PURIFICATION OF PROTEINS
The present invention relates to a selectively soluble polymer capable of binding to one or more constituents in a mixture containing various biological materials and the methods of using such a polymer to purify a biomolecule from such a mixture. The polymer is soluble in the mixture under a certain set of process conditions such as pH or temperature and is rendered insoluble and precipitates out of solution upon a change in the process conditions. While in its solubilized state, the polymer is capable of binding to a selected entity within the stream such as impurities (DNA, RNA, host cell protein, endotoxins, etc) in a cell broth and remains capable of binding to that entity 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 and further processed.
1 ) A method for purifying a biomolecule from a mixture containing impurities comprising:
a. providing the mixture at a set of conditions,
b. adding one or more polymers solubilizable in said mixture under the set of conditions, the one or more polymers being capable of binding to one or more of the impurities in the mixture,
c. mixing the one or more solubilized polymers throughout the mixture;
d. precipitating the one or more polymers and one or more bound impurities out of solution by changing the set of conditions in the mixture; and
e. recovering the biomolecule.
2 ) The method of claim 1 wherein the one or more polymers are solubilized in carrier liquid before addition to the mixture.
3 ) The method of claim 1 wherein the one or more polymers is caused to be soluble in the aqueous solution due to a pH level and the polymer is precipitated by a pH change.
4 ) The method of claim 1 wherein the one or more polymers is caused to be soluble in the aqueous solution due to a pH level below 7.
5 ) The method of claim 1 wherein the one or more polymers is caused to be soluble in the mixture due to a temperature of the one or more polymers and the mixture and the one or more polymers precipitates by a change in the temperature of the one or more polymers and mixture.
6 ) The method of claim 1 wherein the biomolecule is a protein.
7 ) The method of claim 1 wherein the biomolecule is an antibody.
8 ) The method of claim 1 wherein the biomolecule is a recombinant antibody.
9 ) The method of claim 1 wherein the biomolecule is a monoclonal antibody.
10 ) The method of claim 1 wherein the biomolecule is a recombinant monoclonal antibody.
11 ) The method of claim 1 wherein the biomolecule is selected from the group consisting of a polyclonal antibody, a humanized antibody and an antibody fragment.
12 ) The method of claim 1 wherein said biomolecule is an antibody fragment selected from the group consisting of Fab, Fab ! , f(ab ! ) 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 said 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, HER4 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 said biomolecule is an antibody 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-α, 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-αvβ3; 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 said 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 H 2/C H 3 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 H 2/C H 3 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-l-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; atrial 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 and thereof.
20 ) The method of claim 1 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 selected from the group consisting of poly(N-isopropylacrylamide), agarose, dextran, polyethylene oxide, cationic polyelectrolytes and anionic polyelectrolytes.
22 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in temperature.
23 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in temperature and are selected from the group consisting of poly(N-isopropylacrylamide), agarose Hydroxyalkylcellulose and polyethylene oxide.
24 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in pH.
25 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in pH and are selected from the group consisting of cationic polyelectrolytes and anionic polyelectrolytes.
26 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in pH and are cationic polyelectrolytes selected from the group consisting of chitosan, polyvinylpyridine, copolymers of vinylpyridine, primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers.
27 ) The method of claim 1 wherein the one or more polymers are precipitated by a change in pH and are anionic polyelectrolytes selected from the group consisting of copolymers of acrylic acid and methyl methacrylate and copolymers of methacrylic acid and methyl methacrylate.
28 ) The method of claim 1 wherein the one or more polymers are rendered soluble by the condition of temperature and the mixture is maintained at the desired temperature for 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.
29 ) The method of claim 1 wherein the one or more polymers are solubilized and precipitated by a change in pH, are cationic polyelectrolytes selected from the group consisting of chitosan, polyvinylpyridine, copolymers of vinyl pyridine, primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers and are soluble at a first pH and are insoluble at a second pH.
30 ) The method of claim 1 wherein the one or more polymers are solubilized and precipitated by a change in pH, are cationic polyelectrolytes selected from the group consisting of chitosan, polyvinylpyridine, copolymers of vinyl pyridine, primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers.
31 ) The method of claim 1 wherein the one or more polymers are solubilized and precipitated by a change in pH, are anionic polyelectrolytes selected from the group consisting of copolymers of acrylic acid and methyl methacrylate and copolymers of methacrylic acid and methyl methacrylate and are soluble at a first pH and are insoluble at a second pH.
32 ) The method of claim 1 wherein the one or more polymers are added to a carrier liquid under conditions to cause the one or more polymers to go into solution and the carrier liquid containing the one or more polymers in solution is added to the mixture through a static mixer.
33 ) A method for purifying a biomolecule from a mixture containing a host cell protein as an impurity comprising subjecting said mixture to:
a. providing a mixture of a biomolecule and a host cell protein as an impurity,
b. conducting a purification step by adding a carrier liquid containing one or more polymers solubilized in said carrier liquid to the mixture, the polymer being soluble under a set of conditions within the carrier liquid and the mixture and being capable of binding to the impurity,
c. allowing the one or more polymers to mix with the constituents of the mixture;
d. precipitating the one or more polymers and host cell protein of the mixture out of solution by changing the set of conditions in the mixture which causes the one or more polymers to be insoluble;
e. filtering the precipitated one or more polymers from the mixture, and
f. recovering the biomolecule at a purity at least 1 LRV better than the initial mixture.
34 ) The method of claim 1 further comprising the recovered biomolecule is formulated in a pharmaceutically acceptable carrier.
35 ) The method of claim 1 further comprising the recovered biomolecule is formulated in a pharmaceutically acceptable carrier a purpose selected from the group consisting of research, diagnostic and therapeutic purposes.
36 ) The method of claim 1 wherein the one or more polymers are added in excess to the mixture and recovered as the precipitate.
37 ) The method of claim 1 further comprising subjecting the recovered biomolecule to a second purification step in which a stimuli responsive soluble polymer which is capable of binding to the biomolecule is added to a mixture containing the recovered biomolecule from the method of claim 1 under conditions to cause the polymer to be in solution, the conditions are changed so as to cause the polymer to precipitate out of solution with the recovered biomolecule, recovering the precipitate, eluting the biomolecule from the precipitate and recovering the biomolecule.
38 ) The method of claim 1 further comprising subjecting the recovered biomolecule to a second purification step in which a stimuli responsive soluble polymer which is capable of binding to the biomolecule is added to a mixture containing the recovered biomolecule from the method of claim 1 under conditions to cause the polymer to be in solution, the conditions are changed so as to cause the polymer to precipitate out of solution with the recovered biomolecule, recovering the precipitate, washing the precipitate at least one time, eluting the biomolecule from the precipitate and recovering the biomolecule.
39 ) The method of claim 1 wherein the impurity is selected from the group consisting of DNA, RNA, virus, host cell proteins, endotoxins, cells, cell fragments and combination thereof.
40 ) The method of claim 1 wherein the biomolecule is recovered by separating the one or more precipitated polymers and one or more impurities from the mixture.
41 ) The method of claim 1 wherein the one or more polymers are capable of binding with one or more of the impurities in the solubilized and precipitated state.