method for purifying an anti-il-13 antibody from a sample mixture comprising an anti-il-13 antibody

专利摘要:
ISOLATION AND PURIFICATION OF ANTI-IL-13 ANTIBODIES WITH THE USE OF PROTEIN A AFFINITY CHROMATOGRAPHY These are methods for the isolation and purification of anti-IL-13 antibodies, in which the use of an affinity chromatographic step results in an antibody composition sufficiently pure for pharmaceutical uses. The methods described in this document comprise viral pH reduction / inactivation, utrafiltration / diafiltration, affinity chromatography (for example, protein A affinity chromatography), ion exchange chromatography, and hydrophobic chromatography. In addition, the present invention is directed to pharmaceutical compositions that comprise one or more antibodies of the present invention.
公开号:BR112012009289B1
申请号:R112012009289-8
申请日:2010-10-20
公开日:2021-01-05
发明作者:Robert K. Hickman
申请人:Abbvie Inc.；
IPC主号:

专利说明:

Cross-reference to related order
[001] This application claims the benefit of U.S. provisional application no. serial 61 / 253,411, filed on October 20, 2009, which is incorporated herein by reference, in its entirety. Fundamentals of the invention
[002] Human IL-13 is a 17-kDa glycoprotein cloned from activated T cells and is produced by activated T cells of the Th2, ThO and Thl lineage CD4 + T cells, CD8 + cells, and various populations of non-T cells , such as mast cells. (Zurawski and de Vries, 1994 Immunol Today, 15, 19-26). IL-13 promotes the switching of the immunoglobulin isotype to IgE in human B cells (Punnonen, Aversa et al. 1993 Proc Natl Acad Sci USA 90 3730-4) and suppresses the production of inflammatory cytokine in both humans and mice (de Waal Malefyt et al., 1993, J Immunol, 11.6370-81; Doherty et al., 1993, J Immunol, 151, 7151 -60). IL-13 binds to its cell surface receptors, IL-13Rα1 and IL-13Rα2. IL-13Rα1 interacts with IL-13 with a low affinity (KD ~ 10 nM), followed by the recruitment of IL-4R to form the high affinity heterodimeric signaling receptor complex (KD ~ 0.4 nM) (Aman et al., 1996, J Biol Chem, 271,29265-70; Hilton et al., 1996, Proc Natl Acad Sci USA, 93, 497-501). The IL-4P / IL-13Rα1 complex is expressed in many types of cells, such as B cells, monocyte / macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells and airway smooth muscle cells (Graber et al., 1998, Eur J Immunol, 28, 4286-98; Murata et al., 1998, Int Immunol, 10, 1103-10; Akaiwa et al., 2001, Cytokine, 13, 75-84) . Binding of the IL-13Rα1 / IL-4R receptor complex results in the activation of a variety of signal transduction pathways that include the signal transducer and transcription activator (ST AT6) and the insulin receptor substrate 2 pathways (IRS-2) (Wang et al, 1995, Blood, 864218-27; Takeda et al., 1996, J Immunol, 157, 3220-2). The IL-13Rα2 chain alone has a high affinity (KD ~ 0.25 to 0.4 nM) for IL-13 and functions both as a trap receptor that down-regulates IL-13 binding (Donaldson et al., 1998, J Immunol, 161, 2317-24) as a signaling receptor that induces the synthesis of TGF-β and fribose via the AP-I pathway in macrophages and possibly other cell types (Fichtner-Feigl, Strober et al. 2006 Nat Med 12 99-106).
