CN104862280A

CN104862280A - Method for screening target protein CHO expressing cell strain applied to large-scale suspension culture

Info

Publication number: CN104862280A
Application number: CN201510290161.3A
Authority: CN
Inventors: 王海华; 文铁桥
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2015-05-29
Filing date: 2015-05-29
Publication date: 2015-08-26

Abstract

The invention relates to a method for screening a target protein CHO expressing cell strain applied to large-scale suspension culture. According to the method, the comprehensive situation of each cell strain is further understood through an experimental method comprising the steps of continuous culture, ventilation interference to the continuous culture, osmotic pressure interference to the continuous culture and fed-batch culture; finally the cell strain most suitable for large-scale suspension culture according to the comprehensive situation is selected. Through the mutual cooperation of several ways, the method greatly reduces the labor intensity and improves the efficiency and accuracy. Throughout the screening process, the applied ways and methods are quite simple and cheap; such a process can be carried out by most research institutions and enterprises, therefore, the practicality is high.

Description

Be applied to the screening method of the expression target protein Chinese hamster ovary celI strain of extensive suspension culture

Technical field

The present invention relates to a kind of screening method being applied to the expression target protein Chinese hamster ovary celI strain of extensive suspension culture.

Background technology

From the sixties in last century, people just start and utilize mammaliancellculture technology to produce various macromole biological products.And, along with a large amount of development and application of permanent cell strain, and under actual needs and commercial benefits demand, be that the pharmaceutical protein R & D and manufacture of instrument starts to grow up fast, as the R & D and manufacture of virus vaccines, antibody, Interferon, rabbit, hormone, immunomodulator and somatomedin etc. with mammalian cell.

The mammalian cell being usually used in exogenous protein expression has the cells such as CHO, NSO, hybridoma, wherein, Chinese hamster ovary celI derives from the ovary of Chinese hamster, be usually used in biology and medical research, Chinese hamster ovary (Chinese hamster ovary, CHO) cell is the optimum expression system of external source eukaryotic gene, is mammalian cell the most frequently used in bio-pharmaceuticals.

Within 1984, Genentech company utilizes the tissue-type plasminogen activator t-PA of r-CHO cell expressing, becomes the biotech drug that first is used the scale operation of mammaliancellculture technology.Now, Chinese hamster ovary celI has been the most frequently used clone of industrial Restruction albumen.Going on the market and carrying out in the genetically engineered drug of clinical study, about 60-70% is mammalian cell expression product, and expressing cho cell product accounts for the overwhelming majority wherein.

Chinese hamster the earliest for research be in 1919, be the mensuration of carrying out pneumococcus for alternative mouse at that time.1949, Chinese hamster was brought to the U.S., and bred in the lab.Next year, it is found that, Chinese hamster only containing 11 pairs of karyomit(e)s, is therefore considered to the ideal model of radiation cytogenetics and tissue culture.Nineteen fifty-seven, Theodore T Puck obtains a female Chinese hamster, and from obtaining original Chinese hamster ovary line thus, containing proline(Pro) in the growth needs substratum of this cell.Hereafter, Chinese hamster ovary celI due to grow fast and protein yield high and be widely used.

CH0-K1 comes from original Chinese hamster ovary celI system, and compared with original Chinese hamster ovary celI system, it contains less DNA.CH0-K1 sudden change creates CH0-DXB11 (also referred to as CHO-DUKX or CH0-DUK-XB11), it is Tetrahydrofolate dehydrogenase (DHFR) defective type, one of them Tetrahydrofolate dehydrogenase allelotrope (dhfr) site deletion, the dhfr in another one site there occurs missense mutation.Afterwards, people use the cell strain (CHO-pro3) that in original Chinese hamster ovary celI system, a strain dried meat Gas acid relies on, and create CHO-DG44 through sudden change, and allelic two sites of dhfr of this strain cell are all disappearances.CH0-DXB11 and CHO-DG44 is the cell of two strain DHFR defects, their growth needs glycine, secondary Huang he purine and thymidine (being called GHT), this two strains cell Chang Zuowei host cell, for transfection foreign gene.Because foreign gene is transfected in DHFR defect Chinese hamster ovary celI together with dhfr gene, therefore can grow in not containing the substratum of GHT, therefore, GHT defect substratum may be used for screening transfection positive cell.

Use the Chinese hamster ovary celI of DHFR defect as host, the step of expression alien gene is:

1. expression vector establishment: clone's goal gene, on goal gene and dhfr gene integration a to carrier or both are on different carriers.

2. transfection: expression vector is transfected in the Chinese hamster ovary celI of DHFR defect.

3. select: the cell after transfection grows in the substratum of GHT defect, and what survive is dhfr positive cell, usually also containing goal gene.

4. gene amplification: the single copy of the positive cell obtained in previous step usually only containing goal gene, in order to improve gene copy number, add methotrexate (MTX in the medium, the competitive inhibitor of Tetrahydrofolate dehydrogenase) for the dhfr that increases, and and then goal gene is increased.

5. cell screening: pick out the cell that output is high from the cell through gene amplification, and assess its stability.

Above step is traditional screening method.

The core link of this protein drug manufacture of the extensive suspension culture of Chinese hamster ovary celI, be guarantee the research and development of protein drug, clinical application and production and sales consumption basic.Should so think: protein drug, particularly the development of therapeutic monoclonal antibodies and fusion rotein medicine thereof is the main driver promoting this technical development.At home, therapeutic monoclonal antibody and fusion rotein medicine thereof research and development and produce just ground zero, in this respect few of technological accumulation.

At present, the preparation of recombinant antibodies and fusion rotein thereof mainly adopts Chinese hamster ovary celI culture process in the world, is mainly fed batch culture mode, and its unit output generally can reach 3 ~ 5g/L.(reach as high as 13g/L, but this is an example, meanwhile, also has comparatively Important Relations with product itself.) the cultivation scale of large-scale pharmacy corporation more than 200,000 liters as: Amgen company 200000L, Genentech company 250000L etc., domestic recombinant antibodies and fusion rotein production level thereof are lower than 1g/L, and feeding culture scale is no more than 3000L(Zhong Xin state and is good for.Separately, the said firm single tank 5000L scale, the production line of total 30000L scale is in building.), perfusion culture scale is no more than (500L).

The scale that in protein drug production process, single reactor is cultivated often improves 10 times, and its comprehensive production cost just can reduce by more than 20%.Visible, Chinese hamster ovary celI domestic at present cultivates scale and unit output result in high production cost, can not meet the low cost requirement that domestic market will reach, meanwhile, the consigned processing (CMO) that the production cost of high enterprise also constrains for world market is served.

Viable cell density aspect closely-related with unit output, can reach 5X10 abroad ⁷the level of cells/ml, and be the highlyest less than 2X10 ⁷the level of cells/ml, on average at 0.7X10 ⁷the level of about cells/ml.In the culturing process Midst density of Chinese hamster ovary celI and the raising of unit output, not only be subject to the impact of very important culture medium system and supplying technics thereof, also finely control relevant with processing parameter, instant and accurate regulation and control are needed in the process that cultivation scale is amplified, the culturing engineering teacher that experience is not enriched, accomplish that this point is difficult to.In this area, there is a big difference for domestic so-called core technology and world level, and corresponding production technique does not have independent intellectual property right at all, and just indiscriminately imitating or imitating expired patent technology, falls behind relatively.Meanwhile, also have very large relation with the of cell strain own and its screening process, this is also this research Producing reason.

