HK1150995B - High gradient magnet separation of biological material - Google Patents

Description

High gradient magnetic separation of biological materials

Technical Field

The present invention relates to the use of High Gradient Magnetic Separation (HGMS) technology for the separation and purification of biological materials.

Background

Especially in the field of biomedical research, the separation and purification of defined particles from heterogeneous particle suspensions is of great importance for a variety of different analytical methods. In general, the particles to be purified, hereinafter referred to as "target particles", are usually only minimally different from the rest of the particles contained in the suspension, hereinafter referred to as "non-target particles". The target and non-target particles are typically cells or cell fragments, but may be any other biological substance.

Some existing separation methods make use of the magnetic properties of the target particles, wherein the target particles have naturally occurring "intrinsic" magnetic properties, or wherein the target particles are labeled by targeted binding of synthetic magnetic particles prior to the actual separation step.

The intrinsic magnetic particles are, for example, red blood cells, provided that: hemoglobin contained in red blood cells exists in a state of being deoxygenated or oxidized rather than oxygenated. Here, deoxygenation means that no oxygen is carried, while oxygenation means that oxygen is carried. In the latter case, the hemoglobin molecule carries the oxygen molecule by non-covalent, that is to say reversible, binding. Distinguished from this is the oxidation state of hemoglobin, in which an oxygen atom or other oxidizing atom is covalently, i.e., irreversibly, bound to the central iron atom of the hemoglobin molecule. At this time, in the deoxygenated and oxidized forms (but not the unoxygenated forms), the orbitals of the central iron atom contained in the hemoglobin molecule carry unpaired electrons. The unpaired spins of these electrons achieve induced magnetic poles in the iron atoms by applying a magnetic field.

However, the applied magnetic field of the conventional type cannot exert a net directed magnetic force on particles containing such iron atoms, because the attractive and repulsive magnetic forces at the north and south poles of the polarized iron atoms are balanced due to the very small diameter of the atoms. Likewise, upon removal of the magnetic field, the particles will lose their polarization. This magnetic property is called "paramagnetism". A particular form of paramagnetism is sometimes referred to as "superparamagnetism". However, there is no completely clear distinction between these two forms, and therefore in the following the term "paramagnetic" or "paramagnetic" will encompass the terms "superparamagnetic" and "superparamagnetic".

Likewise, synthetic particles useful for magnetic labeling of target particles are paramagnetic and, much like oxidized hemoglobin, typically contain very little oxidized iron or another magnetizable substance. It is furthermore desirable that the synthetic particles are so small that they form stable colloids in suspension, in other words they do not settle over a long period of time (months to years), and therefore the diameter of the synthetic particles is typically 30-200 nm. Commercially available particles of this type are sold, alone or coupled to antibodies, for example, by Chemicell GmbH (Eresburgstrasse 22-23, D-12103 Berlin, Deutschland), micromodule Partikeltec technology GmbH (Friedrich-Barnewitz-Str.4, D-18119 Rostock, Deutschland), oder Miltenyi Biotech GmbH (Friedrich Ebert Stra. beta.e 68, D-51429 Bergisch Gladbach, Deutschland).

A feasible method for purifying paramagnetic particles, that is to say separating paramagnetic particles from a suspension of particles, is to generate a very high magnetic field gradient. A sufficiently high magnetic field gradient results in: the north and south poles of the paramagnetic particles experience a difference in attractive and repulsive forces and thus produce a net magnetic force directed. This technique is known under the name High Gradient Magnetic Separation (HGMS).

Here, a distinction needs to be made between so-called "internal" and "external" high gradient magnetic separators. The first introduction to the HGMS technology involved an internal separator. They can be found in Obertouffer (IEEE Transactions on Magnetics, Mag-9, No.3, September 1973: 303-. The ferromagnetic material in a suitable non-magnetic container serving as the separation chamber is introduced into a strong, uniform magnetic field, which may be generated by an electromagnet or a permanent magnet in the shape of a horseshoe (dipole magnet). In this case, the ferromagnetic material is generally referred to as a "matrix"; it may be filiform (wire or fiber), spherical (ball-shaped), or of a different shape, for example it may be constituted by a punched sheet of steel. The ferromagnetic material of the matrix acquires a magnetization corresponding to its magnetic susceptibility X by an externally applied field. Starting from the surface of the matrix material, a magnetic field gradient is thus generated which can reach more than 100 tesla per cm, wherein the magnitude of the gradient is inversely proportional to the diameter of the wire-like or spherical elements used. An "external" high gradient magnetic separator, achieving similar high gradients by a technically more complex special arrangement of magnets outside the intrinsic separation chamber, is disclosed for example in WO 98/055236, WO 99/019071 or US 6.241.894B 1. As an important difference, the necessity of the matrix being located in the separation chamber is eliminated.

Today, internal high gradient magnetic separators are most widely spread in biomedical research. Specific embodiments of such separators are described in US 4.664.796 and US 5.200.084.

US 5.200.084 discloses a device and a method specifically directed to the purification of very small amounts of biological material in recesses of microtiter plates. In this, purification up to 83% of CD4 cells labelled with paramagnetic second particles from Peripheral Blood Mononuclear Cells (PBMCs) is described.

To the knowledge of the inventors, only one technical embodiment of an internal high gradient magnetic separator is currently used to achieve high purification rates, as disclosed in WO 96/26782 and EP 0.942.766B 1. In order to avoid non-specific binding of non-target particles to the matrix, which binding would impair the purification result in the separators described in the mentioned publications, the separation chambers of which have a matrix coated with a polymer. As described in detail in WO 96/26782, the polymer is applied to the substrate in several steps. According to the information given by the authors, the separating chamber thus produced is characterized in that the polymer forms a strong, closed, liquid-and ion-impermeable coating containing less than 1% water on the ferromagnetic material of the matrix; and the polymer-coated matrix fills 60-70% of the total volume of the described separation chamber.

In addition to reducing non-specific binding of non-target particles to the matrix, according to the mentioned disclosure, the coating is believed to also avoid damage (physical damage) of the biological material to be separated due to direct contact with the ferromagnetic matrix material, as well as to exclude chemical reaction of the ferromagnetic matrix material with a buffer solution used for suspending the biological material, since the released ions may also cause damage (chemical damage) of the biological material to be separated. However, both of these injuries will be considered to be postulated because no scientifically reliable knowledge can be provided for this purpose. In addition, Paul et al, Clinical and Laboratory Haematology, 7, 1985: 43-53, which in turn reported that the morphology and viability of blood cells and blood cell fragments passed through the untreated stainless steel matrix of the HGMS column were not impaired.

