白的样品中；和
(b)对所得样品进行层析，所述层析任选用含有氨基胍的层析缓冲液进行。
在本发明的一个实施方案中，所述含有重组蛋白的样品为发酵上清液。优选地，通过离心、固液分离、加热灭活、中空纤维超滤或/和深层过滤澄清分离常规技术，从发酵液获得澄明上清液。
在本发明的一个实施方案中，采用离心机进行固液分离，可以迅速将发酵菌体与上清液分离，以保持发酵液的一致性；将发酵液固液分离后，可在辛酸钠等热稳定剂的条件下，对发酵上清液进行55℃～68℃之间的加热，再次进行固液分离；然后采用300～500KDa的膜包或中空纤维膜或0.1～2μm之间膜孔径的中空纤维进行澄清处理。澄清处理可在加热灭活的前后分别处理一次。
在本发明的另一个实施方案中，上述步骤(a)中所述氨基胍的浓度为2-100mmol/g(重组蛋白)，优选氨基胍浓度为3-80mmol/g(重组蛋白)。
在本发明的一个实施方案中，所述中长链脂肪酸选自辛酸、癸酸、肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的 一种或多种及其盐。在一个实施方案中，所述中长链脂肪酸的浓度为2-300mmol/g(重组蛋白)。优选地，所述中长链脂肪酸的浓度为6-150mmol/g(重组蛋白)。
在本发明的一个实施方案中，所述层析包括阳离子交换层析和疏水层析。
在其它实施方案中，所述重组蛋白也可以为G-CSF、GLP-1、干扰素、生长激素、白介素及其类似物，以及上述蛋白与白蛋白的融合蛋白。
与现有技术不同，本申请发明人意想不到地发现，向含有重组人白蛋白的样品中加入氨基胍和中长链脂肪酸，然后进行阳离子层析和疏水层析(任选用含有氨基胍的层析缓冲液进行)，可以产生若干意外效果。第一，氨基胍可以阻止和减少以往阳离子交换介质容易产生二聚体、多聚体和杂聚体的现象；中长链脂肪酸作为主动强配体抑制了大部分的宿主蛋白、色素和糖类物质与白蛋白的相互作用。因此，本发明的实施方案使得阳离子交换层析中普遍存在的聚合体、杂聚体、色素和宿主蛋白大大降低。第二，氨基胍和中长链脂肪酸的存在抑制了45KDa分子量重组白蛋白碎片之间的相互二硫键聚合，以便于暴露更多的不能折叠的蛋白碎片疏水区域，使疏水层析更加彻底地去除重组人白蛋白的小分子碎片和大量的疏水杂质，以及脱去带有强疏水结构的酵母色素。
在本发明的一个实施方案中，所述阳离子交换层析的层析平衡液、洗涤液或洗脱缓冲液中氨基胍的浓度为1-200mmol/L，优选层析平衡液、洗涤液或洗脱缓冲液中氨基胍的浓度为1-150mmol/L。
在本发明的一个实施方案中，所述阳离子交换层析的层析平衡液和洗涤液的pH在4.0-6.0之间，优选pH在4.0-5.5之间；洗脱液的pH在7.0-9.5之间，优选pH在7.0-8.5之间。
在本发明的一个实施方案中，阳离子交换层析的层析平衡液和洗涤液的电导率不高于15ms/cm，优选不高于10ms/cm；洗脱液的 电导率不高于30ms/cm，优选不高于25ms/cm。
在本发明的一个实施方案中，所述阳离子交换层析的层析平衡液和洗涤液为磷酸或醋酸或Tris缓冲溶液，优选磷酸或醋酸缓冲液。
在本发明的一个实施方案中，所述阳离子交换层析的介质基材是聚丙烯酸酯基材、聚苯乙烯-二乙烯基苯基材、琼脂糖基材、改性纤维素基材。
在本发明的一个实施方案中，所述阳离子交换层析的介质偶联有疏水性阳离子配基，所述疏水性阳离子配基包括高耐盐的Sepharose Capto MMC。
在本发明的一个实施方案中，所述阳离子交换层析的介质选自Uni-SP系列、UniGel-SP系列、NanoGel-SP系列、MonoMix-HC SP系列、MonoMix-MC SP系列、琼脂糖的Sepharose系列或Bestarose系列。
在本发明的一个实施方案中，所述疏水层析的介质为经亲水改造的琼脂糖、聚丙烯酸酯、聚苯乙烯-二乙烯基苯基材微球，其偶联有Phenyl或Butyl系列的疏水配基；或者疏水层析的介质为亲水性聚甲基丙烯酸酯基质，其偶联有Phenyl或Butyl系列的疏水配基。
