CN105777897A - Method for pre-treating CHO (Chinese hamster ovary) cell harvesting liquid - Google Patents
Method for pre-treating CHO (Chinese hamster ovary) cell harvesting liquid Download PDFInfo
- Publication number
- CN105777897A CN105777897A CN201610154299.5A CN201610154299A CN105777897A CN 105777897 A CN105777897 A CN 105777897A CN 201610154299 A CN201610154299 A CN 201610154299A CN 105777897 A CN105777897 A CN 105777897A
- Authority
- CN
- China
- Prior art keywords
- liquid
- harvesting liquid
- chitosan
- concentration
- cell harvesting
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for pre-treating CHO (Chinese hamster ovary) cell harvesting liquid. The method includes adding mixed solution with chitosan and alanine into cell harvesting liquid; regulating the pH (potential of hydrogen) of the harvesting liquid until the pH of the harvesting liquid reaches 5-7 and then adding calcium chloride into the cell harvesting liquid; stirring the calcium chloride and the cell harvesting liquid and then centrifugally filtering the cell harvesting liquid. The method has the advantages that the turbidity of the centrifuged harvesting liquid is reduced and is lower than 30 NTU, the concentration of DNA (deoxyribonucleic acid) is reduced by 95% at least, the concentration of HCP (host cell proteins) is reduced by 50% at least, and the proportion of HMW (high molecular weight) single-antibody polymers is reduced by 60% at least; the method is simple and feasible, and the purification efficiency can be improved.
Description
Technical field
The present invention relates to monoclonal antibody purification art, it particularly relates to the pre-treating method of a kind of Chinese hamster ovary celI harvest liquid.
Technical background
In modern biotechnology pharmaceutical field, utilize the monoclonal antibody that animal cell culture technology produces that the development of medical and health care system is played more and more important effect.Chinese hamster ovary celI cultivates harvest liquid through steps such as clarification, filtration, pure preparations, prepares monoclonal antibody.But, the impurity such as the cell debris that comprises in cell harvesting liquid, host cell proteins (HCP), DNA, monoclonal antibody polymer (HMW), not only increase the difficulty of clarification filtration, but also the cost of downstream purification processes can be increased.The pre-treatment common method of cell harvesting liquid includes: two-stage deep layer membrane filtration technique or centrifugation are in conjunction with one-level in-depth filtration technology.In-depth filtration film price is much more expensive, and filtration flux is relatively low, and the membrane filtration cost therefore separating Chinese hamster ovary celI culture fluid is very high.Owing to after membrane filtration, the impurity concentration such as feed liquid turbidity, HCP, DNA is significantly high, feed liquid is big to the infringement in filler service life when crossing ProteinA post so that downstream purification relatively costly.
At present, existing bibliographical information by adding flocculant or filter aid, adjustment solution ph in cell harvesting liquid, it is possible to reduces the content of turbidity and harvest liquid amplifying nucleic acid, but can not optimize turbidity, DNA, HCP, HMW and membrane flux simultaneously.Removing more impurity and the method optimizing more parameters in pretreatment process, the Expression product for antibody engineering has real economy benefit.
Summary of the invention
The defect existed for prior art or deficiency, it is an object of the invention to provide the pre-treating method of a kind of Chinese hamster ovary celI harvest liquid, and the pretreatment stage at cell harvesting liquid removes more impurity, advantageously reduces the pressure of subsequent purification, also have compressed cost.
Concrete, the invention provides the pre-treating method of a kind of Chinese hamster ovary celI harvest liquid, comprise the following steps:
1) joining in cell harvesting liquid by the mixed solution of chitosan-containing and alanine, regulating harvest liquid pH is 5～7；
2) calcium chloride is joined in cell harvesting liquid, centrifugal filtration after stirring.
In some embodiments, in harvest liquid, the concentration of chitosan is 0.6～0.8g/L.
In some embodiments, chitosan is non-water-soluble chitosan.
In other embodiments, water-insoluble chitosan is pharmaceutical grade chitosan, and deacetylation is more than 95%.Dilute sulfuric acid or dilute hydrochloric acid can be used during use first to dissolve water-insoluble chitosan.
In some embodiments, in harvest liquid, the concentration of alanine is 0.1～1g/L.
In some embodiments, in harvest liquid, the concentration of calcium chloride is 3～6g/L.
In some embodiments, step 1) pre-treatment time harvest liquid be suitable for pH be 5～6.
The technology of the inventive method has the beneficial effect that:
(1) this method technique is simple, after fermentation liquid processes by this method, harvest liquid be centrifuged after haze reduction to below 30NTU；
(2) harvest liquid DNA concentration reduces more than 95%, and host cell proteins HCP concentration reduces by more than 50%, and HMW ratio reduces more than 60%.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being further described in detail, the examples of implementation provided are only for illustrating the present invention, but not in order to limit the range of application of the present invention.
