CN110004108A - Novel animal cell culture amplification method based on three-dimensional viscous shear space - Google Patents
Novel animal cell culture amplification method based on three-dimensional viscous shear space Download PDFInfo
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Abstract
The present invention is based on the novel animal cell culture amplification methods of three-dimensional viscous shear space, include the following steps: that (1) passes through the shear environment in CFD approach quantitative analysis different scales reactor first, and is verified by laser particle tachymeter PIV method；(2) then it has been determined that shear parameters can be used for characterizing the shear environment in reactor according to laboratory cell cultivation results, and as amplification standard；(3) and then according to the association analysis that Spodoptera frugiperdaSf9 amount of viable cell in different scales reactor and three features are sheared, optimal three-dimensional shearing rate operating space is established；(4) shearing rate operating space is converted to speed of agitator according to the correlation of the shearing rate of foundation and end of blade speed can operating space, and experimental verification has been carried out on different scales reactor, cell is finally successfully realized from experimental size to production-scale amplification.
Description
Technical field
The present invention relates to a kind of animal cell culture amplification methods, especially shear-sensitive type cell amplification method.
Background technique
Zooblast has stronger advantage in terms of protein expression and modification, and the culture that suspends is that a kind of efficiently production is anti-
The training method of body, vaccine and protein medicaments, wherein such as baculovirus expression system of the expression platform based on insect cell
System (BEVS) is widely used in the production of vaccine.However, animal cell culture is in production amplification process with reactor scale
With the variation of structure, the environment such as mass transfer, mixing and shearing in reactor can have greatly changed.For this purpose, related scholar mentions
Many amplification methods, including theoretical analysis, half theory method, orthodox regression and empirical method are gone out.Tescione etc. is being carried out
Discovery uses different amplification strategies during Chinese hamster ovary celI amplification research, such as specific volume power (P/V), volume oxygen transfer
Coefficient (kLA) and oxygen transfer rate (OTR), final cell growing state are widely different.In carrying out stem cell incubation,
Borys etc. has studied seven kinds of common amplification tactful (average speed, average shear rate, average energy dissipative shock wave, Reynolds numbers, leaf
Hold speed, power input and maximum shear rate) influence to process, as a result, it has been found that the speed of agitator that distinct methods are calculated
It differs greatly, cell growth and coherent condition also generate greatest differences.Hydrodynamic characteristic in amplification process in reactor
(including mass transfer, mixing and shearing etc.) shows strong structure and Scale Efficiency, traditional similar based on single parameter
Amplification method usually cannot achieve in reactor the consistent of crucial flow field characteristic.A large number of studies show that the flow field in reactor is special
Property or hydrodynamic characteristic be to influence cell physiological metabolism principal element, maintain crucial the similar of hydrodynamic parameters to be expected into
For a kind of efficient bioprocess amplification method.
Insect cell is very sensitive to shearing, and especially after transfected virus, therefore ventilates and stir caused shearing ring
Border variation is usually the main reason for leading to amplification failure.Since shearing is difficult to quantitative description, many scholars use and indirectly cut
Cut parameter such as agitating paddle end of blade speed, energy absorbing device, specific volume power input (P/V), energy absorbing device/circulation time
Function (EDCF), maximum or average shear rate etc. are used as amplification standard[14-17].However, a large number of studies show that cutting in reactor
Environment is cut with height heterogencity, for stirring type bioreactor, the shearing rate in blade region is usually than tank body area
High several orders of magnitude.Therefore, single shear parameters are difficult to characterize the shear environment in reactor.How quantitative description is tested
Shear environment in scale reaction device and to carry out it in commercial scale reactor to reappear be the key that realize production amplification institute
?.
