FREEZE-DRYING APPARATUS AND METHOD

Abstract

A process for freeze drying a material. The process has the steps of (a) placing material to be freeze-dried in a vessel, the vessel comprising a container having a base, and at least one wall defining an opening at one end; (b) applying a membrane to the at least one wall to thereby cover the opening, wherein the membrane comprises an aperture which, in use, is aligned with the opening; and (c) subjecting the vessel to a lyophilisation procedure, wherein the membrane is in place on the container during the lyophilisation procedure.

Claims

1. A process for freeze drying a material, said process comprising: (a) placing the material in a vessel, the vessel comprising a container having a base, and at least one wall defining an opening at one end; (b) applying a membrane to the at least one wall to thereby cover the opening, wherein the membrane comprises an aperture which, in use, is aligned with the opening; and (c) subjecting the vessel to a lyophilisation procedure, wherein the membrane is in place on the container during the lyophilisation procedure.

2. A process according to claim 1, wherein the membrane comprises a metal foil.

3. A process according to claim 1, wherein the aperture has an area of from about 2 mm.sup.2 to about 13 mm.sup.2.

4. A process according to claim 1, wherein the step of applying the membrane to the at least one wall of the container comprises sealing the membrane to the at least one wall of the container.

5. A process according to claim 4, wherein sealing the membrane to the at least one wall of the container is selected from heat-sealing and adhesive sealing.

6. A process according to claim 4, wherein the membrane is reversibly sealed to the at least one wall of the container.

7. A process according to claim 1, comprising subjecting the vessel to a centrifugation process after step (b), wherein the membrane is in place on the container during the centrifugation process.

8. A process according to claim 1, further comprising (d) introducing inert gas into the vessel after the lyophilisation procedure, wherein the membrane is in place on the container during the introduction of the inert gas into the vessel.

9. A process according to claim 8, wherein the inert gas is selected from nitrogen gas, argon gas and dried air.

10. A process according to claim 8, wherein subjecting the vessel to a lyophilisation procedure further comprises subjecting the vessel to a lyophilisation procedure in a freeze-drying apparatus, and wherein the process further comprises (e) transferring the vessel containing the freeze-dried material and the inert gas from the freeze-drying apparatus to a dry cabinet, wherein the membrane is in place on the container during the transfer.

11. A process according to claim 10, wherein no additional closure covers the opening of the container during the (e) transferring.

12. A process according to claim 10, further comprising (f) in the dry cabinet, packaging the vessel into a non-porous receptacle with a desiccant and sealing the receptacle.

13. A process according to claim 12, wherein no additional closure covers the opening of the container when the vessel is packaged in the non-porous receptacle.

14. A process according to claim 12, wherein the non-porous receptacle comprises a metallised polymer than has a vapour transmission rate lower than the native polymer.

15. A process according to claim 1, wherein the material comprises one or more components that are required for carrying out a chemical or biochemical reaction.

16. A process according to claim 15, wherein the chemical or biochemical reaction is a nucleic acid amplification reaction.

17. A process according to claim 16, wherein the nucleic acid amplification reaction is a polymerase chain reaction or reverse-transcriptase polymerase chain reaction.

18. A vessel for use in a freeze-drying procedure, the vessel comprising a container having a base, and at least one wall defining an opening at one end; a membrane configured to cover the opening of the container, wherein the membrane is provided with an aperture which, in use, is aligned with the opening of the container.

19. A vessel according to claim 18, wherein the membrane comprises a metal foil.

20. A vessel according to claim 18, wherein the aperture has an area of from about 2 mm.sup.2 to about 13 mm.sup.2

21. A vessel according to claim 18, wherein the membrane is removably attachable to the container.

22. A vessel according to claim 21, wherein the membrane is attached to the container by a heat seal or by adhesive.

23. A vessel according to claim 18, comprising multiple containers, each of which is provided with a membrane or a portion of a membrane.

24. A vessel according to claim 23, wherein the multiple containers are joined together to form a single unit.

25. A vessel according to claim 24, wherein the multiple containers comprise individual or groups of multiple containers in separate units.

26. A vessel according to claim 18, wherein a single membrane is provided for the multiple containers, arranged such that a portion of the membrane covers an opening of each container in the vessel, and each portion of membrane is provided with an aperture.

27. A vessel according to claim 26, wherein a boundary between two adjacent portions of membrane is provided with an area of weakness to facilitate separating the portions of membrane.

28. A vessel according to claim 27, wherein the multiple containers comprise separate individual or groups of containers and wherein the boundary between two adjacent portions of membranes may be aligned with a boundary between adjacent separate individual or groups of containers.

29. A vessel according to claim 18, wherein the membrane comprises at least one alignment feature to aid alignment in an analytical apparatus during a subsequent analysis procedure.

30. A vessel according to claim 29, comprising multiple containers and a single membrane, arranged such that a portion of the membrane covers an opening of each container in the vessel, and each portion of membrane is provided with an aperture, wherein each portion comprises a separate alignment feature.

