US 201400.46038A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0046038A1 SHHARA (43) Pub. Date: Feb. 13, 2014
(54) METHOD OF PURIFYING PROTEIN Publication Classification (71) Applicant: KYOWA HAKKO KIRIN CO, LTD., (51) Int. Cl. (US) C07K L/18 (2006.01) (52) U.S. Cl. (72) Inventor: Takashi ISHIHARA, Tokyo (JP) CPC ...... C07K 1/18 (2013.01) (73) Assignee: KYOWA HAKKO KIRIN CO.,LTD, USPC ...... 530/388.1:530/416:530/350 Tokyo (JP) (57) ABSTRACT (21) Appl. No.: 13/826,195 The present invention relates to a method for purifying a (22) Filed: Mar 14, 2013 protein by separating the protein from impurities in a non Related U.S. Application Data adsorption mode using an activated carbon. In particular, the AV present invention relates to a method for purifying an anti (60) Provisional application No. 61/680,433, filed on Aug. body using the activated carbon instead of protein A affinity 7, 2012. chromatography. Patent Application Publication Feb. 13, 2014 Sheet 1 of 22 US 2014/0046038A1
FIG. 1
0 Mab A 8 Mab B Mab C & 6 w is2 4 &r O ?. 2
O Cuiture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 2 of 22 US 2014/0046038A1
FG. 2
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s & Mab A s 20 & S 5 »&& . &E. 9. 10 « st s x« & O5 &x Culture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 3 of 22 US 2014/0046038A1
FIG. 3
1, 000, 000
100, 000 g , 10,000
s a 1,000 S. . OO g O
Culture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 4 of 22 US 2014/0046038A1
ES Culture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 5 of 22 US 2014/0046038A1
FIG. 5
20 r
Mab A is 16 - x: O Mab B. s & (2) Mab C.
i.S 8 &» s «
Cuiture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 6 of 22 US 2014/0046038A1
FIG. 6
1,000,000 gr.
Mab A 100,000 “ S“ O Mab B. s Mab C. 8. 2 10,000 .C. 5 1,000 . g 00
O O & x Culture supernatant Purified product Patent Application Publication Feb. 13, 2014 Sheet 7 of 22 US 2014/0046038A1
FIG. 7
Protein A Aid Cation total C CCy Activated rate ry ca?ion Process Patent Application Publication Feb. 13, 2014 Sheet 8 of 22 US 2014/0046038A1
FIG. 8
Protein A Anior Cation iota e8: Activated fate y Cabot Process Patent Application Publication Feb. 13, 2014 Sheet 9 of 22 US 2014/0046038A1
FIG. 9
O) Cittire Protein A Aid Catio supernatant ActivatedC caibo Process Patent Application Publication Feb. 13, 2014 Sheet 10 of 22 US 2014/0046038A1
, r Culture Protein A Anior Cation supernatant of Activated Cator Process Patent Application Publication Feb. 13, 2014 Sheet 11 of 22 US 2014/0046038A1
FIG 11
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FIG. 12
Culture Protein A Arion Cation supernatant of Activated carbor Process Patent Application Publication Feb. 13, 2014 Sheet 13 of 22 US 2014/0046038A1
1,000,000 g
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10,000
1,000
1.O.
Culture Protein A Arion Cation supernatant or Activated Caro Process Patent Application Publication Feb. 13, 2014 Sheet 14 of 22 US 2014/0046038A1
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1,000,000
100,000 c 8 es 10,000
aa.a 1,000 . s 1CO wa O O 1.
1. Citute Protein A Arior Cation supernatant or Activated carbor Process Patent Application Publication Feb. 13, 2014 Sheet 15 of 22 US 2014/0046038A1
FIG. 15
Monomer Degradation product Patent Application Publication Feb. 13, 2014 Sheet 16 of 22 US 2014/0046038A1
FG 16
Monomer -->
i H chain
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F.G. 17
Mab A ya. A via yies 3 Protein A Activated Protein A Activated Cathon carbon Patent Application Publication Feb. 13, 2014 Sheet 18 of 22 US 2014/0046038A1
F.G. 18
2 Patent Application Publication Feb. 13, 2014 Sheet 19 of 22 US 2014/0046038A1
O
8
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2 Patent Application Publication Feb. 13, 2014 Sheet 20 of 22 US 2014/0046038A1
FIG. 20
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FIG 2
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FG. 22
800,000
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METHOD OF PURIFYING PROTEIN ingredient, which is a challenging problem. In other words, a reduction in the protein purification cost is demanded in this TECHNICAL FIELD field. 0009. On the other hand, it is known that enzymes secreted 0001. The present invention relates to a method for puri from host cells are included in a culture broth containing the fying a protein and a method for preparing a protein compris protein of interest, and the protein of interest is degraded, ing the purification method. In particular, the present inven modified, oxidized, or reduced by these enzymes during the tion relates to a method for purifying an antibody and a protein purification process. For this reason, addition of method for preparing an antibody comprising the purification enzyme inhibitors during the protein purification has been method. considered to prevent degradation, modification, oxidation, or reduction of the protein of interest (Patent Document 2). BACKGROUND ART However, when the enzyme inhibitors are used during the protein purification, an additional process of removing the 0002 Development of genetic recombination technolo inhibitors is required, and moreover, certain inhibitors may gies has provided drugs including a variety of proteins as an affect the quality of the purified protein. Therefore, it cannot active ingredient. In particular, numerous drugs including be said that the addition of inhibitors is the best way. Removal antibodies as an active ingredient have been recently devel of host cell-derived enzymes is considered as one of the oped and commercialized. In addition, efficient production of drastic methods to solve the problems, but it is essential to use these proteins in large-scale has become a more important chromatography. There is no simple method of removing issue in biopharmaceutical industry. these enzymes. 0003 Generally, such proteins are produced by culturing 0010 Activated carbon is an inexpensive natural material recombinant cells in which a vector including a gene encod having extensive non-specific adsorption properties, and used ing a protein of interest is inserted. The culture broth includes as an adsorbent or as a decolorant in the industrial fields. Such impurities such as a wide variety of medium-derived compo as the production of chemicals and foods, sewage or waste nents, host cell-derived components, protein-derived water treatment, water filtration, and production of Small by-products or the like, in addition to the protein of interest. molecule drugs. However, due to the extensive non-specific Thus, it is a very difficult and challenging task to achieve both adsorption properties, it has been thought that there are diffi the purification of the protein of interest by removing impu culties in the use of activated carbon for high performance rities to meet purity requirements for protein drugs as well as protein purification such as separation of the above-men the efficient production of the protein of interest in large tioned impurities. Thus, a method of purifying a protein using scale. the activated carbon has not been known yet. 0004. In general, the protein purification method is carried out by a combination of different modes of chromatography. CITATION LIST Chromatography is to separate the protein of interest from impurities, for example, based on charge, hydrophilicity, Patent Documents molecular size or the like. 0005. In particular, when the protein of interest is an anti 0011 Patent Document 1 Japanese Patent Publication body, Protein A affinity chromatography or Protein Gaffinity No. He15-504579 chromatography is used as one of chromatography for puri 0012 Patent Application 2 International Patent Publica fying the antibody, by using binding property of Protein A or tion No. 2009/009523 Protein G to the specific region of antibody such as Fc chain (Patent Document 1). SUMMARY OF THE INVENTION 0006. However, Protein A affinity supports generally used are very expensive in comparison to ion exchange Supports or Problems to be Solved by the Invention hydrophobic supports, and a vast amount of Supports are needed for large-scale purification of antibodies in industrial 0013 An object of the present invention is to provide a drug productions or the like, resulting in an inevitable purification method which can lower production cost or increase in the production costs. reduce labor than the conventional protein purification meth 0007 Further, protein A affinity chromatography or pro ods, and has impurity separation properties higher than or tein G affinity chromatography is generally carried out in an equivalent to the conventional protein purification methods, adsorption mode of specifically adsorbing the antibody of in particular, as an alternative to protein A affinity chroma interest onto the Support, washing the adsorbing Support to tography for antibody purification, and a method for prepar separate impurities, and finally eluting the antibody of inter ing a protein comprising the purification method. est from the support. In this regard, buffers used in the wash ing and eluting steps are different from each other, Scale-up of Means for Solving the Problems chromatography apparatus brings out enlargement or com 0014. The present inventors have made many efforts to plexity of the accompanying production facilities such as Solve the above objects. As a result, they surprisingly found a buffer tank, and moreover, manipulations become compli method for purifying a protein by separating the protein from cated. All of these factors are the cause of increasing produc impurities using an inexpensive activated carbon in a non tion costs. absorption mode, in particular, a method for purifying an 0008 For these reasons, the drugs including proteins as an antibody using the activated carbon instead of protein Aaffin active ingredient require much higher production costs than ity chromatography, thereby completing the present inven drugs including Small-molecule compounds as an active tion. US 2014/0046038A1 Feb. 13, 2014
0015 The present invention relates the following (1) to the polymer contents of the culture supernatant (Culture (14) supernatant) and the final purified product (Purified product) 0016 (1) A method for purifying a protein, wherein the are represented. protein is separated from impurities using an activated carbon 0033 FIG. 2 shows the degradation product contents of to obtain the protein with a low content of impurities. the Supernatant and the final purified product in non-adsorp 0017 (2) The purification method described in (1), tion mode comprising activated carbon purification of Mab A. wherein the protein has a molecular weight of 30000 or more. Mab Band Mab C. The vertical axis represents the degrada 0018 (3) The purification method described in (1) or (2), tion product content (%), the black color represents Mab A. wherein the protein is a glycoprotein. the white color represents Mab B, and the grey color repre sents Mab C. From left, the degradation product contents of 0019 (4) The purification method described in (3), the culture Supernatant (Culture Supernatant) and the final wherein the glycoprotein is an antibody. purified product (Purified product) are represented. 0020 (5) The purification method described in any one of 0034 FIG. 3 shows the host cell protein contents of the (1) to (4), wherein the protein is a genetically modified pro Supernatant and the final purified product in non-adsorption tein. mode comprising activated carbon purification of Mab A. 0021 (6) The purification method described in any one of Mab Band Mab C. The vertical axis represents the host cell (1) to (5), wherein the impurities are any one of host cell protein content per 1 mg of protein (ng/mg), the black color proteins, protein-derived polymers, protein-derived degrada represents Mab A, the white color represents Mab B, and the tion products, or DNAs. grey color represents Mab C. From left, the host cell protein 0022 (7) The purification method described in any one of contents of the culture Supernatant (Culture Supernatant) and (1) to (6), wherein the method is carried out in a non-adsorp the final purified product (Purified product) are represented. tion mode. 0035 FIG. 4 shows the polymer contents of the superna 0023 (8) The purification method described in any one of tant and the final purified product in non-adsorption mode (1) to (7), wherein the separation is carried out at pH 3 to 8. comprising activated carbon purification of Mab A. Mab B 0024 (9) The purification method described in any one of and Mab C. The vertical axis represents the polymer content (1) to (8), wherein the activated carbon is an activated carbon (%), the black color represents Mab A, the white color rep from wood. resents Mab B, and the grey color represents Mab C. From 0025 (10) The purification method described in any one of left, the polymer contents of the culture supernatant (Culture (1) to (9), wherein the activated carbon has an average supernatant) and the final purified product (Purified product) micropore diameter of 0.5 to 5 nm. are represented. 