Stirring device and method for concentrating macromolecules

FIELD OF THE INVENTION

[0001] The present invention relates to a stirring device and a centrifuge method for concentrating macromolecules from a solution.

BACKGROUND OF THE INVENTION [0002] For macromolecular solutions (e.g. protein solutions), it is often required to remove solvent in order to increase the concentration of the macromolecules (e.g. proteins or nucleic acids). A method to concentrate macromolecules is to spin the solution in a centrifuge and use the centrifugal force to separate the macromolecules from the solution by using a filter unit with a semi-permeable membrane e.g. ultrafiltration for proteins (see US 5,647,990 A).

[0003] The macromolecular particles of the solution are not able to penetrate the semipermeable filter membrane, because of a smaller pore size of the membrane compared to the size of the macromolecules. This leads to an increased concentration of macromolecules, relative to the lowered amount of solution volume on one side of the mem- brane. Throughout the centrifugation, the centrifugal force also generates a concentration-gradient (also known as concentration polarization) of the macromolecules, as a result of the higher weight of the macromolecules, compared with the molecules of the solution, which are lighter and therefore equally distributed. The concentration-gradient becomes the highest proximal to the surface of the membrane. Consequently, there exists a higher local concentration on the surface of the membrane, as the macromole- cules cannot pass the membrane and are pushed towards the membrane. [0004] High local concentrations caused by a concentration-gradient, often increase the tendency for the formation of macromolecular aggregation (e. g. protein aggregation), as the macromolecules are not stable in higher concentration and therefore lose their solubility. These aggregates lead to membrane fouling, which can be described as clogging of the membrane. As a consequence, soluble macromolecules are lost and the filtration process is slowed down.

[0005] There are already established methods to partly overcome the effect of membrane fouling. For example, in one method the so-called "cross-flow filtration" reduces a concentration polarization with a continuous flow of the solution perpendicular to the membrane surface. This approach causes shear forces which removes macromole- cules and aggregates from the surface of the membrane. The disadvantages are the complicated handling of the apparatus and the use of vacuum instead of a centrifuge as the driving force for the ultrafiltration.

[0006] There is thus a need for an alternative, easy to implement approach to avoid membrane fouling during the centrifugation process of a solution comprising macromol- ecules.

OBJECTS AND SUMMARY OF THE INVENTION

[0007] The present invention addresses this need and provides a sealing cap adapted to a centrifuge tube with a concentrator device comprising a membrane wherein said sealing cap comprises an electric motor, a battery, which is placed in the centrifuge, and a stirring element which allows for homogenization of a macromolecular solution during the process of being concentrated by means of spinning the concentrator device in a centrifuge, wherein the electric motor is powered by the battery. The use of this sealing cap comprising a stirring element decreases the concentration polarization and therefore high local concentrations of macromolecules close to the membrane. It also increases the solubility of the macromolecules in solution, e.g. proteins. The higher solu- bility of the macromolecules inhibits the tendency of the formation of aggregates, which otherwise would clog the membrane, leading to membrane fouling. As a consequence, the following advantages are expected in most macromolecular solutions:

[0008] An increase of the final yield of concentrated macromolecules can be accomplished, as a result of lower formation of aggregates and decreased retention time on the membrane surface of the macromolecule.

[0009] There is a limit to which macromolecular solution can be concentrated until there is no gain in concentration anymore. This is simply due to the fact, that at such high concentrations, the tendency of the formation of aggregates overcomes the concentrating process by lowering the solution volume throughout the centrifugation. With the homogenizing sealing cap according to the invention (also sometimes referred to as homogenizer), the maximal possible concentration magnitude can be increased, because in a more homogeny solution the lower local concentration allows a higher concentration of the macromolecules.

