CA2333747C - Separation of protein monomers by use of ion-exchange chromatography - Google Patents
Separation of protein monomers by use of ion-exchange chromatography Download PDFInfo
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- CA2333747C CA2333747C CA002333747A CA2333747A CA2333747C CA 2333747 C CA2333747 C CA 2333747C CA 002333747 A CA002333747 A CA 002333747A CA 2333747 A CA2333747 A CA 2333747A CA 2333747 C CA2333747 C CA 2333747C
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- antibody
- exchange
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- monomer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
- C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
Abstract
A method is disclosed for separating a polypeptide monomer from a mixture comprising dimers and/or multimers. The method comprises applying the mixture to either a cation-exchange chromatography resin or an anion-exchange chromatography resin and eluting the mixture at a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers and/or multimers present in the mixture.
Description
Separatioa of Profiein Monomen by ase of Ion-E'acehange Chromatogrsphy Bskaound of the lanwon Field of the Invention This invention relatea to a procass for separating polypeptide monomers from dimers and/or other maltimers using ion-exchange cbromatography.
Lgggjnon of Ea*gmuQ and Related Art Attempta to purify mrthentic, properly folded protain from rowmbinant hosts have been fYustrated due to the tereiary structure of the molecule. In this regard, purification of to the rocombinantly produced molecule often yields a heterogeneous mixture that consists largely of inactive, misfolded, in oluble, andlor soluble dimers, multimers, and disulfido-linked aggregates. Odw abecrant molecules, such as fragments, nicked, oxidized, and Slywaylated forms, may also be present. Thus, purifieation is difficult and yields ofthe authentic monomer are often low. See, e.g., E2liot et al., 1. Prntein Chem. Q:
is (1990).
Diffa=t techniques have been used to cornxt these problema. For example, Chang and Swartz, PmWgEol_ nQ: in vivo and in vitrn (Ameacan Chemical Society, 1993), pp. 178-188 descn'bc a method for solubilizing aggregated IGF-I
produced in E.
coli, using low conccaitrations of urea and dithiothreitol (D'Y'!'j in an alkaline bufifix; U.S.
20 Pat. No. 5,231,178 describes a method for the putification of corroctly folded, monomeric IGF-I frorn P.postr,ris using a combinadon of cation exchange, hydrophobic intaaction, and gel filtration chromatogXaphy. WO 96/40776 descn'bes a method for producin,g authentie properiy folded IGF from yeast using a first cation exehange chromatography with the ycast cell medium, dmtaturing and duomatogtaphy, and performing reverse phasa 25 high performance liquid chro11natography.
Separation of protein and peptide monomers from their dimers, tetramers, and muttimers presenis a serious challCnge to the separntions scaentist. Sizo-excWon chromatography (SEC) and Tangmtial-Flow Filtration (TFF) (U.S. Pat Nos.
5,256,294 and 5,490,937) have beea used tbr separating monomors from aggXegates but have 30 limitations. SEC can separate monomas from multimers, and in some casea monoumn - I-tom dimers. The main linritations of SEC arc 1) limited load volumes (typically 5% of the bed volume) requiring large columns or multiple cycles, 2) and load protein concentration (low coneentration fved stocks require pre-conc.entration or multYple cycles on the column. Higher protein conocntrations can be more viscous, thereby reducing the S efficiency of the separation). Historically TFF can separateprotein multimers that arc tcn-fold larger than the monomer. U.S. Pat. No. 5,256,294.
U.S. Pat. Nos. 4,228,154 and 5,250,663 disclose separations of albumin from mixtures. U.S. Pat. No. 4,228,154 describes use of both eation-exchange and anion-exchange ehromatography steps for the purification without separation of monoma from inultimers.
Japanese patent application H7-285885 de8cn'bes a process for separating immunoglobin monomer from a mixture containing monomer and polymers, wherein the mixture of immunoglobins is applied to a cation-mchange resin. 7his proccss involvcs a filtration, but not an elution of any material.
There is a need for soparating monomers from dimers and multimers that is satisfactory, requires the use of only one ion-exchange step, and does not have the limitations of SEC or TFF.
Summa of tltc Invention Accordingly, this invention provides a method for scparating a polypeptide 2o monomer from a mixture comprising dimers and/or multimers, wherein the method comprises applying the mixturc to either a cation-exchange or an anion-exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein ifthe reain is anion-exchange, the pH ofthe buffer is about 6-9, and eluting the mixture at a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers and/or multimers present in the mixture.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the - la -antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of the antibody, where the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, where if the resin is cation-exchange, the pH of the buffer is about 4-7, and where if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the ion-exchange chromatography step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody, where the method comprises the step of applying the mixture to an anion-exchange chromatography resin in a buffer, where the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the anion-exchange chromatography step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody, where the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, where if the resin is cation-exchange, the pH of the buffer is about 4-7, and where if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the cation-exchange step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM; and where the separated monomer has a purity of greater than 99.5%.
In an alternative aspect, the invention provides use of an ion-exchange resin for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody by ion-exchange chromatography in a buffer, comprising applying the mixture to the resin, and eluting the mixture with a gradient of about 0-1M of an elution salt; wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM.
-lb-In this study it is demonstrated that ion-exchange chromatography-either anion or cation--is an effective means to separate protein or polypcptide monomers from their dimers and/or multimers. Separations are performed using either step or lincar gradient elution. Ion exchange has several advantages over the SEC
and TFF rnethods described above. First, separation is independent of polypeptide concentration in the load and therefore no pre-concentration is rcquired. Second. resins can be loaded to greater than 30 mg polypeptide/mL resin and still achieve excellent separations. Third. ion-exchange resins are inexpensive and easy to use. Typical separations achieve enrichment of monomcr to greater than 99.5% purity and yields in excess of 90SE.
Brief Descriotion of the Drawin~s Figures lA and 1B show separation of U266 IgE monomcr from dimers and multimers on a RESOURCE QTM anion-exchange column. The column was equilibrated in 25 mM
Tris/pH 8, and eluted with a aradient from 0 to 0.5 M sodium chloride over 10 column volumes. Fig. 1 A is full-scale: Fig. 1 B is a close-up view to show the dimers and multimers.
Figures 2AI. 2A2. 2B, and 2C show separation of anti-IgE monoclonal antibody monomer from dimcrs and multimers. Figs 2A1 and 2A2 were run on a RESOURCE QTm anion-exchange column. Fig. 2A1 is full-scale: Fig. 2A2 is a close-up view to show the dimers and multimers. Fig.
2B is a run on Q-SEPHAROSE
FAST-FLOWTM resin. Fig. 2C is a plot of monomer and dimer/multimer observed in fractions, where the open dots are monomer and the solid dots are dimer. The monotner and dimer/multimcr were determined using a SUPERDEX 200 HR7"*' 10/30 analytical size-exclusion column (Pharmacia Biotech). In all cases the columns were equilibrated in 25 mM Tris/pH 8. The gradient used in the Fig. 2A panels was 0 to 0.5 M sodium chloride over 40 column volumes. The gradient used for Fig. 2B (Q-SEPHAROSE FAST-FLOWTM) was 0.05 to 0.2 M
NaCI ovcr 10 column volumes.
Figures 3A-C show separation of BSA monomer and dimer on a RESOURCE Q'4' anion-exchange column at pH 8. The column was equilibrated in 25 mM Tris/pH 8. and eluted with a gradient from 0.125 to 0.275 M sodium chloride over 40 column volumes. Fig. 3A is purified monomer.
Fig. 3B is purified ditner, and Fig. 3C is a commercial preparation of BSA (Bayer) that contains both monomer and dimer.
Figures 4A-C show separation of BSA monomer and dimer on a RESOURCE QT M anion-exchange column at pH 6. The column was equilibrated in 20 ntM sodium phosphate/pH 6.
and elutcd with a linear gradient from 0 to 0.5 M sodium chloride over 10 column volumes. Fig. 4A is purified monomer, Fig. 4B is purified dimer. and Fig. 4C is a commercial preparation of BSA (Bayer) that contains both mononter and dinter.
