CN209362021U - A kind of multiplexing convection current chromatographic system - Google Patents
A kind of multiplexing convection current chromatographic system Download PDFInfo
- Publication number
- CN209362021U CN209362021U CN201822092505.1U CN201822092505U CN209362021U CN 209362021 U CN209362021 U CN 209362021U CN 201822092505 U CN201822092505 U CN 201822092505U CN 209362021 U CN209362021 U CN 209362021U
- Authority
- CN
- China
- Prior art keywords
- peristaltic pump
- fluid reservoir
- multiplexing
- convection current
- macropore
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The utility model discloses a kind of multiplexing convection current chromatographic systems, belong to Protein purification techniques field.The multiplexing convection current chromatographic system includes charging fluid reservoir 1, peristaltic pump I, tangential flow filtration device, circulation fluid fluid reservoir, peristaltic pump II, peristaltic pump III, waste filtrate fluid reservoir, peristaltic pump IV, peristaltic pump V, macropore continuous chromatography device and central processing unit.The utility model additionally provides a kind of method using multiplexing convection current chromatographic system purifying protein.The system integrates the formula of the concentration of cell supernatant, buffer exchange, purifying and final product, realizes the single stepping that final product purifying is recovered to by cell liquid.The system can also further integrate the realization such as ultraviolet detector, pH, conductivity gauge to the real-time monitoring of whole process of purification, the final laboratory established efficiently, easy, economic, pilot-scale antibody producing platform, have great application prospect in various protein purification pilot scale platforms.
Description
Technical field
The utility model relates to a kind of multiplexing convection current chromatographic systems, belong to Protein purification techniques field.
Background technique
Tangential flow filtration (tangential flow filtration, abbreviation TFF) is a kind of novel thickening filtration skill
Art refers to liquid flow direction and the perpendicular filtered version of filtering direction.Compared to traditional dead-end filtration (dead end),
Liquid flowing generates shearing force in filter media surface in tangential flow filtration system, reduces the accumulation of cake layer or gel layer,
It ensure that the stable rate of filtration, treatment effeciency can be effectively improved.The separation method, which is based on size, molecular weight or other differences, to be made
The component in liquid solution or suspension is separated with film.Tangential flow filtration technology has using simple, quick, efficient, scale
Can zoom in or out, synchronize and carry out concentration and filter wash, it is economical time saving the advantages that.Tangential flow filtration technology can gather closed follow
Circulation road obtains efficient, sterile and quick technique, therefore tangential flow filtration technology is usually used in the separation of bioactive substance,
The production of biological products, blood product and vaccine.
Affinity chromatography is a kind of common Protein purification techniques, using can tie with bioactive substance (such as antibody) specificity
The medium of conjunction makes in liquid phase corresponding bioactive substance selective binding in stationary phase as stationary phase, so as to liquid phase
In other impurities separate, achieve the purpose that separating-purifying.Macropore continuous chromatography (affinity monolith
Chromatography, abbreviation AMC) it is a kind of novel affinity chromatography technology, stationary phase is individually continuous more by one
Porous materials are constituted.Common stationary phase material includes agarose (agarose), silica (silica), methacrylic acid contracting
Water glyceride/ethylene glycol dimethacrylate (glycidyl methacrylate/ethylene dimethacrylate)
And Cryogels etc..Macropore continuous chromatography has lot of advantages, such as can be fabricated to diversified forms and be filled in pillar, capillary
In pipe and microfluidic device；Back pressure is low, and liquid phase can flow through at a high speed, achievees the purpose that quick separating and analysis；Stationary phase can
To carry out modification as needed.
Monoclonal antibody is to be generated by hybridoma, determined just for single antigen a certain on complex antigen molecule
Determine the specific antibody of cluster.Monoclonal antibody has the advantages that specific height, homogeneity are high, is applied to numerous areas rapidly,
Including laboratory medicine diagnostic reagent, radio-immuno-image technology, protein purification and cancer target, autoimmune disease,
The disease treatments such as infectious diseases and graft-rejection.Existing frequently-used monoclonal antibody-purified system is AKTA protein purification
System, the system have the advantages that high degree of automation, and automatic loading, purifying, sample collection, sample analysis may be implemented in one
Body.But the drawbacks of system be a kind of chromatographic column can only be connected when purifying every time, such as Protein A chromatographic column, Yin/Yang from
Sub- exchange chromatography column, hydrophobic interaction chromatography column etc..When Monoclonal Antibody Cell fermentation broth contents complexity, need using 3-4 kind
Chromatographic column is purified, and then needs each chromatographic column use to terminate using AKTA protein purification system monoclonal antibody purification
Afterwards, rechange another chromatographic column, and require first to adjust sample condition using new chromatographic column every time, as conductivity,
PH etc..Therefore, existing monoclonal antibody technology of preparing is complicated, time-consuming, causes the price of monoclonal antibody also relatively high.