[0010] During classical concentration processes based on centrifugation, membrane fouling takes place which can partly be overcome by an increase of the centrifuge speed. This also increases the centrifugal forces and more solution can be pushed through the membrane. However, high centrifugal forces can have a negative effect on the native folding of macromolecules, e.g. the tertiary structure of proteins. With the use of the homogenizing sealing cap according to the invention, a concentration process can be accomplished with lower centrifugal speed due the decreased amount of membrane fouling and the structure of the macromolecules can be better preserved. In the prior art, there is no description of a cross-flow like filtration system connected with concentrating macromolecules based on centrifugal methods. [0011] Furthermore, after the usage of concentrator devices such as filter units in concert with the homogenizing sealing cap according to the invention there is a lower degree of membrane fouling and theses concentrator devices, e.g. filter units, can be cleaned more easily because the pores of the membrane are not as much clogged as in case of use of concentrator devices without the homogenizing sealing cap. Consequently, concentrator devices can be reused more often.

[0012] In a preferred embodiment of the sealing cap of the present invention, said electric motor and said stirring element are connected via a rotational core.

[0013] In a further preferred embodiment, said stirring element is connected with a magnetic stirrer, which is capable of moving the stirring element.

[0014] In yet another preferred embodiment, said sealing cap is a screw cap or a plug cap fitting on a centrifuge tube.

[0015] In a particularly preferred embodiment, said screw cap or said plug cap is characterized by a sealed housing, which comprises on one side an on/off switch, on a further side the stirring element and inside of the housing the electric motor.

[0016] In yet another embodiment, the present invention relates to a sealing cap as defined herein above, wherein the battery is placed in the sealing cap or is separately placed within another centrifugal inlet, or fixed onto the centre of the centrifuge's rotor, e.g. via a magnet or a supporting frame and connected via wires with the electric motor. It is particularly preferred that the battery is a rechargeable battery.

[0017] In a further preferred embodiment of the sealing cap according to the present invention, said concentrator device as mentioned above comprises at least one membrane and a dead stop, in which a portion of the macromolecular solution remains un- filtered. [0018] In an additional, preferred embodiment said concentrator device is positioned in the central part of the centrifuge tube.

[0019] In certain embodiments of the sealing cap according to the present invention the stirring element as defined herein above is a stirring staff having a shape selected from the group comprising a paddle blade shape, a ribbon blade shape, a turbine vortex blade shape, an umbrella type blade shape, an anchor blade shape, a flat blade turbine tube shape, a spiral propeller blade shape and a high shear homogenizer shape. In a preferred embodiment of the sealing cap according to the present invention the stirring element as defined herein above is a stirring staff having a coiled spiral shape. [0020] In a further particularly preferred embodiment said stirring element is vertically oriented. In yet another embodiment said stirring element reaches into the dead stop of the concentrator device.

[0021] In an additional, preferred embodiment, the stirring staff as defined herein above is modular exchangeable, preferably through a plugging mechanism, a magnetic mechanism or a screwing mechanism.

[0022] In a further aspect the present invention relates to the use of the sealing cap as defined herein above for homogenizing a macromolecular solution during the spinning of a centrifuge tube with a concentrator device.

[0023] In a preferred embodiment of said use the homogenization takes place during the process of concentrating macromolecules from said solution via filtration across a membrane present in the concentrator device.

[0024] In a further aspect the present invention relates to an assembly for homogenization of a macromolecular solution during the process of being concentrated by means of spinning a concentrator device in a centrifuge, comprising a centrifuge tube, a con- centrator device, which comprises at least one membrane and a dead stop, in which a portion of the macromolecular solution remains unfiltered, and a sealing cap as defined herein above.

[0025] In a preferred embodiment of said assembly (i) the concentrator device is positioned in the central part of the centrifuge tube to obtain two chambers, one with an open side and one entirely sealed with the tube and a membrane of the concentrator device; (ii) the stirring element is positioned on one side of the sealing cap; and/or (iii) the sealing cap seals the open side of the centrifuge tube while the stirring element is inside of the centrifuge tube and the on/off switch outside of the centrifuge tube.

[0026] In a further aspect the present invention relates to the use of an assembly as defined herein above for effectively concentrating macromolecules from a solution.