[003] Several studies conducted in preclinical animal models for asthma indicate that IL-13 plays an important role in asthma. These data include resistance to asthma in IL-13 knockout mice, as well as inhibition of asthma phenotype with IL-13 antagonists (soluble IL-13 receptors, anti-IL-13 mAbs, etc.) in several mouse models (Wills- Karp and Chiara- monte, 2003, Curr Opin Pulm Med, 9 21-7; Wills- Karp, 2004, Immunol Rev, 202 175-90). Multiple studies have shown that pharmacological administration of recombinant IL-13 to the lungs of mice, as well as guinea pigs, induces airway mucus hypersecretion, eosinophilia and airway hyperresponsiveness ("AHR"; Grunig et al. , 1998, Science, 282, 2261-3; Wills-Karp et al., 1998, Science, 282, 2258-61; Kibe et al, 2003, Am J Respir Crit Care Med, 167, 50-6; Vargaftig and Singer , 2003, Am J Physiol Lung Cell Mol Physiol, 284, L260-9; Vargafig and Singer, 2003, Am J Respir Cell Mol Biol, 28, 410-9). These effects of IL-13 are reproduced in transgenic mouse systems with constitutive or inducible IL-13 expression (Zhu et al., 1999, J Clin Invest, 103, 779-88; Zhu et al., 2001, Am J Respir Crit Care Med, 164, S67- 70; Lanone et al., 2002, J Clin Invest, 110463-74). Chronic transgenic overexpression of IL-13 also induces subepithelial fibrosis and emphysema. Mice deficient in the IL-13 (and IL-4) STAT6 signaling molecule cease to develop allergen-induced AHR and mucus overproduction (Kuperman et al, 2002, Nat Med, 8, 885-9). Studies using soluble IL-13 receptor fusion protein (sIL-13Rα2Fc) have demonstrated the essential role of this cytokine in airway disease induced by experimental allergen ovalbumin (OVA) (Grunig et al., 1998 , Science, 282, 2261-3; Wilis-Karp et al., 1998, Science, 282, 2258-61; Taube et al., 2002, J Immunol, 169, 6482-9). The effectiveness of anti-IL-13 treatment has also been demonstrated in a chronic model of murine asthma. In addition to displaying characteristics of hypersecretion of mucus and AHR, this model of chronic asthma demonstrates several attributes of human disease that are absent in the most acute models. These include eosinophilia of the lung tissue located in inter-epithelial spaces, as well as smooth muscle fibrosis, as measured by increases in collagen deposition. The chronic asthma model is induced with repeated aerosol challenge with OVA in mice sensitive to OVA 1x / week for a total of 4 weeks. The anti-IL-13 antibody administered for the final 2 weeks of OVA challenge (from day 36 with efficacy readings assessed on day 53 of the study) significantly inhibited AHR, lung inflammation, goblet cell hyperplasia, hypersecretion mucus and airway fibrosis (Yang et al., 2005, J Pharmacol Exp Ther, 313, 8-15). IL-13 is involved in the pathogenesis of human asthma as high levels of IL-13 mRNA and protein have been detected in the lungs of asthmatic patients, which correlate with the severity of the disease (Huang et al. , 1995, J Immunol, 155, 2688-94). In addition, genetic polymorphisms of human IL-3, which lead to elevated levels of IL-13, have been identified and are associated with asthma and atopy (Heinzmann et al., 2000, Hum Mol Genet, 9, 549-59; Hoerauf et al, 2002, Microbes Infect, 4, 37-42; Vercelli, 2002, Curr Opin Allergy Clin Immunol, 2, 389-93; Heinzmann et al., 2003, J Allergy Clin Immunol, 112 , 735-9; Chen et al., 2004, J Allergy Clin Immunol, 114, 553-60; Vladich et al., 2005, J Clin Invest, 115, 747-54), and elevated levels of IL-13 have been detected in the lungs of asthma patients (Huang et al., 1995, J Immunol, 155, 2688-94; Arima et al., 2002, J Allergy Clin Immunol, 109, 980-7; Berry et al., 2004, J Allergy Clin Immunol, 114, 1106-9). A genetic lineage between IL-13 and asthma has also been demonstrated, as individuals with a polymorphism in the IL-13 gene that causes higher plasma levels of IL-13 are at increased risk for atropy and asthma (Wills- Karp, 2000, Respir Res, 1, 19-23).
[004] Due to the role of human IL-13 in a variety of human disorders, therapeutic strategies have been designed to inhibit or neutralize IL-13 activity. In particular, antibodies have been sought that bind to, and neutralize, IL-13 as a means to inhibit IL-13 activity. However, there is a need in the art for improved methods of producing and purifying such antibodies for pharmaceutical use. The present invention addresses this need. Summary of the invention
[005] In certain embodiments, the present invention is directed to isolated, purified antibodies and antibody fragments that bind to IL-13, as well as pharmaceutical compositions comprising such antibodies and fragments. In certain embodiments, the invention pertains to isolated antibodies or antigen binding parts thereof, which bind to human IL-13. The isolated anti-IL-13 antibodies of the present invention can be used in a clinical setting, as well as in research and development. In certain embodiments, the present invention is directed to an anti-IL-13 antibody that comprises the light and heavy chain sequences identified in Figure 1.