Figures 5A and 5B show separation of anti-IgE monoclonal antibody monomer from dirners and multimers on a RESOURCE STM cation-exchange column at pH 6. The column was equilibrated in 20 mM
sodium phosphate/pH 6. and elutcd with a linear gradient from 0 to 0.05 M
sodium chloride over 30 column volutrxs. Fig. 5A is the chrornatogram from the separation, and Fig. SB is a plot of monomer and dimer/multimer observed in fractions using the same rnethod described in Figure 2, where the open dots are monomer and the solid dots are dimer.
Figures 6A and 6B show separation of BSA monomer and dimcr on a RESOURCE STM
cation-exchange column at pH 4.3. The column was equilibrated in 20 mM sodium acetate/pH 4.3. then cluted with a Qradient from 0 to 1 M sodiurri chloride over 20 column volumes. Fig. 6A is purified monomer, and Fig. 6B is purified dimer.
Detailed Description of the Preferred Embodiments Definitions As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. Preferably, the polypeptides are "exogenous," meanins! that they are "heterologous." i.e.. foreign to the host cell being utilized, such as a human protein produced by E. coli.
However, they may also be derived from a native source in which they are present naturally.
Examples of mammalian polypeptides include molecules such as, e.g., renin, a'Trowth hormone.
inciudinLy human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; 1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin:
thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone;
glucagon; clotting factors such as factor VIIIC, factor IX. tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial naturietic factor; lung surfactant: a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); hombesin; thrombin: hemopoietic growth factor:
tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; retaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase: DNase; inhibin; activin: vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF; cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- I. TGF- 2. TGF-3, TGF- 4. or TGF- 5; insulin-like erowth factor-I and -II (IGF-I and IGF-II);
des(I-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins: CD proteins such as CD-3, CD-4.
CD-8, and CD-19; erythropoietin:
osteoinductive factors: immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; serum albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA); colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g.. IL-1 to IL-10; anti-HER-2 antibody; superoxide dismutase; T-cell receptors; surface membrane proteins: decay accelerating factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins:
homing receptors; addressins; regulatory proteins; antibodies; and fragments of any of the above-listed polypeptides.
The preferred pofypeptides of interest are manunalian polypeptides. Examples of such mammafian polypeptides include enzymes, hormones, cytokines, albumins, chemokines, immunotoxins, viral components, antibodies. neurotrophins, and antigens. Suitable such polypeptides encompass polypeptides such as HSA, BSA, anti-IgE, anti-CD20, anti-IgG, t-PA, gpl20, anti-CDlla, anti-CD18, anti-VEGF, VEGF, TGF-beta, activin, inhibin, anti-HER-2, DNase, IGF-I, IGF-II, brain IGF-I, growth hormone, relaxin chains. growth hormone releasing factor, insulin chains or pro-insulin, NGF, NT-3, BDNF, and urokinase. Particularly preferred mammalian polypeptides include, e.g., t-PA, gp120 (IIIb), anti-HER-2, anti-CDI la, anti-CD18, anti-VEGF, VEGF, BSA, HSA, anti-CD20, anti-IgE, anti-IgG, DNase, IGF-I, IGF-II. TGF-beta, IGFBP-,. IGFBP-2, IGFBP-l, growth hormone, NGF, NT-3, NT-4. NT-i. and NT-6. The polypeptide is more preferably an antibody or a serum albumin, more preferably. anti-IgE, anti-IgG, anti-Her-2.
anti-CDlla. anti-CD18, anti-CD20, anti-VEGF, BSA, or HSA.
For purposes herein, the "mixture" contains monomers and either dimers or multimers or both dimers and multimers. Typically, the mixture is a biological fluid, which denotes any fluid derived from or containing cells, cell components, or cell products. Biological fluids include, but are not limited to. fermentation broth, cell culture supernatants, cell lysates, cleared cell lysates. cell extracts, tissue extracts, blood, plasma, serum, sputum, semen, mucus, milk, and fractions thereof. This definition includes cell-conditioned culture medium, which denotes a nutrient medium in which cells have been cultured and which contains cell products.
For purposes herein, "ion-exchange chromatography resin" refers to chromatography medium for anion- or cation-exchange separation.
As used herein, "elution salt" refers to an alkaline earth, alkali metal. or ammonium salt, i.e., a salt having a cation from the alkaline earth or alkali metal elements or an ammonium cation and having an inorganic or organic (hydrocarbon-based) anion. Examples of such salts include sodium chloride. ammonium chloride.
sodium citrate, potassium citrate, potassium chloride, magnesium chloride, calcium chloride. sodium phosphate.
calcium phosphate, arnmonium phosphate, magnesium phosphate, potassium phosphate, sodium sulfate, ammonium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, etc.
Preferred salts herein are chlorides or sulfates. The most preferred salt herein is sodium chloride.
As used herein, "muitimers" refer to n-mers where n is 3-10, i.e., polymers that are not dimers but exclude aggregates. In contrast to multimers, aggregates have a value for n of greater than 10, and/or a molecular weight of greater than 2 million daltons, and/or are species contained in the excluded volume of analytical size-exclusion chromatography columns such as SUPEROSE 6TM
(Pharmacia).
Modes for Carrying out the Invention This invention relates to a method of separating monomers of polypeptides from their dimers or multimers or both. The method involves placing the mixture of monomers and dimers and/or multimers, from whatever source, in an equilibration buffer at a pH in the range of about 4 and 9 depending on whether the resin used for chromatographic separation is a cation- or anion-exchange resin. The resulting mixture is loaded onto either a cation-exchange or anion-exchange chromatography resin to capture all the n-mers (monomers, dimers, trimers, tetramers, etc.) present in the mixture. For ion-exchange column chromatography. ligands of general affinity can be used to achieve the desired selectivities and binding properties. The loading takes place in a buffer at a pH of about 6-9 if the resin is anion-exchange and about 4-7 if the resin is cation-exchange. The exact pH will depend, for example, on the isoelectric point of the polypeptide.
If the resin is a cation-exchange resin, prior to loading the mixture, the matrix can be equiiibrated using several column volumes of a dilute, weak acid (e.g., four column volumes of 20 mM acetic acid, pH 3, or of 20 mM phosphoric acid, pH about 2.8). Following equilibration, the mixture is added and the column can be washed one to several times, prior to elution of the mixture, also using a weak acid solution such as a weak acetic acid or phosphoric acid solution. The buffer used for this purpose depends on, e.g., the polypeptide and the anionic or cationic nature of the resin. For anion-exchange, preferably the buffer is TRIS or phosphate buffer; for cation-exchange, the buffer is preferably acetate or phosphate buffer.
Lgggjnon of Ea*gmuQ and Related Art Attempta to purify mrthentic, properly folded protain from rowmbinant hosts have been fYustrated due to the tereiary structure of the molecule. In this regard, purification of to the rocombinantly produced molecule often yields a heterogeneous mixture that consists largely of inactive, misfolded, in oluble, andlor soluble dimers, multimers, and disulfido-linked aggregates. Odw abecrant molecules, such as fragments, nicked, oxidized, and Slywaylated forms, may also be present. Thus, purifieation is difficult and yields ofthe authentic monomer are often low. See, e.g., E2liot et al., 1. Prntein Chem. Q:
is (1990).
Diffa=t techniques have been used to cornxt these problema. For example, Chang and Swartz, PmWgEol_ nQ: in vivo and in vitrn (Ameacan Chemical Society, 1993), pp. 178-188 descn'bc a method for solubilizing aggregated IGF-I
produced in E.
coli, using low conccaitrations of urea and dithiothreitol (D'Y'!'j in an alkaline bufifix; U.S.
20 Pat. No. 5,231,178 describes a method for the putification of corroctly folded, monomeric IGF-I frorn P.postr,ris using a combinadon of cation exchange, hydrophobic intaaction, and gel filtration chromatogXaphy. WO 96/40776 descn'bes a method for producin,g authentie properiy folded IGF from yeast using a first cation exehange chromatography with the ycast cell medium, dmtaturing and duomatogtaphy, and performing reverse phasa 25 high performance liquid chro11natography.
Separation of protein and peptide monomers from their dimers, tetramers, and muttimers presenis a serious challCnge to the separntions scaentist. Sizo-excWon chromatography (SEC) and Tangmtial-Flow Filtration (TFF) (U.S. Pat Nos.