The method of shear environment in quantitative description bioreactor includes theoretical analysis, experimental method and numerical simulation
Method.By theory analysis, numerous studies are special by average shear rate and speed of agitator, the Ventilation Rate and rheology in reactor
Property is associated.It (such as specific structure of reactor and is stirred however, theoretical analysis is only applicable to certain specific status of criterion
Mix structure), and average or maximum shearing rate can only be assessed.And by being based on Particle Image Velocity (PIV) or laser
The experimental method of Doppler range rate measurement (LDV) can obtain the information of flow in reactor, and then local shearing letter is calculated
Breath.Wu etc. establishes the empirical correlation of shearing rate and agitating paddle flow number in reactor by theory analysis and LDV experiment
Formula.Some scholars also establish the experimental provision of quantification shearing rate and for studying mammalian cell and insect cell pair
The sensibility of shearing.In addition, some scholars propose to characterize using indirect experimental method or parameter (such as than oxygen consumption rate)
Shear the damage to cell.However, experiment research is only applicable to the reactor of experimental size, and shearing rate is to reactor
Structure has very strong dependency, and the data that laboratory obtains are not used to the assessment of shearing rate in commercial scale reactor.In recent years
Come, Fluid Mechanics Computation method (CFD) is widely used in the research of reactor flow field characteristic, compared with above two method, tool
There are very strong scalability and economy.In addition, the shearing information in the available reactor three-dimensional space of CFD approach, is conducive to
The statistical analysis of every shear parameters.Liu et al. analyzes Carthamus using the CFD approach based on Euler-Lagrange
Shear environment that tinctorius L. cell is undergone in reactor simultaneously successfully simulates cell under complicated shear environment
Death and growing state.Collignon etc. establishes the death and EDCF (description of cell by the method that big whirlpool CFD is simulated
Cell experience shearing force size and frequency) correlation.It is damaged however, these methods are concentrated mainly on research shearing with cell
Relationship between wound, without proposing perfect shear-sensitive type cell amplification method.
Summary of the invention
It is based on the similar animal cell culture amplification method of shear environment the purpose of the present invention is to propose to a kind of, to overcome
Above-mentioned the deficiencies in the prior art.The specific technical solution of the present invention is:
Novel animal cell culture amplification method based on three-dimensional viscous shear space, includes the following steps:
(1) first by the shear environment in CFD approach quantitative analysis different scales reactor, and pass through PIV method
It is verified；
(2) then it has been determined that three feature shear parameters can be used for characterizing in reactor according to laboratory cell cultivation results
Shear environment, and as amplification standard；
(3) and then according to Spodoptera frugiperdaSf9 amount of viable cell in different scales reactor and three features
The association analysis of shearing establishes optimal three-dimensional shearing rate operating space；
(4) shearing rate operating space is converted to speed of agitator according to the correlation of the shearing rate of foundation and end of blade speed can
Operating space, and experimental verification has been carried out on different scales reactor, finally it is successfully realized Spodoptera
Frugiperda Sf9 cell is from experimental size to production-scale amplification.
Further, the reactor is the reactor of 7.5-1000L.
Further, described from experimental size to production-scale amplification is amplification from 7.5-42L to 30-1000L.
The medicine have the advantages that insect baculovirus expression system (BEVS) is widely used in the production of various vaccines,
However insect cell usually hinders the high susceptibility of shearing the amplification of its production process.For this purpose, originally having researched and proposed one kind
Animal cell culture amplification method based on three-dimensional viscous shear space.Firstly, right herein by Fluid Mechanics Computation method (CFD)
Shear environment in experimental size (7.5L and 42L) and production scale (30L, 90L, 350L and 1000L) reactor is determined
Amount analysis, and result is verified by laser particle tachymeter (PIV), and then establish every shearing in reactor
Parameter (including paddle area shearing rate, tank field shearing rate, average shear rate and maximum shear rate) is quantified with agitating paddle end of blade speed
Relational expression.The pass of shear parameters in the Spodoptera frugiperda Sf9 cell culture result and tank obtained by experiment
Connection analysis finds that single shear parameters can not successfully realize the production amplification of incubation, and uses three feature shearing ginsengs
Number, the Optimum Operation that the area Ji Jiang shearing rate, tank field shearing rate and average shear rate can establish shearing rate in three dimensions are empty
Between, when three characteristic parameters in reactor are located in the space, then the amplification of cell cultivation process can be achieved.Based on this
Method establishes the optimal speed of agitator behaviour of production scale reactor according to the correlation of obtained shearing rate and end of blade speed
Make space, and verified on production scale reactors at different levels, amplification is finally successfully realized on 1000L reactor, carefully
Intracellular growth rate and amount of viable cell are consistent with pilot plant test (final viable count about 700 × 106cells/mL).The amplification plan
Slightly hope the production amplification process for being applied to other shear-sensitive type cells.
