DE102005034327B3 - Device, useful for affinity separation, comprises non-magnetic separator container with space, fluid inlet and outlet, separation matrix for magnetizable particle in the space, magnetic field on the matrix and a rolling tool for pouring - Google Patents

Abstract

Device, for affinity separation by means of magnetic particles, comprises a non-magnetic separator container (1) with a space (9), at least one fluid inlet and outlet, a separation matrix for magnetizable particle in the space, a magnetic field (3) on the matrix and a tool for rolling the pouring, where the matrix comprises a pouring (2) from homogeneous magnetizable molded articles and the tool comprises a tilting- and/or a slewable device for the space.

Description

The The invention relates to a device for affinity separation by means of magnetic Microsorbents, in particular in biological suspensions according to the first Patentanpruchs.

such Devices are used for the selective separation of suspended or dissolved substances, e.g. Proteins from a solution. In addition, magnetic microparticles are added to the suspension (Microsorbents) which has a selective affinity to a or more of the aforementioned substances and these on bind. After the connection, the microsorbents with the selectively bound substances deposited in a magnetic field. Finally Washing and elution steps take place which bind the bound substance in purified and often in concentrated form release.

was standing The technique of bioproduct processing is usually an adsorption chromatography in a chromatographic column. However, this sets a complete Removal of all suspended solids from the suspension advance, allowing the chromatography precipitation, centrifugation and often also precede microfiltration stages. Although this causes usually particle-free solutions win, leads this multi-step approach to long process times as well a reduction of the product yield.

A Reduction of the number of processing steps now offers this Potential the high proportion of refurbishment costs to the total cost of a To reduce bioprocessing. The reduction of the share of refurbishment costs could thereby by lower investment costs as well as by an increase of the Product yield can be effected.

A possibility to reduce the number of processing steps offers the direct and selective separation of bioproducts from cell suspensions, Cell cultures and cell digestions, e.g. using special affinity matrices, immobilized antibody or receptors. The one for this on a laboratory or industrial scale investigated methods such as "fluid-bed" adsorption, EBA (expanded bed adsorption) and "Aqueous Two Phase "extraction could not find broad acceptance so far, so that from sides The bio-industry has a strong need for a robust process for one direct, primary Bioproduct purification exists.

One Interesting approach to direct, primary bioproduct purification is the use of magnetic micro sorbents in combination with suitable magnetic technology. In the field of bioanalytics and the Medical diagnostics become magnetic microparticles (Microsorbents, Magnetbeads) have long been successful for the isolation of e.g. nucleic acids or proteins applied [1, 2, 3]. It will be in a first Step magnetic particles (beads) into a container (e.g., mixing vessel) with the brought the substance containing suspension in contact. Then be the loaded magnetic particles by applying a magnetic field to a wall surface in container pulled and held. In a next step, the rest of the suspension with applied magnetic field, i. without the trapped particles removed from the vessel and by a rinsing liquid replaced. After deactivation of the magnetic field, a suspension takes place the particle into the rinse solution, the Removal of spent rinse solution as well subsequently the desorption or elution of the biosubstance by resuspension of Particles in an elution buffer. Finally, the elution buffer discharged together with the biosubstance, while the magnetic beads again withheld magnetic field become.

While the specific binding of solutes, especially biomolecules, on Magnetbeads and the reagents for the subsequent rinsing and elution steps in principle transferable to the technical scale are, lets a separation of magnetic microparticles with diameters of approx. 1 μm on a pilot or technical scale certainly not over the simple attachment of a permanent magnet to the outer wall a stirrer accomplish. While due to the small particle size and magnetic drum separators for such Separations are not suitable, is the applicability of so-called high gradient magnetic separators for the industrial separation of magnetic microparticles from the Mineral processing and environmental technology [4, 5].