Utility model content
To solve that a kind of chromatographic column, the albumen of purified fractions complexity can only be connected when AKTA protein purification system purifies every time
When need each chromatographic column use after, rechange another chromatographic column, also to readjust sample condition leads to this
The time-consuming and laborious problem of method complex procedures, the utility model provide a kind of multiplexing convection current chromatographic system (multiplexed
Convective chromatography, abbreviation MCC) and its for protein purification method, the system combination egg in downstream
White purification step realizes the single stepping for being recovered to final product purifying by cell liquid, can be applied to laboratory and pilot scale
The antibody producing platform of scale.The technical solution adopted is as follows:
The purpose of this utility model is to provide a kind of multiplexing convection current chromatographic system, the multiplexing convection current chromatographic system include into
Expect fluid reservoir 1, peristaltic pump I2, tangential flow filtration device 3, circulation fluid fluid reservoir 4, peristaltic pump II5, peristaltic pump III6 discard filter
Liquid fluid reservoir 7, peristaltic pump IV8, macropore continuous chromatography device 9, peristaltic pump V13 and central processing unit；Wherein: the charging liquid storage
Tank 1 is connected by peristaltic pump I2 with the feed inlet of tangential flow filtration device 3；The tangential flow filtration device 3 discharges there are two setting
Mouthful, respectively recycle filtrate discharge port and waste filtrate discharge port；The waste filtrate discharge port is by peristaltic pump III6 and gives up
Filtrate fluid reservoir 7 is abandoned to be connected；The recycling filtrate discharge port is divided into two branches, and a branch passes through peristaltic pump V13 and circulation
The feed inlet of liquid fluid reservoir 4 is connected, another branch passes through the combined feed mouth phase of peristaltic pump IV8 and macropore continuous chromatography device 9
Even；The discharge port of the circulation fluid fluid reservoir 4 is connected by peristaltic pump II5 with the feed inlet of tangential flow filtration device 3；It is described big
Total discharge port of hole continuous chromatography device 9 is connected with the feed inlet of circulation fluid fluid reservoir 4；The central processing unit controls peristaltic pump
I2, peristaltic pump II5, peristaltic pump III6, peristaltic pump IV8 and peristaltic pump V13 are started and stopped.
Preferably, the multiplexing convection current chromatographic system is that single channel is multiplexed convection current chromatographic system, wherein macropore continuous chromatography
Device 9 includes a macropore continuous chromatography column.
It is highly preferred that the macropore continuous chromatography column is selected from hydrophobic chromatography column, affinity column, molecular sieve, anion friendship
Chromatographic column is changed, any one in cation-exchange chromatography post and composite chromatographic chromatographic column.
Preferably, the multiplexing convection current chromatographic system is that multichannel is multiplexed convection current chromatographic system, wherein macropore continuous chromatography
Device 9 includes multiple macropore continuous chromatography columns being connected in parallel.
It is highly preferred that the multiple macropore continuous chromatography column being connected in parallel is selected from hydrophobic chromatography column, affinity column divides
Son sieve, anion exchange chromatography, any one in cation-exchange chromatography post and composite chromatographic chromatographic column be connected in parallel or
It is arbitrarily a variety of to be connected in parallel.
Preferably, the charging fluid reservoir 1 is used to store depositing for the crude protein solution of processing to be purified, buffer and albumen
Liquid storage.
Preferably, pressure sensor 10 is equipped on the pipeline between 3 feed inlet of peristaltic pump I2 and tangential flow filtration device,
And/or pressure sensor 10 is equipped on the pipeline between 3 feed inlet of peristaltic pump II5 and tangential flow filtration device, and/or compacted
It is equipped with pressure sensor 10 between dynamic pump IV8 and the combined feed mouth of macropore continuous chromatography device 9, and/or is filled in tangential flow filtration
It sets and is equipped with pressure sensor 10, all pressure sensors 10 and centre between 3 waste filtrate discharge port and peristaltic pump III6
It manages device data to be connected, the pressure signal of acquisition is sent to central processing unit.
Preferably, it is equipped with pH and conductivity sensor 12 between peristaltic pump I2 and charging fluid reservoir 1, and/or wriggled
It pumps and is equipped with pH and conductivity sensor 12 between II5 and the discharge port of circulation fluid fluid reservoir 4；The pH and conductivity sensor 12
With central processing unit data connection, its signal collected is sent to central processing unit by pH and conductivity sensor 12.
Preferably, the multiplexing convection current chromatographic system further includes weighing device 11, and the weighing device 11 is stored up with circulation fluid
Flow container 4 connects, for weighing the weight of circulation fluid fluid reservoir 4 and/or its solution stored.
It is highly preferred that the weighing device 11 is electronic scale.
