EP3501660A1 - Method and device for the separation of substances by centrifugation - Google Patents

Abstract

The present invention relates to a method for separating substances, in particular for separating the components of suspensions, emulsions and gas mixtures, and suitable devices for carrying out the same. According to the invention, the separating method is carried out by conventional centrifugation using suitable separating devices and/or centrifugation containers in the presence of at least one acceleration sensor close proximity to the material to be centrifuged.

Description

The present invention relates to a process for the separation of substances, in particular for the separation of the constituents of suspensions, emulsions and gas mixtures, as well as suitable devices for carrying it out.

Centrifuges are often used for substance separation, which work by exploiting the inertia. The operation is based on the centrifugal force, which comes about due to a uniform circular motion of the material to be centrifuged (Zentrifugiergut) and brings about the desired separation of substances. A similar method uses the centrifugal separator, for the application of the inventive method is equally suitable.

Centrifuges use the mass inertia in the centrifuging compartment for separating substances. Higher density particles or media are separated from the lower density components due to their higher inertia. This process is much faster than the sedimentation by gravity or even possible at all, since opposing forces such as the adhesion, the thermal molecular motion or the viscosity are overcome.

If different centrifuging conditions are used for the desired substance separation of a given substance mixture using the same centrifuge and a centrifuge container of the same specification, considerable separation errors may occur, especially in the medical-diagnostic field, since the separation of the components of different density has taken place only partially. Practical applications for this are, for example, the separation of whole blood into serum or plasma and cell components. The separated components such as plasma and serum are used, for example, in diagnostics for the determination of disease markers on. In this case, compliance with the centrifugation conditions is indispensable for the comparability of the measurement results, since even small differences can influence the measurement results of modern methods. The current development in cell-based immunotherapy against cancer has shown that centrifugation processes are also relevant in the pharmaceutical sector. For example, in the newly approved T cell-based drug CTL019 (Novartis, CAR-T technology), centrifugation to prepare the T cells is imperative for the preparation of T cells. The verifiability of whether these necessary for the conditioning centrifugation processes within the scope of the previously made specification (validation) have taken place, is given exactly only on Zentrifugationsgerät or Zentrifugationsbehältnis and only directly after the centrifugation process. This circumstance requires confidence in regular maintenance, proper use and truthful manufacturer information. Particularly in decentralized processes with multiple users, a large number of errors or irregularities may occur that may not occur in the course of many certification and auditing measures, such as GMP (Good Manufacturing Practice) or GLP (Good Laboratory Practice ).

There is therefore a need for the provision of a method as well as suitable devices with which the results of a separation of substances of the aforementioned type can be recorded directly in situ in real time and can be checked comparatively easily and inexpensively.

The object is achieved by the method and by the separating device of the independent claims. Advantageous embodiments are set forth in the subclaims and in the following detailed description.

, wobei v die Sedimentationsgeschwindigkeit der Partikelsphäre, d den Durchmesser der Sphäre, p die Partikeldichte, L die Dichte des umgebenden Mediums, n die Viskosität des umgebenden Mediums, und g die Beschleunigungskraft bezeichnen.As already mentioned, the quality of the separation of particles in suspension by acceleration forces is mainly due to the particle size and the density differences between the substances dependent. By applying accelerating forces, larger volume, higher density particles are separated from lower volume and / or lower density particles during centrifugation, and the time to desired separation or sedimentation can be shortened by the application of higher acceleration forces. By the Stokes equation, the sedimentation velocity of particles can be calculated very well based on 5 parameters $$v=\frac{d^2\left(p-L\right)G}{18\eta }$$

where v denotes the sedimentation velocity of the particle sphere, d the diameter of the sphere, p the particle density, L the density of the surrounding medium, n the viscosity of the surrounding medium, and g the acceleration force.