5,256,294 and 5,490,937) have beea used tbr separating monomors from aggXegates but have 30 limitations. SEC can separate monomas from multimers, and in some casea monoumn - I-tom dimers. The main linritations of SEC arc 1) limited load volumes (typically 5% of the bed volume) requiring large columns or multiple cycles, 2) and load protein concentration (low coneentration fved stocks require pre-conc.entration or multYple cycles on the column. Higher protein conocntrations can be more viscous, thereby reducing the S efficiency of the separation). Historically TFF can separateprotein multimers that arc tcn-fold larger than the monomer. U.S. Pat. No. 5,256,294.
U.S. Pat. Nos. 4,228,154 and 5,250,663 disclose separations of albumin from mixtures. U.S. Pat. No. 4,228,154 describes use of both eation-exchange and anion-exchange ehromatography steps for the purification without separation of monoma from inultimers.
Japanese patent application H7-285885 de8cn'bes a process for separating immunoglobin monomer from a mixture containing monomer and polymers, wherein the mixture of immunoglobins is applied to a cation-mchange resin. 7his proccss involvcs a filtration, but not an elution of any material.
There is a need for soparating monomers from dimers and multimers that is satisfactory, requires the use of only one ion-exchange step, and does not have the limitations of SEC or TFF.
Summa of tltc Invention Accordingly, this invention provides a method for scparating a polypeptide 2o monomer from a mixture comprising dimers and/or multimers, wherein the method comprises applying the mixturc to either a cation-exchange or an anion-exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein ifthe reain is anion-exchange, the pH ofthe buffer is about 6-9, and eluting the mixture at a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers and/or multimers present in the mixture.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the - la -antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of the antibody, where the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, where if the resin is cation-exchange, the pH of the buffer is about 4-7, and where if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the ion-exchange chromatography step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody, where the method comprises the step of applying the mixture to an anion-exchange chromatography resin in a buffer, where the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the anion-exchange chromatography step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM.
In an alternative aspect, the invention provides a method for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody, where the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, where if the resin is cation-exchange, the pH of the buffer is about 4-7, and where if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, where the monomer is separated from the dimers or multimers or both present in the mixture in the cation-exchange step; where if citrate is used as buffer, it is employed at a concentration of below 20 mM; and where the separated monomer has a purity of greater than 99.5%.
In an alternative aspect, the invention provides use of an ion-exchange resin for separating an antibody monomer from a mixture comprising monomers of the antibody and dimers of the antibody or n-mers of the antibody where n is 3-10 or both dimers and n-mers of said antibody by ion-exchange chromatography in a buffer, comprising applying the mixture to the resin, and eluting the mixture with a gradient of about 0-1M of an elution salt; wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM.
-lb-In this study it is demonstrated that ion-exchange chromatography-either anion or cation--is an effective means to separate protein or polypcptide monomers from their dimers and/or multimers. Separations are performed using either step or lincar gradient elution. Ion exchange has several advantages over the SEC
and TFF rnethods described above. First, separation is independent of polypeptide concentration in the load and therefore no pre-concentration is rcquired. Second. resins can be loaded to greater than 30 mg polypeptide/mL resin and still achieve excellent separations. Third. ion-exchange resins are inexpensive and easy to use. Typical separations achieve enrichment of monomcr to greater than 99.5% purity and yields in excess of 90SE.
Brief Descriotion of the Drawin~s Figures lA and 1B show separation of U266 IgE monomcr from dimers and multimers on a RESOURCE QTM anion-exchange column. The column was equilibrated in 25 mM
Tris/pH 8, and eluted with a aradient from 0 to 0.5 M sodium chloride over 10 column volumes. Fig. 1 A is full-scale: Fig. 1 B is a close-up view to show the dimers and multimers.
Figures 2AI. 2A2. 2B, and 2C show separation of anti-IgE monoclonal antibody monomer from dimcrs and multimers. Figs 2A1 and 2A2 were run on a RESOURCE QTm anion-exchange column. Fig. 2A1 is full-scale: Fig. 2A2 is a close-up view to show the dimers and multimers. Fig.
2B is a run on Q-SEPHAROSE
FAST-FLOWTM resin. Fig. 2C is a plot of monomer and dimer/multimer observed in fractions, where the open dots are monomer and the solid dots are dimer. The monotner and dimer/multimcr were determined using a SUPERDEX 200 HR7"*' 10/30 analytical size-exclusion column (Pharmacia Biotech). In all cases the columns were equilibrated in 25 mM Tris/pH 8. The gradient used in the Fig. 2A panels was 0 to 0.5 M sodium chloride over 40 column volumes. The gradient used for Fig. 2B (Q-SEPHAROSE FAST-FLOWTM) was 0.05 to 0.2 M
NaCI ovcr 10 column volumes.
Figures 3A-C show separation of BSA monomer and dimer on a RESOURCE Q'4' anion-exchange column at pH 8. The column was equilibrated in 25 mM Tris/pH 8. and eluted with a gradient from 0.125 to 0.275 M sodium chloride over 40 column volumes. Fig. 3A is purified monomer.
Fig. 3B is purified ditner, and Fig. 3C is a commercial preparation of BSA (Bayer) that contains both monomer and dimer.
Figures 4A-C show separation of BSA monomer and dimer on a RESOURCE QT M anion-exchange column at pH 6. The column was equilibrated in 20 ntM sodium phosphate/pH 6.
and elutcd with a linear gradient from 0 to 0.5 M sodium chloride over 10 column volumes. Fig. 4A is purified monomer, Fig. 4B is purified dimer. and Fig. 4C is a commercial preparation of BSA (Bayer) that contains both mononter and dinter.
Figures 5A and 5B show separation of anti-IgE monoclonal antibody monomer from dirners and multimers on a RESOURCE STM cation-exchange column at pH 6. The column was equilibrated in 20 mM
sodium phosphate/pH 6. and elutcd with a linear gradient from 0 to 0.05 M
sodium chloride over 30 column volutrxs. Fig. 5A is the chrornatogram from the separation, and Fig. SB is a plot of monomer and dimer/multimer observed in fractions using the same rnethod described in Figure 2, where the open dots are monomer and the solid dots are dimer.
Figures 6A and 6B show separation of BSA monomer and dimcr on a RESOURCE STM
cation-exchange column at pH 4.3. The column was equilibrated in 20 mM sodium acetate/pH 4.3. then cluted with a Qradient from 0 to 1 M sodiurri chloride over 20 column volumes. Fig. 6A is purified monomer, and Fig. 6B is purified dimer.
Detailed Description of the Preferred Embodiments Definitions As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. Preferably, the polypeptides are "exogenous," meanins! that they are "heterologous." i.e.. foreign to the host cell being utilized, such as a human protein produced by E. coli.
However, they may also be derived from a native source in which they are present naturally.
Examples of mammalian polypeptides include molecules such as, e.g., renin, a'Trowth hormone.
inciudinLy human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; 1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin:
thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone;
glucagon; clotting factors such as factor VIIIC, factor IX. tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial naturietic factor; lung surfactant: a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); hombesin; thrombin: hemopoietic growth factor:
tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; retaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase: DNase; inhibin; activin: vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF; cardiotrophins (cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- I. TGF- 2. TGF-3, TGF- 4. or TGF- 5; insulin-like erowth factor-I and -II (IGF-I and IGF-II);
des(I-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins: CD proteins such as CD-3, CD-4.
CD-8, and CD-19; erythropoietin:
osteoinductive factors: immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; serum albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA); colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g.. IL-1 to IL-10; anti-HER-2 antibody; superoxide dismutase; T-cell receptors; surface membrane proteins: decay accelerating factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins:
homing receptors; addressins; regulatory proteins; antibodies; and fragments of any of the above-listed polypeptides.
The preferred pofypeptides of interest are manunalian polypeptides. Examples of such mammafian polypeptides include enzymes, hormones, cytokines, albumins, chemokines, immunotoxins, viral components, antibodies. neurotrophins, and antigens. Suitable such polypeptides encompass polypeptides such as HSA, BSA, anti-IgE, anti-CD20, anti-IgG, t-PA, gpl20, anti-CDlla, anti-CD18, anti-VEGF, VEGF, TGF-beta, activin, inhibin, anti-HER-2, DNase, IGF-I, IGF-II, brain IGF-I, growth hormone, relaxin chains. growth hormone releasing factor, insulin chains or pro-insulin, NGF, NT-3, BDNF, and urokinase. Particularly preferred mammalian polypeptides include, e.g., t-PA, gp120 (IIIb), anti-HER-2, anti-CDI la, anti-CD18, anti-VEGF, VEGF, BSA, HSA, anti-CD20, anti-IgE, anti-IgG, DNase, IGF-I, IGF-II. TGF-beta, IGFBP-,. IGFBP-2, IGFBP-l, growth hormone, NGF, NT-3, NT-4. NT-i. and NT-6. The polypeptide is more preferably an antibody or a serum albumin, more preferably. anti-IgE, anti-IgG, anti-Her-2.
anti-CDlla. anti-CD18, anti-CD20, anti-VEGF, BSA, or HSA.