[0027] In yet another aspect the present invention relates to a method for avoiding membrane fouling during concentrating macromolecules from a solution, comprising spinning a centrifuge tube with a concentrator device, which comprises at least one membrane and a dead stop, in which a portion of the macromolecular solution remains unfiltered, wherein said method comprises the step of stirring said solution during said spinning process.

[0028] In a further aspect the present invention relates to a method for effectively concentrating macromolecules from a solution, comprising spinning a centrifuge tube with a concentrator device which comprises at least one membrane and a dead stop, in which a portion of the macromolecular solution remains unfiltered in a centrifuge, wherein said centrifuge tube is covered by a sealing cap adapted to the centrifuge comprising an electric motor, a battery, which is placed in the centrifuge, and a stirring element as mentioned herein above and wherein said solution is stirred during the process of being concentrated by means of spinning the concentrator device in said centrifuge and wherein the electric motor is powered by the battery.

[0029] In preferred embodiments of said methods the stirring is performed during (i) the entire process of being concentrated by means of spinning the concentrator device in said centrifuge, (ii) during about 20 to 75% of the time of said spinning process, or (iii) during the last about 25% of the time of said spinning process.

[0030] I n a further preferred embodiment of said methods, the stirring homogenizes said solution to sweep the portion of the solution which remains unfiltered in a dead stop of the concentrator device in a direction towards at least a part of the at least one membrane of said concentrator device.

[0031] I n a final aspect the present invention relates to a concentrated macromolecular solution obtainable with the method as described herein a bove, wherein said solution is essentially free of macromolecular aggregates. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows a schematic cross-section of an embodiment of the homogenizing sealing cap according to the invention including an adapter unit (1), which connects the sealing cap with a concentrator device such as a filter unit (2).

[0033] Figure 2 shows a tilted top view representation of the same embodiment of the homogenizing sealing ca p according to the invention as depicted in Figure 1, without adapter unit 1 and filter unit 2. Shown is a housing (3) with its lid (8), as well as the stirring staff (13).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] Although the present invention will be described with respect to particular em- bodiments, this description is not to be construed in a limiting sense.

[0035] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. [0036] As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise

[0037] In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

[0038] It is to be understood that the term "comprising" is not limiting. For the pur- poses of the present invention the term "consisting of" or "essentially consisting of" is considered to be a preferred embodiment of the term "comprising of". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

[0039] Furthermore, the terms "(i)", "(ii)", "(iii)" or "(a)", "(b)", "(c)", "(d)", or "first", "second", "third" etc. and the like in the description or in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms relate to steps of a method or use there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks etc. between such steps, unless otherwise indicated.