0026 (11) A method for preparing a protein, comprising 0036 FIG. 5 shows the degradation product contents of the purification method of any one of (1) to (10). the Supernatant and the final purified product in non-adsorp tion mode comprising activated carbon purification of Mab A. 0027 (12) The preparation method described in (11), Mab Band Mab C. The vertical axis represents the degrada wherein protein A chromatography is not used. tion product content (%), the black color represents Mab A. 0028 (13) The preparation method described in (11) or the white color represents Mab B, and the grey color repre (12), comprising any one of anion exchange chromatography, sents Mab C. From left, the degradation product contents of cation exchange chromatography, hydrophobic interaction the culture Supernatant (Culture Supernatant) and the final chromatography, and multimodal chromatography. purified product (Purified product) are represented; 0029 (14) The preparation method described in any one of 0037 FIG. 6 shows the host cell protein contents of the (11) to (13), comprising at least one adsorption-mode chro Supernatant and the final purified product in non-adsorption matography. mode comprising activated carbon purification of Mab A. 0030 (15) A protein that is prepared by the method of any Mab Band Mab C. The vertical axis represents the host cell one of (11) to (14). protein content per 1 mg of protein (ng/mg), the black color represents Mab A, the white color represents Mab B, and the Effect of the Invention grey color represents Mab C. From left, the host cell protein 0031. The present invention provides a purification contents of the culture Supernatant (Culture Supernatant) and method which can lower production cost or reduce labor than the final purified product (Purified product) are represented. the conventional protein purification methods, and has impu 0038 FIG. 7 shows the recovery rate of each process and rity separation properties higher than or equivalent to the the total recovery rate in the Mab A purification. The vertical conventional protein purification methods, in particular, as an axis represents the recovery rate of each process (%) or the alternative to protein A affinity chromatography for antibody total recovery rate (%), the white color represents purification purification, and a method for preparing a protein comprising comprising protein A affinity chromatography, the black the purification method. The protein prepared by the present color represents purification comprising activated carbon invention is useful as a drug. treatment. From left, the recovery rate of MabS elect SuRe treatment or activated carbon treatment (Protein A or Acti vated carbon), the recovery rate of Q Sepharose treatment BRIEF DESCRIPTION OF THE DRAWINGS (Anion), the recovery rate of POROS XS treatment (Cation), 0032 FIG. 1 shows the polymer contents of the superna and the total recovery rate (Total recovery rate) are repre tant and the final purified product in non-adsorption mode sented. comprising activated carbon purification of Mab A. Mab B 0039 FIG. 8 shows the recovery rate of each process and and Mab C. The vertical axis represents the polymer content the total recovery rate in the Mab B purification. The vertical (%), the black color represents Mab A, and the white color axis represents the recovery rate of each process (%) or the represents Mab B, the grey color represents Mab C. From left, total recovery rate (%), the black color represents purification US 2014/0046038A1 Feb. 13, 2014
comprising protein A affinity chromatography, and the grey 0045 FIG. 14 shows the host cell protein contents of the color represents purification comprising activated carbon purification intermediate and the final purified product in the treatment. From left, the recovery rate of MabSelect SuRe Mab B purification. The vertical axis represents the content of treatment or activated carbon treatment (Protein A or Acti the host cell protein per 1 mg of protein (ng/mg). The black vated carbon), the recovery rate of Q Sepharose treatment diamond represents purification comprising protein Aaffinity (Anion), the recovery rate of POROS XS treatment (Cation), chromatography, and the white square represents purification and the total recovery rate (Total recovery rate) are repre comprising activated carbon treatment. From left, the host sented. cell protein contents of the clarified solution A (Supernatant), 0040 FIG. 9 shows the polymer contents of the purifica the MabSelect SuRe eluate or the activated carbon eluate tion intermediate and the final purified product in the Mab A (Protein A or Activated carbon), the Q Sepharose eluate (An purification. The vertical axis represents the polymer content ion), and the final Mab B purified product (Cation) are rep (%). The black circle represents purification comprising pro resented. tein A affinity chromatography, and the white triangle repre 0046 FIG. 15 shows SDS-PAGE of the Mab B culture sents purification comprising activated carbon treatment. supernatant. From left, (A) the clarified solution, (B) the From left, the polymer contents of the clarified solution A Supernatant that was maintained under addition of activated (Supernatant), the MabSelect SuRe eluate or the activated carbon for 24 hours and then the activated carbon was carbon eluate (Protein A or Activated carbon), the Q removed therefrom, and (C) the Supernatant that was main Sepharose eluate (Anion), and the final Mab A purified prod tained for 24 hours without addition of activated carbon are uct (Cation) are represented. represented. 0041 FIG. 10 shows the degradation product contents of 0047 FIG. 16 shows SDS-PAGE of the Mab D culture the purification intermediate and the final purified product in Supernatant. From left, (A) the Supernatant, (B) the Superna the Mab A purification. The vertical axis represents the deg tant that was treated with addition/removal of activated car radation product content (%). The black circle represents bon and maintained for 24 hours, and (C) the Supernatant that purification comprising protein A affinity chromatography, was only treated with removal and maintained for 24 hours and the white triangle represents purification comprising acti are represented. vated carbon treatment. From left, the degradation product 0048 FIG. 17 shows DNA contents per 1 mg protein in the contents of the clarified solution A (Supernatant), the Mab final purified product of activated carbon purification and the Select SuReeluate or the activated carbon eluate (Protein A or purified product of Protein Apurification, with respect to Mab Activated carbon), the Q Sepharose eluate (Anion), and the A and Mab B. The vertical axis represents the content of DNA final Mab A purified product (Cation) are represented. per 1 mg of protein (pg/mg). From left, the DNA contents per 0042 FIG. 11 shows the polymer contents of the purifica 1 mg protein in the Mab A purified product of Protein A tion intermediate and the final purified product in the Mab B purification that was obtained by Comparative Example 1 purification. The vertical axis represents the polymer content (Mab A Protein A), the final Mab A purified product of acti (%). The black diamond represents purification comprising vated carbon purification that was obtained by Example 7 protein A affinity chromatography, and the white square rep (Mab A activated carbon), the Mab B purified product of resents purification comprising activated carbon treatment. Protein A purification that was obtained by Comparative From left, the polymer contents of the clarified solution B Example 2 (Mab B Protein A), and the final Mab B purified (Supernatant), the MabSelect SuRe eluate or the activated product of activated carbon purification that was obtained by carbon eluate (Protein A or Activated carbon), the Q Example 8 (Mab Bactivated carbon) are represented. Sepharose eluate (Anion), and the final Mab B purified prod 0049 FIG. 18 shows the reduction rate of host cell pro uct (Cation) are represented. teins of activated carbon eluate at each pH by treatment of 0043 FIG. 12 shows the degradation product contents of Mab B with activated carbon. The vertical axis represents the the purification intermediate and the final purified product in reduction rate of host cell protein of activated carbon eluate the Mab B purification. The vertical axis represents the deg (HCPLRV). From left, the reduction rates of host cell protein radation product content (%). The black diamond represents of activated carbon eluate at pH 4, pH 5, pH 6, pH 7, and pH purification comprising protein A affinity chromatography, 8 are represented. and the white square represents purification comprising acti 0050 FIG. 19 shows the relative antibody concentration vated carbon treatment. From left, the degradation product of activated carbon eluate at each pH by treatment of Mab B contents of the clarified solution B (Supernatant), the Mab with activated carbon. The vertical axis represents the relative Select SuReeluate or the activated carbon eluate (Protein A or antibody concentration of activated carbon eluate (%), when Activated carbon), the Q Sepharose eluate (Anion), and the the antibody concentration of activated carbon eluate at pH 7 final Mab B purified product (Cation) are represented. is regarded as 100. From left, the relative antibody concen 0044 FIG. 13 shows the host cell protein contents of the trations of activated carbon eluate at pH 4, pH 5, pH 6, pH 7. purification intermediate and the final purified product in the and pH 8 are represented. Mab Apurification. The vertical axis represents the content of 0051 FIG. 20 shows the polymer content of activated the host cell protein per 1 mg of protein (ng/mg). The black carbon eluate by treatment of Mab B with different types of circle represents purification comprising protein A affinity activated carbon. The vertical axis represents the polymer chromatography, and the white triangle represents purifica content of activated carbon eluate (%). From left, the polymer tion comprising activated carbon treatment. From left, the contents of activated carbon eluates in the culture Superna host cell protein contents of the clarified solution A (Super tant, SHIRASAGI PSHIRASAGI DO-2, and SHIRASAGI natant), the MabSelect SuRe eluate or the activated carbon DO-5 are represented. eluate (Protein A or Activated carbon), the Q Sepharose eluate 0.052 FIG. 21 shows the degradation product content of (Anion), and the final Mab A purified product (Cation) are activated carbon eluate by treatment of Mab B with different represented. types of activated carbon. The vertical axis represents the US 2014/0046038A1 Feb. 13, 2014 degradation product content of activated carbon eluate (%). 