Purification of immunoglobulins
| Title: | Purification of immunoglobulins |
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| Patent Number: | 9,044,740 |
| Publication Date: | June 02, 2015 |
| Appl. No: | 12/594002 |
| Application Filed: | March 20, 2008 |
| Abstract: | The present invention relates to a separation matrix comprised of a porous or non-porous support to which ligands have been immobilized, wherein said ligands comprise at least one aliphatic sulfamide. The invention also relates to a chromatography column that contains the described separation matrix, as well as to a method of isolating immunoglobulins, such as IgG, Fab fragments, fusion proteins containing immunoglobulins etc, by adsorption to a separation matrix that comprises the aliphatic sulfamide ligands of the invention. |
| Inventors: | Maloisel, Jean-Luc (Uppsala, SE); Johansson, Bo-Lennart (Uppsala, SE) |
| Assignees: | GE Healthcare Bio-Sciences AB (Uppsala, SE) |
| Claim: | 1. A separation matrix comprised of a support to which at least one ligand has been covalently immobilized, optionally via a spacer arm, wherein said ligand comprises one aliphatic sulfamide group described by the following formula: —N(R 1)—S(O) 2 —N(R 2)(R 3) (I) wherein R 1 , R 2 and R 3 are, independently from each other, either hydrogen or linear, optionally branched carbon chains, and wherein the content of aliphatic sulfamide groups is 113-337 micromol/ml. |
| Claim: | 2. The matrix of claim 1 , wherein the support is porous. |
| Claim: | 3. The matrix of claim 1 , wherein R 1 is hydrogen. |
| Claim: | 4. The matrix of claim 1 , wherein R 2 and/or R 3 are C1-C3 carbon chains. |
| Claim: | 5. The matrix of claim 1 , wherein R 1 ═H and R 2 ═R 3 ═CH 3 having the following structure [chemical expression included] |
| Claim: | 6. The matrix of claim 1 , wherein the sulfamide groups are present as repetitive units of a polymer covalently immobilized to the support. |
| Claim: | 7. The matrix of claim 1 , which comprises sulfamide ligands coupled to a support via spacer arms, which optionally comprises one or more nitrogen atoms. |
| Claim: | 8. The matrix of claim 1 , further comprising ion exchange ligands or hydrophobic interaction chromatography (HIC) ligands. |
| Claim: | 9. The matrix of claim 1 , wherein the support is a cross-linked polysaccharide. |
| Claim: | 10. The separation matrix of claim 1 in a solution phase. |
| Claim: | 11. A chromatography column packed with the separation matrix of claim 1 . |
| Claim: | 12. The chromatography column of claim 11 , which is substantially sterile. |
| Claim: | 13. The chromatography column of claim 11 , which is a disposable product. |
| Claim: | 14. A process of preparing the separation matrix of claim 1 , said method comprises a first step of covalently immobilizing amines to a support and a subsequent step of sulfamoylating said amines to provide aliphatic sulphamide ligands. |
| Claim: | 15. The process of claim 14 , wherein the amines and/or polyamines are covalently immobilized to the support via spacer arms. |
| Claim: | 16. A method of isolating at least one immunoglobulin from a liquid, which method comprises the steps of: (a) providing a liquid that comprises at least one immunoglobulin: (b) contacting said liquid with aliphatic sulfamide ligands to adsorb one or more immunoglobulins to the matrix of claim 1 containing said aliphatic sulfamide ligands; and, optionally, (c) contacting said matrix with an eluent to release one or more immunoglobulins; and (d) recovering at least one immunoglobulin. |
| Claim: | 17. The method of claim 16 , wherein the liquid provided in step (a) also comprises one or more other proteins. |
| Claim: | 18. The method of claim 16 , wherein the separation matrix of step (b) is provided in a chromatography column. |
| Claim: | 19. The method of claim 16 , wherein step (b) is performed at a neutral or close to neutral pH. |
| Claim: | 20. The method of claim 16 , wherein step (c) is a gradient elution performed by passing an eluent of decreasing pH across the matrix. |
| Claim: | 21. The method of claim 16 , wherein the immunoglobulins recovered in step (d) are human or humanised immunoglobulins. |
| Claim: | 22. The method of claim 21 , wherein the immunoglobulins recovered in step (d) are immunoglobulin G (IgG). |
| Claim: | 23. A method of determining the quantity of an immunoglobulin, which method encompass the method of claim 16 and in addition a step (e) of determining the amount of immunoglobulin spectrophotometrically. |
| Patent References Cited: | 4725355 February 1988 Yamamoto et al. 8685248 April 2014 Glad et al. 2003/0027809 February 2003 Kano et al. 2006/0063822 March 2006 Weinstein et al. 2010/0151581 June 2010 Glad et al. WO 96/09116 March 1996 WO 03/046063 June 2003 WO 2005/061543 July 2005 WO 2006/001771 January 2006 WO 2007/064281 June 2007 |
| Other References: | DuBois, “Amination of aryl sulfamate esters. A convenient general synthesis of aliphatic sulfamides,” J. Org. Chem., 1980, 45 (26), pp. 5373-5375. cited by examiner Reitz et al., “The role of sulfamide derivatives in medicinal chemistry: a patent review (2006-2008),” Expert Opin. Ther. Patents, Oct. 2009, vol. 19, No. 10, pp. 1449-1453. cited by examiner Winum et al., “Therapeutic potential of sulfamides as enzyme inhibitors,” Med. Res. Rev., 2006; vol. 26, pp. 767-792. cited by examiner Arshady, R., “Styrene based polymer supports developed by suspension polymerization”, Chimica e L'Industria, 70 (9), 70-75 (1988). cited by applicant Dahri, L., et al., “Affinity of Human Anti-Factor VIII Antibodies for Functional Polystyrene Supports”, Journal of Molecular Recognition, 9(5-6), 401-406 (1996). cited by applicant Dourges, M., et al., “Affinity chromatography of fibroblast growth factors on substituted polystyrene”, Journal of Chromatography, 526(1), 35-45 (1990). cited by applicant Hjerten, S., “The Preparation of Agarose Spheres for Chromatography of Molecules and Particles”, Biochim Biophys Acta, 79(2), 393-398 (1964). cited by applicant Knudsen, K., et al., “Sulfone-Aromatic Ligands for Thiophilic Adsorption Chromatography: Purification of Human and Mouse Immunoglobulins”, Analytical Biochemistry, 201, 170-177 (1992). cited by applicant Liu, Y., et al., “Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma”, Journal of Chromatography B, 792(2), 177-185 (2003). cited by applicant Porath, J., et al., “Thiophilic adsorption—a new method for protein fractionation”, FEBS Letters, 185(2), 306-310 (1985). cited by applicant Porath, J., et al., “A New Kind of ‘Thiophilic’ Electron-Donor-Acceptor Adsorbent”, Makromol. Chem., Makromol. Symp., 17, 359-371 (1988). cited by applicant Schwarz, A., et al., “Novel heterocyclic ligands for the thiophilic purification of antibodies”, Journal of Chromatography B, 664, 83-88 (1995). cited by applicant |
| Primary Examiner: | Yakovleva, Galina |
| Attorney, Agent or Firm: | Parks Wood LLC |
| Accession Number: | edspgr.09044740 |
| Database: | USPTO Patent Grants |
| Language: | English |
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