Claims for Patent: 8,569,464

Summary for Patent: 8,569,464

Title:	Purification of proteins
Abstract:	The present invention relates to a selectively soluble polymer capable of binding to a desired molecules in an unclarified mixture containing various biological materials and the methods of using such a polymer to purify a molecule from such a mixture. The polymer is soluble in the mixture under a certain set of process conditions such as pH or temperature and/or salt concentration and is rendered insoluble and precipitates out of solution upon a change in the process conditions. The polymer is capable of binding to the desired molecule (protein, polypeptide, etc) and remains capable of binding to that molecule even after the polymer is precipitated out of solution. The precipitate can then be filtered out from the remainder of the stream and the desired biomolecule is recovered such as by elution and further processed.
Inventor(s):	Moya; Wilson (Concord, MA), Jaber; Jad (Nashua, NH)
Assignee:	EMD Millipore Corporation (Billerica, MA)
Application Number:	12/316,708
Patent Claims:	1. A method for purifying a biomolecule from a mixture containing soluble and insoluble impurities, comprising: a. providing the mixture at a set of conditions, b. adding one or more polyvinyl pyridine polymers or copolymers to said mixture, soluble in said mixture under the set of conditions and capable of reversibly and selectively binding to the biomolecule, and capable of irreversibly binding to said insoluble impurities, c. mixing the one or more solubilized polymers throughout the mixture; d. precipitating the one or more polymers, insoluble impurities and said biomolecule out of solution by changing the set of conditions in the mixture; e. separating the precipitate from the mixture, and f. selectively eluting the biomolecule from the precipitate. 2. The method of claim 1 wherein in step b, the polymer is added to a solution under a set of conditions within the solution and the solution containing the solubilized polymer is added to the mixture. 3. The method of claim 1 further comprising step g. wherein the biomolecule is recovered from the mixture. 4. The method of claim 1 wherein the one or more polymers is solubilized at a selected pH level and the polymer is precipitated at a pH different from that selected pH level. 5. The method of claim 1 wherein the one or more polymers is precipitated by a change in pH that varies by 0.1 pH units (+/-) from the pH level at which the polymer is soluble in the mixture. 6. The method of claim 1 wherein the one or more polymers solubilizes at a selected temperature of the mixture and the one or more polymers precipitates by a change in the temperature of the mixture. 7. The method of claim 1 wherein the biomolecule is a protein. 8. The method of claim 1 wherein the biomolecule is a recombinant protein. 9. The method of claim 1 wherein the biomolecule is an antibody. 10. The method of claim 1 wherein the biomolecule is a monoclonal antibody. 11. The method of claim 1 wherein the biomolecule is an antibody selected from the group consisting of a recombinant antibody, a recombinant monoclonal antibody, a polyclonal antibody, a humanized antibody and an antibody fragment. 12. The method of claim 1 wherein the biomolecule is an antibody fragment selected from the group consisting of Fab, Fab.sup.1, f(ab.sup.1).sub.2 and Fv fragments, single-chain antibody molecules diabodies, linear antibodies, bispecific antibodies and multispecific antibodies formed from antibody fragments. 13. The method of claim 1 wherein the biomolecule is an antibody that specifically binds to an antigen selected from the group consisting of CD3, CD4, CD8, CD19, CD20, CD34, CD40, EGF receptor, HER2, HER3, HERO receptor, LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM, av/b3 integrin, CD11a, CD18, CD11b, VEGF, IgE, flk2/flt3 receptor, obesity (OB) receptor, mpl receptor, CTLA-4 and polypeptide C. 14. The method of claim 1 wherein biomolecule is selected from the group consisting of anti-HER2; anti-CD20; anti-IL-8; anti-VEGF; anti-PSCA; anti-CD11a; anti-IgE; anti-Apo-2 receptor; anti-TNF-.alpha., anti-Tissue Factor(TF); anti-CD3; anti-CD25; anti-CD34; anti-CD40; anti-tac; anti-CD4; anti-CD52; anti-Fc receptor; anti-carcinoembryonic antigen (CEA) antibodies; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-CD33; anti-CD22; anti-EpCAM; anti-GpIIb/IIIa; anti-RSV; anti-CMV; anti-HIV; anti-hepatitis; anti-avp3; anti-human renal cell carcinoma; anti-human 17-1A; anti-human colorectal tumor; anti-human melanoma; anti-human squamous-cell carcinoma; and anti-human leukocyte antigen (HLA) antibodies. 15. The method of claim 1 wherein biomolecule is an antibody selected from the group consisting of anti-HER2 receptor, anti-VEGF, anti-IgE, anti-CD20, anti-CD11a, and anti-CD40 antibodies. 