[006] Certain embodiments of the invention are directed to methods of purifying anti-IL-13 antibodies, or antigen binding parts thereof, from a sample matrix to provide antibodies substantially free of protein from host cell ("HCPs") and leached protein A. In certain respects, the sample matrix (or simply "sample") comprises a cell line employed to produce anti-IL-13 antibodies of the present invention. In particular aspects, the sample comprises a cell line used to produce human anti-IL-13 antibodies.
[007] In certain embodiments, the present invention provides a method of purifying IL-13 antibodies that comprises a primary recovery step to, among other things, remove cells and cell fragments. In certain embodiments of the method, the primary recovery step includes one or more depth filtration or centrifugation steps. For example, and not as a limitation, such centrifugation steps can be performed at approximately 7000 x g to approximately 11,000 x g. In addition, certain modalities of the method described above include a depth filtration step, such as a delipid depth filtration step (lipid removal).
[008] In certain modalities, the primary recovery sample is subjected to an affinity chromatography step. The affinity chromatography step comprises subjecting the primary recovery sample to a column comprising a suitable affinity chromatographic support. Non-limiting examples of such chromatographic supports include, but are not limited to, protein A resin, protein G resin, affinity supports that comprise the antigen against which the antibody of interest has been detached, and affinity supports. that they comprise an Fc-binding protein. Protein A resin is useful for affinity purification and antibody isolation (IgG). In one aspect, a protein A column is equilibrated with a suitable buffer prior to loading the sample. An example of a suitable buffer is a Tris / NaCl buffer, pH around 7.2. After this equilibration, the sample can be loaded onto the column. After loading the column, the column can be washed once or multiple times using, for example, the equilibration buffer. Other washes that include washes using different buffers can be used before column elution. The protein A column can then be eluted using a suitable elution buffer. An example of a suitable elution buffer is an acetic acid / NaCl buffer, pH around 3.5. The eluate can be monitored using techniques well known to those skilled in the art. For example, the absorbance in OD280 can be followed. The eluted fraction (s) of interest can then be prepared for further processing.
[009] In certain embodiments of the present invention, a low pH adjustment step follows protein A affinity chromatography. In such modalities, the protein A eluate comprising the alleged anti-IL-13 antibody, or part thereof of antigen binding thereof, it is subjected to a pH adjustment to a pH of about 3 to about 4. In certain aspects, the pH is adjusted to about 3.5. The low pH, among other things, promotes the reduction and / or inactivation of pH sensitive viruses that may be contaminating the sample. After an appropriate period of time, the pH is adjusted to between about 4.5 and about 6.0, which includes, but is not limited to, about 5.0, and the sample is subjected to further purification steps .
[0010] In certain embodiments, an ion exchange step follows protein A affinity chromatography or a low pH adjustment step. This ion exchange step can consist of a cation or anion exchange or a sequential combination of both. This step can consist of a single ion exchange procedure or it can include multiple ion exchange steps, such as a cation exchange step followed by an anion exchange step or vice versa. In one aspect, the ion exchange step consists of a one-step procedure. In another aspect, the ion exchange stage involves a two-stage ion exchange process. A cation exchange column is a column whose stationary phase comprises anionic groups. An example of such a column is a Fractogel ™ SO3-. This ion exchange capture chromatography step facilitates the isolation of antibodies from a sample. A suitable anion exchange column consists of a column whose stationary phase comprises cationic groups. An example of such a column is a Q Sepharose ™ column. An alternative is a Pall Mustang Q membrane cartridge. One or more ion exchange steps additionally isolate antibodies by reducing impurities, such as host cell proteins and DNA, and, where applicable, protein of affinity matrix. This anion exchange procedure is a continuous flow chromatography mode in which the antibodies of interest do not interact or bind to the anion exchange resin (or solid phase). However, many impurities do not interact and bind to the anion exchange resin. In a particular aspect, the ion exchange step consists of anion exchange chromatography.