Present invention will be further explained below with reference to the attached drawings and examples.
Detailed description of the invention
Fig. 1 is the six kinds of reactor basic structures used, and wherein 7.5L and 42L is used for experimental study, and 30~1000L is used for
Production.
Fig. 2 is used for the structure of reactor of PIV experiment, wherein (A, the dot at three different heights indicate velocity contrast's
Sample point), at speed cloud atlas comparing result (B, left one side of something are PIV as a result, right one side of something is CFD analog result) and different height
Local velocity's comparing result (C-E).
Fig. 3 .CFD simulation is compared with the average shear rate that empirical equation is calculated.
The relationship of shearing rate (SSR) and agitating paddle end of blade speed in the different reactor that Fig. 4 is simulated, wherein (A: as
30L push type blade (APP) is added as a comparison in ear type (EE) paddle area shearing rate；B: three oblique blade profile (PBT) paddle area shearings
Rate；C: pusher blade (APP) paddle area shearing rate；D: tank body shearing rate；E: ensemble average shearing rate；F: maximum shear rate).
The distribution situation of Fig. 5 maximum shear rate is analyzed, wherein (A: as ear type blade EE；B: pusher blade APP；C: group
Mould assembly blade APP+PBT).
The relationship of shear parameters and final viable cell density under the conditions of Fig. 6 different operation.Wherein (blue dot indicates to live
The comparatively ideal operating condition of cell density, red spots indicate the lower operating condition of viable cell density；Horizontal dotted line indicates reason
The viable cell density threshold value thought；Two vertical dotted lines indicate the boundary of amplification standard).
The distribution of three-dimensional manipulating space (cubical area of black wire frame composition) and each experimental implementation point that Fig. 7 is established
(red spots indicate poor operating condition, and blue dot indicates preferable operating condition, and green dot indicates production amplification
The operating point used；Two groups of numbers beside dot respectively indicate reactor scale and speed of agitator).
Cell growth status of each scale reaction device of Fig. 8 under different speeds of agitator, wherein (A: mini-reactor；B: compared with
Commercial scale reactor).
Fig. 9 .Spodoptera frugiperda Sf9 cell is in low sheraing environment, wherein (A, 1000L-30rpm) and suitable
The cellular morphology (being 84h sampling microscopic examination result) of (B, 1000L-48rpm) under suitable shear environment.
Specific embodiment
1. the novel animal cell culture amplification method based on three-dimensional viscous shear space, includes the following steps:
(1) first by the shear environment in CFD approach quantitative analysis different scales reactor, and pass through PIV method
It is verified；
(2) then it has been determined that three feature shear parameters can be used for characterizing in reactor according to laboratory cell cultivation results
Shear environment, and as amplification standard；
(3) and then according to Spodoptera frugiperdaSf9 amount of viable cell in different scales reactor and three features
The association analysis of shearing establishes optimal three-dimensional shearing rate operating space；
(4) shearing rate operating space is converted to speed of agitator according to the correlation of the shearing rate of foundation and end of blade speed can
Operating space, and experimental verification has been carried out on different scales reactor, finally it is successfully realized Spodoptera
Frugiperda Sf9 cell is from experimental size to production-scale amplification.
