WO2020083372A1 - 一种半连续流的磁珠纯化系统 - Google Patents

一种半连续流的磁珠纯化系统 Download PDF

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WO2020083372A1
WO2020083372A1 PCT/CN2019/113258 CN2019113258W WO2020083372A1 WO 2020083372 A1 WO2020083372 A1 WO 2020083372A1 CN 2019113258 W CN2019113258 W CN 2019113258W WO 2020083372 A1 WO2020083372 A1 WO 2020083372A1
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liquid storage
liquid
storage tank
magnetic bead
magnet
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PCT/CN2019/113258
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English (en)
French (fr)
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冯在东
陈国栋
贺瑞娜
钱红
白涛
朱金鑫
谢涛
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南京金斯瑞生物科技有限公司
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Publication of WO2020083372A1 publication Critical patent/WO2020083372A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the invention relates to a semi-continuous flow magnetic bead purification system, which is mainly used in the field of biological purification, for example, for small-scale and large-scale extraction of proteins and nucleic acids.
  • the magnetic bead purification method generally uses a purification system to separate the magnetic beads by a magnetic field to achieve the purpose of separating and purifying substances such as cells, proteins, or nucleic acids. Compared with the commonly used precipitation method, centrifugal method, and column membrane method, the magnetic bead purification method has the characteristics of high extraction efficiency, fast separation speed, and simple equipment required.
  • the magnetic bead purification method based on magnetic adsorption relies on the combination of magnetic principle and incubation to quickly and efficiently enrich the target protein in the sample, effectively avoiding the pretreatment and loading of the sample by the resin.
  • the disadvantages such as the limitation of the system have got rid of the limitation of packing and flow rate.
  • the purification method using magnetic separation is easier to automate and can meet the needs of high-throughput purification for fast, automated, multi-channel simultaneous processing.
  • the purification devices currently on the market generally handle relatively low throughput and require a lot of manual operations to assist the purification process.
  • the current operations are all open and open operations, it is difficult to prevent external environmental pollution, nor can it avoid damage to the environment caused by the volatilization of reagents during the purification process.
  • the magnetic rack of the prior art can meet the needs of some customers, there are still many deficiencies, such as: (1) the flux is small and cannot meet the needs of large-scale purification; (2) it needs to be transferred to a centrifuge tube or other container , The process is complicated, and there is a risk of damaging the sample; (3) additional equipment is required to achieve automation, increase additional cost, and occupy additional space; (4) the shape is fixed and cannot be flexibly adapted to the shape of the container.
  • the large-scale purification devices used in the market all adopt the method of resin purification, which requires deep filtration, consumes a large amount of disposable consumables, and requires a long time investment of many people.
  • a semi-continuous flow magnetic bead purification system which includes: a shaking device; a frame structure, the frame structure is mounted on the shaking device; and is mounted on the frame structure Liquid storage device, magnet and magnet actuator; and control system; wherein, the liquid storage device is connected to the liquid source and the waste liquid tank through the liquid circuit system; the control system is connected to the liquid circuit system, magnet actuator and shaking device Connect and control the hydraulic system, magnet actuator and shaking device.
  • control system controls the liquid circuit system to realize the filling and discharging of the liquid storage device.
  • the liquid storage device includes a plurality of liquid storage tanks, and when the magnets work, the magnets are arranged in groups at the bottom of each liquid storage tank, and each group of magnets includes a V-shape or other matching tank body shape Two or more magnets arranged.
  • the liquid storage tank has a tapered bottom, and the pipeline in the liquid storage tank is divided into two paths to spray the solution to positions corresponding to two magnets in a group of magnets, respectively.
  • the liquid storage tank includes a sampling needle interface and an exhaust port.
  • the shaking device is a digital planar circumferential shaking device.
  • the frame structure includes a fixed base plate, a liquid storage tank fixed pressure plate, an upper support wrench, a lower support plate, and side supports, wherein the fixed base plate is mounted to the shaking device, and the liquid storage tank fixed pressure plate and upper support
  • the wrench and the lower support wrench are used to install the liquid storage tank
  • the side support is used to fix and fix the liquid storage tank fixing platen, the upper support wrench and the lower support wrench support to the fixed base plate.
  • the frame structure includes a fixed base plate, side supports, a lower support plate, a limit plate, an upper support wrench, a beam tube frame, a buckle, and a fixed pressure plate of a liquid storage tank, wherein the fixed base plate is mounted to the shaking device ,
  • the liquid storage tank fixed pressure plate, upper support wrench, limit plate and lower support wrench are used to install the liquid storage tank
  • the side support is used to fix the liquid storage tank pressure plate, upper support wrench, limit
  • the board and the lower bracket are supported and fixed to the fixed base plate, the beam tube frame is fixed above the upper bracket, and the tower buckle is fixed on the side support.
  • a method for purifying a biological sample comprising injecting a biological sample containing a target component and magnetic beads capable of binding the target component into the storage solution of the above-mentioned semi-continuous flow magnetic bead purification system Steps of purification treatment in the device.