Chinese hamster ovary celI (Chinese hamster ovary cell) cell strain in following example of the present invention is purchased from invitrogen company；Expressing the cell strain used by recombinant human tumor necrosis factor's receptor-Fc recombiant protein (rhTNFR-Fc) antibody is autonomous structure.In the embodiment of the present invention, rhTNFR-Fc antibody is based on patent US2005/0069979A fermentation preparation.
Embodiment 1
In 1L harvest liquid, add chitosan 0.6g, alanine 0.1g, be after 5.0 with 0.8M salt acid for adjusting pH value, in harvest liquid, add calcium chloride 3g.Being stirred 30 minutes at 15 DEG C by above-mentioned harvest liquid, 15 DEG C, centrifugal 15 minutes of 4000 × g, survey turbidity, the percentage composition of HCP concentration and HMW in feed liquid after DNA concentration and ProteinA step in detection initial gross separation feed liquid, detection method is as follows, and result is in Table 1.
The detection method of DNA, HCP and HMW three:
DNA detection adopts quantitative real-time PCR.Enzyme activition and denaturation condition are 95 DEG C, 5min.PCR reaction condition is 95 DEG C, 15s；60 DEG C, 60s；40 circulations.
HCP detection adopts sandwich ELISA method.Rotating speed 180rpm, incubation temperature 37 DEG C, OD value detection wavelength 450/630nm.
HMW adopts high performance liquid chromatography.Chromatographic column TSK-gelG3000SW7.8*300mm (5 μm), mobile phase 150mM phosphate buffer, 100%A phase, flow velocity 0.5ml/min, temperature 25 DEG C, pH7.0, detects time 30min, wavelength 280nm.
Embodiment 2
In 1L harvest liquid, add chitosan 0.8g, alanine 0.2g, be after 7.0 with 1M salt acid for adjusting pH value, in harvest liquid, add calcium chloride 6g.Being stirred 30 minutes at 15 DEG C by above-mentioned harvest liquid, 15 DEG C, centrifugal 15 minutes of 4000 × g, survey turbidity, the percentage composition of HCP concentration and HMW in feed liquid after DNA concentration and ProteinA step in detection initial gross separation feed liquid, detection method is with embodiment 1, and result is in Table 1.
Embodiment 3
In 1L harvest liquid, add chitosan 0.6g, alanine 1g, be after 6.0 with 0.8M salt acid for adjusting pH value, in harvest liquid, add calcium chloride 6g.Being stirred 30 minutes at 15 DEG C by above-mentioned harvest liquid, 15 DEG C, centrifugal 15 minutes of 4000 × g, survey turbidity, the percentage composition of HCP concentration and HMW in feed liquid after DNA concentration and ProteinA step in detection initial gross separation feed liquid, detection method is with embodiment 1, and result is in Table 1.
Comparative example 1
The pH value regulating cell harvesting liquid with 0.8M hydrochloric acid is 7.0, adds calcium chloride, the final concentration of 3g/L of calcium chloride in cell harvesting liquid.Being stirred 30 minutes at 15 DEG C by above-mentioned harvest liquid, 15 DEG C, centrifugal 15 minutes of 4000 × g, survey turbidity, the percentage composition of HCP concentration and HMW in feed liquid after DNA concentration and ProteinA step in detection initial gross separation feed liquid, detection method is with embodiment 1, and result is in Table 1.
Comparative example 2
In 1L harvest liquid, add chitosan 0.8g, be 6.0 with 0.8M salt acid for adjusting pH value.Above-mentioned harvest liquid is stirred 30 minutes by harvest liquid at 15 DEG C, 15 DEG C, centrifugal 15 minutes of 4000 × g, survey turbidity, the percentage composition of HCP concentration and HMW in feed liquid after DNA concentration and ProteinA step in detection initial gross separation feed liquid, detection method is with embodiment 1, and result is in Table 1.
Table 1 testing result table
| Turbidity (NTU) after centrifugal | DNA concentration (g/L) | HCP concentration (g/L) | HMW percentage composition (%) | |
| Comparative example 1 | 71.06 | 19275 | 1366.24 | 0.74 |
| Comparative example 2 | 13.21 | 92 | 705.37 | 1.49 |
| Embodiment 1 | 2.01 | 63 | 536.18 | 0.66 |
| Embodiment 2 | 25.18 | 113 | 851.08 | 0.71 |
| Embodiment 3 | 10.87 | 81 | 643.64 | 0.43 |
As seen from the above table, in harvest liquid, add chitosan, alanine, calcium chloride and regulate solution ph, it is possible to reducing the content of the turbidity of feed liquid, DNA, HCP and HMW simultaneously.
Claims (7)
1. the pre-treating method of a Chinese hamster ovary celI harvest liquid, it is characterised in that comprise the steps:
1) joining in cell harvesting liquid by the mixed solution of chitosan-containing and alanine, regulating harvest liquid pH is 5～7；
2) calcium chloride is joined in cell harvesting liquid, centrifugal filtration after stirring.
