CN109134645A - A kind of cell concentration feed-batch culture technique - Google Patents
A kind of cell concentration feed-batch culture technique Download PDFInfo
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- CN109134645A CN109134645A CN201811065813.3A CN201811065813A CN109134645A CN 109134645 A CN109134645 A CN 109134645A CN 201811065813 A CN201811065813 A CN 201811065813A CN 109134645 A CN109134645 A CN 109134645A
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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Abstract
The present invention relates to field of cell culture, and in particular to a kind of cell concentration feed-batch culture technique, cell cultivation process carries out fluid infusion and changes liquid, wherein changes during liquid, only cell is trapped, and target product is discharged with liquid.Culture process of the invention, when changing liquid, entrapped cell, the not product of entrapped cell secretion, target product is discharged outside reactor together with cells and supernatant, it is compared with existing concentration feed-batch culture technique, the basic indifference of target product preferable for self stability, but the target product poor for self stability can greatly improve its yield.
Description
Technical field
The present invention relates to field of cell culture, in particular to a kind of cell concentration feed-batch culture technique.
Background technique
Monoclonal antibody can be used for the diagnosing and treating of disease, have very huge application value.Since monoclonal is anti-
Body molecular weight is larger, needs complicated posttranslational modification.And microbial cell can not accurately carry out posttranslational modification, because
The production of this current monoclonal antibody relies primarily on mammalian cell expression.
Industry is broadly divided into four kinds for the mode of mammaliancellculture at present: batch cultivation (batch), feed supplement
Batch cultivation (fed-batch), concentration feed-batch culture (concentration fed-batch) and perfusion culture
(perfusion).Four kinds of training methods are each advantageous and disadvantage:
1) batch cultivation is fairly simple, but antibody expression amount is lower, and production cost is higher, is not suitable for industrialization production.
2) feed batch fermentation be at present using most common production method, have it is easy to control, antibody expression amount compared with
Height, the moderate advantage of production cost.But project lower for some expression quantity, the cost of the culture process still seem compared with
It is high.Simultaneously for some unstable antibody or protein product, which is just no longer applicable in.
3) concentration feed-batch culture is there is presently no being widely used, and only the higher company of a small number of technical levels is using,
With unit tank volume high production efficiency, advantage at low cost.But the technique is since incubation time is longer, antibody or albumen one
It directly is trapped within reactor, so to some unstable antibody or albumen, which just seems not applicable.
4) perfusion culture starts to be concerned and use recently, which has high production efficiency, low excellent of production cost
Gesture, especially for unstable antibody or albumen, which has very big advantage.But the technique is at present not
It is widely used, mainly since the technique first three technique that compares seems extremely complex, increases the difficulty of production,
It is very difficult to implement in the industrial production.Simultaneously because the technique is continuous receipts sample, also lead to a complete production week
Phase generates more batches of harvest antibody or albumen, so that may have larger difference between criticizing.
In view of this, the present invention is specifically proposed.
Summary of the invention
To solve the problems, such as that the above mainstream cell culture process exists, the present invention provides a kind of new cell culture works
Skill, the cell culture process be improved on the basis of feed-batch culture (concentration fed-batch) is concentrated and
Innovation, concentration feed-batch culture are all to be trapped in cell and target product in reactor, and culture process of the invention only will
In the reactor, target product is discharged outside reactor cell retention together with cells and supernatant, mends with existing concentration
Material culture process is compared, and can be improved antibody producing efficiency, and substantially increases the stability production of unstable antibody.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of cell concentration feed-batch culture technique, cell cultivation process carry out fluid infusion and change liquid, wherein it changes during liquid,
Only cell is trapped, and target product is discharged with liquid.
Cell concentration feed-batch culture technique provided by the invention is in concentration feed-batch culture (concentration fed-
Batch it improving and innovates on the basis of), concentration feed-batch culture is all to be trapped in cell and target product in reactor,
Reactor is discharged in cells and supernatant without containing target product, to achieve the purpose that concentrated product, but which results in targets
There is the long period in product, increase the relevant impurity of target product in the reactor, simultaneously for some unstable productions
Product, the culture process are just no longer applicable in.
