WO2018053797A1 - 一种初步处理cho细胞培养液的纯化方法 - Google Patents

一种初步处理cho细胞培养液的纯化方法 Download PDF

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WO2018053797A1
WO2018053797A1 PCT/CN2016/099851 CN2016099851W WO2018053797A1 WO 2018053797 A1 WO2018053797 A1 WO 2018053797A1 CN 2016099851 W CN2016099851 W CN 2016099851W WO 2018053797 A1 WO2018053797 A1 WO 2018053797A1
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cell culture
cho cell
culture medium
liquid
chitosan
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French (fr)
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鄢成伟
杨彬
孙文正
蒋金龙
颜旭
邓崇飞
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Sunshine Lake Pharma Co Ltd
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Sunshine Lake Pharma Co Ltd
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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

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  • the present invention relates to the field of monoclonal antibody purification, and in particular to a method for purifying a CHO cell culture solution.
  • the invention flocculates the cells, cell debris, HCP and other impurities in the CHO cell liquid by flocculation and acid precipitation, increases the particle size of the particles in the liquid solution, reduces the turbidity of the liquid after centrifugation, and can significantly increase the membrane filtration flux. Reduce membrane filtration costs.
  • the method of flocculation combined with acid precipitation can adsorb negatively charged impurities such as HCP and DNA, improve the service life of the Protein A column, and reduce the cost of the downstream purification process.
  • the technical problem to be solved by the present invention is to provide a purification method for preliminary treatment of CHO cell culture solution, which can reduce the turbidity of the liquid solution, increase the membrane filtration flux and reduce the membrane filtration cost under the premise of ensuring the yield and quality of the monoclonal antibody. It can reduce the content of impurities such as HCP and DNA, improve the service life of Protein A, save production costs and improve production efficiency.
  • the present invention provides a method for purifying a CHO cell culture solution, which comprises the following steps:
  • the flocculating agent is selected from the group consisting of chitosan, polyaluminum chloride, or a combination thereof.
  • the final concentration of chitosan is 0.2-0.5 g/L and the final concentration of polyaluminum chloride is 20-70 mg/L in the CHO cell culture solution.
  • step 2) adjusts the pH to 5.0 to 7.0.
  • step 2) the agitation time is 30 minutes.
  • the clarification method is selected from the group consisting of centrifugation, static separation, membrane filtration, or a combination thereof.
  • the centrifugation preferably, has a rotational speed of 4000 x g, a time of 15 to 20 minutes, and a temperature of 15 to 25 °C.
  • the invention utilizes a combination method of chitosan, polyaluminum chloride and pH to flocculate cells, cell debris, HCP and DNA and other impurities in the cell liquid, has obvious effects, simple process, and residual chitosan and polychlorination in the liquid solution.
  • the aluminum can be further removed in the downstream process, and the final chitosan residue can be less than 1 ppm.
  • the CHO cell (Chinese hamster ovary cell) cell line in the following examples of the present invention was purchased from Invitrogen; the cell line for expressing the recombinant human tumor necrosis factor receptor-Fc recombinant protein (rhTNFR-Fc) antibody was constructed autonomously.
  • the rhTNFR-Fc antibody of the present invention is prepared by fermentation based on the patent US 2005/0069979A.
  • DNA detection was performed using real-time fluorescent quantitative PCR. Enzyme activation and pre-denaturation conditions were 95 ° C for 5 min. The PCR reaction conditions were 95 ° C, 15 s; 60 ° C, 60 s; 40 cycles.
  • the HCP assay uses a sandwich ELISA method.
  • the rotation speed was 180 rpm
  • the incubation temperature was 37 ° C
  • the OD value detection wavelength was 450/630 nm.
  • HMW uses high performance liquid chromatography.
  • the pH of the cell harvested liquid was adjusted to 7.0 with 0.8 M hydrochloric acid, and polyaluminum chloride was added to the cell harvested liquid, and the final concentration of polyaluminum chloride was 40 mg/L.
  • the above harvested liquid was stirred at 15 ° C for 30 minutes, centrifuged at 4,000 ⁇ g for 15 minutes at 15 ° C, turbidity was measured, and DNA in the preliminary separation liquid was detected.
  • the concentration and the percentage of HCP and HMW in the feed after the Protein A step were the same as in Example 1. The results are shown in Table 1.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Separation Of Suspended Particles By Flocculating Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了初步处理CHO细胞培养液的纯化方法，包括以下步骤：1)收获CHO细胞培养液；2)添加絮凝剂，调节pH值后，搅拌；3)澄清步骤2)所述CHO细胞培养液。利用壳聚糖、聚氯化铝和pH的组合方法，絮凝细胞液中细胞、细胞碎片、HCP和DNA等杂质，而且料液中残留的壳聚糖和聚氯化铝可在下游工艺中进一步去除，最终壳聚糖残留量可低于1ppm。

