CENTRIFUGE ROTOR, CENTRIFUGE OR ULTRACENTRIFUGE COMPRISING CENTRIFUGE ROTOR, SAMPLE RETRACTION NEEDLE, METHOD FOR IN-SITU SAMPLE RETRACTION FROM A CENTRIFUGE TUBE

Abstract

Centrifuge rotor comprising a rotor assembly adapted for centrifugal rotation around a rotation centre axis, with a plurality of centrifuge tube beds, each with a longitudinal axis, wherein each centrifuge tube bed comprises a tubular cavity which is defined by a cavity sidewall and a cavity bottom which together act as a bearing surface for the outer surface of a centrifuge tube when received in said centrifuge tube bed, wherein at least one of the plurality of cavity bottoms comprises at least one extraction aperture, which connects the tubular cavity to the exterior of the rotor assembly, preferably wherein at least one closure device is removably fastened to the rotor assembly to seal each of the at least one extraction apertures in an airtight manner.

Claims

1. Centrifuge rotor (1) comprising: a rotor assembly (10) adapted for centrifugal rotation around a rotation centre axis (14), with a plurality of centrifuge tube beds (16), each with a longitudinal axis (160), wherein each centrifuge tube bed (16) comprises a tubular cavity (162) which is defined by a cavity sidewall (162a) and a cavity bottom (162b), wherein at least one of the plurality of cavity bottoms (162b) comprises at least one extraction aperture (18), which connects the tubular cavity (162) to the exterior of the rotor assembly (10), characterized in that the cavity sidewall (162a) and the cavity bottom (162b) together act as a bearing surface for the outer surface of a centrifuge tube (4) when received in said centrifuge tube bed (16).

2. Centrifuge rotor (1) of claim 1, wherein the rotor assembly (10) is a fixed angle rotor formed by a rotor body (12), wherein the plurality of centrifuge tube beds (16) are formed as tubular cavities (162) within the rotor body (12) and wherein the at least one extraction aperture (18) extends through the rotor body (12).

3. Centrifuge rotor (1) of claim 1, wherein the rotor assembly (10) is a swing bucket rotor comprising a plurality of rotor buckets (13) pivotable connected to a rotating stem (11), wherein the plurality of rotor buckets (13) comprises at least one tubular cavity (162) to form the centrifuge tube bed (16) and wherein the at least one extraction aperture (18) extends through at least one body of the respective rotor bucket (13).

4. Centrifuge rotor (1) of claim 1, wherein at least one closure device (2) is removably fastened to the rotor assembly (10) to seal each of the at least one extraction apertures (18) in an airtight manner, wherein each of the at least one closure devices (2) comprises a shaft (22) extending along a shaft axis (22a) from a first end (221) to a second end (222), wherein the shaft (22) is adapted to the geometry of the extraction aperture (18), wherein the first end (221) forms a partial area of the cavity bottom (162b) when the closure device (2) is fastened to the rotor assembly (10) and wherein the shape of the first end (21) is adapted to the shape of the cavity bottom (162b) to form a smooth transition of the surface of the first end (221) and the surrounding surface of the cavity bottom (162b).

5. Centrifuge rotor of claim 4, wherein each of the plurality of closure devices (2) comprises a limit stop (24) at the second end (222) to restrict the movement of the closure device (2) in the direction of the shaft axis (22a), when the closure device (2) is fastened to the rotor assembly (10) to ensure the correct alignment of the first end (221) with the surrounding surface of the cavity bottom (162b).

6. Centrifuge rotor of claim 4, wherein each of the plurality of closure devices (2) comprises at least one elastomeric seal (28), which is received in a groove (26) of the closure device (2) and deformed when the closure device (2) is fastened to the rotor assembly (10) to form an airtight seal with the rotor assembly (10).

7. Centrifuge rotor of claim 4, wherein at least a partial area of the shaft comprises external threads (27), wherein the extraction aperture (18) comprises internal threads (27) adapted to the shape and position of the external threads (27), so that the closure device (2) is fastened to the rotor assembly (10) by the adapted threads (27).

8. Centrifuge rotor of claim 4, wherein the second end (222) of the shaft (22) comprises at least one interlocking element (29) which protrudes at least partially in radial direction of the shaft (22) and wherein the rotor assembly (10) comprises at least one cutout (19a) and/or at least one pocket (19b) adapted to receive said at least one interlocking element (29) to form an interlocking structure when the closure device (2) is fastened to the rotor assembly (10).