[0040] It is to be understood that this invention is not limited to the particular meth- odology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0041] As has been set out above, the present invention concerns in one aspect a seal- ing cap adapted to a centrifuge tube with a concentrator device, comprising an electric motor, a battery, which is placed in the centrifuge, and a stirring element which allows for homogenization of a macromolecular solution during the process of being concentrated by means of spinning the concentrator device in a centrifuge and wherein the electric motor is powered by the battery. The sealing cap comprising a stirring element according to the present invention creates agitation of a macromolecular solution within a concentrator device simultaneous to the centrifugation process. The stirring leads to a circulation of the macromolecular solution and generates cross-flow like shear force, which homogenizes, mixes or disperses the solution and reduces the formation of a concentration-gradient. [0042] The term "macromolecule" or "macromolecular" as used herein refers to a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. Examples of macromolecules to be used in the context of the present invention are polypeptides or proteins, biological material derived from cells or cell substructures, colloids, nucleic acids, branched biopolymers such as carbohydrates, or synthetic macromolecules such as synthetic polymers or inorganic polymers. A preferred macromolecule is a polypeptide or protein. A "macromolecular solution" as described herein means a liquid solution comprising a macromolecule as defined herein above. The liquid may for instance be an aqueous liquid, e.g. comprise water, or a buff- ered solution. The term "solution" as used herein means a mixture composed of a solvent, i.e. a liquid, e.g. water, and a solute, i.e. a macromolecule dissolved in the solvent. The solution may comprise more than one solute type, e.g. two or more different macromolecule species, or, in addition to the macromolecule species, further molecules, such as stabilizers, ions, small organic molecules, buffer molecules etc. In one embodiment, the solution may comprise only polypeptides/proteins as macromolecules. In further embodiments, the solution may comprise polypeptides/proteins and carbohydrates or synthetic macromolecules or additional solutes such as stabilizers, ions, small organic molecules or buffer molecules. In certain embodiments, the solution is "homog- enously" mixed, i.e. the ingredients are evenly distributed, or an activity is applied whose final result is an even distribution of the ingredients. The "homogeneity" of the solution may typically be influenced by the centrifugal forces and/or filtration activity applied to it during the process of spinning the concentrator device in a centrifuge. As a result, a gradient of macromolecules within the solution may appear which can effectively be reversed and transformed into an even distribution of the macromolecules in the solution when stirring said solution during the centrifugation process according to the embodiments of the present invention. The term "homogenization" as used herein refers to the use of a stirring element for stirring and mixing a solution, e.g. a macromo- lecular solution, or suspension to achieve an increase of the homogeneity or of the degree of even distribution of ingredients of a solution or suspension. The "homogenization" of a solution may happen during a centrifugation process. One example of an envisaged centrifugation process is a density gradient centrifugation. Density gradient centrifugation is one of the physical separation processes of particles or macromolecules using sedimentation in a density gradient. Density gradients may, for example, be obtained due to the presence of filter or membrane barriers in the concentrator device during centrifugation, wherein said filter or membrane barrier does not allow a macro- molecule to pass through.

[0043] Different dissolved macromolecules are sorted in a centrifuge according to their sedimentation speed or density under the influence of centrifugal forces. During spinning a macromolecular solution in a centrifuge tube in a centrifuge, a concentration gradient of macromolecules dissolved in the solution is generated, wherein the density increases from top to bottom. Without wishing to be bound by theory, it is assumed that said concentration gradient is mainly generated by the effects of the least one membrane present in the concentrator device. Centrifugal forces add upon said gradient formation.

[0044] In the context of a "homogenization" of a solution in a centrifuge tube during the spinning process in a centrifuge, the generated concentration gradient of the macromolecules may be reduced or reverted. Accordingly, the homogenization of the solution during a centrifugation process can also be a partial homogenization. The term "homogenize" can also be understood in the sense of "stirring" or "mixing". In certain embodiments, the homogenization constitutes a counter movement to the forces exerted by the filtration or pore size exclusion of macromolecules at the membrane or filter unit and/or by the centrifugation. If molecule movement inducing activities are applied at the same time, i.e. homogenization at one hand and centrifugation at the other hand, a clearing of the membrane zone may be obtained, which allows smaller molecules to pass the membrane without obstruction or with a reduced obstruction by macromolecules due to their lower local concentration or lower density at the membrane. The macromolecules may, at the same time, still be present in a form of a concentration gradient with the highest concentration at the membrane, albeit with a reduced concentration in comparison to a situation without homogenization activity.