Description

一种初步处理CHO细胞培养液的纯化方法 技术领域
本发明涉及单抗纯化领域，具体来说，涉及一种初步处理CHO细胞培养液的纯化方法
技术背景
在现代生物制药领域中，利用动物细胞培养技术生产的单克隆抗体对医疗卫生事业的发展起着越来越重要的作用。动物细胞培养液经过澄清过滤、纯化制剂等步骤，制得单克隆抗体。然而，细胞培养液中所包含的细胞碎片、宿主细胞蛋白(HCP)、DNA、高分子聚合物(HMW)等杂质，不仅加大了澄清过滤的难度，而且还会增加下游纯化处理的成本。在产品的成本构成中，后处理成本占总成本的40％～80％。
目前，分离细胞和培养液的方法包括：两层深层膜过滤技术或离心沉降结合一层深层膜过滤技术等。通过深层膜过滤技术获得浊度较低的料液，需要使用孔径较小(可能低于小于0.2um)的深层过滤膜，而该深层过滤膜的价格非常昂贵，且过滤通量也较低，因此分离CHO细胞培养液的膜过滤成本非常高。由于膜过滤后料液中HCP和DNA等杂质的浓度很高，料液在过Protein A柱时对填料使用寿命的损害大，使得下游纯化的成本较高。
本发明通过絮凝结合酸沉的方式，絮凝CHO细胞液中细胞、细胞碎片、HCP等杂质，增大料液中颗粒的粒径，降低离心后料液浊度，可以显著提高膜过滤通量，降低膜过滤成本。同时，絮凝结合酸沉的方法，可吸附带负电的HCP和DNA等杂质，提高Protein A柱的使用寿命，降低下游纯化工艺的成本。
发明内容
本发明要解决的技术问题是提供一种初步处理CHO细胞培养液的纯化方法，在保证单克隆抗体产量和质量的前提下，降低料液浊度，提高膜过滤通量，降低膜过滤成本，并且能减少HCP、DNA等杂质含量，提高Protein A的使用寿命，节约生产成本，提高生产效率。
具体的，本发明提供了一种初步处理CHO细胞培养液的纯化方法，包括以下步骤：
1)收获CHO细胞培养液；
2)添加絮凝剂，调节pH值后，搅拌；
3)澄清步骤2)所述CHO细胞培养液。
根据本发明一些实施例，所述絮凝剂选自壳聚糖、聚氯化铝或其组合。
根据本发明一些实施例，在CHO细胞培养液中，壳聚糖的终浓度为0.2-0.5g/L，聚氯化铝终浓度为20-70mg/L。
根据本发明一些实施例，步骤2)调节pH值为5.0～7.0。
根据本发明一些实施例，步骤2)搅拌时间为30分钟。
根据本发明一些实施例，所述澄清方法选自离心、静置分离、膜过滤或其组合。
根据本发明一些实施例，所述离心，优选地，转速为4000×g，时间为15～20分钟，温度为15～25℃。
本发明利用壳聚糖、聚氯化铝和pH的组合方法，絮凝细胞液中细胞、细胞碎片、HCP和DNA等杂质，效果显著，工艺简单，而且料液中残留的壳聚糖和聚氯化铝可在下游工艺中进一步去除，最终壳聚糖残留量可低于1ppm。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例子仅为了阐明本发明，而非为了限制本发明的应用范围。
本发明以下实施例中的CHO细胞(中国仓鼠卵巢细胞)细胞株购自invitrogen公司；表达重组人肿瘤坏死因子受体-Fc重组蛋白(rhTNFR-Fc)抗体所用的细胞株为自主构建。本发明实施例中rhTNFR-Fc抗体是基于专利US 2005/0069979A发酵制备而来。
实施例1
向1L收获液中添加壳聚糖0.2g，聚氯化铝20mg，用0.8M盐酸调节pH值为5.0。将上述收获液在15℃下搅拌30分钟，15℃、4000×g离心15分钟，测浊度，检测初步分离料液中DNA浓度及Protein A步骤后料液中HCP浓度和HMW的百分含量，结果见表1。
DNA、HCP和HMW三者的检测方法：
DNA检测采用实时荧光定量PCR法。酶激活和预变性条件是95℃，5min。PCR反应条件是95℃，15s；60℃，60s；40个循环。