9. Centrifuge rotor of claim 4, wherein the shaft has a diameter (22d) that lies in the range between 0.5 mm up to 8.0 mm and which lies in the preferred range between 0.5 mm and 2.0 mm.

10. Centrifuge rotor of claim 1, wherein the cavity bottom (162b) is formed by a narrowing surface in the direction towards the lower end of the rotor assembly (10) and the at least one extraction aperture (18) is located at the lowest point of the test tube bed (16) with respect to the rotation centre axis (14).

11. Centrifuge or ultracentrifuge comprising: a centrifuge rotor according to claim 1.

12. Method for in-situ sample retraction from a centrifuge tube (4) housed in a centrifuge tube bed (16) of a centrifuge rotor (1) of claim 1, the method comprising the steps: A) conducting the centrifugation of the samples contained in the centrifuge tubes (4) and housed in the centrifuge rotor (1); B) transferring the centrifuge rotor (1) to a rotor stand, disassembling the centrifuge rotor (1) C) choosing a first centrifuge tube (4) housed in the centrifuge rotor (1) of the plurality of centrifuge tubes (4) from which the content should be extracted; D) generating at least one venting hole in the area of the top of the chosen first centrifuge tube (4); E) opening a respective extraction aperture (18) of a centrifuge tube bed (16) in which the first centrifuge tube (4) is located by removing a respective closure device (2); F) introducing a sample retraction needle (3) through the extraction aperture (18) towards the outer wall of the centrifuge tube (4), piercing the centrifuge tube wall and inserting the sample retraction needle (3) into the first centrifuge tube to generate a fluid connection to the inner volume of the centrifuge tube (4); and G) extracting the content of the centrifuge tube (4) through the sample retraction needle (3).

13. Method according to claim 12, wherein the sample retraction from the centrifuge tube (4) is caused by displacing the sample under pressure with compressed gas or by sample suction out of the centrifuge tube (4) by using an appropriate pump.

14. Method according to claim 12, wherein the sample is displaced from the centrifuge tube (4) by evenly dosing a low density liquid through the venting hole in centrifuge tube.

15. Method according to claim 14, wherein using a high-performance liquid chromatography, HPLC-type pump or peristaltic pump or syringe pump that enable for sample displacement from centrifuge tube (4).

16. Method according to claim 12, wherein the contents extracted by the sample retraction needle (3) is fed to at least one of the devices chosen from the group of a UV-VIS detection system, a fluorescence detection system, a light scattering detection system, a device for conducting a high performance liquid chromatography and/or an automated fraction collector.

17. Method according to claim 12, wherein the first centrifuge tube (4) is placed in a centrifuge tube stand prior to step D).

18. (canceled)

19. An automated system for in-situ sample extraction comprising: a centrifuge or an ultracentrifuge and at least an extraction needle, wherein the system is configured to conduct the method for in-situ sample retraction according to claim 12.

20. Method according to claim 12 further comprising the step: H) repeating steps C) to G) on at least a further centrifuge tube (4) housed in the centrifugation rotor (1).

21. Method according to claim 14 wherein the low density liquid is non-water-miscible.

Description

[0061] In the following, exemplary embodiments of the devices according to the current invention will be described with respect to the attached figures:

[0062] FIG. 1 shows the cross-section of a centrifuge rotor in the configuration of a fixed angle rotor, wherein a cut through an exemplary centrifuge tube bed with an extraction aperture which is closed by a closure device is shown,

[0063] FIG. 2 shows the cross-section of a centrifuge rotor in the configuration of a swing bucket rotor, the figure shows a partial cut through a centrifuge tube bed of a swing bucket with an extraction aperture foreseen in the bottom in the centrifuge tube bed sealed by a closure device,

[0064] FIG. 3A shows a first exemplary embodiment of a closure device,

[0065] FIG. 3B shows a second exemplary embodiment of a closure device,

[0066] FIG. 3C shows the closure device of FIG. 3B during the installation process to a rotor assembly of a centrifuge rotor,

[0067] FIG. 4 shows an exemplary embodiment of a sample retraction needle,

[0068] FIG. 5 shows a schematic representation of a sample retraction needle which is inserted to a centrifuge tube housed in a centrifuge rotor to extract a sample from aforesaid centrifuge tube,

[0069] FIG. 6 shows a result of sample displacement from centrifuge tube by evenly dosing a low density liquid by high performance liquid chromatography pump through a venting aperture in centrifuge tube. The displaced sample is fed to an ultraviolet-visual detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system, and

[0070] FIG. 7 shows a result of sample suction from centrifuge tube with high performance liquid chromatography pump through an extraction aperture in centrifuge tube. The displaced sample is fed to an ultraviolet-visual detection system, a fluorescence detection system, a light scattering detection system, and a conductivity detection system.

