WO2023134773A1 - 纯化方法 - Google Patents

纯化方法 Download PDF

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Publication number
WO2023134773A1
WO2023134773A1 PCT/CN2023/072567 CN2023072567W WO2023134773A1 WO 2023134773 A1 WO2023134773 A1 WO 2023134773A1 CN 2023072567 W CN2023072567 W CN 2023072567W WO 2023134773 A1 WO2023134773 A1 WO 2023134773A1
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WO
WIPO (PCT)
Prior art keywords
liquid
magnetic
magnetic beads
purification
separation container
Prior art date
Application number
PCT/CN2023/072567
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English (en)
French (fr)
Inventor
刘显昱
陈春花
廖元明
白涛
张昊
钱红
贺瑞娜
孟凯特
Original Assignee
南京金斯瑞生物科技有限公司
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Application filed by 南京金斯瑞生物科技有限公司 filed Critical 南京金斯瑞生物科技有限公司
Publication of WO2023134773A1 publication Critical patent/WO2023134773A1/zh

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Classifications

    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Definitions

  • This description relates to the technical field of biomacromolecule purification, in particular to a purification method.
  • the purification method of biomacromolecules may include chromatography column purification and magnetic bead purification.
  • the chromatographic column purification method may include: using a centrifuge to centrifuge and filter the culture solution, and using a disposable sterile filter to vacuum filter the culture solution; The upper opening of the column is added into the chromatographic column, so that the culture solution flows to the lower opening of the chromatographic column.
  • the purified target in the culture medium can be mixed with the filler and remain in the chromatographic column; the washing liquid is added into the chromatographic column from the upper opening of the chromatographic column, and the uncombined
  • the impurities mixed with the filler can be mixed with the eluent and discharged from the lower opening of the chromatography column; the eluent is added to the chromatography column from the upper opening of the chromatography column, and the eluent can separate the purification target from the filler, making the purification
  • the target substance is mixed in the eluent, which flows out through the lower opening of the chromatographic column, and the purified target substance can be obtained by collecting the eluent.
  • the magnetic bead purification method may include: incubating the cell disruption solution and magnetic beads in a centrifuge tube, then placing the centrifuge tube in a magnetic separator, and removing the supernatant with a pipette after the solution becomes clear liquid. Then add washing buffer solution to the centrifuge tube, pipette the tip repeatedly for 5-10 times, then place the centrifuge tube on the magnetic separator, and suck up the supernatant with a pipette after the solution becomes clear. Repeat the above steps 2 times to complete the washing.
  • a purification method is provided, aiming at reducing the purification cost, improving the purification speed and the purification effect.
  • One of the specifications of this specification is to provide a purification method, the purification method comprising: magnetic beads and purification Mix the standard; transfer the magnetic beads and the purification target to a separation container; separate the magnetic beads from impurities; pass the magnetic beads and the purification target through the filter in the separation container separation, the filter element is provided with filter holes, and the diameter of the filter holes is smaller than the diameter of the magnetic beads.
  • both ends of the separation container are provided with openings.
  • the transferring the magnetic beads and the purification target into the separation container includes: adsorbing the magnetic beads by a magnetic element; moving the magnetic element to the upper opening of the separation container; The magnetic beads are separated from the magnetic member so that the magnetic beads enter the separation container.
  • the separating the magnetic beads from the magnetic bar so that the magnetic beads enter the separation container includes: separating the magnetic beads from the magnetic member by washing.
  • the magnetic member includes a magnetic bar
  • the separating the magnetic beads from the magnetic member to allow the magnetic beads to enter the separation container further includes: reducing the magnetic bar and the magnetic bar. The adsorption force between the magnetic beads is such that the magnetic beads are separated from the magnetic element under the action of gravity.
  • the magnetic rod includes an electromagnetic rod.
  • the electromagnetic rod When the electromagnetic rod is in the energized state, the electromagnetic rod has magnetism, and when the electromagnetic rod is in the de-energized state, the electromagnetic rod loses its magnetism;
  • the reducing the adsorption force between the magnetic rod and the magnetic bead includes: adjusting the magnetic rod to the power-off state.
  • the magnetic member further includes a magnetic bar sleeve sheathed outside the magnetic bar, and the magnetic bar can move relative to the magnetic bar sleeve along the sheathing direction; the reduction of the The adsorption force between the magnetic rod and the magnetic beads includes: moving the magnetic rod relative to the magnetic rod casing along the sheathing direction so that the magnetic rod is away from all the particles enriched on the surface of the magnetic rod casing. The magnetic beads.
  • the separating the magnetic beads from impurities includes: washing with a first liquid to separate the magnetic beads from the impurities.
  • the separating the magnetic beads from the purification target through the filter in the separation container includes: adding a second liquid to the separation container to wash all the particles on the filter.
  • the magnetic beads are used to separate the magnetic beads from the purification target; the second liquid filtered by the filter is collected.
  • adding a second liquid to the separation container to wash the magnetic beads on the filter element to separate the magnetic beads from the purification target comprises: detecting the separation Whether the liquid level position in the container reaches the set first liquid level threshold; if the liquid level position reaches the set first liquid level threshold, then reduce the liquid intake of the second liquid or increase the The output volume of the second liquid.
  • the method further includes: detecting whether the liquid level in the separation container reaches a set second liquid level threshold, the first liquid level threshold is greater than the second liquid level threshold; if The liquid level has not reached When the set threshold value of the second liquid level is reached, the liquid addition amount of the second liquid is increased or the liquid discharge amount of the second liquid is decreased.
  • before adding the second liquid into the separation container to wash the magnetic beads on the filter to separate the magnetic beads from the purification target it includes: detecting The volume of the first liquid added to the separation container; when the volume of the first liquid reaches a set volume threshold, stop adding the first liquid.
  • the second liquid into the separation container before adding the second liquid into the separation container to wash the magnetic beads on the filter element to separate the magnetic beads from the purification target, it further includes: Detecting the impurity content in the first liquid from the separation container; when the impurity content in the first liquid reaches a first content threshold and the detection time threshold remains unchanged, stop adding the first liquid.
  • the collecting the second liquid filtered by the filter element includes: detecting whether the content of the purification target substance in the second liquid from the separation container reaches a second content threshold; If the content of the purified target substance does not reach the second content threshold, the second liquid is collected into a waste liquid temporary storage container; if the content of the purified target substance reaches the second content threshold, the second liquid is collected Collect into collection container.
  • the collection container includes a plurality; the collecting the second liquid into the collection container includes: detecting whether the current liquid level position of the collection container reaches a third liquid level threshold; When the liquid level position of the collection container reaches the third liquid level threshold, the second liquid is collected into the other collection containers.
  • the collection of the second liquid is stopped.
  • the collecting the second liquid into the collection container further includes: adding a third liquid into the collection container, the third liquid is used to neutralize the acidic environment in the collection container .
  • Figure 1 is an exemplary flow chart of a purification method according to some embodiments of the present specification
  • Fig. 2 is the ultraviolet light absorption peak diagram of antibody elution shown in the embodiment according to the specification;
  • Fig. 3 is an antibody electrophoresis diagram shown according to some embodiments of the specification.
  • Fig. 4 is a schematic structural diagram of a purification device according to some embodiments of the specification.
  • Fig. 5 is an exemplary flowchart of purification based on purification equipment according to some embodiments of the present specification.
  • purification refers to separating the purified target substance (eg, target antibody) in a crude sample (eg, culture fluid) from other impurities to achieve purification.
  • the purification target may include biomacromolecules (such as proteins, antibodies, and nucleic acids, etc.).
  • the purification method uses magnetic beads to mix with the purification target in the culture medium, and the magnetic beads mixed with the purification target will be transferred to a separation container.
  • the magnetic beads Since the magnetic beads cannot pass through the filter holes of the filter element arranged in the separation container, they will be trapped in the separation container. Then wash the magnetic beads by adding washing liquid to the separation container to remove impurities on the magnetic beads (i.e. substances other than the purified target), and the removed impurities can be mixed in the washing liquid and pass through the filter
  • the filter holes are discharged from the separation container. Then add an eluent to the separation container to wash the magnetic beads to remove the purification target on the magnetic beads, and the removed purification target can be mixed in the eluent and discharged from the separation container through the filter holes of the filter element. Finally, the discharged eluate is collected and processed to obtain the purified target substance.
  • magnetic beads can refer to the Combined magnetically responsive nanoparticles.
  • the magnetic beads can be coated with silicon base, PS base (ie polystyrene polymer material) or agarose.
  • the material of the magnetic core of the magnetic bead may be Fe 3 O 4 .
  • the diameter of the magnetic beads may range from 1 nm to 500 um. In some embodiments, the diameter of the magnetic beads may range from 3nm to 400um. In some embodiments, the diameter of the magnetic beads may range from 5nm to 300um. In some embodiments, the diameter of the magnetic beads may range from 50 nm to 200 um.
  • functional groups can be bound to the magnetic beads, and the functional groups can be combined with specific biomacromolecules. Therefore, various applications can be performed according to the combination of different functional groups. For example, using magnetic beads to combine with cells, so as to achieve the purpose of extracting specific cells. Another example is the use of magnetic beads to combine with proteins and antibodies to achieve protein and antibody extraction.
  • the functional group may include but not limited to protein A, protein G, Ni, GSH, oligo probe, streptavidin, antibody, etc. single or multiple, or a sandwich structure.
  • mixing may refer to mixing the purified target substance in the culture medium with the filler.
  • the two are mixed by physical means (for example, allowing the target object of purification to enter the three-dimensional network pores of the filler and retain it).
  • the two are combined through chemical bonds or through ion exchange, electrostatic adsorption, and the like.
  • the cleaning solution may include Na 2 HPO 4 , KH 2 PO 4 , NaCl, KCl, and the like.
  • the washing liquid may be different according to the type of the purification target, for example, when the purification target is nucleic acid, the washing liquid may include tris(hydroxymethyl)methane, mannitol, etc.
  • the eluent may include pure water, Tris-HCl, glycine, citrate, or sodium acetate.
  • the eluent can separate the purification target from the active groups of the magnetic beads, thereby separating the purification target from the magnetic beads.
  • the eluent can be different according to the type of the purification target, for example, when the purification target is nucleic acid, the eluent can also include pure water or Tris-HCl.
  • FIG. 1 is an exemplary flowchart of a purification method according to some embodiments of the present specification. As shown in Figure 1, the purification method 100 comprises the following steps:
  • Step 110 mixing the magnetic beads with the purified target.
  • the target compound before purifying the target compound, the target compound may be preliminarily separated from the culture medium.
  • magnetic beads can be used to mix with the purification target, so as to facilitate the transfer of the purification target.
  • the mixing of the magnetic beads and the purification target may include binding the purification target to the functional groups on the magnetic beads.
  • the magnetic beads and the culture medium can be put into a specific container, and then the specific container can be mixed uniformly, so that the magnetic beads can be mixed with the purified target substance in the culture medium.
  • a specific container for containing magnetic beads and culture solution may be called a mixing container.
  • the mixing operation can refer to the mixing container such as shaking, rotating, The act of shaking, etc., to mix or combine the substances contained in the mixing vessel.
  • the mixing container can be manually mixed by rotating, shaking, or the like.
  • the mixing container can be mixed by a mixing component.
  • operations such as vibration, rotation, shaking, etc. are performed on the mixing container by the mixing component.
  • the mixing container can be mixed using a shaking platform.
  • the shaking platform can be used to place mixing containers.
  • the vibration platform can move in the form of reciprocating swing, rotation, etc., and drives the mixing container placed on the vibration platform to move.
  • the magnetic beads and the culture medium can be directly mixed manually.
  • Step 120 transferring the magnetic beads and the purification target to a separation container.
  • the magnetic beads after the magnetic beads are mixed with the purification target, the magnetic beads can be transferred to a separation vessel.
  • both ends of the separation container are provided with openings, including an upper opening and a lower opening.
  • the filter element in the separation container is used to filter substances that enter from a certain opening (for example, an upper opening) of the separation container and are discharged from another opening.
  • the filter element is plate-shaped.
  • the thickness direction of the plate filter element is the same or approximately the same as the height direction of the separation container.
  • the cross-sectional shape of the plate-shaped filter element along its thickness direction is the same or approximately the same as the cross-sectional shape of the internal cavity of the separation container along its height direction, and the size is similar, so that the material to be filtered cannot be separated from the filter element and the separation container. gaps between the inner walls.
  • the thickness direction of the filter element may be the same or approximately the same as the thickness direction of the separation container.
  • the magnetic beads can enter the separation container from the upper opening of the separation container. Since the diameter of the filter holes of the filter element is smaller than the diameter of the magnetic beads, the magnetic beads will be trapped in the separation container by the filter element.
  • the pore diameter of the filter hole is larger than the diameter of the purification target (for example, a biomacromolecule) and impurities (for example, a biomacromolecule that does not belong to the purification target and other substances except the biomacromolecule), so that After being separated and mixed with magnetic beads as impurities and as purification targets, they can be discharged through the filter hole and the lower opening of the separation container.
  • the washing solution mixed with impurities can flow out from the lower opening of the separation container through the filter hole, while the magnetic beads will continue to accumulate on the filter element and be washed.
  • the filter element may be a frit.
  • the sieve plate can be detached from the separation container so that the sieve plate can be cleaned or replaced.
  • transferring the magnetic beads and the purification target to the separation container may include: adsorbing the magnetic beads through a magnetic piece; moving the magnetic piece to the upper opening of the separation container; separating the magnetic beads from the magnetic piece to make the magnetic The beads enter the separation vessel.
  • the magnetic piece is magnetic, and when the magnetic piece extends into the culture solution, the The magnetic beads can be enriched on the surface of the submerged part of the magnetic part under the action of magnetic force. At this time, the magnetic piece is taken out from the liquid. Since the magnetic force between the magnetic piece and the magnetic beads overcomes the gravitational effect of the magnetic beads, the magnetic beads will move with the magnetic piece to separate from the culture medium. The magnetic beads are mixed, so the purified target is separated from the culture medium.
  • the magnets may include bar magnets.
  • the magnetic rod is magnetic and can absorb the magnetic beads in the culture medium.
  • the magnetic member may include a magnetic rod and a magnetic rod casing sheathed on the magnetic rod, and the magnetic rod may move relative to the magnetic rod casing along the sheathing direction.
  • the outer cover of the magnetic rod may refer to a shell structure sheathed outside the magnetic rod.
  • one end of the magnetic rod casing has an opening, and the other end is closed.
  • the magnetic rod can be inserted into the magnetic rod casing from the open end of the magnetic rod casing and move along the sheathing direction.
  • the sleeve direction may be the length direction of the magnetic bar.
  • the nesting direction may be the same or approximately the same as the height direction of the mixing container.