  • the range of biological samples in which the liquid storage device is injected into the storage device for purification treatment is 5mL-100L, preferably 100mL-50L, more preferably 200mL-10L.
  • the semi-continuous flow magnetic bead purification system of the present invention manual intervention can be reduced, the consistency of purification results can be ensured, and a shorter purification time can be achieved while achieving a better purification effect.
  • FIG. 1a shows the system assembly of the magnetic bead purification system according to the present invention.
  • Fig. 1b shows another system assembly of the magnetic bead purification system according to the present invention.
  • Fig. 2a shows a 6-channel liquid storage tank in the magnetic bead purification system according to the present invention.
  • Fig. 2b shows another 6-channel liquid storage tank in the magnetic bead purification system according to the present invention.
  • Figure 3a shows a single channel liquid storage tank in a magnetic bead purification system according to the present invention.
  • Fig. 3b shows another single-channel liquid storage tank in the magnetic bead purification system according to the present invention.
  • Figure 4 shows the magnet and actuator in the magnetic bead purification system according to the present invention.
  • Fig. 5 shows a shaking device in the magnetic bead purification system according to the present invention.
  • Figure 6a shows the support frame in the magnetic bead purification system according to the present invention.
  • Fig. 6b shows another support frame in the magnetic bead purification system according to the present invention.
  • FIG. 7 shows a pump valve control system in the magnetic bead purification system according to the present invention.
  • Fig. 8 shows a liquid circuit system in the magnetic bead purification system according to the present invention.
  • the magnetic bead purification system may include a shaking device, as well as the entire liquid storage system, a magnet, and its actuator disposed on the shaking device.
  • the magnetic bead purification system may further include a collection needle assembly, a sample collection area, a pump valve control box, a support frame module, a storage tank, a magnet, and an actuator and Shake the device.
  • the collection needle assembly is set on the upper side of the sample collection area, the collection needle assembly is connected with the pump valve control box through the pipeline, the sample collection area is placed on the upper side of the pump valve control box, and the pump valve control box is connected with the storage through the pipeline
  • the liquid tank is connected, a magnet and an actuator are arranged on the lower side of the liquid storage tank, the liquid storage tank, the magnet and the actuator are placed above the shaker, and the liquid storage tank is fixed on the support frame.
  • the volume of the biological sample is greater than the volume of the single tank of the storage tank, after the adsorption is pumped once, the remaining biological sample is pumped again after the supernatant is removed, and the protein is realized by pumping the biological sample into the storage device for multiple consecutive accumulations Or capture of other target components.
  • the magnetic beads that capture the protein are then washed, eluted, and regenerated. Purification of more biological samples is handled by pumping and adsorption of biological samples in the form of semi-continuous flow.
  • Fig. 6a shows a frame structure used in a magnetic bead purification system according to an embodiment of the present invention.
  • the frame structure is responsible for supporting the liquid storage device and the magnet, and the magnet moving device.
  • the frame structure may include components such as a fixed base plate, a fixed pressure plate for a liquid storage tank, an upper puller, a lower puller, and a side support.
  • the fixed substrate is used for mounting to the shaking device.
  • the fixed pressure plate of the liquid storage tank, the upper support plate and the lower support plate are used to fix the liquid storage tank, etc.
  • the side supports are used to support and fix these plates to the fixed base plate.
  • the magnet and its actuator can be mounted on the fixed substrate.
  • FIG. 6b shows a frame structure in another embodiment of the present invention.
  • the frame structure may include, for example, a fixed base plate, side supports, a lower support plate, a limit plate, an upper support wrench, a tube rack, and a tower Buckle, liquid storage tank fixed pressure plate.
  • the fixed substrate is used for mounting to the shaking device.
  • the fixed pressure plate, upper support plate, limit plate and lower support plate of the liquid storage tank are used to fix the liquid storage tank, and the position of the liquid storage tank is only limited to an angle to ensure that the bottom of the liquid storage tank is parallel to the magnet.
  • the side supports are used to support and fix these plates to the fixed base plate.
  • the magnet and its actuator can be mounted on the fixed substrate.
  • the beam tube rack is fixed above the upper supporting plate, and the pipeline can be reasonably arranged in the corresponding position of each liquid storage tank.
  • the tower buckle and the limit plate are fixed on the side support for limiting and fixing the liquid storage tank.
  • the fixed pressure plate of the liquid storage tank can be quickly disassembled and disassembled by the buckle design, which is convenient for taking out and placing the liquid storage tank. At the same time, after being pressed by the buckle, it can ensure that the whole device has less noise during the shaking of the shaker and the vibration of the entire system is smaller .
  • the upper pallet, limit plate, and lower pallet are fixed on the side support, while ensuring the overall tilt angle, which can provide a suitable horizontal thrust when cooperating with the shake of the shaker circle, so that the magnetic beads are shaken at a lower Under the bed speed, at the same time ensure that it is fully shaken.
  • the beam tube rack can reasonably arrange the pipeline in the corresponding position of each liquid storage tank.
  • the liquid storage tank, the magnet and its actuator are arranged on the shaking device through the frame structure.