The general suitability of high-gradient magnetic separators for the separation of magnetic microparticles, also in the context of bioproduct processing, has already been demonstrated several times [3, 6]. In addition to the actual high-gradient magnetic separation system (HGMS system) in particular the realization of a circuit with lines and valves falls on, in which the magnetic bead suspension can be pumped. Namely, the necessary rinsing and elution steps can be carried out efficiently only in a suspended and non-agglomerated or bound state of the particles. Consequently, in the course of the overall process, the particles often have to be rinsed out again after separation in the magnetic separator and effectively deagglomerated by means of a washing or eluting solution. Subsequently, the suspension is again through the Magnetsepa Pumped pumped and thereby freed from the particles. In the illustrated construction, the washing and elution steps occur within a cycle. However, it was found that insufficient backwashing of the particles from the separator is a serious problem of the process. While backwashing in the case of pure particle model solution is still possible with efficiencies of over 90%, the presence of real biosuspensions often leads to backwashing efficiencies below about 70%. The reason for this is a tendency of the particles to adhere to the deposition matrix and the particles to one another (agglomerates) due to the biological substances. As a remedy, various ways of improving such as ultrasound or mechanical shocks were tested, but only partially remedied. In addition, the problem of agglomeration of the micron particles often remains even after the separation of the particles from the separator matrix.

When Deposition matrix becomes ordinary a wire mesh used.

Further in [8] and [9] a deposition matrix with a spherical packing is created made of very small, coated spheres for separation magnetic labeled cells or biomolecules proposed.

A measure to improve the particle backwashing a ball bed as a deposition matrix is described in [7]. It is about around a carousel magnetic separator whose annular deposition matrix by a ballast is formed. To clean the matrix is a continuous small part of the balls over an outlet opening stripped, externally washed and over conveyor belt back again brought into the matrix housing.

These external washing, however, is expensive and in case of necessity a quick backwash the entire matrix impractical. In addition, the described guarantees Ball migration through a sieve bottom no effective deagglomeration the rinsed off Particle agglomerates. Purpose of the device described in [7] it's just cleaning the balls and not the deagglomeration of particle dressings.

In the abovementioned Abscheidematrices the detachment of the separated particles from the separation elements (balls) by means of a flow or a spray the ball packing Based on this, the object of the invention to propose a device that can handle magnetic Microsorbents for Separations in the preperative and technical scale easier and implemented more efficiently, and to a lesser extent also especially for a use for Suspensions with biological substances with basically increased adhesion tendency.

The Device should be both the separation and in particular enable the effective resuspension of the magnetic microparticles, and without complex circuits and valve circuits.

The Task is performed by a device with the characteristics of the first Patent claim solved. The under claims give advantageous embodiments the device again.

The Device comprises a non-magnetic separator with a separation space having at least one fluid inlet and at least one Fluid outlet, a deposition matrix for magnetizable particles in the separation space, comprising a bed of similar magnetizable moldings and a magnetic field acting on the deposition matrix. The separation space is preferably oblong, has two Ends on, preferably at both ends by a non-magnetic fluid-permeable wall as a fluid inlet or fluid outlet is limited. The magnetic field is suitable Agents such as e.g. activate a process control or a switch and deactivatable, for example by removal of permanent magnets or by switching off electromagnets.

In the through the separation space durchzuseitende and selectively cleaned off Suspension are the aforementioned magnetic particles as micro sorbents then suspended. Preferably, these particles have one for certain substances to be filtered out have high affinity, so that they are preferred, i.e. selectively attach or attach to the particle. This can for example, by a specific assembly of microsorbents take place, e.g. above functionalization of the surface (e.g., activation).

The fluid pervious Walls are permeable for all potential fluids to be treated, i. Liquids, Suspensions, aerosols but also particle streams such as dusts without liquid phases, but not permeable for the Moldings, the as a batch form the deposition matrix. Preferably, the walls have openings (holes, gaps etc.), whose cross-sections are dimensioned so that they from the moldings are not permeable in any Projketionsrichtung. Preferably they have a width, which is the smallest cross-sectional dimensions clearly below.

An essential feature of the invention um includes means for circulating the bed in the separation tank. The circulation causes rubbing of the moldings with each other and on the Separationsraumwandung and thus a particularly efficient way of separating the magnetizable particles from the mold body surfaces and the deagglomeration of the detached particle dressings. The separation is particularly effective when the moldings each have a completely outwardly directed molding surface, which are accessible to the other moldings in a circulation as Reibeflächen. The shaped bodies therefore preferably also have no pores, openings, holes, deeper depressions, surface roughness or other depressions or undercuts.