At present, many research units and enterprise, be also value very much for cell strain screening, focus mainly concentrates on expression amount situation and the quality condition of protein drug.Simultaneously, also can the effect that cell culture medium and feeding strategy are correlated be paid close attention to, this respect mainly with the consistent preliminary culture process optimized without personalization under small-scale condition, each candidate cell strain is compared, pick out the cell strain that albumen conforms to quality requirements and expression amount is the highest of acquisition, and determine the basic conditions such as its activity.

But, can, when cell strain screens, the adaptability for each cell strain scale amplification culture and process optimization compares aspect, or adapt in the future scale amplification culture and technique thereof for cell strain and optimize further and require aspect, no matter be bibliographical information, or rarely have appearance in practical study.

In cell cultures scale amplification process and process optimization thereof, need to compare, or the requirement that can produce mainly contains: under very clear and definite equal conditions, the comparison of the overall unit output of cell mass and lactic acid accumulation situation, to the tolerance of shearing force, to requirement and the difference of the situations such as the tolerance of osmotic pressure.

Simultaneously, at each cell strain after screening in early stage, when running into the candidate cell strain still having more basic condition similar, if carry out the follow-up comparison of scale amplification process and process optimization thereof again, will produce great time and fund cost, therefore, be to carry out follow-up comparison substantially in real work, but according to garbled data in early stage closely, existing each subsequent cell strain is similar to the selection of random fashion.Like this, in fact selected cell strain might not be best suited for the cell strain in follow-up extensive suspension culture, numerous items can be allowed to lose and obtain as far as possible close to best achievement chance.

Therefore, need a kind of screening method being applied to the expression target protein Chinese hamster ovary celI strain of extensive suspension culture, so that from numerous candidate cell strain that basic condition is similar screening in early stage, select the cell strain that comprehensive condition is more suitable for extensive suspension culture, namely by cell strain that this screening mode obtains, by than the cell strain selected by being similar to random fashion in this stage, obtain higher protein expression output when carrying out extensive suspension culture.

Summary of the invention

The object of the present invention is to provide a kind of screening method being applied to the expression target protein Chinese hamster ovary celI strain of extensive suspension culture.

For achieving the above object, the invention provides following technical scheme:

Be applied to a screening method for the expression target protein Chinese hamster ovary celI strain of extensive suspension culture, it is characterized in that the concrete steps of the method are:

A. conventional screening methods is used to obtain several candidate cell strains, concise and to the point step is as follows: the cell strain of employing is Chinese hamster ovary celI, cultivate the expression carrying out target protein at serum free suspension, according to actual needs, filter out the satisfactory cell strain of Qp value and preserve as seed;

B. the cell strain obtained step a carries out continous way and cultivates screening, and concrete steps are as follows:

B-1. get after cell strain that step a filters out carries out resuscitation, be inoculated in respectively in serum free medium, the density of inoculation is 1 ~ 5X10 ⁵cells/ml, obtains cell suspending liquid, carries out follow-up non-interfering parallelly cultivate process;

B-2. each for step b-1 gained cell suspension is carried out shake-flask culture, shaking table speed 50 ~ 150rpm, gas concentration lwevel 3 ~ 8%, culture temperature 35 ~ 37 DEG C, cultivate after 48 ~ 92 hours, under centrifugal condition is 800 ~ 1500rpm, centrifugation 3 ~ 8 minutes, getting cell precipitation is inoculated in serum free medium, and obtaining inoculum density is 3 ± 0.15X10 ⁵the cell suspension of cells/ml;

B-3. each cell suspension of b-2 step gained is carried out shake-flask culture, shaking table speed 50 ~ 150rpm, gas concentration lwevel 3 ~ 8%, culture temperature 35 ~ 37 DEG C, reaches 2 ~ 3X10 to viable cell density ⁶cells/ml;

B-4. each for step b-2 gained cell suspension is carried out centrifugation, centrifugal condition is 800 ~ 1500rpm, 3 ~ 8 minutes, collects supernatant, and under-25 ~-15 DEG C of conditions, sealing is preserved;

B-5. be suspended from serum free medium by each for step b-4 gained cell precipitation, the amount of this serum free medium is identical with the serum free medium volume used in step b-2;

B-6. each for step b-5 gained suspension is carried out shake-flask culture, shaking table speed 50 ~ 150rpm, gas concentration lwevel 3 ~ 8%, culture temperature 35 ~ 37 DEG C, cultivate after 20 ~ 28 hours, sampling metering viable cell density;

B-7. get step b-6 gained suspension repeating step b-4 to step b-6, reach stationary value to viable cell density, enter the stationary phase that continous way is cultivated, maintain and operate 3 ~ 10 days stationary phase;

B-8. detect the unit output of the target protein of every 20 ~ 28 hours in each supernatant liquor, filter out the satisfactory cell strain of protein units output according to actual needs;

C. carrying out ventilation to the suspension of each cell strain that step b filters out interferes continous way to cultivate screening, and concrete steps are as follows:

C-1. pass into the condition of air at the flow velocity of 20 ~ 30ml/min under, repeating step b-4 to step b-6, after reaching stationary phase to viable cell density 3 ~ 10 days;

C-2. adjust air flow, amplification is 20 ~ 30ml/min, and repeating step b-4 is to step b-6, after reaching stationary phase to viable cell density 3 ~ 10 days;

C-3. repeating step c-2, until cell mass is all dead;

C-4. according to can the number of times of repeating step c-2 and actual needs, satisfactory cell strain be filtered out;

D. each cell strain filtered out step c carries out osmotic pressure and interferes continous way to cultivate screening, and concrete steps are as follows:

D-1. the operation of steps performed b-1 to b-7, does not preserve supernatant;

D-2. adjust its osmotic pressure value, amplification is 40 ± 10mOsm/kg, and under new osmotic pressure condition, repeating step b-4, to step b-6, does not preserve supernatant, after reaching stationary phase to viable cell density 3 ~ 10 days;

D-3. repeating step d-2, until cell mass is basic all dead;

D-4. according to can the number of times of repeating step d-2 and actual needs, satisfactory cell strain be filtered out;

E. fed batch culture screening is carried out to each cell strain filtered out in steps d, finally filter out satisfactory cell strain.

Compared with prior art, the invention has the beneficial effects as follows: the present invention is by the mode of actual experiment, probe into and set up feasibility, validity, the practicality that the Chinese hamster ovary celI strain expressing target protein is applied to the screening method of extensive suspension culture, and finally established simple, cheap, effective screening method.At each cell strain after screening in early stage, when running into the candidate cell strain still having more basic condition similar, use the mode that continous way is cultivated, culture environment can be allowed to be in stable state for a long time, interference experiment can be passed through like this, single condition is tested, finally equally effectively obtain data clearly, on this basis clear and definite screening is carried out to each cell strain, with in this stage by compared with the selection that is similar to random fashion and carries out, its comprehensive condition of the cell strain selected is more suitable for extensive suspension culture, namely when carrying out extensive suspension culture, higher protein expression output can be obtained.Finally, by the comparison of the extensive suspension culture of bio-reactor, demonstrate this process of application result of carrying out screening and be better than original selection.Above process, specify that the feasibility of the screening method of foundation, validity.

It should be noted that and use continous way to cultivate, ventilation interferes continous way to be cultivated, and osmotic pressure interferes continous way to be cultivated, and carrying out test and comparison to each condition, is the core of this screening method.Meanwhile, fed batch culture is in whole method, and be also indispensable, both complement each other, and reaches the comprehensive condition that each cell strain of overall understanding is suitable for for large scale culturing.

In addition, merge mutually again between a few person and make up respective relative merits, by working in coordination of several mode, greatly reducing labour intensity, improve efficiency and accuracy.In whole screening process, the methods of utilization are all very simple and cheap, and such process, all have the ability to implement for most of research institution and enterprise, practicality is very strong.