31. A vessel according to claim 18, comprising a material which has been freeze-dried in situ in the container.

32. A vessel according to claim 31, comprising an inert gas in the container.

33. A vessel according to claim 31, wherein the material comprises reagents useful in a nucleic acid amplification reaction, such as a polymerase chain reaction.

34. A system for use in a freeze-drying procedure, the system comprising: two or more vessels, each vessel comprising: one or more container, each container having at least one wall defining an opening at one end; and a membrane, divided into membrane portions, each membrane portion being configured to cover the opening of a container, wherein each membrane portion is provided with an aperture which, in use, is aligned with the opening of the container; and a support for receiving the two or more vessels and being configured to retain the two more vessels in alignment with one another, the support having at least one alignment feature configured to engage with a corresponding alignment feature of the membrane to thereby align the membrane relative to the two or more vessels.

35. A system according to claim 34, wherein the alignment feature of the support comprises one or more male member and the corresponding alignment feature of the membrane comprises one or more female member.

36. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0060] FIG. 1 is cross section of a freeze-drying vessel;

[0061] FIG. 2 is a plan view of a membrane of the freeze-drying vessel according to FIG. 1;

[0062] FIG. 3 is a flow diagram illustrating a freeze-drying process;

[0063] FIG. 4 is a flow diagram illustrating the process following freeze-drying;

[0064] FIG. 5 is a schematic illustration showing transfer of the vessel from freeze-drying apparatus to dry cupboard;

[0065] FIGS. 6A-6C are plan and cross-sectional views of a mandrel;

[0066] FIG. 7 is an exploded side view showing a mandrel, plate, vials and foil;

[0067] FIGS. 8A-8C are plan and cross-sectional views of a plate; and

[0068] FIG. 9 is a plan view of a membrane.

[0069] A vessel 10 for freeze drying a material is illustrated in FIG. 1. The vessel 10 has a container 12 which has a base at its lower end 22 and an opening 24 at an upper end. In this embodiment, the container is a PCR tube of the type referred to as an “Eppendorff™” or tube and has an upper cylindrical body section and a lower conical body section. The tube is made of plastics material, in this embodiment polypropylene. The tube typically has a volume of approximately 0.5 ml

[0070] The opening 24 is covered by membrane 14, in the form of a metal foil. In this embodiment, the membrane is aluminium foil. The membrane is secured to the walls of the upper cylindrical body section surrounding the opening 22 by a heat seal. The membrane 14 is provided with an aperture 16, having a diameter of approximately 1 mm. FIG. 2 is a plan view of the membrane 14 of the vessel of FIG. 1, showing the aperture 16.

[0071] FIG. 3 is a flow diagram showing the main steps of the freeze-drying method. Firstly, the material 18 to be freeze-dried is dispensed into the container 26. The material 18 typically comprises reagents required to carry out a polymerase chain reaction on a particular target nucleic acid. The reagents may typically comprise primers, enzymes, salts and probes.

[0072] The membrane is then applied to cover the opening of the container 28. The membrane is fixed in place by heat sealing. The membrane is applied after dispensing and prior to centrifugation, freeze-drying and transfer to a dry cabinet.

[0073] The vessel may be subjected to an optional centrifugation step 30, followed by a lyophilisation procedure 32, which takes place in a freeze-drying apparatus. The lyophilisation procedure is well known and will not be described in detail.

[0074] The procedure following lyophilisation is shown in FIG. 4. Once the lyophilisation process is complete, the vacuum inside the freeze-drying apparatus is broken with an inert gas, thereby back-filling the vessel with the inert gas 34. In this embodiment, the inert gas is nitrogen.

[0075] In step 36, the vessel is transferred from the freeze-drying apparatus to a dry cabinet. The dry cabinet is filled with nitrogen gas and has moisture level of 1-2% V/V.

[0076] As discussed above, the membrane is applied to the container before both centrifugation and the lyophilisation procedure takes place. The aperture in the membrane is sufficiently sized to retain the liquid effectively prior to drying (for example in the centrifugation step) and to allow sufficient venting for the cakes to dry during the lyophilisation process. The aperture also allows back-filling of the vessel with nitrogen and allows a “blanket” of dry nitrogen to be retained within the vessel. Furthermore, it mechanically retains the freeze-dried cake. These last two advantages are particularly important during the transfer of the vessel from freeze-drying apparatus to dry cabinet. The aperture is sufficiently small that very little nitrogen is lost from the vessel during this short transition, protecting the freeze-dried material from the humidity and oxygen in the air. No additional closure is required to cover the opening of the container during this transfer.