It is highly preferred that the weighing device 11 is weight sensor, weight sensor acquisition circulation fluid fluid reservoir 4 with
Its weight signal collected is sent to central processing unit by the weight information of its solution stored.
The utility model additionally provides a kind of method using above-mentioned multiplexing convection current chromatographic system purifying protein, this method packet
Include following steps:
1) the crude protein solution of processing to be purified is delivered in tangential flow filtration device 3 and is filtered, waste filtrate by
Peristaltic pump III6 is delivered in waste filtrate fluid reservoir 7, recycles filtrate flows into circulation fluid fluid reservoir 4, circulation fluid fluid reservoir 4
It is transmitted back in tangential flow filtration device 3 after middle recycling filtrate is weighed by peristaltic pump II5, repeats above-mentioned filter process to reaching both
Determine concentrating degree, completes the concentration of first stage；
2) solution fed in fluid reservoir 1 is replaced with into buffer, the buffer in fluid reservoir 1 will be fed by peristaltic pump I2
It is delivered in tangential flow filtration device 3 and carries out buffer exchange, by the waste liquid after displacement by wriggling in replacement process
Pump III6 be delivered in waste filtrate fluid reservoir 7, the recovered liquid after displacement is delivered in circulation fluid fluid reservoir 4, it is weighed after by
Peristaltic pump II5 is transmitted back in tangential flow filtration device 3, is repeated above-mentioned replacement process and is completed buffer exchange to set pH and electricity
Conductance；
3) solution in tangential flow filtration device 3 and circulation fluid fluid reservoir 4 macropore continuous chromatography device 9 is delivered to carry out
Removal of impurities；
4) the storage liquid that the solution in charging fluid reservoir 1 is replaced with to albumen, will clean by macropore continuous chromatography device 9
Solution afterwards is delivered to again carries out repetitive cycling to complete the storage liquid of secondary concentration and albumen in tangential flow filtration device 3
Displacement；
5) after completing secondary concentration, solution is collected up to protein solution after purification.
It is highly preferred that described method includes following steps:
1) the crude protein solution storage of processing to be purified is started by central processing unit and is wriggled in charging fluid reservoir 1
I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 are pumped, peristaltic pump IV8 is closed, the place to be purified in fluid reservoir 1 will be fed
The crude protein solution of reason, which is delivered in tangential flow filtration device 3, to be filtered, during the filtration process by waste filtrate by peristaltic pump
III6 is delivered in waste filtrate fluid reservoir 7, recycling filtrate be delivered in circulation fluid fluid reservoir 4, it is weighed after by peristaltic pump II5
It is transmitted back in tangential flow filtration device 3, repeats above-mentioned filter process to set concentrating degree is reached, complete the dense of first stage
Contracting；
2) solution fed in fluid reservoir 1 is replaced with into buffer, the buffer in fluid reservoir 1 will be fed by peristaltic pump I2
It is delivered in tangential flow filtration device 3 and carries out buffer exchange, by the waste liquid after displacement by wriggling in replacement process
Pump III6 be delivered in waste filtrate fluid reservoir 7, the recovered liquid after displacement is delivered in circulation fluid fluid reservoir 4, it is weighed after by
Peristaltic pump II5 is transmitted back in tangential flow filtration device 3, is repeated above-mentioned replacement process and is completed buffer exchange to set pH and electricity
Conductance；
3) peristaltic pump II5 and peristaltic pump IV8 is started by central processing unit, peristaltic pump V13 is closed, so that tangential flow filtration
Solution in device 3 and circulation fluid fluid reservoir 4 enters macropore continuous chromatography device 9 and cleans, and fills by macropore continuous chromatography
Solution after setting 9 removal of impurities, which enters in circulation fluid fluid reservoir 4, to be stored；
4) peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 are started by central processing unit, closes and wriggles
IV8 is pumped, the solution in charging fluid reservoir 1 is replaced with to the storage liquid of albumen, what will be stored in circulation fluid fluid reservoir 4 passes through macropore
It is secondary dense to complete that solution after the removal of impurities of continuous chromatography device 9 is delivered to progress repetitive cycling processing in tangential flow filtration device 3
The displacement of the storage liquid of contracting and albumen, the liquid of final process are stored by circulation fluid fluid reservoir 4；
5) peristaltic pump I2, peristaltic pump II5, peristaltic pump III6, peristaltic pump V13 and peristaltic pump are closed by central processing unit
IV8 collects the solution in circulation fluid fluid reservoir 4 to get protein solution after purification.
Preferably, the crude protein solution of the processing to be purified is Monoclonal Antibody Cell fermentation liquid.It is highly preferred that step
5) concentration of the secondary concentration to concentrate is 1~5mg/mL.