  • the liquid storage tank is connected with the reagent bottle and the waste liquid barrel through the pipeline in the liquid circuit system.
  • the liquid storage device of the present invention can be made from a single channel to 100 channels; preferably, the number of channels is 1-50; more preferably, the number of channels is 1-20.
  • the liquid storage tank of the semi-continuous magnetic bead purification system can have a unique shape (for example, the lower part is tapered), which can more effectively exert the adsorption effect of the magnet and improve the purification efficiency.
  • the unique conical cone conforms to the shaking device for mixing and mixing, so that the magnet can be fully mixed and can be compatible with the smallest working liquid volume in the storage tank to the greatest extent.
  • each liquid storage tank is set to three ports, which are a liquid inlet port, a liquid drain port and a spare port.
  • the liquid inlet port is used to add reagents to the liquid tank and the liquid drain port It is used to discharge liquid, and the spare interface is used to replace air.
  • the annular liquid inlet pipe in the liquid storage tank shown in Figure 3a can be divided into two pipes, and the liquid is injected into the liquid storage tank at the same time, and the two solutions are sprayed onto the two surfaces where each group of magnets is located. Improve the effect of magnetic beads washing.
  • each group of magnets includes multiple magnets, the liquid inlet lines may also be provided accordingly. This improves the recovery rate of the magnetic beads.
  • the pipeline in the container can be made of stainless steel, which can be easily processed into a more suitable angle, and it can be cleaned at the same time, which improves flexibility, cleanliness and reuse rate, and reduces costs.
  • the pipeline connection at the top of each liquid storage tank can also be configured as a sample needle interface and an exhaust port, which can be used Used for inlet and outlet.
  • the sample injection needle shown in Figure 3b is a single-hole needle.
  • the sample injection needle is used for adding reagents, removing waste liquid, and blowing bubbles.
  • the sampling needle is made of non-magnetic material, and the surface is specially coated to ensure that it exhibits non-sticky characteristics to the medium in the storage tank, reducing the risk of hanging liquid, but also reducing the difficulty of cleaning and reducing the risk of cross-contamination.
  • the port of the sampling needle is designed to be appropriate in size. Under the condition of ensuring the liquid flow rate, the load pressure of the liquid circuit system is reduced to avoid the risk of excessive pressure of the liquid circuit system caused by the port being too small.
  • the storage device can use commonly used centrifuge bottles, and the tapered shape can increase the height of the liquid surface during purification and elution of magnetic beads, which can reduce the problems caused by the volume decrease from gettering to elution, and make the effective use of the magnet Big.
  • the inclined angle of the liquid storage device can ensure that the magnetic beads can fully shake the magnetic beads when the shaking device shakes, which improves the mixing effect.
  • the magnets in the magnet mechanism are grouped into two groups and are installed in a V-shape.
  • the V-shaped magnet is fixed on the drive screw.
  • the screw and the nut on the screw convert the circular motion of the screw into the linear motion of the magnet device.
  • the bearing seat is used to support the screw.
  • the limit sensor is used by the control system to detect Magnet zero position. The magnet will move to the position sensed by the limit sensor every time it is turned on. Then as the moving zero point of the magnet device.
  • each group of magnets includes two or more magnets arranged in a V-shape or other matching tank shape.
  • the drive screw is rotated to drive the connected V-shaped magnet holder to move back and forth along the direction of the linear guide, thereby driving the magnet to move back and forth, thereby moving away from or approaching the liquid storage tank.
  • the shaking device can be a digital shaking device that can be quickly started and stopped.
  • a RS232 or other communication port can be used to directly control the start and stop of the shaking device.
  • the shaking speed of the previous process can be inherited into the controller of the control system to achieve full automatic control; and can ensure that the shaking amplitude and shaking time are stable, ensuring the consistency of the results of multiple experiments.
  • the rocking device adopts a planar circumferential rocking device, and the angle of the tank body can be well controlled to achieve the mixing effect required in different steps by controlling the rotation speed of the rocking device.
  • the liquid circuit system uses a porous valve structure, which can reduce the risk of cross-contamination between reagents.
  • the pipeline adopts a pressure-resistant pipeline that does not stick to magnetic beads, which can reduce the loss of magnetic beads.
  • the pump adopts a diaphragm pump or a plunger pump with adjustable speed, which reduces the risk of magnetic bead breakage, and at the same time can adjust the liquid flow rate of the pipeline.
  • the reagent bottle can be connected to the multi-way switching valve through the corresponding pipeline, the multi-way switching valve is connected to the multi-way shunt module, and the multi-way shunt module is connected to the three-way solenoid valve And then connect the pump.
  • the front of the pump can be connected to another three-way solenoid valve, and finally to the corresponding storage tank.
  • the control system can adopt the combined control method of touch screen and controller, write the control process into the touch screen, and perform the decomposition action of the lower computer, which improves the interactive ability of the touch screen and can display more flexible processes through the interface.
  • one-touch operation can reduce the user's operating frequency. This improves the efficiency of personnel.
  • An embodiment of the present invention includes a two-way valve, a three-way valve, a quick connector, a shunt block, a bubble detector, a pump and other liquid circuit devices.