All patents and publications mentioned so far are hereby incorporated by reference.

The separation chamber described is, above all, costly for the substrate coating, since its production process is time-consuming. Such separation chambers are commercially available from Miltenyi Biotech GmbH (cited above).

Such a separation chamber achieves a high degree of purification, in particular when used for purifying synthetic paramagnetic particle-labeled particles from a particle suspension.

The separation chamber with the coated substrate was investigated for intrinsic (native) paramagnetic particles. Herein, malaria-infected red blood cells are used as intrinsically paramagnetic particles. The causative agent of malaria, a parasite of the genus Plasmodium (Plasmodium) from the protozoan kingdom, selectively attacks red blood cells and has the property of oxidizing the central iron atom of the free heme molecule produced in the infected red blood cells to ferric iron. As mentioned above, it is paramagnetic. Therefore, malaria-infected red blood cells should be separable from uninfected and oxygenated red blood cells in the separation chamber of the high gradient magnetic separator. This was first noted in 1981 by Paul et al (Lancet, July11, 1981: 70-71). The commercially available separation chamber with coated substrate should achieve up to now more than 80% purification (Uhlemann et al, MACS & more 2000; 4 (2): 7-8; Trang et al, Malaria Journal 2004; 3: 1-7).

However, the cited studies only deal with one of the four known pathogenic malaria pathogens occurring in humans, namely Plasmodium falciparum (Plasmodium falciparum), the causative agent of tropical malaria, and another malaria pathogen in rodents, Plasmodium berghei (Plasmodium berghei). Other scientific studies on a total of approximately 120 other known plasmodium species are not yet available. However, it is known to the present inventors that the purification of red blood cells infected with Plasmodium vivax, a pathogen of Plasmodium vivax, also occurring in humans, using commercially available separation chambers does not always give satisfactory results.

As is apparent from the above explanation, further improvement of the purification efficiency of the HGMS technique would be extremely useful for the biomedical research field. This is also suitable in terms of cost-effectiveness, since the known sophisticated separation chambers are not available in many fields solely for reasons of price, in particular in the study of malaria pathogens, which are particularly influential in countries with a low medical and research budget.

Disclosure of Invention

It is therefore an object of the present invention to provide an HGMS separation column which achieves better purification results in a cost-effective manner.

This object is solved by an HGMS separation column according to claim 1 and a method according to claim 10, wherein an internal HGMS is used. The solution of the present invention is based on the principle of solving the problem of non-specific binding of non-target particles to the matrix of the HGMS separation column by means of a buffer solution for equilibration of the separation column and for suspension of the biological material to be separated having the properties set out in the claims. This allows the elimination of complex and costly coatings, since the buffer solution saturates the non-specific binding sites of the separation column. Such non-specific binding sites are to be understood here as being sites at which particles bind independently of their magnetic properties, and at which sites the particles therefore bind mechanically, electrically, chemically, physically or otherwise. Thus, non-target particles will be screened independent of magnetic gradients, which will lead to incomplete purification or separation results. The biological material is preferably cells, cell aggregates or cell components which have intrinsic paramagnetism or which can be directly or indirectly labelled with paramagnetic or superparamagnetic particles.

An advantage of the solution according to the invention is that the buffer solution can be provided relatively simply and cost-effectively. Time-consuming, material-and cost-intensive pretreatment of the separation column and of the matrix contained therein can be dispensed with. This simplification has the advantage here of even improving the purification results compared with conventional separation columns with a complex coating of the substrate.

Preferably, the buffer solution has a density that matches the density of the particles of biological material to be separated to such a high degree that the gravitational forces acting on the particles are substantially compensated for, so that the particles almost float in the buffer solution, and/or the buffer solution has a high viscosity, so that the buffer solution can flow through the separation column in layers at a flow rate suitable for the separation process. In this way, the target particles can remain free of the disruptive influence of gravity in the region of action of the high-gradient magnetic field for such a long time that they precipitate out to a very high degree. In this way, a particularly high degree of separation and purification is obtained.

Preferably, the magnet is a permanent magnet or an electromagnet, which is shaped in such a way that the separator column can be arranged within a particularly homogeneous magnetic field generated by the magnet, and by spatial separation and/or switching off, the separator column can optionally be under the influence of the magnetic field or not. By adjusting the shape, the magnetic field can be brought close to the matrix, thereby providing a sufficient external magnetic field strength. Furthermore, the magnet must be strong enough to produce a strong high gradient magnetic field by focusing the field strength with the substrate. By spatial separation or switching off, the target particles can be fixed and released in a targeted manner, depending on whether the liquid flowing out of the separation column should contain the target particles in the respective working step.

Preferably, the substrate is uncoated and/or has an ordered or disordered filiform, spherical or other shape of material, in particular rust-resistant, magnetic stainless steel or steel wool. Uncoated substrates are particularly cost-effective and, according to the invention, no coating is necessary. However, it is alternatively still conceivable to provide a coating in order to further improve the separation result and the purification result, wherein the coating may not be completely or less high-performing than conventionally. The relatively wide mesh arrangement of the matrix prevents physical or chemical damage to the biological material to be separated, such as damage to sensitive cells, which can be effectively avoided in the experiments described below.

Preferably, the storage vessel is connected to the separation column so that the buffer solution can be introduced into the separation column at an adjustable flow rate, with or without passing through the inflow limiting means, and/or the separation column has outflow limiting means which are capable of influencing the outflow therefrom and the flow rate inside the separation column. The connection between the storage container and the separation column can be provided in a fitting manner, for example by a pipe, but can also be provided in a free-dripping manner. The flow rate can thus be adapted to the biological material, to the buffer solution and to the specific working step, wherein a complete blocking of the inflow of buffer solution can be achieved, for example in order to arrange the idle phase in a separation column for equilibration, or in working steps where no buffer solution is required.

The buffer solution comprises a base solution and at least one macromolecule. For example, the buffer solution contains 80 to 99.8% by weight of the base solution and 0.2 to 20% by weight of the macromolecule.

The buffer solution preferably contains a base solution whose ionic strength is adjusted in dependence on the macromolecules in order to compensate for the aggregation effect of the macromolecules on the biological material, wherein the base solution has an isotonic concentration of cationic sodium, potassium, magnesium or calcium and anionic chloride, phosphate, sulfate or carbonate, in particular a phosphate-buffered saline solution or sucrose solution or a mixture thereof. The base solution should also be matched in its properties, such as pH, according to the mechanism described below, and also matched to the biological material and the macromolecules used to saturate the non-specific binding sites, in order to obtain better results. Here, undesired aggregation of biological material can be prevented particularly well if the isotonic (physiological) phosphate-buffered saline solution is completely or partially replaced by an isotonic phosphate-buffered sucrose solution. Other sugar solutions than sucrose solutions are also contemplated.