[0045] The term "concentrator device" as used herein refers to any device which is ca- pable of filtering or selecting a macromolecule from a solution comprising said macro- molecule. In an embodiment, said concentrator device is a filter unit, optionally with a suitable adaptor to accommodate it to different centrifuge types. The filtering process applied to said concentration device may be based on principles of microfiltration, ultrafiltration, nanofiltration or reverse osmosis. In a preferred embodiment, the principle of ultrafiltration is employed for the selection or retention of macromolecules such as proteins or biological material. The concentrator device may preferably by capable of concentrating a solution with macromolecules wherein said macromolecules are proteins, e.g. a solution which comprises exclusively one or more proteins, or a solution which comprises proteins in addition to further compounds or elements, e.g. as characterized herein above. In further embodiments the concentration of other non-proteine- ous macromolecules such as nucleic acids and/or sugars or carbohydrates etc. is also envisaged. The membranes of the concentrator device, e.g. filter units, are typically made of porous and polymeric materials. Examples of such materials include polyether- sulfone (PES), Hydrosart ^® (stabilized cellulose based membrane) or Gaskets PVDF Spacer Polypropylene Sealing compound. Different polymeric materials may lead to different pore-sizes of a membrane, which are capable of concentrating molecules with a certain molecular weight cutoff (MWCO). Typical MWCO values envisaged by the present in- vention are: 3 kDa 5 kDa, 10 kDa, 30 kDa, 50 kDa and 100 kDa. The present invention further envisages also values below 3kDa and above 100 kDa, as well as any numerical value in between 3 kDa and lOOkDa. An average pore-size of about 5 to 20%, preferably of about 10 % of the diameter of the targeted macromolecule may be used in certain embodiments to avoid the passage of too many macromolecules. In certain embodi- ments the pore-size may be adjusted to the exact identity of the solute, e.g. the size, form, charge, concentration etc. of the macromolecule to be filtered. The pore-size typically comprises a distribution of about 20 %; however, other distributions are also envisaged by the present invention. In certain embodiments, the concentrator device, e.g. filter-unit can withstand high centrifugal forces, e.g. up to 10.000 g. Such high forces may, for example, be present in fixed angle centrifuges which can provide for faster concentration processes and are, among other centrifugation forms and formats, envisaged by the present invention. The sealing cap comprising a stirring element according to the present invention will be working in concert with a variety of commercially available and well established filter-units. Non-limiting examples of such filter units comprise Vi- vaspin ^® ultrafiltration concentrators, Millipore Amicon ^® concentrators or Macrosep ^® Advance Centrifugal Devices.

[0046] The design of the concentrator device may, in certain embodiments, resemble that of the patent US 5,647,990. For example, the two membranes of the concentrator device may have a large size to enhance the flow rate of the solution and may be oriented in an acute angle to each other mainly to reduce the membrane fouling effect. With the use of the sealing cap comprising a stirring element according to the present invention both, membrane fouling and flow rates, are improved and therefore, the membrane size can be reduced and the angle to each other can be flatter. The present invention therefore envisages a variety of designs, which are based on US 5,647,990, yet with angles which differ by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% in comparison to those of US 5,647,990. In specific embodiments, the concentrator device may have an angle between its two membranes of e.g. 60° to 90°, which may vary, pref- erably be increased, by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or more than 80%.

[0047] In further embodiments the membrane size of the concentrator device may be drastically reduced in comparison to standard concentration devices, e.g. those of US 5,647,990. The lower membrane size, e.g. a membrane area which is reduced by about 30 to 50%, preferably reduced by about 40 % in comparison to the membrane size of a standard concentration device as disclosed in US 5,647,990 advantageously leads to a reduced aggregation and higher yields of the concentrated macromolecular solutions. A flatter angle of the membranes, as described above, will also allow to accommodate more solution into the filter units, as the sample reservoir of the filter unit will be signif- icantly higher.

[0048] The present invention further envisages the use of concentrator devices such as filter units with different form factors and for different volumes. To use many concentrator devices in concert with the sealing cap comprising a stirring element according to the present invention modular adapter units may be used which allow to switch be- tween different concentrator devices. The adapter units may feature cavities in which smaller and differently formed concentrator devices can be placed, to ensure the exact fit for the sealing cap comprising a stirring element according to the present invention in respect to the concentrator devices. The adapter unit may also harbour a sample reservoir to collect the retentate, which represents the fluid which does not pass through the filter membrane.

[0049] Typically, an adapter unit connects the stirring element with different concen- trator devices such as filter-units, which may vary in size and volume of macromolecular solution they can maximal accommodate. Typical maximal volumes are: 0.5 mL, 2 mL, 4 mL, 6 mL, 15 mL, 20 mL and 100 mL. Also envisaged are all suitable volumes between 0.5 ml to 100 mL, volumes smaller than 0.5 mL or larger than 100 mL, e.g. 150 mL or 200 mL or more. [0050] The agitation (or mixing) may, in certain embodiments, be accomplished with an autonomic, battery powered, electrical stirring element or device which works as a "homogenizer". In further embodiments, the stirring is accomplished by a "stirring staff" like assembly.