HCP检测采用夹心ELISA方法。转速180rpm，孵育温度37℃，OD值检测波长450/630nm。
HMW采用高效液相色谱法。色谱柱TSK-gel G3000SW 7.8*300mm(5μm)，流动相150mM磷酸盐缓冲液，100％A相，流速0.5mL/min，温度25℃，pH 7.0，检测时间30min，波长280nm。
实施例2
向1L收获液中添加壳聚糖0.5g，聚氯化铝40mg，用1M盐酸调节pH值为7.0。将上述收获液在15℃下搅拌30分钟，15℃、4000×g离心15分钟，测浊度，检测初步分离料液中DNA浓度及Protein A步骤后料液中HCP浓度和HMW的百分含量，检测方法同实施例1，结果见表1。
实施例3
向1L收获液中添加壳聚糖0.5g，聚氯化铝70mg，用0.8M盐酸调节pH值为6.0。将上述收获液在15℃下搅拌30分钟，15℃、4000×g离心15分钟，测浊度，检测初步分离料液中DNA浓度及Protein A步骤后料液中HCP浓度和HMW的百分含量，检测方法同实施例1，结果见表1。
对比例1
用0.8M盐酸调节细胞收获液的pH值为7.0，向细胞收获液中加入聚氯化铝，聚氯化铝终浓度40mg/L。将上述收获液在15℃下搅拌30分钟，15℃、4000×g离心15分钟，测浊度，检测初步分离料液中DNA 浓度及Protein A步骤后料液中HCP浓度和HMW的百分含量，检测方法同实施例1，结果见表1。
对比例2
向1L收获液中添加壳聚糖0.5g，用0.8M盐酸调节pH值为6.0。收获液中将上述收获液在15℃下搅拌30分钟，15℃、4000×g离心15分钟，测浊度，检测初步分离料液中DNA浓度及Protein A步骤后料液中HCP浓度和HMW的百分含量，检测方法同实施例1，结果见表1。
表1 检测结果表
  离心后浊度(NTU) DNA浓度(g/L) HCP浓度(g/L) HMW百分含量(％)
实施例1 22.5 120 820.18 0.83
实施例2 10.9 60 506.08 0.50
实施例3 15.58 81 550.64 0.95
对比例1 98.06 1523 8866.24 0.75
对比例2 84.21 492 2405.37 1.49
由上表可见，向收获液中添加壳聚糖、聚氯化铝并调节溶液pH值，能够同时降低料液的浊度、DNA、HCP和HMW的含量。

Claims (7)

  1. 一种初步处理CHO细胞培养液的纯化方法，其特征在于，包括如下步骤：
    1)收获CHO细胞培养液；
    2)添加絮凝剂，调节pH值后，搅拌；
    3)澄清步骤2)所述CHO细胞培养液。
  2. 根据权利要求1所述的方法，其特征在于，所述絮凝剂为壳聚糖、聚氯化铝或其组合。
  3. 根据权利要求2或3所述的方法，其特征在于，所述壳聚糖的终浓度为0.2～0.5g/L，所述聚氯化铝终浓度为20～70mg/L。
  4. 根据权利要求1所述的方法，其特征在于，所述步骤2)调节pH值为5.0～7.0。
  5. 根据权利要求1所述的方法，其特征在于，步骤2)搅拌时间为30分钟。
  6. 根据权利要求1所述的方法，其特征在于，所述澄清为离心、静置分离、膜过滤，或其组合。
  7. 根据权利要求7所述的方法，其特征在于，所述离心，转速为4000×g，时间为15～20分钟，温度为15～25℃。
PCT/CN2016/099851 2016-09-23 2016-09-23 一种初步处理cho细胞培养液的纯化方法 Ceased WO2018053797A1 (zh)

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