For purposes herein, the "mixture" contains monomers and either dimers or multimers or both dimers and multimers. Typically, the mixture is a biological fluid, which denotes any fluid derived from or containing cells, cell components, or cell products. Biological fluids include, but are not limited to. fermentation broth, cell culture supernatants, cell lysates, cleared cell lysates. cell extracts, tissue extracts, blood, plasma, serum, sputum, semen, mucus, milk, and fractions thereof. This definition includes cell-conditioned culture medium, which denotes a nutrient medium in which cells have been cultured and which contains cell products.
For purposes herein, "ion-exchange chromatography resin" refers to chromatography medium for anion- or cation-exchange separation.
As used herein, "elution salt" refers to an alkaline earth, alkali metal. or ammonium salt, i.e., a salt having a cation from the alkaline earth or alkali metal elements or an ammonium cation and having an inorganic or organic (hydrocarbon-based) anion. Examples of such salts include sodium chloride. ammonium chloride.
sodium citrate, potassium citrate, potassium chloride, magnesium chloride, calcium chloride. sodium phosphate.
calcium phosphate, arnmonium phosphate, magnesium phosphate, potassium phosphate, sodium sulfate, ammonium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, etc.
Preferred salts herein are chlorides or sulfates. The most preferred salt herein is sodium chloride.
As used herein, "muitimers" refer to n-mers where n is 3-10, i.e., polymers that are not dimers but exclude aggregates. In contrast to multimers, aggregates have a value for n of greater than 10, and/or a molecular weight of greater than 2 million daltons, and/or are species contained in the excluded volume of analytical size-exclusion chromatography columns such as SUPEROSE 6TM
(Pharmacia).
Modes for Carrying out the Invention This invention relates to a method of separating monomers of polypeptides from their dimers or multimers or both. The method involves placing the mixture of monomers and dimers and/or multimers, from whatever source, in an equilibration buffer at a pH in the range of about 4 and 9 depending on whether the resin used for chromatographic separation is a cation- or anion-exchange resin. The resulting mixture is loaded onto either a cation-exchange or anion-exchange chromatography resin to capture all the n-mers (monomers, dimers, trimers, tetramers, etc.) present in the mixture. For ion-exchange column chromatography. ligands of general affinity can be used to achieve the desired selectivities and binding properties. The loading takes place in a buffer at a pH of about 6-9 if the resin is anion-exchange and about 4-7 if the resin is cation-exchange. The exact pH will depend, for example, on the isoelectric point of the polypeptide.
If the resin is a cation-exchange resin, prior to loading the mixture, the matrix can be equiiibrated using several column volumes of a dilute, weak acid (e.g., four column volumes of 20 mM acetic acid, pH 3, or of 20 mM phosphoric acid, pH about 2.8). Following equilibration, the mixture is added and the column can be washed one to several times, prior to elution of the mixture, also using a weak acid solution such as a weak acetic acid or phosphoric acid solution. The buffer used for this purpose depends on, e.g., the polypeptide and the anionic or cationic nature of the resin. For anion-exchange, preferably the buffer is TRIS or phosphate buffer; for cation-exchange, the buffer is preferably acetate or phosphate buffer.
Ion-exchange chromatography is typically carried out at a temperature of about 18-25 C. preferably about 20 C (room temperature). The preferred column loading is about I ml resin per 20-30 mg total polvpeptide.
Following adsorption of the n-mer molecules to the ion exchanger, the rnixture is eluted by contacting the resin with an elution salt having an appropriate ionic strength to displace the monomer from the matrix. An elution salt gradient is used of about 0 to I M. The gradient may be linear or stepwise. Preferably the gradient is from about 0 to 500 mM elution salt, more preferably 50 to 200 mM elution salt, and most preferably, 0 to 50 mM elution salt. Preferably the elution salt is a sodium salt, such as sodium chloride, although other elution salts and concentration gradients, known to those of skill in the art, also find use herein. The quantity of elution buffer can vary widely and will generally be in the range of about 2 to 40 column volumes. preferably 10 to 40 column volumes. Following elution, the eluate can,be assayed for total monomeric concentration.
Suitable cation-exchange resins herein include a wide variety of materials known in the art, including those capable of binding polypeptides over a wide pH range. For example, carboxymethylated, sulfonated, agarose-based, or polymeric polystyrene/divinyl benzene cation-exchange matrices are particularly preferred.
Other useful matrix materials include, but are not limited to, celluiose matrices, such as fibrous, microgranular, and beaded matrices; dextran, polyacrylate, polyvinyl, polystyrene, silica, and polyether matrices; and composites. These matrices include, for example, CM52 CELLULOSETM (Whatman, Inc.); S-HYPERDTM and CM SPHERODEXTM (Secpracor); SP SEPHAROSE FFTM, DEAE SEPHAROSE FFTM, CM-SEPHAROSETM, and RESOURCE STM (Amersham Pharmacia Biotech AB); and JT BAKER CSxTM (J.T.
Baker, Inc.), as well as those containing the functional ligand R-S03, preferably sulfopropyl resins.
such as TOYOPEARL SP550CTM
(Tosohaas) and FRACTOGEL EMDTM SO3- -650 (m) (Merck). Other suitable materials for use in cation-exchange chromatography are within the knowledge of those skilled in the art.
Anion-exchange chromatography is carried out using media appropriate therefor, as are known in the art. Suitable media include, e.g., polymeric polystyrene/divinyi benzene resins and agarose-based resins, as well as agarose beads. dextran beads, polystyrene beads, media that comprise an insoluble, particulate support derivatized with tertiary or quaternary amino groups., and supports derivatized with trimethylaminoethyl groups. Examples of suitable such media include DE92TM (diethylaminoethvl cellulose, Whatman): DEAE-CELLULOSET'" (Sigma), BAKERBOND ABX 40 muTM (J.T. Baker, Inc.); DEAE resins such as FRACTOGEL EMD DEAE-650TM (EM Separations), FRACTOGEL EMD TMAE-650 (S) T~ (EM
Science, Gibbstown, NJ), TSK gel DEAE-SPWTM (Tosohaas), DEAE-SEPHAROSE CL-6BT' and chelating SEPHAROSETV' (Amersham Pharmacia Biotech AB), DEAE MERE SEP. I000TM
(Millipore), and DEAE
SPHERODEXTM (Sepracor); RESOURCE QTx' and Q SEPHAROSETM (QSFF) (Amersham Pharmacia Biotech AB); MACRO-PEP QTM (Bio-Rad Laboratories, Hercules, CA); Q- HYPERD7"
(BioSepra. Inc., Marlborough, Mass); and the like. Other suitable anion-exchange chromatography materials, as well as the selection and use of these materials for the present application, are conventional in the art.
Purified fractions of monomer obtained from the ion-exchange chromatography may be further processed by subjecting them to any appropriate technique designed for downstream processing and purification. This will depend largely on the type of poiypeptide and its intended use. Only one ion-exchange step is necessary to effect the desircd separation of monomer from dimers and/or multimers in a mixture, although the invention does not exclude using more such steps if desired in the upstream or downstream processing of the polypeptide.
The invention will be more fully understood by reference to the following examples. They should not.
however, be construed as limiting the scope of the invention.
EXAMPLE I
This example shows the separation of anti-IgE monomers and bovine serum albumin tnonomers from dimers and multimers.