For example, the buffer solution contains 0.2 to 10% by weight of gelatin, 9.5 to 10% by weight of sucrose, 80 to 95% by weight of distilled water and sodium phosphate for buffering, and/or 3 to 10% by weight of bovine serum albumin, 0.85 to 0.95% by weight of sodium chloride and 89 to 98% by weight of distilled water and sodium phosphate for buffering, and/or 0.5 to 20% by weight of hydrolyzed collagen, 5 to 10% by weight of sucrose, 0.1 to 0.9% by weight of sodium chloride and sodium phosphate for buffering.

The macromolecules may advantageously comprise natural or synthetic polyelectrolytes or polyampholytes, which may be strong or weak, in particular the synthetic polyelectrolyte Orotan 1850 or the organic polyelectrolyte D-glucuronic acid, and/or have an isoelectric point which is capable of causing a charge corresponding to the charge of the particles of biological material to be separated at the pH of the basic solution, and/or a molecular weight of 10,000 to 100,000kDa, in particular 30,000 to 70,000 kDa. It has been demonstrated that: such buffer solutions give particularly good results.

Furthermore, the macromolecule may preferably comprise globular proteins, in particular albumin, bovine or human serum albumin, ovalbumin, lactalbumin or vegetable albumin, β -lactoglobulin, κ -casein, histone, protamine, globulin, prolamine or gluten, in concentrations of 3 to 7% by weight, in particular 4 to 5% by weight. Alternatively or additionally, the macromolecule further comprises a more preferably filamentous protein, in particular gelatin, bovine gelatin, porcine gelatin or teleost gelatin, in a concentration of 0.3 to 1.5 wt.%, in particular 0.4 to 0.8 wt.%, having a low gel strength of 150Bloom or less, in particular 75Bloom or less. Furthermore, enzymatically hydrolyzed collagen (collagen hydrolysate) may alternatively or additionally be used, preferably in a concentration of 0.3 to 20 wt.%, in particular 1 to 10 wt.%.

These kinds of macromolecules, especially at the specified concentrations and with the resulting viscosity, are exceptionally well suited for saturating non-specific binding sites, and here, having a suitable flow rate and density results in conditions under which the target particles can adhere particularly well to the substrate.

In the method of the present invention, each of the buffer solutions mentioned at appropriate places in the specification, particularly each of the buffer solutions described in the sub-claims of the apparatus, may be used as the buffer solution.

The matrix and the separation column surrounding the matrix are equilibrated here, preferably by pre-incubation with a clean buffer solution, i.e. a buffer solution free of biological material, before the suspension is flowed through, more precisely over a sufficiently long period of time to saturate the non-specific binding sites in the matrix, in particular over a period of 3 to 20 minutes or 5 to 10 minutes, wherein the matrix is kept continuously covered with buffer solution during the equilibration process. It is further preferred here that the suspension has a concentration of the biological material which is critical for the separation process, which also depends on the buffer solution used. According to the knowledge of the inventors, the stated time interval is sufficient to saturate the non-specific binding sites sufficiently beforehand. This balance of readiness prevents: initially upon introduction of the suspension into the matrix, unwanted particles bind to non-specific binding sites of the suspension that have not been fully saturated with buffer solution. Throughout the process, the matrix remains covered so that non-specific binding sites are not released and the flow rate remains constant as this would be detrimental to the separation results.

For separating the unwanted biological material, the eluent, i.e. the fluid leaving the matrix, is preferably caught after the throughflow, wherein, with the magnetic field still activated, after the suspension has been introduced into the matrix, the matrix is throughflowed through with additional pure buffer solution until it is ensured that the suspension has completely left the matrix, and wherein, during the throughflow, the matrix is always completely covered with buffer solution. In this case, the target particles are interfering substances which are to be removed and which adhere to the matrix until the originally desired components of the biological material are completely washed out of the matrix. The trapped eluent no longer contains the target particles magnetically adsorbed to the matrix, the target particles are separated, the concentration of target particles is significantly reduced in the eluent, or in the ideal case, the eluent no longer contains the target particles. Subsequently, the target particles remaining in the matrix can be removed separately or used for other purposes by: the target particles are washed out in an own working step.

Optionally, for purifying the desired biological material, after introduction of the suspension into the matrix, the matrix is flowed through with additional pure buffer solution with the external magnetic field still activated for a period of time until the suspension completely leaves the matrix, and then, with spatial separation or with the external magnetic field deactivated by switching off, the matrix is flushed with additional pure buffer solution and the eluate thus produced is caught there, wherein the matrix remains covered with buffer solution throughout the process. In this case, the fluid leaving the matrix during the activated magnetic field phase is free of the mainly required biological material, which can be discarded or used elsewhere. Only if the washing-out process then takes place in the absence of a magnetic field does the matrix re-liberate the target particles, which are contained in the caught eluate with a very significantly increased degree of purification during the subsequent washing-out of the matrix. During this flushing, the buffer solution may be the same as in the actual HGMS stage with the magnetic field activated, but may also be a different buffer solution. In this case, the target particles are contained in another buffer solution of the eluent, or in a mixture of two buffer solutions.

Preferably, the captured eluate is centrifuged and the process is preferably repeated one or several times using the centrifuged biological material without the liquid phase of the eluate. Thus, when used in the separation step, the biological material remains free of interfering target particles to be separated, and in the opposite case, during the purification process, the target particles remain in pure form, free of buffer solution of the suspension. This procedure can be carried out anew, provided that the high degree of separation or degree of purification which has usually been obtained after one pass is not sufficient. This is particularly true when the substrate is overloaded during the separation process, otherwise only a significantly lower improvement is expected compared to the first pass.

The method of the invention can also be modified with similar features and exhibit similar advantages here, as given by way of example and not exclusively in the sub-claims that follow the independent claims.