[0071] Referring to FIG. 1, an exemplary embodiment of a centrifuge rotor 1 in the configuration of a fixed angle rotor is shown. The centrifuge rotor comprises a rotor assembly 10 which is formed by a rotor body 12. The rotor assembly 10 in the form of the fixed angle rotor body 12 is adapted for centrifugal rotation around a rotation centre axis 14 shown in FIG. 1 as dashed line. As is understood to the person skilled in the art, the rotor body 12 is rotationally symmetrical with respect to the rotation centre axis 14. In FIG. 1, a cross-section of a single one of the plurality of centrifuge tube beds 16 is shown with a longitudinal axis 160 of the centrifuge tube bed 16. The centrifuge tube bed 16 is defined by a tubular cavity 162 adapted to receive and support a centrifuge tube 4 during the centrifugation process. The tubular cavity 162 is formed in the material of the rotor body 12 and delimited by a cavity side wall 162a and a cavity bottom 162b. The cavity side wall 162a and the cavity bottom 162b together act as a bearing surface for the outer surface of a centrifuge tube 4 which can be housed in said tubular cavity 162 of the centrifuge tube bed 16. As can be taken from FIG. 1, the cavity bottom 162b comprises in the example of the FIG. 1 a single extraction aperture 18 which is closed in FIG. 1 by a closure device 2. In the exemplary embodiment according to FIG. 1, the cavity bottom 162b is formed to have a narrowing shape towards the bottom 10b of the centrifuge rotor assembly 10. The top of the rotor assembly 10 is marked with the reference sign 10t.

[0072] The cavity bottom 162b is formed in the example of FIG. 1 by a hemisphere.

[0073] The tubular cavities 162 of the centrifuge tube beds 16 of centrifuge rotor 1 are formed within the rotor body 12 of the fixed angle rotor and wherein the at least one extraction aperture 18 is formed as a hole to extend through the rotor body 12 to fluidly connect the tubular cavity 162 with the surrounding of the rotor assembly 10.

[0074] The FIG. 2 shows an alternate configuration of a centrifuge rotor 1 wherein the rotor assembly 10 is formed by a swing bucket rotor which comprises a plurality of rotor buckets 13 which are pivotally connected to a rotating stem 11 wherein the plurality of rotor buckets 13 comprises at least one tubular cavity 162 to form at least one centrifuge tube bed 16 and wherein the at least one extraction aperture 18 extends through the body of the respective rotor bucket 13. The tubular cavity 162 is also formed in the material of the rotor buckets 13. In FIG. 2, only one cut-through of a rotor bucket 13 is shown wherein a single exemplary centrifuge tube 4 is received. The swing bucket rotor as shown in FIG. 2 is configured to rotate around the central rotation axis 14, wherein each of the plurality of swing buckets 13 is pivotally connected to the rotating stem 11 through a swing out axis 15. The longitudinal axis 160 of the centrifuge bed 16 in FIG. 2 is shown in the unswung or static condition wherein the aforesaid axis 160 extends approximately parallel to the rotation centre axis 14 of the rotating stem 11.

[0075] The extraction aperture 18 which is formed in the housing or material defining the swing bucket 13 is closed and sealed in an airtight manner by a closure device 2.

[0076] The FIG. 3A shows a first exemplary embodiment of a closure device 2 wherein the closure device 2 is formed as a capping screw which comprises a shaft 22 which extends along a shaft axis 22a from a first end 221 to a second end 222 wherein the first end 221 forms a partial area of the cavity bottom 162b in a situation when the closure device 2 is fastened to the rotor assembly 10 as can be derived from the examples shown in the FIGS. 1 and 2. As can be taken from FIG. 2, the first end 221 is adapted to the shape of the cavity bottom 162b to form a smooth transition of the surface of the first end 221 and the surrounding surface of the cavity bottom 162b. The closure device 2 as shown in the configuration of FIG. 3 further comprises a limit stop 24 at the second end 222 to restrict the movement of the closure device 2 in the direction along the shaft axis 22a when the closure device 2 is fastened to the rotor assembly 10 to ensure the correct alignment of the first end 221 with the surrounding surface of the cavity bottom 162b. The closure device 2 as shown in FIG. 3 comprises a groove 26 to receive one elastomeric seal 28 shown in FIG. 3 configured as an O-ring. The elastomeric seal 28 is received in aforesaid groove 26 of the closure device and deformed when the closure device 2 is fastened to the rotor assembly 10. When the closure device 2 is fastened to the rotor assembly 10, the shown closure device 2 according to FIG. 3 mates with the outer surface of the rotor body 12 or the body of the rotor bucket 13 at least in the area of the limit stop 24 and elastically deforms the elastomeric seal 28 to define an airtight seal. As can also be taken from the exemplary embodiment of the closure device 2, a partial area of the shaft 22 comprises external threads 27 wherein the extraction aperture 18 comprises internal threads which are adapted to the shape and position of the external threads 27 so that the closure device can be fastened to the rotor assembly 10 by the adapted threads 27.