  • the magnetic rod when the magnetic rod is matched with the magnetic rod jacket (that is, the magnetic rod jacket is sleeved outside the magnetic rod) and extends below the liquid level of the mixing container, the adsorption force provided by the magnetic rod will make the magnetic beads rich.
  • the magnetic rod jacket can protect the magnetic rod to avoid corrosion of the magnetic rod caused by direct contact with the culture medium.
  • the magnetic rod casing since the magnetic rod casing can move relative to the magnetic rod along the sheathing direction, the magnetic rod casing can be separated from the magnetic rod for easy cleaning or replacement.
  • the magnetic bead can be separated from the magnetic rod housing by utilizing the feature that the magnetic rod housing can move relative to the magnetic rod. This is described in more detail in other embodiments of this specification, and will not be repeated here. .
  • the magnetic parts when the magnetic beads are adsorbed and transferred by the magnetic parts, the magnetic parts can be manually operated to move.
  • the magnetic part including the magnetic bar and the magnetic bar casing is taken as an example for illustration.
  • the operator can control the movement of the magnetic rod and the magnetic rod cover by holding the magnetic rod and the magnetic rod cover.
  • the movement of the magnetic member may be controlled by other means, devices or components (eg, a control device).
  • the control device can control the rod magnet to move along the first track, control the rod magnet cover to move along the second track, and control the rod magnet and the rod cover to move along the third track simultaneously. More details about the control device can be found in other embodiments of this specification.
  • the extension direction of the first track and the second track may be the same or approximately the same as the height direction of the mixing container.
  • the extension direction of the third track may be the same or approximately the same as the direction from the upper opening of the mixing container to the upper opening of the separation container.
  • the magnetic member does not have to move along a specific track. For example, the movement of the magnetic rod and the magnetic rod casing can be controlled by the mechanical arm, and there is no need to rely on the track.
  • the magnetic beads enriched on the magnetic piece can be separated from the magnetic piece by means of external force and/or reducing the adsorption force of the magnetic piece to the magnetic beads. Separation, so that the magnetic beads enter the separation container from the upper opening of the separation container.
  • the magnetic beads can be directly actuated to separate the magnetic beads from the magnetic member.
  • the toggle lever toggles the magnetic beads.
  • the toggle lever can be manually controlled, for example, the operator holds the toggle lever to toggle.
  • the toggle lever may be controlled by other devices, devices or components (eg, a robotic arm).
  • separating the magnetic beads from the magnetic member so that the magnetic beads enter the separation container may include: separating the magnetic beads from the magnetic member by washing. For example, if the magnetic beads are washed with flowing liquid, the liquid will generate an impact force on the magnetic beads, and the impact force may cause the magnetic beads to fall off the magnetic member.
  • the magnetic beads may be washed with a first liquid, wherein the first liquid may be the washing liquid in the foregoing embodiments.
  • washing the magnetic beads with the washing solution can not only wash the magnetic beads from the magnetic parts, but also remove the unbonded magnetic beads attached to the surface of the magnetic beads or between adjacent magnetic beads. The mixed impurities are separated from the magnetic beads, effectively improving the purification efficiency.
  • the magnetic beads can be washed manually.
  • the operator manually controls the spray pipe to spray a specific liquid to wash the magnetic beads, or holds a container containing the first liquid and pours out the first liquid to wash the magnetic beads.
  • the magnetic beads can be detached from the magnetic member by controlling the washing component to wash the magnetic beads.
  • the rinse assembly may include a rinse spray head disposed above the separation vessel.
  • the flushing nozzle can communicate with an external pump valve assembly, and the flushing nozzle can be controlled to spray a specific liquid by controlling the pump valve assembly.
  • a plurality of flushing nozzles can be provided to improve flushing efficiency.
  • apart from washing to separate the magnetic beads from the magnetic element it can also be achieved by utilizing the characteristics of the magnetic element itself.
  • the magnetic beads can be separated from the magnetic parts under the action of gravity by reducing the adsorption force between the magnetic parts (for example, magnetic rods) and the magnetic beads.
  • the magnetic rod may comprise an electromagnetic rod.
  • the electromagnetic rod When the electromagnetic rod is in the energized state, the electromagnetic rod is magnetic; when the electromagnetic rod is in the de-energized state, the electromagnetic rod loses its magnetism.
  • the adsorption force between the electromagnetic rod and the magnetic bead can be reduced by adjusting the energized state of the electromagnetic rod, so that the magnetic bead is separated from the electromagnetic rod.
  • the electromagnetic rod when it is necessary to use the electromagnetic rod to absorb the magnetic beads, the electromagnetic rod can be adjusted to the energized state, and the electromagnetic rod is magnetic when it is in the energized state, so the magnetic beads can be adsorbed.
  • the magnetic rod when it is necessary to separate the magnetic beads from the magnetic rod, the magnetic rod can be adjusted to a power-off state.
  • the magnetism disappears, so the magnetic force between the magnetic rod and the magnetic bead also changes accordingly.
  • the magnetic beads can be separated from the electromagnetic rod under the action of gravity.
  • the relative movement of the magnetic rod and the magnetic rod sheath can be used to change the magnetic rod and the concentration of the magnetic rod enriched on the surface of the magnetic rod sheath.
  • the distance between the magnetic beads thereby changing the strength of the magnetic force between the two.
  • the magnetic rod cover with magnetic beads and the magnetic rod embedded in the magnetic rod cover move to the upper opening of the separation container, the magnetic rod can be moved relative to the magnetic rod cover along the sleeve direction (for example, slide along the first track) to keep the magnetic bar away from the magnetic beads enriched on the outer surface of the magnetic bar.
  • Step 130 separating the magnetic beads from impurities.
  • the impurities in the culture solution ie, substances other than the purified target substance
  • the magnetic beads can be washed by the first liquid to separate impurities from the magnetic beads.
  • the first liquid may include a washing liquid. For more details about the washing liquid, please refer to the description of other embodiments in this specification.
  • washing the magnetic beads with the first liquid to separate the impurities from the magnetic beads may include: (1) washing the magnetic beads with the first liquid before the magnetic beads are separated from the magnetic parts, so as to remove Beads are rinsed off the magnetic piece. In the process of separating the magnetic beads from the magnetic parts, the impurities combined with the magnetic beads will also be washed and removed; (2) After the magnetic beads are separated from the magnetic parts and enter the separation container, since the diameter of the filter hole of the filter part is smaller than The diameter of the magnetic beads, so the magnetic beads cannot pass through the filter pores and will be trapped by the filter in the separation vessel. At this time, the first liquid can be used to wash the magnetic beads accumulated on the filter element, so as to further remove the impurities attached to the magnetic beads.
  • the removed impurities may be mixed in the first liquid. Since the pore diameter of the filter hole of the filter element is larger than the diameter of the impurities, the first liquid and the impurities can pass through the filter holes and be discharged through the lower opening of the separation container. The magnetic beads will be trapped in the cavity of the separation container by the filter element, so as to realize the separation of impurities and magnetic beads.
  • flushing may be performed manually.
  • the operator manually controls the liquid spray pipe to spray the first liquid to wash the magnetic beads.
  • the washing component can be controlled to spray out the first liquid to wash the magnetic beads.
  • Step 140 separating the magnetic beads from the purification target through the filter element in the separation container, the filter element is provided with filter holes, and the diameter of the filter holes is smaller than the diameter of the magnetic beads.
  • the purified target on the magnetic beads can be detached from the magnetic beads.
  • a second liquid can be added to the separation container to wash the magnetic beads accumulated on the filter element.
  • the second liquid may be an eluent, including but not limited to glycine, citrate, or sodium acetate.
  • the purified object can be mixed in the second liquid after being separated from the magnetic beads.
  • the second liquid and the purification target can be discharged through the filter hole and through the lower opening of the separation container, and the magnetic beads will be trapped in the cavity of the separation container by the filter element.
  • the second liquid filtered by the filter element and mixed with the target substance for purification can be collected.
  • the second liquid in the separation container can be drained through a drain line communicating with the opening below it.
  • the second liquid can be collected by a collection assembly.
  • the collection assembly may include a liquid filling port and a collection container for collecting the second liquid meeting the collection requirements.
  • the liquid filling port can communicate with the lower opening of the separation container through the liquid discharge pipe, and discharge the second liquid into the collection container.
  • the collection requirement mentioned here may include whether the content of the purification target in the second liquid reaches a set value (for example, the second content threshold). Regarding this, more detailed descriptions can be found in other embodiments of this specification.
  • the first liquid mixed with impurities discharged during the cleaning process can also be collected.
  • the collection assembly may also include a waste liquid temporary storage container.
  • the waste liquid temporary storage container may be used to collect the first liquid discharged through the filter hole through the lower opening of the separation container during the cleaning process.
  • the lower opening of the separation container communicates with the liquid filling port, and the first liquid discharged from the liquid filling port can be discharged into the waste liquid temporary storage container.
  • the waste liquid temporary storage container can also be used to collect the second liquid that does not meet the collection requirements during the elution treatment.
  • a third liquid may be added to the collection container containing the second liquid.
  • the third liquid may include a neutralizing liquid.
  • the neutralizing solution may include a buffer with a higher pH.
  • the neutralizing solution can be used to neutralize the acidic environment in the eluate in the collection container to neutral pH, avoiding the long-term acidity of the antibody in the collection container The environment is damaged.
  • the purification methods or steps in one or more embodiments of the present specification can be performed manually.
  • the operator can manually control (that is, hold) the magnetic member to extend into the mixing container to absorb the magnetic beads.
  • the operator can manually control the liquid filling port to move to the upper opening of the collection container or the waste liquid temporary storage container.
  • the purification methods or steps in one or more embodiments of this specification can be controlled by specific devices, components or equipment.
  • the above methods or steps can be executed by a control device.
  • the control device may include a control unit and a drive unit.
  • the control unit may be used to receive work instructions, and the work instructions may be input by an operator to instruct the control device to drive one or more components or parts in the foregoing embodiments to perform corresponding functions in corresponding steps.
  • the control unit can generate corresponding instructions according to the work instructions, and send the instructions to the drive unit.
  • the driving unit may be drivingly connected to one or more components or components in the foregoing embodiments.
  • the drive unit can receive instructions from the control unit, and drive corresponding components or components to perform specific functions according to the instructions.
  • the control unit may generate the first instruction according to the work instruction and send it to the drive unit.
  • the first command corresponds to the vibration platform, and can instruct the vibration platform to rotate and vibrate the mixing container according to the set speed within the set working time.
  • drive unit receiving After the first instruction the shock platform can be driven to work.
  • the control unit may generate a second instruction according to the work instruction and send it to the driving unit.
  • the third instruction corresponds to the flushing assembly, and may instruct the flushing nozzle of the flushing assembly to spray a specific liquid at a certain rate.
  • the drive unit After the drive unit receives the second instruction, it can control the flushing nozzle to work according to the instruction of the second instruction.
  • the driving unit can drive the pump valve assembly communicated with the flushing nozzle to work, and the first liquid is sprayed out through the flushing nozzle to flush the magnetic beads.
  • the control device can be controlled by a host computer.
  • the upper computer may refer to a computer that issues manipulation commands (for example, work instructions).
  • the upper computer can connect/communicate with the control equipment, and instruct the drive unit to drive the corresponding components or parts to work by sending work instructions to the control unit.
  • the upper computer can be manually controlled, for example, an operator can manually operate the upper computer to issue a work order.
  • the host computer can control one or more components or components in the foregoing embodiments through a control program or software.
  • the host computer may include a display device, such as a display screen. The display screen can be used to present the detection results of one or more detection components in a specific form (for example, numbers, images, etc.).
  • the purification method provided in this specification may also include one or more detection steps.
  • the detecting step mentioned here may refer to determining one or more parameters in the step. Including but not limited to: the liquid level position of the liquid in the separation container, the liquid level position of the liquid in the collection container, the content of specific substances in the liquid in the drain line connected to the separation container (for example, the purification target in the second liquid substance content, impurity content in the first liquid) and so on.
  • the first liquid and the second liquid to the separation container to wash and elute the magnetic beads.
  • the liquid level in the separation container needs to be controlled at Appropriate range.
  • the position of the liquid level in the separation container can be detected and the position of the liquid level can be controlled and adjusted according to the detection result.
  • this specification will take the second liquid as an example for illustration.
  • the liquid level in the separation container reaches a set first liquid level threshold. If the position of the liquid level reaches the set first liquid level threshold, then reduce the liquid intake volume of the second liquid or increase the liquid output volume of the second liquid. In this embodiment, when the liquid level reaches the first liquid level threshold, it can be considered that the liquid level in the separation container is too high, which is not conducive to the separation of the magnetic beads and the purification target, so the liquid level needs to be lowered.
  • Reducing the feed amount of the second liquid may refer to reducing the volume of the second liquid added to the separation container. Exemplarily, it can be realized by reducing the flow rate of the liquid ejected from the flushing nozzle.
  • Increasing the discharge volume of the second liquid may refer to increasing the volume of the second liquid discharged from the lower opening of the separation container. Exemplarily, it can be realized by increasing the flow rate of the liquid discharged from the liquid discharge pipeline.
  • the first detection component may include a first liquid level sensor.
  • the first liquid level sensor may be arranged at the first position of the separation container to detect the relationship between the liquid level position and the first position. Wherein the height of the first position may correspond to the first liquid level threshold.
  • the liquid level in the separation container can be detected whether the liquid level in the separation container reaches the set second liquid level threshold. If the position of the liquid level does not reach the set second liquid level threshold, then reduce the liquid output volume of the second liquid or increase the liquid intake volume of the second liquid. In this embodiment, when the liquid level does not reach the second liquid level threshold, it can be considered that the liquid level in the separation container is too low, which is not conducive to the separation of the magnetic beads and the purification target, so the liquid level needs to be increased.
  • the first detection assembly may include a second level sensor.
  • a second liquid level sensor may be arranged at the second position of the separation container to detect the relationship between the liquid level position and the second position. Wherein the height of the second position may correspond to the second liquid level threshold.
  • the magnetic beads before separating the magnetic beads from the purification target, it is necessary to detect whether the impurities are completely separated from the magnetic beads. After it is determined that the impurities on the magnetic beads have been removed, the magnetic beads can be washed with the second liquid to ensure that no residual impurities on the magnetic beads are mixed in the second liquid or that the content of impurities is negligible.
  • the completion of washing can be determined based on the volume of the first liquid that has been added to the separation vessel.
  • stop adding the first liquid In some embodiments, the volume of the first liquid that has been added to the separation vessel can be detected. When the volume of the first liquid reaches the set volume threshold (which may be referred to as the first volume threshold), it can be considered that washing has been completed.
  • the volume of the first liquid added to the separation container can be detected by the second detection component.