0063 Specific examples thereof may include Chinese From left, the degradation product contents of activated car hamster ovary cells (CHO cells), mouse myeloma cells such bon eluates in the culture supernatant, SHIRASAGI P. as NSO cell and SP2/0 cell, rat myeloma cells such as YB2/0 SHIRASAGI DO-2, and SHIRASAGI DO-5 are represented. cell and IR983F cell, Syrian hamster kidney-derived BHK 0053 FIG.22 shows the host cell protein content of acti cells, human myeloma cells Such as Namalwa cell, embryo vated carbon eluate by treatment of Mab B with different stem cells, amphicytula or the like. types of activated carbon. The vertical axis represents the host 0064. A medium for culturing the protein-producing cells cell protein content of activated carbon eluate (ng/mg). From may be any medium, as long as it is Suitable for culturing each left, the host cell protein contents of activated carbon eluates of the cells, and examples of the medium for culturing animal in the culture supernatant, SHIRASAGI P, SHIRASAGI cells may include typical media used for culturing animal DO-2, and SHIRASAGI DO-5 are represented. cells. For example, any medium of a serum-containing medium, a medium containing no animal-derived component EMBODIMENTS FOR CARRYING OUT THE Such as serum albumin or serum fraction, a serum-free INVENTION medium or a protein-free medium may be used, and prefer 0054 The present invention relates to a method for puri ably, the serum-free medium or the protein-free medium is fying a protein, in which the protein is separated from impu used. rities using an activated carbon to obtain a protein having a 0065 Specifically, for example, RPMI1640 medium The lower content of impurities. Journal of the American Medical Association, 199, 519 0055. In the present invention, examples of the protein (1967), Eagle MEM medium Science, 122, 501 (1952), may include natural or non-natural proteins having no Sugar Dulbecco's modified MEM (DMEM) medium Virology, 8, chain, natural or non-natural glycoproteins, derivatives 396 (1959), 199 medium Proceeding of the Society for the thereof or the like. The glycoproteins or derivatives thereof Biological Medicine, 73, 1 (1950), F12 medium Proc. Natl. may be compositions comprising molecules different in their Acad. Sci. USA, 53, 288 (1965), Iscove’s Modified Dul Sugar chains. becco medium (IMDM medium) J. Experimental Medicine, 0056. The protein may be a protein having a molecular 147, 923 (1978), EX-CELL302 medium, EX-CELL-CD weight of preferably 30000 or higher, and more preferably CHO medium, and EX-CELL 325 medium (which are manu 50000 or higher. factured by SAFC bioscience Inc.), CD-CHO medium and 0057 Specific examples thereof may include erythropoi CD DG44 medium (which are manufactured by Invitrogen etin, darbepoetin, antithrombin (O. or B form, or mixtures Corp.) or ISCD-CHO medium (manufactured by Irvine Sci thereof), interferons, interleukins, protein S, tissue plasmino entific Sales Co., Inc.), modified media thereof, mixed media gen activator, factor VII, factor VIII, factor IX, thrombo thereof, concentrated media thereof or the like is used, and modulin, glucocerebrosidase, C.-galactosidase, C-L-idu preferably RPMI1640 medium, DMEM medium, F12 ronidase, acidic C-glucosidase, Granulocyte Colony medium, IMDM medium, EX-CELL302 medium, CD-CHO Stimulating Factor (G-CSF), Granulocyte Macrophage medium, or ISCD-CHO medium is used. Colony Stimulating Factor (GM-CSF), thrombopoietin or 0066. If necessary, physiologically active substances or Megakaryocyte Growth and Development Factor (MGDF), nutrient factors essential for growth of the protein-producing fibroblast growth factor (FGF), epidermal growth factor cells may be added. These additives may be previously (EGF), insulin-like growth factor (IGF), brain-derived neu included in the medium prior to cultivation, or further prop rotrophic factor (BDNF), ciliary neurotrophic factor (CTNF). erly Supplied to the culture liquid as an additive medium oran Glial-CellDerived Neurotrophic Factor (GDNF), or antibod additive solution during cultivation. The further Supplying ies and, derivatives thereof, or the like, preferably antibodies, method may be carried out using any form of one solution or and more preferably monoclonal antibodies. mixtures of two or more solutions by any of continuous or 0058 Examples of the antibodies may include mouse anti intermittent Supply. bodies, llama antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibodies with modified Fc 0067. The protein-producing transgenic non-human ani regions or the like. Examples of the molecular type may mals, plants or insects may be non-human animals, plants or include IgG, IgM, IgA, Ig), IgE, Fab., Fc, Fc-fusion proteins, insects in which the protein-encoding gene is integrated into VH, VL, VHH, Fab', scFv, scFab, scDb, scDbFc or the like. their cells. Examples of the non-human animals may include 0059. In the purification method of the present invention, a mouse, rat, guinea pig, hamster, rabbit, dog, sheep, pig, goat, protein-containing aqueous solution that includes a protein of cattle or monkey. Examples of the plants may include interest and impurities is provided. tobacco, potato, tomato, carrot, soybean, brassica, alfalfa, 0060 Examples of the protein-containing aqueous solu rice, wheat, barley, corn or the like. tion may include a composition obtained from the living 0068 Examples of the method for producing the protein body, such as plasma, serum, breast milk, or urine, a culture containing aqueous Solution may include those described in broth of protein-producing cells or bacteria Such as E. coli, International Patent Publication No. 2008/120801, Japanese which are obtained by a genetic recombination technique or a Publication No. Hei3-198792, International Patent Publica cell fusion technique, a composition obtained from trans tion No. 2010/018847, International Patent Publication No. genic non-human animals, plants or insects, a composition 2007/06224.5, International Patent Publication No. 2007/ obtained by cell-free protein synthesis, or the like. 114496 or the like. 0061 Examples of the protein-producing cell may include 0069. Further, in the present invention, the protein-con a transformed cell in which a gene encoding a protein of taining aqueous solution includes a protein-containing aque interest is integrated in a host cell, or the like. ous solution obtained from the purification process, in addi 0062) Examples of the host cell may include cell lines of tion to those obtained from the living body, Such as plasma, animal cells, plant cells, yeast cells or the like. urine or the like. Specific examples thereof may include a US 2014/0046038A1 Feb. 13, 2014 cell-free liquid, a precipitate-free liquid, an alcohol fraction, milk, or urine, the protein-containing aqueous Solution a salting-out fraction, a chromatography eluate or the like. obtained from transgenic non-human animals, plants or 0070 The cell-free liquid may be a liquid that is prepared insects, the protein-containing aqueous solution obtained by removing cells from the protein-containing aqueous solu from cells established by the genetic recombination tech tion obtained from the living body. Such as plasma, serum, nique, the protein-containing aqueous solution obtained by breast milk, or urine, the protein-containing aqueous Solution cell-free protein synthesis or the protein-containing aqueous obtained from transgenic non-human animals, plants or Solution obtained from the purification process, so as to pre insects, the protein-containing aqueous solution obtained cipitate proteins. from cells established by the genetic recombination tech 0076. The chromatography eluate may be a protein eluate nique, the protein-containing aqueous solution obtained from that is prepared by adsorbing the protein-containing aqueous the purification process, or the like. Specific examples thereof Solution obtained from the living body, such as plasma, may include Solutions that are obtained by removing cells serum, breast milk, or urine, the protein-containing aqueous from a cell culture broth by centrifugation, cross-flow filtra Solution obtained from transgenic non-human animals, plants tion (Tangential flow filtration), filtration using a depth filter, or insects, the protein-containing aqueous solution obtained filtration using a membrane filter, dialysis, combinations from cells established by the genetic recombination tech thereof or the like. nique, the protein-containing aqueous solution obtained by 0071 Specific examples of the depth filter may include a cell-free protein synthesis or the protein-containing aqueous Millistak+ HC depth filter, a Millistak+ DE depth filter, a Solution obtained from the purification process onto a Support Millistak+ CE depth filter (manufactured by Merck millipore or a membrane used in the chromatography so as to elute it Corp.), a SUPRAP depth filter (manufactured by Pall Corp.), using a proper elution Solution, or by non-adsorbing it. a Sartoclear PB depth filter, a Sartoclear PC depth filter (manufactured by Sartorius Corp.), a Zeta plus SP depth filter, 0077. The support or the membrane used in the chroma a Zeta plus AP depth filter, a Zeta plus LA depth filter, a Zeta tography may include an affinity Support, an ion exchange plus-Delipid depth filter, a Zeta plus ZA depth filter or a Zeta Support, an ion exchange membrane, a gel filtration Support, plus EXT charged depth filter (manufactured by Sumitomo a hydrophobic interaction Support, a reverse phase Support, a 3M Ltd., but are not limited thereto. hydroxyapatite Support, a fluoroapatite Support, a cellulose 0072 The precipitate-free liquid may be a liquid that is Sulfate Support, an agarose Sulfate Support, a multimodal prepared by performing flocculation or two-phase separation Support or the like. of the protein-containing aqueous solution obtained from the 0078. The ion exchange support or the ion exchange mem living body, such as plasma, serum, breast milk, or urine, the brane may be a Support or a membrane that is prepared by protein-containing aqueous Solution obtained from trans directly or indirectly immobilizing a molecule having an ion genic non-human animals, plants or insects, the protein-con exchange group, Such as a Sulfate group, a methyl Sulfate taining aqueous solution obtained from cells established by group, a Sulfophenyl group, a Sulfonpropyl group, a car the genetic recombination technique, the protein-containing boxymethyl group, a quaternary ammonium group, a quater aqueous solution obtained by cell-free protein synthesis or nary aminoethyl group, a diethylaminoethyl group or the like the protein-containing aqueous Solution obtained from the onto a base Support or a membrane, for example, a polymer or purification process by low-pH treatment or by addition of a derivative thereof (including crosslinked polymer) Such as caprylic acid, an organic solvent, polyethylene glycol, a Sur cellulose, Sepharose, agarose, chitosan, an acrylic acid poly factant, a salt, an amino acid, a polymer or the like, and then mer or a styrene-divinylbenzene copolymer, a polymer con by removing precipitates therefrom. Examples of the method sisting of silica particles, glass particles, ceramic particles, or for removing precipitates may include centrifugation, cross surface-treated particles thereof. Specific examples thereof flow filtration (Tangential flow filtration), filtration using a may include Q Sepharose XL. Q Sepharose FF, DEAE depth filter, filtration using a membrane filter, dialysis, com Sepharose FF, ANX Sepharose FF, Capto Q, Capto DEAE, binations thereof or the like. Capto Q ImpRes (which are manufactured by GE Healthcare 0073. The pH of the low-pH treatment is preferably pH 3 Ltd., Inc.), TOYOPEARL GigaCap Q-650, TOYOPEARL to 6, and adjusted by addition of an acid such as hydrochloric SuperQ-650 (which are manufactured by TOSOH Corp.), acid, acetic acid, citric acid, phosphoric acid or the like. Fractogel DEAE, Fractogel TMAE, Fractogel TMAE Hicap, 0074 The alcohol fraction may be a fraction that is pre Eshmuno Q (which are manufactured by Merck millipore pared by adding alcohol or the like to the protein-containing Corp.), Cellufine MAX-Q (manufactured by JNC Corp.), aqueous solution obtained from the living body, such as Mustang Q (manufactured by Pall Corp.), Sartobind Q, Sar plasma, serum, breast milk, or urine, the protein-containing tobind STIC (which are manufactured by Sartorius Corp.), SP aqueous solution obtained from transgenic non-human ani Sepharose FF, CM Sepharose FF, SP Sepharose XL, Capto S mals, plants or insects, the protein-containing aqueous solu (which are manufactured by GE Healthcare Ltd., Inc.), Poros tion obtained from cells established by the genetic recombi 50 HS, Poros 50 XS (which are manufactured by Applied nation technique, the protein-containing aqueous Solution Biosystems Inc.), Eshmuno S, Fractogel COO, Fractogel obtained by cell-free protein synthesis or the protein-contain SO, Fractogel SE Hicap (which are manufactured by Merck ing aqueous solution obtained from the purification process. millipore Corp.), TOYOPEARL GigaCap S-650, TOYOPE Specific examples thereof may include fractions obtained by ARL GigaCap CM-650 (which are manufactured by TOSOH low temperature ethanol fraction or the like. Corp.), Cellufine MAX-S (manufactured by INC Corp.), 0075. The salting-out fraction may be a fraction that is Mustang S (manufactured by Pall Corp.) or Sartobind S prepared by adding a salt Such as ammonium Sulfate, sodium (manufactured by Sartorius Corp.), DIAION PK, DIAION Sulfate, Sodium citrate, Sodium chloride, potassium chloride PA, DIAION CR, DIAION CR, DIAION AMP (which are or the like to the protein-containing aqueous solution manufactured by Mitsubishi Chemical Corp.) or the like, but obtained from the living body, Such as plasma, serum, breast are not limited thereto. US 2014/0046038A1 Feb. 13, 2014