Preferably takes the fill, comprising the shaped bodies and the spaces between between the moldings, for one better recyclability not the entire Separationsraumvolumen, but a maximum of 95 %, preferably between 20 and 70%, more preferably between 25 and 50%.

The Device is therefore particularly suitable for use for affinity separation of biological substances by means of magnetic micro sorbents in real biosuspensions. Alone through the mechanical and thus particularly efficient cleaning of the abrading moldings increases namely the backwash efficiencies below about 70% (cf state of the art in connection with biological substances) to more than 95 %, despite the aforementioned increased adhesion tendency of particulate matter in the presence of biological substances.

The fill must for a fluid (esp. For the Suspension and the rinsing liquid) good flow through be, i. the moldings allowed to occupy only a certain volume fraction in the bed. preferably, are the molding surfaces not flat, but have an outwardly directed with respect to the shaped body bulge on.

Preferably are the moldings in particular with regard to the aforementioned upheavals Balls of a magnetizable material, wherein the molding surface preferably hardened is or preferably a wear protection layer and a general low wettability and low affinity for the suspension to be passed through, especially the substances contained therein.

The Use of spheres as a shaped body for a bed, the can be used as a deposition matrix in the invention is for the reasons mentioned above the bulk flowability and especially the cleanability particularly advantageous.

The Device has an operating state with activated magnetic field and a cleaning condition preferably, but not absolutely necessary, with deactivated magnetic field.

in the ongoing operation (operating state) is an admission the molded body by the applied magnetic field, wherein the separation space with the bed is traversed by the fluid to be cleaned with the magnetic particles. As a result, the magnetic particles attach themselves to them these bound substances, e.g. biological substances or molecules to the molding surfaces.

The Cleaning of the molding surfaces in Cleaning condition happens in the proposed device by replacement the particle in a flushing fluid within the closed separation vessel, i. without flow alone due to the relative speed between the freely moving Separation elements (shaped bodies) and the solution due to a rearrangement of the bed. The moldings do not leave the separation room. This is a Spülfuid in filled the separation space, where at the same time the original Biosuspension displaced is and the particles held by the magnetic field to the molding become. Subsequently a closure of the Valves (fluid inlet and fluid outlet) to the separation chamber and a deactivation of the magnetic field. By the aforementioned means a revolution the molded body become the on the moldings adhering magnetic particles together with the bound substances sheared off from the mold body surfaces and the rinse fluid added. additionally the breakup occurs detached particles associations due to the countless ball collisions during the circulation, comparable to Principle of a ball mill. Subsequently there is a derivative of the flushing fluid, the magnetic field in the area of the bed previously again for particle retention has been activated.

the same Procedure as for the rinse fluid is repeated with an elution fluid to give a desired Desorb protein (target protein) from the particles and thus to win in a suitable form.

The Means of upheaval the bed include means for relative movement of the moldings to each other and cause the Separationsrauminnenwandungen.

The simplest variant for these means is based on a tilting and / or a rotating device for the entire separation space, optionally at the Separationsrauminnenwandungen attached mixing blades accelerate a circulation. The circulation of the bed is made to the relevant part solely by a change in the orientation of the bed to the direction of gravity. A prerequisite for an effective circulation is a comparatively small volume fraction of the bed in the separation chamber volume, preferably between 10 and 60%, preferably between 20 or 25 and 50%, while the remaining empty volume fraction in the separation volume serves as an escape space for the bed. If this is too small, ie the separation space filled to over 70% with moldings or the bed and thus too full, the moldings impede increasingly in their individual movements and thus the Umwälzungseffizienz. In addition, the sliding friction activity of the molded bodies also reduces against each other and thus also the cleaning efficiency.

alternative The abovementioned means for circulating the bed at least comprise two components in the separation space, which move relative to each other carry out. In this case, at least one of these components through the Separationsraumwandung be formed. Through these measures the circulation takes place active by the aforementioned relative movement, whereby by the components a shift to the moldings of the Transferred bed and the aforementioned influence of gravity at most one plays a minor role. The meaning of the aforementioned bulk-free Alternative space for the moldings in Separation room is for a high circulation efficiency not so relevant anymore. Consequently, the volume fraction of the bed in the separation volume between 10 or 20% and preferably 50%, 60%, 70% or 80 % can be set. The means preferably comprise a rotatable Stirring element or a screw conveyor in the separation room.