  • the second detection component may include a first flow sensor, and the first flow sensor may detect the volume of the first liquid added to the separation container.
  • the volume of the first liquid reaches the second volume threshold, it can be determined that washing has been completed.
  • the second volume threshold may be equal to the first volume threshold.
  • the volume of the first liquid discharged from the lower opening of the separation container can also be detected by the second detection component.
  • the second detection component may include a second flow sensor, and the second flow sensor may detect the volume of the first liquid discharged from the lower opening of the separation container.
  • the volume of the first liquid added to the separation container or the first liquid discharged from the lower opening of the separation container may not be limited to be detected by the flow sensor.
  • the operation A person can manually measure the volume of the first liquid added to the separation container through the container with the scale.
  • the operator may use a container with a measurement to contain the first liquid that is the same or approximately the same as the first volume threshold, and directly add the first liquid in the container to the separation container.
  • the level of impurities in the first liquid from the separation vessel can be detected.
  • the impurity washing process after adding the first liquid to remove or wash the impurities on the magnetic beads, the impurities will be mixed in the first liquid. It can be understood that as the washing continues, the impurities on the magnetic beads will become less and less, and the content of impurities mixed in the first liquid will gradually decrease and tend to be stable.
  • the threshold may be referred to as a baseline
  • the impurity content remains unchanged or hardly changes for a certain period of time (for example, the detection time threshold) , indicating that the impurities on the magnetic beads have been completely separated from the magnetic beads or the remaining impurities are negligible, that is, the washing is complete.
  • the impurity content in the first liquid can be detected by a third detection component.
  • the third detection component refers to an instrument or device for detecting the content of a certain substance in a liquid. It can be used to detect the impurity content in the first liquid from the separation vessel.
  • the lower opening of the separation container is connected with a drain line, and the first liquid filtered through the filter element can be discharged from the lower opening of the separation container through the drain line or discharged into other components (for example, a waste liquid collection container)
  • the third detection component can be arranged on the discharge pipeline, and the impurity content in the first liquid can be determined by detecting the first liquid in the discharge pipeline communicated with the separation container.
  • the third detection component may include a content detection sensor communicated with the drainage pipeline.
  • the type of assay sensor is related to the type of impurity to be detected.
  • the content detection sensor may include an ultraviolet absorption detector. The ultraviolet absorption detector can detect the impurity content according to the principle that the intensity of the ultraviolet light absorption of the substance to be detected is proportional to the concentration of the substance to be detected.
  • the ultraviolet absorption detector can detect the ultraviolet absorption value (or optical density value) of the liquid at the ultraviolet absorption peak at 280nm, and then determine the concentration of the purified protein in the first liquid according to the ultraviolet absorption value. content of other proteins.
  • the first content threshold may be represented by an ordinate value corresponding to posi:1, and the ordinate value corresponding to posi:1 is 5.
  • the temporary waste liquid storage container can be used to collect the first liquid, so the impurity content in the first liquid collected in the temporary waste liquid storage container can be detected by the content detection sensor.
  • the second liquid it is possible to detect whether the content of the purification target in the second liquid from the separation vessel The second content threshold is reached. If the content of the purified target substance does not reach the second content threshold, the second liquid is collected into a waste liquid temporary storage container. If the content of the purified target substance reaches the second content threshold, the second liquid is collected into a collection container.
  • a second liquid is added to separate the purified target substance (eg, target protein) from the magnetic beads, and the purified target substance will be mixed in the second liquid.
  • the amount of purified target compound detached from the magnetic beads is small, and the content of the purified target compound mixed in the second liquid may not meet the requirements.
  • this part of the second liquid will be discharged into the waste liquid temporary storage container as waste liquid.
  • the detached purification target substance increases continuously, and the content of the purification target substance mixed in the second liquid also continuously increases.
  • the second content threshold it indicates that the elution has met the purification requirement, and the second liquid can be collected, so it can be collected into a collection container.
  • the content of the purified target substance in the second liquid from the separation container can be detected by a third detection component.
  • the content of the purified target substance in the second liquid in the drain line connected to the separation container can be detected by the third detection component.
  • the ultraviolet absorption detector can detect the ultraviolet light absorption value of the second liquid at a specific ultraviolet light absorption peak (for example, the ultraviolet light absorption peak is 280nm), so as to determine the content of the antibody in the second liquid according to the ultraviolet light absorption value .
  • the second content threshold may be represented by an ordinate value corresponding to posi:2, and the ordinate value corresponding to posi:2 is 100.
  • the collection container comprises a plurality. It may be detected whether the current liquid level position of the collection container reaches the third liquid level threshold. When the liquid level position of the current collection container reaches the third liquid level threshold, the second liquid is collected into other collection containers.
  • the current collection container may refer to a collection container that is currently collecting the second liquid.
  • the liquid filling port can move to the upper opening of other collection containers to continue collecting the second liquid, so as to prevent the liquid in the current collection container from overflowing.
  • the liquid filling port can be controlled to stop adding liquid, which can also prevent the liquid in the current collection container from overflowing.
  • the position of the liquid level in the collection container can be detected by the fourth detection component.
  • the fourth detection component may refer to an instrument or device that detects whether the liquid level in the container reaches a set liquid level. In some embodiments, the fourth detection component can detect whether the liquid level in the collection container reaches the third liquid level threshold.
  • whether the liquid level in the waste liquid temporary storage container reaches the fourth liquid level threshold can be detected by the fourth detection component.
  • the liquid in the waste liquid temporary storage container reaches the fourth liquid level threshold the liquid can be discharged to an external container with a larger volume through the discharge pipeline arranged at the bottom of the container, so as to avoid the waste liquid from overflowing.
  • the fourth detection component can be relatively fixed to the liquid filling port, and move with the movement of the liquid filling port, so as to monitor the liquid level position in the waste liquid temporary storage container or collection container corresponding to the liquid filling port. detection.
  • the fourth detection component may include one or more liquid level sensors as described in the foregoing embodiments. In some specific embodiments, the fourth detection component may include an ultrasonic liquid level sensor.
  • whether to stop collecting the second liquid can be determined based on the content of the purified target substance in the eluent (ie, the second liquid). In some embodiments, it can be determined whether the content of the purification target in the second liquid reaches the third content threshold. When the content of the purified target reaches the third content threshold, it may be determined to stop collecting the second liquid.
  • the third content threshold may refer to the lowest value of the content of the purification target substance in the second liquid meeting the collection requirement. In some embodiments, the third content threshold may exceed the second content threshold.
  • the third content threshold exceeds the second content threshold, if the purification target content reaches the third content threshold, it can be judged whether the purification target content in the second liquid is in a downward trend (for example, it can be based on several consecutive moments corresponding to Purification target substance content value (such as 10 values), determine the current variation trend of purification target substance content) If yes, then determine to stop collecting the second liquid; if not, continue to collect the second liquid.
  • Purification target substance content value such as 10 values
  • the third content threshold may be less than the second content threshold.
  • the third content threshold may be represented by an ordinate value corresponding to posi:3, and the ordinate value corresponding to posi:3 is 80.
  • the purification process may be deemed to be complete.
  • one or more pipelines and magnetic beads in the foregoing embodiments may also be cleaned and sterilized.
  • the rinsing component for example, the rinsing nozzle
  • the rinsing component can be controlled to add other liquids (including but not limited to PBS cleaning solution, pure water, sodium hydroxide solution) into the separation container.
  • PBS cleaning solution pure water or sodium hydroxide solution
  • parts such as the magnetic rod jacket are soaked in sodium hydroxide solution for disinfection.
  • the detection step in this specification can be completed manually by an operator.
  • the operator can directly use the liquid level detection sensor to detect the liquid level in the collection container.
  • the detection steps in this specification can be executed under the control of a control device.
  • the control device may control the first detection component to detect whether the liquid level in the separation container reaches a set threshold.
  • Fig. 2 is an ultraviolet light absorption peak diagram showing antibody elution according to an embodiment of the specification.
  • Figure 2 shows that when the antibody is purified based on the purification method provided by the specification, the liquid from the separation container (for example, from the first liquid and the second liquid in the drain pipe communicating with the lower opening of the separation container) UV absorption peak curves of substances (eg, impurities in the first liquid, purified objects in the second liquid).
  • the liquid from the separation container for example, from the first liquid and the second liquid in the drain pipe communicating with the lower opening of the separation container
  • UV absorption peak curves of substances eg, impurities in the first liquid, purified objects in the second liquid.
  • One or more steps in the embodiment shown in FIG. 2 can be controlled by the control device in the previous embodiment. Part or component implementation.
  • the magnetic beads used in the purification process are Sepharose Protein A magnetic beads.
  • the object of purification is a monoclonal antibody.
  • the abscissa coordinate in Fig. 2 is the elapsed time of the elution treatment.
  • the ordinate is the UV absorption value of the second liquid at a specific UV absorption peak, and the larger the value of the ordinate, the higher the content of the monoclonal antibody in the second liquid.
  • posi: 1 (position 1) on the curve can represent the ultraviolet light absorption value (i.e. the first content threshold) of the first liquid discharged through the lower opening of the separation container at the end of the cleaning process, where posi: 1 corresponds to the ordinate
  • the value is 5.
  • the UV absorption value of the first liquid reaches posi: 1 and tends to be stable (for example, the detection time threshold remains unchanged), it means that the washing is over.
  • the second liquid can be added to the separation container to wash the magnetic beads.
  • posi: 2 may represent the set initial ultraviolet light absorption value (ie, the second content threshold).
  • the ultraviolet light absorption value reaches the initial ultraviolet light absorption value, it indicates that the content of the antibody in the second liquid meets the collection requirement, and the second liquid can be collected at this time. According to the curve, it can be seen that within the time range corresponding to posi:1 to posi:2, the ultraviolet light absorption value does not reach the set initial ultraviolet light absorption value.
  • This part of the second liquid will be discharged as waste liquid (for example, discarded or collected into a waste liquid temporary storage container).
  • posi: 3 (position 3) may represent the set end ultraviolet light absorption value (ie the third content threshold). According to the curve, within the time range corresponding to posi: 2 to posi: 3, the ultraviolet light absorption value reaches the set initial ultraviolet light absorption value, so this part of the second liquid will be collected (for example, collected to collection container).
  • the ultraviolet light absorption value of the second liquid is less than the end ultraviolet light absorption value, it indicates that the antibody content in the second liquid no longer meets the collection requirement, and at this time, the collection of the second liquid can be stopped.
  • the speed of the UV absorption peak curve of the antibody from posi:2 to the peak posi:4 (position 4) and the speed from the peak posi:4 to posi:3 are faster, indicating During the detachment process, the antibody can be separated from the magnetic beads in a short time.
  • the decrease in the ultraviolet light absorption curve is small, and even after a long period of elution (for example, from the abscissa value of 360 to the abscissa value of 480), its ultraviolet light absorption curve is also similar to that of The axis of abscissa is approximately horizontal, and the change of ultraviolet light absorption value is not obvious.
  • FIG. 3 is an electrophoresis diagram of an antibody according to some embodiments of the present specification.
  • Figure 3 shows the eluate obtained after the antibody is purified and washed based on the purification method provided in this manual, the eluate obtained after the elution treatment, and the eluate and eluate obtained based on the Resin purification method
  • the electrophoresis results refers to the directional migration of charged biomacromolecules (such as antibodies) under the action of electric field force.
  • small biomolecules migrate faster and biomacromolecules migrate slower in the molecular sieve of the SDS-PAGE gel.
  • Biomacromolecules with different molecular weights have different migration speeds.
  • Molecular sieves can be used to separate biomacromolecules of different molecular weights.
  • Coomassie Brilliant Blue staining experimenters can visually judge how many biomacromolecules of different molecular weights are contained in each lane. The approximate content of a certain biomacromolecule can be judged by the area of the stained area in the lane and the depth of the staining.
  • the Resin purification method uses AKTA pure25 purification equipment to purify the antibody. It specifically includes: diluting the fermented liquid to obtain a culture liquid with a concentration of 0.044 mg/ml and a volume of 800 ml. In some embodiments, the concentration and volume of the culture solution used in the Resin method purification experiment and the purification experiment based on the purification method provided in this description are the same.
  • the eppendorf 5920R high-speed centrifuge was used to centrifuge the crude sample (ie, the culture medium) to precipitate impurities such as cell debris, and the supernatant was taken and filtered with a Millipore 0.22 ⁇ m funnel-type disposable filter to further remove impurities. Finally, the filtrate was purified by column purification using AKTA pure25 purification equipment and GE Mabselect SuRe 5ml Prepacked Resin chromatography column to obtain purified antibodies.
  • lane 1 (ie, the area marked with a dotted rectangle in the lower rectangle marked with 1) represents the electrophoresis result of Marker.
  • the Marker is a standard, which is a mixture of several biomacromolecules (such as proteins) with known molecular weights. Each band at a different position in the lane represents a known molecular weight of the biomacromolecule. As a reference, the experimenter can determine the approximate molecular weight by comparing the sample bands in the remaining lanes with the Marker bands.
  • swimming lane 2 represents the electrophoresis result of the washing solution obtained after washing treatment based on the purification method provided in this specification.
  • swimming lane 3 represents the electrophoresis result of the combined reagent of the eluate obtained after elution treatment based on the purification method provided in this specification.
  • swimming lane 4 represents the electrophoresis result of the washing solution obtained based on the Resin purification method.
  • Lane 5 represents the electrophoresis result of eluate 1 obtained by the Resin method.
  • Lane 6 represents the electrophoresis result of eluate 2 obtained by the Resin method.
  • the samples in lanes 5 and 6 were collected before and after the Resin purification method experiment and were not combined Two tubes of eluent. Wherein, the sample volume in lane 1 is 5ul, and the sample volume in other lanes is 10ul.
  • the Resin purification method needs to centrifuge and filter the culture medium, and this process takes about 30 minutes. Transfer the centrifuged and filtered culture solution to a chromatography column and perform column treatment (i.e., add washing liquid to the chromatography column to separate impurities from the filler, and add eluent to separate the purified target from the filler), This process takes about 4 hours. However, it only takes 2.5 hours to purify the same volume and type of culture fluid based on the purification method provided in this manual. Among them, it takes about 2 hours to mix the magnetic beads and the purified target.
  • the time-consuming referred to here may refer to the time required to execute the purification method provided in this specification by other components, devices or equipment (eg, control equipment). Therefore, the purification method provided based on this description greatly shortens the time-consuming experiments and significantly improves the purification efficiency.
  • the time required for the mixing of the magnetic beads and the purified target can be determined according to the needs. The longer it takes to mix the magnetic beads with the purified target, the less the purified target remains in the culture medium, and the higher the recovery rate of the purified target (that is, the amount of the purified target mixed with the magnetic beads is equal to the amount of the purified target in the culture medium).