007.9 The affinity support may be a support that is pre are manufactured by Bio-Rad Inc.) or the like, but are not pared by directly or indirectly immobilizing a molecule hav limited thereto. In addition, examples of the fluoroapatite ing an affinity for the protein of interest, for example, heparin, support may include CFT Ceramic Fluoroapatite (manufac protein A, protein G protein L or the like, onto the above base tured by Bio-Rad Inc.) or the like, but are not limited thereto. Support, and specific examples thereof may include Heparin I0084 Examples of the cellulose sulfate support or the Sepharose 6 Fast Flow (manufactured by GE Healthcare Ltd., agarose Sulfate Support may include Cellufine Sulfate, Cel Inc.), Procep-heparin (manufactured by Merck millipore lufine sulfate m, Cellufine sulfate c, Cellulofine sulfate m, Corp.), TOYOPEARL AF-Heparin-650 (manufactured by Cellulofine sulfate c, Cellufine sulfate m or Cellufine sulfate TOSOH Corp.), Heparin HyperD (manufactured by Pall c (which are manufactured by JNC Corp.), Capto DeVirS Corp.), MabSelect, Protein A Sepharose FF, MabSelect Xtra, (manufactured by GE Healthcare Ltd., Inc.) or the like, but MabSelect SuRe, MabSelect SuRe LX, Protein G Sepharose are not limited thereto. FF, Capto L (which are manufactured by GE Healthcare Ltd., I0085. The multimodal support may be a support that is Inc.), Prosep VA Hicapacity, Prosep VA Ultra, Prosep Ultra prepared by directly or indirectly immobilizing two or more plus (which are manufactured by Merck millipore Corp.) or types of functional groups having different selectivity, pref the like. erably, the above ion exchange group and the above hydro 0080 Examples of the gel filtration support may include a phobic interaction group, onto the above base Support, and Support composed of a polymer consisting of dextran, allyl specific examples thereof may include Capto adhere, Capto dextran, N,N'-methylenebisacrylamide, cellulose, agarose, MMC (which are manufactured by GE Healthcare Ltd., Inc.), styrene, divinylbenzene, polyvinyl alcohol, silica, chitosan or HEA HyperCel, PPA HyperCel, MEP HyperCel (which are the like, and specific examples thereofmay include Sephacryl manufactured by Pall Corp.), TOYOPEARL MX-Trp-650M S series, Sepharose series, Sephadex series, Superdex series, (manufactured by TOSOH Corp.) or the like, but are not Sephacryl series (which are manufactured by GE Healthcare limited thereto. Ltd., Inc.), TOYOPEARL HW series, TSKgel PW series I0086. In the present invention, if the protein is an antibody, (which are manufactured by TOSOH Corp.), Biogel Agarose, the protein-containing aqueous solution may be preferably a Biogel P Polyacrylamide (which are manufactured by Bio protein-containing aqueous solution that is obtained without Rad Inc.), Cellufine GH, Cellufine GCL (which are manufac using affinity chromatography, and more preferably, a pro tured by JNC Corp.), Trisacryl GF05, Trisacryl GF2000, tein-containing aqueous solution that is obtained without Ultrogel AcA (which are manufactured by Pall Corp.) or using protein A affinity chromatography. Fractogel BioSEC (manufactured by Merck millipore Corp.) or the like, but are not limited thereto. I0087 Further, if insoluble materials such as particles or 0081. The hydrophobic interaction support may be a Sup the like are present in the protein-containing aqueous solu port that is prepared by directly or indirectly immobilizing a tion, they are removed inadvance, and the resulting insoluble hydrophobic molecule, for example, methyl group, ethyl free solution may be provided in the purification method of group, propyl group, isopropyl group, butyl group, tert-butyl the present invention. Examples of the method of removing group, octyl group, ether group, phenyl group or the like onto insoluble materials such as particles may include centrifuga the above base Support, and specific examples thereof may tion, cross-flow filtration (Tangential flow filtration), filtra include Phenyl Sepharose 6 Fast Flow (high-sub), Phenyl tion using a depth filter, filtration using a membrane filter, Sepharose 6 Fast Flow (low-sub), Octyl Sepharose 4 Fast dialysis, or combinations thereof. If necessary, the after-men Flow, Butyl Sepharose 4 Fast Flow (which are manufactured tioned pH, conductivity, buffer, protein concentration of the by GE Healthcare Ltd., Inc.), TOYOPEARL Hexyl-650, protein-containing aqueous Solution, or protein addition TOYOPEARL Butyl-650, TOYOPEARL Phenyl-650, amount per unit volume of the activated carbon are adjusted, TOYOPEARL Ether-650, TOYOPEARL PPG-600, TOYO and then the resulting protein-containing aqueous solution PEARL Butyl-600, TOYOPEARL Super Butyl-550 (which may be provided in the purification method of the present are manufactured by TOSOH Corp.), Mactro-Prep t-Butyl, invention. Macro-Prep Methyl (which are manufactured by Bio-Rad I0088. Examples of the method of adjusting the pH, the Inc.), QMA Spherosil, Methyl Ceramic HyperD (which are conductivity, the buffer, the protein concentration, or the pro manufactured by Pall Corp.), Fractogel Phenyl(S), Fractogel tein addition amount per unit volume of the activated carbon Propyl(S) (which are manufactured by Merck millipore may include ultrafiltration using an ultrafiltration membrane Corp.), phenyl-Cellufine (manufactured by JNC Corp.), or the like. DIAION HP DIAION SP (which are manufactured by Mit I0089. The ultrafiltration membrane includes a positively subishi Chemical Corp.), butylated Chitopearl, phenylated or negatively charged ultrafiltration membrane, in addition to Chitopearl (which are manufactured by FUJIBO Holdings, the typical ultrafiltration membranes, and specific examples Inc.) or the like. thereof may include Pelicon 3 Ultracel membrane, Pellicon 3 0082. The reverse phase support may be, for example, a biomax membrane, Pellicon 2 Ultracel membrane, Pellicon 2 Support that is prepared by directly or indirectly immobilizing biomax membrane (which are manufactured by Merck mil a hydrocarbon group onto a solid-phase matrix. Examples of lipore Corp.), omega membrane (manufactured by Pall the hydrocarbon group may include trimethyl group, butyl Corp.), Kvick membrane (manufactured by GE Healthcare group, phenyl group, octyl group, octadecyl group, terminus Ltd., Inc.) or the like, but are not limited thereto. modified functional group thereof or the like. Specific 0090. In the present invention, the impurities may include examples thereof may include RESOURCE RPC series, host cell proteins (HCP), protein-derived polymers, protein SOURCE RPC series (which are manufactured by GE derived degradation products, protein-derived modification Healthcare Ltd., Inc.) or the like, but are not limited thereto. products resulting from denaturation, removal of Sugar chain 0083. Examples of the hydroxyapatite support may components, oxidation, deamidation or the like, DNAS, include CHT Ceramic Hydroxyapatite Type I, Type II (which medium-derived components, culture additives or enzymes US 2014/0046038A1 Feb. 13, 2014
secreted from host cells, and preferably host cell proteins, SHIRASAGI Gx, SHIRASAGI G, SHIRASAGI GH, protein-derived polymers, protein-derived degradation prod SHIRASAGI FAC-10, SHIRASAGI M, SHIRASAGI P. ucts, or DNAs. SHIRASAGI PHC, SHIRASAGI Gc, SHIRASAGI GH, 0091 Examples of the enzymes secreted from host cells SHIRASAGI GM, SHIRASAGI GS, SHIRASAGI GT, may include glycolytic enzymes, proteolytic enzymes, oxi SHIRASAGIGAA, SHIRASAGIGOC, SHIRASAGIGOX, dation/reduction enzymes or the like. SHIRASAGI APRC, SHIRASAGI TAC, SHIRASAGI 0092 Specific examples of the glycolytic enzymes may MAC, SHIRASAGIXRC, SHIRASAGINCC, SHIRASAGI include neuraminidase (sialidase), galactosidase, glycanase SRCX, SHIRASAGI We, SHIRASAGI LGK, SHIRASAGI or the like. Specific examples of the proteolytic enzymes may KL, SHIRASAGIWH, SHIRASAGIW, SHIRASAGIWHA, include serine protease, esterase, cysteine protease, trypsin SHIRASAGI LH, SHIRASAGI KL, SHIRASAGI LGK, like protease, aminopeptidase, aspartic protease, cathepsin or SHIRASAGI MAC-W, SHIRASAGI S, SHIRASAGI Sx, the like. Specific examples of the oxidation/reduction SHIRASAGI X2M, SHIRASAGI X7000, SHIRASAGI enzymes may include thioredoxin-related enzymes such as X7100, SHIRASAGI DX7-3, MOLSIEVON (which are thioredoxin reductase or the like. Specific examples of the manufactured by Japan EnviroChemicals, Ltd.), ACF. Taiko amino acid isomerizing enzyme may include transglutami (which are manufactured by Fuji Chemical Corp.), GLC nase or the like. (manufactured by KURARAY CHEMICAL CO., Ltd.), 0093. The activated carbonused in the purification method Taiko S, Taiko K, Taiko Q (which are manufactured by of the present invention may be any one, as long as it is FUTAMURA Chemical CO., Ltd.), GAC, CN, CG, CAP/ Suitable for the drug preparation, and one type of activated CGP. SX, CA (which are manufactured by Norit Japan Co., carbon may be used alone, or two or more types of activated Ltd.) or the like. carbon may be used alone or in combination. 0101 Examples of the means of the purification method of 0094. Examples of the activated carbon may include min the present invention may include, but are not particularly eral-based activated carbon, plant-based activated carbon or limited to, a batch method, a membrane treatment method, the like. Specific examples of the mineral-based activated column chromatography or the like. Depending on each carbon may include coal-based activated carbon, petroleum means, a suitable form of the activated carbon is selected. If based activated carbon or the like, and specific examples of necessary, a particle form or the like prepared by encapsulat the plant-based activated carbon may include wood-based ing the activated carbon in a porous polymer or a gel, a activated carbon, coconut-shell-based activated carbon or the membrane or cartridge form or the like prepared by adsorb like, and preferably wood-based activated carbon. ing, fixing or molding the activated carbon using a support 0095. The raw material of the activated carbon may be any such as polypropylene or cellulose, or fiber or the like. Spe one, as long as it is carbonaceous, and examples thereof may cific examples thereof may include a CUNO activated carbon include wood materials such as sawdust, charcoal, ash, peat filter cartridge, a Zeta plus activated carbon filter cartridge moss, peat or wood chip, coconut-shell, coals such as lignite, (manufactured by Sumitomo 3M Ltd.), a Millistak+ activated brown coal or anthracite, coal pitch, petroleum pitch, oil carbon filter (manufactured by Merck millipore Corp.), a carbon, rayon, acrylonitrile or phenol resin or the like. SUPRA AKS filter (manufactured by Pall Corp.), Adol 0096. The preparation method of the activated carbon is (manufactured by UNITIKA Ltd.), a K filter, an activated not particularly limited, but examples thereof may include a carbon sheet (which are manufactured by TOYOBO CO., chemical liquid activation method of adding and penetrating Ltd), Hemax (manufactured by KURARAY Co., Ltd.), a chemical Such as Zinc chloride orphosphoric acid at a high Hemosorba (manufactured by Asahi Kasei Medical Co., temperature and performing carbonization at a high tempera Ltd.), Hemocolumn (manufactured by TERUMO Corp.), ture, or a gas activation method of reacting carbonized raw Hecellose (manufactured by TEIJIN Ltd.) or the like. materials and gas such as water vapor, carbon dioxide, air