Drawings

The invention will be described with respect to other features and advantages in connection with the embodiments and with reference to the accompanying drawings. In the illustration of the drawings:

FIG. 1 shows a schematic front view of the structure of an embodiment of an HGMS apparatus and separation column of the present invention;

FIGS. 2A/B show a first exemplary flow cytometric analysis of the purification of the present invention, in which an isotonic phosphate buffered sucrose solution containing 0.75% gelatin was used as a buffer solution (FIG. 2A: Plasmodium falciparum (P. falciparum) culture before passing through the HGMS column; FIG. 2B: eluate after passing through, washing and removing the separation column from the magnetic field);

FIGS. 3A/B show a diagram depicting the use of Phosphate Buffered Saline (PBS) containing Bovine Serum Albumin (BSA) as a buffer solution according to the second example of FIG. 2;

FIGS. 4A/B show flow cytometric analysis as a third example of a dot plot (FIG. 4A: proportion of CD8 positive leukocytes in a suspension of Peripheral Blood Mononuclear Cells (PBMCs) before purification; FIG. 4B: eluate from a separation column after PBMCs have passed, washed and the separation column has been moved away from the magnetic field); as in the first example, an isotonic phosphate buffered sucrose solution containing 0.75% gelatin was used as a buffer solution; and

fig. 5A/B show a fourth example according to fig. 4, but as in the second example, Phosphate Buffered Saline (PBS) containing Bovine Serum Albumin (BSA) was used as the buffer solution.

Detailed Description

Fig. 1 shows a schematic front view of an embodiment of the purification apparatus of the present invention. The purification device comprises a separation column 1, a liquid storage container 11 from which a buffer solution can be supplied to the separation column 1, a permanent magnet or electromagnet 7 for generating a strong magnetic field, and a specially prepared buffer solution, which will be explained in further detail below.

The separation column 1 has a housing and a matrix 2 arranged in the housing. The housing is constructed of a non-magnetic material and has at least one liquid inlet 6 and at least one liquid outlet 3. The liquid outlet 3 is provided with means 4 for influencing the flow rate of the buffer solution in the separation column 1, which means 4 are provided with a multi-tap 4 and a throughflow limiting means 5. Alternatively, other known means for regulating the flow rate in the separation column may be used.

The matrix 2 is constructed in such a way that inside the separation column 1a high magnetic gradient is created by externally arranged magnets, as required for HGM separation. For this purpose, the substrate 2 contains ferromagnetic material, such as stainless steel, magnetic stainless steel, etc., which are shaped in an ordered or disordered manner, in the form of filaments, spheres or other shapes. For an example of an embodiment of matrix 2, further listed below in connection with experimental purification.

The separation column 1 with the substrate 2 contained in the separation column 1 is located in a strong, preferably homogeneous, magnetic field, which may be generated by a permanent magnet or electromagnet 7. The magnetic field may be switched on or off by, for example, removing the separation column from the magnetic field of the permanent magnet, or by switching off the electromagnet 7.

The liquid storage vessel 11 has a liquid inlet region 12, a reservoir region and a liquid outlet 10 from which buffer solution can be brought into the liquid inlet 6 of the separation column 1. The liquid outlet 10 of the liquid reservoir 11 is provided with means for influencing the outflow rate of the liquid contained in the liquid reservoir 11, which means have a one-way tap 8 and a flow restriction device 9. As with the liquid outlet 3 of the separation column 1, other known means for regulating the outlet flow rate from the liquid storage vessel 11 are also contemplated herein. Further, single taps or multiple taps may also be used and/or interchanged, as desired.

The buffer solution mentioned above and used according to the invention serves for equilibrating the separation column 1 and suspending the biological material to be separated. The buffer solution may comprise pure water and one or several different kinds of macromolecules having the property of being able to saturate the non-specific binding sites of the separation column 1 and of the matrix 2 comprised therein at a sufficient concentration. Furthermore, the density of the buffer solution is preferably sufficiently similar to the density of the particles to be separated so that the effect of gravity on the particles can be largely or even possibly completely subtracted out, thus keeping the particles in suspension. In this way, during the slow flow in the separation column, sedimentation of the particles to be separated is avoided. Furthermore, the buffer solution has a viscosity which produces a flow rate in the separation column 1 with laminar flow properties suitable for the separation process. Finally, the buffer solution may contain different ions such as, but not limited to, cations such as sodium, potassium, magnesium and calcium, or anions such as chloride, phosphate, sulfate and carbonate. The list mentioned here is not meant to exclude other cations or anions.

The macromolecules used in the buffer solution should be large enough to effectively saturate the non-specific binding sites, but not so large that these macromolecules severely increase the viscosity of the buffer solution at the concentrations used. Thus, the macromolecule preferably has a molecular weight of 10,000 to 100,000kDa, more preferably 30,000 to 70,000 kDa. Advantageously, the macromolecules added carry a charge corresponding to the charge of the particles to be separated, since this preferably involves competitive inhibition of the non-specific binding of the particles and macromolecules to the material of the matrix 2 to be separated. For example, if the particle to be separated is a cell whose cell wall is predominantly negatively charged, then it is preferred to select a negatively charged macromolecule.

Such positively or negatively charged macromolecules are known as polyelectrolytes. In addition, a subset of polyelectrolytes are referred to as polyampholytes. This is referred to as a polyelectrolyte with both positive and negative functional groups. The net charge of the polyampholyte can be easily deduced knowing its isoelectric point and the pH of the buffer solution around the molecule: if the pH of the buffer solution is below the isoelectric point of the polyampholyte, the net charge of the buffer solution will be in the positive region. When the pH of the buffer solution is above the isoelectric point, the opposite is true, so the net charge is in the negative region. If the pH of the buffer solution is at or very close to the isoelectric point of the polyampholyte, the buffer solution is neutral and therefore does not carry a net charge.

Polyelectrolytes which have proven particularly advantageous within the scope of the invention for separating cellular material are, for example, macromolecules from the protein (proteome). The proteome is divided into two subgroups, globulins (globular) and filamentous proteins (fibrillar). However, the conceptual division between the two mentioned subgroups, strictly speaking, should be regarded as universal, since intermediate forms between globular and filamentous proteins also exist. Thus, hereinafter, the terms "globular" and "filamentous" will comprise two subgroups and transitional forms of the protein.

Now, globular and filamentous proteins dissolved in liquids have the property of binding reversibly and irreversibly to solid surfaces, a phenomenon which is mostly adsorption or adhesion. In the case of binding to stainless steel surfaces, scientific publications indicate that, after contacting the stainless steel surface with proteins dissolved in a liquid, a monolayer (monolayer layer) of protein molecules on the stainless steel surface is produced (Nakanishi et al, Journal of Bioscience and Bioengineering, 91, 2001: 233-. The thickness of the monolayer varies within the range of the hydrodynamic diameter (stokes-Radius) of the corresponding protein molecule, i.e. within the range of a few nanometers, but no strict planar symmetry of the monolayer has been found so far. However, it can be assumed that the shape of the single layer is not entirely regularly constituted. Pradier et al indicate that gaps may even exist between the deposited protein molecules, i.e., the stainless steel Surface may not be completely covered by protein molecules (Surface and Interface Analysis, 34, 2002: 50-54). Further studies did not reveal whether this monolayer formation had a positive or negative effect on Corrosion of the stainless steel surface (Omanovic und Roscoe, Langmuir, 15, 1999: 8315-. In summary, this must be triggered by the fact that water molecules and dissolved ions are never completely eliminated by the monolayer. However, in the case of the present invention, it was found that: the formation of such a monolayer is extremely effective in preventing non-specific binding of non-target particles to the matrix of the HGMS-separator; furthermore, no physical or chemical damage of the biological material to be separated was observed.