The utility model multiplexing convection current chromatographic system includes tangential flow filtration system (TFF) and macropore continuous chromatography (AMC),
According to the complexity of Monoclonal Antibody Cell fermentation broth contents, single channel multiplexing convection current chromatographic system and multichannel can be developed
It is multiplexed convection current chromatographic system, the simple situation of Monoclonal Antibody Cell fermentation broth contents is multiplexed using single channel to flow chromatography system
The single stepping that final product purifying is recovered to by cell liquid can be achieved in system；The feelings of Monoclonal Antibody Cell fermentation broth contents complexity
Condition can realize the single stepping that final product purifying is recovered to by cell liquid using multichannel multiplexing convection current chromatographic system.
Single channel is multiplexed convection current chromatographic system can be by a tangential flow filtration device, macropore continuous chromatography column, five
A peristaltic pump, three pressure sensors, two pH/ conductivity sensors, a circulation fluid liquid storage canister, a charging liquid storage
Tank, a waste filtrate fluid reservoir group are shaped to circulation stream.Outer Lian Yitai weighing device and a central processing unit control follow
Ring liquid stream is moved and peristaltic pump on and off.
Multichannel is multiplexed convection current chromatographic system can be by a tangential flow filtration device, multiple macropore continuous chromatography columns, five
A peristaltic pump, three pressure sensors, two pH/ conductivity sensors, a circulation fluid fluid reservoir, a charging fluid reservoir,
One waste filtrate fluid reservoir outlet group is shaped to circulation stream.Outer Lian Yitai weighing device and a central processing unit control follow
Ring liquid stream is moved and peristaltic pump on and off.
The utility model multiplexing convection current chromatographic system can be used for purifying protein, be especially adapted for use in monoclonal antibody, benefit
With the method for the utility model multiplexing convection current chromatographic system processing monoclonal antibody are as follows: pass through Monoclonal Antibody Cell fermentation liquid
Feed inlet enters multiplexing convection current chromatographic system, initially enters tangential flow filtration system and carries out sample concentration；In sample concentration to one
When determining degree, buffer exchange is carried out from feed inlet addition buffer；By tangential flow filtration system concentration and buffer exchange
Sample afterwards enters macropore continuous chromatography and carries out one or more steps removal of impurities；By macropore continuous chromatography processing after, sample again into
Enter tangential flow filtration system to be concentrated.During secondary concentration, from feed inlet addition monoclonal antibody store liquid, to sample into
Row storage liquid displacement；Sample is after storage liquid replaces and is concentrated to specific concentration range, tangential flow filtration system and macropore
Continuous chromatography system is offline, and sample is collected by collecting pipe, stored.
Buffer components are determined according to the adsorbing medium of macropore continuous chromatography in the utility model.
The crude protein solution of processing to be purified in the utility model refers to that the fermented cells of acquisition are broken through centrifugation, cell
The crude protein solution obtained after preliminary purification after broken, centrifugation, filtering.
The storage liquid of albumen refers to the solution for storing albumen after purification in the utility model.
Macropore continuous chromatography column refers to the protein chromatographic column based on macropore continuous chromatography technology, the i.e. color in the utility model
Composing column is the purification column for combining macropore continuous chromatography technology with protein chromatographic, such as hydrophobic chromatography column, affinity column, molecule
Sieve, anion exchange chromatography, cation-exchange chromatography post, composite chromatographic chromatographic column or other be based on macropore continuous chromatography skill
The protein chromatographic column of art.
The utility model the utility model has the advantages that
Tangential flow filtration device plays inspissation, buffer solution displacement and albumen storage liquid displacement in the utility model
Effect, macropore continuous chromatography device play purification, and Tthe utility model system takes full advantage of chromatographic column high throughput, resistance to pressure
By force, in conjunction with tangential flow filtration system, collect the formula of the concentration of cell supernatant, buffer exchange, purifying and final product in
One；Meanwhile the system combination purification step in downstream, no replacement is required chromatographic column are readjusted condition, are realized by cell
Liquid is recovered to the single stepping of final product purifying, can achieve time saving and energy saving, economical and efficient purifying purpose.In addition, this is
System can further integrate the realization such as ultraviolet detector, pH, conductivity gauge to the real-time monitoring of whole process of purification, and final foundation is high
Effect, easy, economic laboratory, pilot-scale antibody producing platform have very big answer in various protein purification pilot scale platforms
Use prospect.
Tthe utility model system has preferable purification effect, such as handles monoclonal antibody protein, before purification cell fermentation
In liquid, host protein (Host cell protein, HCP) content is 300,000ppm, DNA content 30,000ppm.By
After octanoic acid-allantoin-centrifugation-filtering pretreatment, HCP content is reduced to 500ppm hereinafter, being multiplexed by single channel to flow chromatography
After purification, HCP content is less than 100ppm to system, and DNA content is less than 10ppm；IgG1 purity is 95% or more, and the rate of recovery is higher than
85%.
