Purification of proteins using hydrophobic interaction chromatography

Abstract

The present invention is directed to methods for purifying a protein of interest, e.g., an antibody, from a sample comprising the protein of interest and at least one impurity, e.g., an aggregate, by employing a hydrophobic interaction chromatography (HIC) method that allows for binding of both the protein of interest and the at least one impurity under strong binding conditions. The present invention is based, at least in part, on the finding that both flow through and bind-elute techniques can be combined to achieve greater purification and recovery of a protein of interest, e.g., an antibody, under isocratic wash conditions and strong binding conditions.

What is claimed is: 
     
         1 . A method for producing a preparation comprising a protein of interest and having a reduced level of at least one impurity, said method comprising:
 (a) contacting a sample comprising the protein of interest and at least one impurity, to a hydrophobic interaction chromatography (HIC) media, in the presence of a load buffer such that (i) a portion of the protein of interest binds to the HIC media and (ii) a substantial portion of the at least one impurity binds to the HIC media;   (b) collecting a flow through fraction comprising the protein of interest unbound to the HIC media;   (c) washing the HIC media with a wash buffer that is substantially the same as the load buffer such that a substantial portion of the protein of interest bound to the HIC media is released from the media; and   (d) collecting a wash fraction comprising the protein of interest released from the HIC media,   wherein each of the flow through and wash fractions comprise the protein of interest and have a reduced level of the at least one impurity.   
     
     
         2 . The method of  claim 1 , wherein the portion of the protein of interest binds to the HIC media at
 (a) a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or 300; or   (b) at a Kp of greater than 20.   
     
     
         3 . The method of  claim 1 , wherein the protein of interest is adalimumab. 
     
     
         4 . The method of  claim 1 , wherein a substantial portion of the impurity bound to the HIC media remains bound upon washing with the wash buffer. 
     
     
         5 . The method of  claim 1 , wherein the flow through and/or wash fractions are substantially free of the at least one impurity. 
     
     
         6 . The method of  claim 1 , wherein the at least one impurity is an aggregate of the protein of interest, optionally, a multimer, a dimer, a trimer, a tetramer, an oligomer or other high molecular weight species. 
     
     
         7 . The method of  claim 1 , wherein the protein of interest is adalimumab and the at least one impurity is an aggregate of adalimumab, optionally, selected from the group consisting of multimer 1, multimer 2 and multimer 3. 
     
     
         8 . The method of  claim 1 , wherein the impurity is a process-related impurity or a product-related substance. 
     
     
         9 . The method of  claim 8 , wherein the process-related impurity is selected from the group consisting of a host cell protein, a host cell nucleic acid, a media component, and a chromatographic material. 
     
     
         10 . The method of  claim 8 , wherein the product-related substance is selected from the group consisting of a charge variant, an acidic variant, a basic variant, a lysine variant species, an aggregate of the protein of interest, a fragment of the protein of interest, an Fc fragment of the protein of interest, a Fab fragment of the protein of interest, a modified protein, a deamidated protein, and a glycosylated protein. 
     
     
         11 . The method of  claim 1 , wherein the impurity is an acidic species (AR), optionally, selected from the group consisting of AR1, AR2, a charge variant, a structure variant, a fragmentation variant, a process-related impurity and a product-related impurity. 
     
     
         12 . The method of  claim 1 , wherein the protein of interest is an antibody or antigen-binding fragment thereof, a soluble protein, a membrane protein, a structural protein, a ribosomal protein, an enzyme, a zymogen, an antibody molecule, a humanized antibody or antigen-binding portion thereof, a human antibody or antigen-binding portion thereof, a chimeric antibody or antigen-binding portion thereof, a multivalent antibody, a cell surface receptor protein, a transcription regulatory protein, a translation regulatory protein, a chromatin protein, a hormone, a cell cycle regulatory protein, a G protein, a neuroactive peptide, an immunoregulatory protein, a blood component protein, an ion gate protein, a heat shock protein, an antibiotic resistance protein, a functional fragment of any of the preceding proteins, an epitope-containing fragment of any of the preceding proteins, and combinations thereof. 
     
     
         13 . The method of  claim 1 , further comprising repeating steps (a)-(d) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 times using the flow through fraction, wash fraction, or combination thereof having a reduced level of the at least one impurity 
     
     
         14 . The method of  claim 1 , wherein the flow through fraction and the wash fraction are combined. 
     
