Affinity Chromatography

Abstract

This invention relates to a method of removing a chemical entity from a liquid using affinity chromatography. The method involves passing an elongate solid phase through a conduit through which the liquid also flows.

Claims

1. A method comprising: passing an elongate body comprising a liquid input port and a liquid outlet port through a conduit to remove a chemical entity from a liquid; wherein the liquid from which the chemical entity is removed passes along the conduit from the liquid input port to the liquid output port in the opposite direction to the elongate body; wherein the conduit being is configured such that the liquid contacts the elongate body; and wherein attached to the elongate body is an affinity entity having an affinity for the chemical entity; and washing the elongate body to remove products present having lower affinity for the affinity entity than the chemical entity.

2. The method of claim 1, wherein one or both the elongate body and the liquid is subjected to sonication as it passes through the conduit.

3. The method of claim 1, wherein washing the elongate body comprises passing the elongate body through a wash conduit to remove the products present on the elongate body having lower affinity for the affinity entity than the chemical entity; wherein the wash conduit comprises a wash liquid input port and a wash liquid outlet port; wherein a wash liquid passes along the wash conduit from the wash liquid input port to the wash liquid output port in the opposite direction to the elongate body; and wherein the wash conduit is configured such that the wash liquid contacts the elongate body.

4. The method of claim 3, wherein one or both the elongate body and the wash liquid is subjected to sonication as it passes through the wash conduit.

5. The method of claim 1 further comprising recovering the chemical entity from the elongate body.

6. The method of claim 5, wherein recovering the chemical entity comprises passing the elongate body through a displacement conduit to displace the chemical entity from the affinity entity; wherein the displacement conduit comprises a displacement liquid input port and a displacement liquid outlet port; wherein a displacement liquid passes along the displacement conduit from the displacement liquid input port to the displacement liquid output port in the opposite direction to the elongate body; and wherein the displacement conduit is configured such that the displacement liquid contacts the elongate body.

7. The method of claim 6, wherein one or both the elongate body and the displacement liquid is subjected to sonication as it passes through the displacement conduit.

8. The method of claim 5 further comprising recovering the chemical entity from the displacement liquid that is recovered from the displacement liquid output port.

9. The method of claim 1 further comprising recovering the products having lower affinity from the wash.

10. A method comprising: passing an elongate body comprising a liquid input port and a liquid outlet port through a plurality of conduits to remove a chemical entity from a liquid; wherein the liquid from which the chemical entity is removed passes along each conduit from the liquid input port to the liquid output port in the opposite direction to the elongate body; wherein each conduit is configured such that the liquid contacts the elongate body; and wherein attached to the elongate body is an affinity entity having an affinity for the chemical entity; washing the elongate body comprising passing the elongate body through a plurality of wash conduits to remove the products present on the elongate body having lower affinity for the affinity entity than the chemical entity; and recovering the chemical entity by passing the elongate body through a plurality of displacement conduits to displace the chemical entity from the affinity entity.

11. The method of claim 1, wherein the elongate body comprises an elongate body portion and a plurality of enclosures distributed along the length of the elongate body portion; wherein each enclosure being formed of a material comprising: a chemically inert mesh; and a plurality of solid phase particles within the enclosure to which the affinity entity is attached to the solid phase particles; wherein the size of holes in the mesh and the size distribution of the solid phase particles are selected such that the solid phase particles do not pass through the mesh.

12. The method of claim 1 further comprising providing a system comprising: an affinity module comprising the conduit; and a first service module operably connected to a first side of the affinity module, the first service module for one or both supplying the liquid to, and receiving the liquid from, the affinity module; wherein the system is configured for passing the elongate body being a solid phase body through the affinity module via the conduit.

13. The method of claim 1 further comprising regenerating the affinity entity.

14. The method of claim 1, wherein the chemical entity is selected from the group consisting of a protein, a nucleic acid, an alkaloid, an antibody, a peptide and an oligosaccharide.

15. The method of claim 1, wherein the affinity entity comprises a metal ion.

16. The method of claim 15 further comprising recovering the chemical entity from the elongate body being a solid phase body by contacting the solid phase body with a wash solution comprising imidazole.

17. The method of claim 1, wherein the affinity entity comprises an antibody, protein, lectin, antigen or anti-antibody.

18. The method of claim 1, wherein washing the elongate body comprises washing the elongate body being a solid phase body by contacting the solid phase body with a wash solution comprising a buffer.

19. A material comprising: an elongate body; and an affinity entity.

20. The material of claim 19, wherein the elongate body comprises an elongate body portion and a plurality of enclosures distributed along the length of the elongate body portion, the enclosures being formed of a material comprising a chemically inert mesh; wherein the affinity entity is attached to the elongate body; wherein within each enclosure of the elongate body, the affinity entity is attached to a plurality of solid phase particles; and wherein the size of holes in the mesh and the size distribution of the solid phase particles are selected such that the solid phase particles do not pass through the mesh.