Accompanying drawing explanation

Fig. 1 is that cell strain 12 and cell strain 15 carry out feed supplement written instructions cultivation viable cell density data comparison diagram in the production tank suspension culture of 50L scale, (bio-reactor Mobius CellReady 50L).

Fig. 2 is that cell strain 12 and cell strain 15 carry out feed supplement written instructions culture units yield data comparison diagram in the production tank suspension culture of 50L scale, (bio-reactor Mobius CellReady 50L).

Embodiment

Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.

Embodiment 1

In the embodiment of the present invention, the Chinese hamster ovary celI strain expressing target protein is applied to the screening method of extensive suspension culture, and concrete steps comprise:

1. tentatively prepare: testing cell strain used is Chinese hamster ovary celI (DHFR system, CHO-DXB11 type), express FC-fusion rotein, totally 15 strain clones.

This 15 strain clone is obtained by prerequisite activity, and its process brief introduction is as follows:

Screening step process traditionally, carries out vector construction, transfection, and GHT defect is selected, and then carries out gene amplification under MTX pressure, finally screens in 96 orifice plates with limiting dilution assay again.(substratum that period uses is, and not containing the DMEM/F12 of GHT, and adds the FBS of 10%.）

96 orifice plates (20 blocks of plates) are used when screening, obtain mono-clonal, then cultivate, cultivate after 10 days, get the expression amount that culture supernatant ELISA method measures FC-fusion rotein again, overall expression amount is obtained relatively high and distinguish 72 little strain clones from totally 1920 plate holes, this 72 strain clone is inoculated in (3 blocks of plates) in 24 orifice plates, proceed the comparison screening of individual cells daily output protein content (Qp), therefrom obtain Qp value higher clone 15 strain, each cell strain is numbered from 01 ~ 15, this 15 strain clone is carried out liquid nitrogen cryopreservation after amplification cultivation.

Due to this 15 strain clone Qp value all relatively, therefore, screening mode traditionally carries out the words selected, general random is selected, or select that strain clone that expression data is relatively high, it should be noted that these select the clone obtained, its expression data does not have difference in essence, this also time the phenomenon that often runs in most of project, there is randomness when this also allows screening process reach this step.

Cell strain 15 is chosen to be Cells for production strain, prepares the follow-up phase of the project that is applied to.

2. the screening that cell strain serum free medium adapts to and batch-type is cultivated

The acquired 15 strains Chinese hamster ovary celI strain of expressing FC-fusion roteins containing carrying out in blood serum medium cultivating, transfection, selection, screening, amplification, frozen.Because Chinese hamster ovary celI is in industrial large scale culturing application, substantially all carries out serum free suspension cultivation, therefore, first need to carry out serum free medium adaptation to this 15 strain cell.

2.1 serum free mediums of expressing the Chinese hamster ovary celI strain of target protein adapt to

2.1.1 serum free medium adaptive process

Can relate to two kinds of substratum in serum free medium adaptive process, wherein, be not containing the DMEM/F12 of GHT containing blood serum medium, MTX concentration 100nmol/L, and the FBS adding 10%; Serum free medium is that experimenter has by oneself, is applicable to the basic medium W027 of the extensive suspension culture of Chinese hamster ovary celI, and wherein, MTX concentration is also 100nmol/L.

1), after 15 strains being expressed the Chinese hamster ovary celI recovery of target protein, be inoculated in containing in blood serum medium respectively, inoculum density is 1 ± 0.05X10 ⁵cells/ml.

2) be placed in by cell suspension in T-25 Tissue Culture Flask, inoculation volume is 8ml, and every strain cell inoculates 2 bottles.

3) 15 strain cell numberings are respectively: 01 ~ 15, and the different culturing bottle of homophyletic cell is respectively A0 and B0.

4) will altogether be positioned in CO2gas incubator and carry out quiescent culture by 30 bottles of cells, gas concentration lwevel be 5.0%.

After inoculating Tissue Culture Flask, now, owing to being also the use of containing blood serum medium, therefore cell is adherent growth substantially, is namely affixed on bottom Tissue Culture Flask and grows, and also can contain and a small amount of suspension cell in supernatant.Cultivate after 72 to 96 hours, cell by full for growth basic bottom whole T-25 Tissue Culture Flask, now, can be carried out operation of going down to posterity by cell.

1) under the microscope, two culturing bottles of every strain cell are observed, determining not abnormal, after there is no significant difference, then get arbitrarily wherein one bottle operate.

2) first supernatant is moved in 15ml centrifuge tube together with the suspension cell in supernatant.

3) with 1ml cell dissociation buffer (containing pancreatin and EDTA), digest the attached cell in Tissue Culture Flask, digestion time about 1 to 2 minute, now, the cell be affixed on bottom Tissue Culture Flask can come off into suspension.

4) supernatant in 15ml centrifuge tube and suspension cell are wherein moved in Tissue Culture Flask, carry out piping and druming mixing operation.

5) moved to by cell suspension in 15ml centrifuge tube, carry out centrifugally operated, centrifugal condition is 1000rpm, 5 minutes.

6) after centrifugal end, obtain cell precipitation, be suspended from by cell precipitation in fresh culture, the substratum now used is 50% containing blood serum medium and 50% serum free medium.

7) with substratum, the viable cell density in cell suspension is adjusted to 2 ± 0.1X10 ⁵cells/ml, is inoculated in T-25 Tissue Culture Flask, and inoculation volume is 8ml, every strain 2 bottles, and the different culturing bottle numbering of homophyletic cell is respectively A1 and B1.

8) will altogether be positioned in CO2gas incubator and carry out quiescent culture by 30 bottles of cells, gas concentration lwevel be 5.0%, and culture temperature is 37 DEG C.

Cultivate after 48 to 72 hours, then carry out identical operation, unlike, specifically substratum finally used is adjusted to 25% containing blood serum medium and 75% serum free medium, then, proceeds to cultivate.Then, so cyclically carry out cultivating and operate, progressively adjusting the ratio between two kinds of substratum.Actually operating situation is in table 1.

In whole adaptive process, cell can from most adherent growth state, and change whole suspension growth state gradually into, this process is incremental, therefore, there will be the situation that adhered state cell and suspended state cell coexist.In this research, carry out again going down to posterity after all cells are all collected mixing, when the supernatant containing suspension cell rejoins in culturing bottle, suspension cell group in former supernatant can be digested to the cell of dispersion by pancreatin in culturing bottle equally, can guarantee like this to obtain the good cell of deployment conditions at every turn.

2.1.2 suspend after expressing the Chinese hamster ovary celI serum free medium adaptation of target protein effectiveness comparison

After cyclical operation several times, obtain 15 strains can in serum free medium W027 the cell of suspension growth, but in this 15 strain cell, also there is difference, in this stage, do not pay close attention to their growth and expression, focal point is placed on adherently to be changed on suspension effect, when they are after the 8th operation, it is grown in Tissue Culture Flask and observes, find that every strain cell still has the attached cell of different ratios to occur in growth amplification, in suspension cell, agglomerating situation also has difference simultaneously, and table 2 is observations.Each cell strain suspension effect after concluding and being adapted to by serum free medium sorts, in table 3.

"+" is evaluation of estimate, and more multilist shows that suspension effect is relatively better.