在本发明的一个实施方案中，所述疏水层析的介质选自UniHR Phenyl系列、NanoHR Phenyl系列、UniHR Butyl系列、NanoHR Butyl系列、MonoMix-MC Butyl系列、MonoMix-MC Phenyl系列、琼脂糖的Sepharose系列或Bestarose系列。
对于阳离子交换层析和疏水层析，优选亲水性表面改性的聚丙烯酸酯或聚苯乙烯-二乙烯基苯或聚甲基丙烯酸酯基材微球偶联的阳离子配基或疏水配基分离介质。
本发明采用的聚丙烯酸酯聚合物微球亲水改造的基材分离介质还例如是默克密理博公司生产的聚甲基丙烯酸酯的
Figure PCTCN2020134385-appb-000001
型亲水改进的离子交换、疏水作用和亲和层析填料产品的基材，包括但不 限制于如下系列，阳离子交换介质
Figure PCTCN2020134385-appb-000002
EMD SO 3 -(S)Resin或
Figure PCTCN2020134385-appb-000003
SO 3 -(strong CEX)或
Figure PCTCN2020134385-appb-000004
SE Hicap(strong CEX)或
Figure PCTCN2020134385-appb-000005
S(strong CEX)等。
本发明包括但不限制于相同基材介质所合成后亲水改性的阳离子和疏水分离介质。
本发明优选如上所述的默克密理博公司生产的聚甲基丙烯酸酯的
Figure PCTCN2020134385-appb-000006
型亲水改性基材的分离介质和苏州纳微科技股份有限公司或赛分科技有限公司(Sepax Technologies,Inc)生产的上述聚丙烯酸酯或聚苯乙烯-二乙烯基苯基材微球经亲水改造的偶联分离介质；最优选苏州纳微科技股份有限公司和赛分科技有限公司(Sepax Technologies,Inc)生产的上述聚丙烯酸酯或聚苯乙烯-二乙烯基苯基材微球经亲水改造的偶联分离介质。
本发明的各个层析步骤之间的置换缓冲液和浓缩过程，可以采用分离孔径在分子量1KDa到30KDa之间的中空纤维膜和平板膜包等装置与设备实施，包括但不限制于使用顺序和交叉使用。
本发明的层析步骤之间的置换缓冲液，也可以采用Sephadex G25或Superdex G75进行换液处理。
另外，本申请发明人还意想不到地发现，在上述层析中，优选采用直径为10μm～150μm的聚丙烯酸酯或聚甲基丙烯酸酯或聚苯乙烯-二乙烯基苯微球，在经亲水包覆改性后，接枝阳离子、疏水配基，例如阳离子交换介质的Uni-SP系列、UniGel-SP或NanoGel-SP系列、MonoMix-HC SP或MonoMix-MC SP系列；疏水层析的UniHR Phenyl系列、NanoHR Phenyl系列、UniHR Butyl系列以及NanoHR Butyl系列，MonoMix-MC Butyl系列或MonoMix-MC Phenyl系列，该类分离纯化介质保留了聚丙烯酸酯或聚甲基丙烯酸酯或聚苯乙烯-二乙烯基苯自身或涂层的一定量的疏水性，使其在配合含有氨基胍和中长链脂肪酸的重组人白蛋白的层析中更加容易除去色素和宿主蛋白。
在本发明的一个实施方案中，所述疏水层析的pH在6.0-8.5之间，优选pH在6.5-8.0之间。
在本发明的一个实施方案中，所述疏水层析的电导率不高于30ms/cm，优选不高于25ms/cm。
在本发明的一个实施方案中，所述疏水层析的上样溶液中氨基胍的浓度为1-100mmol/g重组蛋白。
在本发明的一个实施方案中，所述氨基胍为其盐形式，优选其盐酸盐。
本发明所述的方法主要应用在酵母表达发酵上清液纯化工艺的前期，有利于在富集发酵液的过程中，提高纯化精度，以便于在后期的精制纯化中提高效率，增加收率，减少成本。
本发明所述的方法也可以应用在重组人白蛋白的其他工艺当中，包括但不限于采用应用在精制工艺，中间纯化工艺。
本发明的一些具体实施方案采用的聚丙烯酸酯或聚甲基丙烯酸酯或聚苯乙烯-二乙烯基苯聚合亲水改性微球，其孔径在