MATERIAIS AND METHODB-Resim:
Phatmacia Q-SEPHAROSE FAST FLOWT"'. 4mL to 235L bed volumes evaluated Pharmacia RESOURCE S and RESOURCE QT~: I mL prepacked columns JT Baker CSxTm. 0.46 X Scm, 51A particles Prot-aim:
A. Humanized anti-IgE monoclonal antibodies (IgG1) available from Genentech.
Inc.: pI - 7.5.
desigaated as E25 and E26. WO 93/04173 pubiishcd March 4, 1993 describes humanized anti-IgE antibodies wherein a murine antibody directed against human IgE (MaE)1) was used to provide the CDR regions that were substituted into an IgGI ittununoglobulin frantework (rhuMaE25). See also Cacia et al.. Biochemistrv. 35:
1897-1903 (1996) for studies and further descriptions of E-25.
B. Monoclonal anti-IgE antibody prcpared from the culture supetttatants of an immortalized human myeioma cell line U266B I (ATCC TIB 196) using affinity chromatography purification on an isolated anti-IgE
antibody (Genentech MAEI). Specifically, five BALBA; female mice, age six weeks. vrere immunized in their foot pads with 10 pg of purified IgE in Ribi's adjuvant. Subsequent injections were done in the same manner at one and three weeks after the initial immunizations. Three days after the final injection, the inguinal and popliteal lymph nodes were removed and pooled. and a single cell suspension was made by passing the tissue through steel gauze. The cells wcre fused at a 4:1 ratio with mouse myeloma P3X63-Ag8.653 (ATCC CRL
1580) in high glucose (DMEM) containing 50% w!v polyethylene glycol 4000.
Alternatively. the immunizations were done in a similar manner except that 30 pg of IgE per injection were used and 1gE
fragment 315-347 (Kabat) was assayed as a prefusion boost; or injections were given subcutaneously in two doses of 100 pg and a final booster of 50 g, and spleen cells were used for the fusions.
The fused cells were then plated at a density of 2xI05 per well in 96-well tissue culture plates. After 24 hours HAT selective medium hypoxanthinelaminopteriNthymidirte, Sigma.
ifH0262) was added. Of 1440 wells plated. 365 contained growing cells after HAT selection.
Fifteen days after the fusion, supernatants were tesmd for the ptr.sence of antibodies specific for human , IgE using an enzyme-linked immunosorbent assay (EUSA). 17te ELISA was performed as follows, with all incubations done at room temperature. Test plates (Nuac Imtnttnoplaea) were coated for 2 hours with rat anti-mousa lgG (Boehringer Mannlteim, N605-500) at I pglml in 50 mM sodium carbonate buffer. pH 9.6, then blocked with 0.5% bovine serum albumin in phosphate buffeted saline (PBS) for 30 minutes. then washed four timea with PBS contnining 0.05% TWEEN 20T"A (PBST). Test supernatants were added and incubated two hours with shaking, then washed four times with PBST. Human 1gE (purified from U266 cells as described above) was added at 0.5 Ng/ml and incubated for one hour with shaking, then washed four times in PBST.
Horseradish-peroxidase-conjugated goat anti-human IgE (Kirkegarrd & Perrv I_abs, #14-10-04, 0.5 mg/ml) was added at a 12500 dilution and incubated for one hour, then washed four times with PBST. The plates were developed by adding 100 Uwell of a solution containing 10 mg of o-phenylenediamine dihydrochioride (Sigma, #P8287) and 10 i of a 30% hydrogen peroxide solution in 25 ml phosphate citrate buffer, pH 5.0, and incubating for 15 minutes. The reaction was stopped by adding 100 l/weil of 2.5 M sulfuric acid. Data were obtained by reading the plates in an automated ELISA plate reader at an absorbance of 490 nm. For one antibody, 365 supernatants were tested and 100 were specific for human IgE.
Similar frequencies of IgE
specificity were obtained when screening for the other antibodies.
C. Bovine serum Albumin: pI 4.7 and 4.9 (Radola, Biochim. Biophys. Acta, 295:
412-428 (1973)) =Bayer Corp. P/N 81-024-2, "Bovine Albumin, Sulfhydryl Modified" (BSA Mix, blocked) =ICN Biomedical Inc. P/N 810013, "Albumin Bovine" (BSA Mix, native) =BSA monomer and dimer prepared in house from Bayer BSA (BSA Monomer and BSA
Dimer. respectively) Chromatography Svstems:
Hewlett-Packard I 090TM HPLC
PharmaciaT'l FPLC
Detection at 215 or 280 nm Buffers: (see Table I for details) Purified water Tris=HCl Sodium acetate Sodium chloride Sodium phosphate Sodium citrate and citric acid Sample Preparation:
Samples were diluted with the buffer used for equilibration (indicated in Table I below) to assure pH
and conductivity matched starting column conditions. All samples were 0.2-pm filtered prior to loading.
Chromatoeraphv:
Samples were introduced to the column using either an automatic or manual injector. All runs were performed at room temperature. Fractions were collected manually or with a PHARMACIA FRAC 100r"~
collector.
Chromatographic separation performance was evaluated by comparing elution profiles of BSA stock reagent and purified BSA monomer and dimer; the same was done for IgE and the monoclonal antibodies (MAb). Separation of IgE and MAb from their dimers and multimers was further evaluated by analyzing elution fraction using analytical size-exclusion chromatography. Plots of MAb MW forms vs. Fraction number were created. Recovery of IgE and MAb was determined spectrophotometrically by measuring absorbance at 280nm.
Following adsorption of the n-mer molecules to the ion exchanger, the rnixture is eluted by contacting the resin with an elution salt having an appropriate ionic strength to displace the monomer from the matrix. An elution salt gradient is used of about 0 to I M. The gradient may be linear or stepwise. Preferably the gradient is from about 0 to 500 mM elution salt, more preferably 50 to 200 mM elution salt, and most preferably, 0 to 50 mM elution salt. Preferably the elution salt is a sodium salt, such as sodium chloride, although other elution salts and concentration gradients, known to those of skill in the art, also find use herein. The quantity of elution buffer can vary widely and will generally be in the range of about 2 to 40 column volumes. preferably 10 to 40 column volumes. Following elution, the eluate can,be assayed for total monomeric concentration.
Suitable cation-exchange resins herein include a wide variety of materials known in the art, including those capable of binding polypeptides over a wide pH range. For example, carboxymethylated, sulfonated, agarose-based, or polymeric polystyrene/divinyl benzene cation-exchange matrices are particularly preferred.
Other useful matrix materials include, but are not limited to, celluiose matrices, such as fibrous, microgranular, and beaded matrices; dextran, polyacrylate, polyvinyl, polystyrene, silica, and polyether matrices; and composites. These matrices include, for example, CM52 CELLULOSETM (Whatman, Inc.); S-HYPERDTM and CM SPHERODEXTM (Secpracor); SP SEPHAROSE FFTM, DEAE SEPHAROSE FFTM, CM-SEPHAROSETM, and RESOURCE STM (Amersham Pharmacia Biotech AB); and JT BAKER CSxTM (J.T.
Baker, Inc.), as well as those containing the functional ligand R-S03, preferably sulfopropyl resins.
such as TOYOPEARL SP550CTM
(Tosohaas) and FRACTOGEL EMDTM SO3- -650 (m) (Merck). Other suitable materials for use in cation-exchange chromatography are within the knowledge of those skilled in the art.
Anion-exchange chromatography is carried out using media appropriate therefor, as are known in the art. Suitable media include, e.g., polymeric polystyrene/divinyi benzene resins and agarose-based resins, as well as agarose beads. dextran beads, polystyrene beads, media that comprise an insoluble, particulate support derivatized with tertiary or quaternary amino groups., and supports derivatized with trimethylaminoethyl groups. Examples of suitable such media include DE92TM (diethylaminoethvl cellulose, Whatman): DEAE-CELLULOSET'" (Sigma), BAKERBOND ABX 40 muTM (J.T. Baker, Inc.); DEAE resins such as FRACTOGEL EMD DEAE-650TM (EM Separations), FRACTOGEL EMD TMAE-650 (S) T~ (EM
Science, Gibbstown, NJ), TSK gel DEAE-SPWTM (Tosohaas), DEAE-SEPHAROSE CL-6BT' and chelating SEPHAROSETV' (Amersham Pharmacia Biotech AB), DEAE MERE SEP. I000TM
(Millipore), and DEAE
SPHERODEXTM (Sepracor); RESOURCE QTx' and Q SEPHAROSETM (QSFF) (Amersham Pharmacia Biotech AB); MACRO-PEP QTM (Bio-Rad Laboratories, Hercules, CA); Q- HYPERD7"
(BioSepra. Inc., Marlborough, Mass); and the like. Other suitable anion-exchange chromatography materials, as well as the selection and use of these materials for the present application, are conventional in the art.