Detailed description of the invention
Fig. 1 is structural schematic diagram of the single channel multiplexing to flow chromatography；
Fig. 2 is Purification: Principles figure of the single channel multiplexing to flow chromatography；
Fig. 3 is structural schematic diagram of the multichannel multiplexing to flow chromatography；
Fig. 4 is Purification: Principles figure of the multichannel multiplexing to flow chromatography；
In figure: 1, feed fluid reservoir；2, peristaltic pump I；3, tangential flow filtration device；4, circulation fluid fluid reservoir；5, peristaltic pump
II；6, peristaltic pump III；7, waste filtrate fluid reservoir；8, peristaltic pump IV；9, macropore continuous chromatography device；10, pressure sensor；
11, weighing device；12, pH and conductivity sensor；13, peristaltic pump V.
Specific embodiment
The utility model is described further combined with specific embodiments below, but the utility model is not by the limit of embodiment
System.
Embodiment 1
As shown in Figure 1, present embodiments providing a kind of single channel multiplexing convection current chromatographic system includes charging fluid reservoir 1, it is compacted
Dynamic pump I2, tangential flow filtration device 3, circulation fluid fluid reservoir 4, peristaltic pump II5, peristaltic pump III6, waste filtrate fluid reservoir 7 are compacted
Dynamic pump IV8, macropore continuous chromatography device 9, peristaltic pump V13 and central processing unit；Wherein: the charging fluid reservoir 1 passes through wriggling
Pump I2 is connected with the feed inlet of tangential flow filtration device 3；The tangential flow filtration device 3 is set there are two discharge port, is respectively returned
Receive filtrate discharge port and waste filtrate discharge port；The waste filtrate discharge port passes through peristaltic pump III6 and waste filtrate fluid reservoir
7 are connected；The recycling filtrate discharge port is divided into two branches, a branch by peristaltic pump V13 and circulation fluid fluid reservoir 4 into
Material mouth is connected, another branch is connected by peristaltic pump IV8 with the combined feed mouth of macropore continuous chromatography device 9；The circulation fluid
The discharge port of fluid reservoir 4 is connected by peristaltic pump II5 with the feed inlet of tangential flow filtration device 3；The macropore continuous chromatography dress
9 total discharge port is set to be connected with the feed inlet of circulation fluid fluid reservoir 4；The central processing unit controls peristaltic pump I2, peristaltic pump
II5, peristaltic pump III6, peristaltic pump IV8 and peristaltic pump V13 are started and stopped.
It is that single channel is multiplexed convection current chromatographic system that the present embodiment, which is multiplexed convection current chromatographic system, wherein macropore continuous chromatography device
9 include a macropore continuous chromatography column.Macropore continuous chromatography column is selected from hydrophobic chromatography column, affinity column, molecular sieve, anion
Displacement chromatography column, any one in cation-exchange chromatography post and composite chromatographic chromatographic column.Tangential flow filtration device 3 includes cutting
To flowing through filter column.
The charging fluid reservoir 1 of the present embodiment is used to store crude protein solution, buffer and the albumen of processing to be purified respectively
Store liquid.
Pipe as a kind of preferred embodiment of the present embodiment, between 3 feed inlet of peristaltic pump I2 and tangential flow filtration device
On the road, on the pipeline between 3 feed inlet of peristaltic pump II5 and tangential flow filtration device, in peristaltic pump IV8 and macropore continuous chromatography
Pressure sensor 10 is equipped between the combined feed mouth of device 9 and between waste filtrate discharge port and peristaltic pump III6；Institute
There is pressure sensor 10 to be connected with central processing unit data, collected pressure signal is delivered to central processing unit.
As a kind of preferred embodiment of the present embodiment, between peristaltic pump I2 and charging fluid reservoir 1 and in peristaltic pump
PH and conductivity sensor 12 are equipped between II5 and the discharge port of circulation fluid fluid reservoir 4；PH and conductivity sensor 12 are in
Its signal collected is sent to central processing unit by central processor data connection, pH/ conductivity sensor.
As a kind of preferred embodiment of the present embodiment, the multiplexing convection current chromatographic system further includes weighing device 11, described
Weighing device 11 is connect with circulation fluid fluid reservoir 4, for weighing the weight of solution in circulation fluid fluid reservoir 4.Weighing device 11 can
Think electronic scale, directly weighs the weight of circulation fluid fluid reservoir 4 and its solution stored；Weighing device 11 can also be weight
Quantity sensor, weight sensor acquires the weight information of circulation fluid fluid reservoir 4 and its solution stored, its is collected heavy
Amount signal is sent to central processing unit.
The single channel multiplexing convection current chromatographic system technical schematic diagram of the present embodiment is as shown in Figure 2.