The Brownian molecular motion counteracts the sedimentation velocity. For very small particles and small differences in density, therefore, very high acceleration forces must act to ensure successful separation of the particles. By applying centripetal forces about a fixed axis, these high acceleration forces can be achieved, so that even separations of molecules that differ only in the isotopic distribution, are possible. This technique, commonly referred to as centrifugation, allows the separation of cells, organelles, membranes and molecules in the biological or biotechnological field. A rough classification of centrifugation applications is made by the achievable acceleration forces (expressed in multiples of g, the gravitational acceleration). While up to 8,000 g are used in low-speed centrifugation, up to 22,000 g are used for high-speed centrifugation and up to 700,000 g for ultra-centrifugation.

In addition to direct sedimentation, especially the low speed centrifugation is also used for accelerated filtration or other Size exclusion method used. In addition, in the solid phase extraction of, for example, nucleic acids by means of so-called 'spin columns' centrifugation processes are used to speed up the process (see, for example, BioRad (http://www.bio-rad.com/en-us/product/empty-spin-columns).

An extension of the methods involves differential centrifugation, which uses the sequential application of different centrifugation conditions to separate several substances from the same mixture. Further, rate zonal centrifugations and isopycnic centrifugations are used. Both techniques use adjuvants that form gradients. At rate zonal, the density of the gradient is lower than the materials to be separated, and therefore it is necessary to stop the centrifugation at a defined time to achieve the separation of different particle sizes. In the case of isopycnic centrifugation, the density of the gradient is certainly higher than that of the substances to be separated, so that the particles collect in a band of the gradient of the same density or the same sedimentation rate.

In centrifuges usually fixed-angle rotors, swing-bucket rotors or specialized rotor systems are used, which are referred to herein as separation devices. The rotors can be designed directly for receiving a mixture to be separated, or they are designed to accommodate containers such as tubes of different size and texture (polypropylene, usually up to 20,000 g (fixed angle rotor) at higher g-forces rather thermosets (e.g., polycarbonate).

Beckmann Coulters (Sorvall and other brands, the largest manufacturer of ultracentrifuges), Hettich, Thermo, Eppendorf, and NuAire are among the best-known manufacturers of centrifuges.

Typical centrifugation containers, such as centrifuge tubes, have a volume of between 5 .mu.l and 5000 ml, wherein they are usually conical, spherical or elliptical in shape. The However, the present invention is not limited to this exemplary selection of sizes and shapes, but is equally suitable for use with containers of other sizes and shapes.

Conventional centrifuge tubes are usually made of thermoplastic or thermosetting plastics by injection molding (in particular stretch blow molding or injection blow molding), wherein the temperatures can reach up to 220 ° C in the normal injection molding process. The injection blow molding process uses lower processing temperatures in the range of approx. 100-120 ° C.

In the context of the present invention, it is proposed to carry out the desired material separation with centrifuges whose rotors and / or containers, in particular tubes, are equipped with at least one RFID transponder, this transponder preferably representing a component of an RFID system which also comprises a receiver, and Accelerometer (accelerometer) includes, wherein the acceleration sensor may alternatively be provided as a component of a so-called inertial measurement unit (Engl. 'Inertial Measurement Unit', IMU).

RFID (Radio Frequency Identification) refers to a technology for transceiver systems for the automatic and contactless identification and localization of objects with radio waves. A known RFID system thus consists of a transponder, which is located on or in the article and contains a distinctive code, and a reader for reading this identifier. According to the invention, the transponder is arranged in the separating device of a centrifuge, in particular on the rotor and / or on the centrifuge tube and thus in the immediate vicinity of the material to be separated, wherein the arrangement of the transponder alternatively also take place in the rotor and / or in the centrifugation tube or, if necessary, in the material to be separated can. Although according to the invention preferred RFID systems of the Type, ie, with the code of the transponder and reader for reading the identifier, can be used, in particular provided with an RFID transponder centrifugation tube can be configured as disposable or reusable article. Since it is possible to embed RFID transponders circuit stable and heat-resistant in polymers via a special printing process, it is inventively preferred to use centrifugation tubes made of plastic, which have been prepared by the aforementioned injection molding process with storage of such heat-resistant RFID transponder made of polymers.