Purified fractions of monomer obtained from the ion-exchange chromatography may be further processed by subjecting them to any appropriate technique designed for downstream processing and purification. This will depend largely on the type of poiypeptide and its intended use. Only one ion-exchange step is necessary to effect the desircd separation of monomer from dimers and/or multimers in a mixture, although the invention does not exclude using more such steps if desired in the upstream or downstream processing of the polypeptide.
The invention will be more fully understood by reference to the following examples. They should not.
however, be construed as limiting the scope of the invention.
EXAMPLE I
This example shows the separation of anti-IgE monomers and bovine serum albumin tnonomers from dimers and multimers.
MATERIAIS AND METHODB-Resim:
Phatmacia Q-SEPHAROSE FAST FLOWT"'. 4mL to 235L bed volumes evaluated Pharmacia RESOURCE S and RESOURCE QT~: I mL prepacked columns JT Baker CSxTm. 0.46 X Scm, 51A particles Prot-aim:
A. Humanized anti-IgE monoclonal antibodies (IgG1) available from Genentech.
Inc.: pI - 7.5.
desigaated as E25 and E26. WO 93/04173 pubiishcd March 4, 1993 describes humanized anti-IgE antibodies wherein a murine antibody directed against human IgE (MaE)1) was used to provide the CDR regions that were substituted into an IgGI ittununoglobulin frantework (rhuMaE25). See also Cacia et al.. Biochemistrv. 35:
1897-1903 (1996) for studies and further descriptions of E-25.
B. Monoclonal anti-IgE antibody prcpared from the culture supetttatants of an immortalized human myeioma cell line U266B I (ATCC TIB 196) using affinity chromatography purification on an isolated anti-IgE
antibody (Genentech MAEI). Specifically, five BALBA; female mice, age six weeks. vrere immunized in their foot pads with 10 pg of purified IgE in Ribi's adjuvant. Subsequent injections were done in the same manner at one and three weeks after the initial immunizations. Three days after the final injection, the inguinal and popliteal lymph nodes were removed and pooled. and a single cell suspension was made by passing the tissue through steel gauze. The cells wcre fused at a 4:1 ratio with mouse myeloma P3X63-Ag8.653 (ATCC CRL
1580) in high glucose (DMEM) containing 50% w!v polyethylene glycol 4000.
Alternatively. the immunizations were done in a similar manner except that 30 pg of IgE per injection were used and 1gE
fragment 315-347 (Kabat) was assayed as a prefusion boost; or injections were given subcutaneously in two doses of 100 pg and a final booster of 50 g, and spleen cells were used for the fusions.
The fused cells were then plated at a density of 2xI05 per well in 96-well tissue culture plates. After 24 hours HAT selective medium hypoxanthinelaminopteriNthymidirte, Sigma.
ifH0262) was added. Of 1440 wells plated. 365 contained growing cells after HAT selection.
Fifteen days after the fusion, supernatants were tesmd for the ptr.sence of antibodies specific for human , IgE using an enzyme-linked immunosorbent assay (EUSA). 17te ELISA was performed as follows, with all incubations done at room temperature. Test plates (Nuac Imtnttnoplaea) were coated for 2 hours with rat anti-mousa lgG (Boehringer Mannlteim, N605-500) at I pglml in 50 mM sodium carbonate buffer. pH 9.6, then blocked with 0.5% bovine serum albumin in phosphate buffeted saline (PBS) for 30 minutes. then washed four timea with PBS contnining 0.05% TWEEN 20T"A (PBST). Test supernatants were added and incubated two hours with shaking, then washed four times with PBST. Human 1gE (purified from U266 cells as described above) was added at 0.5 Ng/ml and incubated for one hour with shaking, then washed four times in PBST.
Horseradish-peroxidase-conjugated goat anti-human IgE (Kirkegarrd & Perrv I_abs, #14-10-04, 0.5 mg/ml) was added at a 12500 dilution and incubated for one hour, then washed four times with PBST. The plates were developed by adding 100 Uwell of a solution containing 10 mg of o-phenylenediamine dihydrochioride (Sigma, #P8287) and 10 i of a 30% hydrogen peroxide solution in 25 ml phosphate citrate buffer, pH 5.0, and incubating for 15 minutes. The reaction was stopped by adding 100 l/weil of 2.5 M sulfuric acid. Data were obtained by reading the plates in an automated ELISA plate reader at an absorbance of 490 nm. For one antibody, 365 supernatants were tested and 100 were specific for human IgE.
Similar frequencies of IgE
specificity were obtained when screening for the other antibodies.
C. Bovine serum Albumin: pI 4.7 and 4.9 (Radola, Biochim. Biophys. Acta, 295:
412-428 (1973)) =Bayer Corp. P/N 81-024-2, "Bovine Albumin, Sulfhydryl Modified" (BSA Mix, blocked) =ICN Biomedical Inc. P/N 810013, "Albumin Bovine" (BSA Mix, native) =BSA monomer and dimer prepared in house from Bayer BSA (BSA Monomer and BSA
Dimer. respectively) Chromatography Svstems:
Hewlett-Packard I 090TM HPLC
PharmaciaT'l FPLC
Detection at 215 or 280 nm Buffers: (see Table I for details) Purified water Tris=HCl Sodium acetate Sodium chloride Sodium phosphate Sodium citrate and citric acid Sample Preparation:
Samples were diluted with the buffer used for equilibration (indicated in Table I below) to assure pH
and conductivity matched starting column conditions. All samples were 0.2-pm filtered prior to loading.
Chromatoeraphv:
Samples were introduced to the column using either an automatic or manual injector. All runs were performed at room temperature. Fractions were collected manually or with a PHARMACIA FRAC 100r"~
collector.
Chromatographic separation performance was evaluated by comparing elution profiles of BSA stock reagent and purified BSA monomer and dimer; the same was done for IgE and the monoclonal antibodies (MAb). Separation of IgE and MAb from their dimers and multimers was further evaluated by analyzing elution fraction using analytical size-exclusion chromatography. Plots of MAb MW forms vs. Fraction number were created. Recovery of IgE and MAb was determined spectrophotometrically by measuring absorbance at 280nm.
RESULTS:
The results are summarized in Table I below.
Table I
MONOMER- DIM ER/M ULTIMER SEPARATIONS
RESIN PROTEIN PH EQUILIBRATION ELUTION COMMENTS
BUFFER
Anion-Exchange QSFF "' MAb 8 Tris-HCI linear gradient: 0 to Good separation 500 mM NaCI
linear gradient: 50 to Best separation 200 mM NaCI
step gradients to 200. Separation works 175, 150. 125 mM
NaCl Resource Q' MAb 8 Tris-HCI
Resource QTM U266 IgE 8 Tris-HCI Removed aggregates and multimers Resource Q BSA 8 Tris-HCI linear gradient: 0 to Good separation Monomer IM NaCI.