[0011] The affinity chromatography eluent is prepared for ion exchange chromatography by adjusting the pH and ionic intensity of the sample buffer. For example, the affinity eluate can be adjusted to a pH of about 4.5 to about 8.5 in a 1 M Tris buffer. Before loading the sample (the affinity eluate) onto the ion exchange column, the column can be balanced with the use of a suitable buffer. An example of a suitable buffer is a Tris NaCl buffer with a pH of about 4.5 to about 8. After equilibration, the column can be loaded with the affinity eluate. After loading, the column can be washed once or multiple times with a suitable buffer. An example of a suitable buffer is the balancing buffer itself. The continuous flow collection can start, for example: as the absorbance (OD280) rises above about 0.2 AU.
[0012] In certain modalities, a first and second stage of ion exchange is performed after primary recovery or, otherwise, in the absence of an affinity chromatography stage. In certain modalities, the ion exchange sample is subjected to an intermediate filtration step, before the first ion exchange step, between the two ion exchange steps, or both. In certain aspects, this filtration stage comprises ultrafiltration / capture diafiltration ("UF / DF"). Among other things, such filtration facilitates the concentration and buffer exchange of anti-IL-13 antibodies and antigen binding parts thereof.
[0013] Certain embodiments of the invention provide a method comprising one or more steps of hydrophobic interactive chromatography ("HIC"). A suitable HIC column consists of one whose stationary phase comprises hydrophobic groups. A non-limiting example of such a column is a Fenil HP Sepharose ™ column. In certain circumstances, anti-IL-13 antibodies will form aggregates during the isolation / purification process. The inclusion of one or more HIC steps facilitates the reduction or elimination of such aggregations. HIC also helps to remove impurities. In certain embodiments, the HIC stage employs a high salt buffer to promote the interaction of anti-IL-13 antibodies (or aggregations thereof) with the hydrophobic column. Anti-IL-13 antibodies can then be eluted using lower concentrations of salt.
[0014] In certain embodiments, the HIC eluate is filtered using a viral removal filter, such as, but not limited to, a Ultipor DV50 ™ filter (Pall Corporation, East Hills, N.Y.). Alternative filters, such as Viresolve ™ filters (Millipore, Billerica, Mass.); Zeta Plus VR ™ filters (CUNO; Meriden, Conn.) and Planova ™ filters (Asahi Kasei Pharma, Planova Division, Buffalo Grove, 111.) can also be used in such modalities.
[0015] In certain embodiments, the invention is directed to one or more pharmaceutical compositions comprising an isolated anti-IL-13 antibody or antigen binding part thereof and an acceptable carrier. In one aspect, the composition additionally comprises one or more antibodies or antigen binding part thereof in addition to the anti-IL-13 antibody. In another aspect, the compositions additionally comprise one or more pharmaceutical agents.
[0016] The purity of the antibodies of interest in the resulting sample product can be analyzed using methods well known to the elements skilled in the art, for example, size exclusion chromatography, Poros ™ A HPLC assay, HCP ELISA, protein A ELISA, and western blot analysis. Brief description of the drawings
[0017] Figure 1 shows the heavy and light chain variable region sequences of a non-limiting example of an anti-IL-13 antibody.
[0018] Figure 2 presents a flowchart of the example cell culture process, which includes set points, control tests during the process and action limits.
[0019] Figure 3 presents a comparison of alternative cell culture process flow strategies.
[0020] Figure 4 presents a flowchart of the primary recovery capture chromatography process, which includes set points, control tests during the process and action limits.
[0021] Figure 5 presents a comparison of alternative catch flow and primary recovery strategies.
[0022] Figure 6 presents a flow chart of the process of fine purification, which includes set points, control tests during the process and action limits.
