WO2023024214A1 - 纯化重组人血清白蛋白的融合蛋白的方法 - Google Patents

纯化重组人血清白蛋白的融合蛋白的方法 Download PDF

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WO2023024214A1
WO2023024214A1 PCT/CN2021/121807 CN2021121807W WO2023024214A1 WO 2023024214 A1 WO2023024214 A1 WO 2023024214A1 CN 2021121807 W CN2021121807 W CN 2021121807W WO 2023024214 A1 WO2023024214 A1 WO 2023024214A1
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solution
fusion protein
exchange chromatography
chromatography
protein
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French (fr)
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成健伟
李绍奎
田新生
李自强
张筠
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北京伟德杰生物科技有限公司
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    • 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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • 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
    • 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
    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

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  • the invention belongs to the field of biological medicine purification methods, in particular to a method for purifying fusion proteins expressing recombinant human serum albumin (HSA) in mammalian cells.
  • HSA human serum albumin
  • Human serum albumin is a very important natural protein in blood circulation, accounting for half or more of the total serum protein content.
  • the molecular weight is 66KDa, it cannot be filtered by the glomerulus under normal circumstances, and its half-life in serum is about 3 weeks. It has good internal environment compatibility and low immunogenicity.
  • Studies have shown that using human serum albumin as a carrier, the fusion protein expressed by fusing a therapeutic protein or polypeptide with human serum albumin not only maintains the biological activity of the original protein or polypeptide, but also significantly reduces the clearance of the drug in the body Rate, thereby prolonging the half-life of the drug, greatly reducing the frequency of drug administration to patients, and improving patient compliance.
  • Interleukin 2 is a cytokine, mainly produced by T lymphocytes, which can activate T cells, promote B cell differentiation and secrete antibodies, and play an important role in the body's immune response, anti-tumor, anti-viral infection, etc. Is a natural immune booster.
  • interleukin-2 approved for clinical use is mainly used for the treatment of cancer (metastatic renal cancer, melanoma) and autoimmune diseases.
  • cancer metalastatic renal cancer, melanoma
  • autoimmune diseases mainly used so far. The main reason may be that the half-life of self-plasma is only a few minutes in the body, and it is difficult to achieve a stable effective plasma concentration, so the expected immune effect cannot be achieved.
  • the invention establishes a brand-new purification method which not only improves product quality, protein yield and reduces production cost, but also is a robust, reliable and scalable purification method.
  • the purpose of the present invention is mainly to provide a method for purifying mammalian cells (CHO) to express HSA fusion protein, effectively remove various impurities, improve protein purity and yield, reduce production costs, and make the quality of the final product conform to pharmaceutical research and application.
  • CHO mammalian cells
  • the present invention is mainly achieved through the following technical solutions: the cell culture supernatant containing the fusion protein is purified through acidification precipitation, cation exchange chromatography, anion exchange chromatography, and hydrophobic chromatography in sequence.
  • the acidification precipitation step can remove most of the host cell protein (HCP) and DNA
  • the cation exchange chromatography step is used to capture and separate the fusion protein
  • the anion exchange chromatography is used to further remove impurities such as host proteins and polymers.
  • the chromatography step is used to further increase the purity of the target protein while continuing to further reduce the level of trace impurities.
  • the method of the present invention also includes steps of virus inactivation and filtration, so as to effectively control the virus content in the final product.
  • the method for purifying the fusion protein of recombinant human serum albumin of the present invention comprises the following steps:
  • the fusion protein further comprises a cytokine; preferably, the cytokine is interleukin-2.
  • calcium ions are added to the cell supernatant, and the calcium ions can make the precipitation more complete; preferably, the calcium ions are in the form of calcium salts; more preferably, the The calcium salt is calcium chloride.
  • the acidic solution is selected from one or more of phosphoric acid, hydrochloric acid, acetic acid and citric acid, and this type of acidic solution has a good ability to remove aggregates, degradation products , DNA and the effect of impurities such as host cell proteins; preferably, the acidic solution is phosphoric acid; preferably, adjust the pH of the supernatant to 4.5-6.0, preferably pH 5.0-5.5 for acidification, and filter to obtain the acidified supernatant .
  • the cation exchange chromatography is high-resolution strong cation exchange chromatography
  • the chromatographic medium is a medium containing a sulfopropyl ligand; the positively charged ligand and the protein The negatively charged amino acids on the surface interact to achieve the purpose of adsorption.
  • the chromatographic medium is selected from one of SP HP, NanoGel-50SP, Capto MMC, Toyopearl SP-650M, SP Bestarose HP, and this type of medium has high capacity, high resolution, high protein yield, etc.
  • the chromatographic medium is SP HP.
  • the equilibration buffer in the cation exchange chromatography of the purification method of the present invention, can be selected from phosphate, acetate, citrate, 2-(N-morpholino)ethanesulfonic acid (MES ) etc.
  • the working pH is 5.0-6.0, preferably pH5.0-5.5; preferably, the equilibrium buffer is sodium acetate solution, the pH is 5.0-6.0; more preferably, the equilibrium buffer contains 10- 150mM sodium chloride solution in acetic acid.
  • the elution buffer is phosphate, acetate, citrate, 2-(N-morpholino)ethanesulfonic acid (MES) etc.
  • the pH containing a certain concentration of sodium chloride, and the pH is 5.0-6.0, preferably pH 5.0-5.5; preferably, the elution buffer is a solution containing 20-100 mM sodium acetate and 50-500 mM sodium chloride, with a pH of 5.0-6.0.
  • the column retention time is 6-8min.
  • the sample load is 20-30g/L filler. During chromatography, the acidified supernatant was filtered and loaded directly without any adjustment.
  • the virus inactivation in the purification method of the present invention is to use an acidic solution to adjust the pH of the first eluent to acidic in the presence of a stabilizer, and then neutralize it to neutral with an alkaline solution.