  • the quick connector is connected to the two-way valve through the pipeline.
  • the two-way valve is connected to the distribution block through the pipeline to shunt the liquid to the corresponding three-way valve.
  • the three-way valve is connected to the pump through the pipeline. Bubble detector, the outlet of the pump is finally connected to the corresponding storage tank through the pipeline.
  • the two-way valve and three-way valve are mainly used to open and close the pipeline and control the flow direction of the medium; the quick joint can be used to easily disassemble the pipeline, which is convenient for assembly and after-sales maintenance; the shunt block can distribute the same medium to the liquid storage tanks in different channels ;
  • the bubble sensor can accurately detect the presence or absence of bubbles in the pipeline, which can ensure the accuracy of liquid addition and discharge; the pump serves as the power source for the liquid circuit work, and controls the liquid system to add liquid, discharge liquid, and collect samples.
  • the semi-continuous magnetic bead purification system according to the present invention has its own control system, which can reduce manual intervention. Customized program design can ensure the consistency of purification results.
  • the optimized process flow can achieve the best purification effect and the shortest time.
  • the present invention provides a method for purifying biological samples, including injecting a biological sample containing a target component and magnetic beads capable of binding the target component into the semi-continuous-flow magnetic beads The steps of the purification process in the storage device of the purification system.
  • the range of biological samples in which the liquid storage device is injected into the storage device for purification treatment is 5mL-100L, preferably 100mL-50L, more preferably 200mL-10L.
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, it passes through the liquid circuit system. Remove the supernatant, the control system controls the magnet device, moves the magnet to a position away from the liquid storage device, pumps 20% NaOH into the liquid storage device through the liquid system, and stands still for 30 minutes, during which the control system will pass Turn on the shaking device at the set speed to ensure that the magnetic bead solution can be mixed evenly.
  • control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is removed through the fluid system.
  • the control system controls the magnet device and moves the magnet away from the liquid storage device PBS is pumped into the liquid storage device through the liquid circuit system, during which the control system will open the shaking device at a preset speed to ensure that the magnetic bead solution can be mixed evenly.
  • step b) Repeat step a) 2 to 3 times, the equilibration time is 10-15 minutes, and the equilibration solution is replaced twice during the period until the pH is the same as the equilibration solution
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is removed through the liquid system. The control system controls the magnet device and moves the magnet to a position away from the liquid storage device.
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is removed through the liquid system. The control system controls the magnet device and moves the magnet to a position away from the liquid storage device.
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is collected through the liquid circuit system.
  • the control system controls the magnet device, moves the magnet to a position away from the liquid storage device, and pumps 20% NaOH into the liquid storage device through the liquid circuit system. During this period, the control system will open the shaking device at a preset speed. Ensure that the magnetic bead solution can be mixed evenly.
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is removed through the fluid system. The control system controls the magnet device and moves the magnet to a position away from the liquid storage device.
  • the control system controls the magnet device and moves the magnet directly below the bottom of the liquid storage device. After the adsorption is completed, the supernatant is removed through the liquid system. The control system controls the magnet device and moves the magnet to a position away from the liquid storage device.
  • An exemplary application example of the semi-continuous flow magnetic bead purification system according to the present invention is as follows.
  • Experimental group pump 5ml magnetic beads into the storage tank of the semi-continuous flow magnetic bead purification system according to the present invention, equilibrate with PBS three times, then pump 200ml of cell fermentation broth into the magnetic bead mixture and shake After the device was incubated for 1 hour, it was washed three times with PBS, washed once with water, and then eluted three times with an elution buffer (buffer).
  • PBS elution buffer
  • Control group equilibrate 5ml of resin pre-packed column with AKTA, then load 200ml of cell fermentation broth, then wash 5cv with PBS, elute and collect the sample.
  • the total time test is as follows:
  • the semi-continuous flow magnetic bead purification system saves 2-5 times the time compared to the traditional resin purification method, and it is easy to increase the throughput. Taking 300ml as an example, the purification of 6 samples is completed, and the resin purification takes 20 hours, while the semi-continuous flow equipment based on magnetic bead purification can be completed in 4 hours.
  • the recovery rate, endotoxin content and purity are comparable to the traditional resin purification.