Culture process of the invention, when changing liquid, entrapped cell, not entrapped cell secretion product, target product with
Cells and supernatant be discharged outside reactor together, compared with existing concentration feed-batch culture technique, for self stability
The basic indifference of preferable target product, but the target product poor for self stability can greatly improve its yield.
Further, the step of cell cultivation process carries out fluid infusion and changes liquid are as follows:
Cell inoculation culture the 2-3 days, fluid infusion, changes liquid and fluid infusion for 3-6 days, carries out changing liquid and fluid infusion daily later
Operation.
Different according to the culture demand of different cells, fluid infusion is different with the liquid time is changed.
It is inoculated into production equipment and is cultivated when cell quantity is met the requirements.
Further, the cell inoculation is cultivated into production equipment, and the production equipment includes square vase, shaking flask, reaction
Device etc..
Further, the density of the inoculation is 1 × 105Cells/mL to 9 × 106cells/mL。
I.e. inoculum density is required according to detailed programs, generally 100,000 cells/mL to 1,000,000 cells/mL.
Further, the fluid replacement volume is the 3% ± 0.5% of initial incubation volume.
Changing liquid is to be replaced according to former volume of culture, also, the culture medium changed before and after liquid is equivalent.This equally may be used
Be it is identical, be also possible to it is similar, as long as meet or be conducive to cell normal growth.
Further, the cell retention includes following manner: centrifugation, hollow-fibre membrane retention or tangential Flow Technique.
It does not need abandon processing after cell retention, cell density is directly added into new cell liquid without adjustment
Liquid is changed, culture is continued.
In the present invention, the cells and supernatant containing target product can be discharged from reactor, and be stored in 4-8
DEG C, whole process keeps sterile；The cells and supernatant containing target product of discharge can also be purified in time, then
Then sterile storage is purified in 4-8 DEG C after merging all cells and supernatants after culture terminates, or
Product after purifying is merged, final target product is obtained.
Further, the target product includes any processing mode in following:
Under aseptic process mode by the fluid storage of discharge 2-8 DEG C under the conditions of；
4-8 DEG C is stored in after being sterile filtered after the liquid concentration of discharge.
Further, the cell is hybridoma, the Chinese hamster ovary cell for carrying heterogenous expression carrier.
Further, the target product includes antigen, antibody.
Further, the antigen includes HBsAg.
Further, the antibody includes the antibody being adapted to dengue fever, PD-1 antibody.
Further, the cell is hybridoma or the Chinese hamster ovary cell of HBsAg expression etc., the benefit
The lactoalbumin hydrolysate that material solution used is 100g/L；
The cell is the Chinese hamster ovary cell for expressing PD-1 antibody, and solution used in the feed supplement is
Efficient feedTM C+。
The culture solution of different cells is different, if the culture solution of hybridoma is Hybridoma SFM culture medium, China
The culture solution of hamster ovary cell is Dynamis culture medium, and certainly, other are suitable for the culture medium of cell culture of the invention
Also within the scope of the present invention.
Further, concentration of glucose is controlled during the entire process of cell culture between 1-6g/L.Such as different thin
In born of the same parents, control concentration of glucose can 1-3g/L, control concentration of glucose 1-4g/L, control concentration of glucose in 1-5g/
L, control concentration of glucose is in 2-6g/L, control concentration of glucose in 2-5g/L etc..
Further, the total time of cell culture is 7-15 days.
Further, the collected liquid further includes the steps that separating and purify, and obtains target product.
Further, the separation is successively carried out by the way of centrifugation and filter membrane.
Cell culture process provided by the invention is compared to existing technology, it is preferred that emphasis is it is changed during liquid to cell
Different from the processing of target product, subsequent separation and purification step are carried out according to prior art conventional method.