The present embodiment additionally provides a kind of method using above-mentioned multiplexing convection current chromatographic system purifying protein, and this method includes
Following steps:
1) the crude protein solution of processing to be purified is stored in charging fluid reservoir 1 such as Monoclonal Antibody Cell fermentation liquid
In, peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 are started by central processing unit, close peristaltic pump IV8,
The crude protein solution of the processing to be purified fed in fluid reservoir 1 is delivered in tangential flow filtration device 3 and is filtered, is being filtered
Waste filtrate is delivered in waste filtrate fluid reservoir 7 by peristaltic pump III6 in the process, recycling filtrate is delivered to circulation fluid liquid storage
It in tank 4, is transmitted back in tangential flow filtration device 3 after weighed by peristaltic pump II5, repeats above-mentioned filter process to reaching set dense
Contracting degree completes the concentration of first stage；
2) solution fed in fluid reservoir 1 is replaced with into buffer, the buffer in fluid reservoir 1 will be fed by peristaltic pump I2
It is delivered in tangential flow filtration device 3 and carries out buffer exchange, by the waste liquid after displacement by wriggling in replacement process
Pump III6 be delivered in waste filtrate fluid reservoir 7, the recovered liquid after displacement is delivered in circulation fluid fluid reservoir 4, it is weighed after by
Peristaltic pump II5 is transmitted back in tangential flow filtration device 3, is repeated above-mentioned replacement process and is completed buffer exchange to set pH and electricity
Conductance；
3) peristaltic pump II5 and peristaltic pump IV8 is started by central processing unit, peristaltic pump V13 is closed, so that tangential flow filtration
Solution in device 3 and circulation fluid fluid reservoir 4 enters macropore continuous chromatography device 9 and carries out edulcoration purification, when pollutant absorption becomes
Nearly balance, macropore continuous chromatography is offline, and the solution after the removal of impurities of macropore continuous chromatography device 9 enters in circulation fluid fluid reservoir 4
Storage；
4) peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 are started by central processing unit, closes and wriggles
IV8 is pumped, the solution in charging fluid reservoir 1 is replaced with to the storage liquid of albumen, what will be stored in circulation fluid fluid reservoir 4 passes through macropore
It is secondary dense to complete that solution after the removal of impurities of continuous chromatography device 9 is delivered to progress repetitive cycling processing in tangential flow filtration device 3
The displacement of the storage liquid of contracting and albumen, the liquid of final process are stored by circulation fluid fluid reservoir 4；
5) when MAb concentration reaches 1mg/mL, by central processing unit close peristaltic pump I2, peristaltic pump II5,
Peristaltic pump III6, peristaltic pump V13 and peristaltic pump IV8, the solution collected in circulation fluid fluid reservoir 4 are molten to get albumen after purification
Liquid.
Embodiment 2
As shown in figure 3, the present embodiment the difference from embodiment 1 is that: multiplexing convection current chromatographic system be multichannel multiplexing pair
Flow chromatography system, wherein macropore continuous chromatography device 9 includes 2 macropore continuous chromatography columns being connected in parallel.Wherein 2 parallel connections connect
The macropore continuous chromatography column connect is respectively anion exchange chromatography and hydrophobic chromatography column.It is also possible to selected from hydrophobic chromatography column,
Affinity column, molecular sieve, anion exchange chromatography, any group in cation-exchange chromatography post and composite chromatographic chromatographic column
It closes.
The multichannel multiplexing convection current chromatographic system technical schematic diagram of the present embodiment is as shown in Figure 4.
The purification process of the present embodiment the difference from embodiment 1 is that: when MAb concentration reaches 5mg/mL,
TFF system is offline, and sample treatment terminates, and final purification of samples is collected by collecting pipe, stored.
Embodiment 3
By taking monoclonal antibody IgG1 as an example, instruction sheet channel multiplexing convection current chromatographic system is achievable dense from cell fermentation liquid
Contract, be recovered to the single stepping of purifying.
Cell culture: monoclonal antibody IgG1 is by Chinese hamster ovary cell (Chinese Hamster Ovary, CHO)
Expression generates, and antibody is at 5 litersIt is produced in B stirring-type glass biological reaction device, using the culture side of batch feeding
Formula collects cell fermentation liquid after culture 15~30 days.Cell culture medium is protein-free medium CD CHO (Life
) and protein-free medium TechnologiesThe mixture of PF-CHO (GE Healthcare), the two volume ratio are 1:
1。
The pretreatment of cell fermentation liquid: cell fermentation liquid pH is adjusted to 5.3, octanoic acid and allantoin is added, makes octanoic acid and allantois
Final mass concentration of the element in said mixture is respectively 0.4% and 1%.Mixture is stirred at room temperature 2 hours, then 4000 ×
G, be centrifuged 20min under room temperature, collect supernatant via 0.22 μm of filter membrane (Rapid-Flow Filters,
Thermo Scientific) filtering, collect filtered fluid.