[0077] The FIG. 3B shows a second exemplary embodiment of a closure device 2 wherein the closure device 2 is configured to form an interlocking structure with the rotor assembly 10 to realize a fixation. The closure device 2 as shown in FIG. 3B differs from the device as shown in FIG. 3A that no external threads 27 are provided in the area of the shaft 22 to realize the fixation of the closure device 2 to the rotor assembly 10. The closure device 2 according FIG. 3B comprises two interlocking elements 29 to enable a fastening of the closure device 2 to the rotor assembly 10. The interlocking elements 29 can be provided in addition or alternative to the external treads 27 as described with respect to FIG. 3A. The closure device 2 according to FIG. 3B comprises two elastomeric seals 28, wherein one of the seals 28 is arranged to be received in a groove 26 in the area of the shaft 22 and a further elastomeric seal 28 is arranged in a groove 26 located in the area of the second end 222 at a mating surface of the limit stop 24 with the rotor assembly 10. The second 222 of the locking device 2 comprises in the area of the limit stop 24 on a surface facing away from the rotor housing a recess 222a whereby the closure device 2 is enabled to be rotated around the shaft axis 22a to fasten or unfasten the closure device 2 to the rotor housing 10.

[0078] As is shown in FIG. 3C detailing the closure device 2 during the fastening process to the rotor housing 10. The FIG. 3C shows a top view on the second end 222 of the closure device 2. During the fastening of the closure device 2 the device 2 is placed in the area of the extraction aperture 18, wherein the interlocking elements 29 are placed in respective cutouts 19a of the rotor housing 10. To fasten the closure device 2 to the rotor housing 10 the closure device is rotated around the shaft axis 22a to move the interlocking elements 29 into the adapted pockets 19b of the rotor housing 10. The interlocking elements 29 and the cutouts 19a as well as the pockets 19b are adapted to each other to form an interlocking structure and thereby to realize a secure fastening of the closure device 2 to the rotor housing.

[0079] The FIG. 4 shows an exemplary embodiment of a sample retraction needle 3 wherein the sample retraction needle 3 comprises a cannula 30 which extends along an axial axis 300 from a distal end 30d to a proximal end 30p wherein the proximal end 30p is formed by a sharp-closed tip 31 to seal the proximal end 30p of the cannula 30 of the sample retraction needle 3. In the embodiment as shown in FIG. 4, the sample retraction needle 3 comprises four drainage holes 32 which are positioned in the area of the lateral side wall 33 of the cannula 30. In the schematic view of FIG. 3 only three of the four drainage holes 32 are shown to be evenly distributed at the lateral side wall 33 around the axial axis 300 in circumferential direction of the cannula 30.

[0080] As can also be taken from FIG. 4, the needle cannula 30 has a conical shape along the axial axis 300 wherein the outer diameter of the cannula 30 reduces in the direction towards the sharp-closed tip 31. The sharp-closed tip 31 is formed by a circular cone wherein the front surface of the proximal end 30p is formed by the tip of the cone. The ground surface of the cone is adapted to the shape of the cannula end.

[0081] The cannula 30 of the retraction needle 3 can be formed by a hollow stainless steel tube and the cannula 30 can have an outer diameter in the area of the distal end 30d in the range of 0.7 to 1.5 mm. The distal end 30d of the cannula 3 is connected to a needle base 34 wherein the needle base 34 has a diameter 34d which is dimensioned to be larger than the opening of the extraction aperture 18 of the centrifuge rotor 1 according to the present invention. The needle base 34 is thereby configured to act as a limiting device to limit the maximum insertion depth I of the sample retraction needle 3 in the centrifuge tube bed 16.