     
         15 . The method of  claim 1 , wherein
 (a) the portion of the protein of interest that binds to the HIC media is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the protein of interest in the sample;   (b) the substantial portion of the protein of interest released from the HIC media upon washing with the wash buffer is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the amount of protein of interest bound to the HIC media;   (c) the accumulative yield of the protein of interest in the flow through fraction and/or wash fraction is at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%;   (d) the accumulative yield of the protein of interest in any one flow through fraction and/or wash fraction is at least about 4%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85, at least about 90%, at least about 95% or about 100%;   (e) the substantial portion of the at least one impurity that binds to the HIC media is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the at least one impurity in the sample;   (f) the reduced level of the at least one impurity of the flow through fraction and/or wash fraction is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the at least one impurity in the sample;   (g) the accumulative aggregate reduction of the at least one impurity in any one flow through fraction and/or wash fraction is at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, at least about 10.0%, or at least about 20.0%;   (h) the accumulative aggregate reduction of the at least one impurity in the flow through fraction and/or wash fraction is at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1.0%, at least about 2.0%, at least about 3.0%, at least about 4.0%, at least about 5.0%, at least about 10.0%, or at least about 20.0%; and/or   (i) the level of the at least one impurity is reduced by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the at least one impurity in the sample.   
     
     
         16 . The method of  claim 1 , wherein
 (a) the at least one impurity binds to the HIC media at a Kp of greater than 250, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, or greater than 1000;   (b) the protein of interest and the at least one impurity have a Kp ratio less than 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2;   (c) the K d  for the binding of the protein of interest to the HIC media is at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, or at least about 0.6;   (d) the K d  for the binding of the at least one impurity to the HIC media is less than or equal to about 0.001, about 0.005, about 0.01, about 0.02, about 0.05, about 0.1, about 0.15 or about 0.2;   (e) the K d  for the binding of the protein of interest to the HIC media is less than 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 times the K d  for the binding of the at least one impurity to the HIC media;   (f) the protein of interest has a Qmax of at least about 20, at least about 30, at least about 40, at least about 50, at least about 60 or at least about 100; and/or   (g) the at least one impurity has a Qmax of at least about 2, at least about 5, at least about 10, at least about 20, at least about 30 or at least about 40.   
     
     
         17 . The method of  claim 1 , wherein the HIC media either (i) comprises at least one hydrophobic ligand, optionally selected from the group consisting of alkyl-, aryl-ligands, and butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands; and/or (ii) is selected from the group consisting of CaptoPhenyl, Phenyl Sepharose™ 6 Fast Flow with low or high substitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl, Macro-Prep™ Methyl, Macro-Prep™ t-Butyl, WP HI-Propyl (C3)™, Toyopearl™ ether, Toyopearl™ phenyl, Toyopearl™ butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, and Tosoh Hexyl. 
     
     
         18 . The method of  claim 1 , wherein
 (a) the load buffer and/or wash buffer comprise a salt selected from the group consisting of a sulfate salt, a citrate salt, ammonium sulfate, sodium sulfate, sodium chloride, ammonium chloride, sodium bromide and a combination thereof;   (b) the load buffer and/or wash buffer comprise a cation selected from the group consisting of Ba 2+ , Ca 2+ , Mg 2+ , Li + , Cs + , Na + , K + , Rb + , NH 4   +  and a combination thereof;   (c) the load buffer and/or wash buffer comprise an anion selected from the group consisting of PO 4   3− , SO 4   2− , CH 3 CO 3   − , Cl − , Br − , NO 3   − , ClO 4   − , I − , SCN −   and a combination thereof;   (d) the load buffer and/or wash buffer comprise a salt having a concentration of between about 50 mM and 2000 mM;   (e) the load buffer and/or wash buffer have a pH between about 4.0 and 8.5 or between about 5.0 and 7.0;   (f) the load buffer and/or wash buffer have a pH of about 4.0, about 4.5, about 5.0, about 5.5, about 6, about 6.5, about 7.0, about 7.5, about 8.0, or about 8.5;   (g) the load buffer and/or wash buffer are the same;   (h) the load buffer and/or wash buffer are substantially the same; and/or   (i) the salt concentration and/or the pH of the wash buffer are within about 20%, 15%, 10% or 5% of the salt concentration and/or pH of the loading buffer.   
     
