WO2018053797A1 - Procédé de purification dans le traitement primaire d'un milieu de culture de cellules cho - Google Patents

Procédé de purification dans le traitement primaire d'un milieu de culture de cellules cho Download PDF

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Publication number
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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WIPO (PCT)
Prior art keywords
cell culture
cho cell
culture medium
liquid
chitosan
Prior art date
Application number
PCT/CN2016/099851
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English (en)
Chinese (zh)
Inventor
鄢成伟
杨彬
孙文正
蒋金龙
颜旭
邓崇飞
Original Assignee
广东东阳光药业有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by 广东东阳光药业有限公司 filed Critical 广东东阳光药业有限公司
Priority to PCT/CN2016/099851 priority Critical patent/WO2018053797A1/fr
Publication of WO2018053797A1 publication Critical patent/WO2018053797A1/fr

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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

Definitions

  • 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.

Landscapes

  • 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

L'invention concerne un procédé de purification dans un traitement primaire d'un milieu de culture de cellules CHO, comprenant les étapes suivantes consistant à: 1) récolter un milieu de culture de cellules CHO; 2) ajouter un floculant, ajuster une valeur de pH et agiter; et 3) clarifier le milieu de culture de cellules CHO de l'étape 2). Le procédé de purification utilise un procédé de combinaison de chitosane, de chlorure de polyaluminium et de pH pour floculer des cellules, des débris cellulaires, les HCP, l'ADN et d'autres impuretés dans le milieu de culture cellulaire. De plus, le chitosane résiduel et le chlorure de polyaluminium peuvent être encore éliminés dans un traitement en aval, ce qui peut réduire la quantité de résidu de chitosane final à moins de 1 ppm.
PCT/CN2016/099851 2016-09-23 2016-09-23 Procédé de purification dans le traitement primaire d'un milieu de culture de cellules cho WO2018053797A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/099851 WO2018053797A1 (fr) 2016-09-23 2016-09-23 Procédé de purification dans le traitement primaire d'un milieu de culture de cellules cho

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/099851 WO2018053797A1 (fr) 2016-09-23 2016-09-23 Procédé de purification dans le traitement primaire d'un milieu de culture de cellules cho

Publications (1)

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WO2018053797A1 true WO2018053797A1 (fr) 2018-03-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116199735A (zh) * 2023-03-29 2023-06-02 无锡药明生物技术股份有限公司 一种高效处理高固含量细胞液的澄清收获方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090162919A1 (en) * 2007-12-21 2009-06-25 Aurora Biofuels, Inc. Methods for concentrating microalgae
CN101591625A (zh) * 2009-07-02 2009-12-02 上海海洋大学 一种高效经济的光合细菌浓缩方法
CN103275877A (zh) * 2013-06-09 2013-09-04 新奥科技发展有限公司 一种裂殖壶菌的收集方法
WO2015163783A1 (fr) * 2014-04-25 2015-10-29 Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Procédé pour obtenir une substance de érythropoïétine recombinante humaine et érythropoïétine recombinante humaine sous forme de nanocapsules utilisant la substance obtenue par le procédé de l'invention
CN105777897A (zh) * 2015-03-20 2016-07-20 广东东阳光药业有限公司 一种cho细胞收获液的前处理方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090162919A1 (en) * 2007-12-21 2009-06-25 Aurora Biofuels, Inc. Methods for concentrating microalgae
CN101591625A (zh) * 2009-07-02 2009-12-02 上海海洋大学 一种高效经济的光合细菌浓缩方法
CN103275877A (zh) * 2013-06-09 2013-09-04 新奥科技发展有限公司 一种裂殖壶菌的收集方法
WO2015163783A1 (fr) * 2014-04-25 2015-10-29 Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Procédé pour obtenir une substance de érythropoïétine recombinante humaine et érythropoïétine recombinante humaine sous forme de nanocapsules utilisant la substance obtenue par le procédé de l'invention
CN105777897A (zh) * 2015-03-20 2016-07-20 广东东阳光药业有限公司 一种cho细胞收获液的前处理方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116199735A (zh) * 2023-03-29 2023-06-02 无锡药明生物技术股份有限公司 一种高效处理高固含量细胞液的澄清收获方法

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