The mentioned upper limits of the volume fraction are preferably possible if the moldings Balls are.

The Invention is based on embodiments and the following figures explained. Show it

1 a schematic side view of a manually operable first embodiment, for example with electromagnets,

2 a plan view of a permanent magnet based embodiment with a schematic representation of a mechanical circulation device and

3 a schematic perspective view of another embodiment.

1 shows a side view of a first embodiment of the device for affinity separation. The separator tank 1 consists of a non-magnetic material, such as plastic, and is characterized in that the elongated separation space 9 with a bed 2 at both ends by a lid 4 can be sealed with respect to liquid leakage. The separation room 9 is at its upper and lower end respectively by a non-magnetic perforated plate 5 (fluid-permeable wall) limited by this example between the lid 4 and the separator tank 1 is clamped. The perforated plate is characterized in that the dimensions of the holes are chosen so that the retention of moldings of the bed 2 (Deposition matrix) is ensured, however, liquid passage can take place.

The bed 2 itself consists of a plurality of similar soft magnetic moldings, for example balls with a diameter of 1-5 mm, which in the embodiment, the separation space 9 to fill 10-50%.

The lids 4 are provided with at least one hole each for the fluid inlet and fluid outlet (inlet or outlet for the separation space). These are constructive as connecting pieces 6 . 7 with valves 14 . 15 placed on the lids, so that fluids such as liquids or suspensions in the Separatorbe container can be inserted and removed. In addition, the upper lid includes at least a second bore with outlet 8th , which serves as an air outlet for pressure equalization in the separator and can also be closed by a valve.

The separator tank 1 is in upright position with the bed during operation 2 filled container portion in the region of a magnetic field 3 , eg within a coil of an electromagnet, a superconducting magnet or between the poles of a permanent magnet.

For operation (operating state) is first an equilibration buffer, for example a protein binding buffer in the separation space 9 filled. If this is completely filled, the air valve is at the outlet 8th closed and fed to the separator, the Biorohsuspension with the magnetic particles, which are loaded with the target product on affinity ligands, while discharged via the lower valve of Equilibirierungspuffer via a pump. Here, the magnetic field is turned on, so that the magnetic particles are retained within the bed. After the original in the separation room 9 present fluids is replaced by (eg Bioroh-) suspension, as long as still Biorohsuspension can be supplied with magnetic particles until the particle absorption capacity of the bed is almost exhausted. At this point, the separation process is stopped and then the biorohine suspension still inside the separation chamber from the lower valve 15 discharged while over the upper valve 14 at the same time washing buffer (rinsing liquid) is introduced.

Thereafter, the fluid inlet and outlet via the valves 14 and 15 closed and the separator with the magnetic field switched off manually or by a device from the coil or the pole pieces 16 (see. 2 ).

By the means 11 for circulating the bed, such as by the rotation of a stored shaft 10 that have a non-magnetic cuff 13 is flanged (cf. 2 ), or manually, the bed is rearranged by the separator several times by 180 ° about its central axis by rotation 12 is moved in the vertical. This results in a shift of the forming the body molding due to gravity and thereby to their intensive mixing. The mutual contact of the moldings with each other and with the washing buffer leads to an effective detachment of the deposited magnetic particles. In addition, the separation bodies break up any microsorbent agglomerates and at the same time serve as agitating tools to ensure complete resuspension of the particles. After this wash, the separator is re-inserted into the coil or between the pole pieces 16 brought and turned on the magnetic field to retain the magnetic particles. By replacing the used rinsing liquid with fresh rinsing liquid while simultaneously discharging the used rinsing liquid, the first washing step is completed.

The the aforementioned rinsing procedure is preferably repeated 1-2 times as part of a purification process. After the 2-3 Washes (rinses) is those in the container remaining rinsing liquid replaced by an elution buffer containing the biological target product releases from the magnetic particles. Further Elution steps are optional. By the final discharge of the elution buffer, the product is purified and useful solids-free solution in front.