[0023] Figure 7 presents a comparison of alternative thin purification flow strategies. Detailed description of the invention
[0024] The present invention is directed to antibodies that bind to IL-13. In one aspect, the invention belongs to isolated antibodies, or antigen binding parts thereof, that bind to human IL-13. The anti-IL-13 antibody isolated from the present invention can be used in a clinical setting, as well as in research and development. The present invention also pertains to methods for the purification of anti-IL-13 antibodies, or antigen binding parts thereof. Suitable anti-IL-13 antibodies that can be purified in the context of the present invention are disclosed in PCT application no. PCT US2007 / 019660, which is hereby incorporated by reference, in its entirety, which includes the antibody which has subsequently been identified as ABT-308. The exemplary light and heavy chain sequences of the anti-IL-13 antibody are shown in Figure 1. The present invention also relates to pharmaceutical compositions comprising anti-IL-13 antibodies or binding parts of antigen described in this document. For clarity and not as a limitation, this detailed description is divided into the following subparts: 1. Definitions; 2. Antibody generation; 3. Production of antibody; 4. Purification of antibody; 5. Methods for testing the purity of the sample; 6. Additional modifications; 7. Pharmaceutical compositions; and 8. Uses of antibody. 1. Definitions
[0025] In order that the present invention can be more readily understood, certain terms are defined first.
[0026] The term "antibody" includes an immunoglobulin molecule that comprises four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain comprises a variable heavy chain region (abbreviated herein as HCVR or VH) and a heavy chain constant (CH) region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determination regions (CDRs), intercalates with regions that are more conserved, called structure regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminal to the carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0027] The term "antigen binding part" of an antibody (or "antibody part") includes fragments of an antibody that maintain the ability to specifically bind to an antigen (for example, hIL-13). It has been shown that the antigen binding function of an antibody can be performed by fragments of an entire antibody. Examples of binding fragments included in the term "antigen binding part" of an antibody include (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the articulation region; (iii) an Fd fragment comprising the VH and CH1 domains; (iv) an Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546, whose full instruction is here incorporated by reference), which comprises a VH domain; and (vi) an isolated complementarity determination region (CDR). Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made as a single chain protein, in which the VL and VH regions join to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883, whose integral instructions are hereby incorporated by reference). Such single chain antibodies are also intended to be included in the term "antigen binding part" of an antibody. Other forms of single chain antibodies, such as diabodies, are also included. Diabodies consist of bispecific bispecific antibodies, in which the VH and VL domains are expressed in a single polypeptide chain, but with the use of a linker that is too short to allow pairing between the two domains in the same chain thus forcing the domains to pair with complementary domains from another chain and creating two antigen binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1 121-1123, whose full instructions are hereby incorporated by reference). In addition, an antibody or antigen-binding part thereof may be part of a larger immunoa- sion molecule, formed by covalent or non-covalent association of the antibody or part of antibody with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin nucleus region to make a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6: 93-101, whose integral instruction is incorporated herein into reference title) and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make divalent and bi-tinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31 : 1047-1058, whose full instructions are hereby incorporated by reference). The antibody parts, such as Fab and F (ab ') 2 fragments, can be prepared from total antibodies using conventional techniques, such as digestion for papain or pepsin, respectively, of total antibodies. In addition, antibodies, antibody parts and immunoadhesion molecules can be obtained using standard DNA combining techniques, as described in this document. In one aspect, the antigen binding parts consist of complete domains or pairs of complete domains.
[0028] The phrase "human interleukin 13" (abbreviated herein as hIL-13, or IL-13), for use in the present invention, refers to a 17-kDa glycoprotein cloned from activated T cells ( Zurawski and de Vries, 1994 Immunol Today 15 19-26) and which is produced by activated T cells of the Th2 lineage. T ThO and Thl CD4 + cells, CD8 + T cells, and various non-T cell populations, such as mast cells, also produce IL-13 (Zurawski and de Vries, 1994 Immunol Day 15 19-26). The function of IL-13 includes promoting the switching of the immunoglobulin isotype to IgE in human B cells (Punnonen, Aversa et al. 1993 Proc Natl Acad Sci USA 90 3730-4) and suppressing the production of inflammatory cytokine in both humans and mice (de Waal et al., 1993 J Immunol 151 6370-81; Doherty et al., 1993 J Immunol 151 7151-60). IL-13 binds to cell surface receptors identified as IL-13Rα1 and IL-13Rα2. The IL-13Rα1 receptor interacts with IL-13 with a low affinity (KD ~ 10 nM), followed by the recruitment of IL-4R to form the high affinity heterodimeric signaling receptor complex (KD ~ 0.4 nM ) (Aman et al., 1996 J Biol Chem 271 29265-70; Hilton et al., 1996 Proc Natl Acad Sci USA 93 497-501). The IL-4R / IL-13Rα1 complex is expressed in many types of cells, such as B cells, monocyte / macrophages, dendritic cells, eosinophils, basophils, fibroblasts, endothelial cells, airway epithelial cells and smooth