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Abstract

本发明公开了一种半连续流的磁珠纯化系统，其包括：摇动装置；框架结构，所述框架结构安装在所述摇动装置上；安装在所述框架结构上的储液装置、磁铁及磁铁执行装置；和控制系统；其中，所述储液装置通过液路系统与液体源及废液桶连接；所述控制系统与液路系统、磁铁执行装置和摇动装置连接，并控制液路系统、磁铁执行装置和摇动装置。

Description

一种半连续流的磁珠纯化系统 技术领域
本发明涉及一种半连续流的磁珠纯化系统，主要应用于生物纯化领域，例如用于蛋白和核酸的小规模和大规模提取。
背景技术
磁珠纯化法通常使用纯化系统通过磁场分离磁珠而达到分离和纯化细胞、蛋白或核酸等物质的目的。与常用的沉淀法、离心法、柱膜法相比，磁珠纯化法具有提取效率高、分离速度快、所需设备简单等特点。
与树脂纯化(Resin)相比，基于磁力吸附的磁珠纯化法依靠磁力原理和孵育的结合方式可以快速有效的富集样品中的目标蛋白，有效避免了树脂对样品预处理和对上样方式的限制等弊端，摆脱了装柱和流速等的限制。采用磁分离的纯化方法较易实现自动化，可满足快速、自动化、多通道同时处理的高通量纯化需求。
目前市面上存在的纯化装置普遍处理的通量都比较小，并且需要大量人工操作来辅助纯化流程。另外，目前操作都是敞口开放式操作，很难防止外部环境污染，也不能避免纯化过程中试剂的挥发对环境造成的伤害。现有技术的磁力架，虽可以满足部分客户需求，但仍存在诸多不足，如：(1)通量较小，不能满足大规模纯化的需求；(2)需要转移到离心管或者其它容器中，工序复杂，且有损坏样本的风险；(3)需要额外的装置才能实现自动化，增加额外的成本，且占用额外的场地；(4)形状固定，不能灵活适应容器的外形。
另外市面上采用的大规模纯化装置都是采用树脂纯化的方式，需要深层过滤，消耗大量的一次性耗材，需要多人次长时间投入。
发明内容
根据本发明的一方面，提供一种半连续流的磁珠纯化系统，其特征在于，包括：摇动装置；框架结构，所述框架结构安装在所述摇动装置上；安装在所述框架结构上的储液装置、磁铁及磁铁执行装置；和控制系统；其中，所述储液装置通过液路系统与液体源及废液桶连接；所述控制系统与液路系统、磁铁执行装置和摇动装置连接，并控制液路系统、磁铁执行装置和摇动装置。
优选的，所述控制系统控制所述液路系统实现对所述储液装置的加液和排液。
优选的，所述储液装置包括多个储液罐，当所述磁铁工作时，所述磁铁成组布置在每个储液罐底部，每组磁铁包括成V字形或其他配合罐体形状所布置的两个或多个磁铁。
优选的，所述储液罐具有锥形底部，所述储液罐中的管路分为两路以分别喷洒溶液到与一组磁铁中两个磁铁相对应的位置。
优选的，所述储液罐包括加样针接口和排气口。
优选的，所述摇动装置为数字式平面圆周摇动装置。
优选的，所述框架结构包括固定基板、储液罐固定压板、上托扳、下托扳和侧支撑，其中所述固定基板安装到所述摇动装置，所述储液罐固定压板、上托扳和下托扳用于安装所述储液罐，所述侧支撑用于将所述储液罐固定压板、上托扳和下托扳支撑固定到所述固定基板。
优选的，所述框架结构包括固定基板、侧支撑、下托板、限位板、上托扳、束管架、塔扣和储液罐固定压板，其中所述固定基板安装到所述摇动装置，所述储液罐固定压板、上托扳、限位板和下托扳用于安装所述储液罐，所述侧支撑用于将所述储液罐固定压板、上托扳、限位板和下托扳支撑固定到所述固定基板，所述束管架固定于上托板上方，所述塔扣固定于侧支撑上。
根据本发明的另一方面，提供了一种纯化生物样品的方法，包括将含有目标成分的生物样品和能够结合所述目标成分的磁珠注入到上述半连续流的磁珠纯化系统的储液装置中进行纯化处理的步骤。
优选的，其中单次注入所述储液装置进行纯化处理的生物样品范围为5mL-100L,优选为100mL-50L,更优选为200mL-10L。
根据本发明的半连续流的磁珠纯化系统，可以减少人工的干预，可以确保纯化结果的一致性，可以在达到较好的纯化效果的同时耗时较短。
附图说明
附图中示例性地示出了根据本发明的优选实施例的磁珠纯化系统及部件。各附图只是示例性说明，其比例不必要一致。
图1a示出了根据本发明的磁珠纯化系统的系统总成。
图1b示出了根据本发明的磁珠纯化系统的另一个系统总成。
图2a示出了根据本发明的磁珠纯化系统中的6通道储液罐。
图2b示出了根据本发明的磁珠纯化系统中的另一个6通道储液罐。
图3a示出了根据本发明的磁珠纯化系统中的单通道储液罐。
图3b示出了根据本发明的磁珠纯化系统中的另一个单通道储液罐。
图4示出了根据本发明的磁珠纯化系统中的磁铁和执行机构。
图5示出了根据本发明的磁珠纯化系统中的摇动装置。
图6a示出了根据本发明的磁珠纯化系统中的支撑架。
图6b示出了根据本发明的磁珠纯化系统中的另一个支撑架。
图7示出了根据本发明的磁珠纯化系统中的泵阀控制系统。
图8示出了根据本发明的磁珠纯化系统中的液路系统。
具体实施方式