[0051] In preferred embodiments, the stirring staff of the stirring element is dipping deep into the macromolecular solution within the sample reservoir of the centrifugal filter unit. In such a position it is directed along the centrifugal force vector of the centrifuge.

[0052] The sealing cap comprising a stirring element according to the present invention may be used with any suitable centrifuge known to the skilled person. In preferred em- bodiments, the used centrifuge comprises a swinging bucket rotor, which avoids force vectors generated by centrifugal forces within a rotor to break or damage the stirring staff. In further embodiments, also rotors with a fixed angle are envisaged. In these embodiments, the stirring elements is provided in a more stable configuration which withstands high centrifugal forces. [0053] To enable a continuous stirring throughout the centrifugation process, the sealing cap comprising a stirring element may comprise an electronic motor and an autonomous energy source, which is accomplished by a rechargeable battery. [0054] The lowered membrane fouling allows to increase the speed of the concentrating process significantly. There also is no need anymore to stop the centrifuge in order the manually remove aggregates from the membrane e.g. with the agitation of a pipette. Latter process is quite time consuming, as the breaking and the acceleration of the cen- trifuge's rotor is often very slow. The presently claimed methods thus comprise a concentration activity in which the spinning of the centrifuge is not paused or stopped before the concentration of the macromolecules has reached the intended value. In specific, optional embodiments, the spinning may be stopped or decelerated, e.g. in order to allow for a more effective homogenization, in order to add or remove elements to the solution, to generate or decompose gradients within the solution etc. The claimed methods also provide for a repetition of the concentration activity, e.g. 2, 3, 4, 5 or more times in dependence of the size of the macromolecules, the size of the concentrator units, the speed and architecture of the centrifuge, the presence of elements in the solution, the presence of gradients, the pore size etc. [0055] Highly viscous macromolecular solutions can often not be well concentrated as a result of a strong decline in the filtration speed due to membrane fouling. With the use of the homogenizer according to the present invention, e.g. in the form of the sealing cap comprising a stirring element as defined herein above or the assembly comprising said sealing cap as mentioned herein above, it is expected that highly viscous solu- tions can be filtrated more easily and faster. The term "highly viscous solution" as used herein refers to a solution with a dynamic viscosity higher than about 0.0020 Ns/m ² , equal to an aqueous solution containing about 20 % glycerol (v/v; at 293.15 K; based on the parameterisation in Cheng (2008) Ind. Eng. Chem. Res. 47 3285-3288). The present invention thus envisages in a specific embodiment the filtration and concentration of solutions with a dynamic viscosity of at least about 0.0020 Ns/m ² to about 0.0084 Ns/m ² , equal to an aqueous solution containing about 50 % glycerol (v/v; 293.15 K) and still acceptable concentration speed, e.g. of doubling the start concentration in at least 30 minutes. Also envisaged is the filtration and concentration of solutions with a dynamic viscosity of more than 0.0084 Ns/m ² , e.g. up to 0.5 Ns/m ² . [0056] Additionally, the shear forces produced by the homogenizer according to the present invention, e.g. in the form of the sealing cap comprising a stirring element as defined herein above or the assembly comprising said sealing cap as mentioned herein above, can also be used to clean the concentrator device, e.g. a filter-unit as described herein. In a specific embodiment, the cleaning may be carried out together with a suitable cleaning reagent, e.g. water, a buffer and, in specific embodiments, additionally with isopropanol or ethanol.

[0057] In certain embodiments it is envisaged that the sealing cap comprising a stirring element works independent from any outside energy source, as the homogenization and the concentration process takes place within a closed centrifuge. Therefore, a small and compact rechargeable battery may be provided, which derives the energy to power a brushless electric motor, which generates a defined rotational speed of the stirring staff, which is coupled to the motor's driving shaft.

[0058] To recharge the battery, a loading station may deliver the power by using either simple metallic contacts or with the usage of contactless induction of electric current. Such a recharger may be connected directly to the electrical grid and may transform the voltage value suitable to recharge the battery (e.g. 4V).