2. material and method
2.1 bioreactor construction
The present invention uses the stirring type bioreactor of six kinds of scales (7.5-1000L), and basic structure is shown in Fig. 1.Two
The reactor (7.5L and 42L) of experimental size is respectively arranged with three leaves " as ear type " agitating paddle (EE), and wherein 42L blade is opposite
Position is on the upper side.30L reactor is equipped with a three leaf push type agitating paddles (APP).Other reactors use identical stirring paddle type
Formula includes a three leaf push type agitating paddles (APP) and a three oblique leaf agitating paddles (PBT), and diameter of stirring paddle is different.
2.2 cell culture processes
The recombinant baculovirus expression system that the present invention uses is Spodoptera frugiperda Sf9, is stored in nothing
Blood serum medium Sf900 II (Invitrogen, USA).Preculture is carried out first in shaking flask, cultivation temperature is 28 DEG C, shaking table
Revolving speed 60rpm is linked into experimental size 7.5L fermentor after culture to exponential phase of growth, then further expansion to 42L.?
In industrial production, shake-flask seed is linked into first in 30L fermentor, is then expanded to 1000L (30L-90L-350L- step by step
1000L).Initial cell density after each fermentor inoculation is 80 × 106cells/mL.Speed of agitator is permanent in incubation
It is fixed, maintain dissolved oxygen level to be not less than 40% by four gas control systems.Viable count is carried out using trypan blue staining, often
A sample detection is three times.
2.3 CFD analogy methods
During carrying out Insect cellculture, the discovery bubble size such as Weidner is not the pass for influencing cellular damage
Key factor, cellular damage are mainly related to gas-liquid interface size.Since the ventilatory capacity that this research uses is very low (< 0.1vvm), gas
Smaller containing rate and gas-liquid interface, shearing rate caused by furthermore ventilating also is difficult quantitative analysis, therefore is carrying out CFD simulation
Fermentation system is reduced to single-phase flow in the process.Since II culture medium of Sf900 is diluter, rheological behavior is close with water phase, therefore simulates
The medium with water of use replaces.Flow field characteristic in reactor uses commercialized 15.0 (ANSYS of CFD software ANSYS CFX
Inc., USA) it is analyzed.Reactor grid is divided using ANSYS ICEM CFD 15.0 (ANSYS Inc., USA),
It is non-structured tetrahedral grid, the grid in blade region carries out local cypher, total grid number about 1,000,000.Every kind of reaction
Device simulates five kinds of speeds of agitator, and corresponding blade end of blade velocity interval is 0.3-1.5m/s, and the smallest stirring Reynolds number is
21000, i.e. flowing reaches complete turbulence state.The simulation of turbulent flow closes Equations of Turbulence using the k- ε model of standard,
The rotation of blade is described using Moving reference (MRF) method.Using time averaging NS equation to momentum and continuity
Equation is described.The velocity component U that the present invention is simulated according to CFDx、UyAnd UzShearing rate (SSR) is calculated,
Equation is as follows:
The torque of monitoring calculation residual sum blade in simulation process, when residual error is less than 10-4And blade torque stabilization then thinks
Iterative process convergence.Derivation is enterprising in the 96 core dawn servers (Sugon Co., Ltd., China) that 5 nodes form
Row.
2.4 PIV experiment methods
The PIV experiment platform that flow field verifying of the present invention uses is identical as report before, mainly by laser emitter
(Leamtech, Nd:YAG, 200mJ, 15Hz), PIV camera (PCO2000,2048 × 2048pixels), synchronizer and
4.7 data processing software of Vidpiv composition.The angle of stirrer paddle is carried out in addition, this research joined a shaft encoder
Locking, to obtain the PIV experiment data of angle parsing.The reactor of PIV flow invariance is 42L acrylic transparent jar, dress
There are two as ear type agitating paddle (EE), basic structure is shown in Fig. 2A.Flow invariance region is the vertical plane among two baffles.
Since blocking for agitating shaft is reflective with tank bottom, measurement region only includes half of tank body straight tube region.Detailed experiment amplification and
Data processing method can be found in the document of the prior art.