From table 2 and table 3, different cell strain just starts to have occurred difference after serum free medium adapts to, but above data still can not screen as carrying out the foundation accepted or rejected, so, after serum free medium has adapted to, 15 strain cells are seeded in T-75 Tissue Culture Flask respectively and have increased, every strain cell amplification to 2 bottle, culturing bottle numbering A9 and B9, treats that every bottle of viable cell density grows to 1X10 ⁶after more than cells/ml, mixed by cell suspension, carry out frozen operation, use serum free medium (containing 10%DMSO) to carry out frozen, often prop up 1ml, viable cell density is about 1X10 ⁷cells/ml, every strain cell obtains 6 backups, is placed in liquid nitrogen container and preserves, and prepares to be used for subsequent experimental research.

The screening that the 2.2 Chinese hamster ovary celI strain batch-types expressing target protein are cultivated

2.2.1 the batch-type expressing the Chinese hamster ovary celI strain of target protein is cultivated

Through previous research, obtain 15 strains and adapt to serum free medium and the Chinese hamster ovary celI strain expressing the expression target protein of FC-fusion rotein, in this stage, by to them under identical batch-type culture condition, carry out growing and the comparison of expression, therefrom select and carry out next stage and study the suitable cell strain of comparing.The substratum that this stage uses is serum free medium W027, and wherein, MTX concentration is 100nmol/L.

15 strain cells are taken turns through 5 respectively and carries out batch-type cultivation, 1st takes turns cell strain 01, cell strain 02, cell strain 03, cell strain 04 is tested, 2nd takes turns cell strain 01, cell strain 05, cell strain 06, cell strain 07 is tested, 3rd takes turns cell strain 01, cell strain 08, cell strain 09, cell strain 10 is tested, 4th takes turns cell strain 01, cell strain 11, cell strain 12, cell strain 13 is tested, 5th takes turns cell strain 01, cell strain 14, cell strain 15 is tested, the wherein control that tests as this of cell strain 01, take turns in experiment each, the data of cell strain 01 are all close, therefore the experimental data obtained in each wheel can carry out unifying to compare.Period, pH controls all to meet the requirements.Concrete operations are as follows.

1) be inoculated in respectively in serum free medium by the cell of recovery, the density of inoculation is 2 ± 0.10X10 ⁵cells/ml.

3) cell strain numbering uses original numbering, and the different culturing bottle of homophyletic cell is respectively A0 and B0.

4) be positioned in CO2gas incubator by cell and carry out quiescent culture, gas concentration lwevel is 5.0%, and culture temperature is 37 DEG C.

5) cultivate after 72 hours, taken out by 2 T-25 Tissue Culture Flask inner cell suspensions of every strain cell, mixing, obtain about 15ml cell suspension, wherein viable cell density is all at 1X10 ⁶more than cells/ml.

6) this cell suspension inoculation is entered in the shaking flask of 125ml specification, and fill into serum free medium to final volume 50ml, shaking flask is positioned on the little shaking table of decolouring, little shaking table is placed in CO2gas incubator, carries out shake-flask culture.Between incubation period, shaking table speed remains that 100rpm is constant, and gas concentration lwevel is always 5.0%, and culture temperature is 37 DEG C.

7) cultivate after 72 hours, sampling counting operation is carried out to each shaking flask, find that viable cell density is all at 1.5X10 ⁶more than cells/ml.

8) get a certain amount of cell suspension, and add serum free medium to cumulative volume 100ml, make viable cell density in the cell suspension of each cell strain of acquisition be 3 ± 0.15X10 ⁵cells/ml.

9) inoculated respectively by the cell suspension of each cell strain in the shaking flask of 2 125ml specifications, the cell suspension in each shaking flask is 45ml, and the different shaking flask of homophyletic cell is numbered C0 and D0.

10) each shaking flask be placed in CO2gas incubator and cultivate, between incubation period, shaking table speed remains that 100rpm is constant, and gas concentration lwevel is always 5.0%, and culture temperature is 37 DEG C.

11) in the training period, can add the sodium hydrogen carbonate solution of trace, to guarantee the pH value that each cell strain keeps more close, and pH value remained in suitable interval, meanwhile, the sodium bicarbonate amount added in each cell strain shaking flask is consistent substantially.PH is an important parameter of Growth of Cells environment, different for its suitable pH of different cell type, for Chinese hamster ovary celI, its suitable growth pH of report is 6.8-7.2 mostly, according to bibliographical information and summary of experience, suitable interval is set as 6.6-7.5, because in the process of cultivating, pH can slowly decline, and the meaning in this interval is exactly, be no more than 7.5 when starting to cultivate, at the end of cultivation, be not less than 6.6.

12) to the remaining cell of each strain, in original shaking flask, carry out amplification operation, namely fill into fresh substratum to final volume 75ml, then continue to cultivate by old terms, at interval of 24 hours sampling countings, when viable cell density in each shaking flask is more than 2X10 ⁶carry out frozen operation during cells/ml, freezing media is serum free medium W027,10%DMSO, and every cell strain can obtain at least 14 backups, and often prop up 1ml, viable cell density is about 1X10 ⁷cells/ml, is placed in liquid nitrogen container and preserves, for use in follow-up study.

13) carry out batch-type culture studies to each shaking flask being numbered C0 and D0, carried out sampling counting operation at interval of about 24 hours, each sample volume is unanimously 0.5ml, obtains instant viable cell density and cell viability data.

14) when each shaking flask inner cell vigor being numbered C0 and D0 is reduced to below 70%, stop this Shake flask batch formula to cultivate operation, centrifugally collect supernatant, carry out expression amount mensuration, will remain supernatant packing, at being placed in-20 DEG C, preservation, for subsequent use.

Carried out 5 altogether and taken turns experiment, 15 strain cells all complete batch-type culture experiment.Every strain cell uses 2 shaking flasks to carry out parallel laboratory test when testing, and in experimentation, the experimental data of every strain cell 2 shaking flasks is all substantially close.Get the mean value of same cell strain parallel laboratory test 2 groups of data again, obtain 15 cell mean data, represent 15 strain cells experimental result separately, wherein each wheel experimental data of cell strain 01 is also more close, so only take out wherein 12 groups of data of taking turns to carry out data processing and analysis.Concrete outcome compares as table 4.

The Chinese hamster ovary celI strain batch-type that target protein respectively expressed by table 4 cultivates viable cell density and unit output data summary table

* viable cell density, unit is 10 ⁵cells/ml.

* unit output, unit is g/L.

2.2.2 the screening that the Chinese hamster ovary celI strain batch-type expressing target protein is cultivated

As can be seen from the data obtained, there is difference when batch-type is cultivated in the cell strain of 15 strains after serum free medium adapts to.Because the object of this research is probing into for large scale culturing screening method, therefore only need to carry out lateral comparison to existing data, determine that whether this mode is feasible again, thus, this 15 strain cell is not needed to quote when carrying out data summarization containing representation of data when cultivating in blood serum medium.Certainly, why selecting this 15 strain cell, is also that it shows basic simlarity, does not have very large difference because they are containing when cultivating in blood serum medium.

For cell strain 02, cell strain 06, cell strain 10, no matter be stand density, or the unit output after batch-type cultivation, both distinguish comparatively large with most cell strain, so this three strains cell is given up.

For cell strain 13, although cell density and most cells strain are distinguished not quite, the unit output after batch culture is lower, so also given up by this strain cell.

Thus, after serum free medium adaptation is carried out to the Chinese hamster ovary celI strain expressing target protein, carry out batch-type cultivation, then, compare through data, cell strain poor for performance can be removed.

Cultivate through serum free medium batch-type, 4 strain performances are filtered out poor from 15 strain cells, the cell larger with other each cell strain gaps, they are given up, leave cell strain 01, research that cell strain 03, cell strain 04, cell strain 05, cell strain 07, cell strain 08, cell strain 09, cell strain 11, cell strain 12, cell strain 14, cell strain 15 carry out next stage.