2. method according to claim 1, it is characterised in that in described harvest liquid, the concentration of chitosan is 0.6～0.8g/L.
3. method according to claim 1 and 2, it is characterised in that described chitosan is non-water-soluble chitosan.
4. method according to claim 3, it is characterised in that described water-insoluble chitosan is selected from pharmaceutical grade chitosan, and deacetylation is more than 95%.
5. method according to claim 1, it is characterised in that in described harvest liquid, the concentration of alanine is 0.1～1g/L.
6. method according to claim 1, it is characterised in that in described harvest liquid, the concentration of calcium chloride is 3～6g/L.
7. method according to claim 1, it is characterised in that step 1) described adjustment harvest liquid pH is 5～6.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510125146 | 2015-03-20 | ||
| CN2015101251463 | 2015-03-20 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105777897A true CN105777897A (en) | 2016-07-20 |
Family
ID=56392833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610154299.5A Pending CN105777897A (en) | 2015-03-20 | 2016-03-17 | Method for pre-treating CHO (Chinese hamster ovary) cell harvesting liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105777897A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018053797A1 (en) * | 2016-09-23 | 2018-03-29 | 广东东阳光药业有限公司 | Purification method for primary treatment of cho cell culture medium |
| CN111218494A (en) * | 2018-11-23 | 2020-06-02 | 鲁南制药集团股份有限公司 | Method for treating CHO cell fermentation broth |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596302A (en) * | 2001-11-28 | 2005-03-16 | 桑多斯有限公司 | Cell culture process |
| CN101061231A (en) * | 2004-08-27 | 2007-10-24 | 惠氏研究爱尔兰有限公司 | Production of TNFR-Ig fusion protein |
-
2016
- 2016-03-17 CN CN201610154299.5A patent/CN105777897A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596302A (en) * | 2001-11-28 | 2005-03-16 | 桑多斯有限公司 | Cell culture process |
| CN101061231A (en) * | 2004-08-27 | 2007-10-24 | 惠氏研究爱尔兰有限公司 | Production of TNFR-Ig fusion protein |
Non-Patent Citations (2)
| Title |
|---|
| 刘美: "GS-CHO细胞培养工艺优化", 《东北农业大学硕士学位论文》 * |
| 孙婕，等: "大豆乳清蛋白提取工艺的研究", 《食品研究与开发》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018053797A1 (en) * | 2016-09-23 | 2018-03-29 | 广东东阳光药业有限公司 | Purification method for primary treatment of cho cell culture medium |
| CN111218494A (en) * | 2018-11-23 | 2020-06-02 | 鲁南制药集团股份有限公司 | Method for treating CHO cell fermentation broth |
| CN111218494B (en) * | 2018-11-23 | 2023-07-14 | 鲁南制药集团股份有限公司 | Method for treating CHO cell fermentation liquid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI670279B (en) | Antibody purification and purity monitoring | |
| JP6783767B2 (en) | How to Purify Recombinant Protein | |
| US20220185868A1 (en) | Bioreactor arrangement and continuous process for producing and capturing a biopol ymer | |
| RU2698654C2 (en) | Fusion protein purification method | |
| Riske et al. | The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery | |
| CN105777896B (en) | A kind of purification process at antibody acidity peak | |
| CN102272146A (en) | Precipitation of biomolecules with negatively charged polymers | |
| JP2016518318A (en) | Method for increasing protein purity using chromatography based on protein A | |
| JP2017515501A (en) | Integrated continuous biomanufacturing method | |
| CN108686634A (en) | The solid phase of hybrid chromatogram purification for protein | |
| CN103497248B (en) | A method for isolating and purifying antibody from cell culture supernatant | |
| CN105777897A (en) | Method for pre-treating CHO (Chinese hamster ovary) cell harvesting liquid | |
| JP2019533724A (en) | Continuous method for reducing heterogeneity of therapeutic proteins | |
| CN111153993B (en) | Preparation method of anti-TNF-alpha monoclonal antibody | |
| CN107964044B (en) | Method for purifying anti-CD 20 monoclonal antibody from milk sample | |
| CN104450656A (en) | Method for preparing and purifying thrombin in rabbit blood | |
| CN106478769A (en) | A kind of purification process of preliminary treatment Chinese hamster ovary celI culture fluid | |
| CN103740797A (en) | Method for preparing high-hydrolysis degree functional oligopeptide by use of high-temperature peanut meal | |
| US12043849B2 (en) | Protein purification method and kit | |
| Jiang et al. | Demonstrating β‐glucan and yeast peptide clearance in biopharmaceutical downstream processes | |
| CN103073616A (en) | Method for removing antibody aggregate by mixed-model chromatographic technology | |
| WO2018053797A1 (en) | Purification method for primary treatment of cho cell culture medium | |
| Buyel et al. | Very‐large‐scale production of monoclonal antibodies in plants | |
| CN103562383A (en) | Low ph protein purification process | |
| US20220162535A1 (en) | Integrated and continuous recombinant protein manufacturing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160720 |