为了更清楚地说明本发明实施例的技术方案，下面将对照附图说明本发明的具体实施方式。显而易见地，下面描述中的附图仅仅是本发明的一些示例性实施例。
根据本发明的一个实施例，提供一种半连续流的磁珠纯化系统。如图1a所示，根据本发明的磁珠纯化系统可以包括摇动装置，以及设置在摇动装置之上的整个储液系统、磁铁及其执行机构。
本发明的另一个实施例如图1b所示，根据本发明的磁珠纯化系统还可以包括收集针组件、样品收集区、泵阀控制箱体、支撑架模块、储液罐、磁铁及执行机构和摇动装置。其中，收集针组件设置在样品收集区上侧，收集针组件通过管路与泵阀控制箱体连接，样品收集区置于泵阀控制箱体上侧，泵阀控制箱体通过管路与储液罐连接，储液罐下侧设置磁铁及执行机构，储液罐与磁铁及执行机构均置于摇床上方，储液罐限位固定于支撑架上。
当生物样品的体积大于储液罐的单罐体积时，一次泵入吸附后，排除上清之后再次泵入剩余的生物样品，通过多次连续累积的将生物样品泵入储液装置中实现蛋白或其他目标成分的捕获。再将捕获蛋白的磁珠进行洗杂、洗脱、再生的过程。通过半连续流形式的生物样品的泵入和吸附来处理更多生物样品的纯化。
将整个储液系统置于摇动装置之上，可以提升生物样品与磁珠混合的效果，同时不需要搅拌桨。配合系统自带的加液管路，可以将挂在容器壁上的生物样品和磁珠很彻底的收集到容器底部。
图6a示出了根据本发明一个实施例的磁珠纯化系统中所使用的框架结构。框架结构负责支撑储液装置和磁铁，以及磁铁移动装置。如图所示，框架结构 例如可以包括固定基板、储液罐固定压板、上托扳、下托扳和侧支撑等部件。固定基板用于安装到摇动装置。储液罐固定压板、上托扳和下托扳用于固定储液罐等。侧支撑用于支撑固定这些板件到固定基板上。磁铁及其执行部件可以安装在固定基板上。
图6b示出了本发明的另一个实施例中的框架结构，如图所示，框架结构例如可以包括固定基板、侧支撑、下托板、限位板、上托扳、束管架、塔扣、储液罐固定压板。固定基板用于安装到摇动装置。储液罐固定压板、上托扳、限位板和下托扳用于固定储液罐，将储液罐的位置唯一的限定在一个角度上，确保储液罐底部与磁铁平行贴合。侧支撑用于支撑固定这些板件到固定基板上。磁铁及其执行部件可以安装在固定基板上。束管架固定于上托板上方，可以将管路合理的布置在各储液罐相应位置。塔扣和限位板固定于侧支撑上，用于限位固定储液罐。储液罐固定压板通过塔扣设计，可以快速拆装，便于取放储液罐，同时通过塔扣压紧后，可以确保整个装置在摇床摇晃过程中噪音更小，整个系统的震动更小。上托板、限位板、下托板固定在侧支撑上，同时保证整体的倾斜角度，可以在配合摇床圆周摇动的时候，提供合适的水平方向的推力，使得磁珠在更低的摇床转速下，同时保证充分摇匀。束管架可以将管路合理的布置在各储液罐相应位置。
储液罐、磁铁及其执行机构等装置都通过框架结构设置在摇动装置上。通过液路系统中的管路将储液罐与试剂瓶和废液桶相连。
根据本发明的优选实施例，例如参考图2a和图3a，本发明的储液装置可以做成单通道到100通道的；优选地，通道数量为1-50；更优选地，通道数量为为1-20。半连续流的磁珠纯化系统自带的储液罐可以具有独特的外形(例如下部为锥形)，更加有效的发挥磁铁的吸附效果，提升纯化的效率。特有的符合摇动装置摇晃混匀的锥形，实现磁铁充分混匀的同时可以最大程度的兼容储液罐中的最小的工作液体体积。2a和图3a所示的每个储液罐顶部管路接口设置为3个接口，分别为进液接口、排液接口及备用接口，进液接口用于向储液罐添加试剂，排液接口用于排出液体，备用接口用于置换空气。图3a所示的储液罐中的环形进液管可以采用一分为二的管路，同时向储液罐中注液，并将两股溶液喷洒到每组磁铁所在的两个面，可以提升磁珠冲洗的效果。在每组磁铁包括多个磁铁的实施例中，进液管路也可以相应地设置。这提高了磁珠的回收率。容器内管路可以采用不锈钢材质，容易加工成比较合适的角度，同时可以进行清洗，提升了灵活度、洁净度和重复利用率，并降低了成本。
根据本发明的另一优选实施例，如图2b和图3b所示，每个储液罐顶部管路接口也可以设置为一个加样针接口和一个排气口，该加样针接口可以用于进液和排液。图3b所示加样针为单孔针，加样针用于添加试剂、排除废液和吹打气泡。加样针选用无磁材料，表面进行特殊镀层处理，确保其对储液罐中的介质展现出不粘的特性，减少挂液的风险，同时也降低了清洗的难度，减少交叉污染的风险。加样针的端口设计成合适大小，在保障液体流速的情况下，降低液路系统的负载压力，避免端口过小造成液路系统压力过大的风险。
储液装置可以采用常用的离心瓶，锥形的外形可以提升磁珠纯化洗脱时液面的高度，可以降低从吸杂到洗脱的体积下降带来的问题，使磁铁的有效利用面积更大。储液装置倾斜的角度可以保证磁珠在摇动装置摇晃的时候能够充分的将磁珠摇晃起来，提升了混匀的效果。