By stand density and final unit output Combined Ration comparatively, can find out, the cell strain that growing state is poor, its final unit output is poor.But from cell strain 13, the good cell strain of growing state, its final unit output also might not be outstanding, so for the evaluation of cell strain, stand density is an index, but final unit output is only prior standard.

3. the screening of cell strain continous way cultivation

The 3.1 Chinese hamster ovary celI strain continous ways expressing target protein are cultivated

For individual plant cell, a complete continous way culturing process is as follows:

1) be inoculated in serum free medium by the cell of recovery, the density of inoculation is 2 ± 0.10X10 ⁵cells/ml.

2) be placed in by cell suspension in T-25 Tissue Culture Flask, inoculation volume is 8ml, and cell inoculates 2 bottles, and culturing bottle numbering is respectively A0 and B0.

3) Tissue Culture Flask is positioned in CO2gas incubator and carries out quiescent culture, and gas concentration lwevel is 5.0%.

4) cultivate after 72 hours, taken out by 2 T-25 Tissue Culture Flask inner cell suspensions, mixing, obtain about 15ml cell suspension, wherein viable cell density is about 1X10 ⁶about cells/ml.

5) this cell suspension inoculation is entered in the shaking flask of 125ml specification, and fill into serum free medium to final volume 50ml, shaking flask is positioned on the little shaking table of decolouring, little shaking table is placed in CO2gas incubator, carries out shake-flask culture.Between incubation period, shaking table speed remains that 100rpm is constant, and gas concentration lwevel is always 5.0%, and culture temperature is 37 DEG C.

6) cultivate after 72 hours, carry out sampling counting operation to each shaking flask, viable cell density will at 1.5X10 ⁶more than cells/ml, sample volume is 0.5ml.

7) all move in 50ml centrifuge tube by the cell suspension in shaking flask, carry out centrifugally operated, centrifugal condition is 1000rpm, 5 minutes.

8) cell precipitation is resuspended in the fresh culture of about 20ml, sampling counting operation is carried out to cell suspension.

9) get a certain amount of cell suspension, and add serum free medium to cumulative volume 100ml, make viable cell density in the cell suspension of acquisition be 3 ± 0.15X10 ⁵cells/ml.

10) be inoculated in respectively by cell suspension in the shaking flask of 2 125ml specifications, the volume of each shaking flask inner cell suspension is 45ml, and shaking flask is numbered C0 and D0.

11) shaking flask is positioned on the little shaking table of decolouring, little shaking table is placed in CO2gas incubator, carries out shake-flask culture.Between incubation period, shaking table speed remains that 100rpm is constant, and gas concentration lwevel is always 5.0%, and culture temperature is 37 DEG C.

12) in first 72 hours, carry out batch-type culture studies to each shaking flask, carried out sampling counting operation at interval of about 24 hours, each sample volume is unanimously 0.5ml, obtains instant viable cell density and cell viability data.

13) cultivate after 72 hours, viable cell density will at 2.0X10 ⁶about cells/ml.

14) now, continue to carry out independent parallel operation to each shaking flask, all move in 50ml centrifuge tube respectively, carry out centrifugally operated by the cell suspension in each shaking flask, condition is 1000rpm, 5 minutes.

15) collect supernatant, and preserve respectively, stand-by, at preservation condition is-20 DEG C, sealing is preserved.

16) respectively by resuspended for the cell precipitation fresh culture obtained after each shaking flask inner cell suspension centrifugally operated, the amount adding fresh culture makes cell suspension final volume be 45ml.

17) respectively cell suspension renewed vaccination is entered in original shaking flask.

18) shaking flask is positioned on the little shaking table of decolouring, little shaking table is placed in CO2gas incubator, carries out shake-flask culture.Between incubation period, shaking table speed remains that 100rpm is constant, and gas concentration lwevel is always 3%, and culture temperature is 37 DEG C.

19) cultivate after 24 hours, carry out sampling counting operation to each shaking flask, sample volume is 0.5ml, obtains instant viable cell density and cell viability data.

20) be cycled to repeat step 14), 15), 16), 17), 18), 19), obtain each shaking flask instant viable cell density of every 24 hours and cell viability data, and can all obtain the cells and supernatant that each shaking flask is about 40ml at every turn, containing the target protein that this shaking flask cell mass produced in incubation time at 24 hours in this supernatant, through detecting, 24 hours unit outputs of the cell mass that this strain cell is formed with this understanding can be obtained.

21), simultaneously, when operating above at every turn, after obtaining supernatant, also can carry out pH detection to the supernatant obtained immediately, obtain pH value instant in each shaking flask, to determine whether to exceed that suitable interval.

22) time length of whole continous way culturing process can experimentally require to determine, as can be seen from content of operation, in such manner, culturing process can continue to carry out for a long time.

In whole continous way culturing process, the serum free medium of use is W027, and wherein, MTX concentration is 100nmol/L.

Due to, in this substratum, nutritive ingredient is sufficient, and at viable cell density higher than 2 ± 0.10X10 ⁶the whole replacement operations carrying out new and old substratum every 24 hours are just started, so when each strain cell is cultivated with this understanding, media environment can keep good state, also can keep good stability during cells/ml.

Under this continous way cultural method, we find, for most cell strain, after viable cell density reaches certain value, namely can not increase significantly, also can not reduce significantly, can keep stable in certain numerical value aspect, the instant numerical value that each operation obtains can not produce with this center value and exceed ± the deviation of 5 ~ 10%, and we think now, and continous way cultivation enters stationary phase.

As can be seen here, when each strain cell is under this culture condition, when there is viable cell density, cell viability and 24 hours unit output difference, substratum and training method can not become the factor affecting this result.By with such culturing process as a setting, carry out follow-up research work.Meanwhile, can the procedure definition described in above step for without interfering continous way to be cultivated, this is relative to increasing for other conditions carry out testing with this understanding again.

3.2 express the Chinese hamster ovary celI strain of target protein without the screening interfering continous way to be cultivated

3.2.1 cultivate without interference continous way

According to the mode in 3.1, carrying out nothing respectively to 11 strain cells interferes continous way to be cultivated, carry out 3 altogether and taken turns experiment, 1st takes turns cell strain 01, cell strain 03, cell strain 04, cell strain 05 is tested, 2nd takes turns cell strain 04, cell strain 07, cell strain 08, cell strain 09 is tested, 3rd takes turns cell strain 04, cell strain 11, cell strain 12, cell strain 14, cell strain 15 is tested, the wherein control that tests as this of cell strain 04, take turns in experiment each, the data of cell strain 04 are all close, therefore the experimental data obtained in each wheel can carry out unifying to compare.Period, pH controls all to meet the requirements.

Carried out 3 altogether and taken turns experiment, 11 strain cells all complete without interfering continous way culture experiment.Every strain cell uses 2 shaking flasks to carry out parallel laboratory test when testing, and in experimentation, the experimental data of every strain cell 2 shaking flasks is all substantially close.Get the mean value of same cell strain parallel laboratory test 2 groups of data again, obtain 11 cell mean data, represent 11 strain cells experimental result separately, wherein each wheel experimental data of cell strain 04 is also more close, so only take out wherein 12 groups of data of taking turns to carry out data processing and analysis.Concrete outcome is more as shown in table 5.

According to viable cell density data, be the stationary phase without interfering continous way to be cultivated to the 12nd day each cell strain changed after liquid from the 5th day, the cells and supernatant that 6th day to the 12nd day obtains is detected, detect the supernatant of each 24 hours sections of each cell strain, measure the protein units output in supernatant and lactic acid generation unit vol, as shown in table 6 Yu table 7.