  • a stabilizing agent such as sugar, amino acid, sugar alcohol and redox substances, etc.
  • the preferred stabilizing agent is sugar and sugar alcohol, more preferably sucrose and sorbitol;
  • the concentration of the stabilizer is 2%-10%, preferably 5%.
  • the acidic solution can be selected from one or more of phosphoric acid, hydrochloric acid, acetic acid and citric acid, preferably phosphoric acid; the concentration of the acidic solution is 0.5M-1.5M; adjust the pH To 3.5-3.8, preferably pH3.6.
  • the alkaline solution used when neutralizing the acidic solution can be selected from one or more of sodium hydroxide, Tris and sodium acetate, preferably Tris-HCl solution; the concentration of the alkaline solution is 1M-3M.
  • the anion exchange chromatography is high-resolution strong anion exchange chromatography
  • the chromatographic medium is a medium containing a quaternary ammonium ligand; preferably, the chromatographic medium is selected from One of Q HP, UniGel-30Q, Toyopearl SuperQ-650M, Q Bestarose HP, these media have the characteristics of high loading, high resolution, low back pressure, etc.; more preferably, the chromatography medium is Q HP.
  • the equilibrium buffer in the anion exchange chromatography can be selected from one or more of phosphoric acid, Tris base, and ethanolamine, with a pH of 6.0-8.0, preferably pH6. 5-7.5; Preferably, the equilibrium buffer is a solution of 20-100mM Tris and 10-150mM sodium chloride, with a pH of 6.0-8.0.
  • the elution buffer in the anion exchange chromatography can be selected from one or more of phosphoric acid, Tris base, ethanolamine, contains a certain concentration of sodium chloride, and has a pH of 6.0-8.0, preferably pH 7.0-7.5 .
  • the elution buffer is a solution containing 20-100mM Tris and no higher than 500mM sodium chloride, with a pH of 6.0-8.0.
  • the column retention time is 7-9min.
  • the sample load is 15-25g/L filler.
  • the chromatographic medium is a medium containing a phenyl ligand, preferably, the chromatographic medium is selected from Capto Phenyl (HS), Phenyl Bestarose HP, Toyopearl Phenyl One of -650M and Phenyl HP; these media have the characteristics of high flow rate and high loading capacity; Capto Phenyl (HS) is preferred.
  • HS Capto Phenyl
  • the equilibrium buffer in the hydrophobic chromatography of the purification method of the present invention, can be selected from phosphate, Tris, citrate, MES, etc., with a pH of 6.0-8.0, preferably pH 7.0-7.5; preferably , the equilibrium buffer is a solution of 10-100mM sodium phosphate and 500-2500mM sodium chloride, with a pH of 6.0-8.0.
  • the elution buffer and the equilibrium buffer can be the same or different; preferably, the elution buffer is a solution of 10-100mM sodium phosphate and 500-2500mM sodium chloride, and the pH is 6.0-8.0 .
  • the column retention time is 3-5min.
  • the sample load is 50-70g/L filler.
  • the method for purifying the fusion protein of recombinant human serum albumin of the present invention comprises the following steps:
  • the cell supernatant containing the fusion protein from upstream, add calcium chloride solution, then use an acidic solution to adjust the pH of the supernatant to acidic, carry out acidification precipitation at room temperature, and filter to obtain the acidification supernatant;
  • the acidified supernatant was subjected to strong cation exchange chromatography to obtain the first eluate containing the fusion protein; a stabilizer was added to the obtained first eluate, and an acidic solution was used to adjust the pH to carry out low pH virus inactivation, and then use
  • the alkaline solution is neutralized to neutral; the obtained neutralized eluate is subjected to strong anion exchange chromatography to obtain a second eluate containing the fusion protein; the obtained second eluate is subjected to strong hydrophobic chromatography , to obtain a solution containing the fusion protein.
  • the chromatographic medium used in the present invention has good repeatability and is easy to scale up production. Compared with some composite chromatographic mediums, it is more conducive to large-scale operation. It is cheap and easy to get, and has a long service life, which further reduces production cost.
  • Fig. 1 is the HPLC purity determination diagram of the culture supernatant in Example 1 of the present invention.
  • Fig. 2 is the SP cation exchange chromatogram spectrum in Example 1 of the present invention.
  • Fig. 3 is the HPLC purity determination chart of the SP eluted protein solution in Example 1 of the present invention.
  • Figure 4 is the Q anion exchange chromatogram in Example 1 of the present invention.
  • Fig. 5 is an HPLC purity measurement chart of the Q-eluted protein solution in Example 1 of the present invention.
  • Fig. 6 is the Capto Pheny hydrophobic chromatogram spectrum in the embodiment 1 of the present invention.
  • Fig. 7 is the HPLC purity measurement spectrum of Capto Pheny penetration protein solution in Example 1 of the present invention.
  • Figure 8 is the electrophoresis (SDS-PAGE) pattern of the purified sample in Example 1 of the present invention
  • M is Marker
  • 1 is the culture supernatant
  • 2 is the supernatant after acidification
  • 3 is the SP eluent
  • 4 is the Q elution Liquid
  • 5 is Capto Pheny penetrating liquid.
  • Fig. 9 is the SP cation exchange chromatography spectrum in Example 2 of the present invention.
  • Figure 10 is the Q anion exchange chromatogram in Example 2 of the present invention.
  • Figure 11 is the Capto Pheny hydrophobic chromatogram in Example 2 of the present invention.
  • Figure 12 is the electrophoresis (SDS-PAGE) pattern of the purified sample in Example 2 of the present invention
  • M is Marker
  • 1 is the culture supernatant
  • 2 is the supernatant after acidification
  • 3 is the SP eluent
  • 4 is the Q elution Liquid
  • 5 is Capto Pheny penetrating liquid.