The currently available RFID transponders (RFID chips) already allow a temperature resistance at 220 ° C for the duration of one hour, which corresponds to the temperature range for injection molding in the context of the production of centrifugation plastic tubes. In the context of the present invention, it is therefore preferable to provide and use separation devices, in particular centrifugation tubes with embedded RFID transponders, which have already been introduced into the injection mold or for the injection blow molding process of the tube into the preform. Particularly preferred is the use and provision of separation devices such as in particular of centrifugation tubes with RFID transponders, which have been introduced into the tail (in the wall of the bottom) of the preform, so that the transponder using the injection blown in the subsequent solidification of the Förmlings in Bottom of the centrifuge tube is permanently fixed. Preferably, the RFID transponder has additional recesses which facilitate orientation and positioning as well as spacing during the injection molding process. Alternatively, and depending on the specific application and the material to be separated is preferred to insert a commercial RFID transponder regardless of its nature and method of preparation prior to the separation process or centrifugation loose in the centrifuge tube, or a tube with a way to accommodate the transponder such as a slot on the bottom or the cap to use, whereby the centrifugation takes place in both cases in the presence of the RFID transponder in the centrifuge tube.

Alternatively or additionally, it is proposed to arrange the equipped with the function of an acceleration sensor RFID transponder in or on a rotor as a separation device. In certain technical Zentrifugationsprozessen no tubes are needed, but it comes directly to the separation of the material in the rotor. Mostly it is about the continuous separation of suspensions. Especially in the biological field, cells are often subjected to continuous flow centrifugation. This is done in the low-speed range up to 1000 g, but due to the size of the rotors depending on the specific positioning of the material to be separated within the rotor can lead to significant differences in the g-force prevailing at the separation material. In the case of the use of rotors equipped with RFID transponders, it is therefore preferable to carry out a multiple punctual monitoring (increased spatial resolution by simultaneous use of several RFID transponders) of the actually prevailing acceleration forces in order to enable an improved yield and viability of cells and cell products ,

In the context of the above discussion, it is pointed out that, especially during the acceleration and deceleration phases of a separation process, differences in the acting acceleration force g occur (see FIG. FIG. 3 ), provided that the distance of the material to be separated to the Zentrifugationsachse (axis of rotation) of the rotor does not change.

, wobei R den Abstand zum Zentrifugationsmittelpunkt in cm, und Q die Drehzahl in rpm ('rounds per minute') bezeichnen.Especially with swing-bucket rotors and the use of gradient systems, it can lead to significant positional differences of the particles during centrifugation, which is why differences in particular in the purification of cells can be observed in terms of viability and yield. The further away a cell is from the axis of centrifugation, the greater the force that acts on it and can cause damage to it. The g-force in a centrifuge (also called Radial Centrifugal Force (RCF)) can be calculated using the set speed and the distance from the centrifugation column (rotation axis) according to the following formula $$\mathit{RCF}=\mathrm{11:18}•r•{\left(Q/1000\right)}^2$$

where R is the distance to the centrifuge center in cm, and Q is the rpm in rpm ('rounds per minute').

A difference in the g-force at an assumed distance of 10 cm of the material to be separated to Zentrifugations- or rotation axis can easily come about both in a centrifuge tube and in a rotor. At a distance of 1 cm and a speed of 5000 rpm results in a RCF of 279.5 g. At 10 cm distance and 5000 rpm results in 2795 g for the RCF. While 280 g are well tolerated by cells, g-forces of over 1000 g can destroy cells and cause them to burst. It is clear that a (locally) multiple introduction of equipped with acceleration sensors RFID transponders in a tube, a rotor, or another separation device can be advantageous and is therefore preferred. This is especially true in the case of a desired differentiated separation of cells and biomolecules.

The present invention will be explained in more detail with reference to embodiments.

example 1

RFID transponders 4 with integrated functionality of an acceleration sensor were purchased from ST Electronics (Geneva, Switzerland) for carrying out the teaching according to the invention. They are temperature resistant up to about 200 ° C, comprise an antenna and have a size of about 3 mm x 3 mm and a thickness of about 1 mm on (s. FIGS. 1 and 3 ). Due to their smaller size, passive RFID transponders 4 were used in the present case, with semipassive transponders also being used as an alternative.