An alternative embodiment of the device shown in FIG 3 , sees a motor-driven non-magnetic auger 17 in the separator room. The sealed against the separator shaft 18 the screw conveyor 17 is from one of the covers (not explicitly shown, but see 1 ) led out. For a particle transfer in this case provides the axially oriented conveying action of the screw so that the separator does not have to be lifted out of the magnetic field. In addition, any very solid microsorbent agglomerates may be easier to break up than by merely changing the position / rotation of the separator.

Literature:

• [1] Olsvik O., Popovic E.S., Cudjoe K.S., Hornes E., Ugelstad J. & Uhlen M .; Magnetic separation techniques in diagnostic microbiology; Clinical Mirobiol. Reviews 7 (1994) 43-54
• [2] Whitesides G.M., Kazlauskas R.J .; Magnetic separation in Biotechnology; Trends in Biotechnology Vol.1, No.5 (1983) 144-148
• [3] O'Brien S.M., Thomas O.R.T., Dunnill P .: Non-porous magnetic chelator supports for protein recovery by immobilized metal affinity adsorption; J. Biotech. 50 (1996) 13-25
• [4] Franzreb, M., Höll, W.H .; Phosphate removal by high-gradient magnetic filtration using permanent magnets; IEEE Trans. Appl. Superconductivity 10, No.1 (2000) 923-926
• [5] Franzreb, M .; Use of magnetic separators for wastewater treatment; Environment 27, No.11-12 (1997) 41-43
• [6] Hubbuch, J.J., Thomas, O.R.T .; High-gradient magnetic affinity separation of trypsin from porcine pancreatin; Biotech. and bioeng. 79, No.3 (2002) 301-313
• [7] Svoboda, J., Magnetic methods for the treatment of minerals, Development in Mineral Processing 8, Elsevier Sc. Publishers, Amsterdam, 1987
• [8th] US 5,711,871
• [9] US 6,417,011

1

Separator

2

fill

3

magnetic field

4

cover

5

perforated sheet

6

upper spigot

7

lower spigot

8th

outlet

9

separation chamber

10

manipulator

11

medium

12

rotation

13

cuff

14

upper Valve

15

lower Valve

16

pole

17

Auger

18

wave

Claims (12)

Magnetic particle affinity separation apparatus comprising a) a non-magnetic separator vessel ( 1 ) with a separation space ( 9 ) with at least one fluid inlet and at least one fluid outlet, b) a deposition matrix for magnetisable particles in the separation space, comprising a bed ( 2 ) of similar magnetizable moldings, c) a magnetic field ( 3 ) acting on the deposition matrix and d) means ( 11 ) for circulating the bed, wherein the means comprise a tilting and / or pivoting device for the separation space. Apparatus according to claim 1, wherein the shaped bodies are balls are. Apparatus according to claim 1 or 2, wherein the shaped body of are made of a soft magnetic iron material. Device according to one of the preceding claims, characterized in that the shaped bodies on their molding surface and / or the inner wall of the separation space ( 9 ) or have a wear-reducing coating. Device according to one of the preceding claims, wherein the particles have a functional coating for affinity separation exhibit. Device according to one of the preceding claims, wherein the separation space is elongate and has two ends, wherein the separation space ( 9 ) at its two ends in each case by a non-magnetic fluid-permeable, but for the molding impermeable wall ( 5 ) is limited. Apparatus according to claim 6, wherein the walls pass through Perforated sheets with breakthroughs are formed, with the breakthroughs are smaller in size than the shaped bodies. Device according to one of the preceding claims, wherein the bed in the separation space occupies a volume fraction of between 10 and 60%. Device according to one of claims 1 to 8, wherein the means at least two relatively movable components in the separation space exhibit. Apparatus according to claim 9, wherein at least one Component is formed by the Separationsraumwandung. Apparatus according to claim 9 or 10, wherein at least a component by a rotatable stirring element or a screw conveyor is formed in the separation space. Device according to one of claims 9 to 11, wherein the bed in the Separation space occupies a volume fraction of between 10 and 95%