  • the ratio of the amount of purified target For example, it takes 15 minutes to mix the magnetic beads with the target antibody to achieve 85% recovery. For another example, it takes 45 minutes to mix the magnetic beads with the target antibody, and the recovery rate of more than 98% can be achieved.
  • the mixing time of the magnetic beads and the target antibody was extended to 2 hours, which may have exceeded the requirement
  • the target antibody recovery rate is higher, so the actual time required to mix the magnetic beads with the purified target may be shorter.
  • the recovery rate of the target antibody is required to reach 85%
  • the actual time required to mix the magnetic beads with the purified target may be about 15 minutes, and the entire purification process does not exceed 1 hour.
  • the recovery rate of the target antibody is required to reach 85%, the total time required for purification does not exceed 2 hours. Compared with the 4.5 hours described in the previous examples, the time-consuming is shortened to a large extent.
  • the purification method provided in this specification can be applied to specific purification equipment, and the purification equipment can be controlled according to the purification method provided in this specification, so as to purify the purification target substance in the culture medium.
  • Fig. 4 is a schematic structural diagram of a purification device according to some embodiments of the present specification.
  • the purification device 10 may include a mixing component 11 , a transfer component 12 and a separation component 13 .
  • the mixing component 11 can be used to mix the magnetic beads with the purification target.
  • the transfer component 12 can be used to transfer the magnetic beads to a specific container, so as to separate the purification target on the magnetic beads.
  • the separation component 13 can be used to accommodate magnetic beads.
  • the mixing assembly 11 may include a shaking platform 111 and a mixing container 112 .
  • the transfer assembly 12 may include a magnetic member 121 .
  • the separation assembly 13 may include a separation container 131 , and a filter element 132 is disposed in the separation container 131 .
  • the magnetic member 121 may include a magnetic rod and a magnetic rod sheath covering the magnetic rod.
  • the transfer assembly 12 may further include a first track for sliding the magnetic bar, a second track for sliding the magnetic bar cover, and a third track for sliding the magnetic bar and the magnetic bar cover.
  • purification apparatus 10 may also include a flushing component.
  • the rinse assembly may include a rinse spray head.
  • purification apparatus 10 may also include collection assembly 14 .
  • the collection assembly 14 may include a collection container 141 and a liquid filling port 142 .
  • the collection assembly 14 may further include a waste liquid temporary storage container 143 .
  • the purification device 10 may further include a detection component 151 .
  • the detection component 15 may include a first detection component 151 , a second detection component 152 , a third detection component 153 and a fourth detection component 154 .
  • one or more components or components of purification apparatus 10 may be the same as or similar to components or components in previous embodiments.
  • the first detection component 151, the second detection component 152, the third detection component 153 and the fourth detection component 154 can be combined with the first detection component, the second detection component, the third detection component and the fourth detection component in the foregoing embodiments.
  • the four detection components are the same.
  • the temporary waste liquid storage container 143 and/or the collection container 1412 and the liquid filling port 142 may be the same as the temporary waste liquid storage container and/or the collection container and the liquid filling port in the foregoing embodiments.
  • the purification device 10 may further include a control component 16, which may be used to control one or more components of the purification device 10 to perform corresponding functions.
  • the control assembly 16 may include a control unit 161 and a drive unit 162 .
  • the control unit 161 and the drive unit 162 can be the same as or similar to the control unit and the drive unit in the foregoing embodiments. Regarding the control unit and the drive unit Further descriptions can be found elsewhere in this specification.
  • Fig. 5 is an exemplary flowchart of purification based on purification equipment according to some embodiments of the present specification.
  • Fig. 5 shows the specific process of controlling the purification device 10 based on the purification method in the previous embodiment to purify the biomacromolecules in the culture fluid, and the process 500 may include the following steps:
  • Step 510 control the mixing component 11 to perform a mixing operation.
  • controlling the mixing component 11 to perform the mixing operation includes: firstly putting a certain amount of culture solution and a certain amount of magnetic beads into the mixing container 112, then fixing the mixing container 112 on the shaking platform 111 and controlling The oscillating platform 111 oscillates the mixing container 112 to combine the magnetic beads with the purified target substance in the culture medium.
  • Step 520 controlling the magnetic member 121 in the transfer assembly 12 to transfer the magnetic beads to the upper opening of the separation container 131 .
  • controlling the magnetic member 121 in the transfer assembly 12 to transfer the magnetic beads to the upper opening of the separation container 131 includes: protruding a part of the magnetic member 121 below the liquid surface of the mixing container 112, using the magnetic member 121 The adsorption force makes the magnetic beads enrich on the surface of the magnetic member 121. Then the magnetic piece 121 is taken out from the mixing container 112 and moved to the upper opening of the separation container 131 .
  • Step 530 controlling the washing component to separate the magnetic beads from the magnetic element 121 , so that the magnetic beads enter the separation container 131 and accumulate on the filter element 132 .
  • Step 540 control the flushing component to add the first liquid into the separation container 131 . Impurities not bound to the beads are removed by washing the beads with the first liquid.
  • Step 550 control the flushing component to add the second liquid into the separation container 131 . Washing the magnetic beads with the second liquid separates the purification targets bound to the magnetic beads from the magnetic beads.
  • Step 560 control the collecting component 14 to collect the first liquid and/or the second liquid from the separation container 131 .
  • the second liquid mixed with the purification target may be collected through the collection component 14 (eg, the collection container 141 ) communicated with the lower opening of the separation container 131 .
  • the collection component 14 can be controlled to collect the first liquid from the separation container 131 .
  • the first liquid mixed with impurities can be collected through the collection component 14 (eg, the waste liquid temporary storage container 143 ) communicated with the lower opening of the separation container 131 .
  • the magnetic member 121 may include a magnetic bar.