[0082] FIG. 5 shows a schematic representation of a sample retraction needle 3 according to the present invention when inserted to a centrifuge tube 4 housed in an exemplary embodiment of a rotor assembly 10 of a fixed angle rotor through a foreseen extraction aperture 18 in a rotor body 12 and inserted to the centrifuge tube 4 to extract the samples and/or fractions of the sample contained in the centrifuge tube 4 after the centrifugation process. FIG. 5 shows the use of a limiting device 9 which has a thickness S wherein the limiting device 9 has a through-hole through which the insertion needle 3 can be guided and inserted through the extraction aperture 18 to the centrifuge tube bed 16 and inserted in the centrifuge tube 4. By the use of the limiting device 9, the retraction needle 3 can be inserted in the centrifuge tube 4 to a desired and previously defined insertion depth I so that the foreseen drainage holes 32 are placed in a desired height H above the lowest point of the centrifuge tube bed 16. The aforementioned configuration enables to insert the extraction needle 3 to a desired insertion depth I and, thereby, to place the drainage holes in a desired height above the bottom of the centrifuge tube 4.

[0083] The FIG. 6 shows an example of a sample displacement from a centrifuge tube by evenly dosing a low density liquid by high performance liquid chromatography pump through a venting aperture in the centrifuge tube.

[0084] Density gradient fractionation was performed on a Sorvall? WX 90+ ultracentrifuge (Thermo Scientific) using 11.5 mL polyethylene UltraCrimp? centrifuge tubes (Thermo Scientific) in a T890 fixed-angle rotor. Samples of Adeno Associated Virus (AAV) were mixed with concentrated cesium chloride to obtain an AAV sample in 3 M cesium chloride. Centrifugation was performed at 53,500 RPM for 24 h at room temperature. The tube was then fixed in a stand and a venting aperture was pierced near the top with a hypodermic needle (23 gauge, 70 mm, B Braun). Another hypodermic needle was used to pierce extraction aperture at the bottom of the centrifuge tube. Tube content was displaced from a centrifuge tube by pumping water at constant flow rate of 1 mL/min through the venting aperture at the top of the centrifuge tube with a HPLC pump of a PATfix? LPG HPLC system (BIA separations). The extraction aperture at the bottom of centrifuge tube was connected directly to the monitor array of a PATfix? LPG HPLC system. This evacuated the tube in order of decreasing density. UV absorbance was monitored at 260 nm (solid trace). Intrinsic fluorescence was monitored at an excitation wavelength of 280 nm and an emission wavelength of 348 nm with a fluorescence detector (Shimadzu, dashed trace). Light scattering was monitored at a 90? angle with a DAWN? HELEOS II multi-angle light scattering detector (Wyatt Technology, dash-dotted black trace). Caesium chloride density is represented by the conductivity profile (dotted trace). The higher the conductivity, the higher the density of the cesium chloride.

[0085] The FIG. 7 shows an example of a sample suction from a centrifuge tube with high performance liquid chromatography pump through an extraction aperture in centrifuge tube.

[0086] Density gradient fractionation was performed on a Sorvall? WX 90+ ultracentrifuge (Thermo Scientific) using 11.5 mL polyethylene UltraCrimp? centrifuge tubes (Thermo Scientific) in a T890 fixed-angle rotor. Samples of Adeno Associated Virus (AAV) were mixed with concentrated cesium chloride to obtain an AAV sample in 3 M cesium chloride. Centrifugation was performed at 53,500 RPM for 24 h at room temperature. The tube was then fixed in a stand, a venting aperture was pierced near the top with a hypodermic needle (23 gauge, 70 mm, B Braun) and left open to the normal atmospheric pressure. Another hypodermic needle was used to pierce extraction aperture at the bottom of the centrifuge tube. Tube content was extracted from a centrifuge tube at constant flow rate of 1 m/min through the extraction aperture at the bottom of the centrifuge tube with a HPLC pump of a PATfix? LPG HPLC system (BIA separations). The HPLC pump directed the content of the centrifuge tube to the monitor array of of a PATfix? LPG HPLC system. This evacuated the tube in order of decreasing density. UV absorbance was monitored 260 nm (solid trace). Intrinsic fluorescence was monitored at an excitation wavelength of 280 nm and an emission wavelength of 348 nm with a fluorescence detector (Shimadzu, dashed trace). Light scattering was monitored at a 90? angle with a DAWN? HELEOS II multi-angle light scattering detector (Wyatt Technology, dash-dotted trace). Caesium chloride density is represented by the conductivity profile (dotted trace). The higher the conductivity, the higher the density of the cesium chloride.