3. results and discussion
Verifying of 3.1 PIV experiments to CFD model
The present invention verifies CFD model by the PIV flow invariance experiment in 42L reactor, tests stirring for use
Mixing revolving speed is 50rpm.The speed cloud atlas that comparative analysis PIV experiment and CFD approach obtain can be seen that two methods acquisition
Flow field is very close, and a biggish axial circulation (Fig. 2 B) is integrally formed in reactor side.For further comparative analysis reaction
Qi Nei local velocity has selected reactor axis to compare and analyze (Fig. 2 C- to the local average speed at three different heights
E).From the results, it was seen that the blade discharge area peak velocity that simulation obtains is slightly above experiment value, the average speed of other positions
It spends very close with experiment value.The researchs such as Singh also indicate that k-ε turbulence model and other models (SST, SSG-RSM and SAS-
SST tubulence energy size can preferably be predicted by) comparing, but it predicts that the speed in obtained blade region can be higher.Liu et al. is same
The blade area peak velocity that sample discovery k-ε turbulence model is predicted can be higher.Wu etc. is pointed out, even if using high-precision big
Whirlpool analogy method, the paddle area peak velocity simulated also can be higher.It is found, experimental data and is simulated by statistical analysis
To data there was no significant difference that (p value at Z=0.29m, 0.21m and 0.11m is respectively 0.346,0.192 and 0.259, T-
test).It can be seen that the flow field data that the CFD approach that the present invention uses is simulated have enough precision, velocity field is used
The shearing rate being calculated is also believable.
The quantitative analysis results of 3.2 shear environments
The present invention uses the CFD approach after experimental verification to carry out analysis simultaneously to the flow field situation in different scales reactor
Obtain shearing relevant information.By simulating the shear strength under different speeds of agitator, can establish performance variable (such as speed of agitator,
End of blade speed etc.) and shear parameters between correlation.For the accuracy for further verifying simulation, the present invention obtains simulation
Single-layer agitator under the conditions of average shear rate and the average shear rate that is calculated of empirical equation reported in the literature carry out
Comparative analysis, calculation method are as follows:
WhereinFor average shear rate (s-1), NQFor the quasi- number of nondimensional axial flow, can be calculated by axial flow velocity
It arrives[34], N is speed of agitator (s-1)。
Flow number (N of the 7.5L and 42L reactor that simulation obtains as ear type agitating paddle (EE)Q) it is respectively 0.4 He
0.5, and the flow number of 30L reactor push type blade (APP) is 0.3.Due to the stirrer paddle area phase of 7.5L reactor
To smaller (connector for being primarily due to blade and wheel hub is relatively long), therefore its flow number is smaller compared with 42L reactor.Zhu
The flow number of the EE blade of equal reports is higher (~0.7), be primarily due to stirring paddle structure that it uses (blade angle,
The connector length of the wheel hub of blade) and tank body pattern (whether with the presence of baffle) and larger difference of the present invention.It can be with from Fig. 3
Find out, the average shear rate simulated and the result that empirical equation is calculated are very close, and average shear rate and stirring
Revolving speed is in preferable linear relationship, also identical as document report, and the shearing rate for illustrating that simulation obtains is reliable.This research is also
It was found that although 7.5L and 42L reactor uses closely similar stirring pattern (only there are larger differences for installation site),
Finally obtained average shear rate but differs larger, and traditional empirical equation is used (only to turn average shear rate with stirring
Speed is associated) average shear rate of two kinds of reactors that is calculated can be identical.It can be seen that average shear rate not only with
Blade type is related, and reactor scale or blade installation site can have an impact it.