3.2.2 without the screening interfering continous way to be cultivated

The information of following needs can be obtained from data:

1) without interfering continous way to cultivate stationary phase, all closely, and the viable cell density of cell strain 07 and cell strain 11 is relatively low for the viable cell density of most cells strain.

2) stationary phase each cell strain 24 hours unit outputs uneven, wherein cell strain 07 is relatively low compared with major part with the unit output of cell strain 11, in all the other cell strains, unit output is up to 0.14g/L, minimum 0.10g/L, be cell strain 15 and cell strain 14 respectively, both differ 1.4 times.

3), in this 11 strain cell, the final unit output cell strain order from high to low that batch-type is cultivated is: 15 > 04=12 > 09=11 > 05 > 07 > 01=03=08 > 14.

4), in this 11 strain cell, cultivating 24 hours unit outputs without interfering continous way by height cell strain order is on earth: 15 > 12 > 04=09 > 01=03=05 > 08=14 > 11 > 07.

5) stationary phase each cell strain 24 hours lactic acid generate unit voies relatively high be cell strain 09, relatively low is cell strain 07 and cell strain 11.Here it should be noted that, in serum free medium, the content of lactic acid is zero.

6) stationary phase each cell strain 24 hours lactic acid to generate unit voies uneven equally, but remove relatively high cell strain 09, after relatively low cell strain 07 and cell strain 11, all the other each strain data relatively.

3.2.2.1 the comparison of oxygen demand and supply

As can be seen from these information, 24 hours unit outputs of cell strain 07 and cell strain 11 and lactic acid growing amount lower be because their viable cell density is relatively low, but, when batch-type is cultivated the stand density of cell strain 07 and cell strain 11 and all the other each strain gaps little, be so, that what reason causes this result? this is the oxygen demand inconsistent generation due to each cell strain.

Oxygen is needed to supply during Growth of Cells, when oxygen supply is inadequate, Growth of Cells can be affected, continous way cultivation results is interfered from current nothing, the result of impact is exactly that viable cell density cannot increase, and this just makes cell strain 07 relatively low compared with other each strains with the viable cell density of cell strain 11.

This without under interference continous way training method, in each shaking flask, oxygen-supplying amount is consistent, so the cell mass of this 11 strain cell is when stationary phase, their oxygen-consumption differs not too large in same level.Therefore, in current experiment, when there is larger gap in viable cell density, although can not conclude, whether the cell mass that viable cell density is high has obtained sufficient oxygen supply, but, its oxygen undersupply of cell strain that viable cell density is relatively low can be concluded.

Certainly, it is hypothetical for obtaining such conclusion:

1) each cell strain compared is in batch-type is cultivated, and their upgrowth situation is similar, illustrates that cell enlargement trend is roughly the same.

2) each cell strain compared, when batch-type is cultivated and cultivate without interference continous way, viable cell density gap is comparatively large, and at least the viable cell density in the latter period apparently higher than the former in period, when this illustrates that batch-type is cultivated, not by the impact of oxygen supply condition.

3) need nutrient in the composition of substratum sufficient, the obstacle of the growth of the relatively low cell strain of viable cell density can not be become, the substratum used can arrive such requirement, and, cultivate without interfering continous way and nutritive ingredient when cultivating also can well be allowed to keep sufficient.Meanwhile, in this experiment, the viable cell density of all the other cell strains and the viable cell density of lower cell strain have a long way to go, and this also describes indirectly, and in the culture environment of the cell mass that density is lower, nutritive ingredient is sufficient.

4) idea in order to again verify at the 3rd, after cell strain 07 and cell strain 11 cultivate the 12nd day stationary phase without interference continous way, proceed to cultivate to this two strains cell, between the 12nd day to the 14th day, in 48 hours, do not carry out substratum replacing, find that viable cell density and cell viability do not change, meanwhile, 48 hours unit outputs are also about 2 times of 24 hours unit outputs.Draw thus, for this two strains cell, during cultured continuously mode before, can guarantee that culture environment Middle nutrition composition is sufficient all the time.

5) from 3 3.2.2), 4) can find out in information, after removing cell strain 07 and cell strain 11, for batch-type being cultivated and cultivating without interference continous way, the cell strain throughput data sequence drawn by these two kinds of methods is basically identical, and this just can prove the reliability of two kinds of methods each other.

6) according to bibliographical information and summary of experience, generally when lactic acid concn is lower than 20mmol/L(1.8g/L) time, do not affect the growth of cell, when lactic acid concn is between 20 ~ 40mmol/L(1.8 ~ 3.6g/L) time can reduce the output of albumen, when higher than 40mmol/L(3.6g/L) Shi Caihui produces damage to the growth of cell.In this experiment, except the lactic acid content in cell strain 09 culture supernatant is more than except 1.8g/L, all the other are all lower than this limit, and in the culture supernatant of cell strain 07 and cell strain 11, the content of lactic acid compares other each cell strains, is relatively low.So for cell strain 07 and cell strain 11, its viable cell density is relatively low, not because this reason of lactic acid production causes.

As can be seen here, in candidate cell strain, if there is obvious oxygen demand gap, and throughput gap is little, so, selects oxygen demand relatively less, gives up oxygen demand relatively many.So, in this stage, cell strain 07 and cell strain 11 are given up, retain 9 remaining strain cells, continue follow-up experimental study.

3.2.2.2 the comparison of lactic acid generation

Find in this experiment, in the 9 strain cells remained, each cell strain is when cultivating stationary phase without interference continous way, and the lactic acid of 24 hours generates unit vol major part all between 1.5 ~ 1.8g/L, in addition, the data of cell strain 09 are then free on outside this scope and reach 2.3g/L.

From data, feel that the lactic acid growing amount of each cell strain is all in good level, but, it should be noted that this is the data obtained in cultivating without interference continous way, the technology pattern of most large scale culturing is fed batch culture, and three scopes about lactic acid mentioned in literary composition are based on fed batch culture, this has just been related to the situation in each stage in fed batch culture, so these data can not be used for the judgement of adiabatic condition.But, still can from document and practical experience, recognize that lactic acid generates unit vol to the importance in this process, so, the comparative selection that can be undertaken between each cell strain by these data.

Because 24 hours unit outputs of cell strain 09 and other each strain cell relative mistakes are apart from little, but the lactic acid growing amount of 24 hours is by contrast with other, and relative mistake distance of each strain cell is larger, so, the index that screening is investigated can be it can be used as, for the lactic acid accumulation problem run into during cell large scale culturing alleviates obstacle.In this stage, given up by cell strain 09, retain 8 remaining strain cells, remaining cell strain is 01,03,04,05,08,12,14,15, continues follow-up experimental study.

The screening that the 3.3 Chinese hamster ovary celI strain ventilations expressing target protein interfere continous way to be cultivated

3.3.1 ventilation interferes continous way to be cultivated

On original basis without interfering continous way to be cultivated, by transforming the shaking flask used, the ventilation can carried out when cultivating in various degree is interfered, like this, the tolerance of each cell strain to ventilation intensity can be understood, thus, understand each cell strain and the tolerance of shearing force is distinguished, carry out the comparison of each cell strain tolerance shearing force ability, filter out the cell strain that more can tolerate shearing force comparatively speaking.

For individual plant cell, a complete ventilation interferes continous way culturing process as follows:

1) then carry out follow-up operation according to complete without interfering continous way training method to start, wherein substratum, sampling counts, condition and the operation such as keep sample, and all interfere continous way to be cultivated with nothing and are consistent.