  • the magnetic bar can be controlled to slide along the first track, so as to standardize the movement path of the magnetic bar and improve the stability during the movement.
  • the magnetic rod and the magnetic rod casing can be controlled to move along the first track and the second track respectively.
  • the magnetic rod and the magnetic rod casing can be controlled to move along the third track.
  • the magnetic piece 121 when the magnetic piece 121 is located at the upper opening of the separation container 131, it can pass The washing assembly disposed at the upper opening of the separation container 131 washes the magnetic beads to separate the magnetic beads from the magnetic member 121 .
  • the magnetic force of the magnetic member 121 can also be reduced to reduce the adsorption force of the magnetic member 121 to the magnetic beads, so that the magnetic beads are separated from the magnetic member 121 under the action of gravity.
  • reducing the magnetic force of the magnetic member 121 to reduce the adsorption force of the magnetic member 121 to the magnetic beads please refer to the description of other embodiments of this specification, which will not be repeated here.
  • the third detection component 153 can be used to detect the content of impurities in the first liquid from the separation container 131 . For example, determine the impurity content therein by detecting the first liquid in the drain line communicated with the separation container 131, when the impurity content reaches the first content threshold and maintains the detection time threshold unchanged, it indicates that the washing is completed, and the addition can be stopped. first liquid.
  • the content of the purified target substance in the second liquid from the separation container 131 can be detected by the third detection component 153 .
  • the second liquid in the drain line communicating with the separation container 131 to determine the content of the purification target therein when the content of the purification target reaches the second content threshold, it indicates that the second liquid can be collected.
  • the content of the purified target substance reaches the third content threshold and the content of the purified target substance is in a downward trend it indicates that the purified target substance separated from the magnetic beads is less, and it can be considered that the purified target substance remaining on the magnetic beads is less or completely No residue.
  • the purification process can be considered as finished.
  • the second liquid may be collected into the waste liquid temporary storage container 143 .
  • the second liquid may be collected into the collection container 141 .
  • the content of the purified target substance reaches the third content threshold and the content of the purified target substance is on a downward trend (for example, it can be determined based on the values of the purified target substance content corresponding to several consecutive moments (such as 10 values), determine the current
  • the second liquid can be collected in the waste liquid temporary storage container 143.
  • the possible beneficial effects of the purification method in the embodiment of this specification include but are not limited to: (1) Compared with the Resin purification method, there is no need to centrifuge and filter the culture medium, but to use magnetic beads to mix with the purification target to make The purification target is separated from the culture medium, which not only simplifies the purification steps, but also saves the time required for purification; (2) Compared with the Resin purification method, there is no need to use a disposable filter to vacuum filter the chromatographic column, not only Simplifies the purification steps and saves the cost of consumables in the purification process; (3) Use magnetic beads to mix with the purified target in the culture medium, and use magnetic parts to transfer the magnetic beads to achieve preliminary filtration of the purified target and the culture medium , can avoid the blockage caused by the culture medium; (4) There is no need to set up additional magnetic beads in the separation container to absorb and purify the target substance, and it is unnecessary to control the motor to drive the permanent magnet close to and away from the magnetic bead filter column to control the magnetic field

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Abstract

本说明书一个或多个实施例涉及一种纯化方法，纯化方法包括：将磁珠与纯化目标物混合；将磁珠与纯化目标物转移至分离容器内；将磁珠与杂质分离；通过分离容器中的过滤件将磁珠与纯化目标物分离，过滤件开设有过滤孔，过滤孔的孔径小于磁珠的直径。

Description

纯化方法
优先权信息
本发明要求2022年01月17日提交的申请号为202210049124.3的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本说明书涉及生物大分子纯化技术领域，尤其涉及一种纯化方法。
背景技术
生物大分子(例如，抗体、蛋白、核酸等)的纯化是分子生物学、医学研究中的重要环节之一。纯化的效果直接影响研究和诊断的进程和结果。然而，目前的纯化方法不仅耗费时间长、耗材成本高，并且纯化效果也仍有进一步提升空间。在一些实施例中，生物大分子的纯化方法可以包括层析柱纯化法以及磁珠纯化法。在一些实施例中，层析柱纯化方法可以包括：利用离心机对培养液进行离心和过滤，利用一次性无菌滤器对培养液进行真空抽滤；将已真空抽滤的培养液从层析柱的上方开口加入层析柱中，使培养液流向层析柱的下方开口。在此过程中，培养液中的纯化目标物可以与填料混合并滞留在层析柱中；将洗杂液从层析柱的上方开口加入层析柱中，滞留在层析柱中的未与填料混合的杂质可以混合在洗杂液从层析柱的下方开口排出；将洗脱液从层析柱的上方开口加入层析柱中，洗脱液可以将纯化目标物与填料分离，使得纯化目标物混合在洗脱液中，经由层析柱的下方开口流出，收集该洗脱液即可得到提纯的纯化目标物。在一些实施例中，磁珠纯化方法可以包括：将细胞破碎液和磁珠在离心管中孵育，然后将离心管置于磁分离器中，待溶液变澄清后，用移液器移出上清液。然后向离心管中加入清洗缓冲液，使用移液器枪头反复吹打5-10次，再将离心管置于磁分离器上，待溶液变澄清后，用移液器吸取上清液。重复上述步骤2次，洗杂完成。然后将洗脱缓冲液加入到离心管中，使用移液器枪头轻轻吹打3-5次，混匀，将离心管置于磁分离器上，待溶液变澄清后，用移液器吸取上清液，即为目的蛋白组分。然而，上述纯化方法不仅耗费时间长、耗材成本高，并且纯化效果也不佳。
针对上述问题，提供一种纯化方法，旨在降低纯化成本、提高纯化速度和纯化效果。
发明内容
本说明书的之一在于提供一种纯化方法，所述纯化方法包括：将磁珠与纯化目 标物混合；将所述磁珠与所述纯化目标物转移至分离容器内；将所述磁珠与杂质分离；通过所述分离容器中的过滤件将所述磁珠与所述纯化目标物分离，所述过滤件开设有过滤孔，所述过滤孔的孔径小于所述磁珠的直径。
在一些实施例中，所述分离容器的两端均设置有开口。
在一些实施例中，所述将所述磁珠与所述纯化目标物转移至分离容器内包括：通过磁性件吸附所述磁珠；将所述磁性件移动至所述分离容器的上方开口；将所述磁珠与所述磁性件分离以使所述磁珠进入所述分离容器内。
在一些实施例中，所述将所述磁珠与所述磁棒分离以使所述磁珠进入所述分离容器内，包括：通过冲洗方式将所述磁珠与所述磁性件分离。
在一些实施例中，所述磁性件包括磁棒，所述将所述磁珠与所述磁性件分离以使所述磁珠进入所述分离容器内，还包括：减小所述磁棒与所述磁珠之间的吸附力，以使所述磁珠在重力作用下与所述磁性件分离。
在一些实施例中，所述磁棒包括电磁棒，当所述电磁棒处于通电状态时，所述电磁棒具有磁性，当所述电磁棒处于断电状态时，所述电磁棒失去磁性；所述减小所述磁棒与所述磁珠之间的吸附力包括：将所述电磁棒调整至所述断电状态。
在一些实施例中，所述磁性件还包括套设在所述磁棒外的磁棒外套，所述磁棒可沿所述套设方向相对所述磁棒外套移动；所述减小所述磁棒与所述磁珠之间的吸附力包括：将所述磁棒沿所述套设方向相对所述磁棒外套移动以使所述磁棒远离富集在所述磁棒外套表面的所述磁珠。
在一些实施例中，所述将所述磁珠与杂质分离包括：通过第一液体冲洗以将所述磁珠与所述杂质分离。
在一些实施例中，所述通过所述分离容器中的过滤件将所述磁珠与所述纯化目标物分离包括：向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离；收集经所述过滤件过滤的所述第二液体。
在一些实施例中，所述向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离包括：检测所述分离容器中的液面位置是否达到设定的第一液面阈值；若所述液面位置达到设定的所述第一液面阈值，则降低所述第二液体的进液量或提高所述第二液体的出液量。
在一些实施例中，所述方法还包括：检测所述分离容器中的液面位置是否达到设定的第二液面阈值，所述第一液面阈值大于所述第二液面阈值；若所述液面位置未达 到设定的所述第二液面阈值，则提高所述第二液体加液量或降低所述第二液体的排液量。
在一些实施例中，在所述向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离之前包括：检测已加入到所述分离容器中所述第一液体的体积；当所述第一液体的体积达到设定的体积阈值时，停止加入所述第一液体。
在一些实施例中，在所述向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离之前，还包括：检测来自所述分离容器的所述第一液体中的杂质含量；当所述第一液体中的杂质含量达到第一含量阈值且维持检测时间阈值不变时，停止加入所述第一液体。
在一些实施例中，所述收集经所述过滤件过滤的所述第二液体包括：检测来自所述分离容器的所述第二液体中的纯化目标物含量是否达到第二含量阈值；若所述纯化目标物含量未达到所述第二含量阈值，将所述第二液体收集至废液暂存容器中；若所述纯化目标物含量达到所述第二含量阈值，将所述第二液体收集至收集容器中。
在一些实施例中，所述收集容器包括多个；所述将所述第二液体收集至收集容器中包括：检测当前所述收集容器的液面位置是否达到第三液面阈值；当当前所述收集容器的液面位置达到所述第三液面阈值时，将所述第二液体收集至其他所述收集容器中。
在一些实施例中，当所述纯化目标物含量达到所述第三含量阈值时，停止收集所述第二液体。
在一些实施例中，所述将所述第二液体收集至收集容器中还包括：向所述收集容器中加入第三液体，所述第三液体用于中和所述收集容器中的酸性环境。
附图说明
本说明书将以示例性实施例的方式进一步说明，这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的，在这些实施例中，相同的编号表示相同的结构，其中：
图1是根据本说明书一些实施例所示的纯化方法的示例性流程图；
图2是根据本说明书实施例所示的抗体洗脱紫外光吸收峰图；
图3是根据本说明书一些实施例所示的抗体电泳图；
图4是根据本说明书一些实施例所示的纯化设备的简易结构示意图；
图5是根据本说明书一些实施例所示的基于纯化设备进行纯化的示例性流程图。
具体实施方式