[0077] Once the vessel is in the dry cabinet, it is placed into a non-porous receptacle 38, along with a desiccant and the receptacle is sealed (for example heat-sealed). The non-porous receptacle is typically made from metallized boPET, such as a Mylar™ bag. No additional closure is required, so the fiddly procedure of applying closures in the dry cabinet is eliminated. If a zip-lock bag is used, a heat seal may be preferably made post packing outside the cabinet to eliminate all other processes from the cabinet.

[0078] As humidity and oxygen is kept out of the vessel during the transfer from freeze-drying apparatus to dry cabinet, the vessel can be stored in the non-porous receptacle without the need for an additional cap, which simplifies the procedure.

[0079] FIG. 5 shows the transfer of the vessel 10 from the freeze-drying apparatus 40, where freeze-drying and back-filling with nitrogen gas take place, to the dry cabinet 42 where the final stages of packaging take place. The membrane is applied to the container prior to placing it in the freeze-dryings apparatus and remains in place in the freeze-drying apparatus, during the transfer, and in the dry cabinet, without any further closure being required.

[0080] The freeze-dried material packaged in this way can be stored for between 1.5 to 5 years, depending on the reagent formulation.

[0081] The freeze-dried material can be rehydrated, for example by introduction of a liquid sample that contains, or is suspected of containing, the target nucleic acid. This may be done by removing or piercing the membrane, for example with a pipette containing the liquid sample. It is advantageous if the membrane is removably attachable to the container, allowing it to be peeled off prior to use. The vessel may then be subject to conditions under which the polymerase chain reaction occurs, in particular thermal cycling conditions. Alternatively, the resulting reaction may contain multiple reactions that may be dispensed into other vessels after dissolution either prior to, or after the formation of a complete reaction through the addition of other reagents and/or template.

[0082] In one embodiment, multiple vessels are clustered together for the freeze-drying procedure but separated for storage. This is achieved by multiple containers held together in a support or “mandrel”. For example, the containers may comprise individual wells, tubes or vials; strips (for example strips of 8 or 12 wells, tubes or vials); or blocks (i.e. multiple strips) which are mounted in a multiwell plate which is in turn mounted in the mandrel. A one-piece membrane is applied to the top of the multiple containers and heat sealed in place.

[0083] FIG. 6A-6C show the mandrel used to support the cluster of multiple vessels. FIG. 6A is a plan view, FIG. 6B is a cross section along AA and FIG. 6C is a cross section along BB. The mandrel 44 has a supporting surface 46 for receiving a multiwell plate and a peripheral rim 45 for engagement with the plate. Four location pins 48 locate both the multiwell plate and membrane in the desired position.

[0084] FIG. 7 shows an exploded side view of the mandrel 44, showing the multiwell plate 50 which is held in position by pins 48 cooperating with apertures (not shown) on the plate 50. Vials 52 are supported in the multiwell plate 50. Finally, FIG. 7 shows a membrane 54, in the form of a metal foil. This is provided with four apertures for cooperation with location pins 48 to ensure precise location of the membrane.

[0085] FIG. 8A-C illustrate the multiwell plate for use with the mandrel of FIG. 6A-C. the multiwell plate 50 is provided with four apertures 56 to cooperate with mandrel pins 46. The multiwell plate 50 is also provided with a peripheral flange 58, which, in use, hooks over rim 45 of the mandrel.

[0086] FIG. 9 shows a plan view of the membrane 54. In this embodiment, the membrane is an aluminium foil and is provided with four alignment apertures 70, which cooperate with the alignment pins of the mandrel. The membrane has multiple apertures 64 which, when aligned by the mandrel, line up with the individual vials. The membrane has lines of weakness 62, which will allow the membrane to be broken apart in line with the containers. In the embodiment illustrated in FIG. 9, the lines of weakness are provided by perforations.

[0087] The membrane is labelled, for example via printing, text or bar codes to identify wells and their contents. Each portion may be labelled, so that the label remains when the individual containers are separated.

[0088] The membrane may be provided with foil features to assist with orientation of individual wells, strips and blocks in subsequent analysers. Foil features include chamfers or other cut features that allows visual or mechanical orientation.

[0089] Once the membrane has been sealed in place, for example by heat-sealing, the combined plate, containers and membrane are removed from the mandrel and undergo the freeze-drying process as described above.

[0090] Once the freeze-drying process is complete, the individual wells, strips or blocks are separated, making use of the lines of weakness in the membrane. They are then stored as required in Mylar™ packaging.

[0091] The vessel and method of use has several advantages. Once the membrane is applied to the container, it stays in place for centrifugation, freeze-drying, back-filling with inert gas, transfer and final packaging, with no extra closure being required. The process removes the time-consuming and fiddly operations in the dry cabinet by removing the need to add closures post processing. Furthermore, the end product has improved stability as it has reduced exposure to humidity and oxygen. The process of the present invention simplifies workflows, human time, and cabinet time. Furthermore, the end user experience during subsequent PCR analysis is improved, as this this entails the simple removal of the membrane from the container, addition of liquids and lids before amplification.