  • Fig. 13 is the comparative pattern of electrophoresis (SDS-PAGE) of the purified sample in Examples 1 and 2 of the present invention; M is Marker, 1 is the Capto Pheny breakthrough liquid 1 of embodiment 1, and 2 is the Capto Pheny breakthrough liquid of embodiment 2 2.
  • Embodiment 1 the method for the fusion protein of purifying recombinant human serum albumin (HSA) and interleukin 2 (IL-2)
  • Supernatant pretreatment Obtain the cell supernatant from the upstream, add calcium chloride solution to make the final concentration of the supernatant 10mM, then adjust the pH of the supernatant to 5.0 with 1M phosphoric acid, and let stand at room temperature for 1 hour Left and right, the precipitate and some impurities were removed by deep filtration to obtain the acidified supernatant.
  • Equilibrium buffer 50mM sodium acetate + 100mM sodium chloride, pH5.0.
  • Elution buffer 50mM sodium acetate + 300mM sodium chloride, pH5.0.
  • the acidified supernatant obtained in step 1 was directly loaded into the sample, the loading capacity of the sample was 20g/L filler, and the retention time of the sample was 6-8min.
  • the arrow marks the target protein.
  • Elution buffer E 25mM Tris+150mM sodium chloride, pH7.4.
  • step 3 Load the protein neutralization solution obtained in step 3, with a loading capacity of at least 15 g/L filler, and a sample retention time of 7-9 minutes.
  • the arrow marks the target protein.
  • Capto Phenyl (HS) hydrophobic chromatography column Use the Capto Phenyl (HS) hydrophobic chromatography column to further refine the eluted protein solution obtained in step 4, remove some stubborn impurities in the penetration mode, and obtain a high-quality fusion protein solution.
  • the specific operation steps are as follows:
  • Equilibrium buffer 20mM phosphate + 1000mM sodium chloride, pH7.0.
  • the elution buffer is the same as the equilibration buffer.
  • the sample can be loaded, and the target protein peak that penetrates is collected, and the UV at the beginning of collection is 100mAU, and the loading capacity of the sample is 50-70g/L filler , the loading retention time is 3-5min.
  • the arrow marks the target protein.
  • Embodiment 2 the method for the fusion protein of purifying recombinant human serum albumin (HSA) and interleukin 2 (IL-2)
  • Supernatant pretreatment Obtain the cell supernatant from the upstream, add calcium chloride solution to make the final concentration of the supernatant 10mM, then adjust the pH of the supernatant to 5.5 with 1M phosphoric acid, and let stand at room temperature for 1 hour Left and right, the precipitate and some impurities were removed by deep filtration to obtain the acidified supernatant.
  • Equilibrium buffer 25mM sodium acetate + 50mM sodium chloride, pH5.5.
  • Elution buffer 25mM sodium acetate + 200mM sodium chloride, pH5.5.
  • the acidified supernatant obtained in step 1 was directly loaded into a sample with a loading capacity of 30 g/L filler and a retention time of 6-8 min.
  • the arrow marks the target protein.
  • step 3 The crude target protein solution obtained in step 2 is subjected to low pH virus inactivation, while removing some impurities.
  • the specific operation process is as follows:
  • Equilibrium buffer 50mM Tris+30mM sodium chloride, pH6.5.
  • Elution buffer 50mM Tris+130mM sodium chloride, pH6.5.
  • the arrow marks the target protein.
  • Equilibrium buffer 100mM sodium phosphate + 1000mM sodium chloride, pH7.0.
  • the elution buffer is the same as the equilibration buffer.
  • the sample can be loaded, and the target protein peak that penetrates is collected, and the UV at the beginning of collection is 100mAU, and the loading capacity of the sample is 50-70g/L filler , the sample retention time is 3-5min.
  • the arrow marks the target protein.
  • SDS-PAGE electrophoresis pattern of the purified sample in Example 2 of the present invention is shown in Figure 12, wherein, M is Marker, 1 is the culture supernatant, 2 is the supernatant after acidification, 3 is the SP eluent, and 4 is Q Eluent, 5 is Capto Pheny breakthrough solution.
  • the purification method of the present invention can obtain high-quality target protein after the supernatant of the fusion protein expressed by CHO mammalian cells is purified by acidification—SP—Q—Capto Phenyl (HS), with a purity of more than 99.5%.
  • the protein yield is more than 45%, which is in line with pharmaceutical research and application.
  • the removal of impurities is an important content in the development of biotechnology drug purification process, especially the removal of host proteins.
  • host protein residues There are no clear regulations on host protein residues in the world.
  • the usual host protein residue content is 100ng/mg protein.
  • the present invention particularly compares the removal effects of different purification processes on host proteins.
  • Comparative Example 1 reduces the steps of hydrophobic chromatography. Compared with Example 1, Comparative Example 2 adopts different chromatographic media for its hydrophobic chromatography. After the fusion protein is purified, Comparative Example 1 and Comparative Example 1 In ratio 2, the HCP residual content is relatively high, but in embodiment 1, the purification effect of the fusion protein is very good, and most of the host proteins are basically removed.
  • the removal of process-related impurities and product-related impurities in the fusion protein shows the same purification effect, which proves the feasibility and stability of the technical solution of the present invention.
  • the supernatant of fusion protein expressed in CHO mammalian cells can be purified by acidification precipitation, strong cation exchange chromatography, virus inactivation, strong anion exchange chromatography, and hydrophobic chromatography to obtain high-quality target protein with high purity. More than 99.5%, the protein yield is more than 45%, in line with pharmaceutical research and application standards.
  • the technical solution of the present invention can greatly increase protein yield and purity, shorten production cycle, reduce production cost, and improve work efficiency.
  • the most direct economic benefit is that it is easy to scale up production and has good stability.