为了更清楚地说明本说明书实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本说明书的一些示例或实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图将本说明书应用于其它类似情景。除非从语言环境中显而易见或另做说明，图中相同标号代表相同结构或操作。
本领域技术人员可以理解，本说明书中的“第一”“第二”等术语仅是用于区别不同设备、模块或参数等，既不代表任何特定技术含义，也不表示他们之间的必然逻辑顺序。
如本说明书和权利要求书中所示，除非上下文明确提示例外情形，“一”、“一个”、“一种”和/或“该”等词并非特指单数，也可包括复数。一般说来，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法或者设备也可能包含其它的步骤或元素。
本说明书涵盖任何由权利要求定义的在本说明书的精髓和范围上做的替代、修改、等效方法以及方案。进一步，为了使公众对本说明书有更好的了解，在下文对本说明书的细节描述中，详尽描述了一些特定的细节部分。对本领域技术人员来说没有这些细节部分的描述也可以完全理解本说明书。
本说明书一些实施例提供了一种对培养液中的纯化目标物进行纯化的方法。在一些实施例中，纯化是指将粗样品(例如，培养液)中的纯化目标物(例如，目标抗体)与其他杂质分离，实现提纯。在一些实施例中，纯化目标物可以包括生物大分子(例如蛋白、抗体以及核酸等)。例如，当纯化目标物为抗体时，可以称为目标抗体。又例如，当纯化目标物为细胞时，可以称为目标细胞。在一些实施例中，该纯化方法利用磁珠与培养液中的纯化目标物混合，而混合有纯化目标物的磁珠将会被转移至分离容器内。由于磁珠无法通过设置在分离容器内的过滤件的过滤孔，因此将会被截留在分离容器内。然后通过向分离容器内加入洗杂液对磁珠进行冲洗来移除磁珠上的杂质(即除纯化目标物以外的物质)，而被移除的杂质可以混合在洗杂液中通过过滤件的过滤孔从分离容器排出。再通过向分离容器内加入洗脱液冲洗磁珠来移除磁珠上的纯化目标物，被移除的纯化目标物可以混合在洗脱液中通过过滤件的过滤孔从分离容器排出。最后对排出的洗脱液进行收集、处理即可得到提纯后的纯化目标物。
在本说明书中，磁珠(或称为超顺磁性纳米微球)可以是指能够与生物大分子 相结合的具有磁响应性的纳米粒子。在一些实施例中，磁珠可以由硅基、PS基(即聚苯乙烯高分子材料)或琼脂糖等包裹。示例性的，磁珠的磁核的材料可以是Fe3O4。在一些实施例中，磁珠的直径的取值范围可以包括1nm～500um。在一些实施例中，磁珠的直径的取值范围可以包括3nm～400um。在一些实施例中，磁珠的直径的取值范围可以包括5nm～300um。在一些实施例中，磁珠的直径的取值范围可以包括50nm～200um。在一些实施例中，磁珠上可以结合功能基团，功能基团可以与特定的生物大分子进行结合。因此可以根据结合不同功能基团来进行各种应用。例如，利用磁珠与细胞进行结合，从而实现提取特定细胞的目的。又例如，利用磁珠与蛋白、抗体进行结合，实现蛋白、抗体的提取。在一些实施例中，功能基团可以包括但不限于Protein A、Protein G、Ni、GSH、oligo探针、链霉亲和素、抗体等单种或多种或夹心结构。
在一些实施例中，混合可以是指使培养液中的纯化目标物与填料进行混杂。例如，两者通过物理方式混合(例如，让纯化目标物进入填料的三维网状孔径里面滞留)。又例如，两者通过化学键相结合或者通过离子交换、静电吸附等方式相结合等。
在一些实施例中，洗杂液可以包括Na2HPO4、KH2PO4、NaCl和KCl等。在一些实施例中，洗杂液可以根据纯化目标物的种类不同而不同，例如，当纯化目标物为核酸时，洗杂液可以包括三(羟甲基)基甲烷、甘露醇等。在一些实施例中，洗脱液可以包括纯水、Tris-HCl、甘氨酸、柠檬酸盐或者醋酸钠等。在一些实施例中，洗脱液可以让纯化目标物与磁珠的活性基团脱离，从而使纯化目标物与磁珠分离。在一些实施例中，洗脱液可以根据纯化目标物的种类不同而不同，例如，当纯化目标物为核酸时，洗脱液还可以包括纯水或Tris-HCl。
图1是根据本说明书一些实施例所示的纯化方法的示例性流程图。如图1所示，该纯化方法100包括以下步骤：
步骤110、将磁珠与纯化目标物混合。
在一些实施例中，在对纯化目标物提纯之前，可以将纯化目标物与培养液初步分离。在一些实施例中，可以利用磁珠与纯化目标物混合，以便于转移纯化目标物。在本说明书实施例中，磁珠与纯化目标物混合可以包括纯化目标物与磁珠上的功能基团进行结合。
在一些实施例中，可以将磁珠与培养液放入特定容器中，然后对特定容器进行混匀操作，使得磁珠与培养液中的纯化目标物混合。为了描述方便，用于收容磁珠和培养液的特定容器可以称为混匀容器。混匀操作可以是指对混匀容器进行如震荡、旋转、 摇晃等形式的操作，从而使得容纳在混匀容器中的物质混合或结合。
在一些实施例中，可以以旋转、摇晃等形式手动对混匀容器进行混匀。在一些实施例中，可以通过混合组件对混匀容器进行混匀操作。例如，通过混合组件对混匀容器进行如震荡、旋转、摇晃等形式的操作。在一些实施例中，可以利用震荡平台对混匀容器进行混匀。震荡平台上可以用于放置混匀容器。震荡平台可以以包括往复摆动、旋转等在内的形式进行运动，并带动放置在震荡平台上的混匀容器进行运动。
在另一些实施例中，可以通过手动方式直接对磁珠和培养液进行混合。例如，将磁珠与培养液放入混匀容器中，然后直接对磁珠和培养液进行包括不限于搅拌等操作。
步骤120、将磁珠与纯化目标物转移至分离容器内。
在一些实施例中，当磁珠与纯化目标物混合后，可以将磁珠转移至分离容器中。
在一些实施例中，分离容器的两端均设置有开口，包括上方开口和下方开口。分离容器中的过滤件用于将从分离容器的某一开口(例如，上方开口)进入，从另一开口排出的物质进行过滤。在一些实施例中，过滤件的形状呈板状。板状过滤件的厚度方向与分离容器的高度方向相同或近似相同。板状过滤件沿其厚度方向的横截面形状与分离容器的内部腔体沿其高度方向的横截面形状相同或近似相同，且尺寸相近，以使得所要过滤的物质无法从过滤件与分离容器的内壁之间的缝隙通过。在一些实施例中，过滤件的厚度方向可以与分离容器的厚度方向相同或近似相同。在一些实施例中，磁珠可以从分离容器的上方开口处进入分离容器内部。由于过滤件的过滤孔的孔径小于磁珠的直径，因此磁珠将会被过滤件截留在分离容器内部。在一些实施例中，过滤孔的孔径大于纯化目标物(例如，生物大分子)以及杂质(例如，不属于纯化目标物的生物大分子以及除生物大分子以外的其他物质)的直径，以使得作为杂质和作为纯化目标物与磁珠分离混入到特定液体中后可以经过过滤孔通过分离容器的下方开口排出。例如，混合杂质的洗杂液可以通过经由过滤孔从分离容器的下方开口流出，而磁珠将会继续堆积在过滤件上被冲洗。
在一些实施例中，过滤件可以为筛板。在一些实施例中，筛板可以相对分离容器拆卸，以便可以对筛板进行清洗或更换。
在一些实施例中，将磁珠与纯化目标物转移至分离容器内可以包括：通过磁性件吸附磁珠；将磁性件移动至分离容器的上方开口处；将磁珠与磁性件分离以使磁珠进入分离容器内。
在一些实施例中，磁性件具有磁性，当磁性件伸入到培养液中后，培养液中的 磁珠可以在磁力作用下富集在磁性件没入液面以下部分的表面。此时将磁性件从液体中取出，由于磁性件与磁珠之间的磁力克服了磁珠的重力作用，因此磁珠将会随着磁性件移动从而与培养液分离，又由于纯化目标物与磁珠混合，因此纯化目标物与培养液分离。
在一些实施例中，磁性件可以包括磁棒。磁棒具有磁性，可以吸附培养液中的磁珠。
在另一些实施例中，磁性件可以包括磁棒以及套设在磁棒外的磁棒外套，磁棒可以沿套设方向相对磁棒外套移动。其中，磁棒外套可以是指套设在磁棒外的壳体结构。示例性的，磁棒外套的一端具有开口，另一端封闭。磁棒可以从磁棒外套的开口端嵌入磁棒外套内，并沿套设方向移动。在一些实施例中，套设方向可以是磁棒的长度方向。在一些实施例中，套设方向可以与混匀容器的高度方向相同或近似相同。在本实施例中，当磁棒与磁棒外套配接(即磁棒外套套设在磁棒外)并伸入混匀容器的液面以下时，磁棒提供的吸附力会使得磁珠富集在磁棒外套的表面。在一些情况下，磁棒外套可以对磁棒进行保护，避免磁棒直接与培养液接触对磁棒造成腐蚀。在另一些情况下，由于磁棒外套能够相对磁棒沿套设方向运动，因此可以将磁棒外套与磁棒分离，便于清洗或更换。还有一些情况下，可以利用磁棒外套能够相对磁棒移动的特点，实现磁珠与磁棒外套的分离，关于此，在本说明书其他实施例中有更详细的描述，此处不再赘述。
在一些实施例中，在利用磁性件吸附、转移磁珠时，可以手动操作磁性件进行移动。为了方便描述，以磁性件包括磁棒和磁棒外套为例进行说明。示例性的，操作人员可以手拿磁棒和磁棒外套控制磁棒和磁棒外套移动。在一些实施例中，可以利用其它装置、设备或组件(例如，控制设备)控制磁性件实现移动。在一些具体实施例中，控制设备可以控制磁棒沿第一轨道运动，控制磁棒外套沿第二轨道运动，以及控制磁棒和磁棒外套同时沿第三轨道运动。关于控制设备的更多细节，可以在本说明书其他实施例中找到。其中，第一轨道、第二轨道的延伸方向可以与混匀容器的高度方向相同或近似相同。第三轨道的延伸方向可以与从混匀容器的上方开口至分离容器的上方开口方向相同或近似相同。在一些实施例中，磁性件不是必须沿特定的轨道进行移动。例如，可以通过机械臂控制磁棒和磁棒外套进行移动，并不需要依靠轨道。
在一些实施例中，当磁性件移动至分离容器的上方开口处之后，可以利用外力和/或减小磁性件对磁珠的吸附力等方式将富集在磁性件上的磁珠与磁性件分离，以使得磁珠从分离容器的上方开口进入分离容器内。
在一些实施例中，可以直接拨动磁珠，使磁珠与磁性件分离。例如，通过控制 拨动杆拨动磁珠。在一些实施例中，拨动杆可以通过手动控制，例如，操作人员手拿拨动杆拨动。在一些实施例中，拨动杆可以其他装置、设备或组件(例如，机械臂)控制。
在一些实施例中，将磁珠与磁性件分离以使磁珠进入分离容器内可以包括：通过冲洗方式将磁珠与磁性件分离。例如，利用流动的液体对磁珠进行冲洗，液体会对磁珠产生冲击力，该冲击力可以使得磁珠从磁性件上掉落下来。在一些实施例中，可以利用第一液体对磁珠进行冲洗，其中第一液体可以为前述实施例中的洗杂液。在一些情况下，利用洗杂液对磁珠进行冲洗不仅可以将磁珠从磁性件上冲洗下来，同时洗杂液还可以将附着在磁珠表面或者相邻磁珠之间的未与磁珠混合的杂质与磁珠分离，有效提高纯化效率。
在一些实施例中，可以手动冲洗磁珠。例如，操作人员手动控制喷液管喷出特定液体对磁珠进行冲洗或手拿装有第一液体的容器倒出第一液体冲洗磁珠。
在一些实施例中，可以通过控制冲洗组件对磁珠进行冲洗使磁珠从磁性件上脱离。在一些实施例中，冲洗组件可以包括设置在分离容器的上方的冲洗喷头。在一些实施例中，冲洗喷头可以与外部的泵阀组件连通，通过控制泵阀组件来控制冲洗喷头喷出特定的液体。在一些实施例中，可以通过设置多个冲洗喷头，以提高冲洗效率。
在一些实施例中，除了通过冲洗的方式将磁珠与磁性件分离之外，还可以利用磁性件本身的特性来实现。示例性的，可以通过减小磁性件(例如，磁棒)与磁珠之间的吸附力以使磁珠在重力作用下与磁性件分离。
在一些实施例中，磁棒可以包括电磁棒。当电磁棒处于通电状态时，电磁棒具有磁性；当电磁棒处于断电状态时，电磁棒失去磁性。在一些实施例中，可以通过调整电磁棒的通电状态来减小电磁棒与磁珠之间的吸附力，以使磁珠与电磁棒分离。示例性的，当需要利用电磁棒吸附磁珠时，可以将电磁棒调整至通电状态，电磁棒处于通电状态时具有磁性，因此可以对磁珠进行吸附。在另一示例中，当需要将磁珠与电磁棒分离时，可以将电磁棒调整至断电状态，电磁棒处于断电状态时磁性消失，因此电磁棒与磁珠之间的磁力作用也随之消失，此时磁珠可以在重力作用下与电磁棒分离。
在另一些实施例中，当磁性件包括磁棒和套设在磁棒外的磁棒外套时，可以利用磁棒与磁棒外套的相对移动来改变磁棒与富集在磁棒外套表面的磁珠之间的距离，从而改变两者之间的磁力强度。示例性的，当吸附有磁珠的磁棒外套和嵌设在磁棒外套内的磁棒移动至分离容器的上方开口处时，可以将磁棒沿套设方向相对磁棒外套移动(例如，沿着第一轨道滑动)，以使磁棒远离富集在磁棒外套表面的磁珠。磁棒与磁珠之间 的距离越远，磁棒与磁珠之间的磁力作用也就越小。随着磁棒逐渐远离磁珠，磁棒与磁珠之间的磁力作用逐渐减弱，直至磁棒与磁珠之间的磁力作用无法克服磁珠的重力作用时，磁珠将会从磁棒上脱离。
步骤130、将磁珠与杂质分离。
在一些实施例中，培养液中的杂质(即除纯化目标物以外的物质)无法与磁珠混合，但可能会聚集在相邻磁珠之间的缝隙中或者附着在磁珠表面。因此在提纯纯化目标物之前，需要将这些杂质从磁珠上移除。在一些实施例中，可以通过第一液体冲洗磁珠以将杂质与磁珠分离。在一些实施例中，第一液体可以包括洗杂液。关于洗杂液的更多细节可以参见本说明书的其他实施例的描述。在一些实施例中，通过第一液体对磁珠进行冲洗将杂质与磁珠分离可以包括：(1)在磁珠还未与磁性件分离之前，利用第一液体对磁珠进行冲洗，以将磁珠从磁性件上冲洗下来。在磁珠与磁性件分离的过程中，与磁珠结合的杂质也会被冲洗移除；(2)在磁珠与磁性件分离并进入分离容器内后，由于过滤件的过滤孔的直径小于磁珠的直径，因此磁珠无法通过过滤孔，将会被过滤件截留在分离容器中。此时可以利用第一液体对堆积在过滤件上的磁珠进行冲洗，将附着在磁珠上的杂质进一步清除。在一些实施例中，利用第一液体冲洗磁珠时，被移除的杂质可以混合在第一液体中。由于过滤件的过滤孔的孔径大于杂质的直径，因此第一液体以及杂质可以经由过滤孔并通过分离容器的下方开口排出。而磁珠将会被过滤件截留在分离容器的腔体中，实现杂质与磁珠的分离。
在一些实施例中，可以通过手动方式进行冲洗。例如，操作人员手动控制喷液管喷出第一液体冲洗磁珠。在另一些实施例中，可以控制冲洗组件喷出第一液体冲洗磁珠。
步骤140、通过分离容器中的过滤件将磁珠与纯化目标物分离，过滤件开设有过滤孔，过滤孔的孔径小于磁珠的直径。
在一些实施例中，当磁珠上的杂质被移除之后，可以将磁珠上的纯化目标物从磁珠上脱离下来。在一些实施例中，可以向分离容器中加入第二液体，对堆积在过滤件上的磁珠进行冲洗。在一些实施例中，第二液体可以是指洗脱液，包括但不限于甘氨酸、柠檬酸盐或者醋酸钠等。关于洗脱液的更多描述，可以参见本说明书其他实施例的描述。纯化目标物与磁珠分离之后可以混合在第二液体中。由于过滤孔的孔径大于纯化目标物的直径，因此第二液体与纯化目标物可以经由过滤孔并通过分离容器的下方开口排出，而磁珠将会被过滤件截留在分离容器的腔体中。
在一些实施例中，可以收集经过滤件过滤且混合有纯化目标物的第二液体。在一些实施例中，分离容器中的第二液体可以通过与其下方开口连通的排液管路排出。在一些实施例中，可以通过收集组件收集第二液体。在一些实施例中，收集组件可以包括加液口和用于收集满足收集要求的第二液体的收集容器。加液口可以通过排液管道与分离容器的下方开口连通，将第二液体排到收集容器中。这里所说的收集要求可以包括第二液体中的纯化目标物的含量是否达到设定的值(例如，第二含量阈值)。关于此，可以在本说明书其他实施例中找到更详细的描述。
在一些实施例中，还可以对洗杂过程中排出的混合有杂质的第一液体进行收集。在一些实施例中，收集组件还可以包括废液暂存容器。废液暂存容器可以用于在洗杂处理中收集经由过滤孔通过分离容器的下方开口排出的第一液体。例如，分离容器的下方开口与加液口连通，加液口排出的第一液体可以排入到废液暂存容器。在一些实施例中，废液暂存容器还可以用于在洗脱处理中收集不满足收集要求的第二液体。
在一些实施例中，可以向盛装有第二液体的收集容器加入第三液体。第三液体可以包括中和液。在一些实施例中，中和液可以包括pH值偏高的缓冲液。在一些应用场景中，当纯化目标物的类型为抗体时，中和液可以用于将收集容器中的洗脱液中的酸性环境中和成pH中性，避免收集容器中的抗体长期处于酸性环境受到破坏。
在一些实施例中，本说明书一个或多个实施例中的纯化方法或步骤可以通过手动的方式执行。示例性的，操作人员可以手动控制(即手持)磁性件伸入混匀容器内吸附磁珠。在另一示例中，操作人员可以手动控制加液口移动到收集容器或废液暂存容器的上方开口。
在一些实施例中，本说明书一个或多个实施例中的纯化方法或步骤可以由特定的装置、组件或设备进行控制执行。在一些实施例中，上述方法或步骤可以通过控制设备进行执行。在一些实施例中，控制设备可以包括控制单元和驱动单元。控制单元可以用于接收工作指令，工作指令可以由操作人员输入，用于指示控制设备驱动前述实施例中的一个或多个组件或部件在相应步骤中执行相应功能。在一些实施例中，控制单元可以根据工作指令生成对应的指令，并将指令发送至驱动单元。在一些实施例中，驱动单元可以与前述实施例中的一个或多个组件或部件驱动连接。驱动单元可以接收来自控制单元的指令，并根据指令驱动对应的组件或部件执行特定功能。示例性的，控制单元可以根据工作指令生成第一指令并发送至驱动单元。第一指令与震荡平台对应，可以指示震荡平台在设定的工作时间内，按照设定转速对混匀容器进行旋转震荡。驱动单元接收 到第一指令后，可以驱动震荡平台进行工作。在另一示例中，控制单元可以根据工作指令生成第二指令并发送至驱动单元。第三指令与冲洗组件对应，可以指示冲洗组件的冲洗喷头按照一定的速率喷出特定的液体。驱动单元接收到第二指令后，可以控制冲洗喷头按照第二指令的指示进行工作。示例性的，驱动单元可以驱动与冲洗喷头连通的泵阀组件工作，通过冲洗喷头喷出第一液体对磁珠进行冲洗。
在一些实施例中，可以通过上位机对控制设备进行控制。上位机可以是指发出操控命令(例如，工作指令)的计算机。上位机可以与控制设备连接/通讯，通过将工作指令发送给控制单元，指示驱动单元驱动相应的组件或部件进行工作。在一些实施例中，上位机可以被手动操控，例如，操作人员可以手动操作上位机发出工作指令。在另一些实施例中，上位机可以通过控制程序或软件对前述实施例中的一个或多个组件或部件进行控制。在一些实施例中，上位机可以包括显示装置，例如显示屏。显示屏可以用于将一个或多个检测组件的检测结果以特定的形式(例如，数字、图像等)呈现。