Filtered fluid (about 5 liters) is placed in charging fluid reservoir 1, enters 3 (slipstream of tangential flow filtration device via peristaltic pump I
Filter column, TFF), waste filtrate is dense by tangential flow filtration device 3 through 7 discharge system of peristaltic pump III6 waste filtrate fluid reservoir
The sample of contracting enters in circulation fluid fluid reservoir 4 weigh after be again introduced into TFF column and be concentrated.When circulation fluid volume is concentrated into about 3
When rising, Tris-HCl buffer (pH 7.5) is added in charging fluid reservoir 1, Tris-HCl buffer enters TFF column to concentration
Sample carry out buffer exchange.Circulation fluid controls macropore continuous chromatography device 9 after buffer exchange, by central processing unit
(single channel is multiplexed convection current chromatographic system, and macropore continuous chromatography column uses anion exchange chromatography) front end peristaltic pump IV8 is opened,
Circulation fluid enters macropore continuous chromatography device 9 (anion-exchange column) and starts IgG1 purifying, and circulation fluid is all from the continuous color of macropore
After spectral apparatus 9 (anion-exchange column) outflow, it is offline that purifying terminates AMC system.It is further dense to enter TFF system by IgG1 after purification
Contracting, while addition PBS buffer solution (pH 7.2-7.4,10mM) is right as monoclonal antibody storage liquid in charging fluid reservoir 1
IgG1 carries out storage liquid displacement.When IgG1 concentration reaches 3mg/mL, TFF system is offline, and sample treatment terminates, final purified sample
Product are collected by collecting pipe, are stored.Host protein content is detected by Generation III CHO HCP kit, DNA content
Pass through numeric type PCR instrument QX100TM Droplet DigitalTMPCR System (Bio-Rad) detection.
Experimental result: before purification in cell fermentation liquid, host protein (Host cell protein, HCP) content is 300,
000ppm, DNA content 30,000ppm.After octanoic acid-allantoin-centrifugation-filtering pretreatment, HCP content is reduced to
500ppm or less.After purification by single channel multiplexing convection current chromatographic system, HCP content is less than 100ppm, and DNA content is less than
10ppm；IgG1 purity is 95% or more, and the rate of recovery is higher than 85%.Meet existing food and drug administration (Food and
Drug administration, FDA) requirement to monoclonal antibody protein product.
Although the utility model has been disclosed in the preferred embodiment as above, it is not intended to limit the utility model, and is appointed
What person skilled in the art can do various changes and modification, therefore without departing from the spirit and scope of the utility model
The protection scope of the utility model should subject to the definition of the claims.
Claims (8)
1. a kind of multiplexing convection current chromatographic system, which is characterized in that the multiplexing convection current chromatographic system includes charging fluid reservoir (1),
Peristaltic pump I (2), tangential flow filtration device (3), circulation fluid fluid reservoir (4), peristaltic pump II (5), peristaltic pump III (6) discard filter
Liquid fluid reservoir (7), peristaltic pump IV (8), macropore continuous chromatography device (9), peristaltic pump V (13) and central processing unit；Wherein: described
Charging fluid reservoir (1) is connected by peristaltic pump I (2) with the feed inlet of tangential flow filtration device (3)；The tangential flow filtration device
(3) discharge port there are two setting, respectively recycling filtrate discharge port and waste filtrate discharge port；The waste filtrate discharge port passes through
Peristaltic pump III (6) is connected with waste filtrate fluid reservoir (7)；The recycling filtrate discharge port is divided into two branches, and a branch is logical
It crosses peristaltic pump V (13) to be connected with the feed inlet of circulation fluid fluid reservoir (4), another branch is connected by peristaltic pump IV (8) and macropore
The combined feed mouth of continuous chromatogram arrangement (9) is connected；The discharge port of the circulation fluid fluid reservoir (4) passes through peristaltic pump II (5) and tangential
The feed inlet for flowing filter device (3) is connected；Total discharge port of the macropore continuous chromatography device (9) and circulation fluid fluid reservoir (4)
Feed inlet be connected；The central processing unit controls peristaltic pump I (2), peristaltic pump II (5), peristaltic pump III (6), peristaltic pump IV
(8) it is started and stopped with peristaltic pump V (13).
2. multiplexing convection current chromatographic system according to claim 1, which is characterized in that the multiplexing convection current chromatographic system is single
Channel multiplexing convection current chromatographic system, wherein macropore continuous chromatography device (9) includes a macropore continuous chromatography column.
3. multiplexing convection current chromatographic system according to claim 2, which is characterized in that the macropore continuous chromatography column, which is selected from, to be dredged
Water chromatographic column, affinity column, molecular sieve, anion exchange chromatography, cation-exchange chromatography post and composite chromatographic chromatographic column
In any one.
4. multiplexing convection current chromatographic system according to claim 1, which is characterized in that the multiplexing convection current chromatographic system is more
Channel multiplexing convection current chromatographic system, wherein macropore continuous chromatography device (9) includes multiple macropore continuous chromatography columns being connected in parallel.