or 0102 Depending on the protein of interest and the means combustion gas at a high temperature. of the purification method, packing density, granularity, rigid 0097. The form of the activated carbon may be any one, as ity, drying loss, residue on ignition, specific Surface area, pore long as it is Suitable for the drug preparation, and examples volume, or pH, or the like of the used activated carbon may be thereof may include a particle form of activated carbon, Such properly selected. as pulverized carbon, granular carbon, spherical carbon, pel 0103) The purification method of the present invention is let carbon or the like, a fibrous form of activated carbon such preferably carried out in a non-adsorption mode. The non as fiber, cross fiber or the like, a specialized form of activated adsorption mode means that the protein-containing aqueous carbon Such as a sheet form, a compact, a honeycomb shape Solution is contacted with the activated carbon, and the pro or the like, powder activated carbon or the like. tein of interest is not adsorbed onto the activated carbon to 0098. The positively or negatively charged activated car recover a non-adsorption fraction. In detail, the pH, the con bonor activated carbon modified with a surface modifier such ductivity, the buffer, the protein concentration of the protein as polyhydroxyethylmethacrylate (PHEMA), heparin, cellu containing aqueous Solution, the protein addition amount per lose, polyurethane or the like may be also included in the unit Volume of the activated carbon, temperature, or contact activated carbon of the present invention. time with the activated carbon are adjusted in advance, and 0099. An average micropore diameter of the activated car then contacted with the activated carbon. Thus, the protein of bon may be, but is not particularly limited to, typically 0.1 to interest is not adsorbed onto the activated carbon, but impu 20 nm, preferably 0.5 to 5 nm, and more preferably 1 to 3 nm. rities are adsorbed onto the activated carbon, thereby recov 0100 Specific examples thereof may include Carboraffin, ering the non-adsorption fraction having the protein with a strong SHIRASAGI, purified SHIRASAGI, specialized low content of impurities. SHIRASAGI, SHIRASAGI A, SHIRASAGI C, 0104. The pH of the protein-containing aqueous solution SHIRASAGIC-1, SHIRASAGI DO-2, SHIRASAGI DO-5, to be contacted with the activated carbon is preferably 2 to 9 SHIRASAGI DO-11, SHIRASAGI DC, SHIRASAGI DO, and more preferably 3 to 8. In particular, if the protein is an US 2014/0046038A1 Feb. 13, 2014 antibody, the pH of the protein-containing aqueous solution may be used in combinations of a plurality of types and to be contacted with the activated carbon is preferably 2 to 8, numbers thereof. The purification method to be used in com more preferably 3 to 7, much more preferably 4 to 6, and most bination with the activated carbon may be carried out either preferably 4 to 5. In addition, examples of the salt constituting before or after the purification method using the activated the protein-containing aqueous solution may include phos carbon. phate, citrate, acetate. Succinate, maleate, borate, Tris(base), 0110. The support or membrane used in the chromatogra HEPES, MES, PIPES, MOPS, TES, Tricine or the like. The phy to be used in combination with the activated carbon may concentration thereof is preferably 0.01 mol/L to 0.5 mol/L. include those similar to the above-mentioned affinity Support, For example, these salts may be also used in combinations of an ion exchange Support, an ion exchange membrane, a gel 0.01 mol/L to 0.5 mol/L, preferably of 0.01 mol/L to 0.5 filtration Support, a hydrophobic interaction Support, a mol/L of other salts, such as sodium chloride, potassium reverse phase Support, a hydroxyapatite Support, a fluoroapa chloride, calcium chloride, sodium citrate, sodium Sulfate, tite Support, a cellulose Sulfate Support, an agarose Sulfate ammonium sulfate or the like. The buffer components, for Support, a multimodal Support or the like. example, amino acids such as glycine, alanine, arginine, 0111. In the present invention, if the protein is an antibody, serine, threonine, glutamic acid, aspartic acid, histidine or the the chromatography to be used in combination with the acti like, Sugars such as glucose, Sucrose, lactose, sialic acid or the vated carbon may be preferably a preparation method com like, or derivatives thereof may be used in combinations. prising no affinity chromatography, and more preferably a 0105. The temperature of the protein-containing aqueous preparation method comprising no protein A affinity chroma solution to be contacted with the activated carbon is prefer tography. If the protein is an antibody, examples of the chro ably from 4°C. to 60° C., more preferably from 10°C. to 50° matography to be used in combination with the activated C., and much more preferably from 20°C. to 40°C. carbon may include ion exchange chromatography, multimo 0106. In the present invention, the non-adsorption fraction dal chromatography or combinations thereof. of the activated carbon is recovered, so as to obtain the protein 0112 The chromatography to be used in combination with with a low content of impurities in a highyield. In detail, with the activated carbon may be carried out in an adsorption mode respect to the content of the impurities, the content of the host or in a non-adsorption mode, depending on the purpose. Pref cell proteins is preferably 100000ng or less per 1 mg of the erably, at least one of the chromatography to be used in protein, more preferably 10000ng or less per 1 mg of the combination with the activated carbon is carried out in the protein, and much more preferably 1000 ng or less per 1 mg adsorption mode. of the protein, the content of the protein-derived polymers is 0113. The adsorption mode in the chromatography means preferably 5% or less, more preferably 4% or less, and much that an aqueous solution provided in the chromatography is more preferably 3% or less, the content of the protein-derived contacted with the corresponding Support or membrane, the degradation products is preferably 10% or less, more prefer protein of interest is adsorbed onto the corresponding Support ably 5% or less, much more preferably 4% or less, and most or membrane, if necessary, washing is performed, and then preferably 3% or less. The recovery rate is preferably 50% or the protein of interest is eluted using a buffer of which pH, higher, more preferably 60% or higher, and the reduction rate conductivity, buffer components, salt concentration or addi of the host cell proteins (HCPLRV) is preferably 1 or higher, tive or the like is altered, thereby recovering the adsorption more preferably 1.5 or higher and much more preferably 2 or fraction. The non-adsorption mode in the chromatography higher. means that an aqueous Solution provided in the chromatog 0107. In the present invention, the recovery rate of the raphy is contacted with the corresponding Support or mem protein with a low content of impurities and the content of brane, the protein of interest is not adsorbed onto the corre impurities may be determined by the analysis method typi sponding Support or membrane, thereby recovering the non cally used for protein purification. For example, the recovery adsorption fraction. rate may be determined by absorbance or affinity HPLC such 0114. In the protein purification method of the present as protein A, the content of the host cell proteins may be invention, all the chromatographies to be used in combination determined by ELISA (Enzyme-Linked Immunosorbent with the activated carbon may be, for example, a protein Assay), Western blotting, an electrochemiluminescence purification method that is carried out in the non-adsorption assay or the like, the content of the protein-derived polymers mode (All negative chromatography). or the protein-derived degradation products may be deter 0.115. In the present invention, if the protein is an antibody, mined by gel filtration HPLC, ion exchange HPLC, polyacry the purification method using the activated carbon is carried lamide gel electrophoresis, a light scattering method, an ultra out, and Subsequently, the non-adsorption mode-ion centrifugal method or the like, DNAs were determined by exchange chromatography is carried out, followed by the Pico-green, Threshold, QPCR or the like. adsorption mode-cation exchange chromatography, or the 0108 Further, the present invention relates to a method for purification method using the activated carbon is carried out, preparing a protein, comprising the step of separating the and Subsequently, the adsorption mode-cation exchange protein from impurities using the activated carbon to obtain chromatography is carried out, followed by non-adsorption the protein with a low content of impurities. mode-anion exchange chromatography. 0109. In the preparation method of the present invention, a 0116. The conditions of the aqueous solution provided in purification method to be used in combination with the acti the chromatography to be used in combination with the acti vated carbon may be any one as long as it is suitable for drug vated carbon or the buffer used in washing are properly preparation, and examples thereof may include chromatog selected with respect to the pH, conductivity, buffer compo raphy, alcohol fraction, removal of precipitates, salting out, nents, salt concentration, additives or the like. buffer exchange, concentration, dilution, filtration, virus 0117. In the selection of the chromatographic conditions, inactivation, virus removal or the like. These purification differences in the physicochemical characteristics between methods to be used in combination with the activated carbon the protein of interest and the compounds desired to be sepa US 2014/0046038A1 Feb. 13, 2014
rated, for example, differences in isoelectric point, charge, were passed through the column. The column non-adsorbed hydrophobicity, molecular size, or steric structure or the like fraction was pooled as a Q Sepharose eluate. may be utilized. The elution method of the adsorption mode 0.124 Approximately 10 mmol/L of sodium citrate was may include a one step elution method of using a buffer added to the resulting Q Sepharose eluate, and adjusted to pH having a specific salt concentration or pH to reduce affinity 7.0 with hydrochloric acid, and then applied to a multimodal between the protein of interest and the Support, a stepwise chromatography column (manufactured by GE Healthcare method of eluting the protein of interest by changing the salt Ltd., Inc., Capto adhere, 10 mm IDX20 cm) that was equili concentration or pH in a stepwise manner, or a gradient brated with an equilibration buffer prepared by adjusting 10 method of eluting the protein of interest by continuously mmol/L Tris buffer (pH 8.0) to pH 7.0 with a citric acid changing the salt concentration or pH. Solution. After completion of the application, the equilibra 0118. Examples of the salt constituting the buffer may tion buffer was passed through the column. A part of the include phosphate, citrate, acetate. Succinate, maleate, borate, column non-adsorbed fraction was pooled as a Capto adhere Tris(base), HEPES, MES, PIPES, MOPS, TES, Tricine or the eluate. like. These salts may be used in combinations with other salts, 0.125. The resulting Capto adhere eluate was adjusted to for example, sodium chloride, potassium chloride, calcium pH 4.5 with acetic acid, and then passed through the activated chloride, Sodium citrate, Sodium sulfate or ammonium Sul carbon filter (manufactured by CUNO Ltd., Zeta carbon filter, fate. The buffer components, for example, amino acids Such 25 cm), and pooled as an activated carbon eluate B. The as glycine, alanine, arginine, serine, threonine, glutamic acid, resulting activated carbon eluate B was used as a final Mab A aspartic acid or histidine or the like, Sugars such as glucose, purified product. Sucrose, lactose, Sialic acid or the like, or derivatives thereof 0.126 The contents of polymers and degradation products or the like may be used in combinations. in the final Mab A purified product were analyzed by gel 0119. In the preparation method of the present invention, filtration HPLC, and the content of host cell proteins therein