Compared with prior art, the invention has the benefit that
(1) technique of the invention, being compared to feed-batch culture (fed-batch) can be improved antibody producing efficiency, reduce
Antibody producing cost.
(2) target product is constantly pumped out into cell production equipment in technical process provided by the invention, and is stored in 2-
8 DEG C, for unstable target product, being compared to concentration feed-batch culture (concentration fed-batch) has more
Big advantage.
(3) technique of the invention is compared to perfusion culture (perfusion), and technology difficulty substantially reduces.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described.
Fig. 1 is viable cell density figure in the cell cultivation process of groups processing different in the embodiment of the present invention 1；
Fig. 2 is viable cell density figure in the cell cultivation process of groups processing different in the embodiment of the present invention 2；
Fig. 3 is viable cell density figure in the cell cultivation process of groups processing different in the embodiment of the present invention 3.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument
Person is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
Cell strain used is hybridoma made of murine myeloma cell is merged with mouse B cell, can secrete and be used for
Diagnose the monoclonal antibody (DEN-30DN) of dengue fever.
Cell passage is carried out after cell recovery when cell density reaches when inoculation requires by 300,000 cell/ml of inoculation
Start inoculated and cultured, basis culture used is Hybridoma SFM culture medium, and the supplemented medium is the hydrolysis of 100g/L
Lactoprotein, control concentration of glucose is between 1-5g/L in whole process.
Control 1: using feed-batch culture (fed-batch), carries out feed supplement, daily feed supplement initial incubation when culture was to the 2nd day
The 3% of volume, culture to end in the 7th day are cultivated.
Control 2: it using concentration feed-batch culture (concentration fed-batch), is mended when culture was to the 2nd day
Material, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture is to 72h, injects fresh culture, then feed supplement 3%；
It carries out changing liquid and feed-batch culture daily later, culture terminates for 15 days, and antibody is trapped in the reactor in whole process.
Implementation group: cell culture process (cell concentration feed-batch culture) of the invention is used, is carried out when culture was to the 2nd day
Feed supplement, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture was to the 3rd day, injects fresh culture, then feed supplement
3%；Carry out changing liquid and feed-batch culture daily later, culture terminates for 15 days, and antibody is discharged reaction with changing liquid in whole process
Device.The cell culture supernatant of discharge is stored in inside sterile liquid storing bag, and is stored at 4-8 DEG C.
Implementation group sample is first mixed after culture, is then centrifuged all samples 20 minutes through 10000g, it
Afterwards again through 0.45 μm of membrane filtration.Final DEN-30DN monoclonal antibody is obtained through the purifying of mono- step of Protein G.
Other also obtain target product according to corresponding separation and purification step.
Viable cell density measurement result in cell cultivation process is as shown in Figure 1.The expression of target product such as table 1
It is shown.
1 target product expression of table summarizes
Pass through embodiment 1, it can be seen that new process provided by the invention is more raw with higher antibody than feed-batch culture technique
Produce efficiency.Since the Dengue Antibody stability is good, so new process is opposite similar compared with concentration feed-batch culture effect.
Embodiment 2
Cell strain used is Chinese hamster ovary cell (CHO), and will contain can express hepatitis B surface antigen (HBsAg)
Plasmid is transferred in the CHO cell line, and the stabilization cell clone of energy HBsAg expression is obtained after screening.
Cell passage is carried out after cell recovery when cell density reaches when inoculation requires by 500,000 cell/mL of inoculation
Starting inoculated and cultured, basis culture used is Dynamis culture medium, and the supplemented medium is the lactoalbumin hydrolysate of 100g/L,
Concentration of glucose is controlled in whole process between 1-6g/L.
Control 1: using feed-batch culture (fed-batch), carries out feed supplement, daily feed supplement initial incubation when culture was to the 3rd day
The 3% of volume, culture to end in the 15th day are cultivated.
Control 2: it using concentration feed-batch culture (concentration fed-batch), is mended when culture was to the 3rd day
Material, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture was to the 5th day, injects fresh culture, then feed supplement
3%；It carries out changing liquid and feed-batch culture daily later, culture terminates for 15 days.Antibody is trapped in the reactor in whole process.