5. multiplexing convection current chromatographic system according to claim 4, which is characterized in that the multiple macropore being connected in parallel connects
Continuous chromatographic column is selected from hydrophobic chromatography column, affinity column, molecular sieve, anion exchange chromatography, cation-exchange chromatography post and
Any one in composite chromatographic chromatographic column is connected in parallel or any a variety of is connected in parallel.
6. multiplexing convection current chromatographic system according to claim 1, which is characterized in that the charging fluid reservoir (1) is for storing up
Deposit the crude protein solution of processing to be purified, the storage liquid of buffer and albumen.
7. multiplexing convection current chromatographic system according to claim 1, which is characterized in that in peristaltic pump I (2) and tangential flow filtration
Pipeline between device (3) feed inlet is equipped with pressure sensor (10), and/or fills in peristaltic pump II (5) and tangential flow filtration
The pipeline set between (3) feed inlet is equipped with pressure sensor (10), and/or fills in peristaltic pump IV (8) and macropore continuous chromatography
It sets and is equipped with pressure sensor (10) between the combined feed mouth of (9), and/or discharge in the waste filtrate of tangential flow filtration device (3)
Pressure sensor (10), all pressure sensors (10) and central processing unit data phase are equipped between mouth and peristaltic pump III (6)
Even, the pressure signal of acquisition is sent to central processing unit.
8. multiplexing convection current chromatographic system according to claim 1, which is characterized in that in peristaltic pump I (2) and charging fluid reservoir
(1) be equipped with pH and conductivity sensor (12) between, and/or peristaltic pump II (5) and circulation fluid fluid reservoir (4) discharge port it
Between be equipped with pH and conductivity sensor (12)；The pH and conductivity sensor (12) and central processing unit data connection, pH and
Its signal collected is sent to central processing unit by conductivity sensor (12).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201822092505.1U CN209362021U (en) | 2018-12-13 | 2018-12-13 | A kind of multiplexing convection current chromatographic system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201822092505.1U CN209362021U (en) | 2018-12-13 | 2018-12-13 | A kind of multiplexing convection current chromatographic system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN209362021U true CN209362021U (en) | 2019-09-10 |
Family
ID=67832571
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201822092505.1U Active CN209362021U (en) | 2018-12-13 | 2018-12-13 | A kind of multiplexing convection current chromatographic system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN209362021U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111318077A (en) * | 2018-12-13 | 2020-06-23 | 中国科学院青岛生物能源与过程研究所 | Multiplex convection chromatographic system and method for purifying protein by using same |
-
2018
- 2018-12-13 CN CN201822092505.1U patent/CN209362021U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111318077A (en) * | 2018-12-13 | 2020-06-23 | 中国科学院青岛生物能源与过程研究所 | Multiplex convection chromatographic system and method for purifying protein by using same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3828143B2 (en) | Improved tangential flow filtration method and apparatus | |
| CN101340960B (en) | Device for chromatographic separations | |
| Zydney | New developments in membranes for bioprocessing–A review | |
| US9321805B2 (en) | Downstream bioprocessing device | |
| AU756832B2 (en) | Purification of biological substances | |
| CN105107228B (en) | The continuous process of biological products | |
| US6596172B1 (en) | Purification of biological substances | |
| CN104395341B (en) | The purifying of biomolecule | |
| EP0730639B1 (en) | Cell separation device | |
| US20130115588A1 (en) | Integrated bioreactor and separation system and methods of use therof | |
| JP2017502666A (en) | Equipment for cell culture | |
| EP2675540A1 (en) | System and process for biopolymer chromatography | |
| HUE032538T2 (en) | Devices and Methods for Integrated Continuous Manufacturing of Biological Molecules | |
| KR20210149117A (en) | Continuous Production of Recombinant Proteins | |
| CN209362021U (en) | A kind of multiplexing convection current chromatographic system | |
| US10843104B2 (en) | System and process for biopolymer chromatography | |
| CN111318077A (en) | Multiplex convection chromatographic system and method for purifying protein by using same | |
| CN114317227A (en) | Exosome purification method and all-in-one machine device thereof | |
| JP2023145471A (en) | In-line product concentration to reduce volumetric load flow rate and increase productivity of bind and elute chromatography purification | |
| CN214286794U (en) | Biological particle purification device | |
| CN214300076U (en) | Membrane carrier purification device for biological particles | |
| CN109320603A (en) | A kind of system and method for serialization purification antibody | |
| CN105688442B (en) | A kind of column chromatography and membrane filtration integrated system | |
| CN115094030B (en) | Purification method of clinical-grade high-purity exosome | |
| CN106317166A (en) | Device for separating proteins by using high-rate filtration and chromatography mixed mechanism |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR01 | Patent grant | ||
| GR01 | Patent grant |