Figure PCTCN2020134385-appb-000007
之间，微球直径在10μm～150μm之间。
本发明的一些具体实施方案采用的聚丙烯酸酯或聚苯乙烯-二乙烯基苯聚合物组成的10μm～150μm之间的微球属于高强度聚合物材料柱床装填在100mm～800mm之间。优选250mm～600mm之间。
本发明的一些具体实施方案采用的聚丙烯酸酯或聚苯乙烯-二乙烯基苯聚合物微球直径分布均匀，反压相对较低，进行连续流层析的分离纯化方式。
本发明所述的层析条件可按照通用的指导手册进行一定的调节和变更。
本发明所述的层析之间可以采用其他一些常规的方法进行透析、超滤和巴氏灭活等步骤，不影响本发明的实施效果。
对于重组人白蛋白的纯化，任选地，在上述层析最终完成后，可采用100KDa和/或30KDa和/或10KDa的膜包进行截留大分子聚 集体和去除小分子物质，进行继续纯化，置换缓冲液并浓缩为浓度大于20％的重组人白蛋白原液。另外，表达重组人蛋白的宿主细胞包括但不限于酵母、包含糖酵母属系的酿酒酵母、克鲁维酵母属系、汉逊酵母属系和毕赤酵母属系。
除非另外定义，本文所用的所有技术和科学术语具有本领域技术人员通常理解的相同含义。在冲突的情况下，以本申请说明书为准。下面描述优选的方法和材料，但是与本文所述那些类似或等同的方法和材料可用于实施或测试本发明。本文公开的材料、方法和实例仅是说明性的，而非旨在限制。
在本说明书中，术语“重组人白蛋白”也可以称为“重组人血清白蛋白”和/或“重组人血白蛋白”和/或“rHA”和/或“rHSA”。术语“人血清白蛋白”是指从人血清中提取出来的人白蛋白，也可以称为“人血白蛋白”和/或“HSA”和/或“HA”和/或“pdHSA”。
附图说明
图1示出了采用不含有氨基胍的纯化工艺，进行第一步的富集纯化时的聚体产生情况。
图2示出了根据本发明的一个实施方案的方法，添加氨基胍可以阻止和减少二聚体、多聚体和杂聚体的产生。
图3示出了无中长链脂肪酸添加情况下的疏水层析收集液HPLC-C4检测图谱；
图4示出了含有中长链脂肪酸情况下的疏水层析收集液HPLC-C4检测图谱，表明中长链脂肪酸作为主动强配体抑制了大部分的宿主蛋白、色素和糖类物质与白蛋白的相互作用。
图5和图6分别示出了根据实施例4的方法，用不含有和含有氨基胍以及中长链脂肪酸的两种纯化工艺进行纯化的抗HCP-Western Blotting还原图谱和非还原图谱。泳道1-6分别为阳离子交换收集溶液-1、阳离子交换收集溶液-3、阳离子交换收集溶液-4、阳离子交换 收集溶液-5、阳离子交换收集溶液-6和阳离子交换收集溶液-7(上样体积均为0.14ul)，其中数字标号为纯化过程亚批编号，-2批实验过程中留样进行其他实验；-1/-3/-4为不含氨基胍生产工艺生产的样品，-4/-5/-6按照本发明添加氨基胍及中长链脂肪酸生产工艺的样品。
图7示出了根据实施例4的方法，采用相同的阳离子交换层析收集液，进行两种纯化工艺的对比检测而获得的非还原电泳SDS-PAGE图谱。本实验取同一批次阳离子交换层析收集液，分别采用不含有氨基胍和中长链脂肪酸和含有氨基胍和中长链脂肪酸两种纯化工艺对蛋白进行相应纯化，聚体产生情况对比；-1标识为不加氨基胍和中长链脂肪酸的纯化生产工艺纯化的样品，2按照本发明添加氨基胍及中长链脂肪酸生产工艺纯化的样品。泳道1-10的上样情况如下：
1    阳离子交换层析收集液       0.2ul
2    疏水层析上样流穿-1         5ul
3    疏水层析目标收集溶液-1     0.2ul
4    疏水层析洗涤液-1           1ul
5    疏水层析清洗液-1           5ul
6    疏水层析上样流穿1#样-2     5ul
7    疏水层析上样流穿2#样-2     0.5ul
8    疏水层析目标收集溶液-2     0.2ul
9    疏水层析洗涤液-2           2ul
10   疏水层析清洗液-2           2ul