BSA Dimer linear gradicnts: 150 Excellent separation to 550, 250 to 550mM NaCI
BSA Mix, native BSA Mix, step gradients: Some separation but blocked 0.3/0.6, 0.38/0.6. fine control required 0.4M/0.6M NaCI
Resource Q' BSA 6 sodium citrate linear gradient: Does not bind in citrate Monomer pH 6 BSA Dimer BSA Mix, blocked Resource ~
Q' BSA 6 sodium phosphate linear gradient: 0 to Good separations Monomer 0.5M NaCI in 10 CVs BSA Dimer BSA Mix, blocked Cation Exchange Resource SN' BSA 6 sodium citrate linear gradient: 0 to Does not bind in citrate Monomer 0.5M NaCI in 10 pH 6 CVs BSA Dimer MAb Resource STM MAb 6 sodium phosphate linear gradient: 0 to Equivalent to Q
0.05M NaCl in 20 separation CVs Loaded to 16.5 mg/mL
Resource Stm BSA 4.6 NaOAc buffer linear gradient: 0 to Proteins somewhat Monomer IM NaCI/40 CVs resolved BSA Dimer BSA Mix, blocked BSA 4.3 NaOAc buffer linear gradient: 0 to Better resolution than RESIN PROTEIN PH EQUILIBRATION ELUTION COMMENTS
BUFFER
Monomer IM NaCI/20 CVs pH 4.6 BSA Dimer BSA Mix, blocked JT Baker BSA 4.6 NaOAc buffer linear gradient: 0 to Proteins somewhat CSxTM Monomer IM NaCI/12 CVs resoived BSA Dimer JT Baker BSA 4.3 NaOAc buffer linear gradient: 0 to Proteins somewhat CSxTM Monomer lM NaCI/12 CVs resolved BSA Dimer Separations were evaluated using polymeric polystyrene/divinyl benzene resins (RESOURCE Q and ST'"), a silica-based resin (JT BAKER CSXTM), and an agarose-based resin (Q-SEPHAROSE FAST FLOWTM, QSFF). While separations were accomplished using any of these resins, separations worked especiallv well on Q-SEPHAROSE FAST FLOWT't. RESOURCE Qt'", and RESOURCE STM . The separation of BSA monomer and dimer from both suppliers looked very similar, suggesting the "Sulfhydryl Modified" material from Bayer did not alter the protein such that the species were easier to separate. It can be seen that phosphate buffer at pH
6 worked well, but no protein bound to the cation- or anion-exchange columns when 20 mM citrate buffer at pH
6 was used as equilibration buffer. Citrate buffer would be expected to work for both anion- and cation-exchange at a lower concentration, e.g., about 5 mM.
Recovery of monomeric IgE and MAbs to IgE on anion-exchange resins was typically greater than 90% at greater than 99.5% purity. Figures IA and 1B show anion-exchange (RESOURCETM, Q) chromatograms in the separation of IgE monomers from dimers and multimers.
Figures 2A1 and 2A2 show anion-exchange (RESOURCET"' Q) chromatograms in the separation of anti-IgE MAb monomers from dimers and multimers. Fig. 2B shows an anion-exchange (Q-SEPHAROSE FAST-FLOWTM) chromatogram in the separation of anti-IgE MAb monomers from dimers and multimers. SEC (SUPERDEX
200 HR 10/3OT") was used as an analytical method to determine the amount of monomer and multimer in samples from the ion-exchange separation, and Figure 2C shows the SEC analysis of fractions from Figure 2B. Separation of BSA
monomer from dimer was readily achieved on anion-exchange resins at pH 8 and pH 6. See Figures 3A-C and 4A-C for chromatograms in the separation of BSA monomers from dimers and multimers by anion-exchange (RESOURCETIM Q) at pH 8 (Tris buffer) and at pH 6 (phosphate buffer), respectively.
Recovery and purity of MAb monomer from the cation-exchange resin was comparable to that of the anion-exchange resin. Figures 5A-B show cation-exchange (RESOURCET'm S) chromatograms in the separation of anti-IgE MAb monomers from dimers and multimers at pH 6 (phosphate buffer). Separations of BSA on cation-exchange resins could be performed at pH 4.6 and 4.3, 4.3 being somewhat better. Figures 6A-B show cation-exchange (RESOURCETM S) chromatograms in the separation of BSA
monomers from dimers and multimers at pH 4.3 (acetate buffer).
In summary, mixtures of polypeptide mers were subjected to cation- or anion-exchange chromatography using a variety of resins and under a variety of pH and elution salt conditions, and successful separation was achieved. Based on results from four proteins with basic and acidic isoelectric points (two IgGi MAbs, IgE and serum albumin). the method demonstrates general applicability to separation of polypeptidc monomers from their dimers and multimers.
The results are summarized in Table I below.
Table I
MONOMER- DIM ER/M ULTIMER SEPARATIONS
RESIN PROTEIN PH EQUILIBRATION ELUTION COMMENTS
BUFFER
Anion-Exchange QSFF "' MAb 8 Tris-HCI linear gradient: 0 to Good separation 500 mM NaCI
linear gradient: 50 to Best separation 200 mM NaCI
step gradients to 200. Separation works 175, 150. 125 mM
NaCl Resource Q' MAb 8 Tris-HCI
Resource QTM U266 IgE 8 Tris-HCI Removed aggregates and multimers Resource Q BSA 8 Tris-HCI linear gradient: 0 to Good separation Monomer IM NaCI.
BSA Dimer linear gradicnts: 150 Excellent separation to 550, 250 to 550mM NaCI
BSA Mix, native BSA Mix, step gradients: Some separation but blocked 0.3/0.6, 0.38/0.6. fine control required 0.4M/0.6M NaCI
Resource Q' BSA 6 sodium citrate linear gradient: Does not bind in citrate Monomer pH 6 BSA Dimer BSA Mix, blocked Resource ~
Q' BSA 6 sodium phosphate linear gradient: 0 to Good separations Monomer 0.5M NaCI in 10 CVs BSA Dimer BSA Mix, blocked Cation Exchange Resource SN' BSA 6 sodium citrate linear gradient: 0 to Does not bind in citrate Monomer 0.5M NaCI in 10 pH 6 CVs BSA Dimer MAb Resource STM MAb 6 sodium phosphate linear gradient: 0 to Equivalent to Q
0.05M NaCl in 20 separation CVs Loaded to 16.5 mg/mL
Resource Stm BSA 4.6 NaOAc buffer linear gradient: 0 to Proteins somewhat Monomer IM NaCI/40 CVs resolved BSA Dimer BSA Mix, blocked BSA 4.3 NaOAc buffer linear gradient: 0 to Better resolution than RESIN PROTEIN PH EQUILIBRATION ELUTION COMMENTS
BUFFER
Monomer IM NaCI/20 CVs pH 4.6 BSA Dimer BSA Mix, blocked JT Baker BSA 4.6 NaOAc buffer linear gradient: 0 to Proteins somewhat CSxTM Monomer IM NaCI/12 CVs resoived BSA Dimer JT Baker BSA 4.3 NaOAc buffer linear gradient: 0 to Proteins somewhat CSxTM Monomer lM NaCI/12 CVs resolved BSA Dimer Separations were evaluated using polymeric polystyrene/divinyl benzene resins (RESOURCE Q and ST'"), a silica-based resin (JT BAKER CSXTM), and an agarose-based resin (Q-SEPHAROSE FAST FLOWTM, QSFF). While separations were accomplished using any of these resins, separations worked especiallv well on Q-SEPHAROSE FAST FLOWT't. RESOURCE Qt'", and RESOURCE STM . The separation of BSA monomer and dimer from both suppliers looked very similar, suggesting the "Sulfhydryl Modified" material from Bayer did not alter the protein such that the species were easier to separate. It can be seen that phosphate buffer at pH
6 worked well, but no protein bound to the cation- or anion-exchange columns when 20 mM citrate buffer at pH
6 was used as equilibration buffer. Citrate buffer would be expected to work for both anion- and cation-exchange at a lower concentration, e.g., about 5 mM.
Recovery of monomeric IgE and MAbs to IgE on anion-exchange resins was typically greater than 90% at greater than 99.5% purity. Figures IA and 1B show anion-exchange (RESOURCETM, Q) chromatograms in the separation of IgE monomers from dimers and multimers.
Figures 2A1 and 2A2 show anion-exchange (RESOURCET"' Q) chromatograms in the separation of anti-IgE MAb monomers from dimers and multimers. Fig. 2B shows an anion-exchange (Q-SEPHAROSE FAST-FLOWTM) chromatogram in the separation of anti-IgE MAb monomers from dimers and multimers. SEC (SUPERDEX
200 HR 10/3OT") was used as an analytical method to determine the amount of monomer and multimer in samples from the ion-exchange separation, and Figure 2C shows the SEC analysis of fractions from Figure 2B. Separation of BSA
monomer from dimer was readily achieved on anion-exchange resins at pH 8 and pH 6. See Figures 3A-C and 4A-C for chromatograms in the separation of BSA monomers from dimers and multimers by anion-exchange (RESOURCETIM Q) at pH 8 (Tris buffer) and at pH 6 (phosphate buffer), respectively.
Recovery and purity of MAb monomer from the cation-exchange resin was comparable to that of the anion-exchange resin. Figures 5A-B show cation-exchange (RESOURCET'm S) chromatograms in the separation of anti-IgE MAb monomers from dimers and multimers at pH 6 (phosphate buffer). Separations of BSA on cation-exchange resins could be performed at pH 4.6 and 4.3, 4.3 being somewhat better. Figures 6A-B show cation-exchange (RESOURCETM S) chromatograms in the separation of BSA
monomers from dimers and multimers at pH 4.3 (acetate buffer).