Some proteins which have proven particularly valuable in the context of the present invention will be elucidated further below, without limiting the invention to these particularly suitable proteins.

In this connection, albumin, such as bovine or human serum albumin, should particularly preferably be mentioned as representative of the globular protein subgroup. Within the group of albumins, serum albumins of any other species, and also other albumins, such as but not exclusively ovalbumin, lactalbumin or vegetable albumin, may also be used, but are not preferred.

For bovine or human serum albumin in a phosphate buffered isotonic (physiological) saline solution, concentrations from 3% to 7%, more preferably from 4% to 5%, within the scope of the present invention, have been found to be optimal for the isolation process of cellular material. Significantly lower concentrations (0.1% to 1%), such as are commonly added to physiological buffers in cell studies, do not produce the desired results in the context of the present invention. On the one hand, this can be interpreted as: this relatively low albumin concentration is not effective in saturating the non-specific binding sites of the separation column 1 with uncoated matrix 2 used in the present invention. On the other hand, these low albumin concentrations do not give the necessary density of the buffer solution, which enables the particles to be separated to remain suspended and prevent gravity-induced sedimentation even at slow flow rates.

Among the subgroup of filamentous proteins, gelatin is particularly preferred in the context of the present invention. Due to the favourable isoelectric point of gelatin at about pH4.5 to pH5.6 and thus the negative charge of gelatin at neutral pH, it has been found that among gelatins used for the separation of cellular material in the physiological pH range, gelatin class B (bovine gelatin) is particularly preferred. A particularly advantageous concentration range for the isolation process of cellular material is found to be from 0.3% to 1.5%, more preferably from 0.4% to 0.8% for bovine gelatin. Other gelatins, such as class a gelatin (porcine gelatin) or teleost fish gelatin (fish gelatin), or any other gelatin, as well as enzymatically hydrolyzed collagen (collagen hydrolysate) as another subset of gelatins, may also be used, but are not preferred because these gelatins have a less favorable isoelectric point, at least for separations in the physiological pH range. In general, among the different gelatins of group B mentioned and the other gelatins of the group indicated, preference is given to gelatins having a low gel strength (Bloom strength), preferably below 150Bloom, more preferably below 75 Bloom.

Other macromolecules of the protein class that can be used in the context of the invention do not exclude other globular proteins, such as beta-lactoglobulin or kappa-casein, or other globular proteins from the histone or protamine, globulin, prolamine and gluten subgroups, and also filamentous proteins, among others.

In other embodiments of the invention, it is envisaged to use macromolecules from organic or synthetic polyelectrolytes, for example synthetic polyelectrolyte Orotan 1850^TM(Rohm and Haas, Philadelphia, PA, USA) or the organic polyelectrolyte D-glucuronic acid.

Some of the mentioned macromolecules, which can be added to the buffer solution to reduce the non-specific binding of the biological material to be separated to the matrix material, in combination with the particular buffer solution, lead to aggregation of the biological material to be separated in suspension. The tendency of red blood cells to aggregate in suspension is positively correlated with the molecular weight and concentration of the added macromolecules, as well as the ionic strength of the buffer solution. Ionic strength is understood as the total charge concentration of dissolved ions in the buffer solution. The low ionic strength can counteract the aggregation caused by the added macromolecules, and even prevent it completely. Considering the hydrodynamic diameter (Stokes radius) which can be deduced from the molecular weight and from the measurement of the intrinsic viscosity, it is possible to predict the aggregation-promoting effect on the suspension of red blood cells more precisely than when considering the molecular weight of a specific macromolecule alone (in comparison with Jan and Chien (Journal of general Physiology, 61, 1973: 638-. In the following, the term "molecular weight" of a macromolecule will always represent the hydrodynamic diameter (stokes radius) of the macromolecule that can be deduced therefrom.

In the case of the present invention, it has proved advantageous if: using this knowledge, and adjusting the ionic strength of the buffer solution to effectively prevent aggregation of the biological material to be separated. For example, the present inventors have found that blood cells aggregate particularly after being suspended in a phosphate buffered saline solution containing gelatin. In order to prevent such aggregation when using gelatin for the separation of blood cells, it has proved to be particularly advantageous to replace the physiological phosphate-buffered saline solution partly or completely with a phosphate-buffered physiological sucrose solution.

From the correlation described between the particle aggregation and specific properties of the buffer solution (in particular the concentration and molecular weight of the macromolecules, and the ionic strength of the buffer solution), it is possible to predict which combination of macromolecules and buffer solution is particularly suitable for the separation of the particular biological material to be separated, and to select a buffer solution containing macromolecules whose physicochemical properties match the separation conditions required for the particular particles. In the context of the present invention, the term physicochemical properties of the buffer solution encompasses the viscosity, density, ionic strength, osmotic pressure and pH of the liquid, but also the type, molecular weight, charge and concentration of macromolecules dissolved in the liquid, and other physicochemical parameters of the liquid which can be influenced both by the macromolecules and by the surrounding macromolecules.

Hereinafter, the purification method of the present invention will be explained step by step in more detail.

In the first step, the separation column 1 is equilibrated with a buffer solution to saturate the non-specific binding sites. Here, equilibration is carried out by pre-incubating the separation column 1 with the above-described buffer solution (hereinafter referred to as buffer solution a) suitable for the separation process of the particular particle suspension under investigation for a sufficiently long period of time adapted to the macromolecules used in the buffer solution. Typically, this period is 3 to 20 minutes, more typically 5 to 10 minutes, but in rare cases, either a short or a long period is also required.

After equilibration, the biological material to be separated is suspended in a buffer solution a and fed into the inlet region 6 of the separation column 1, wherein the separation column 1 is located in the uniform magnetic field of a horseshoe magnet or electromagnet 7. At the same time, the liquid outlet 3 of the separation column 1 is opened with the aid of the multi-tap 4. Here, care must be taken to keep the substrate 2 always covered with the buffer solution. The biomaterial flows through the separation column 1 and target particles, either intrinsically magnetic or previously labeled with synthetic paramagnetic particles, adhere to the substrate 2. However, all non-magnetic particles, pass unhindered through the separation column 1, exiting at the liquid outlet 3.