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Abstract

提供了纯化重组人血清白蛋白的融合蛋白的方法，所述方法包括以下步骤：将所述融合蛋白上清液依次经过酸化沉淀、阳离子交换层析、阴离子交换层析、疏水层析进行精细纯化，基本上去除融合蛋白中的工艺相关杂质和产品相关杂质，可获得符合药学研究和应用的高质量产品，有效地解决了大规模生产时蛋白纯度低、收率低的问题。

Description

纯化重组人血清白蛋白的融合蛋白的方法 技术领域
本发明属于生物医药纯化方法领域，具体涉及用于纯化哺乳动物细胞表达重组人血清白蛋白(HSA)的融合蛋白的方法。
背景技术
人血清白蛋白(HSA)占血清总蛋白含量一半及以上，是血液循环中的一个非常重要的天然蛋白。分子量为66KDa，正常情况下不能被肾小球滤过，在血清中半衰期达3周左右，内环境相容性好，免疫原性低。经研究表明，使用人血清白蛋白作为一种载体，将治疗性蛋白或多肽与人血清白蛋白融合所表达的融合蛋白既保持原有蛋白或多肽的生物活性，也明显降低药物在体内的清除速率，从而延长药物半衰期，大大减少了病人给药频率，提高病人的顺应性。
白介素2(IL-2)是一种细胞因子，主要是由T淋巴细胞产生，能激活T细胞，促进B细胞分化及分泌抗体，对机体免疫应答和抗肿瘤、抗病毒感染等有重要作用，是一种天然的免疫增强剂。当前，临床上批准使用的白介素2主要用于治疗癌症(转移性肾癌、黑色素癌)和自身免疫疾病。然而，至今没有得到实质性及突破性的进展，主要原因可能是自身血浆半衰期较短，在体内仅为数分钟，很难达到一个稳定的有效血浆浓度，也就达不到预期的免疫效果。
目前为止，实现细胞因子长效化的方法主要有三个：PEG修饰、脂质体包封和融合蛋白技术。PEG修饰蛋白技术产率低，蛋白与受体结合活性较低。脂质体包封技术复杂，且脂质体到达体内靶区或受体时无法出现高浓度富集，影响疗效。HSA融合蛋白技术是通过基因工程和蛋白质工程技术，融合表达细胞因子-HSA片段，其片段与受体结合后形成复合物，在体内不易降解。此融合技术延长半衰期，产率 高，疗效好且免疫原性低，是治疗性细胞因子长效化发展的方向。
然而，目前针对HSA融合蛋白的纯化方法大多数较为复杂，产物纯度不高，蛋白收率低，宿主细胞蛋白含量高，成本高且不易放大，这是大部分融合蛋白纯化所面临的难题。特别是通过哺乳动物细胞(CHO)培养分泌HSA融合蛋白进行纯化时研究发现，该融合蛋白在培养过程中会产生一些不同程度的降解物和聚集物，这种现象在大规模培养中比摇瓶培养中更为明显，它们的存在，对融合蛋白活性造成不良影响，由于理化性质与融合蛋白极为相似，采用一些常规层析方法很难去除，尤其宿主细胞蛋白更难去除，给纯化方法带来极大挑战。
考虑到生物药物安全性，监管机构对用于人施用的蛋白施用严格的纯化和质量要求，标准要求治疗性蛋白产品基本上无杂质(聚集体、降解物、变体、DNA、宿主细胞蛋白、培养基组分、病毒污染物、内毒素等)，此时纯化方法的优劣直接影响到产品质量、产品的成本，这是生物药成功的关键。本发明建立一种全新的纯化方法不仅仅提高了产品质量、蛋白收率、降低生产成本，而且还是稳健的、可靠的和可放大的纯化方法。
发明内容
本发明的目的主要是提供纯化哺乳动物细胞(CHO)表达HSA融合蛋白的方法，有效的去除各种杂质，提高蛋白纯度及收率，降低生产成本，使最终产品质量符合药学研究与应用。
为了达到上述目的，本发明主要是通过以下技术方案来实现的：将含有融合蛋白的细胞培养上清液依次经过酸化沉淀、阳离子交换层析、阴离子交换层析、疏水层析进行纯化。其中，酸化沉淀步骤可以去除大部分宿主细胞蛋白(HCP)和DNA，阳离子交换层析步骤用于将融合蛋白捕获分离出来，阴离子交换层析用于进一步去除宿主蛋白、多聚体等杂质，疏水层析步骤用于进一步提高目的蛋白的纯度，同时 继续将极微量的杂质含量进一步降低。优选地，本发明的方法中还包括病毒灭活和过滤步骤，以有效的控制终产品中的病毒含量。
具体地，本发明的纯化重组人血清白蛋白的融合蛋白的方法包括以下步骤：
将含有所述融合蛋白的细胞培养上清液用酸性溶液调节pH至酸性，进行酸化沉淀，过滤获得酸化上清液；将获得的所述酸化上清液进行阳离子交换层析，获得第一洗脱液；经病毒灭活后，将所述第一洗脱液进行阴离子交换层析，获得第二洗脱液；将所述第二洗脱液进行疏水层析，获得含有所述融合蛋白的溶液。
在一些实施方案中，所述融合蛋白还包含细胞因子；优选地，所述细胞因子为白介素2。
在一些实施方案中，在酸化沉淀前，向所述细胞上清液中加入钙离子，钙离子可以使沉淀更完全；优选地，所述钙离子为钙盐的形式；更优选地，所述钙盐为氯化钙。
在一些实施方案中，本发明纯化方法的酸化沉淀中，所述酸性溶液选自磷酸、盐酸、醋酸和柠檬酸中的一种或多种，这类酸性溶液具有良好地去除聚集体、降解物、DNA及宿主细胞蛋白等杂质的效果；优选地，所述酸性溶液为磷酸；优选地，调节上清液pH至4.5-6.0、优选为pH5.0-5.5进行酸化，过滤获得酸化上清液。