[0059] Further electronic components may be provided to stabilize the fast rotation and adjust the revolutions per minute, e.g. an electronic transistor circuit, comprising a voltage regulator or a potentiometer.

[0060] Additionally, the sealing cap comprising a stirring element may comprise an on/off switch and a rotary switch to adjust the resistance of the potentiometer to vary the rotational speed.

[0061] The rotational speed of the stirring staff, to generate the optimal homogeniza- tion of a macromolecular solution, can be determined empirically with a diverse set of different macromolecules. Typically, rotational speeds of, e.g. 5000 - 10000 rpm such as 5,000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or any other suitable rotational speed may be used. In specific embodiments, the speed can also be adapted to the mac- romolecule to be concentrated, the type of centrifuge or rotor used, or the viscosity of the solution and may vary from the typical speed of 5000 to 10000 rpm. The empirical determination may help to optimize the results of the concentration process in terms of speed und final yield of the macromolecules.

[0062] The stirring staff may have any suitable form known to the skilled person. The design of the stirring staff may, in one preferred example, be that of a coiled spiral (also called helical spiral), which transports the molecules of a macromolecular solution towards the membrane, as a consequence of its rotation and creates a circulating flow of the solution. Additionally, the helical design minimizes the shear forces between the molecules of the macromolecular solution and the stirring staff. Due to the low shear forces of the stirring element having a helical or coiled spiral shape, an advantageously low heat generation within the solution occurs. A spiral shape acts, in principle, like an Archimedean screw, thus transporting the molecules towards the filter area. In certain embodiments, it is preferred that a heating-up of the macromolecular solution according to the invention is avoided in case of macromolecules which are not heat resistant, e.g. in the case of proteins which have the tendency to form insoluble aggregates. In a particularly preferred embodiment, the coiled spiral shape has the form of a twist drill bit with varying helical angle, varying Point angle and varying Lip relief angle. Further details would be known to the skilled person. In further embodiments, the stirring staff may have the form of a simple propeller. A stirring staff with such a simple propellerlike design, generates tangential force vectors which may lead to increased input of heat into the solution. Such a design may preferably be employed with macromolecules which are heat resistant or in case heat is required in order to deliberately block the activity of undesired macromolecules, e.g. enzymes. It may further be preferred in situations in which a cooling or freezing module is employed during the centrifugation, or where the solution has been precooled before starting the centrifugation process. In a specific embodiment the propeller shape of the stirring element is not employed in case a cooling or freezing module is not working during the centrifugation or the macromol- ecules are not heat resistant. In further embodiments, the propeller shape of the stirring element is used in situations, in which concomitantly a cooling or freezing module is employed during the centrifugation or in which the macromolecules are not heat re- sistant. It is generally preferred that a heating-up of the macromolecular solution is avoided in case of macromolecules which are not heat resistant, e.g. in the case of proteins which have the tendency to form insoluble aggregates during the centrifugation process. This can be achieved by the use of suitable cooling or freezing modules and/or the use of suitable stirring elements as mentioned above. [0063] The dead stop feature of a filter unit stops the sample concentrating to dryness as has been described in U.S. Pat. No. 4,632,761. Typically, in a concentration device an area exists, which does not have a penetrable membrane surface being located beneath the membrane. This area may, in certain embodiments of the invention, be used to stabilize the movement of the stirring staff during fast rotations. Accordingly, in specific embodiments, the stirring staff may have a thin extension which reaches down to the dead stop area of the filter unit. The extension can, for example, touch the wall of the dead stop area without harming the membrane area above. This allowed touching of the dead stop wall can, for example, happen if the swinging bucket rotor of the centrifuge does not swing out exactly along the force vector of the centrifugal force, which may be due to the fact that the swinging bucket is not well counterbalanced.

