GB2439050A

GB2439050A - Disposable chromatography device

Info

Publication number: GB2439050A
Application number: GB0611593A
Authority: GB
Inventors: Zhanren Zhang
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-06-13
Filing date: 2006-06-13
Publication date: 2007-12-19
Also published as: GB0611593D0

Abstract

Readily available disposable tubes or bottles, a e.g. centrifuge tubes and universals are employed as the tube body for making an inexpensive disposable chromatography device for purification of biological materials under gravity, vacuum or centrifugation conditions. One or more than one holes b is produced in the bottom or the side wall close to the bottom. Filter material c, is then filled in without empty space. Chromatography medium d of choice is packed above the filter material.

Description

I

DISPOSABLE CHROMATOGRAPHY DEVICE

Introduction

Liquid chromatography is one of the most popular approaches in purification and preparation of biological samples (e.g. proteins and DNAs). It generally consists of two parts: porous or non-porous chromatography media and the special device to hold the media. The special device is commonly called chromatography column.

Liquid phase (i.e. mobile phase) containing the biological material is passed through the media by force. For large scale purifications, chromatography column is very expensive and required to be used many times. In cases of purification of small quantities of biological samples or the purification being done in batch operation, fast purification can be achieved in the following operational modes by addressing the liquid / media solid separation: gravity flow operation, centrifugation operation and vacuum operation. Special chromatography device which can withhold chromatography media but allow free pass of the liquid phase is required.

Background art

The disposable chromatography device used for purification of biological\materials under gravity flow, vacuum or centrifugation is generally in a column shape. It consists of a column with a disc-shaped filter mesh (or membrane) attached in the bottom. There is empty space between the mesh and the liquid outlet.

There are a few drawbacks for such kind of disposable columns. First, it is expensive to make. Special template for the column and the filter mesh has to be cast for individual manufacturer. It is not very economic for disposable applications. The filter mesh must be sealed to the column body without any leakage of chromatography particles. Second, the packed bed height is very shallow, which likely promotes channelling. As a consequence, the biological material can bypass the chromatography media without being captured. The penalty is that the utilisation of packed chromatography media is poor (i.e. waste of precious chromatography media and poor recovery of biological materials). It is particularly the case when small quantity of chromatography particles (e.g. 50 -150.tl) is packed to a micro disposable chromatography column. Therefore, development of inexpensive disposable chromatography device with improved utilisation efficiency of packed chromatography media is still desired.

It is the object of the present invention to disclose a method for making a novel disposable chromatography device and the device so made.

Disclosure of invention

In accordance with the invention a novel method is thus provided for the fabrication of an inexpensive disposable chromatography device with the benefit of higher utilisation of the chromatography media.

In accordance with the invention, a preferred embodiment of such a disposable chromatography device is described with reference to the accompanying Figure 1.

The inexpensive disposable chromatography device consists of four parts: (a) Tube Body, (b) hole(s) in the bottom or the side wall close to the bottom, (c) filter mesh and (d) chromatography media.

In accordance with the invention, any tube or bottle (named as Tube Body) commonly available in the market place can be modified as the main body of the disposable chromatography device. If it is used under centrifugal force, the Tube Body is required to have the size to be held into a larger centrifuge tube (named Collector) and possesses the mechanical strength for centrifugation. Such type of tube or bottle is extensively used in most of the biological laboratories worldwide and is already manufactured in massive scale. The cost is therefore very low.

In accordance with the invention, the inexpensive disposable chromatography device is made as described herein. One or more than one hole in the bottom or side wall is made in the Tube Body first. One or more than one hole is made in the top lid of the Tube Body if there is a top lid. Any material that has filtration functionality and possesses none or very low non-specific binding capacity to a given biological material can be filled into the bottom of the Tube Body as a filter mesh.

Chromatography media of selection is then loaded above the filter mesh. The chromatography device so made can be used in the same way as those available in the market place. In brief, the device is inserted into the holding tube (i.e. Collector).

Biological material is then loaded on top of the media. Under centrifugal force, the liquid passes through the packed media, the filter mesh, the holes and, is subsequently collected in the Collector. Meanwhile, the target molecule is captured to the media that is held by the filter mesh underneath. After washing step, the device is transferred to a fresh Collector. The elution buffer is loaded onto the top of the media. The bound material is recovered under centrifugal force and collected to the Collector.

In accordance with the invention, the utilisation efficiency of the packed chromatography media can be improved by the following device designs. As it is stated above, the packed bed height in the commercial disposable device is very shallow, It is because column shape design is adopted in such a device, i.e. the inner diameter is constant from the top to the bottom mesh section. To increase the packed bed height with much reduction of possible channelling problem, one approach in accordance with the invention is to reduce the inner diameter of the section where the chromatography media is packed. For example, a 50% reduction of the inner diameter can make the packed bed height four times higher. There is another benefit with such an arrangement. Higher bed height produces higher flow resistance (i.e. higher pressure drop). It is a linear relationship. Therefore, liquid is forced through the particles at slower rate, i.e. at longer liquid / solid contact time. All these translate into higher binding capacity of a given volume of chromatography media. Depending on the volume of chromatography media loaded, the ideal diameter of this section may vary. By reducing the porosity of the filter material and / or the holes in the tube bottom, longer liquid / solid contact time can be achieved under the same centrifugal force. Certainly, a balance between speed of purification and utilisation efficiency of chromatography media needs to be considered.