Different shear parameters and end of blade speed have further been carried out association analysis by the present invention, the shearing including stirring region
Rate (uses impeller SSR or SSRimpIndicate), tank body area shearing rate (remove the part in blade region, with tank SSR or
SSRtankIndicate), the ensemble average shearing rate (average value of all areas, with average SSR or SSRavgIndicate) and maximum cut
Rate is cut (with maximum SSR or SSRmaxIndicate), correlation is as follows:
SSRt=KstUT (3)
Wherein KS,iFor model coefficient (m-1), it is mainly related to agitating paddle and tank structure, subscript i expression imp, tank,
Avg and max, UTAgitating paddle end of blade speed (m/s).Fig. 4 describes every shear parameters and end of blade in different scales reactor
Relationship between speed.Obviously, when being combined using identical agitating paddle or identical agitating paddle, in identical end of blade speed item
Under part, as all shear parameters of the increase of reactor scale are gradually reduced.And when the agitating paddle difference of use, shearing rate
Situation of change will be different.For example, Tank SSR and overall the averaged SSR of 30L reactor is significantly lower than 42L
With 90L reactor (Fig. 4 D and 4E).However, the maximum shear rate in different scales reactor is without apparent regular (Fig. 4 F), it is main
If because maximum shear has very strong dependence to agitating paddle pattern.By the distribution situation (figure for analyzing maximum shear rate
5) as can be seen that the maximum shear rate for accounting for reactor total volume about 0.01% is concentrated mainly near blade edge or blade
At special narrow structure (connecting shaft of such as blade and wheel hub, Fig. 5 A).A large number of studies show that cell experience high-shear region
Probability is relatively low, and the time undergone is short, and maximum shear rate is not to cause the most critical factor of cellular damage.
Table 1 lists the model parameter K that equation 3 is obtained by regression analysisS,iValue, the phase of all model regression analyses
Relationship number (R2) all close to 0.99.Model proposed by the present invention can be used for shearing determining for relevant parameter in other similar reactors
Amount analysis provides important reference data for the amplification research of shear-sensitive type cell.
The regression coefficient of the shearing relevant parameter of table 1.
The analysis of 3.2 amplification key factors
Highest viable cell density under the conditions of 2. different operation of table
For Spodoptera frugiperda Sf9 cell, the ideal living cell density of industrialization amplification is carried out
(viable cell density) should be greater than 600 × 106cells/mL.The present invention is ground on the reactor of various scales first
The growing state of cell under different speeds of agitator is studied carefully, final viable cell density the results are shown in Table 2.Pass through the optimization of bench scale
Research, it is determined that the best speed of agitator of 7.5L, 30L and 42L reactor, however the extensive reaction for 90L to 1000L
Device gradually determines that the best speed of agitator of each stage reactor is extremely uneconomic using conventional optimization method.Due to the cell
The control of incubation whole process dissolved oxygen is 40% or more, and it (is 1- than oxygen consumption rate that the metabolism of insect cell is usually very slow
10mmol/109Cells/day is 1-4mmol/10 than glucose consumption rate9cells/day)[37], therefore oxygen is transmitted and is mixed
Conjunction is not to influence the key factor of Spodoptera frugiperda Sf9 cell amplification, amplifies successful key and is to put
The control of shearing rate during big.
The present invention obtains the interior parameters relevant to shearing of reactor under each operating condition by CFD approach, including
Paddle area shearing rate (impeller SSR), tank field shearing rate (tank SSR), ensemble average shearing rate (average SSR), most
Big shearing rate (maximum SSR), end of blade speed (tip velocity) and specific volume power (P/V), and by it to final
Viable cell density mapped (see Fig. 6).By analysis as can be seen that no matter using which kind of shear parameters as amplification standard,
Viable cell density is greater than 600 × 106The shearing that (the blue dot in the region Fig. 6 I) is established under the operating condition of cells/mL can
Operating area (two vertical line regions in Fig. 6) includes the lower operating point of the viable cell density (red in Fig. 6 region II
Dot), when especially with maximum shear rate, end of blade speed and specific volume power as amplification standard, include in region II
Poor outcome it is more, i.e., single shear parameters cannot function as amplification standard.The main reason for causing this result is reaction
Shear environment in device is that extremely non-uniform, single shear parameters can not true shearing ring in accurate description reactor
Border.