根据本发明的优选实施例，例如参考图4，磁铁机构中的磁铁每两块组成一组，呈V字形安装。这样在磁力线从一个磁铁进入到另一个磁铁的时候可以提升磁铁吸附磁珠的效果。V型磁铁固定在传动丝杠上面，通过丝杠和丝杠上的螺母将丝杠的圆周运动转化成磁铁装置的直线运动，轴承座用来支撑丝杠，限位传感器由控制系统用来检测磁铁零点位置。每次开机磁铁会移动到限位传感器所感应到的位置。然后作为磁铁装置的移动零点。根据其他实施例，每组磁铁包括成V字形或其他配合罐体形状所布置的两个或多个磁铁。通过控制执行电机的旋转，带动传动丝杠旋转来驱动连在一起的V型磁铁架沿着直线导轨的方向来回运动，从而带动磁铁来回运动，由此远离或者接近储液罐。
如图5所示，摇动装置可以采用能够快速启动和停止的数字式摇动装置，例如可以用RS232等通讯口直接控制摇动装置的启动和停止。这样就可以将前期工艺摸索的摇晃转速继承到控制系统的控制器中，实现全程自动控制；并且可以确保摇晃幅度和摇晃时间是稳定的，保证了多次实验结果的一致性。摇动装置采用平面圆周摇动装置，配合罐体的角度可以很好地通过控制摇动装置的转速来实现不同步骤中所需要的混合效果。
液路系统采用多孔阀结构，可以降低试剂之间交叉污染的风险。管路采用不沾磁珠的耐压管路，可以降低磁珠的损失。泵采用可以调速的隔膜泵或者柱塞泵，降低了磁珠破碎的风险，同时可以调整管路的液体流速。
在如图7所示的泵阀控制系统中，通过相应的管路，试剂瓶可以连接到多通切换阀，多通切换阀连接到多通分流模块，多通分流模块连接到三通电磁阀，然后连接泵。泵前端可以再连接另外的三通电磁阀，最后到相应的储液罐中。
控制系统可以采用触摸屏和控制器的组合控制方式，将控制工艺写入触摸屏中，由下位机分解动作执行，提升了触摸屏的交互能力，可以将更灵活的工艺通过界面的形式展现出来。同时一键式操作可以降低了用户的操作频率。这提升了人员的工作效率。
本发明的一个实施例，如图8所示的液路系统中，包括两通阀、三通阀、快速接头、分流块、气泡检测器、泵等液路器件。快速接头通过管路与两通阀连接，两通阀通过管路与分流块连接，将液体分流至相应三通阀，三通阀通过管路与泵进行连接，在此段管路中安装有气泡检测器，泵的出口通过管路最终连接到相应的储液罐中。两通阀、三通阀主要用来开闭管路、控制介质流向；采用快速接头可以轻松拆卸管路，便于装配及售后维护；分流块可以将同种介质分配到不同通道的储液罐中；气泡传感器能够精确检测管路中有无气泡，可以确保加液，排液精准性；泵作为液路工作的动力源，控制液路系统的加液、排液、收集样品。
根据本发明的半连续流的磁珠纯化系统自带控制系统，可以减少人工的干预。定制化的程序设计，可以确保纯化结果的一致性。优化的工艺流程，可以在达到最好的纯化效果的同时耗时最短。
基于上述半连续流的磁珠纯化系统，本发明提供了一种纯化生物样品的方法，包括将含有目标成分的生物样品和能够结合所述目标成分的磁珠注入到上述半连续流的磁珠纯化系统的储液装置中进行纯化处理的步骤。
优选的，其中单次注入所述储液装置进行纯化处理的生物样品范围为5mL-100L,优选为100mL-50L,更优选为200mL-10L。
根据本发明的半连续流的磁珠纯化系统的示例性操作如下所述。
1、磁珠孵育
a)将20％乙醇保存的磁珠溶液混匀，通过液路系统泵入储液装置中，控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，通过液路系统将20％NaOH泵入储液装置中，静置30分钟，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠溶液能够混合均匀。
2、磁珠平衡
a)30分钟以后，控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，通过液路系统将PBS泵入储液装置中，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠 溶液能够混合均匀。
b)重复步骤a)2～3次，平衡时间10-15分钟，期间更换2次平衡液，至pH与平衡液相同
3、洗杂
a)控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，
b)通过液路系统将PBS泵入储液装置中，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠溶液能够混合均匀。
c)重复步骤a)～b)2～3次
4、洗脱
a)控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，
b)通过液路系统将洗脱液泵入储液装置中，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠溶液能够混合均匀。
c)控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清收集。
d)重复步骤a)～c)2次
5、磁珠再生
a)控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，通过液路系统将20％NaOH泵入储液装置中，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠溶液能够混合均匀。