在一些实施方案中，本发明的纯化方法中，所述阳离子交换层析为高分辨率强阳离子交换层析，层析介质为含磺丙基配基的介质；带正电荷的配基与蛋白质表面带负电荷的氨基酸相互作用，达到吸附目的。优选地，所述层析介质选自SP HP、NanoGel-50SP、Capto MMC、Toyopearl SP-650M、SP Bestarose HP中的一种，这类介质具有高载量、高分辨率、高蛋白收率等特点，更优选地，所述层析介质为SP HP。
在一些实施方案中，本发明所述纯化方法的阳离子交换层析中，平衡缓冲液可以选自磷酸盐、醋酸盐、柠檬酸盐、2-(N-吗啉代)乙磺 酸(MES)等，工作pH为5.0-6.0，优选为pH5.0-5.5；优选地，所述平衡缓冲液为醋酸钠溶液，pH为5.0-6.0；更优选的，所述平衡缓冲液中含有10-150mM氯化钠的醋酸溶液。所述阳离子交换层析中，洗脱缓冲液为含有一定浓度氯化钠的磷酸盐、醋酸盐、柠檬酸盐、2-(N-吗啉代)乙磺酸(MES)等，pH为5.0-6.0，优选为pH5.0-5.5；优选地，所述洗脱缓冲液为含有20-100mM醋酸钠和50-500mM氯化钠的溶液，pH为5.0-6.0。柱留时间为6-8min。上样载量为20-30g/L填料。层析时，酸化后的上清液过滤直接上样，无需任何调节。
在一些实施方案中，本发明纯化方法中的病毒灭活为在稳定剂的存在下使用酸性溶液调节所述第一洗脱液的pH至酸性，然后用碱性溶液中和至中性。向洗脱液中加入某些稳定性物质作为稳定剂，诸如糖、氨基酸、糖醇及氧化还原类物质等，优选的稳定剂为糖和糖醇，更优选为蔗糖和山梨糖醇；所述稳定剂的浓度为2％-10％，优选为5％。在低pH病毒灭活中，所述酸性溶液可选自磷酸、盐酸、醋酸和柠檬酸中的一种或多种，优选为磷酸；所述酸性溶液的浓度为0.5M-1.5M；调节pH至3.5-3.8，优选pH3.6。中和酸性溶液时所用的碱性溶液可以选自氢氧化钠、Tris和醋酸钠中的一种或多种，优选Tris-HCl溶液；所述碱性溶液的浓度为1M-3M。
在一些实施方案中，本发明的纯化方法中，所述阴离子交换层析为高分辨率强阴离子交换层析，层析介质为含季铵基配基的介质；优选地，所述层析介质选自Q HP、UniGel-30Q、Toyopearl SuperQ-650M、Q Bestarose HP中的一种，这几种介质都具有高载量、高分辨率、反压低等特点；更优选地，所述层析介质为Q HP。
在一些实施方案中，本发明纯化方法中，所述阴离子交换层析中的平衡缓冲液可选自磷酸、Tris碱、乙醇胺中的一种或多种，pH为6.0-8.0，优选为pH6.5-7.5；优选地，所述平衡缓冲液为20-100mM Tris和10-150mM氯化钠的溶液，pH为6.0-8.0。所述阴离子交换层析中 的洗脱缓冲液可选自磷酸、Tris碱、乙醇胺中的一种或多种，含有一定浓度的氯化钠，pH为6.0-8.0，优选为pH7.0-7.5。优选地，洗脱缓冲液为含有20-100mM Tris和不高于500mM氯化钠的溶液，pH为6.0-8.0。柱留时间为7-9min。上样载量为15-25g/L填料。
在一些实施方案中，本发明纯化方法的疏水层析中，层析介质为含苯基配基的介质，优选地，所述层析介质选自Capto Phenyl(HS)、Phenyl Bestarose HP、Toyopearl Phenyl-650M、Phenyl HP中的一种；这几种介质都具有高流速、高载量等特点；优选为Capto Phenyl(HS)。
在一些实施方案中，本发明纯化方法的疏水层析中，平衡缓冲液可以选自磷酸盐、Tris、柠檬酸盐、MES等，pH为6.0-8.0，优选为pH7.0-7.5；优选地，所述平衡缓冲液为10-100mM磷酸钠和500-2500mM氯化钠的溶液，pH为6.0-8.0。所述疏水层析中，洗脱缓冲液与平衡缓冲液可以相同，也可以不同；优选地，洗脱缓冲液为10-100mM磷酸钠和500-2500mM氯化钠的溶液，pH为6.0-8.0。柱留时间为3-5min。上样载量为50-70g/L填料。
在一些实施方案中，本发明的纯化重组人血清白蛋白的融合蛋白的方法包括以下步骤：
从上游获得含有所述融合蛋白的细胞上清液，加入氯化钙溶液，然后使用酸性溶液调节上清液pH至酸性，室温条件下进行酸化沉淀，过滤获得酸化上清液；将获得的所述酸化上清液进行强阳离子交换层析，获得含有融合蛋白的第一洗脱液；向获得的第一洗脱液中加入稳定剂，使用酸性溶液调节pH进行低pH病毒灭活，然后用碱性溶液中和至中性；将获得的中和后的洗脱液进行强阴离子交换层析，获得含有融合蛋白的第二洗脱液；将获得的第二洗脱液进行强疏水层析，获得含有所述融合蛋白的溶液。
本发明纯化方法的有益效果如下：
(1)CHO细胞大规模培养产物依次经过酸化沉淀，高分辨高载 量性质的强阳离子交换层析介质、强阴离子交换层析介质、高载量的疏水层析介质的分步纯化后，基本上去除融合蛋白中的工艺相关杂质和产品相关杂质，最终获得符合药学研究和应用的高质量产品，有效地解决了大规模生产时蛋白纯度低，收率低的难题。
(2)本发明所使用的层析介质重复性好且易于放大生产，较于某些复合层析介质更有利于大规模上的操作，价格低廉易得，使用周期长，也进一步降低了生产成本。
附图说明
下面将结合附图及实施例对本发明进一步说明，其中电泳图中所涉及的M均表示蛋白Marker，层析图及SEC-HPLC图中箭头表示目的蛋白峰。