muscle cells airways (Graber et al, 1998 Eur J Immunol 28 4286-98; Murata et al., 1998 Int Immunol 10 1103-10; Akaiwa et al., 2001 Cytokine 13 75-84). Binding of the IL-13 Rα1 / IL-4R receptor complex results in the activation of a variety of signal transduction pathways that include signal transducer and transcription activator (ST AT6) and the insulin receptor substrate pathways -2 (IRS-2) (Wang et al., 1995 Blood 864218-27; Takeda et al., 1996 J Immunol 157 3220-2). The IL-13Rα2 chain alone has a high affinity (KD ~ 0.25 to 0.4 nM) for IL-13 and functions both as a trap receptor that negatively regulates IL-13 binding (Donaldson, Whitters et al. 1 98 J Immunol 161 2317-24), and as a signaling receptor that induces the synthesis of TGF-b and fibrosis via the AP-I pathway in macrophages and possibly other cell types (Fichtner-Feigl et al. , 2006 Nat Med 12 99-106). Nucleic acid encoding IL-13 is available as GenBank accession number NM_002188 and the polypeptide sequence is available as GenBank accession number NP_002179. The term human IL-13 is intended to include recombinant human IL-13 (rh IL-13), which can be prepared by standard recombinant expression methods.
[0029] The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably in this document. These terms, which are recognized in the art, refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable region of an antibody's light and heavy chain , or an antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH publication No. 91-3242, whose full instructions are hereby incorporated by reference). For the variable region of heavy chain, the hypervariable region is in the range from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2 and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region is in the range from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2 and amino acid positions 89 to 97 for CDR3.
[0030] The term "human antibody" includes antibodies that have variable and constant regions that correspond to human germline immunoglobulin sequences, as described by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, fifth edition, U.S. Department of Health and Human Services, NIH publication No. 91-3242). The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by site-specific or random mutagenesis in vitro or by somatic mutation in vivo), for example , in CDRs and in particular CDR3. Mutations can be introduced using the "selective mutagenesis approach". The human antibody can have at least one position replaced by an amino acid residue, for example, an activity-enhancing amino acid residue that is not encoded by the human germline immunoglobulin sequence. The human antibody can have up to twenty positions with amino acid residues that are not part of the human germline immunoglobulin sequence. In other modalities, up to ten, up to five, up to three or even two positions are replaced. In one embodiment, these substitutions are within the CDR regions. However, the term "human antibody", for use in the present invention, is not intended to include antibodies in which CDR sequences derived from the germline of another species of mammal, such as a mouse, have been used. grafted into human structure sequences.
[0031] The phrase "selective mutagenesis approach" includes a method of enhancing the activity of an antibody by individually selecting and mutating CDR amino acids in at least one appropriate selective mutagenesis position, hypermutation and / or contact position . A "selectively mutant" human antibody consists of an antibody that comprises a mutation at a selected position using a selective mutagenesis approach. In another aspect, the selective mutagenesis approach is intended to provide a preferred mutation method for individual amino acid residues selected in the heavy chain variable region CDR1, CDR2 or CDR3 (further in this document H1, H2 and H3, respectively), or the CDR1, CDR2 or CDR3 of the light chain variable region (hereinafter referred to as L1, L2 and L3, respectively) of an antibody. Amino acid residues can be selected from selective mutagenesis positions, contact positions or hypermutation positions. The individual amino acids are selected based on their position in the variable region of heavy or light chain. It should be understood that a hypermutation position can also consist of a contact position. In one respect, the selective mutagenesis approach consists of a "targeted approach". The language "targeted approach" is intended to include a method of mutating individual amino acid residues selected in the heavy chain variable region CDR1, CDR2 or CDR3 or in the light chain variable region CDR1, CDR2 or CDR3 targeted way, for example, a "group-oriented approach" or "CDR-oriented approach". In the "group-oriented approach", individual amino acid residues, in particular groups, are targeted for selective mutations that include groups I (which include L3 and H3), II (which include H2 and L1) and III (which include L2 and H1), with the groups being listed in order of preference for targeting. In the "CDR-targeted approach", the individual amino acid residues, in particular, CDRs, are targeted for selective mutations in the order of preference for targeting as shown below: H3, L3, H2, L1, H1 and L2 . The selected amino acid residue is subjected to mutation, for example, to at least two other amino acid residues, and the effect of the mutation on antibody activity is determined. Activity is measured as a change in antibody binding specificity / affinity, and / or antibody neutralizing potency. It should be understood that the selective mutagenesis approach can be used for the optimization of any antibody derived from any source that includes phage display transgenic animals with human IgG germline genes, human antibodies isolated from human B cells. The selective mutagenesis approach can be used on antibodies that cannot be further optimized with the use of phage exposure technology. It should be understood that antibodies from any source that include phage display transgenic animals (phage display) with human IgG germline genes, human antibodies isolated from human B cells can be subjected to retromutation before or after the selective mutagenesis approach.