After sufficient addition of the biological material to be separated, the cleaning of the separation column 1 is performed. This process is used to wash away non-magnetic non-target particles remaining in the separation column 1. With the aid of the one-way tap 8, the liquid outlet 10 of the liquid storage container 11 containing buffer solution a is opened, so that pure buffer solution a is now passed through the separation column 1 as washing solution. Here, it must be noted again that: the holding substrate 2 is always covered with the buffer solution. The amount of buffer solution a to be used for washing is adapted according to the size of the separation column 1. The amount of buffer solution A required to be sufficient for washing the separation column 1 having a volume of 3ml is about 30-60ml, and for other sizes of separation columns, it must be adjusted accordingly.

After the cleaning of the separation column 1 is completed, the separation column 1 and the liquid outlet 3 of the liquid storage container 11 are closed. Moving the separation column 1 away from the magnetic field of the horseshoe magnet 7 or, in case an electromagnet 7 is used, switching off the magnetic field. In the absence of a magnetic field, the target particles are released from the matrix 2 and may be washed out by washing the separation column 1 in a forward or reverse direction using the buffer solution a, or any other buffer solution because the separation is performed at this time.

The method is suitable for purifying target particles, since the target particles are contained in an eluate obtained by elution of the column, and for removing the target particles from the biological material to be separated. In the second case, a washing solution is required from the separation column 1, which washing solution contains the biological material depleted of target particles. Then, the washing solution is caught during the introduction of the biological material suspended in the buffer solution a into the separation column 1 and the subsequent washing of the separation column 1 with the pure buffer solution a. Here, important to note are: the substrate 2 does not have to be overloaded with too high a quantity of target particles, since after the capacity of the substrate 2 has been exhausted, unbound target particles can come out of the liquid outlet 3 of the separation column 1 together with the remaining biological material to be separated.

If higher purification is desired, the process can be repeated with the purified target particles obtained. The same applies to the washing solution which is caught after the throughflow of the separation column 1 in the case of an attempt to remove the target particles more purely from the biological material to be separated. However, in most cases, this is no longer necessary because of the high degree of purification according to the invention.

The following test examples shall further illustrate the invention, but are not intended to limit the applicability of the test examples accordingly.

Example 1

Use of isotonic phosphate buffered sucrose solution containing gelatin for treatment of malaria infectionInsects Plasmodium (plasmodium) infected red blood cells of (p. falciparum) cultures And (5) purifying.

Materials:

buffer solution A₁: isotonic phosphate buffered sucrose solution containing 0.75% gelatin

Stainless steel wool 1g

Single-way tap

Three-way tap

20G syringe needle

3ml disposable syringe

10ml disposable syringe

50ml disposable syringe

1 neodymium horseshoe magnet

a) Preparation of purification kit

Manufacture of separation columns

A 3ml disposable syringe used as the separation column 1 was filled to two thirds of the total volume of the 3ml disposable syringe with 1g of stainless steel wool as the matrix 2. Here, it is to be noted that a large number of stainless steel wool fibers are located in the longitudinal direction of the syringe body. A three-way tap 4 and a 20G syringe needle as a flow restriction device 5 are connected to the liquid outlet 3 of the syringe body. The upper third of the disposable syringe, which is not filled with stainless steel wool, serves as the inlet region 6 of the separation column 1.

Equilibration of the separation column

The thus-fabricated separation column was reversely filled with a buffer solution through a three-way tap 4 in a vertical position. The air bubbles are expelled as completely as possible by tapping with a finger, but among other things, it has been found that: the remaining smaller air bubbles do not negatively affect the separation process. At this time, the separation column 1 was positioned between both poles of the horseshoe magnet 7 so that the entire substrate 2 was exposed to a uniform magnetic field and equilibrated for 10 minutes.

Assembly and positioning of liquid storage containers

A one-way tap 8 provided with an 18G needle as a flow restriction device 9 was installed at the liquid outlet 10 of a 50ml disposable syringe serving as a liquid storage container 11. A 50ml disposable syringe serving as a liquid storage container 11 is then placed over the separation column 1 so that the liquid flowing out of the needle drops into the inlet region 6 of the separation column 1.

b) Preparation and execution of a separation process

Preparation of plasmodium falciparum (p. falciparum) culture for purification of hemoglobus cells infected with malaria pathogen

50 μ l of a culture of red blood cells containing 13.51% parasitemia P.falciparum were resuspended in 450 μ l of RPMI medium and oxygenated for 10 min under room air. The culture was then centrifuged and resuspended in 5ml of buffer solution A₁In (1).

Execution of the separation procedure

After 10 minutes of equilibration of the separation column 1, its liquid outlet 3 is opened, while the resuspended cells are slowly introduced into the inlet region of the separation column 1, so that the matrix 2 remains covered with liquid at all times. After complete addition of the resuspended culture, the cell containing 45ml of buffer solution A is opened₁The liquid outlet 10 of the liquid storage container 11. Here, the variation of the flow rate in the separation column 1 caused by the operation of the liquid outlet 3 of the separation column 1 should be avoided, and care should be taken to keep the matrix 2 of the separation column 1 always buffered with the buffer solution A₁And (6) covering. After the addition of the buffer solution A from the liquid storage container 11 is completed₁The separation column 1 is then moved away from the magnetic field by terminating the flow of liquid in the separation column 1 by closing the liquid outlet 3. Will be filled with buffer solution A₁The 10ml disposable syringe was connected to three-way tap 4 and the separation column 1 was back-flushed. The eluate was collected in a suitable container and the suspension was separated at 1500gHeart 5 min. The supernatant was discarded and the pellet was resuspended in 300. mu.l of phosphate buffered saline.

c) Analysis and results

Cells were then stained with acridine orange and flow cytometric analysis was performed as described elsewhere (Bhakdi et al, Cytometry A2007 (71A) 662-667). Figure 2 shows the results of the purification using a histogram of flow cytometry analysis. A) Plasmodium falciparum (p. falciparum) cultures prior to passage through a high gradient magnetic separation column. M1: normal red blood cells, M2: red blood cells (13.51%) infected with plasmodium falciparum (p. falciparum). B) Passing through the culture with 45ml of buffer solution A₁After washing and removal of the separation column 1 from the magnetic field, the eluent from the separation column 1. M1: normal red blood cells, M2: red blood cells infected with p.falciparum (p.falciparum) were purified to 99.54%. Accordingly, blood smears stained with Giemsa showed only red blood cells infected with malaria pathogen. Malaria pathogens from purified red blood cells can be cultured without problems for several days.