The it is proposed of 3.3 three-dimensional shearing manipulation space amplification standards
Since the shear environment in reactor has height heterogencity, traditional one-dimensional shear parameters are difficult accurate description
Shear environment in reactor, using it as amplification standard also just can not successfully realize the amplification of cell cultivation process.As a result,
The present invention is proposed using the three feature shear parameters (area Ji Jiang shearing rate SSR in reactorimp, tank field shearing rate SSRtank
With ensemble average shearing rate SSRavg) amplification standard as shear-sensitive type cell, which facilitates the shearing of lab scale
Ring more accurately copies in commercial scale reactor.In the case that the present invention studies, the shearing rate SSR of tank fieldtankWith it is whole
Body average shear rate SSRavgIt is close, however when blade biggish using relative size, the difference of the two can be larger, therefore is
Expand the applicability of this method, the present invention does not use two-dimensional shearing manipulation space, but it is empty to choose three-dimensional shearing manipulation
Between be used as amplification standard.The boundary in the three-dimensional manipulating space is greater than 600 × 10 by viable cell density6The operation item of cells/mL
The SSR obtained under partimp、SSRtankAnd SSRavgVariation range determine, in order to increase the redundancy of this method, the present invention will be each
The boundary of a dimension expands 20% on the basis of available data.Fig. 7 is the shearing rate operating space established using this method
(black box region), it can be seen that be greater than 600 × 10 comprising all amount of viable cell in the space6The operation item of cells/mL
Part, and the lower operating condition of amount of viable cell is all discharged outside.It can also be seen that in conjunction with 2 data of table when operating point deviates
When the operating space is remoter, viable cell density is lower.
The shearing rate three-dimensional manipulating space of above-mentioned foundation limits three shear parameters in amplification process, passes through
The correlation (equation 3) of the shear parameters proposed above and agitating paddle end of blade speed can convert shearing rate operating space to
Speed of agitator operating space, i.e. speed of agitator optimum range.The best speed of agitator range of each reactor being calculated is shown in Table 3,
All in all with the increase of reactor scale, best speed of agitator is gradually decreased.Best speed of agitator and reactor scale are simultaneously
There is no apparent linear relationship, it can be seen that use conventional methods and be difficult to determine suitable speed of agitator.And use the present invention
The three-dimensional parameter amplification method of proposition can the preferable speed of agitator of accurate prediction commercial scale reactor, and it can be operated
Range is reduced within 10rpm, has good directive significance for amplification process.
Table 3. can opereating specification and experimental verification point by the speed of agitator that shearing manipulation space is calculated
The experimental verification of 3.4 novel amplification methods
According to the optimal speed of agitator (table 3) that three-dimensional amplification policy calculation obtains, carried out on the reactor of different scales
The culture experiment of Spodoptera frugiperda Sf9 cell, the viable cell density in incubation are shown in Fig. 8.From result
It can be seen that cell growth rate differs greatly under different shear environments, cell density highest is more than 700 × 106Cells/mL,
And it is minimum less than 200 × 106cells/mL.The present invention finally chooses 60rpm, 55rpm and 48rpm in 90L, 350L respectively
The amplification method is verified on 1000 L reactors, and chooses the knot under the conditions of 50rpm, 40rpm and 30rpm respectively
Fruit is as control.Compared with the control, the industrial-scale reactor (90L, 350L and 1000L) obtained using the amplification strategy is interior
Cell growth rate and final cell concentration are significantly increased, and viable cell density reaches 700 × 10 in 110h6cells/mL
(Fig. 8 B), it is very close with lab scale result of study.What comparative analysis 1000L reactor obtained under the conditions of 30rpm and 48rpm
Cellular morphology figure can be seen that (Fig. 9), and (i.e. lower shearing rate) part cell aggregation is agglomerating under lower speed conditions,
And cell dispersion is more uniform under the conditions of 48rpm.The result shows that cell conglomeration can hinder nutrition under too low shear environment
It is lower to eventually lead to viable cell density to influence the growth of cell for the mass transfer of substance.It is contemplated that if speed of agitator is more than
The upper limit of three-dimensional viscous shear space then can cause to damage to cell, and microscopy can find cell fragment, however examine for experimental cost
Consider, the destructive test is not done on 1000L reactor.Three-dimensional shearing manipulation space based on optimization, this research is by lab scale
The shear environment information of acquisition is reappeared in production scale, and is successfully realized Spodoptera frugiperda Sf9
The production of cell is amplified.