The Tube Body can be made of any type of material as long as it is compatible with the biological conditions commonly experienced, such as pH 3-11 and most of the buffer systems. The material should show minimum binding to biological targets and minimum impurity leakage under the operational conditions. For the convenience of modification, the tube material is of plastic in the preferred embodiment. The Tube Body can be capped or non-capped, if it is capped, the cap format can be a push-down style or screw style or other suitable ones. The size of the Tube Body can vary depending on the purification scale, such as 0.2 ml, 0.5 ml, 1.5 ml, 5 ml, 15 ml and so on. In a preferred embodiment, the Tube Body is suspended inside the Collector tube and can be taken out easily. In another embodiment, the Tube Body is suspended inside an intermediate tube with holes in the bottom. They are then suspended in the Collector Tube of right size.

The size of each hole and the number of holes in the bottom or side wall of individual Tube Body depends on the tube size and the purification scale. For microcentrifuge tubes of 0.5 -2 ml, one hole with a size of 0.3 -1mm is recommended. For tubes of larger volumes, more than one hole of similar size can be punched with a uniform distribution, as long as the overall mechanical strength of the tube body is not jeopardised.

The filled filter material can be fibres, particles, stacked membrane or filter papers, sintered meshes or a mixture of the above or any other suitable material. The volume of the filled filter material can vary depending on the nature of the filter material and the application demand. For example, if unclarified biological sample is to be processed in such a device, the filter material should possess pores or voids that are sufficiently big to allow free passing through of cells and cell debris.

The chromatography media loaded to the disposable device can be of any type such as ion-exchangers, affinity chromatography resins and gel filtration media etc. The media format can be particles, monolith, pellets, discs and membranes or other type of adsorbers. The media can be packed in liquid or is packed in dehydrated state.

In cases that the media isn't of particle format, filter mesh may be required or may not.

The construction of such an inexpensive disposable chromatography device was further described in the following examples but not limited to the following scopes.

Example 1

A standard 0.5 ml microcentnfuge tube was chosen and was punched with a needle in the centre of the bottom to generate a hole of approx. 0.5 mm in diameter.

Another three holes of the same size were generated in the top lid in the same manner.

Cotton fibre was then filled to the bottom of the tube with a final packed volume of 50 tl. It worked as a filter.

The 0.5 ml tube, after the above modification, was inserted into a standard 1.5 ml centrifuge tube. 100 tl of distilled water was added into the 0.5 ml tube. After spinning the tubes at 6500 rpm for 10 seconds, the filter material was firmly set in place. The 0.5 ml tube so produced was then ready for accommodation of chromatography media.

Example 2

In this example, a device containing immobilised metal affinity chromatography (IMAC) media was constructed and its application was displayed.

Ni SepFast BG (a chelating chromatography media produced by BioToolomics Ltd) was loaded into the 0.5 ml tube as it was prepared in Example 1.

p.1 of the media slurry at a concentration of 50% was loaded. Insert the device into a fresh 1.5 ml centrifuge tube and spin at 6500 rpm for 10 seconds. The Ni SepFast BO was firmed packed above the cotton filter. The liquid waste was collected to the 1.5 ml tube. No leakage of the particles was noticed. The disposable chromatography device with IMAC resin in was ready for purification of histidine-tagged proteins.

A recombinant hydrolase with a 6 x histidine tag in the C-terminus was expressed in E.coli BL-2 1 DE3. The cells were suspended in 20 mM phosphate buffer in the presence of lysozyme (200 p.g/ml) and incubated for 30 mins at 37 C. Sodium chloride and imidazole was then added to give a final concentration of 500 mM and mM, respectively. After spinning the cell lysate at 13,000 rpm for 2.5 mins, the supernatant containing the target protein was collected.

The disposable chromatography device was placed into a 1.5 ml centrifuge tube. 200 p.1 of the binding buffer (20 mM phosphate, 20 mM imidazole, 500 mM NaC1, pH 7.4) was loaded. After spinning at 6500 rpm for 30 seconds, the device was transferred to another fresh 1.5 ml centrifuge tube. 300 p.1 of the lysate supernatant was then loaded. After spinning for 1.5 mm, the liquid was fully passed through the packed media. The device was transferred to a fresh 1.5 ml centrifuge tube. 200 p.1 of the binding buffer was loaded for washing. It was repeated twice with a spinning time of 1 mm each. The device was transferred to a fresh 1.5 ml centrifuge tube. 150 p.1 of the elution buffer (20 mM phosphate, 500 mM imidazole, 500 mM NaC1, pH 7.4) was loaded. After spinning for 1 mm at 6500 rpm, the bound protein was eluted and collected in the 1.5 ml tube. The elution step was repeated another time.