[0032] The phrase "recombinant human antibody" includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected in a host cell, antibodies isolated from a recombinant human combinatorial antibody library, antibodies isolated from an animal (for example, a mouse) that is transgenic to human immunoglobulin genes (see, for example, Taylor, L, D., et al. (1992) Nucl. Acids Res. 20: 6287-6295, whose integral instruction is hereby incorporated by reference) or antibodies prepared, expressed, created or isolated by any other means involving splicing and sequences of human immunoglobulin gene to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences (see, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH publication No. 91-3242). In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, somatic mutagenesis in vivo) and, thus, the amino acid sequences of the VH and VL of recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. In certain modalities, however, such recombinant antibodies are the result of the selective mutagenesis or retromutation approach, or both.
[0033] An "isolated antibody" includes an antibody that is substantially free from other antibodies that has different antigen specificities (for example, an isolated antibody that specifically binds hIL-13 is substantially free of antibodies that specifically bind antigens in addition to hIL-13). An isolated antibody that specifically binds hIL-13 can bind IL-13 molecules from other species. In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals.
[0034] A "neutralizing antibody" (or an "antibody that neutralized hIL-13 activity") includes an antibody whose binding to hIL-13 results in inhibition of the biological activity of hIL-13. This inhibition of hIL-13 biological activity can be assessed by measuring one or more indicators of hIL-13 biological activity. These indicators of the biological activity of hIL-13 can be evaluated by means of one or more among several standard in vitro or in vivo assays known in the art.
[0035] The term "activity" includes activities such as the binding specificity / affinity of an antibody to an antigen, for example, an anti-hIL-13 antibody that binds to an IL- 13 and / or the neutralizing potency of an antibody, for example, an anti-hIL-13 antibody whose binding to hIL-13 inhibits the biological activity of hIL-13.
[0036] The phrase "surface plasmon resonance" includes an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting changes in protein concentrations within a biosensor matrix, for example, using of the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268- 277, whose full instructions are incorporated here.
[0037] The term "off", for use in the present invention, is intended to refer to the dissociation rate constant for dissociation of an antibody from the antibody / antigen complex.
[0038] The term "Kd", for use in the present invention, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
[0039] The phrase "nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but in one aspect it consists of double-stranded DNA.
[0040] The phrase "isolated nucleic acid molecule", for use in the present invention with reference to nucleic acids encoding antibodies or part of antibodies (for example, VH, VL, CDR3), for example, those that bind hIL-13 and include a nucleic acid molecule in which the nucleotide sequences that encode the antibody or part of antibody are free from other nucleotide sequences that encode antibodies or part of antibodies that bind antigens other than hIL- 13, such other sequences can naturally flank nucleic acid in human genomic DNA. Thus, for example, an isolated nucleic acid of the invention encoding a VH region of an anti-hIL-13 antibody does not contain other sequences encoding other VH regions that bind antigens in addition to, for example, hIL-13. The phrase "isolated nucleic acid molecule" is also intended to include sequences that encode bivalent bispecific antibodies, such as diamonds, in which the VH and VL regions contain no sequences other than the diabody sequences.
[0041] The phrase "recombinant host cell" (or simply "host cell") includes a cell in which a recombinant expression vector has been introduced. It must be understood that such terms are meant to refer not only to the present particular cell, but to the progeny of that cell. Due to the fact that certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included in the scope of the term "host cell", for use in the present invention.