在一些实施例中，在前述步骤的基础上，本说明书提供的纯化方法还可以包括一个或多个检测步骤。这里所说的检测步骤可以是指对步骤中的一个或多个参数进行确定。包括但不限于：分离容器中液体的液面位置、收集容器中液体的液面位置、与分离容器连通的排液管路中液体中的特定物质的含量(例如，第二液体中的纯化目标物含量、第一液体中的杂质含量)等等。
根据本说明书其他实施例的描述，在纯化过程中，需要向分离容器加入第一液体以及第二液体对磁珠进行洗杂和洗脱。在一些实施例中，为了保证杂质以及纯化目标物可以更快地脱离磁珠并与特定的液体混合，需要让特定的液体与磁珠充分接触，因此需要将分离容器内的液面位置控制在合适的范围内。在一些实施例中，可以对分离容器内的液面位置进行检测并根据检测结果对液面位置进行控制调整。为了方便描述，本说明书将以第二液体为例进行说明。
在一些实施例中，可以检测分离容器中的液面位置是否达到设定的第一液面阈值。若液面位置达到设定的第一液面阈值，则降低第二液体的进液量或提高第二液体的出液量。在本实施例中，当液面位置达到第一液面阈值时，可以认为分离容器内的液面位置过高，不利于磁珠与纯化目标物分离，因此需要降低液面位置。降低第二液体的进液量可以是指降低加入到分离容器中的第二液体的体积。示例性的，可以通过降低冲洗喷头喷出的液体流速来实现。提高第二液体的出液量可以是指提高从分离容器的下方开口排出的第二液体的体积。示例性的，可以通过提高排液管路排出的液体流速来实现。
在一些实施例中，可以通过第一检测组件来检测分离容器中的液面位置是否达到设定的第一液面阈值。在一些实施例中，第一检测组件可以包括第一液位传感器。可以将第一液位传感器设置在分离容器的第一位置来检测液面位置与第一位置之间的关系。其中第一位置的高度可以对应于第一液面阈值。
在一些实施例中，可以检测分离容器中的液面位置是否达到设定的第二液面阈值。若液面位置未达到设定的第二液面阈值，则降低第二液体的出液量或提高第二液体的进液量。在本实施例中，当液面位置未达到第二液面阈值时，可以认为分离容器内的液面位置过低，不利于磁珠与纯化目标物分离，因此需要提高液面位置。
在一些实施例中，可以通过第一检测组件来检测液面位置是否达到设定的第二液面阈值。在一些实施例中，第一检测组件可以包括第二液位传感器。可以将第二液位传感器设置在分离容器的第二位置来检测液面位置与第二位置之间的关系。其中第二位置的高度可以对应于第二液面阈值。
在一些实施例中，在将磁珠与纯化目标物分离之前，需要检测杂质是否与磁珠分离完毕。当确定磁珠上的杂质移除完毕后，方可利用第二液体对磁珠进行冲洗，以保证磁珠上没有残留的杂质混合在第二液体中或者杂质的含量可以忽略不计。
在一些实施例中，可以根据已加入到分离容器中的第一液体的体积来确定洗杂是否完成。当洗杂完成时，停止加入第一液体。在一些实施例中，可以检测已加入到分离容器中的第一液体的体积。当第一液体的体积达到设定的体积阈值(可称为第一体积阈值)后，可以认为洗杂已经完成。
在一些实施例中，已加入到分离容器中的第一液体的体积可以通过第二检测组件进行检测。在一些实施例中，第二检测组件可以包括第一流量传感器，第一流量传感器可以检测加入到分离容器中的第一液体的体积。
在一些替代性实施例中，可以通过检测经由过滤件的过滤孔通过分离容器的下方开口排出的第一液体的体积来确定洗杂是否完成。当第一液体的体积达到第二体积阈值时，可以确定洗杂已经完成。在一些实施例中，第二体积阈值可以等于第一体积阈值。
在一些实施例中，从分离容器的下方开口排出的第一液体的体积也可以通过第二检测组件进行检测。在一些实施例中，第二检测组件可以包括第二流量传感器，第二流量传感器可以检测分离容器的下方开口排出的第一液体的体积。
在一些实施例中，加入到分离容器中的第一液体的体积或者从分离容器的下方开口排出的第一液体可以不仅限于通过流量传感器来进行检测。在一些实施例中，操作 人员可以通过具有量度的容器手动测量加入到分离容器中的第一液体的体积。示例性的，操作人员可以采用具有量度的容器盛装与第一体积阈值相同或近似相同的第一液体，并直接将该容器中的第一液体加入到分离容器中。
在一些实施例中，可以通过检测来自分离容器中第一液体中的杂质含量来确定洗杂是否完成。在一些实施例中，可以检测来自分离容器中的第一液体中的杂质含量。当第一液体中的杂质含量达到第一含量阈值并且杂质含量维持一定时间不变时，停止加入第一液体。在洗杂处理中，加入第一液体将磁珠上的杂质移除或冲洗后，杂质会混合在第一液体中。可以理解的是，随着洗杂的不断进行，磁珠上的杂质会越来越少，混合在第一液体中的杂质含量会逐渐降低并趋于稳定。在一些实施例中，当洗杂液中的杂质含量达到第一含量阈值(该阈值可以称为基线)并且杂质含量维持了一定时间(例如，检测时间阈值)都没有发生变化或者几乎没有变化时，表明磁珠上的杂质已完全与磁珠分离或者残余的杂质可以忽略不计，即洗杂完成。
在一些实施例中，第一液体中的杂质含量可以通过第三检测组件来进行检测。第三检测组件是指检测液体中某种物质含量的仪器或设备。可以用于检测来自分离容器的第一液体中的杂质含量。示例性，分离容器的下方开口连接有排液管路，经由过滤件过滤的第一液体可以从分离容器的下方开口通过排液管路排出或者排放到其他部件中(例如，废液收集容器)中，第三检测组件可以设置在排液管路上，通过检测与分离容器连通的排液管路中的第一液体来确定其中的杂质含量。
在一些实施例中，第三检测组件可以包括与排液管路连通的含量检测传感器。在一些实施例中，含量检测传感器的类型与所要检测的杂质类型有关。为了方便描述，本实施例将以蛋白(除纯化蛋白以外的其他蛋白)作为所要检测的杂质为例进行说明。在一些实施例中，含量检测传感器可以包括紫外线吸收检测器。紫外吸收检测器可以根据所要检测的物质对紫外光吸收强弱与所要检测的物质浓度成正比的原理对杂质含量进行检测。在一些实施例中，紫外吸收检测器可以检测液体在紫外光吸收峰为280nm处的紫外光吸收值(或称为光密度值)，进而根据紫外光吸收值确定第一液体中除纯化蛋白以外的其他蛋白的含量。在一些示例性应用场景中，如图2所示，第一含量阈值可以通过posi：1对应的纵坐标值表示，posi：1对应的纵坐标值为5。
在另一些实施例中，可以利用废液暂存容器收集第一液体，因此可以通过含量检测传感器检测收集在废液暂存容器中的第一液体中杂质含量。
在一些实施例中，可以检测来自分离容器的第二液体中的纯化目标物含量是否 达到第二含量阈值。若纯化目标物含量未达到第二含量阈值，则将第二液体收集至废液暂存容器中。若纯化目标物含量达到第二含量阈值，则将第二液体收集至收集容器中。在洗脱处理过程中，加入第二液体将纯化目标物(例如，目标蛋白)与磁珠分离，纯化目标物会混合在第二液体中。洗脱过程的开始阶段，从磁珠上脱离的纯化目标物较少，混合在第二液体中的纯化目标物含量可能没有达到要求。因此这部分第二液体将会作为废液排放到废液暂存容器当中。随着洗脱的不断进行，脱离的纯化目标物不断增多，混合在第二液体中的纯化目标物含量也不断上升。直到其含量达到第二含量阈值时，表明洗脱已经达到纯化要求，可以对第二液体进行收集，因此可以将其收集至收集容器中。
在一些实施例中，来自分离容器的第二液体中的纯化目标物含量可以通过第三检测组件进行检测。在一些实施例中，可以通过第三检测组件检测与分离容器连通的排液管路中第二液体中的纯化目标物含量进行检测。示例性的，紫外吸收检测器可以检测第二液体在特定紫外光吸收峰(例如，紫外光吸收峰为280nm)处的紫外光吸收值，从而根据紫外光吸收值确定第二液体中抗体的含量。在一些示例性应用场景中，如图2所示，第二含量阈值可以通过posi：2对应的纵坐标值表示，posi：2对应的纵坐标值为100。
在一些实施例中，收集容器包括多个。可以检测当前收集容器的液面位置是否达到第三液面阈值。当当前收集容器的液面位置达到第三液面阈值时，将第二液体收集至其他收集容器中。当前收集容器可以是指当前正在收集第二液体的收集容器。在一些情况下，当当前收集容器中的液体达到第三液面阈值时，加液口可以移动至其他收集容器的上方开口继续收集第二液体，以防止当前收集容器中的液体溢出。在一些实施例中，当当前收集容器中的液体达到第三液面阈值时，可以控制加液口停止加液，同样可以防止当前收集容器中的液体溢出。
在一些实施例中，可以通过第四检测组件对收集容器中的液面位置进行检测。第四检测组件可以是指检测容器中液位是否达到设定液位的仪器或设备。在一些实施例中，第四检测组件可以检测收集容器中的液面位置是否达到第三液面阈值。
在一些实施例中，可以通过第四检测组件检测废液暂存容器中的液面位置是否达到第四液面阈值。当废液暂存容器中的液体达到第四液面阈值时，可以将液体通过设置在其底部的排出管路排到外部的容积较大的容器中，避免废液溢出。
在一些实施例中，第四检测组件可以与加液口相对固定，随着加液口的移动而移动，以便于对加液口对应的废液暂存容器或收集容器中的液面位置进行检测。在一些 实施例中，第四检测组件可以包括如前述实施例所描述的一种或多种液位传感器。在一些具体实施例中，第四检测组件可以包括超声波液面传感器。
在一些实施例中，在收集第二液体时，可以通过洗脱液(即第二液体)中的纯化目标物含量确定是否停止收集第二液体。在一些实施例中，可以判断第二液体中的纯化目标物含量是否达到第三含量阈值。当纯化目标物含量达到第三含量阈值时，可以确定停止收集第二液体。第三含量阈值可以是指满足收集要求的第二液体中的纯化目标物含量的最低值。在一些实施例中，第三含量阈值可以超过第二含量阈值。当第三含量阈值超过第二含量阈值时，若纯化目标物含量达到第三含量阈值，可以判断第二液体中的纯化目标物含量是否处于下降趋势(例如，可以基于若干个连续的时刻对应的纯化目标物含量数值(如10个数值)，确定目前的纯化目标物含量变化趋势)若是，则确定停止收集第二液体；若不是，则继续收集第二液体。在一些应用场景中，随着洗脱的不断进行，第二液体中的纯化目标物含量会逐渐上升至最高点然后下降，当纯化目标物含量纯化目标物含量处于下降趋势并达到第三含量阈值时，表明从磁珠上分离的纯化目标物较少，可以认为磁珠上残留的纯化目标物较少或完全没有残留。因此可以停止向分离容器中加入第二液体并结束对第二液体的收集。在一些实施例中，第三含量阈值可以小于第二含量阈值。当第三含量阈值小于第二含量阈值时，若纯化目标物含量达到第三含量阈值，可以直接确定停止收集第二液体。在一些示例性应用场景中，如图2所示，第三含量阈值可以通过posi：3对应的纵坐标值表示，posi：3对应的纵坐标值为80。
在一些实施例中，当停止向分离容器中加入第二液体并结束对第二液体的收集时，可以视为纯化过程结束。
在一些实施例中，当停止向分离容器中加入第二液体并结束对第二液体的收集后，还可以对前述实施例中的一个或多个管路以及磁珠进行清洗、消毒。在一些具体实施例中，可以控制冲洗组件(例如，冲洗喷头)向分离容器中加入其他液体(包括但不限于PBS洗杂液、纯水、氢氧化钠溶液)。例如，加入PBS洗杂液、纯水或氢氧化钠溶液等对分离容器内部、过滤件、排液管路以及磁珠进行清洗、消毒。又例如，将磁棒外套等部件浸泡在氢氧化钠溶液中进行消毒。
在一些实施例中，本说明书中的检测步骤可以通过操作人员手动操作完成。例如，操作人员可以直接采用液面检测传感器检测收集容器中的液面高度。
在一些实施例中，本说明书中的检测步骤可以通过控制设备控制执行。例如，控制设备可以控制第一检测组件检测分离容器中的液面高度是否达到设定的阈值。
图2是根据本说明书实施例所示的抗体洗脱紫外光吸收峰图。图2示出了基于本说明书提供的纯化方法对抗体进行纯化时，来自分离容器的液体(例如，来自与分离容器的下方开口连通的排液管道中的第一液体、第二液体)中的物质(例如，第一液体中的杂质、第二液体中的纯化目标物)的紫外吸收峰曲线。在图2所示的实施例中的一个或多个步骤(例如，洗杂处理以及第一液体的收集、洗脱处理以及第二液体的收集)可以通过前述实施例中的控制设备控制相应的部件或组件执行。在一些实施例中，纯化过程中采用的磁珠为琼脂糖Protein A磁珠。纯化目标物为单克隆抗体。图2中的横轴坐标为洗脱处理所经历的时间。纵轴坐标为第二液体在特定紫外光吸收峰时的紫外光吸收值，纵轴坐标的值越大，表示第二液体中的单克隆抗体的含量越高。曲线上的posi：1(位置1)可以表示洗杂处理结束时经分离容器的下方开口排出的第一液体的紫外光吸收值(即第一含量阈值)，其中，posi：1对应的纵坐标值为5。当第一液体的紫外光吸收值达到posi：1并趋于稳定(例如，维持检测时间阈值不变)时，表示洗杂结束，此时可以向分离容器中加入第二液体对磁珠进行洗脱处理。posi：2(位置2)可以表示设定的起始紫外光吸收值(即第二含量阈值)。当紫外光吸收值达到起始紫外光吸收值时，表明第二液体中的抗体的含量满足收集要求，此时可以对第二液体进行收集。根据曲线可知，在对应于posi：1至posi：2的时间范围内，紫外光吸收值未达到设定的起始紫外光吸收值。这部分第二液体将会作为废液排出(例如，废弃或者收集至废液暂存容器中)。posi：3(位置3)可以表示设定的结束紫外光吸收值(即第三含量阈值)。根据曲线可知，在对应于posi：2至posi：3的时间范围内，紫外光吸收值均达到设定的起始紫外光吸收值，因此这部分第二液体将会被收集(例如，收集至收集容器中)。当第二液体的紫外光吸收值小于结束紫外光吸收值时，表明第二液体中的抗体含量不再满足收集要求，此时可以停止对第二液体进行收集。
继续参考图2，由图2可知，抗体的紫外光吸收峰曲线从posi：2到达峰值posi：4(位置4)的速度以及从峰值posi：4到达posi：3的速度较快，表明在洗脱过程中，抗体可以在较短时间内与磁珠分离。同时，posi：3以后的紫外光吸收曲线降低幅度较小，即使经过较长时间(例如，从横坐标值为360处至横坐标值为480处)的洗脱，其紫外光吸收曲线也与横坐标轴近似水平，紫外光吸收值变化不明显。可以表明在posi：3对应的时间之后的洗脱过程中几乎没有抗体继续从磁珠上分离。这也就说明了磁珠上残留的抗体较少，在posi：3对应的时间之前的洗脱过程较为彻底。因此，根据上述实验结果可知，通过本说明书提供的纯化方法对纯化目标物(如，抗体)进行提纯时，可 以在较短时间内将纯化目标物与磁珠进行分离，有效提高了纯化速度。并且利用本说明书提供的纯化方法可以使纯化目标物与磁珠分离较为彻底，使得磁珠上残留的纯化目标物较少，有效提高纯化效果。
为了进一步验证抗体纯化效果，本说明书还提供与Resin纯化方法对包含有相同抗体的发酵液进行纯化的实验结果的对比。图3是根据本说明书一些实施例所示的抗体电泳图。图3示出了基于本说明书提供的纯化方法对抗体进行纯化洗杂处理后得到的洗杂液、洗脱处理后得到的洗脱液，以及基于Resin纯化方法得到的洗杂液和洗脱液的电泳结果。这里所说的电泳是指带电的生物大分子(如抗体)，在电场力的作用下进行定向迁移。在一些实施例中，在SDS-PAGE凝胶的分子筛中，生物小分子(例如，单糖、核苷酸等)迁移得较快，生物大分子迁移得较慢。分子量不一样的生物大分子具有不同的迁移速度。分子筛可以用于将不同分子量的生物大分子进行分离。通过考马斯亮蓝染色，实验人员可通过肉眼判断每个泳道中该样品包含多少不同分子量的生物大分子。通过泳道中染色区域的面积以及染色的深浅程度可以判断某种生物大分子的大致含量。通过分子量标准品Marker可以大致判断各个泳道中相应生物大分子的分子量大小。在一些实施例中，Resin纯化方法采用AKTA pure25纯化设备对抗体进行纯化。具体包括：对发酵液进行稀释，得到浓度为0.044mg/ml，体积为800ml的培养液。在一些实施例中，Resin法纯化实验以及基于本说明书提供的纯化方法的纯化实验所采用的培养液的浓度、体积相同。采用eppendorf 5920R高速离心机用于将粗样品(即培养液)进行离心，使细胞碎片等杂质沉淀，取上清液，以Millipore 0.22μm漏斗式一次性滤器进行抽滤，进一步去除杂质。最后将滤液使用AKTA pure25纯化设备配合GE Mabselect SuRe 5ml的Prepacked Resin层析柱进行过柱纯化，得到提纯的抗体。
在图3中，泳道1(即标号为1的下方矩形虚线框区域)表示Marker的电泳结果。在一些实施例中，Marker是一种标准品，是几种已知分子量的生物大分子(如蛋白质)的混合物。该泳道中不同位置的每一个条带都代表生物大分子的一个已知分子量。作为参照，实验人员可以通过将其余泳道中的样品条带与Marker条带进行对比判断出其大致的分子量大小。泳道2表示基于本说明书提供的纯化方法经洗杂处理后得到的洗杂液的电泳结果。泳道3表示基于本说明书提供的纯化方法经洗脱处理后得到的洗脱液合并试剂的电泳结果。泳道4表示基于Resin纯化方法得到的洗杂液的电泳结果。泳道5表示基于Resin法得到的洗脱液1的电泳结果。泳道6表示基于Resin法得到的洗脱液2的电泳结果。泳道5和6的样品为该Resin纯化方法实验中前后收集且未进行合并 的两管洗脱液。其中，泳道1中的上样体积为5ul，其余泳道中的上样体积均为10ul。根据图3的泳道3、泳道5和泳道6的结果可知，在泳道3、泳道5和泳道6中均显示有明显的条带。此外，由于泳道3中的黑色区域面积明显较大，因此可以确定泳道3中与抗原结合的抗体更多。同时，经进一步的测试数据显示，基于本说明书提供的纯化方法进行的纯化实验所得到的抗体总量为19.77mg，基于Resin纯化方法进行的纯化实验所得到的抗体总量为18.848mg，前者高于后者，表明基于本说明书提供的纯化方法得到的洗脱液中的目标抗体含量更高，纯化效果更佳。进一步的，根据图3的泳道2和泳道4的结果可知，在泳道2和泳道4中均没有与泳道3、5、6主条带大小一致的条带，因此可以确定基于Resin纯化方法及本说明书提供的纯化方法进行的纯化实验得到的洗杂液的上清液中均无明显目标抗体残留，说明磁珠与目标抗体结合较充分且牢固。