The passive RFID transponder 4 can be due to the small size most easily incorporated into the plastic of the centrifuge tube 1 to be produced. However, passive RFID transponders have no independent power source, which is why they receive appropriate signals before they are used via an RFID reader / transmitter station so that they can send or write data. Then an induction is triggered via the integrated antenna, whereby the RFID transponder 4 is supplied with sufficient current (about 0.35 watts). This amount of flow is sufficient to sufficiently supply the 3-way accelerometer integrated in the RFID transponder 4 for the subsequent centrifuging operation.

In several parallel experiments, an activating transmitter / receiver was mounted both on and in the vicinity of the centrifuge to be used. The centrifuge tubes 1 with passive RFID transponder 4 (receiving range 1-6 m) were brought into range of the transmitter for a few seconds before the respective centrifugation. A successful activation could be checked via the transmitter / receiver display. The centrifugation process was started. The passive RFID transponders 4 sent the data in real time to the transmitter / reader. This transmitted the data of the acceleration measurement to a database. Due to the constant activation of the transponder 4, continuous transmission of the centrifugation data was always ensured, even during longer centrifugation processes.

In the context of the alternative use of semi-passive RFID transponders 4, which have a limited power supply, activation by a transmitter / reader unit prior to centrifugation could be dispensed with. Activation took place via the integrated accelerometer, ie data writes only from a predetermined centrifugal force of 50 g. The reading out of the RFID transponder 4 took place at a later time and at a different location. This application has the advantage that the site does not need to have the prerequisite to read out the RFID transponder data online. Thus, a very flexible use of the present invention with RFID transponders 4 equipped centrifuge tube 1 is possible, even in less technological environments.

Example 2

As a practical application of the inventively proposed RFID accelerometer technology in centrifugation processes, the purification of PBMC (Peripheral Blood Mononuclear Cells) is used here, which represents a precursor of the T cell-based immunotherapy. A common application error is the setting of the speed in revolutions (rpm) instead of the intended radial centrifugal force or acceleration (RCF or g). This can lead to the desired separation of the different cell populations not taking place as desired. For this purpose, the following series of experiments was carried out.

50 ml cell culture tubes 1 (article No. 210261 Greiner BioOne, Frickenhausen, Germany) were prepared using 2-component adhesive 11 (Henkel, Dusseldorf, Germany) with one RFID transponder each with accelerometer 4 (accelerometer: H3LIS331DL, ST-Electronics , Geneva, passive RFID transponder Andy 100 IC, Farsens, San Sebastian, Spain) in cover 2 and bottom 3 provided and sealed accordingly. After complete drying, the tubes 1 were held for about 10 seconds via an Impinj R220 UHF RFID reader (also Farsens) to activate the passive RFID transponder 4. Subsequently, tubes 1 were loaded with 20 ml of Ficoll Paque gradient medium 5 (GE Healthcare, Little Chalfont, UK). Subsequently, carefully 20 ml of EDTA whole blood 6 from a blood donation were added to the tube 1 (s. FIG. 3A ). Part of the samples were used according to the Protocol at 350 g ( Fig. 3B Centrifuged for 45 minutes while another part is centrifuged at 350 rpm ( Fig. 3C ) Was centrifuged for 45 minutes (Eppendorf 5810R with rotor A-4-81, without brake force adjustment). After centrifugation, the expected band pattern of plasma 7, mononuclear cells 8 and granulocytes 9 as well as the sediment of erythrocytes 10 were shown at centrifugation B at 350 g. In contrast, centrifugation C did not show the expected bands for mononuclear cells 8 and polymorphonuclear cells 9 and red streaks from erythrocyte sediment 10 traversed most of the sample. The transmission of the data took place online with the passive RFID unit. The evaluation of the data for centrifugation B showed a maximum centrifugation force g in the plateau of 311 g (+/- 2 g) for the introduced in the bottom 3 RFID transponder 4 (b2), while the RFID transponder 4 in the lid 2 (b1) in Plateau showed a maximum acceleration force of 136 g (+/- 2g) (see FIG. 4 ). Interpolation to the center of the vessel, which corresponds to the position of the mononuclear cells 8, gave a maximum centrifugation force of 233 g (+/- 2 g). In centrifugation C, the maximum centrifugal force registered in the plateau by the soil accelerometer (c2) was 22 g, while the measurement by the accelerometer 4 in the lid 2 (c1) gave only 10 g (see FIG. 4 ). The determined course of the centrifugal forces is for centrifugations B and C in FIG. 4 shown. The low insufficient centrifugation forces in centrifugation C can be clearly explained by the application error and the resulting low acceleration. The deviations in centrifugation B from the set 350 g are given by the distance of the accelerometer 4 from the maximum centrifugation radius of the device and makes even in the bottom area of the vessel more than 10% of the centrifugal force. Another application error, which could be clearly recorded via an integrated accelerometer 4, would be the setting of a brake force on the centrifuge. An applied braking force is not desirable in a gradient centrifugation, as by the centrifugal forces released during deceleration Density bands of the gradient become blurred and no cell separation occurs. The recording by an accelerometer 4 here would indicate a much more abrupt decrease of the centrifuging force compared to an unrestrained leak in the discharge phase after the actual centrifugation (see additional curves in FIG FIG. 4 ).