In summary, mixtures of polypeptide mers were subjected to cation- or anion-exchange chromatography using a variety of resins and under a variety of pH and elution salt conditions, and successful separation was achieved. Based on results from four proteins with basic and acidic isoelectric points (two IgGi MAbs, IgE and serum albumin). the method demonstrates general applicability to separation of polypeptidc monomers from their dimers and multimers.
Claims (35)
1. A method for separating an antibody monomer from a mixture comprising monomers of said antibody and dimers of said antibody or n-mers of said antibody where n is 3-10 or both_dimers and n-mers of said antibody, wherein the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers or multimers or both present in the mixture in said ion-exchange chroma-tography step; wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM.
2. The method of claim 1 wherein the antibody is anti-IgE, anti-IgG, anti-Her-2, anti-CD11a, anti-CD18, anti-CD20, anti-VEGF, or an antibody fragment.
3. The method of claim 1 or 2, wherein the ion-exchange resin is a cation-exchange resin.
4. The method of claim 1 or 2, wherein the ion-exchange resin is an anion-exchange resin.
5. The method of any one of claims 1-4, wherein the gradient is linear.
6. The method of any one of claims 1-4, wherein the gradient is stepwise.
7. The method of any one of claims 1-6, wherein the elution salt is a sodium salt.
8. The method of any one of claims 1-6, wherein the elution salt is sodium chlo-ride.
9. The method of any one of claims 1-8, wherein the gradient is from 0 to 500 mM elution salt.
10. The method of any one of claims 1-8, wherein the gradient is from 50 to mM elution salt.
11. The method of any one of claims 1-8, wherein the gradient is from 0 to 50 mM elution salt.
12. The method of any one of claims 1-11, wherein if citrate is used as buffer, it is employed at a concentration of about 5 mM.
13. The method of any one of claims 1-12, wherein the separated monomer is obtained with a yield of greater than 90%.
14. A method for separating an antibody monomer from a mixture comprising monomers of said antibody and dimers of said antibody or n-mers of said antibody where n is 3-10 or both_dimers and n-mers of said antibody, wherein the method comprises the step of applying the mixture to an anion-exchange chromatography resin in a buffer, wherein the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers or multimers or both present in the mixture in said anion-exchange chromatography step; wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM.
15. The method of claim 14 wherein the antibody is anti-IgE, anti-IgG, anti-Her-2, anti-CD11a, anti-CD18, anti-CD20, anti-VEGF, or an antibody fragment.
16. The method of claim 14 or 15, wherein the gradient is linear.
17. The method of claim 14 or 15, wherein the gradient is stepwise.
18. The method of any one of claims 14-17, wherein the elution salt is a sodium salt.
19. The method of any one of claims 14-17, wherein the elution salt is sodium chloride.
20. The method of any one of claims 14-19, wherein the gradient is from 0 to 500 mM elution salt.
21. The method of any one of claims 14-19, wherein the gradient is from 50 to 200 mM elution salt.
22. The method of any one of claims 14-19, wherein the gradient is from 0 to mM elution salt.
23. The method of any one of claims 14-19, wherein if citrate is used as buffer, it is employed at a concentration of about 5 mM.
24. The method of any one of claims 14-23, wherein the separated monomer is obtained with a yield of greater than 90%.
25. A method for separating an antibody monomer from a mixture comprising monomers of said antibody and dimers of said antibody or n-mers of said antibody where n is 3-10 or both_dimers and n-mers of said antibody, wherein the method comprises the step of applying the mixture to a cation-exchange or anion-exchange chromatography resin in a buffer, wherein if the resin is cation-exchange, the pH of the buffer is about 4-7, and wherein if the resin is anion-exchange, the pH of the buffer is about 6-9, and eluting the mixture with a gradient of about 0-1 M of an elution salt, wherein the monomer is separated from the dimers or multimers or both present in the mixture; wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM; and wherein the separated monomer has a purity of greater than 99.5%.
26. The method of claim 25, wherein the antibody is anti-IgE, anti-IgG, anti-Her-2, anti-CD11a, anti-CD18, anti-CD20, anti-VEGF, or an antibody fragment.
27. The method of claim 25 or 26, wherein the ion-exchange resin is a cation-exchange resin.
28. The method of claim 25 or 26, wherein the ion-exchange resin is an anion-exchange resin.
29. The method of any one of claims 25-28, wherein the gradient is linear.
30. The method of any one of claims 25-28, wherein the gradient is stepwise.
31. The method of any one of claims 25-30, wherein the elution salt is a sodium salt.
32. The method of any one of claims 25-30, wherein the elution salt is sodium chloride.
33. The method of any one of claims 25-32, wherein if citrate is used as buffer, it is employed at a concentration of about 5 mM.
34. Use of an ion-exchange resin for separating an antibody monomer from a mixture comprising monomers of said antibody and dimers of said antibody or n-mers of said antibody where n is 3-10 or both dimers and n-mers of said antibody_by ion-exchange chromatography in a buffer, comprising applying said mixture to said resin, and eluting the mixture with a gradient of about 0-1M of an elution salt;wherein if citrate is used as buffer, it is employed at a concentration of below 20 mM.
35. The use according to claim 34 for separating the antibody monomer with a yield of more than 90%.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| US8760298P | 1998-06-01 | 1998-06-01 | |
| US60/087,602 | 1998-06-01 | ||
| PCT/US1999/011598 WO1999062936A1 (en) | 1998-06-01 | 1999-05-24 | Separation of protein monomers from aggregates by use of ion-exchange chromatography |
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| CA2333747A1 CA2333747A1 (en) | 1999-12-09 |
| CA2333747C true CA2333747C (en) | 2008-02-19 |
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| CA002333747A Expired - Fee Related CA2333747C (en) | 1998-06-01 | 1999-05-24 | Separation of protein monomers by use of ion-exchange chromatography |
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| US (2) | US6620918B2 (en) |
| EP (2) | EP1500661A1 (en) |
| JP (3) | JP2002517406A (en) |
| AT (1) | ATE274522T1 (en) |
| AU (1) | AU750977B2 (en) |
| CA (1) | CA2333747C (en) |
| DE (1) | DE69919701T2 (en) |
| DK (1) | DK1084136T3 (en) |
| ES (1) | ES2228052T3 (en) |
| HK (1) | HK1037879A1 (en) |
| IL (2) | IL139609A (en) |
| PT (1) | PT1084136E (en) |
| WO (1) | WO1999062936A1 (en) |
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- 1999-05-24 EP EP04017970A patent/EP1500661A1/en not_active Withdrawn
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- 1999-05-24 ES ES99925856T patent/ES2228052T3/en not_active Expired - Lifetime
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- 1999-05-24 DK DK99925856T patent/DK1084136T3/en active
- 1999-05-24 WO PCT/US1999/011598 patent/WO1999062936A1/en not_active Application Discontinuation
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|---|---|
| US20020010319A1 (en) | 2002-01-24 |
| EP1500661A1 (en) | 2005-01-26 |
| EP1084136A1 (en) | 2001-03-21 |
| JP2002517406A (en) | 2002-06-18 |
| HK1037879A1 (en) | 2002-02-22 |
| AU750977B2 (en) | 2002-08-01 |
| DE69919701D1 (en) | 2004-09-30 |
| US20040116674A1 (en) | 2004-06-17 |
| ATE274522T1 (en) | 2004-09-15 |
| DK1084136T3 (en) | 2005-01-03 |
| IL139609A (en) | 2006-06-11 |
| CA2333747A1 (en) | 1999-12-09 |
| WO1999062936A1 (en) | 1999-12-09 |
| US6620918B2 (en) | 2003-09-16 |
| AU4205999A (en) | 1999-12-20 |
| IL173432A0 (en) | 2006-06-11 |
| DE69919701T2 (en) | 2005-09-15 |
| IL139609A0 (en) | 2002-02-10 |
| EP1084136B1 (en) | 2004-08-25 |
| JP2006071646A (en) | 2006-03-16 |
| JP2004175806A (en) | 2004-06-24 |
| ES2228052T3 (en) | 2005-04-01 |
| PT1084136E (en) | 2004-12-31 |
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