16. The method of claim 1 wherein the biomolecule is an immunoadhesin. 17. The method of claim 1 wherein the biomolecule is an antibody-like molecule. 18. The method of claim 1 wherein the biomolecule is an antibody-like molecule and the antibody-like molecule is a protein fused to, or conjugated with, a C.sub.H2/C.sub.H3 region. 19. The method of claim 1 wherein the biomolecule is an antibody-like molecule and the antibody-like molecule is a protein fused to, or conjugated with, a C.sub.H2/C.sub.H3 region and said protein is selected from the group consisting of rennin; growth hormones; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagons; factor VIIIC; factor IX; tissue factor; von Willebrands factor; Protein C; atral natriuretic factor; lung surfactant; urokinase; human urine and tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES; human macrophage inflammatory protein (MIP-1alpha); serum albumins; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; beta-lactamase; DNase; IgE, cytotoxic T-lymphocyte associated antigens (CTLAs); inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; Protein A or D; rheumatoid factors; bone-derived neurotrophic factor (BDNF); neurotrophin-3, -4, -5, and -6 (NT-3, NT-4, NT-5, and NT-6), nerve growth factors; platelet-derived growth factor (PDGF); fibroblast growth factors; epidermal growth factor (EGF); transforming growth factors (TGF); insulin like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I) insulin-like growth factor binding proteins (IGFBPs); CD proteins; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons-alpha, -beta, and -gamma; colony stimulating factors (CSFs); interleukins IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigens; transport proteins; homing receptors; addressing; regulatory proteins; integrins; tumor associated antigens; and fragments thereof. 20. The method of claim 3, further comprising the step of incorporating the recovered biomolecule into a pharmaceutical formulation. 21. The method of claim 1 wherein the one or more polymers are precipitated by a change in temperature. 22. The method of claim 1 wherein the one or more polymers are precipitated by a change in pH. 23. The method of claim 1 wherein the mixture is maintained at a temperature for a period of time to maintain the one or more polymers in solution and the mixture temperature is then changed to precipitate out the one or more polymers. 24. The method of claim 1 wherein the one or more polymers are soluble at a pH of less than about 5 and are insoluble at a pH of greater than 5. 25. The method of claim 1 further comprising adding the one or more polymers to a carrier liquid under conditions to cause the one or more polymers to go into solution and adding the carrier liquid and the one or more polymers in solution to said mixture in step b. through a static mixer. 26. The method of claim 3, further comprising formulating the recovered biomolecule in a pharmaceutically acceptable carrier. 27. The method of claim 3, further comprising formulating the recovered biomolecule in a pharmaceutically acceptable carrier for a purpose selected from the group consisting of research, diagnostic and therapeutic purposes. 28. The method of claim 1 wherein the one or more polymers are added in excess to the mixture and recovered as the precipitate. 29. The method of claim 1 further comprising the step in which the biomolecule is recovered from the polymer and the recovered biomolecule has at least 1LRV reduction in impurities over the starting mixture. 30. The method of claim 1 wherein the biomolecule is recovered from the polymer by resolubilizing the biomolecule and polymer in a liquid, precipitating the polymer under conditions that cause the polymer to precipitate without binding to the biomolecule and separating the precipitated polymer from the biomolecule in solution. 31. The method of claim 1, further comprising: step g. of separating the precipitated polymer from the biomolecule. 32. The method of claim 1, wherein said selective elution of said biomolecule from said precipitate forms an elution liquid, and wherein said method further comprises: step g. separating the precipitated polymer from the biomolecule, and wherein said elution liquid is a liquid in a condition suitable for a downstream processing step. 33. The method of claim 1, wherein the eluted biomolecule is recovered and subjected to continuous flow through process steps.

Details for Patent 8,569,464

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Microbix Biosystems Inc.	KINLYTIC	urokinase	For Injection	021846	01/16/1978		2026-12-21
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2026-12-21