figure description

The FIG. 1 (AF) illustrates the respective positioning of a miniaturized RFID accelerometer 4 in the region of the bottom 3 of centrifugal containers 1 of different shape. A) Direct incorporation of the RFID accelerometer 4 without antennas in the centrifugation vessel 1 during the manufacturing process. B) Direct incorporation of the RFID accelerometer 4 with antennas during the manufacturing process. C) introducing and, if necessary, fixing an RFID accelerometer 4 in the interior of a centrifugation tube 1. D) attaching and fixing an RFID accelerometer 4 in an outer recess of a centrifugation vessel 1. E) attaching an RFID accelerometer 4 to the lid outer of a centrifugation vessel 1. F) Attaching an RFID accelerometer 4 to the inside of the lid of a centrifugation vessel 1.

The FIG. 2 (A, B) shows the recorded course of the acceleration in g over the time of the centrifugation, for example within specified parameters (A), as well as the recorded course of the acceleration in g over the time of centrifugation and subsequent termination when a fixed acceleration g ( B).

In the Figures 3 and 4 the results of the experiment set out in Example 2 are presented comparatively.

LIST OF REFERENCE NUMBERS

1

Centrifuge tube, cell culture tube, tube, centrifuge container

2

cover

3

ground

4

Accelerometer, RFID transponder with accelerometer, RFID accelerometer, accelerometer, transponder

5

gradient medium

6

EDTA whole blood

7

plasma

8th

Mononuclear cells

9

Granulocytes, polymorphonuclear cells

10

Sediment from erythrocytes, erythrocyte sediment

11

2-component adhesive

Claims (10)

Method for separating the components of centrifuged goods such as suspensions, emulsions and gas mixtures by conventional centrifugation using suitable separation devices and / or Zentrifugationsbehältnissen, characterized in that the separation takes place in the presence of at least one acceleration sensor (4) in the immediate vicinity of Zentrifugiertengut. Method according to Claim 1, characterized in that the acceleration sensor (4) is capable of measuring and recording the acceleration force acting on the material to be centrifuged and / or of transmitting it to a receiver. Method according to Claim 1 or 2, characterized in that an RFID transponder (4) configured functionally for acceleration measurement is used as the acceleration sensor (4). Method according to one of the preceding claims, characterized in that the acceleration sensor (4) during centrifugation in and / or on the separation device or the Zentrifugationsbehältnis (1) is arranged. Method according to one of the preceding claims, characterized in that a rotor is used as the separating device. Method according to one of the preceding claims, characterized in that a centrifugation tube (1) is used as the centrifugation container (1). Separating device or Zentrifugationsbehältnis, characterized in that it or it is equipped with at least one acceleration sensor (4). Separating device or centrifugation container according to claim 7, characterized in that it or the at least one acceleration sensor (4) comprises as an integral part. Separating device according to claim 7 or 8, characterized in that it is a rotor. Separating device or centrifugation container according to one of claims 7 to 9, characterized in that the acceleration sensor (4) is able to measure and record the acceleration force acting on Zentrifugationsgut and / or to send to a receiver.

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