图1为本发明实施例1中培养上清HPLC纯度测定图谱。
图2为本发明实施例1中的SP阳离子交换层析图谱。
图3为本发明实施例1中SP洗脱蛋白液HPLC纯度测定图谱。
图4为本发明实施例1中的Q阴离子交换层析图谱。
图5为本发明实施例1中Q洗脱蛋白液HPLC纯度测定图谱。
图6为本发明实施例1中的Capto Pheny疏水层析图谱。
图7为本发明实施例1中Capto Pheny穿透蛋白液HPLC纯度测定图谱。
图8为本发明实施例1中纯化样品电泳(SDS-PAGE)图谱；M为Marker，1为培养上清液，2为酸化后上清液，3为SP洗脱液，4为Q洗脱液，5为Capto Pheny穿透液。
图9为本发明实施例2中的SP阳离子交换层析图谱。
图10为本发明实施例2中的Q阴离子交换层析图谱。
图11为本发明实施例2中的Capto Pheny疏水层析图谱。
图12为本发明实施例2中纯化样品电泳(SDS-PAGE)图谱；M为Marker，1为培养上清液，2为酸化后上清液，3为SP洗脱液，4 为Q洗脱液，5为Capto Pheny穿透液。
图13为本发明实施例1和2中纯化样品电泳(SDS-PAGE)对比图谱；M为Marker，1为实施例1的Capto Pheny穿透液1，2为实施例2的Capto Pheny穿透液2。
具体实施方式
下面将结合附图以及具体实施例对本发明技术方案进行更加详细的描述，以下实施例只是本发明实施例的一部分，提供这些实施例是为了能够更透彻的理解，能够将公开的完整内容传达给本领域的技术相关人员。
实施例1：纯化重组人血清白蛋白(HSA)与白介素2(IL-2)的融合蛋白的方法
本实施例的方法主要包括以下步骤：
重组人血清白蛋白(HSA)与白介素2(IL-2)的融合蛋白培养上清液HPLC纯度测定图谱见图1。
1.上清液预处理：从上游获得细胞上清液，加入氯化钙溶液使上清液的终浓度为10mM，其后用1M磷酸调节上清液的pH至5.0，室温静置1小时左右，通过深层过滤去除沉淀及部分杂质，获得酸化上清液。
2.用SP HP高分辨率强阳离子层析柱对步骤1获得的酸化上清液进行捕获纯化，获得大量目标蛋白的同时去除大部分杂质；其具体操作步骤如下：
2.1溶液配制
平衡缓冲液：50mM醋酸钠+100mM氯化钠，pH5.0。
洗脱缓冲液：50mM醋酸钠+300mM氯化钠，pH5.0。
2.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
2.3上样
将步骤1获得的酸化上清液直接上样，上样载量为20g/L填料，上样保留时间为6-8min。
2.4洗涤
上样完成后，用平衡缓冲液冲洗层析柱5-7个柱体积，使未结合或结合相对较弱的组分充分被冲洗下来，冲洗至流出液紫外吸收值显示基线一平，稳定即可。
2.5洗脱
洗涤完成后，继续用洗脱缓冲液冲洗层析柱，使结合的目标蛋白充分洗脱出来，收集紫外280nm处主峰，峰收集参数为：开始收集UV为100mAU，结束收集UV为100mAU。
2.6维护
依次用2M氯化钠溶液、0.5M氢氧化钠溶液、纯化水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图2所示，箭头标志为目标蛋白。
SP洗脱蛋白液HPLC纯度测定图谱见图3。
3.将通过步骤2获得的粗纯目标蛋白溶液进行低pH病毒灭活，同时去除部分工艺相关杂质，其具体操作过程为：
向获得粗纯洗脱的蛋白溶液中分别加入终浓度为5％的山梨糖醇溶液和终浓度为5％的蔗糖溶液，室温条件下混匀，用1M磷酸调节蛋白溶液pH为3.6，静置1.5小时后，用2M Tris-HCl中和溶液pH10.0调节蛋白溶液pH至7.4，紧接着通过深层过滤获得蛋白中和溶液。
4.用Q HP高分辨率强阴离子层析柱对步骤3的蛋白中和溶液进一步层析纯化，去除降解物质及部分其它杂质，其具体操作步骤如下：
4.1溶液配制
平衡缓冲液：25mM Tris+30mM氯化钠，pH7.4。
洗脱缓冲液E：25mM Tris+150mM氯化钠，pH7.4。
4.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
4.3上样
将步骤3获得的蛋白中和溶液上样，上样载量最低为15g/L填料，上样保留时间为7-9min。
4.4洗涤
上样完成后，用平衡缓冲液冲洗层析柱5-7个柱体积，使未结合或结合相对较弱的组分充分被冲洗下来，冲洗至紫外吸收值显示基线一平，稳定即可。
4.5洗脱
洗涤完成后，继续用洗脱缓冲液冲洗层析柱，使结合的目标蛋白充分洗脱出来，收集紫外280nm处主峰，峰收集参数为：开始收集UV为100mAU，结束收集UV为100mAU。
4.6维护
依次用2M氯化钠溶液、0.5M氢氧化钠溶液、纯化水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图4所示，箭头标志为目标蛋白。
Q洗脱蛋白液HPLC纯度测定图谱见图5。
5.用Capto Phenyl(HS)疏水层析柱对步骤4获得的洗脱蛋白液再进一步精细纯化，以穿透模式去除部分顽固性杂质，得到高质量的融合蛋白溶液其具体操作步骤如下：
5.1溶液配制