具体实施方式
本发明的发酵规模在10L、20L、3,000L和10,0000L设备上进行的实施，纯化规模分别在柱直径10cm、45cm、120cm的规模实施，其线性放大良好。本实施例包括但不限制于以上规模。以下实施例结合附图可进一步理解本发明的特点和优势，不以任何方式限 制本发明解释的其余内容。
实施例1 发酵与固液分离
按照专利CN102190722的方法构建毕赤酵母菌种和优化的培养基与培养参数发酵，经300小时发酵，得到12g/L重组人白蛋白的发酵液。将发酵液离心分离菌体，收获上清液，加入加热稳定剂(辛酸钠至终浓度为20mM)，氨基胍至终浓度为30mM，半胱氨酸至终浓度为10mM和N-乙酰色氨酸至终浓度为5mM，64℃加热60分钟灭活蛋白酶。经0.22μm中空纤维膜过滤澄清和注射水洗涤后，用醋酸调pH到4.0～4.5之间。
将UniGel SP装柱，柱床高度400mm，采用50mM HAc+50mM NaCl+10mM氨基胍pH4.1平衡液平衡层析柱，将澄清分离的发酵液(含辛酸钠30mmol/g白蛋白)上样；用平衡液洗涤层析柱；再采用50mM PB+170mM NaCl+10mM氨基胍pH 8.3的洗脱液洗脱目的蛋白；洗脱完成后用1M NaCl+0.5M NaOH溶液和水对介质进行彻底清洁再生。
实施例2 疏水层析
将实施例1中收集的洗脱液直接上样至已平衡的疏水层析柱(UniHR Phenyl-80L，柱床高度400mm)，平衡液为：50mM PB+160mM NaCl pH 7.8+10mM氨基胍，采用平衡液洗涤层析柱，收集重组人白蛋白组分样品，采用0.01M的NaOH和水对介质进行彻底清洁再生。
实施例3 超滤脱去氨基胍
将实施例2收集的蛋白液采用30KDa和/或10KDa的膜包进行溶液置换，脱去氨基胍等稳定剂，继续纯化；置换并浓缩为浓度大于20％的重组人白蛋白原液。
实施例4 本发明的纯化工艺和不含氨基胍、中长链脂肪酸的纯化工艺对比
采用相同的发酵工艺生产所得发酵液，在不加入氨基胍以及中 长链脂肪酸的纯化条件下进行纯化；按照本发明的实施例1和实施例2的方法添加氨基胍和油酸钠的条件下进行纯化，通过纯化效果的对比，液相检测对比(图1、图2、图3、图4)，电泳检测对比(图5、图6、图7)，糖检测结果(表1)对比，差距明显。
Figure PCTCN2020134385-appb-000008
表1
实施例5 蛋白纯化过程中质量控制项检测方法
1、HPLC检测法：层析1的主要检测方法为HPLC-SEC检测法/HPLC-C4检测法：
检测方法：参照《中国药典》2015年版三部通则3121人血白蛋白多聚体测定法，建立相应检测方法测得重组人白蛋白溶液中聚体(含多聚体和二聚体)；
检测方法：参照《中国药典》2015年版三部通则0512高效液相色谱法，建立相应检测方法测得重组人白蛋白溶液中结合色素的蛋白以及部分糖结合蛋白；
2、电泳检测法：疏水层析的主要检测方法为SDS-PAGE电泳：
检测方法：参照《中国药典》2015年版四部(通则0541第五法SDS-聚丙烯酰胺凝胶电泳法)，进行样品检测；
参照《中国药典》2015年版第四部(通则3401免疫印迹法)，进 行相关样品检测。
3、PAS法糖含量的检测：
取供试品溶液加入盐酸溶液，使溶液pH为酸性，加入高碘酸钠充分混合，在室温条件下，使供试品中多糖中顺式乙二醇基氧化成醛，通过冰浴终止反应，加入冰硫酸锌和氢氧化钠使蛋白质沉淀，经离心机8000转离心15分钟后吸取上清液置96孔板中，再加入新鲜配制的乙酰丙酮乙酸铵混合液，置37℃条件下孵育1小时。在全自动酶标仪405nm波长下进行比色计算出糖含量。
虽然本发明某些特征已经在本文中阐释和描述，但本领域技术人员将想到许多修改、替代、变更和等同。因此，应理解的是，所附权利要求书意在涵盖落入本发明真实精神范围之内的所有此类修改和变更。

Claims (21)

  1. 一种纯化重组蛋白的方法，包括：
    (a)将氨基胍和中长链脂肪酸加入含有重组蛋白的样品中；和