Example 2

Using a phosphate-buffered saline solution (PBS) containing Bovine Serum Albumin (BSA) Purification of malaria pathogen (plasmodium) from plasmodium falciparum (p. falciparum) culture Infected red blood cells

Materials:

as listed in example 1, but buffer solution A₁Is buffered by solution A₂(PBS containing 5% BSA).

a) Preparation of purification kit

As described in example 1.

b) Preparation and execution of a separation process

Preparation of plasmodium falciparum (p. falciparum) culture for purification of hemoglobus cells infected with malaria pathogen

As described in example 1. Parasitemia of plasmodium falciparum (p. falciparum) cultures was 14.47% in this experiment. Buffer solution A₁With buffer solution A₂Instead.

Execution of the separation procedure

As described in example 1, the following differences apply:

buffer solution A₁With buffer solution A₂Instead. Centrifugation of the eluate was then carried out at 800g for 5 minutes.

c) Analysis and results

The analysis was carried out analogously to example 1. Figure 3 shows the results of the purification using a histogram of flow cytometry analysis. A) Plasmodium falciparum (p. falciparum) cultures prior to passage through a high gradient magnetic separation column. M1: normal red blood cells, M2: red blood cells (14.47%) infected with plasmodium falciparum (p. falciparum). B) Passing through the culture with 45ml of buffer solution A₂After washing and removal of the separation column 1 from the magnetic field, the eluent from the separation column 1. M1: normal red blood cells, M2: red blood cells infected with p.falciparum (p.falciparum) were purified to 97.03%. Accordingly, blood smears stained with Giemsa showed almost exclusively red blood cells infected with malaria pathogen. Malaria pathogens from purified red blood cells can be cultured further for several days without problems.

Example 3

Purification of suspension with surface antigen from leukocyte (peripheral blood mononuclear cells (PBMCs)) suspension Leukocytes of pro-CD 8 (CD8 positive cells).

Materials:

as listed in example 1.

a) Preparation of purification kit

As described in example 1.

b) Preparation and execution of a separation process

Labeling of CD8 positive cells with antibody-conjugated synthetic paramagnetic particles (microbeads)

Mix 1.5x10⁷Individual human peripheral mononuclear cells (PBMCs) were incubated with monoclonal rat anti-human CD8IgG antibody in PBS/BSA 1% for 30 minutes on ice. Cells were washed twice with the same buffer solution and incubated with anti-rat IgG conjugated microbeads (miltenyi biotech GmbH, loc. cit.) on ice for a further 10 minutes. The cells were washed twice more in the same buffer solution and then mixed with fluorescein-labeled antibodies (PE-anti-CD 8 antibody and FITC-anti-CD 3 antibody, Simultest)^TMBD Biosciences, 2350 prime Drive, san jose, CA USA 95131) were incubated on ice for a further 30 minutes. The cells were then washed twice again with PBS/BSA 1%. The cells were then centrifuged again and resuspended in 1.5ml of buffer solution A₁In (1).

Execution of the separation procedure

As described in example 1.

c) Analysis and results

FIG. 4 shows the results of purification of CD8 positive cells from PBMCs suspensions. A) A cell population of the sample. The upper two quadrants show CD8 positive cells (23.21%). B) Passing through PBMCs with 45ml of buffer solution A₁After washing and removal of the separation column 1 from the magnetic field, the eluent from the separation column 1. The upper two quadrants show CD8 positive cells, purified to 99.17%.

Example 4

Removal (depletion) of CD 8-positive cells from PBMCs suspension

Material

As listed in example 2, but a 25G needle was used as the flow restriction device instead of a 20G needle.

A) Preparation of purification kit

As described in example 1.

B) Preparation and execution of a separation process

Labeling of CD8 positive cells with antibody-conjugated synthetic paramagnetic particles (microbeads)

As described in example 3.

Execution of the separation procedure

As described in example 2, but 15ml of buffer solution A were used₂. For this experiment, what is needed is a liquid that passes through the separation column 1. It was caught in a suitable container, and the cells were centrifuged at 800g for 5 minutes and resuspended in 300. mu.l PBS.

c) Analysis and results

FIG. 5 shows the results of removing CD8 positive cells from PBMCs suspension. A) A cell population of the sample. The upper two quadrants show CD8 positive cells (35.41%). B) Passing through PBMCs with 15ml of buffer solution A₂After washing and removal of the separation column 1 from the magnetic field, the eluent from the separation column. The upper two quadrants show CD8 positive cells, depleted to 0.57%.

All of the above-mentioned publications are incorporated by reference in their entirety into this patent document.

In general, the invention is based on the idea of carrying out a high gradient magnetic separation of biological material using a technical device with consumable material, also called "kit", comprising or comprising:

1.1. a separator column comprising a matrix of ferromagnetic material, suitable for generating a high gradient magnetic field in an external strong homogeneous magnetic field,

1.2. a liquid storage vessel from which the buffer solution A can be introduced into the inlet region of the separation column,

1.3. a permanent magnet or an electromagnet for generating a strong, uniform magnetic field,

1.4. a buffer solution A for equilibrating the separation column and suspending the biological material to be separated,

wherein the buffer solution A has at least one of the following three advantageous properties:

1.5. the buffer solution A contains macromolecules having the property of being able to saturate the non-specific binding sites in the separation column and/or to suspend the biological material to be separated for equilibration of the separation column and/or

1.6. The buffer solution A has a density that is sufficiently similar to the density of the particles to be separated to reduce as much as possible the effect of gravity on the particles and thus keep them suspended in the buffer solution A, and/or

1.7. The buffer solution a has a viscosity that contributes to a flow rate with laminar flow properties suitable for the separation process in the separation column.

Claims (29)

1. High gradient magnetic separation device for separating or purifying magnetic or magnetically labeled biological material, comprising a magnet (7), a separation column (1), a ferromagnetic matrix (2) arrangeable in an inner space of the separation column, and a storage vessel (11) containing a buffer solution for equilibrating the separation column (1) and/or for suspending the biological material, wherein, during operation, a magnetic field generated by the magnet (7) is capable of generating a high gradient magnetic field in the matrix (2), the buffer solution being capable of flowing through the separation column (1) from the storage vessel (11),

characterized in that the buffer solution comprises a base solution and macromolecules capable of saturating the non-specific binding sites of the matrix (2), which are the sites to which the particles bind independently of their magnetic properties.