21. The material of claim 20, wherein the affinity entity comprises a metal ion.

22. The material of claim 20, wherein the affinity entity comprises an antibody, peptide, protein, lectin, antigen or anti-antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0104] FIG. 1 is a depiction of an illustrative apparatus that can be used to carry out the methods of the invention. It is the apparatus used to carry out Example 2.

DETAILED DESCRIPTION

[0105] As used in this specification, the term remove may mean entirely remove or it may mean partially remove. Thus, it may mean that greater than 25% of the chemical entity is removed from the liquid. It may mean that greater than 75% of the chemical entity is removed from the liquid. It may mean that greater than 90% of the chemical entity is removed from the liquid. It may mean that greater than 95% of the chemical entity is removed from the liquid.

[0106] Throughout the description and claims of this specification, the words comprise and contain and variations of them mean including but not limited to, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0107] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0108] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

EXAMPLES

Example 1

Preparation of Immobilized Metal Affinity Chromatography (IMAC) Ribbon

[0109] 1. Prepare a saturated solution of disodium iminodiacetate in DMF/water 12:5 v/v. [0110] 2. Unwind cotton tape (22 mm1 mm6 m) from spool and soak in 20 mlg.sup.1 of 0.1% v/v detergent in water at 80 C. for 1 hour. [0111] 3. Wash the tape: 10 min of manual mixing with a glass, using 20 mlg.sup.1 of distilled water at 80 C. Repeat five times. Dry the tape at room temperature until touch dry. [0112] 4. Dry the cotton by placing into an airtight bottle with DMF and 10% v/v 4 A molecular sieves for 48 hours. [0113] 5. Soak DMF-dried cotton tape in anhydrous DMF at 80 C. for one hour. [0114] 6. Prepare a solution of phosphorous oxychloride (POCl.sub.3) in DMF by slowly adding 24 ml of POCL.sub.3 to 1 litre of anhydrous DMF. [0115] 7. Heat the solution to 90 C. using a water bath (exclude moisture from DMF solution!) and add the cotton tape to the solution and continue to heat at 90 C. for 30 mins. [0116] 8. Remove the tape (should be brown coloured) from the DM F/POCl.sub.3 solution and wash twice with DMF. [0117] 9. Wash the tape 2 with water. [0118] 10. Wash the tape with 5% w/v aqueous NaOH solution. [0119] 11. Wash the tape 2 with water. [0120] 12. Wash the tape with 5% v/v aqueous acetic acid solution. [0121] 13. Wash the tape 2 with water. [0122] 14. Place the chlorinated cotton tape into a flask equipped with a reflux condenser, along with an excess of saturated disodium iminodiacetate solution. [0123] 15. Heat the solution to 105-110 C. for 150 mins with cold water flowing through the condenser. [0124] 16. Wash the cotton tape 5 with distilled water. [0125] 17. Dry the tape at room temperature for 48 hours or until touch dry.

Example 2

Performing Continuous Chromatography Purification of Lactalbumin from Milk

[0126] Skimmed milk was used as a simulant for fermented broth due to milk being very consistent in composition and also having a high concentration of a water soluble protein. A fermented broth may also need ultrasonic treatment (to disintegrate cells and release proteins into solution) and filtration prior to affinity chromatography.

[0127] The apparatus 1 used in example 2 is shown in FIG. 1. The cotton tape 2 was passed sequentially through three conduits 3, 4, 5. The conduits were formed from four PTFE blocks 9 that are shaped so as to provide three chambers. Into each chamber was inserted three inserts 6, each insert having at its end a roller 7. The inserts fit into the chamber so as to form three u-shaped conduits 3, 4, 5. The respective liquids flow into each conduit 3, 4, 5 via an inlet (not shown) just below the exit points 10 of the cotton tape 2 into the respective conduits. The respective liquids flow out of each conduit 3, 4, 5 via an inlet (not shown) just below the entry points 11 of the cotton tape 2 into the respective conduits. Rollers 8 were also provided before and after the conduits to help the tape to pass through the apparatus. [0128] 1. The functionalised cotton tape 2 is placed into an excess of 1 M aqueous copper sulphate solution and allowed to stand for 30 mins. [0129] 2. The cotton tape 2 is then removed from the solution and washed with water until the run off is colourless. [0130] 3. Then the tape is passed through the conduit 3, 4, 5 with the first conduit 3 containing a flow of clarified milk whey, the second conduit 4 (the wash conduit) containing a flow of Buffer A (pH 7 phosphate buffer, 20 mM tris(hydroxymethyl)aminomethane, 0.5M NaCl), and the third channel 5 (the displacement conduit) containing a flow of Buffer B (pH 7 phosphate buffer, 20 mM imidazole, 0.5M NaCl) [0131] 4. The flow of Buffer B and lactalbumin leaving the apparatus 1 is collected.