Method for magnetically separating cells with a pressue contolled elution column system

Abstract

The invention is directed to a method for magnetically separating target and non-target cells from a cell suspension by eluding the target cell suspension and / or the non-target cell suspension by providing a first pneumatic pressure to the separation column, detecting the decline of the pneumatic pressure over time due to elude the cell suspension and removing the remaining pneumatic pressure when a non-linear decline of the pneumatic pressure over time is detected.

Description

METHOD FOR MAGNETICALLY SEPARATING CELLS WITH A PRESSUE CONTOLLED ELUTION COLUMN SYSTEMBACKGROUND

[0001] The present invention is directed to an automated method for eluding cell suspension from a magnetic column.

[0002] Magnetic cell separation is an established technology to separate magnetically labelled cells from non-magnetic cells by utilizing a high gradient magnetic field in which the magnetically labelled target cells are retained. Usually, a suspension of magnetically labelled cells and non-magnetic cells are guided through a column provided with a ferromagnetic matrix, which is subjected to a strong magnetic field. The magnetically labelled cells are retained on the ferromagnetic matrix, whereas the non-magnetic cells are eluted. The magnetically labelled cells may then be harvested by removing the column from the magnetic field and rinsing the cells from the matrix. This technology is for example described in US 5693539 or US5385707.

[0003] The separation process is usually performed without applying pressure i.e. under normal gravity conditions, but enhanced pressure may be applied to speed up the process or to reduce the volume of the liquid. In this respect, US6602422 discloses a micro column system for magnetic cell separation provided with a plunger, which can be used to apply pressure in order to flush out liquid from the column.

[0004] By simply applying pressure to such columns, all liquid will be flushed out of the column, resulting in foaming and / or spraying of the suspension thus creating unwanted aerosols. In an automated process, this needs to be prevented.

[0005] On the other hand, removing the pressure too early will result in loss of cells.

[0006] It was therefore an object of the invention to provide an automated process for magnetically separating cells under pressure with easy to establish pressure control of the process.SUMMARY

[0007] Accordingly, it was the object of the invention to provide a method for magnetically separating target and non-target cells from a cell suspension bya) incubating the cell suspension with magnetically labeled conjugates which are capable of binding specific to the target cells and not to the non-target cells, thereby obtaining magnetically labeled target cells and unlabeled non-target cellsb) providing the incubated cell suspension to a separation column comprising a ferromagnetic matrix positioned in a magnetic-field whereby the magnetically labeled target cells are retained by the magnetic field on the ferromagnetic matrix c) eluding the unlabeled non-target cells from the separation column as non-target cell suspensiond) removing the magnetic field from the separation column and eluding the magnetically labeled target cells from the separation column as target cell suspensioncharacterized in that eluding the target cell suspension and / or the non-target cell suspension is performed by providing a first pneumatic pressure to the separation column, detecting the decline of the pneumatic pressure over time due to eluding the cell suspension and removing the remaining pneumatic pressure when a non-linear decline of the pneumatic pressure over time is detected.

[0008] The decline of the pneumatic pressure over time can be easily monitored by a pressure sensor.

[0009] The first pneumatic pressure is a pressure that is applied at the beginning of the elution step. The pressure then declines due over time to the loss of liquid (i.e. cell suspension) from the separation column and is not re-adjusted to the starting value.

[0010] The first pneumatic pressure is at most 1000 mbar like 500 - 1000 mbar.

[0011] In the first stages of method of the invention, the pneumatic pressure usually declines in over time in a linear manner. At a certain point of the method, which depends on the surface area of the column and the first pressure applied, a sudden decline of pneumatic pressure over time can be detected which is used as indicator to remove the remaining pneumatic pressure, thereby effectively stopping the elution process.

[0012] The “sudden decline of pneumatic pressure over time” may also be in a linear manner, but with a different gradient than the pressure decline in the first stages. All in all, at the time of the “sudden decline of pneumatic pressure over time”, a non-linear decline of the pneumatic pressure over time is detected.

[0013] In practice, the non-linear decline of the pneumatic pressure over time is detected between 100 and 500 mbar per second.

[0014] Preferable the remaining pneumatic pressure when a non-linear decline of the pneumatic pressure over time is detected is at most 200 mbar.

[0015] The non linear decline of the pneumatic pressure can be either manually detected by observed by monitoring the pressure decline over time or may be detected by anappropriate algorithm. In both variants, the non linear decline of the pneumatic pressure triggers the removal of the remaining pneumatic pressure and effectively stops the elution process.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig. 1 shows an example of a pressure decline not according to the invention, with the non-linear decline of the pneumatic pressure over time marked by a vertical line.