    (b)对所得样品进行层析，所述层析任选用含有氨基胍的层析缓冲液进行。
  2. 根据权利要求1所述的方法，其中步骤(a)中所述氨基胍的浓度为2-100mmol/g(重组蛋白)。
  3. 根据权利要求1所述的方法，其中所述中长链脂肪酸选自辛酸、癸酸、肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种及其盐。
  4. 根据权利要求1所述的方法，其中所述中长链脂肪酸的浓度为2-300mmol/g(重组蛋白)，优选6-150mmol/g(重组蛋白)。
  5. 根据权利要求1所述的方法，其中所述层析包括阳离子交换层析和疏水层析。
  6. 根据权利要求5所述的方法，其中阳离子交换层析的层析平衡液、洗涤液或洗脱缓冲液中氨基胍的浓度为1-200mmol/L。
  7. 根据权利要求5所述的方法，其中阳离子交换层析的层析平衡液和洗涤液的pH在4.0-6.0之间，优选pH在4.0-5.5之间；洗脱液的pH在7.0-9.5之间，优选pH在7.0-8.5之间。
  8. 根据权利要求5所述的方法，其中阳离子交换层析的层析平衡液和洗涤液的电导率不高于15ms/cm，优选不高于10ms/cm；洗脱液的电导率不高于30ms/cm，优选不高于25ms/cm。
  9. 根据权利要求5所述的方法，其中阳离子交换层析的层析平衡液和洗涤液为磷酸或醋酸或Tris缓冲溶液，优选磷酸或醋酸缓冲液。
  10. 根据权利要求5所述的方法，其中阳离子交换层析的介质基 材选自聚丙烯酸酯基材、聚苯乙烯-二乙烯基苯基材、琼脂糖基材、改性纤维素基材；优选聚丙烯酸酯或聚苯乙烯-二乙烯基苯亲水改性基材。
  11. 根据权利要求5所述的方法，其中阳离子交换层析的介质偶联有疏水性阳离子配基，所述疏水性阳离子配基包括高耐盐的Sepharose Capto MMC。
  12. 根据权利要求5所述的方法，其中阳离子交换层析的介质选自Uni-SP系列、UniGel-SP系列、NanoGel-SP系列、MonoMix-HC SP系列、MonoMix-MC SP系列、琼脂糖的Sepharose系列或Bestarose系列。
  13. 根据权利要求5所述的方法，其中疏水层析的介质为经亲水改造的琼脂糖、聚丙烯酸酯、聚苯乙烯-二乙烯基苯基材微球，其偶联有Phenyl或Butyl系列的疏水配基；或者疏水层析的介质为亲水性聚甲基丙烯酸酯基质，其偶联有Phenyl或Butyl系列的疏水配基。
  14. 根据权利要求5所述的方法，其中疏水层析的介质选自UniHR Phenyl系列、NanoHR Phenyl系列、UniHR Butyl系列、NanoHR Butyl系列、MonoMix-MC Butyl系列或MonoMix-MC Phenyl系列，琼脂糖的Sepharose系列或Bestarose系列。
  15. 根据权利要求5所述的方法，其中疏水层析的pH在6.0-8.5之间，优选pH在6.5-8.0之间。
  16. 根据权利要求5所述的方法，其中疏水层析的电导率不高于30ms/cm，优选不高于25ms/cm。
  17. 根据权利要求5所述的方法，其中疏水层析的上样溶液中氨基胍的浓度为1-100mmol/g重组蛋白。
  18. 根据前述权利要求中任一项所述的方法，其中所述氨基胍为其盐形式，优选其盐酸盐。
  19. 根据前述权利要求中任一项所述的方法，其中所述重组蛋白 为重组人白蛋白。
  20. 根据前述权利要求中任一项所述的方法，其中所述重组蛋白为G-CSF、GLP-1、干扰素、生长激素、白介素及其类似物，以及上述蛋白与白蛋白的融合蛋白。
  21. 根据前述权利要求中任一项所述的方法，其中所述含有重组蛋白的样品为发酵上清液。
PCT/CN2020/134385 2020-12-08 2020-12-08 纯化重组蛋白的方法 WO2022120547A1 (zh)

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