b)控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，
c)通过液路系统将20％NaOH泵入储液装置中，静置30分钟，在此期间控制系统会通过预先设定的速度打开摇动装置，保证磁珠溶液能够混合均匀。
d)控制系统控制磁铁装置，将磁铁移动到储液装置底部正下方，吸附完成之后，通过液路系统将上清排除，控制系统控制磁铁装置，将磁铁移动到储液装置远离的位置，
e)通过液路系统将3倍磁珠体积的20％乙醇泵入储液装置中，
f)通过液路系统将磁珠混合溶液泵入到磁珠收集罐中。
根据本发明的半连续流的磁珠纯化系统的示例性应用实例如下所述。
实验组：将5ml磁珠泵入到根据本发明半连续流的磁珠纯化系统的储液罐中，用PBS平衡3遍后，将200ml的细胞发酵液泵入到磁珠混合液中，摇动装置孵育1个小时后，用PBS清洗3次，水洗一遍，之后用洗脱缓冲剂(buffer)洗脱3次。
对照组：用AKTA平衡5ml的树脂(resin)预装柱，之后上样结合200ml细胞发酵液，之后用PBS洗杂5cv，洗脱收集样品。
总时间测试如下：
Figure PCTCN2019113258-appb-000001
使用根据本发明的半连续流的磁珠纯化系统，相比于传统的树脂纯化方式，节省2-5倍的时间，同时易于提升通量。以300ml为例，完成6个样品的纯化，树脂纯化需要20个小时，而基于磁珠纯化的半连续流设备可以在4小时内完成。
将上述实验，洗脱后的样品收集进行浓度，纯度和内毒素的测试，测试结果如下表：
Figure PCTCN2019113258-appb-000002
基于半连续的磁珠纯化方式，在回收率，内毒素含量和纯度上与传统的树脂纯化相当。
应当说明的是，上述实施例均可根据需要自由组合。以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干变化和改进，这些变化和改进也应视为落入本发明的保护范围。

Claims (10)

  1. 一种半连续流的磁珠纯化系统，其特征在于，包括：
    摇动装置；
    框架结构，所述框架结构安装在所述摇动装置上；
    安装在所述框架结构上的储液装置、磁铁及磁铁执行装置；和
    控制系统；
    其中，所述储液装置通过液路系统与液体源及废液桶连接；所述控制系统与液路系统、磁铁执行装置和摇动装置连接，并控制液路系统、磁铁执行装置和摇动装置。
  2. 根据权利要求1所述的半连续流的磁珠纯化系统，其特征在于，所述控制系统控制所述液路系统实现对所述储液装置的加液和排液。
  3. 根据权利要求1或2所述的半连续流的磁珠纯化系统，其特征在于，所述储液装置包括多个储液罐，当所述磁铁工作时，所述磁铁成组布置在每个储液罐底部，每组磁铁包括成V字形或其他配合罐体形状所布置的两个或多个磁铁。
  4. 根据权利要求3所述的半连续流的磁珠纯化系统，其特征在于，所述储液罐具有锥形底部，所述储液罐中的管路分为两路以分别喷洒溶液到与一组磁铁中两个磁铁相对应的位置。
  5. 根据权利要求3所述的半连续流的磁珠纯化系统，其特征在于，所述储液罐包括加样针接口和排气口。
  6. 根据权利要求1或2所述的半连续流的磁珠纯化系统，其特征在于，所述摇动装置为数字式平面圆周摇动装置。
  7. 根据权利要求1或2所述的半连续流的磁珠纯化系统，其特征在于，所述框架结构包括固定基板、储液罐固定压板、上托扳、下托扳和侧支撑，其中所述固定基板安装到所述摇动装置，所述储液罐固定压板、上托扳和下托扳用 于安装所述储液罐，所述侧支撑用于将所述储液罐固定压板、上托扳和下托扳支撑固定到所述固定基板。
  8. 根据权利要求1或2所述的半连续流的磁珠纯化系统，其特征在于，所述框架结构包括固定基板、侧支撑、下托板、限位板、上托扳、束管架、塔扣和储液罐固定压板，其中所述固定基板安装到所述摇动装置，所述储液罐固定压板、上托扳、限位板和下托扳用于安装所述储液罐，所述侧支撑用于将所述储液罐固定压板、上托扳、限位板和下托扳支撑固定到所述固定基板，所述束管架固定于上托板上方，所述塔扣固定于侧支撑上。
  9. 一种纯化生物样品的方法，包括将含有目标成分的生物样品和能够结合所述目标成分的磁珠注入到根据权利要求1至8中任一项所述的半连续流的磁珠纯化系统的储液装置中进行纯化处理的步骤。
  10. 根据权利要求9所述的方法，其中单次注入所述储液装置进行纯化处理的生物样品范围为5mL-100L,优选为100mL-50L,更优选为200mL-10L。
PCT/CN2019/113258 2018-10-26 2019-10-25 一种半连续流的磁珠纯化系统 WO2020083372A1 (zh)

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