     
         19 . The method of  claim 1 , wherein
 (a) about 100 g to about 800 g of the sample are contacted per one liter of HIC media;   (b) about 0.2 g to about 120 g of the at least one impurity is contacted per one liter of HIC media;   (c) the sample has a protein concentration of about 2 mg/ml to about 50 mg/ml;   (d) the sample has a protein of interest concentration of about 2 mg/ml to about 50 mg/ml; and/or   (e) the concentration of the at least one impurity in the sample is about 0.01 to about 5.0 mg/ml.   
     
     
         20 . The method of  claim 1 ,
 (a) wherein a precursor sample comprising the protein of interest has been subjected to affinity chromatography to generate the sample; and/or   (b) further comprising subjecting the preparation comprising a protein of interest and having a reduced level of one impurity to affinity chromatography,   optionally wherein affinity chromatography is performed using affinity chromatographic media selected from the group consisting of Protein A, G, A/G, L media, and MabSuRe Protein A media.   
     
     
         21 . The method of  claim 1 ,
 (a) wherein a precursor sample comprising the protein of interest has been subjected to ion exchange chromatography to generate the sample; and/or   (b) further comprising subjecting the preparation comprising a protein of interest and having a reduced level of one impurity to ion exchange chromatography,   optionally wherein ion exchange chromatography is performed using ion exchange chromatography media selected from the group consisting of a cation exchange media and an anion exchange media.   
     
     
         22 . The method of  claim 1 ,
 (a) wherein a precursor sample comprising the protein of interest has been subjected to mixed mode chromatography to generate the sample; and/or   (b) further comprising subjecting the preparation comprising a protein of interest and having a reduced level of one impurity to mixed mode chromatography;   optionally wherein the mixed mode chromatography is performed using CaptoAdhere resin.   
     
     
         23 . The method of  claim 1 ,
 (a) wherein a precursor sample comprising the protein of interest has been subjected to a filtration step to generate the sample; and/or   (b) further comprising subjecting the preparation comprising a protein of interest and having a reduced level of one impurity to a filtration step;   optionally, wherein the filtration step is selected from the group consisting of a depth filtration step, a nanofiltration step, an ultrafiltration step, and an absolute filtration step, or a combination thereof.   
     
     
         24 . The method of  claim 1 , wherein the HIC media has a dynamic binding capacity of at least about 2 g, at least about 5 g, at least about 10 g, at least about 20 g, at least about 30 g, at least about 40 g, at least about 50 g, at least about 60 g, at least about 70 g, at least about 90 g, or at least about 100 g of sample per one liter of media. 
     
     
         25 . A method for producing a preparation comprising adalimumab and having a reduced level of at least one aggregate, said method comprising:
 (a) contacting a sample comprising adalimumab and at least one aggregate, to a HIC media, in the presence of a load buffer such that (i) a portion of the adalimumab in the sample binds to the HIC media and (ii) a substantial portion of the at least one aggregate binds to the HIC media;   (b) collecting a flow through fraction comprising the adalimumab unbound to the HIC media;   (c) washing the HIC media with a wash buffer that is substantially the same as the load buffer such that a substantial portion of the adalimumab bound to the HIC media is released from the media; and   (d) collecting a wash fraction comprising the adalimumab released from the HIC media,   wherein each of the flow through and wash fractions comprise adalimumab and have a reduced level of the at least one aggregate.   
     
     
         26 . The method of  claim 25 , wherein adalimumab binds to the HIC media
 (a) at a Kp of greater than 10, 15, 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250 or 300; or   (b) at a Kp of greater than 20.   
     
     
         27 . The method of  claim 25 , wherein the aggregate is multimer 1, multimer 2 or multimer 3. 
     
     
         28 . A pharmaceutical composition comprising a low-aggregate composition and a pharmaceutical acceptable carrier. 
     
     
         29 . A pharmaceutical composition comprising a low-aggregate composition of adalimumab and a pharmaceutically acceptable carrier. 
     
     
         30 . The pharmaceutical composition of  claim 29 , wherein the composition comprises less than 5%, 4%, 3%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.5%, 0.1% of aggregates.