平衡缓冲液：20mM磷酸盐+1000mM氯化钠，pH7.0。
洗脱缓冲液与平衡缓冲液相同。
5.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
5.3上样
将步骤4获得的洗脱蛋白溶液调整电导率和pH与平衡液一致时，即可上样，收集穿透的目标蛋白峰，开始收集紫外为100mAU，上样载量为50-70g/L填料，上样保留时间为3-5min。
5.4洗脱
上样完成后，继续用平衡缓冲液冲洗层析，使紫外下降到100mAU，停止收集。
5.5维护
依次用注射用水、0.5M氢氧化钠溶液、注射用水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图6所示，箭头标志为目标蛋白。
Capto Pheny穿透蛋白液HPLC纯度测定图谱见图7。
本发明实施例1中纯化样品电泳(SDS-PAGE)结果见图8，其中，M为Marker，1为培养上清液，2为酸化后上清液，3为SP洗脱液，4为Q洗脱液，5为Capto Pheny穿透液。
实施例2：纯化重组人血清白蛋白(HSA)与白介素2(IL-2)的融合蛋白的方法
本实施例的方法主要包括以下步骤：
1.上清液预处理：从上游获得细胞上清液，加入氯化钙溶液使上清液的终浓度为10mM，其后用1M磷酸调节上清液的pH至5.5，室温静置1小时左右，通过深层过滤去除沉淀及部分杂质，获得酸化上清液。
2.用SP HP高分辨率强阳离子层析柱对步骤1获得的酸化上清 液进行捕获纯化，获得大量目标蛋白的同时去除部分杂质，其具体操作步骤如下：
2.1溶液配制
平衡缓冲液：25mM醋酸钠+50mM氯化钠，pH5.5。
洗脱缓冲液：25mM醋酸钠+200mM氯化钠，pH5.5。
2.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
2.3上样
将步骤1获得的酸化上清液直接上样，上样载量为30g/L填料，上样保留时间为6-8min。
2.4洗涤
上样完成后，用平衡缓冲液冲洗层析柱5-7个柱体积，使未结合或结合相对较弱的组分充分被冲洗下来，冲洗至流出液紫外吸收值显示基线一平，稳定即可。
2.5洗脱
洗涤完成后，继续用洗脱缓冲液冲洗层析柱，使结合的目标蛋白充分洗脱出来，收集紫外280nm处主峰，峰收集参数为：开始收集UV为120mAU，结束收集UV为120mAU。
2.6维护
依次用2M氯化钠溶液、0.5M氢氧化钠溶液、纯化水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图9所示，箭头标志为目标蛋白。
3.将通过步骤2获得的粗纯目标蛋白溶液进行低pH病毒灭活，同时去除部分杂质，其具体操作过程为：
向获得粗纯洗脱的蛋白溶液中分别加入终浓度为5％的山梨糖醇溶液和终浓度为5％的蔗糖溶液，室温条件下混匀，用1M磷酸调节 蛋白溶液pH为3.6，静置1.5小时后，用2M Tris-HCl中和溶液pH10.0调节蛋白溶液pH至7.4，紧接着通过深层过滤获得蛋白中和溶液。
4.用Q HP高分辨率强阴离子层析柱对步骤3的蛋白中和溶液进一步层析纯化，去除降解物质及部分其它杂质，其具体操作步骤如下：
4.1溶液配制
平衡缓冲液：50mM Tris+30mM氯化钠，pH6.5。
洗脱缓冲液：50mM Tris+130mM氯化钠，pH6.5。
4.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
4.3上样
将步骤3获得的蛋白中和溶液上样，上样载量为25g/L填料，上样保留时间为7-9min。
4.4洗涤
上样完成后，用平衡缓冲液冲洗层析柱5-7个柱体积，使未结合或结合相对较弱的组分充分被冲洗下来，冲洗至紫外吸收值显示基线一平，稳定即可。
4.5洗脱
洗涤完成后，继续用洗脱缓冲液冲洗层析柱，使结合的目标蛋白充分洗脱出来，收集紫外280nm处主峰，峰收集参数为：开始收集UV为150mAU，结束收集UV为150mAU。
4.6维护
依次用2M氯化钠溶液、0.5M氢氧化钠溶液、注射用水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图10所示，箭头标志为目标蛋白。
5.用Capto Phenyl(HS)疏水层析柱对步骤4获得的洗脱蛋白液再进一步精细纯化，以穿透模式去除部分顽固性杂质，得到高质量的融合蛋白溶液，其具体操作步骤如下：
5.1溶液配制
平衡缓冲液：100mM磷酸钠+1000mM氯化钠，pH7.0。
洗脱缓冲液与平衡缓冲液相同。
5.2平衡
用平衡缓冲液冲洗层析柱3-5个柱体积，直到流出层析柱的缓冲液电导率和pH值与平衡前相同，此时将紫外检测归零。
5.3上样
将步骤4获得的洗脱蛋白溶液调整电导率和pH与平衡液一致时，即可上样，收集穿透的目标蛋白峰，开始收集紫外为100mAU，上样载量为50-70g/L填料，上样保留时间为3-5min。
5.4洗脱
上样完成后，继续用平衡缓冲液冲洗层析，使紫外下降到100mAU，停止收集。
5.5维护
依次用注射用水、0.5M氢氧化钠溶液、注射用水清洗层析柱3个柱体积，最后用2个柱体积20％乙醇保存层析柱，于4℃存放。