the protein with a low content of impurities may be obtained was analyzed by ELISA. in a high recovery rate. In detail, with respect to the content of I0127. The analysis results of the final Mab A purified the impurities, the content of the host cell proteins is prefer product are shown in FIGS. 1, 2, and 3. According to the ably 100 ng or less per 1 mg of the protein, and more prefer present purification method, the Mab A purified product ably 10 ng or less per 1 mg of the protein, the content of the could be obtained, in which the contents of polymers and protein-derived polymers is preferably 3.5% or less, and more degradation products were less than 1%, respectively and the preferably 1% or less, the content of the protein-derived deg content of host cell proteins was less than 10 ng/mg. radation products is preferably 3.5% or less, and more pref erably 1% or less. The recovery rate is preferably 30% or Example 2 more, and more preferably 40% or more. 0120 Hereinafter, the present invention will be described Mab B Purification 1 (Non-Adsorption Mode in more detail with reference to Examples. However, the Purification Comprising Activated Carbon) present invention is not limited to these Examples. I0128 Approximately 600 mL of CHO cell culture super natant containing monoclonal antibodies (Mab B) that were EXAMPLES previously clarified by microfiltration was adjusted to pH 4.5 with acetic acid. The formed precipitates were removed by Example 1 centrifugation and a filter. The resulting clarified solution was neutralized with a Tris Solution, and concentrated to approxi Mab A Purification 1 (Non-Adsorption Mode mately 6-fold using the Pellicon 3 Ultracel membrane (manu Purification Comprising Activated Carbon) factured by millipore Corp., 30 kD, 0.11 m). After concen 0121 Approximately 600 mL of CHO cell culture super tration, the buffer was exchanged with 10 mmol/L Tris buffer natant containing monoclonal antibodies (Mab A) that were (pH 8.0) to obtain a concentrated/buffer-exchanged solution. previously clarified by microfiltration was adjusted to pH 4.5 I0129. Subsequently, Mab B purification comprising acti with acetic acid. The formed precipitates were removed by vated carbon was carried out in a non-adsorption mode by the centrifugation and a filter. The resulting clarified solution was following procedure. First, the resulting concentrated/buffer neutralized with a Tris Solution, and concentrated to approxi exchanged solution was passed through the activated carbon mately 6-fold using a Pellicon 3 Ultracel membrane (manu filter (manufactured by CUNO Ltd., Zeta carbon filter, 25 factured by millipore Corp., 30 kD, 0.11 m). After concen cm), and pooled as an activated carbon eluate A. tration, the buffer was exchanged with 10 mmol/L Tris buffer 0.130. The resulting activated carbon eluate A was applied (pH 8.0) to obtain a concentrated/buffer-exchanged solution. to the anion exchange chromatography column (manufac 0122) Subsequently, Mab A purification comprising acti tured by GE Healthcare Ltd., Inc., Q Sepharose, 11 mm vated carbon was carried out in a non-adsorption mode by the IDX20 cm) that was equilibrated with the equilibration buffer following procedure. First, the resulting concentrated/buffer consisting of 10 mmol/L Tris buffer (pH 8.0). After comple exchanged solution was passed through an activated carbon tion of the application, 5 column volumes of the equilibration filter (manufactured by CUNO Ltd., Zeta carbon filter, 25 buffer were passed through the column. The column non cm), and pooled as an activated carbon eluate A. adsorbed fraction was pooled as a Q Sepharose eluate A. 0123. The resulting activated carbon eluate A was applied I0131) Approximately 10 mmol/L of sodium citrate was to an anion exchange chromatography column (manufactured added to the resulting Q Sepharose eluate A, and adjusted to by GE Healthcare Ltd., Inc., Q Sepharose, 11 mm IDX20 cm) pH 7.0 with hydrochloric acid, and then applied to the mul that was equilibrated with an equilibration buffer consisting timodal chromatography column (manufactured by GE of 10 mmol/L Tris buffer (pH 8.0). After completion of the Healthcare Ltd., Inc., Capto adhere, 10 mm IDx20 cm) that application, 5 column volumes of the equilibration buffer was equilibrated with the equilibration buffer prepared by US 2014/0046038A1 Feb. 13, 2014
adjusting 10 mmol/L Tris buffer (pH 8.0) to pH 7.0 with the factured by GE Healthcare Ltd., Inc., Capto adhere, 10 mm citric acid solution. After completion of the application, the IDX20 cm) that was equilibrated with the equilibration buffer equilibration buffer was passed through the column. A part of prepared by adjusting 10 mmol/L Tris buffer (pH 7.1) to pH the column non-adsorbed fraction was pooled as a Capto 6.0 with the citric acid solution. After completion of the adhere eluate. application, the equilibration buffer was passed through the 0132) The resulting Capto adhere eluate was adjusted to column. A part of the column non-adsorbed fraction was pH 4.5 with acetic acid, and then passed through the activated pooled as a Capto adhere eluate. carbon filter (manufactured by CUNO Ltd., Zeta carbon filter, 0140. The resulting Capto adhere eluate was adjusted to 25 cm), and pooled as an activated carbon eluate B. pH 4.5 with acetic acid, and then passed through the activated 0133. The resulting activated carboneluate B was adjusted carbon filter (manufactured by CUNO Ltd., Zeta carbon filter, to pH 8.0 with the Tris solution, and then filtered using a filter 25 cm), and pooled as an activated carbon eluate B. The to obtain a filtrate. The resulting filtrate was applied to the resulting activated carbon eluate B was used as a final Mab C anion exchange chromatography column (manufactured by purified product. GE Healthcare Ltd., Inc., Q Sepharose, 11 mm IDx20 cm) 0.141. The contents of polymers, degradation products, that was equilibrated with the equilibration buffer consisting and host cell proteins in the final Mab C purified product were of 10 mmol/L Tris buffer (pH 8.0). After completion of the application, 5 column volumes of the equilibration buffer analyzed in the same manner as in Example 1. were passed through the column. The column non-adsorbed 0142. The analysis results of the final Mab C purified fraction was pooled as a Q Sepharose eluate B. The resulting product are shown in FIGS. 1, 2, and 3. According to the Q Sepharose eluate B was used as a final Mab B purified present purification method, the Mab C purified product product. could be obtained, in which the contents of polymers and 0134. The contents of polymers, degradation products, degradation products were less than 1%, respectively and the and host cell proteins in the final Mab B purified product were content of host cell proteins was less than 10 ng/mg. analyzed in the same manner as in Example 1. 0135. The analysis results of the final Mab B purified Example 4 product are shown in FIGS. 1, 2, and 3. According to the present purification method, the Mab B purified product Mab A Purification 2 (Non-Adsorption Mode could be obtained, in which the contents of polymers and Purification Comprising Activated Carbon) degradation products were less than 1%, respectively and the content of host cell proteins was less than 10 ng/mg. 0143 Approximately 100 mL of CHO cell culture super natant containing monoclonal antibodies (Mab A) that were Example 3 previously clarified by microfiltration was adjusted to pH 4.5 with acetic acid. The formed precipitates were removed by Mab C Purification 1 (Non-Adsorption Mode centrifugation to obtain a clarified solution. Purification Comprising Activated Carbon) 0144. Subsequently, Mab A purification comprising acti vated carbon was carried out in a non-adsorption mode by the 0136. Approximately 600 mL of CHO cell culture super following procedure. First, the resulting clarified solution natant containing monoclonal antibodies (Mab C) that were was passed through the activated carbon filter (manufactured previously clarified by microfiltration was adjusted to pH 4.5 by CUNO Ltd., Zeta carbon filter, 25 cm), and pooled as an with acetic acid. The formed precipitates were removed by activated carbon eluate A. centrifugation and a filter. The resulting clarified solution was neutralized with a Tris Solution, and concentrated to approxi 0145 The resulting activated carbon eluate A was applied mately 6-fold using the Pellicon 3 Ultracel membrane (manu to the cation exchange chromatography column (manufac factured by millipore Corp., 30 kD, 0.11 m). After concen tured by millipore Corp., ProRes S, 3 mm IDx20 cm) that was tration, the buffer was exchanged with 10 mmol/L Tris buffer equilibrated with the equilibration buffer consisting of 10 (pH 7.1) to obtain a concentrated/buffer-exchanged solution. mmol/L acetic acid buffer (pH 4.5). After completion of the 0.137 Subsequently, Mab C purification comprising acti application, 7 column volumes of the equilibration buffer vated carbon was carried out in a non-adsorption mode by the were passed through the column. A part of the column non following procedure. First, the resulting concentrated/buffer adsorbed fraction was pooled as a ProRes Seluate. exchanged solution was passed through the activated carbon 0146 The resulting ProRes S eluate was passed through filter (manufactured by CUNO Ltd., Zeta plus EXT charged the activated carbon filter (manufactured by CUNO Ltd., Zeta depth filter, 25 cm), and pooled as an activated carbon eluate carbon filter, 25 cm), and pooled as an activated carbon A. eluate B. 0.138. The resulting activated carbon eluate A was applied 0147 The resulting activated carbon eluate B was diluted to the anion exchange chromatography column (manufac 4-fold using 5 mmol/L Tris buffer (pH 8.0) and then neutral tured by GE Healthcare Ltd., Inc., Q Sepharose, 11 mm ized with the Tris solution, and filtered using a filter. There IDX20 cm) that was equilibrated with the equilibration buffer after, the filtrate was applied to the anion exchange chroma consisting of 10 mmol/L Tris buffer (pH 7.1). After comple tography column (manufactured by GE Healthcare Ltd., Inc., tion of the application, 5 column volumes of the equilibration Q Sepharose, 11 mm IDX20 cm) that was equilibrated with buffer were passed through the column. The column non the equilibration buffer consisting of 10 mmol/L Tris buffer adsorbed fraction was pooled as a Q Sepharose eluate. (pH 8.0). After completion of the application, 5 column vol 0.139. Approximately 10 mmol/L of citric acid/sodium cit umes of the equilibration buffer were passed through the rate was added to the resulting Q Sepharose pooled solution, column. The column non-adsorbed fraction was pooled as a Q and adjusted to pH 6.0 with hydrochloric acid, and then Sepharose eluate. The resulting Q Sepharose eluate was used applied to the multimodal chromatography column (manu as a final Mab A purified product. US 2014/0046038A1 Feb. 13, 2014