Following assays indicated that 0.2 mg of protein was recovered with high purity. The whole purification process took approx. 15 mins.

Example 3

In this example, a gel filtration chromatography media was packed to the device and the desalting performance was then evaluated.

0.1 g of the dry Sephadex G-25 (from GE Healthcare) was loaded into the device as it was prepared in Example 1. After placing the device into a 1.5 ml centrifuge tube, 300.t1 of the equilibration buffer (10 mM Tris/1-IC1, pH 7.6) was added. After spinning for 1 mm at 6500 rpm, the device was transferred to another 1.5 ml centrifuge tube. The media was equilibrated with 300 tl of the buffer again in the same manner as above.

The device was then placed in another fresh 1.5 ml centrifuge tube. 100 t1 of lysozyme (1 mg/mI) dissolved in 10 mM Tris/HC1, 1 M NaCI, p1-I 7.6, was loaded to the device. After spinning for 2 mins at 6500 rpm, the liquid collected in the 1.5 ml tube was analysed for protein concentration and solution conductivity. 100 p.1 of BSA (1 mg/ml) dissolved in 10 mM Tris/HCI, 1 M NaCI, pH 7.6, was desalted the same way as above.

The results indicated that over 95% of the protein was recovered and over 83% of the salt was removed, for both lysozyme and BSA, in a single spin operation.

Claims

Claims 1. A method of manufacturing a disposable chromatography device

comprising of the steps of: (a) selecting a tube body with one end sealed; (b) producing a hole (or holes) in the sealed end and / or the side wall close to the sealed end of said tube body; (c) filling in filter material immediate above and / or besides the hole(s) without empty space; (d) following with chromatography media packed above the filter material.

2. The method of claim 1 in which the tube body is selected from pre-made readily available tubes or bottles e.g. centrifuge tubes, universal tubes and test tubes etc. 3. The method of claim 1 in which the tube body is selected from pre-made readily available tubes or bottles that possess the mechanical strength for use under vacuum conditions.

4. The method of claim I in which the tube body is selected from pre-made readily available tubes or bottles that can be accommodated into a larger centrifuge tube and possess the mechanical strength for use in a centrifuge.

5. The method of claim 1 in which the preferred shape of the tube body is of cylinder shape, V shape or a mixture of both.

6. The method of claim 1 in which the steps (a) and (b) is combined into one step, i.e. a suitable tube body with pre-made hole(s) in the sealed end and / or the side wall close to the sealed end.

7. The method of claim 1 in which the filter material is of fibres, particles, monoliths such as sintered discs, membranes.

8. The method of claim 1 in which the packed chromatography media include ion-exchangers, affinity media, hydrophobic media, gel filtration media but not limited to the ones listed herein.

9. The method of claim 1 in which the chromatography media is in the format of particle, pellet, membrane disc, monolith or other suitable formats and is packed in liquid or in dehydrated state (i.e. dry material without liquid inside the pores).

10. The method of claim I in which the step (d) is conducted immediately after the step (c) or conducted just before an actual purification activity takes place.

ii. A disposable chromatography device that consists of (a) a tube body, (b) a hole (or holes) in the bottom or the side wall closed to the bottom, (c) filter material filled immediately above or besides the hole(s) without empty space, (d) following with chromatography media packed above the filter material.

12. The disposable chromatography column of claim 11 in which the packed chromatography media include ion-exchangers, affinity media, hydrophobic media, gel filtration media but not limited to the ones listed herein.

13. The disposable chromatography device of claim 11 in which the chromatography media is in the format of particle, pellet, membrane disc, monolith or other suitable formats.

14. The disposable chromatography device according to claim 11, the part (c) might be removed if the packed chromatography media is in pellet, monolith or membrane format.

15. The disposable chromatography device of claim 11 in which the chromatography media of choice is packed into the tube just before an actual purification activity takes place.

16. The disposable chromatography device according to claim 11 being used in gravity flow purification of biological materials.

17. The disposable chromatography device according to claim 11 being used for purification of biological materials under vacuum.

18. The disposable chromatography device according to claim 11 being used for purification of biological materials in a centrifuge.

19. A disposable chromatography device that consists of (a) a tube body, (b) a hole (or holes) in the bottom or the side wall closed to the bottom, (c) filter material filled immediately above or besides the hole(s) without empty space.

20. The disposable chromatography device according to claim 19 being used in gravity flow purification of biological materials.

21. The disposable chromatography device according to claim 19 being used for purification of biological materials under vacuum.

22. The disposable chromatography device according to claim 19 being used for purification of biological materials in a centrifuge.