[0064] The material of the stirring staff may be any suitable material known to the skilled person. For example, it may be plastic material, e.g. fluoroplastic. It is preferred that the material is biologically inert and cannot alter the biological activity or the structure of the macromolecules. [0065] In further preferred embodiments, the sealing cap comprising a stirring element homogenizer is designed in a modular fashion. Such a modular design allows to replace a used stirring staff with a new one, which may be helpful in order to avoid cross contamination between different macromolecular solutions. Accordingly, a new stirring staff may be attached to the drive staff of the electric motor via an integrated screw thread.

[0066] Centrifugal forces of the centrifuge and axial forces of the stirring of the homogenizer may require, in certain embodiments, to mount the electric motor on a durable "taper roller bearing", which is made to resist axial as well as radial forces for a very long time.

[0067] Furthermore, some of the final concentrated macromolecular solution may still stick on the stirring staff. An additional adapter may be provided and used to ensure the maximal recover of the concentrated macromolecular solution. This adapter also may comprise a cavity, which has a slightly bigger dimension as the stirring staff. With an additional centrifugation of this assembly, the remains of the macromolecular solution can be recovered, e.g. with a pipet.

[0068] The modular nature of the homogenizer makes it possible to switch to other stirring staff designs, if necessary.

[0069] Additionally, the stirring staff can be provided together with a flywheel proximate to the shaft of the stirring staff. This can avoid vibrations, caused by high rotational speed of the stirring staff.

[0070] In a further embodiment of the invention, the rechargeable battery is separately placed within another centrifugal inlet, within the centrifuge and connected with the motor of the homogenizer via wires to power the electric motor. This would allow to scale up the size and the capacity of the battery and therefore prolong the homogeniza- tion process for multiple concentration processes.

[0071] In a further embodiment of the invention, the rechargeable battery can be separately placed to the centre of the centrifuge's rotor, e.g. with magnetic adhesion or a supporting frame and is connected with the motor of the Homogenizer via wires. This may avoid centrifugal forces potentially affecting the lifespan of certain types of batteries.

[0072] In an even further embodiment of the invention, a wind wheel could serve as an alternative to the battery driven electric motor. This is possible, because the spinning speed of a centrifuge creates strong winds, which can power the wind wheel. The rotation of the wind wheel will be translated to the stirring staff.

[0073] In a further aspect the present invention relates to a concentrated macromo- lecular solution obtainable with the concentrating method as defined herein above. In certain embodiments, said solution is, when the centrifugation process is finished, es- sentially free of macromolecular aggregates. In further embodiments, the biological activity of the concentrated macromolecules within said concentrated macromolecular solution is, when the centrifugation process is finished, about 70%, preferably 80%, more preferably 90%, 95%, 96%, 97%, 98% or most preferably 99% of the biological activity of macromolecules in the macromolecule solution before the concentration process has been performed, i.e. most, almost all or, most preferably, all of said biological activity can be retained.

[0074] Turning now to FIGURE 1, adapter unit 1 is shown, which connects the homogenizing sealing cap according to the invention with a concentrator device such as a filter unit 2. As shown, the homogenizing sealing cap may comprise a housing 3 in which the electric motor 4 is located and its rotational core 5, as well as a rechargeable battery 6, a voltage regulator 7 and a potentiometer 8. The housing may comprise a lid 8, which contains a rotary switch, which modulates the resistance of the potentiometer 9, and an on/off switch 10.

[0075] On top of the housing's base plate a tapered roller bearing 11 may be mounted, through which a drive shaft 12 of an electric motor may be directed. [0076] Onto the drive shaft 12 a stirring staff 13 is directly mounted which may have a thin extension 15, which reaches into the area of the dead stop volume 16 and thereby avoids contacts with the membrane area 14 of the filter unit 2.

[0077] FIGU RE 2 shows a further embodiment of the homogenizing sealing cap as de- picted in Fig. 1, depicted without the adapter unit and the filter unit. As shown the housing 3 is connected to lid 8, as well as the stirring staff 13. The stirring staff 13 has a thin extension 15.

[0078] The figures are provided for illustrative purposes. It is thus understood that the figures are not to be construed as limiting. The skilled person in the art will clearly be able to envisage further modifications of the principles laid out herein.