0148. The contents of polymers, degradation products, previously clarified by microfiltration was adjusted to pH 4.5 and host cell proteins in the final Mab A purified product were with acetic acid. The formed precipitates were removed by analyzed in the same manner as in Example 1. centrifugation to obtain a clarified solution. 014.9 The analysis results of the final Mab A purified 0158. Subsequently, Mab C purification comprising acti product are shown in FIGS. 4, 5, and 6. According to the vated carbon was carried out in a non-adsorption mode by the present purification method, the Mab A purified product following procedure. First, the resulting clarified solution could be obtained, in which the contents of polymers and was passed through the activated carbon filter (manufactured degradation products were less than 1%, respectively and the by CUNO Ltd., Zeta carbon filter, 25 cm), and pooled as an content of host cell proteins was less than 10 ng/mg. activated carbon eluate A. 0159. The resulting activated carbon eluate A was applied Example 5 to the cation exchange chromatography column (manufac tured by millipore Corp., ProRes S, 3 mm IDx20 cm) that was Mab B Purification 2 (Non-Adsorption Mode equilibrated with the equilibration buffer consisting of 10 Purification Comprising Activated Carbon) mmol/L acetic acid buffer (pH 4.5). After completion of the 0150. Approximately 100 mL of CHO cell culture super application, 5 column volumes of the equilibration buffer natant containing monoclonal antibodies (Mab B) that were were passed through the column. A part of the column non previously clarified by microfiltration was adjusted to pH 4.5 adsorbed fraction was pooled as a ProRes Seluate. with acetic acid. The formed precipitates were removed by 0160 The resulting ProRes S eluate was passed through centrifugation to obtain a clarified solution. the activated carbon filter (manufactured by CUNO Ltd., Zeta 0151. Subsequently, Mab B purification comprising acti carbon filter, 25 cm), and pooled as an activated carbon vated carbon was carried out in a non-adsorption mode by the eluate B. following procedure. First, the resulting clarified solution 0.161 The resulting activated carbon eluate B was diluted was passed through the activated carbon filter (manufactured 4-fold using 5 mmol/L Tris buffer (pH 7.0) and then neutral by CUNO Ltd., Zeta carbon filter, 25 cm), and pooled as an ized with the Tris solution, and filtered using a filter. There activated carbon eluate A. after, the filtrate was applied to the anion exchange chroma 0152 The resulting activated carbon eluate A was applied tography column (manufactured by GE Healthcare Ltd., Inc., to the cation exchange chromatography column (manufac Q Sepharose, 11 mm IDX20 cm) that was equilibrated with tured by millipore Corp., ProRes S, 3 mm IDx20 cm) that was the equilibration buffer consisting of 10 mmol/L Tris buffer equilibrated with the equilibration buffer consisting of 10 (pH 7.0). After completion of the application, 5 column vol mmol/L acetic acid buffer (pH 4.5). After completion of the umes of the equilibration buffer were passed through the application, 7 column volumes of the equilibration buffer column. The column non-adsorbed fraction was pooled as a Q were passed through the column. A part of the column non Sepharose eluate. The resulting Q Sepharose eluate was used adsorbed fraction was pooled as a ProRes Seluate. as a final Mab C purified product. 0153. The resulting ProRes Seluate was passed through 0162 The contents of polymers, degradation products, the activated carbon filter (manufactured by CUNO Ltd., Zeta and host cell proteins in the final Mab C purified product were carbon filter, 25 cm), and pooled as an activated carbon analyzed in the same manner as in Example 1. eluate B. 0163 The analysis results of the final Mab C purified 0154 The resulting activated carbon eluate B was diluted product are shown in FIGS. 4, 5, and 6. According to the 4-fold using 5 mmol/L Tris buffer (pH 8.0) and then neutral present purification method, the Mab C purified product ized with the Tris solution, and filtered using a filter. There could be obtained, in which the contents of polymers and after, the filtrate was applied to the anion exchange chroma degradation products were less than 1%, respectively and the tography column (manufactured by GE Healthcare Ltd., Inc., content of host cell proteins was less than 10 ng/mg. Q Sepharose, 11 mm IDX20 cm) that was equilibrated with the equilibration buffer consisting of 10 mmol/L Tris buffer Example 7 (pH 8.0). After completion of the application, 5 column vol umes of the equilibration buffer were passed through the Mab A Purification 3 (Purification Comprising column. The column non-adsorbed fraction was pooled as a Q Activated Carbon) Sepharose eluate. The resulting Q Sepharose eluate was used as a final Mab B purified product. 0164. Approximately 200 mL of CHO cell culture super 0155 The contents of polymers, degradation products, natant containing monoclonal antibodies (Mab A) that were and host cell proteins in the final Mab B purified product were clarified by microfiltration was adjusted to pH 4.5 with acetic analyzed in the same manner as in Example 1. acid. The formed precipitates were removed by centrifuga 0156 The analysis results of the final Mab B purified tion to obtain a clarified solution A. product are shown in FIGS. 4, 5, and 6. According to the 0.165 Subsequently, activated carbon (manufactured by present purification method, the Mab B purified product Japan EnviroChemicals, Ltd, SHIRASAGIP) was added to could be obtained, in which the contents of polymers and approximately 60 mL of the resulting clarified solution A, and degradation products were less than 1%, respectively and the mixed. Thereafter, the mixture was subjected to centrifuga content of host cell proteins was less than 10 ng/mg. tion and filtration using a filter to obtain an activated carbon eluate. Example 6 0166 The resulting activated carbon eluate was diluted 4-fold using 5 mmol/L Tris buffer, and then adjusted to pH 8.0 Mab C Purification 2 (Non-Adsorption Mode with the Tris solution. Thereafter, the resultant was applied to Purification Comprising Activated Carbon) the anion exchange chromatography column (manufactured (O157 Approximately 100 mL of CHO cell culture super by GE Healthcare Ltd., Inc., Q Sepharose, 5 mm IDx20 cm) natant containing monoclonal antibodies (Mab C) that were that was equilibrated with the equilibration buffer consisting US 2014/0046038A1 Feb. 13, 2014 of 10 mmol/L Tris buffer (pH 8.0). After completion of the Example 1, and the Mab B purification intermediate and the application, 5 column volumes of the equilibration buffer final purified productobtained in Example 8 and Comparative were passed through the column. The column non-adsorbed Example 2 was analyzed out as follows. fraction was pooled as a Q Sepharose eluate. 0173 The recovery rate of each purification process and 0167. The resulting Q Sepharose eluate was adjusted to the total recovery rate of the entire purification process were pH 5.0 with the acetic acid solution. Thereafter, the resultant analyzed by protein A affinity HPLC. was applied to the cation exchange chromatography column 0.174. The results of recovery rate of each process and total (manufactured by Applied Biosystems, POROS XS, 5 mm recovery rate in the Protein A purification and the activated IDX20 cm) that was equilibrated with the equilibration buffer carbon purification of Mab A and Mab Bare shown in FIGS. consisting of 10 mmol/L acetic acid buffer (pH 5.0). After 7 and 8. completion of the application, 5 column Volumes of the 0.175. The total recovery rate of the activated carbon puri equilibration buffer were passed through the column. Next, fication was almost equal to that of the protein. A purification, elution was carried out with a salt concentration gradient and the recovery rate was as high as 40% or more. (10-column Volumes) of gradually increasing the salt concen 0176 The contents of the polymers and the degradation tration in a 10 mmol/L acetic acid buffer (pH 5.0) containing products in the purification intermediate and the final purified 0.3 mol/L sodium chloride. A part of the column-eluted frac product were analyzed by gel filtration HPLC. tion was pooled as a POROS XS eluate. The POROS XS 0177. With respect to the purification intermediate and the eluate was used as a final Mab A purified product. final purified product in the protein A purification and the Example 8 activated carbon purification of Mab A and Mab B, the con tents of the polymers are shown in FIGS. 9 and 11, and the Mab B Purification 3 (Purification Comprising contents of the degradation products are shown in FIGS. 10 Activated Carbon) and 12, respectively. 0168 Approximately 225 mL of CHO cell culture super 0.178 Irrespective of the type of monoclonal antibodies, natant containing monoclonal antibodies (Mab B) that were both of the purification methods showed that contents of the clarified by microfiltration was adjusted to pH 4.5 with acetic polymers and the degradation products in the final purified acid. The formed precipitates were removed by centrifuga product (cation) were equivalent. Meanwhile, when the con tion to obtain a clarified solution B. tents of the polymers and the degradation products were 0169. Subsequently, activated carbon (manufactured by compared between the protein A purification process and the Japan EnviroChemicals, Ltd, SHIRASAGIP) was added to activated carbon purification process, the activated carbon approximately 60 mL of the resulting clarified solution B, and purification process showed lower contents of the polymers mixed. Thereafter, the mixture was subjected to centrifuga and the degradation products than the protein A purification tion and filtration using a filter to obtain an activated carbon process, in which the contents were less than 2%. eluate. 0179 The contents of the host cell proteins per 1 mg of 0170 The resulting activated carbon eluate was diluted protein in the purification intermediate and the final purified 4-fold using 5 mmol/L Tris buffer, and then adjusted to pH 8.0 product were analyzed by ELISA. with the Tris solution. Thereafter, the resultant was applied to 0180. With respect to the protein A purification and the the anion exchange chromatography column (manufactured activated carbon purification of Mab A and Mab B, the con by GE Healthcare Ltd., Inc., Q Sepharose, 5 mm IDx20 cm) tents of the host cell proteins per 1 mg of protein in the that was equilibrated with the equilibration buffer consisting purification intermediate and the final purified product are of 10 mmol/L Tris buffer (pH 8.0). After completion of the shown in FIGS. 13 and 14. application, 5 column volumes of the equilibration buffer 0181 Irrespective of the type of monoclonal antibodies, were passed through the column. The column non-adsorbed the protein A purification and the activated carbon purifica fraction was pooled as a Q Sepharose eluate. tion showed that contents of the host cell proteins were 0171 The resulting Q Sepharose eluate was adjusted to equivalent. The contents of the host cell proteins in the final pH 5.1 with the acetic acid solution. Thereafter, the resultant purified products were also equivalent, in which the contents was applied to the cation exchange chromatography column were less than 10 ng/mg of protein. (manufactured by Applied Biosystems, POROS XS, 5 mm 0182. These results showed that the contents of the host IDX20 cm) that was equilibrated with the equilibration buffer cell proteins in the purification intermediates were equivalent consisting of 10 mmol/L acetic acid buffer (pH 5.0). After between the protein A purification and the activated carbon completion of the application, 5 column Volumes of the purification, whereas the activated carbon purification equilibration buffer were passed through the column. Next, showed lower contents of the polymers and the degradation elution was carried out with a salt concentration gradient products, indicating that the proteins with much lower con (10-column Volumes) of gradually increasing the salt concen tent of impurities can be obtained by the activated carbon tration from 10 mmol/Lacetic acid buffer (pH 5.0) containing purification. 0.3 mol/L sodium chloride. A part of the column-eluted frac tion was pooled as a POROS XS eluate. The POROS XS Example 10 eluate was used as a final Mab B purified product. Inhibition of Antibody Degradation by Activated Example 9 Carbon Analysis of Mab A Purified Product and Mab B 0183 The cell culture supernatant containing monoclonal Purified Product antibodies (Mab B) was adjusted to pH 4.5 with acetic acid. 0172. The Mab A purification intermediate and the final The formed precipitates were removed using a filter to obtain purified product obtained in Example 7 and Comparative a clarified solution. US 2014/0046038A1 Feb. 13, 2014