4. conclusion
In order to solve the production scale-up problem of shear-sensitive type zooblast, the invention proposes one kind based on three-dimensional shearing
The amplification method of operating space.First by Fluid Mechanics Computation method to cutting in experimental size and production scale reactor
It cuts environment and has carried out quantitative study, and result is verified by PIV experiment, and then establish items in reactor and cut
Cut the quantitative relation formula of parameter Yu agitating paddle end of blade speed.The result that the average shear rate and simulation that document is calculated obtain
Closely, the accuracy of simulation is further demonstrated.Pass through the pass of shear parameters every in cell culture result and reactor
Connection analysis finds that single shear parameters can not successfully realize amplification, and uses three feature shear parameters SSRimp、
SSRtankAnd SSRavgCan shear environment in accurate description reactor, and a three-dimensional shearing rate is established according to it and is grasped
Make space, which can foreclose all poor experimental results, and all preferable experimental results are included,
Using this three-dimensional shearing rate operating space as amplification standard, it is ensured that three feature shear parameters and lab scale one in amplification process
It causes.It is calculated that production scale reactor is optimal to be stirred according to the correlation of the amplification standard and shearing rate and speed of agitator
Mix the range of speeds.The amplification that Spodoptera frugiperda Sf9 cell has finally been carried out on production scale reactor is real
Test, cell growth rate and final cell density are significantly increased, reached pilot plant test it is horizontal (cell density about 700 ×
106Cells/mL), it is successfully authenticated the feasibility of this amplification method.
It summarizes, zooblast amplification method (three dimensions based on three-dimensional shearing manipulation space that the invention proposes a kind of
On the shearing principle of similitude), and be successfully applied to Spodoptera frugiperda Sf9 cell production amplify (from 7.5L
Experimental size is to 1000L production scale).This amplification method has weight for instructing the production of shear-sensitive type cell to amplify
Want meaning, and can further genralrlization into the production amplification process of similar shear-sensitive type cell.
Claims (5)
1. the novel animal cell culture amplification method based on three-dimensional viscous shear space, which comprises the steps of:
(1) it first by the shear environment in CFD approach quantitative analysis different scales reactor, and is tested the speed by laser particle
Instrument PIV method is verified；
(2) then it has been determined that shear parameters can be used for characterizing the shear environment in reactor according to laboratory cell cultivation results,
And as amplification standard；
(3) and then according to Spodoptera frugiperdaSf9 amount of viable cell in different scales reactor and three features it shears
Association analysis, establish optimal three-dimensional shearing rate operating space；
(4) shearing rate operating space being converted to speed of agitator according to the correlation of the shearing rate of foundation and end of blade speed can operate
Space, and experimental verification has been carried out on different scales reactor, finally it is successfully realized Spodoptera frugiperda
Sf9 cell is from experimental size to production-scale amplification.
2. the novel animal cell culture amplification method according to claim 1 based on three-dimensional viscous shear space, feature exist
In the shear parameters are paddle area shearing rate, tank field shearing rate, average shear rate and maximum shear rate.
3. the novel animal cell culture amplification method according to claim 2 based on three-dimensional viscous shear space, feature exist
In the amplification standard is the quantitative relation formula of shear parameters Yu agitating paddle end of blade speed.
4. the novel animal cell culture amplification method according to claim 2 based on three-dimensional viscous shear space, feature exist
In establishing the optimal of shearing rate in three dimensions using shear parameters paddle area shearing rate, tank field shearing rate and average shear rate
The amplification of cell cultivation process is realized when three characteristic parameters in reactor are located in the space in operating space.
5. the novel animal cell culture amplification method according to claim 2 based on three-dimensional viscous shear space, feature exist
In it is empty to establish the optimal speed of agitator operation of production scale reactor according to the correlation of obtained shearing rate and end of blade speed
Between, realize production amplification.
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