在一些实施例中，基于Resin纯化方法纯化400ml培养液需要耗时4.5小时。其中，Resin纯化方法需要对培养液进行离心和过滤，该过程需要耗时约30分钟。将已离心和过滤的培养液转移至层析柱中并进行过柱处理(即向层析柱中加入洗杂液将杂质与填料分离，以及加入洗脱液将纯化目标物与填料分离)，该过程需要耗时约4小时。而基于本说明书提供的纯化方法纯化同样体积、类型的培养液仅需耗时2.5小时。其中，将磁珠与纯化目标物进行混合需要耗时约2小时。将磁珠转移至分离容器中对磁珠进行洗杂和洗脱处理需要耗时约30分钟。需要说明的是，此处所指的耗时可以是指通过其他组件、装置或设备(例如，控制设备)执行本说明书提供的纯化方法所需要的时间。因此，基于本说明书提供的纯化方法极大地缩短了实验耗时，明显提高了纯化效率。
在一些实施例中，磁珠与纯化目标物混合所需要的时间可以根据需要而定。磁珠与纯化目标物混合所耗费的时间越长，培养液中残留的纯化目标物就越少，纯化目标物的回收率越高(即与磁珠混合的纯化目标物数量与培养液中的纯化目标物数量的比值)。例如，耗费15分钟将磁珠与目标抗体混合，即可达到85％的回收率。又例如，耗费45分钟将磁珠与目标抗体混合，即可达到98％以上的回收率。在一些实施例中，在基于本说明书提供的纯化方法进行纯化的过程中，为了尽可能地确保目标抗体回收率，将磁珠与目标抗体混合的时间延长至2小时，可能已经超出了所要求的目标抗体回收率，因此将磁珠与纯化目标物混合实际所需的时间可能会更短。例如，若要求目标抗体回收率达到85％，则将磁珠与纯化目标物混合实际所需的时间可能大约为15分钟，整个纯化过程不超过1小时。又例如，即使要求目标抗体回收率达到85％，纯化所需的总耗时也不超过2个小时。相对于前述实施例所描述的4.5小时耗时均有较大程度的缩短。
在一些实施例中，本说明书提供的纯化方法可以适用于特定的纯化设备，根据本说明书提供的纯化方法可以对该纯化设备进行控制，以对培养液中的纯化目标物进行纯化。
图4是根据本说明书一些实施例所示的纯化设备的简易结构示意图。如图4所示，在一些实施例中，该纯化设备10可以包括混合组件11、转移组件12以及分离组件13。混合组件11可以用于将磁珠与纯化目标物混合。转移组件12可以用于将磁珠转移至特定容器中，以便于对磁珠上的纯化目标物进行分离。分离组件13可以用于收容磁珠。
在一些实施例中，混合组件11可以包括震荡平台111和混匀容器112。在一些实施例中，转移组件12可以包括磁性件121。分离组件13可以包括分离容器131，分离容器131中设置有过滤件132。
在一些实施例中，磁性件121可以包括磁棒和套设在磁棒外的磁棒外套。转移组件12还可以包括供磁棒滑动的第一轨道、供磁棒外套滑动的第二轨道、供磁棒以及磁棒外套滑动的第三轨道。
在一些实施例中，纯化设备10还可以包括冲洗组件。冲洗组件可以包括冲洗喷头。
在一些实施例中，纯化设备10还可以包括收集组件14。在一些实施例中，收集组件14可以包括收集容器141和加液口142。在一些实施例中，收集组件14还可以包括废液暂存容器143。
在一些实施例中，纯化设备10还可以包括检测组件151。检测组件15可以包括第一检测组件151、第二检测组件152、第三检测组件153以及第四检测组件154。
在一些实施例中，纯化设备10的一个或多个组件或部件可以与前述实施例中的组件或部件相同或相似。示例性的，第一检测组件151、第二检测组件152、第三检测组件153以及第四检测组件154可以与前述实施例中的第一检测组件、第二检测组件、第三检测组件以及第四检测组件相同。在另一示例中，废液暂存容器143和/或收集容器1412、加液口142可以与前述实施例中的废液暂存容器和/或收集容器、加液口相同。
在一些实施例中，纯化设备10还可以包括控制组件16，控制组件16可以用于控制纯化设备10的一个或多个组件执行相应的功能。在一些实施例中，控制组件16可以包括控制单元161和驱动单元162。在一些实施例中，控制单元161和驱动单元162可以与前述实施例中的控制单元和驱动单元相同或者相似，关于控制单元和驱动单元的 更多描述可参见本说明书的其他部分。
图5是根据本说明书一些实施例所示的基于纯化设备进行纯化的示例性流程图。图5示出了基于前实施例中的纯化方法控制纯化设备10，以对培养液中的生物大分子进行纯化的具体过程，该过程500可以包括以下步骤：
步骤510、控制混合组件11进行混匀操作。
在一些实施例中，控制混合组件11进行混匀操作包括：首先将一定量的培养液和一定量的磁珠放入混匀容器112中，然后将混匀容器112固定于震荡平台111并控制震荡平台111对混匀容器112进行震荡，使磁珠与培养液中的纯化目标物结合。
步骤520、控制转移组件12中的磁性件121将磁珠转移至分离容器131的上方开口。
在一些实施例中，控制转移组件12中的磁性件121将磁珠转移至分离容器131的上方开口包括：将磁性件121的一部分伸入到混匀容器112的液面以下，利用磁性件121的吸附力使磁珠富集在磁性件121的表面。然后将磁性件121从混匀容器112中取出并移动至分离容器131的上方开口处。
步骤530、控制冲洗组件将磁珠从磁性件121上脱离，使磁珠进入分离容器131并堆积于过滤件132上。
步骤540、控制冲洗组件向分离容器131内加入第一液体。通过第一液体冲洗磁珠来移除未与磁珠结合的杂质。
步骤550、控制冲洗组件向分离容器131内加入第二液体。通过第二液体冲洗磁珠使得与磁珠结合的纯化目标物与磁珠分离。
步骤560、控制收集组件14收集来自分离容器131的第一液体和/或第二液体。在一些实施例中，可以通过与分离容器131的下方开口连通的收集组件14(例如，收集容器141)收集混合有纯化目标物的第二液体。在一些实施例中，可以控制收集组件14收集来自分离容器131的第一液体。在一些实施例中，可以通过与分离容器131的下方开口连通的收集组件14(例如，废液暂存容器143)收集混合有杂质的第一液体。
在一些实施例中，磁性件121可以包括磁棒。在将磁棒转移至分离容器131的上方开口处的过程中，可以控制磁棒沿第一轨道滑动，以规范磁棒的运动路径，提高运动过程中的稳定性。在一些实施例中，可以控制磁棒和磁棒外套分别沿第一轨道和第二轨道运动。在一些实施例中，可以控制磁棒和磁棒外套沿第三轨道运动。
在一些实施例中，当磁性件121位于分离容器131的上方开口处时，可以通过 设置在分离容器131的上方开口处的冲洗组件对磁珠进行冲洗来将磁珠与磁性件121分离。在一些实施例中，还可以通过减小磁性件121的磁力降低磁性件121对磁珠的吸附力，使磁珠在重力作用下与磁性件121分离。关于通过减小磁性件121的磁力降低磁性件121对磁珠的吸附力的更多细节，可以参见本说明书其他实施例的描述，此处不再赘述。
在一些实施例中，在洗杂过程中，可以通过第三检测组件153检测来自分离容器131中的第一液体中的杂质含量。例如，通过检测与分离容器131连通的排液管路中的第一液体确定其中的杂质含量，当杂质含量达到第一含量阈值并且维持检测时间阈值不变时，表明洗杂完成，可以停止加入第一液体。
在一些实施例中，在洗脱过程中，可以通过第三检测组件153检测来自分离容器131中第二液体中的纯化目标物含量。例如，通过检测与分离容器131连通的排液管路中的第二液体来确定其中的纯化目标物含量，当纯化目标物含量达到第二含量阈值时，表明可以对第二液体进行收集。又例如，当纯化目标物含量达到第三含量阈值且纯化目标物含量处于下降趋势时，表明从磁珠上分离的纯化目标物较少，可以认为磁珠上残留的纯化目标物较少或完全没有残留。因此可以停止向分离容器131中加入第二液体并结束对第二液体的收集。在一些实施例中，当停止向分离容器131中加入第二液体并结束对第二液体的收集时，可以视为纯化过程结束。在一些实施例中，当纯化目标物含量未达到第二含量阈值时，可以将第二液体收集至废液暂存容器143。在一些实施例中，当纯化目标物含量达到第二含量阈值时，可以将第二液体收集至收集容器141。在一些实施例中，当纯化目标物含量达到第三含量阈值且纯化目标物含量处于下降趋势(例如，可以基于若干个连续的时刻对应的纯化目标物含量数值(如10个数值)，确定目前的变化趋势)时，可以将第二液体收集至废液暂存容器143。
本说明书实施例的纯化方法可能带来的有益效果包括但不限于：(1)相较于Resin纯化法而言，无需对培养液进行离心和过滤，而是利用磁珠与纯化目标物混合使得纯化目标物与培养液分离，不仅简化了纯化步骤，还节省了纯化所需的时间；(2)相较于Resin纯化法而言，无需利用一次性滤器对层析柱进行真空抽滤，不仅简化了纯化步骤，还节省纯化过程的耗材成本；(3)利用磁珠与培养液中的纯化目标物进行混合，并利用磁性件将磁珠进行转移，实现纯化目标物与培养液的初步过滤，可以避免培养液造成的堵塞；(4)分离容器中无需设置额外设置其他磁珠来吸附纯化目标物，省去通过控制电机来带动永磁铁靠近和远离磁珠过滤柱从而控制其周围的磁场强度的繁 琐步骤。(5)通过控制相应的部件沿特定的轨道(例如，第一轨道(对应于磁棒)、第二轨道(对应于磁棒外套)、第三轨道(对应于磁棒外套和磁棒)运动，规范了对应的部件的运动路径，提高其运动过程中的稳定性；(6)利用特定液体对富集在磁性件上的磁珠进行冲洗，可以加快磁珠与磁性件分离的速度，从而提高纯化效率；(7)通过检测组件对纯化过程中的各种参数进行检测，例如，通过检测分离容器内的液面位置是否达到设定的位置或通过超声波液面传感器来判断相应液体体积，进而将液面位置控制在合适的范围，可以有效提高纯化效率；(8)利用本发明提供的纯化方法进行纯化时，可以省去对培养液离心和过滤的步骤，避免了离心和过滤时对培养液中的纯化目标物造成损失，有效提高了纯化目标物的回收率和纯化效果。
以上所述仅为本说明书的较佳实施例而已，并不用以限制本说明书，凡在本说明书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本说明书的保护范围之内。

Claims (17)

  1. 一种纯化方法，所述纯化方法包括：
    将磁珠与纯化目标物混合；
    将所述磁珠与所述纯化目标物转移至分离容器内；
    将所述磁珠与杂质分离；
    通过所述分离容器中的过滤件将所述磁珠与所述纯化目标物分离，所述过滤件开设有过滤孔，所述过滤孔的孔径小于所述磁珠的直径。
  2. 根据权利要求1所述的纯化方法，所述分离容器的两端均设置有开口。
  3. 根据权利要求1所述的纯化方法，所述将所述磁珠与所述纯化目标物转移至分离容器内包括：
    通过磁性件吸附所述磁珠；
    将所述磁性件移动至所述分离容器的上方开口；
    将所述磁珠与所述磁性件分离以使所述磁珠进入所述分离容器内。
  4. 根据权利要求3所述的纯化方法，所述将所述磁珠与所述磁棒分离以使所述磁珠进入所述分离容器内，包括：
    通过冲洗方式将所述磁珠与所述磁性件分离。
  5. 根据权利要求3所述的纯化方法，所述磁性件包括磁棒，所述将所述磁珠与所述磁性件分离以使所述磁珠进入所述分离容器内，还包括：
    减小所述磁棒与所述磁珠之间的吸附力，以使所述磁珠在重力作用下与所述磁性件分离。
  6. 根据权利要求5所述的纯化方法，所述磁棒包括电磁棒，当所述电磁棒处于通电状态时，所述电磁棒具有磁性，当所述电磁棒处于断电状态时，所述电磁棒失去磁性；所述减小所述磁棒与所述磁珠之间的吸附力包括：
    将所述电磁棒调整至所述断电状态。
  7. 根据权利要求5所述的纯化方法，所述磁性件还包括套设在所述磁棒外的磁棒外套，所述磁棒可沿所述套设方向相对所述磁棒外套移动；所述减小所述磁棒与所述磁珠之间的吸附力包括：
    将所述磁棒沿所述套设方向相对所述磁棒外套移动以使所述磁棒远离富集在所述磁棒外套表面的所述磁珠。
  8. 根据权利要求1所述的纯化方法，所述将所述磁珠与杂质分离包括：
    通过第一液体冲洗以将所述磁珠与所述杂质分离。
  9. 根据权利要求1所述的纯化方法，所述通过所述分离容器中的过滤件将所述磁珠与所述纯化目标物分离包括：
    向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离；
    收集经所述过滤件过滤的所述第二液体。
  10. 根据权利要求9所述的纯化方法，所述向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离包括：
    检测所述分离容器中的液面位置是否达到设定的第一液面阈值；
    若所述液面位置达到设定的所述第一液面阈值，则降低所述第二液体的进液量或提高所述第二液体的出液量。
  11. 根据权利要求10所述的纯化方法，所述方法还包括：
    检测所述分离容器中的液面位置是否达到设定的第二液面阈值，所述第一液面阈值大于所述第二液面阈值；
    若所述液面位置未达到设定的所述第二液面阈值，则提高所述第二液体加液量或降低所述第二液体的排液量。
  12. 根据权利要求9所述的纯化方法，在所述向所述分离容器中加入第二液体冲洗 位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离之前包括：检测已加入到所述分离容器中所述第一液体的体积；
    当所述第一液体的体积达到设定的体积阈值时，停止加入所述第一液体。
  13. 根据权利要求9所述的纯化方法，在所述向所述分离容器中加入第二液体冲洗位于所述过滤件上的所述磁珠，以将所述磁珠与所述纯化目标物分离之前，还包括：
    检测来自所述分离容器的所述第一液体中的杂质含量；
    当所述第一液体中的杂质含量达到第一含量阈值且维持检测时间阈值不变时，停止加入所述第一液体。
  14. 根据权利要求9所述的纯化方法，所述收集经所述过滤件过滤的所述第二液体包括：
    检测来自所述分离容器的所述第二液体中的纯化目标物含量是否达到第二含量阈值；
    若所述纯化目标物含量未达到所述第二含量阈值，将所述第二液体收集至废液暂存容器中；
    若所述纯化目标物含量达到所述第二含量阈值，将所述第二液体收集至收集容器中。
  15. 根据权利要求14所述的纯化方法，所述收集容器包括多个；所述将所述第二液体收集至收集容器中包括：
    检测当前所述收集容器的液面位置是否达到第三液面阈值；
    当当前所述收集容器的液面位置达到所述第三液面阈值时，将所述第二液体收集至其他所述收集容器中。
  16. 根据权利要求14所述的纯化方法，所述方法还包括：
    当所述纯化目标物含量达到所述第三含量阈值时，停止收集所述第二液体。
  17. 根据权利要求14至16任一项所述的纯化方法，所述将所述第二液体收集至收集容器中还包括：
    向所述收集容器中加入第三液体，所述第三液体用于中和所述收集容器中的酸性环境。
PCT/CN2023/072567 2022-01-17 2023-01-17 纯化方法 WO2023134773A1 (zh)

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US6040192A (en) * 1993-02-01 2000-03-21 Labsystems Oy Method and means for magnetic particle specific binding assay
US6448092B1 (en) * 1994-10-20 2002-09-10 Thermo Labsystems Oy Separation device for microparticles involving a magnetic rod
CN110177617A (zh) * 2017-01-04 2019-08-27 南京金斯瑞生物科技有限公司 高载量耐碱蛋白a磁珠及其使用方法
CN110407911A (zh) * 2018-04-28 2019-11-05 南京金斯瑞生物科技有限公司 磁珠纯化系统
CN112469826A (zh) * 2018-04-27 2021-03-09 (株)爱恩德生物 通过磁珠附着至细胞进行的基于磁性的生物淘选方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040192A (en) * 1993-02-01 2000-03-21 Labsystems Oy Method and means for magnetic particle specific binding assay
US6448092B1 (en) * 1994-10-20 2002-09-10 Thermo Labsystems Oy Separation device for microparticles involving a magnetic rod
CN110177617A (zh) * 2017-01-04 2019-08-27 南京金斯瑞生物科技有限公司 高载量耐碱蛋白a磁珠及其使用方法
CN112469826A (zh) * 2018-04-27 2021-03-09 (株)爱恩德生物 通过磁珠附着至细胞进行的基于磁性的生物淘选方法
CN110407911A (zh) * 2018-04-28 2019-11-05 南京金斯瑞生物科技有限公司 磁珠纯化系统

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