如图11所示，箭头标志为目标蛋白。
本发明实施例2中纯化样品电泳(SDS-PAGE)图谱见图12，其中，M为Marker，1为培养上清液，2为酸化后上清液，3为SP洗脱液，4为Q洗脱液，5为Capto Pheny穿透液。
本发明实施例1和2中纯化样品电泳(SDS-PAGE)对比图谱见图13，其中，M为Marker，1为实施例1的Capto Pheny穿透液1，2为实施例2的Capto Pheny穿透液2。
由以上实施例可知，本发明的纯化方法对CHO哺乳动物细胞表达融合蛋白上清液依次经过酸化—SP—Q—Capto Phenyl(HS)纯化后，可获得高质量目标蛋白，纯度99.5％以上，蛋白收率45％以上，符合药学研究与应用。
实验例1：
对杂质的去除是生物技术药物纯化工艺开发的一个重要内容，特别是宿主蛋白的去除。国际上对宿主蛋白残留目前尚无明确的规定，对于抗体药物通常的宿主蛋白残留含量为100ng/mg蛋白。本发明在纯化工艺开发过程中特别比较了不同的纯化工艺对宿主蛋白的去除效果。
对比例1和对比例2与实施例1的纯化工艺不同，宿主蛋白(HCP)去除效果明显不同，结果见表1。
表1 不同纯化工艺及其宿主蛋白去除效果
实验编号 纯化工艺 蛋白浓度 HCP含量
实施例1 SP HP→Q HP→Capto Pheny 3.95mg/mL 65ng/mg
对比例1 SP HP→Q HP 3.20mg/mL 347ng/mg
对比例2 SP HP→Q HP→Capto adhere 2.28mg/mL 333ng/mg
结论：对比例1与实施例1相比减少了疏水层析步骤，对比例2与实施例1相比其疏水层析采用不同的层析介质，对融合蛋白进行纯化后，对比例1和对比例2中HCP残留含量较高，而实施例1中对融合蛋白的纯化效果很好，基本上除去大部分宿主蛋白。
讨论：
通过上述具体实施例对去除融合蛋白中工艺相关杂质与产品相关杂质，表现了同样的纯化效果，证实了本发明技术方案的可行性及稳定性。特别是对CHO哺乳动物细胞表达融合蛋白上清液依次经过酸化沉淀、强阳离子交换层析、病毒灭活、强阴离子交换层析、疏水层析进行精细纯化后，能获得高质量目标蛋白，纯度99.5％以上，蛋 白收率45％以上，符合药学研究和应用标准。
从经济学考虑出发，本发明的技术方案能大大提高蛋白收率及纯度，缩短生产周期，降低生产成本，提高工作效率，最为直接的经济效益是易于放大生产且稳定性好。
本发明列举的实施例仅是本发明优选的技术方案，本发明中提及到的其它层析介质都可以实现本发明的目的。
以上所述的本发明的保护范围不仅限于上述实施例，如是在本发明的原理条件下进行加以任何改造及变形，理应属于本发明的保护范围。

Claims (10)

  1. 纯化重组人血清白蛋白的融合蛋白的方法，包括以下步骤：
    将含有所述融合蛋白的细胞培养上清液用酸性溶液调节pH至酸性，进行酸化沉淀，过滤获得酸化上清液；将获得的所述酸化上清液进行阳离子交换层析，获得第一洗脱液；经病毒灭活后，将所述第一洗脱液进行阴离子交换层析，获得第二洗脱液；将所述第二洗脱液进行疏水层析，获得含有所述融合蛋白的溶液。
  2. 根据权利要求1所述的方法，其中，所述融合蛋白还包含细胞因子；优选地，所述细胞因子为白介素2。
  3. 根据权利要求1所述的方法，其中，在酸化沉淀前，向所述细胞上清液中加入钙离子；优选地，所述钙离子为钙盐的形式；更优选地，所述钙盐为氯化钙。
  4. 根据权利要求1所述的方法，其中，在所述酸化沉淀中，所述酸性溶液选自磷酸、盐酸、醋酸和柠檬酸中的一种或多种；
    优选地，所述酸性溶液为磷酸。
  5. 根据权利要求1所述的方法，其中，所述阳离子交换层析的层析介质为含磺丙基配基的介质；
    优选地，所述阳离子交换层析中的平衡缓冲液的pH为5.0-6.0；
    优选地，所述阳离子交换层析中的洗脱缓冲液的pH为5.0-6.0。
  6. 根据权利要求1所述的方法，其中，所述病毒灭活为在稳定剂的存在下使用酸性溶液调节所述第一洗脱液的pH至酸性，然后用碱性溶液中和至中性。
  7. 根据权利要求6所述的方法，其中，所述稳定剂选自糖、氨基酸、糖醇和氧化还原剂中的一种或多种；优选地，所述稳定剂为糖和糖醇；更优选地，所述稳定剂为蔗糖和山梨糖醇。
  8. 根据权利要求6所述的方法，其中，所述病毒灭活中所述酸性溶液选自磷酸、盐酸、醋酸和柠檬酸中的一种或多种，优选为磷酸； 和/或，所述碱性溶液选自氢氧化钠、Tris和醋酸钠中的一种或多种，优选为Tris-HCl溶液。
  9. 根据权利要求1所述的方法，其中，所述阴离子交换层析的层析介质为含季铵基配基的介质；
    优选地，所述阴离子交换层析中的平衡缓冲液的pH为6.0-8.0；
    优选地，所述阴离子交换层析中的洗脱缓冲液的pH为6.0-8.0。
  10. 根据权利要求1所述的方法，其中，所述疏水层析的层析介质为含苯基配基的介质，
    优选地，所述疏水层析中的平衡缓冲液的pH为6.0-8.0；
    优选地，所述疏水层析中的洗脱缓冲液的pH为6.0-8.0。
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