2) after entering into stationary phase without the cultivation of interference continous way, keep run stationary phase at least for three days on end.

3) start to carry out ventilation to interfere, be initial air flow with 30ml/min, continous way cultivation is carried out to the cell in shaking flask, after the viable cell densities of initial several days and cell viability fluctuation, the stationary phase kept again at least 3 days is run, and namely in 3 days, fluctuating substantially no longer appears in viable cell density and cell viability.

4) adjust air flow, the amplitude of each adjustment is 30ml/min, and each stationary phase kept at least 3 days is run, then enters the test experiments of next air flow.

5) until instant air flow cannot make cell cultivation process enter stationary phase, namely viable cell density and cell viability enter the passage continuing to reduce, until cell mass is basic all dead.

According to upper type, to after screening in early stage, the 8 strain cells retained are tested, 1st takes turns and tests cell strain 01, cell strain 03, cell strain 04,2nd takes turns and tests cell strain 04, cell strain 05, cell strain 08,3rd takes turns and tests cell strain 04, cell strain 12, cell strain 14, cell strain 15, the wherein control that tests as this of cell strain 04, take turns in experiment each, the data of cell strain 04 are all close, and the experimental data therefore obtained in each wheel can carry out unifying to compare.Period, pH controls all to meet the requirements.

Carried out 3 altogether and taken turns experiment, 8 strain cells all complete ventilation and interfere continous way culture experiment.Every strain cell uses 2 shaking flasks to carry out parallel laboratory test when testing, and in experimentation, the experimental data of every strain cell 2 shaking flasks is all substantially close.Get the mean value of same cell strain parallel laboratory test 2 groups of data again, obtain 8 cell mean data, represent 8 strain cells experimental result separately, wherein each wheel experimental data of cell strain 04 is also more close, so only take out wherein 12 groups of data of taking turns to carry out data processing and analysis.Concrete outcome is more as shown in table 8.

The information of following needs can be obtained from data:

1) the shearing force ability comparative sequence produced is interfered in the resistance to ventilation of each cell strain, is roughly: 12=04=05=08 > 01=14 > > > 03 ≈ 15.

2) for fingerprinting stress ability, wherein cell strain 04, cell strain 05, cell strain 08, cell strain 12 are closely, between cell strain 01, both cell strains 14 are mutual closely, and this two strains cell and above 4 strain cells still can find out gap to some extent, cell strain 03, cell strain 15 gap compared with other each strain cells is larger.

3.3.2 the screening that continous way is cultivated is interfered in ventilation

The shearing force of each cell strain is compared and is incorporated into screening stage, meanwhile, need to set up simple effective method.Because this research is still in exploration stage, the method set up, cannot understand above-described this hidden danger has much on earth, can only carry out lateral comparison to the cell strain of each candidate, therefrom selects relatively most suitable.But time, as the preceding paragraph falls to describing, reach such object, there is deep meaning equally, and also set up good basis for follow-up more deep probing into.

Thus, experimentally data, give up cell strain 03 relatively poor for shear stress tolerance ability, cell strain 15, retain 6 remaining strain cells, remaining cell strain is 01, cell strain 04, cell strain 05, cell strain 08, cell strain 12, cell strain 14, continues follow-up experimental study.

The screening that the 3.4 Chinese hamster ovary celI strain osmotic pressure of expressing target protein interfere continous way to be cultivated

3.4.1 osmotic pressure interferes continous way to be cultivated

At present, in the extensive suspension culture of Chinese hamster ovary celI, the technique of main flow is fed batch culture, and simplified process and the situation of this technique are as follows:

1) according to the step described in 2.2.1, start to carry out batch-type and cultivate operation.

2) when batch-type cultivation proceeds to the 3rd day, viable cell density reaches 2X10 ⁶after more than cells/ml, start to fill into supplemented medium in shaking flask, supplement the nutritive ingredient consumed due to Growth of Cells and protein expression.

3) principle filling into supplemented medium within every 24 hours, to fill into 1 time or repeatedly, the amount of filling into is determined according to culture medium system actual characteristic, the batch-type of viable cell density ratio is cultivated, can increase further, compared with cycle of whole culturing process also cultivates with batch-type, extended, the target protein of acquisition is also along with increase.

4) when batch-type is cultivated, in cultivation in earlier stage, cell normal growth, but arrived the cultivation middle and later periods, due to the approach exhaustion of cell suspension Middle nutrition composition, cell starts death, until whole culture cycle terminates.

5) when cultivating without interference continous way, cultivate early stage, to cultivate situation consistent with batch-type, cell normal growth, then every 24 hours of substratum period is more just entered, because nutritive ingredient is sufficient, and the ambient stable of cell suspension, so now viable cell density still can sustainable growth, until viable cell density is by after the factor restriction of the various complexity such as culture space, dissolved oxygen demand, viable cell density keeps constant, then, enter into stationary phase, in theory, under such circumstances, can datelessly continue without the cycle of interfering continous way to be cultivated.

6) when fed batch culture, equally, in cultivation in earlier stage, cell normal growth, when after beginning feed supplement, enters and cultivates mid-term, now, viable cell density still can sustainable growth, but, along with the growth of viable cell density, although filling into due to supplemented medium, nutritive ingredient remains sufficient, but due to the accumulation of meta-bolites, material is continual to be filled into, environmental degradation in cell suspension, makes cell start death, until whole culture cycle terminates.

On original basis without interfering continous way to be cultivated, by the osmotic pressure of adjustment basic medium W027, the osmotic pressure that can carry out when cultivating in various degree is interfered, like this, the tolerance of each cell strain to osmotic pressure can be understood, carry out the comparison of the resistance to osmotic pressure ability of each cell strain, filter out the cell strain of more ability osmotic pressure comparatively speaking.Period, adjust, do not affect the change of its nutritive ingredient to the osmotic pressure of basic medium W027, meanwhile, the initial infiltration pressure of basic medium W027 is 290mOsm/kg.

For individual plant cell, a complete osmotic pressure interferes culture of continuous cultivation as follows:

3) start to carry out osmotic pressure interference, initial infiltration pressure due to substratum is 290mOsm/kg, so, when osmotic pressure is interfered first, with 330mOsm/kg for the amount of beginning, continous way cultivation is carried out to the cell in shaking flask, after the viable cell densities of initial several days and cell viability fluctuation, the stationary phase kept again at least 3 days is run, and namely in 3 days, fluctuating substantially no longer appears in viable cell density and cell viability.

4) adjust osmotic pressure, the amplitude of each adjustment is 40mOsm/kg, and each stationary phase kept at least 3 days is run, then enters the test experiments of next osmotic pressure.

5) until instant osmotic pressure cannot make cell cultivation process enter stationary phase, namely viable cell density and cell viability enter the passage continuing to reduce, until cell mass is basic all dead.

According to upper type, to after screening in early stage, the 6 strain cells retained are tested, 1st takes turns and tests cell strain 01, cell strain 04, cell strain 05,2nd takes turns and tests cell strain 05, cell strain 08, cell strain 12, cell strain 14, and wherein the control that tests as this of cell strain 05, takes turns in experiment each, the data of cell strain 05 are all close, and the experimental data therefore obtained in each wheel can carry out unifying to compare.Period, pH controls all to meet the requirements.

Carried out 2 altogether and taken turns experiment, 6 strain cells all complete osmotic pressure and interfere continous way culture experiment.Every strain cell uses 2 shaking flasks to carry out parallel laboratory test when testing, and in experimentation, the experimental data of every strain cell 2 shaking flasks is all substantially close.Get the mean value of same cell strain parallel laboratory test 2 groups of data again, obtain 6 cell mean data, represent 6 strain cells experimental result separately, wherein each wheel experimental data of cell strain 05 is also more close, so only take out wherein 12 groups of data of taking turns to carry out data processing and analysis.Concrete outcome compares as table 9.