2. The device according to claim 1, wherein the buffer solution has a density which corresponds as much as possible to the density of the particles of the biological material to be separated in such a way that the gravitational forces acting on the particles are substantially compensated for, as a result of which the particles are almost suspended in the buffer solution, and/or has a high viscosity which enables laminar through-flow through the separation column (1) at a flow rate which is suitable for the separation process.

3. Device according to claim 1 or 2, wherein the magnet (7) is a permanent magnet or an electromagnet which is shaped in such a way that the separation column (1) can be placed in a particularly homogeneous magnetic field generated by the permanent magnet or electromagnet, wherein the separation column (1) can be selectively held under the influence of the magnetic field or not by spatial separation and/or by switching off.

4. Device according to one of claims 1-2, wherein the substrate (2) is uncoated and/or has an ordered or disordered filiform or globular material.

5. The device according to one of claims 1 to 2, wherein the storage container (11) is connected to the separation column (1) such that buffer solution can be introduced into the separation column (1) via flow restriction means (8, 9) at an adjustable flow rate or not via the flow restriction means (8, 9) into the separation column (1), and/or wherein the separation column (1) has flow restriction means (4, 5), which flow restriction means (4, 5) can influence the outflow from the separation column and influence the flow rate inside the separation column (1).

6. The device according to one of claims 1 to 2, wherein the ionic strength of the base solution is adjusted in dependence on the macromolecules such that the aggregating effect of the macromolecules on the biological material is compensated, wherein the base solution has an isotonic concentration of sodium, potassium, magnesium or calcium cations and chloride, phosphate, sulphate or carbonate anions.

7. The device according to one of claims 1-2, wherein the macromolecules comprise natural or synthetic polyelectrolytes or polyampholytes, and/or wherein the isoelectric point of the macromolecules is capable of generating a charge corresponding to the charge of the particles of the biological material to be separated at the pH of the basic solution, and/or wherein the macromolecules have a molecular weight of 10,000kDa to 100,000 kDa.

8. The device of any of claims 1-2, wherein the macromolecules comprise globular proteins at a concentration of 3% to 7% of the buffer solution.

9. The device of any of claims 1-2, wherein the macromolecule comprises a filamentous protein or gelatin at a concentration of 0.3% to 1.5% with a low gel strength of 150Bloom or less.

10. The device of claim 4, wherein the material is magnetic stainless steel.

11. The device of claim 6, wherein the base solution is a phosphate buffered saline solution or a sucrose solution or a mixture thereof.

12. The device of claim 7, the polyelectrolyte is a synthetic polyelectrolyte and the polyampholyte is the organic polyelectrolyte D-glucuronic acid.

13. The device of claim 7, the macromolecule having a molecular weight of 30,000kDa to 70,000 kDa.

14. The device of claim 8, wherein the globular protein is albumin, kappa-casein, histone, protamine, globulin, gluten.

15. The device of claim 8, wherein the concentration of globular protein is between 4% and 5% of the buffer solution.

16. The device according to claim 9, wherein said filamentous protein is hydrolyzed collagen at a concentration of 0.3% to 20% by weight, and said gelatin is at a concentration of 0.4% to 0.8% with a low gel strength of 75Bloom or less.

17. The device according to claim 9, wherein the filamentous protein is hydrolyzed collagen at a concentration of 1% to 10% by weight.

18. The device of claim 9, wherein the gelatin is bovine gelatin, porcine gelatin, or teleost gelatin.

19. The device of claim 16, wherein the gelatin is bovine gelatin, porcine gelatin, or teleost gelatin.

20. Method for the separation or purification of intrinsically magnetic or preplastically magnetically labelled biological material by means of high-gradient magnetic separation, wherein a suspension containing the biological material is passed through a ferromagnetic matrix (2) arranged in an external magnetic field, so that the material adheres to the matrix (2), characterized in that the matrix (2) is uncoated and the biological material is suspended in a buffer solution containing a base solution and macromolecules saturating the non-specific binding sites of the matrix (2) during the passage through the matrix, non-specific binding sites being sites to which particles bind independently of their magnetic properties.

21. Method according to claim 20, wherein the matrix (2) and the separation column (1) surrounding the matrix (2) are equilibrated by pre-incubation with a pure buffer solution, i.e. a buffer solution free of biological material, before the suspension is flowed through, more precisely a sufficient period of time is passed to saturate the non-specific binding sites in the matrix (2), and wherein the matrix (2) remains covered with buffer solution throughout the equilibration process.

22. Method according to claim 20 or 21, wherein for separating unwanted biological material, an elution liquid, i.e. the fluid leaving the matrix (2), is caught after through-flow, wherein after introducing the suspension into the matrix (2), with the magnetic field still activated, the matrix (2) is through-flowed all the way with additional pure buffer solution until it is ensured that the suspension has completely left the matrix (2), and wherein the matrix (2) remains covered with buffer solution all the way through-flow.

23. Method according to claim 20 or 21, wherein, for purifying the desired biological material, after the introduction of the suspension into the matrix (2), the matrix (2) is continuously throughflown with additional pure buffer solution with the magnetic field still activated until it is ensured that the suspension has completely left the matrix (2), and then, with the external magnetic field deactivated by spatial separation or switching off, the matrix (2) is flushed with additional pure buffer solution and, here, the elution liquid, i.e. the fluid leaving the matrix (2), is caught, wherein, throughout the process, the matrix (2) is kept covered with buffer solution.

24. The method of claim 22, wherein the caught eluate is centrifuged and the method is repeated one or several times with the centrifuged biological material free of the eluate liquid phase.

25. The device of claim 8, wherein the globular protein is bovine or human serum albumin, ovalbumin, lactalbumin, or plant albumin.

26. The apparatus of claim 8, wherein the globular protein is a prolamine.

27. The device of claim 8, wherein the globular protein is β -lactoglobulin.

28. Method according to claim 20, wherein the matrix (2) and the separation column (1) surrounding the matrix (2) are equilibrated by pre-incubation with a pure buffer solution, i.e. a buffer solution without biological material, before the suspension is flowed through, the non-specific binding sites in the matrix (2) being saturated over a period of 3 to 20 minutes, and wherein the matrix (2) remains covered with buffer solution throughout the equilibration process.

29. Method according to claim 20, wherein the matrix (2) and the separation column (1) surrounding the matrix (2) are equilibrated by pre-incubation with a pure buffer solution, i.e. a buffer solution without biological material, before the suspension is flowed through, the non-specific binding sites in the matrix (2) being saturated over a period of 5 to 10 minutes, and wherein the matrix (2) remains covered with buffer solution throughout the equilibration process.