[0017] Fig. 2 shows an example of a pressure decline wherein a non-linear decline of pneumatic pressure was detected according to the method of the invention. The remaining pneumatic pressure was removed and the decline is therefore linear over time.DETAILED DESCRIPTION

[0018] The column used in the method of the invention is of minor importance for the process and may comprises a reservoir section with an input opening; a filter section provided with a filter matrix and an output opening. Suitable columns are for example disclosed in US6602422 and / or are available from Miltenyi Biotec B. V. & Co. KG under the tradename “MACS column”.

[0019] The column is preferably made of plastics such as polyamide, polystyrene, polyolefin like polyethylene and polypropylene, polycarbonate, polyoxymethylene, acrylics, like polymethylmethacrylate, PET, poly lactic acid, polyamides or steel, and the like.

[0020] When the matrix is coated with lacquer, is preferably made of a plastic that will bind with lacquer, most preferably a resin such as PCTG (polycyclohexadimethylterephtalate modified with ethylenglycol).

[0021] The column may be made hydrophilic by manufacturing it from a hydrophilic material such as a hydrophilic plastic, or, more preferably, by coating the inside of the column with a hydrophilic material, e.g., polyvinyl pyrrolidone.

[0022] The column system used in the invention is provided with a ferromagnetic matrix. In such ferromagnetic matrix, a high gradient magnetic field may be generated by insertion of at least the part of the column where the ferromagnetic matrix is located into an external magnetic field. The ferromagnetic matrix readily demagnetizes when it is taken out of the field.

[0023] The external magnetic field may be provided by a permanent magnet placed in a yoke or by an electromagnet, which should provide a magnetic field greater than about 0.2 Tesla.

[0024] A porous frit or grid may be positioned adjacent the top end of the matrix. The porous frit / grid is preferably made of glass or plastic or metal mesh and has a pore size greater than or equal to the pore size of the matrix and less than the particle size of the matrix.

[0025] The ferromagnetic material may comprise ferromagnetic balls or other ferromagnetic particles. The ferromagnetic material may be coated with a coating which maintains the relative position of the particles with respect to one another. The ferromagnetic balls or particles preferably have a diameter or size of at least 100 pm, more preferably greater than about 200 pm and less than about 2000 pm, still more preferably greater than about 200 pm and less than about 1000 pm, and most preferably about 280 pm.

[0026] For the process of the invention, the column is provided with a pressure source providing pressurized air or other inert gases like N2 or CO2, preferable sterile, to the column. This can be achieved by an appropriate adapter system. Further, the column is connected to a pressure sensor.EXAMPLE

[0027] In a testing environment, autoMACS® Running Buffer available from Miltenyi Biotec B.V & Co KG was pumped through a LS Column (Miltenyi Biotec B.V & Co KG ) by applying pneumatic pressure. The decline of the pneumatic pressure is monitored over time.

[0028] As can be seen from Fig. 1, the decline of the pneumatic pressure has a nonlinear decline approximately at the point where the column is starting to run dry. The result is heavy foaming and spilling of liquid drops at the outlet opening of the column.

[0029] The experiment repeated with monitoring and adjusting the pneumatic pressure according to the invention. Here, the non-linear decline of the pneumatic pressure over time is detected and the remaining pneumatic pressure removed, at the point where the column would run dry. No foaming or spilling of liquid drops can be observed.

Claims

CLAIMS1. Method for magnetically separating target and non-target cells from a cell suspension bya) incubating the cell suspension with magnetically labeled conjugates which are capable of binding specific to the target cells and not to the non-target cells, thereby obtaining magnetically labeled target cells and unlabeled non-target cells b) providing the incubated cell suspension to a separation column comprising a ferromagnetic matrix positioned in a magnetic-field whereby the magnetically labeled target cells are retained by the magnetic field on the ferromagnetic matrix c) eluding the unlabeled non-target cells from the separation column as non-target cell suspensiond) removing the magnetic field from the separation column and eluding the magnetically labeled target cells from the separation column as target cell suspensioncharacterized in that eluding the target cell suspension and / or the non-target cell suspension is performed by providing a first pneumatic pressure to the separation column, detecting the decline of the pneumatic pressure over time due to eluding the cell suspension and removing the remaining pneumatic pressure when a non-linear decline of the pneumatic pressure over time is detected.

2. Method according to claim 1 characterized in that the first pneumatic pressure is at most 1000 mbar.

3. Method according to claim 1 or 2 characterized in that the remaining pneumatic pressure when a non-linear decline of the pneumatic pressure over time is detected is at most 200 mbar.

4. Method according to any of claims 1 to 3 characterized in that the non-linear decline of the pneumatic pressure over time is detected between 100 and 500 mbar per second.

5. Method according to any of claims 1 to 4 characterized in that removing the magnetic field from the separation column is performed by positioning the separation column out of the magnetic field.

6. Method according to any of claims 1 to 5 characterized in that the magnetic field is removed from the separation column by removing the magnet from the separation column.

7. Method according to any of claims 1 to 4 characterized in that the magnetic field is produced by an electric magnet and removing the magnetic field is achieved by switching off the electric magnet.

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