The information of following needs can be obtained from data:

1) each cell strain resistance to osmotic pressure ability comparative sequence, is roughly: 12 ≈ 05 > 08 > 14 > > 01=04.

2) for resistance to osmotic pressure ability, wherein cell strain 12, cell strain 05, cell strain 08, cell strain 14 are obviously better than cell strain 01, cell strain 04.And between cell strain 12, cell strain 05, cell strain 08, cell strain 14, also have gap, but gap is not fairly obvious, gap so in other words still needs other indexs follow-up to participate in comparing and could accepting or rejecting according to comprehensive condition.

3.4.2 osmotic pressure interferes the screening that continous way is cultivated

In the cell screening stage, just start to pay close attention to osmotic pressure tolerance aspect, in each cell strain, carry out the comparison of resistance to osmotic pressure ability, thus in the candidate cell strain that situation is close when early stage compares, filter out the cell strain that relatively resistance to osmotic pressure ability is stronger, so, to follow-up large scale culturing and amplification further thereof, even to follow-up process optimization, be early-stage preparations with very large meaning, or even may be extremely important Key Strategy.

According to such thinking, by revealing the relatively poor cell strain of osmotic pressure tolerance 01 in this phase table, cell strain 04 gives up, retain 4 remaining strain cells, remaining cell be 05, cell strain 08, cell strain 12, cell strain 14, continue follow-up experimental study.

The conclusion retaining 4 cell strains is obtained according to this by each conditional filtering, if each condition is carried out longitudinal comparison, can find out, cell strain 14 is in ventilation is interfered and osmotic pressure interferes continous way to be cultivated, showing in 4 strain cells of remainder of it all belongs to relatively poor, and the ability of expressing protein is also the poorest, so carry out so relatively after, determine to be given up, leave cell strain 05, cell strain 08, cell strain 12 like this for follow-up experimental study, further screen.

The screening of 4 fed batch cultures

The Chinese hamster ovary celI strain fed batch culture of 4.1 expression target proteins

The operation of fed batch culture pattern has relatively simple, and the cycle is short, and efficiency is high, is convenient to amplify and easily meet FDA/SFDA about a batch advantage for definition.So at present, in the application of reality, the most frequently used training method of the extensive suspension culture of Chinese hamster ovary celI is fed batch culture.

Therefore, carrying out cultivation operation with fed batch culture to candidate cell strain, compare the difference between them, is also the method relatively commonly used.

For individual plant cell, a complete fed batch culture process is as follows:

3) principle filling into supplemented medium is from beginning feed supplement, within every 24 hours, fill into 1 time, the amount of filling into is determined according to culture medium system actual characteristic, supplemented medium is added according to glucose consumption index in substratum, the batch-type of viable cell density ratio is cultivated, can increase further, compared with cycle of whole culturing process also cultivates with batch-type, extended, the target protein of acquisition is also along with increase.

4) fed batch culture research is carried out to each shaking flask being numbered C0 and D0, sampling counting operation is carried out at interval of about 24 hours, each sample volume is unanimously 0.5ml, obtains instant viable cell density and cell viability data, and sample time is for before often starting 24 hours feed supplement cycles.

5) when fed batch culture, equally, in cultivation in earlier stage, cell normal growth, when after beginning feed supplement, enters and cultivates mid-term, now, viable cell density still can sustainable growth, but, along with the growth of viable cell density, although filling into due to supplemented medium, nutritive ingredient remains sufficient, but due to the accumulation of meta-bolites, material is continual to be filled into, environmental degradation in cell suspension, makes cell start death, until whole culture cycle terminates.

6) when each shaking flask inner cell vigor being numbered C0 and D0 is reduced to below 70%, stop this Fed-batch batch experiments to operate, centrifugally collect supernatant, carry out expression amount mensuration, supernatant packing will be remained, preserve at being placed in-20 DEG C, for subsequent use.

According to the fed batch culture method described in literary composition, carry out cultivation operation, period to the remaining 3 strain cells got off, pH controls all to meet the requirements.Every strain cell uses 2 shaking flasks to carry out parallel laboratory test when testing, in experimentation, the experimental data of every strain cell 2 shaking flasks is all substantially close, get the mean value of same cell strain parallel laboratory test 2 groups of data, obtain 3 cell mean data, represent 3 strain cells experimental result separately, carry out data processing and analysis.Concrete outcome is more as shown in table 10.

The information of following needs can be obtained from data:

1) each cell strain cell growth status is more similar, but still has the gap can seen and draw, and from data, order of excellence is cell strain 12 > cell strain 08 > cell strain 05.

2) from the unit output of expressing protein capacity index, this 3 strain cell also can simply be distinguished, each cell strain is in the shaking flask stage, with the fed batch culture of same process, the strong and weak order of cell mass expressing protein ability of each cell strain is cell strain 12 > cell strain 08 > cell strain 05.

The screening of the Chinese hamster ovary celI strain fed batch culture of 4.2 expression target proteins

Fed batch culture is the most frequently used mode of current Chinese hamster ovary celI large scale culturing, under selected substratum and identical supplying technics thereof, test in shaking flask, its result can allow understands each cell strain when large scale culturing, performance under this feed supplement batch technique, thus, for the comparison and selection of the throughput of each cell strain expressing protein.This is also current, uses this technique to carry out the main stream approach of cell strain screening.

According to such thinking, from data, can find out, cell strain 12 is optimum cell strains being applied to follow-up study and large scale culturing production protein drug first.

The checking of the extensive suspension culture of 5 bio-reactor

5.1 bio-reactor suspension culture compare confirmation

Respectively cell strain 12 and cell strain 15 are carried out the production tank suspension culture of 50L scale, then result is compared.

Bio-reactor Mobius CellReady 3L is seeding tank, Mobius CellReady 50L is for producing tank, producing tank original volume is 20L, control condition is: Temp (37 ± 0.2 DEG C), DO (40% ± 10%), pH (6.9 ± 0.05), Stir speed(50 rpm), in production tank suspension culture process, what adopt is fed batch culture method, at interval of about 24 hours sampling monitoring related datas, adds supplemented medium according to glucose consumption index in substratum, from beginning feed supplement, feed supplement interval is about 24 hours.When producing tank inner cell vigor and being reduced to below 70%, stop the fed batch culture of this production tank operate, centrifugally collect supernatant, carry out expression amount mensuration, will remain supernatant packing, at being placed in-20 DEG C, preservation, for subsequent use.Result as Figure 1-Figure 2.Result shows, in the production process of carrying out in bio-reactor Mobius CellReady 50L, the closed cans unit output of cell strain 12 and 15 is respectively 0.88g/L and 0.67g/L, cell strain 12 is higher than the closed cans unit output of cell strain 15 0.21g/L, namely production efficiency improves 31%, and the income that this raising brings in suitability for industrialized production is very huge.Visible, newly screening the cell strain 12 determined and have better production application than the cell strain 15 originally determined, is that cell growth status or closed cans unit output are all significantly improved, and the follow-up scale being simultaneously more conducive to project is amplified.Like this, in time Cells for production strain can be replaced by cell strain 12.

To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.

In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.

Claims

1. be applied to a screening method for the expression target protein Chinese hamster ovary celI strain of extensive suspension culture, it is characterized in that the concrete steps of the method are:

C-3. repeating step c-2, until cell mass is all dead;

D-3. repeating step d-2, until cell mass is basic all dead;