Implementation group: cell culture process (cell concentration feed-batch culture) of the invention is used, is carried out when culture was to the 3rd day
Feed supplement, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture was to the 5th day, injects fresh culture, then feed supplement
3%；It carries out changing liquid and feed-batch culture daily later, culture terminates for 15 days.Antibody is discharged reaction with changing liquid in whole process
Device.The cells and supernatant of discharge slightly exists inside sterile liquid storing bag, and is stored at 4-8 DEG C.
Implementation group sample is first mixed after culture, is then centrifuged all samples 20 minutes through 10000g.It
Afterwards again through 0.45 μm of membrane filtration.Final HBsAg is obtained after hydrophobic and molecular sieve purification.
Other also obtain target product according to corresponding separation and purification step.
Viable cell density measurement result in cell cultivation process is as shown in Figure 2.The expression of target product such as table 2
It is shown.
2 target product expression of table summarizes
Pass through embodiment 2, it can be seen that new process of the invention is imitated than feed-batch culture technique with higher antibody producing
Rate.Since hepatitis B surface antigen stability is poor, so, new process effect is substantially better than concentration feed-batch culture.
Embodiment 3
Cell strain used is Chinese hamster ovary cell (CHO), will combine death protein -1 containing that can express
(PD-1) plasmid of antibody is transferred in the CHO cell line, obtains to express the stabilization cell gram of PD-1 antibody after screening
It is grand.
Cell passage is carried out after cell recovery when cell density reaches when inoculation requires by 500,000 cell/mL of inoculation
Start inoculated and cultured, basis culture used is Dynamis culture medium, and the supplemented medium is Efficient feedTMC+,
Concentration of glucose is controlled in whole process between 1-6g/L.
Control 1: using feed-batch culture (fed-batch), carries out feed supplement, daily feed supplement initial incubation when culture was to the 3rd day
The 3% of volume, culture to end in the 15th day are cultivated.
Control 2: it using concentration feed-batch culture (concentration fed-batch), is mended when culture was to the 3rd day
Material, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture was to the 6th day, injects fresh culture, then feed supplement
3%；It carries out changing liquid and feed-batch culture daily later, culture terminates for 15 days, and antibody is trapped in the reactor in whole process.
Implementation group: cell culture process (cell concentration feed-batch culture) of the invention is used, is carried out when culture was to the 3rd day
Feed supplement, feeding volume are the 3% of initial incubation volume；It carries out changing liquid when culture was to the 6th day, injects fresh culture, then feed supplement
3%；Carry out changing liquid and feed-batch culture daily later, culture terminates for 15 days, and antibody is discharged reaction with changing liquid in whole process
Device.The cells and supernatant of discharge is stored in inside sterile liquid storing bag, and is stored at 4-8 DEG C.
Implementation group sample is first mixed after culture, is then centrifuged all samples 20 minutes through 10000g.It
Afterwards again through 0.45 μm of membrane filtration.Obtain final PD-1 antibody after purification through affine, zwitterion.
Other also obtain target product according to corresponding separation and purification step.
Viable cell density measurement result in cell cultivation process is as shown in Figure 3.The expression of target product such as table 3
It is shown.
3 target product expression of table summarizes
Pass through embodiment 3, it can be seen that new process has higher antibody producing efficiency than feed-batch culture technique.Due to
PD-1 Antibody stability is preferable, so, new process is opposite similar compared with concentration feed-batch culture effect.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of cell concentration feed-batch culture technique, which is characterized in that cell cultivation process carries out fluid infusion and changes liquid, wherein changes
During liquid, only cell is trapped, and target product is discharged with liquid.
2. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the cell cultivation process carries out
Fluid infusion and the step of change liquid are as follows:
Cell inoculation culture the 2-3 days, fluid infusion, changes liquid and fluid infusion for 3-6 days, carries out the operation for changing liquid and fluid infusion daily later.
3. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the cell inoculation to production is set
Standby middle culture, the production equipment includes square vase, shaking flask, reactor；
Further, the density of the inoculation is 1 × 105Cells/mL to 9 × 106cells/mL。
4. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the fluid replacement volume is initial
The 3% ± 0.5% of volume of culture.
5. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the cell retention includes following
Mode: centrifugation, hollow-fibre membrane retention or tangential Flow Technique.
6. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the target product includes following
In any processing mode:
Under aseptic process mode by the fluid storage of discharge 2-8 DEG C under the conditions of；
4-8 DEG C is stored in after being sterile filtered after the liquid concentration of discharge.
7. cell concentration feed-batch culture technique according to claim 1, which is characterized in that the cell is that hybridoma is thin
Born of the same parents, the Chinese hamster ovary cell for carrying heterogenous expression carrier；
Further, the target product includes antigen, antibody；
Further, the antigen includes HBsAg；
Further, the antibody includes the antibody being adapted to dengue fever, PD-1 antibody.
8. according to claim ask 7 described in cell concentration feed-batch culture technique, which is characterized in that the cell be hybridoma it is thin
The Chinese hamster ovary cell of born of the same parents or HBsAg expression, solution used in the feed supplement are the lactoalbumin hydrolysate of 100g/L；
The cell is the Chinese hamster ovary cell for expressing PD-1 antibody, and solution used in the feed supplement is Efficient
feedTMC+；
Further, concentration of glucose is controlled during the entire process of cell culture between 1-6g/L.
9. cell concentration feed-batch culture technique according to claim 1-8, which is characterized in that cell culture it is total
Time is 7-15 days.
10. cell concentration feed-batch culture technique according to claim 1-8, which is characterized in that collected
Liquid further includes the steps that separating and purify, and obtains target product；
Further, the separation is successively carried out by the way of centrifugation and filter membrane.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628407A (en) * | 2019-01-28 | 2019-04-16 | 哈药集团技术中心 | The method of high density long-term cultivation expression anti-vegf Humanized monoclonal antibodies recombinaant CHO cell |
| WO2021008571A1 (en) * | 2019-07-16 | 2021-01-21 | 信达生物制药(苏州)有限公司 | Cell culture method and application thereof based on high-density and continuous inoculation |
| CN112592948A (en) * | 2020-12-16 | 2021-04-02 | 广州汉腾生物科技有限公司 | Perfusion culture method of animal cells |
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| CN1238495C (en) * | 2003-12-31 | 2006-01-25 | 中国人民解放军军事医学科学院生物工程研究所 | Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium |
| MX2009003034A (en) * | 2006-09-21 | 2009-11-18 | Verenium Corp | Phospholipases, nucleic acids encoding them and methods for making and using them. |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238495C (en) * | 2003-12-31 | 2006-01-25 | 中国人民解放军军事医学科学院生物工程研究所 | Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium |
| MX2009003034A (en) * | 2006-09-21 | 2009-11-18 | Verenium Corp | Phospholipases, nucleic acids encoding them and methods for making and using them. |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628407A (en) * | 2019-01-28 | 2019-04-16 | 哈药集团技术中心 | The method of high density long-term cultivation expression anti-vegf Humanized monoclonal antibodies recombinaant CHO cell |
| WO2021008571A1 (en) * | 2019-07-16 | 2021-01-21 | 信达生物制药(苏州)有限公司 | Cell culture method and application thereof based on high-density and continuous inoculation |
| CN114127260A (en) * | 2019-07-16 | 2022-03-01 | 信达生物制药(苏州)有限公司 | Cell culture method of high-density continuous inoculation and its application |
| CN112592948A (en) * | 2020-12-16 | 2021-04-02 | 广州汉腾生物科技有限公司 | Perfusion culture method of animal cells |
| CN112592948B (en) * | 2020-12-16 | 2023-05-09 | 广州汉腾生物科技有限公司 | Perfusion culture method of animal cells |
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