0184 Subsequently, the activated carbon (manufactured an alkali, respectively. The formed precipitates were removed by Japan EnviroChemicals, Ltd, SHIRASAGIP) was added by a filter to obtain pH-adjusted clarified solutions. to the resulting clarified solution, and mixed. After retaining 0194 Subsequently, the activated carbon (manufactured for 24 hours, the Supernatant from which the activated carbon by Japan EnviroChemicals, Ltd, SHIRASAGIP) was added was removed was provided for SDS-PAGE analysis. As a to approximately 10 mL of each of the pH-adjusted clarified control, the solution without addition of the activated carbon Solutions, and mixed. Then, each mixture was centrifuged to was manipulated in the same manner as above, and provided obtain each activated carbon eluate. Each of the pH-adjusted for SDS-PAGE analysis under non-reduction conditions. clarified solutions without addition of the activated carbon 0185. The results of SDS-PAGE analysis are shown in FIG. 15. The control (C) showed stronger bands of the deg was used as each of pH control. radation products than the clarified solution (A), and the 0.195 With respect to each of the pH-adjusted activated addition of the activated carbon (B) caused no strong band of carbon eluates and each of the pH controls, the contents of the the degradation products. host cell proteins per 1 mg of protein were analyzed by 0186 These results indicate that formation of degradation ELISA. products was inhibited by addition of activated carbon. (0196. The reduction rates of host cell proteins (HCPLRV) were calculated using the contents of the host cell proteins per Example 11 1 mg of protein by the following Equation. reduction rate of host cell protein (HCPLRV)=-Logo Inhibition of Antibody Reduction by Activated (content of host cell protein per 1 mg of protein Carbon of activated carbon eluate? content of host cell 0187. The activated carbon (manufactured by Japan Envi protein per 1 mg of protein of control) (Equation) roChemicals, Ltd, SHIRASAGI P) was added to the cell culture Supernatant containing monoclonal antibodies (Mab (0197) The reduction rates of host cell proteins (HCPLRV) D), and mixed. After removing the activated carbon, the at each pH by the activated carbon treatment are shown in Supernatant was maintained under anaerobic conditions for FIG. 18. HCP LRV was 2 or higher at pH 4 and 5. HCP LRV 24 hours. After retaining 24 hours, the Supernatant was pro was in the range of 1-2 at pH 6, pH 7 and pH 8. Therefore, it vided for SDS-PAGE analysis under non-reduction condi was found that the effect of activated carbon treatment on host tions. As a control, the solution without addition of the acti cell protein reduction was higher at pH 4 and 5 than at pH 6. vated carbon was manipulated in the same manner as above, pH 7 and pH 8. and provided for SDS-PAGE analysis. 0198 The antibody concentration of each pH activated 0188 The results of SDS-PAGE analysis are shown in carbon eluate was analyzed by protein A affinity HPLC. FIG. 16. The bands (H chain, L chain) corresponding to 0199 When the antibody concentration of the activated antibody reduction were observed in the control (C), com carbon eluate at pH 7 was regarded as 100, the relative anti pared to the culture Supernatant (A), but no bands (H chain, L body concentration (%) at each pH was shown in FIG. 2019. chain) corresponding to antibody reduction were observed in The antibody concentration at pH 4 was lower than that at the activated carbon treatment (B). other pH, and was approximately 70% of the antibody con 0189 These results indicate that formation of reduced centration at pH 7. At other pH, the antibody concentrations products was inhibited by addition of activated carbon. were within the range of +10% of the antibody concentration at pH 7. With respect to the activated carbon treatment, the Example 12 relative antibody concentrations and the antibody recovery rates at pH 5. pH 6, pH 7 and pH 8 were higher than those at DNA Analysis of Mab A Purified Product and Mab B Purified Product pH 4. 0200. The results of reduction rates of host cell proteins (0190. DNA analysis of the final Mab A purified product and relative antibody concentrations at each pH Suggested obtained in Example 7, the Mab A purified product obtained that it is possible to perform the activated carbon treatment at in Comparative Example 1, the final Mab B purified product any pH of pH 4 to 8, and preferably pH 4 to 6. obtained in Example 8, and the Mab B purified product obtained in Comparative Example 2 was carried out by Threshold method. Example 14 0191 DNA contents per 1 mg of the proteins in the final purified product of the activated carbon purification and in the Effect of Activated Carbon Raw Material in purified product of the Protein A purification with respect to Activated Carbon Purification Mab A and Mab B are shown in FIG. 17. 0.192 The DNA contents of the final purified product and 0201 The CHO cell culture supernatant containing mono the purified product are equivalent between both purifica clonal antibodies (Mab B) clarified by microfiltration was tions, in which the contents were 10 pg/mg or less. adjusted to pH 4.6 with acetic acid. The formed precipitates were removed by centrifugation and filtration using a filter to Example 13 obtain a clarified solution B. 0202 Subsequently, each of the activated carbons listed in pH Effect in Activated Carbon Purification Table 1 was added to approximately 10 mL of the resulting 0193 The CHO cell culture supernatants containing clarified solution B, and mixed. Then, the mixtures were monoclonal antibodies (Mab B) clarified by microfiltration Subjected to centrifugation and filtration using a filter to were adjusted to pH 4, pH 5, pH 6, pH 7, pH 8 with an acid or obtain activated carbon eluates. US 2014/0046038A1 Feb. 13, 2014
TABLE 1. Comparative Example 2 List of activated carbon evaluated Mab B Purification (Purification Comprising Protein A Affinity Chromatography) Name Raw material Manufacturer SHIRASAGIP Wood Japan EnviroChemicals, Ltd. 0208. The clarified solution B obtained in Example 8 was SHIRASAGI DO-2 Coconut-shell Japan EnviroChemicals, Ltd. adjusted to pH 6.4 with the Tris solution. Approximately 60 SHIRASAGIDO-5 Coal Japan EnviroChemicals, Ltd. mL of this solution was applied to the protein A affinity chromatography column (manufactured by GE Healthcare Ltd., Inc., MabSelect SuRe, 5 mm IDx20 cm) that was equili 0203 The contents of the polymers and the degradation brated with the equilibration buffer consisting of 10 mmol/L products and the contents of host cell proteins in each of the Tris buffer (pH 7.0). After completion of the application, the activated carbon eluates were analyzed by gel filtration HPLC column was washed with 5 column volumes of 10 mmol/L and by ELISA, respectively. Tris buffer (pH 7.0) containing 1 mol/L sodium chloride and 0204 The analysis results of each activated carbon eluate the equilibration buffer. Next, elution was carried out using 5 were shown in FIGS. 20, 21, and 22. All of the activated column volumes of 100 mmol/L glycine buffer (pH 3.2). The carbon eluates obtained by SHIRASAGI P, SHIRASAGI column-eluted fraction was pooled as a MabSelect SuRe DO-2 and SHIRASAGI DO-5 showed lower contents of eluate. polymers, degradation products, and host cell proteins than 0209. The resulting MabSelect SuRe eluate was adjusted the culture Supernatant. In particular, the activated carbon to pH 8.0 with the Tris solution. This solution was applied to eluate obtained by using SHIRASAGIP of which raw mate the anion exchange chromatography column (manufactured rial is wood, showed much lower contents of degradation by GE Healthcare Ltd., Inc., Q Sepharose, 5 mm IDx20 cm) products and host cell proteins than other activated carbons. that was equilibrated with the equilibration buffer consisting of 10 mmol/L Tris buffer (pH 8.0). After completion of the Comparative Example 1 application, 5 column volumes of equilibration buffer were passed through the column. The column non-adsorbed frac Mab A Purification (Purification Comprising Protein tion was pooled as a Q Sepharose eluate. A Affinity Chromatography) 0210. The resulting Q Sepharose eluate was adjusted to pH 5.0 with the acetic acid solution. This solution was applied 0205 The clarified solution A obtained in Example 7 was to the cation exchange chromatography column (manufac adjusted to pH 6.4 with the Tris solution. Approximately 60 tured by Applied Biosystems, POROS XS, 5 mm IDx20 cm) mL of this solution was applied to the protein A affinity that was equilibrated with the equilibration buffer consisting chromatography column (manufactured by GE Healthcare of 10 mmol/Lacetic acid buffer (pH 5.0). After completion of Ltd., Inc., MabSelect SuRe, 5 mm IDx20 cm) that was equili the application, 5 column volumes of equilibration buffer brated with the equilibration buffer consisting of 10 mmol/L were passed through the column. Next, elution was carried Tris buffer (pH 7.0). After completion of the application, the out with a salt concentration gradient (10-column Volumes) column was washed with 5 column volumes of 10 mmol/L of gradually increasing the salt concentration in the 10 Tris buffer (pH 7.0) containing 1 mol/L sodium chloride and mmol/L acetic acid buffer (pH 5.0) containing 0.3 mol/L the equilibration buffer. Next, elution was carried out using 5 sodium chloride. A part of the column-eluted fraction was column volumes of 100 mmol/L glycine buffer (pH 3.2). The pooled as a POROS XS eluate. The POROS XS eluate was column-eluted fraction was pooled as a MabSelect SuRe used as a final Mab B purified product. eluate. 0211 Although the present invention has been described 0206. The resulting MabSelect SuRe eluate was adjusted in detail with reference to the specific embodiments, it will be to pH 8.0 with the Tris solution. This solution was applied to apparent to those skilled in the art that various modifications the anion exchange chromatography column (manufactured and changes may be made thereto without departing from the by GE Healthcare Ltd., Inc., Q Sepharose, 5 mm IDx20 cm) Scope and spirit of the invention. that was equilibrated with the equilibration buffer consisting 1. A method for purifying a protein, wherein the protein is of 10 mmol/L Tris buffer (pH 8.0). After completion of the separated from impurities using an activated carbon to obtain application, 5 column volumes of equilibration buffer were the protein with a low content of impurities. passed through the column. The column non-adsorbed frac 2. The purification method according to claim 1, wherein tion was pooled as a Q Sepharose eluate. the protein has a molecular weight of 30000 or more. 0207. The resulting Q Sepharose eluate was adjusted to 3. The purification method according to claim 1, wherein pH 5.0 with the acetic acid solution. This solution was applied the protein is a glycoprotein. to the cation exchange chromatography column (manufac 4. The purification method according to claim 3, wherein tured by Applied Biosystems, POROS XS, 5 mm IDx20 cm) the glycoprotein is an antibody. that was equilibrated with the equilibration buffer consisting 5. The purification method according to claim 1, wherein of 10 mmol/Lacetic acid buffer (pH 5.0). After completion of the protein is a genetically modified protein. the application, 5 column volumes of equilibration buffer 6. The purification method according to claim 1, wherein were passed through the column. Next, elution was carried the impurities are any one of host cell proteins, protein-de out with a salt concentration gradient (10-column Volumes) rived polymers, protein-derived degradation products, or of gradually increasing the salt concentration in the 10 DNAS. mmol/L acetic acid buffer (pH 5.0) containing 0.3 mol/L 7. The purification method according to claim 1, wherein sodium chloride. A part of the column-eluted fraction was the method is carried out in a non-adsorption mode. pooled as a POROS XS eluate. The POROS XS eluate was 8. The purification method according to claim 1, wherein used as a final Mab A purified product. the separation is carried out at pH 3 to 8. US 2014/0046038A1 Feb. 13, 2014 15
9. The purification method according to claim 1, wherein the activated carbon is an activated carbon from wood. 10. The purification method according to claim 1, wherein the activated carbon has an average micropore diameter of 0.5 to 5 nm. 11. A method for preparing a protein, comprising the puri fication method of claim 1. 12. The preparation method according to claim 11, wherein protein A chromatography is not used. 13. The preparation method according to claim 11, com prising any one of anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chroma tography, and multimodal chromatography. 14. The preparation method according to claim 11, com prising at least one adsorption-mode chromatography. 15. A protein that is prepared by the method of claim 11. k k k k k