[0042] The term "modification", for use in the present invention, is intended to refer to the alteration of one or more amino acids in the antibodies or antigen binding parts thereof. The change can be produced by adding, replacing or deleting an amino acid in one or more positions. The change can be produced using known techniques, such as PCR mutagenesis.
[0043] The term "about", for use in the present invention, is intended to refer to ranges approximately 10 to 20% greater or less than the mentioned value. In certain circumstances, a person skilled in the art will recognize that, due to the nature of the mentioned value, the term "about" may mean more or less than a 10 to 20% deviation from this value.
[0044] The phrase "viral reduction / inactivation", for use in the present invention, is intended to refer to a decrease in the number of viral particles in a particular sample ("reduction"), as well as a decrease in activity, for example, but not limited to, in infectivity or ability to replicate, viral particles and a particular sample ("inactivation"). Such decreases in the number and / or activity of the viral particles can be on the order of about 1% to about 99%, which includes about 20% to about 99%, which includes about 30% to about 99%, which includes about 40% to about 99%, which includes about 50% to about 99%, which includes about 60% to about 99%, which includes about 70% to about 99%, which includes about 80% to 99%, and that includes about 90% to about 99%. In certain non-limiting modalities, the amount of virus, if any, in the purified antibody product is less than the ID50 (the amount of virus that will infect 50 percent of a target population) for that virus, that is at least 10 times less than the ID50 for that virus, or at least 100 times less than the ID50 for that virus, or at least 1000 times less than the ID50 for that virus.
[0045] The phrase "contact position" includes an amino acid position in CDR1, CDR2 or CDR3 of the heavy chain variable region or the light chain variable region of an antibody that is occupied by an amino acid that comes into contact with the antigen in one of the twenty-six known antibody-antigen structures. If a CDR amino acid in any of the twenty-six known resolved structures of antibody-antigen complexes comes into contact with the antigen, then this amino acid can be considered as occupying a contact position. Contact positions are more likely to be occupied by an amino acid that comes into contact with antigens than in a non-contact position. In one aspect, a contact position consists of a CDR position that contains an amino acid that comes in contact with the antigen in more than 3 of the 26 structures (> 1.5%). In another aspect, a contact position consists of a CDR position that contains an amino acid that comes in contact with antigen in more than 8 of the 25 structures (> 32%). 2. Antibody generation
[0046] The term "antibody", for use in this section, refers to an intact antibody or an antigen binding fragment thereof.
[0047] The antibodies of the present description can be generated by a variety of techniques, which include immunizing an animal with the antigen of interest, followed by conventional monoclonal antibody methodologies, for example, a hybridization technique standard somatic cells of Kohler and Milstein (1975) Nature 256: 495. -While somatic cell hybridization procedures are preferred, in principle, other techniques for the production of monoclonal antibody can be employed, for example, viral transformation or oncogenic B lymphocytes.
[0048] An animal system for the preparation of hybridomas consists of the murine system. Hybridoma production is a well-established procedure. Immunization protocols and techniques for the isolation of splenocytes immunized for fusion are known in the art. Fusion partners (e.g. murine myeloma cells) and fusion procedures are also known.
[0049] An antibody can be a human, chimeric or humanized antibody. The chimeric or humanized antibodies of the present description can be prepared based on the sequence of a non-human monoclonal antibody prepared as described above. The DNA encoding light and heavy chain immunoglobulins can be obtained from the non-human hybridoma of interest and designed to contain non-murine immunoglobulin sequences (for example, human) using biology techniques standard molecular. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, murine CDR regions can be inserted into a human structure using methods known in the art (see, for example, US patent No. 5,225,539 to Winter, and US patent No. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).
[0050] In a non-limiting embodiment, the antibodies of this description are human monoclonal antibodies. Such human monoclonal antibodies directed against IL-13 can be generated with the use of transgenic or trans-chromosomal mice that carry parts of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include mice mentioned in this document such as HuMAb Mouse® (Medarex, Inc.), KM Mouse® (Medarex, Inc.), and XenoMouse® (Amgen).
[0051] In addition, alternative transchromosomal animal systems that express human immunoglobulin genes are available in the art and can be used to construct antibodies of this description, such as anti-IL-13 antibodies. For example, mice that carry both a human heavy-chain transchromosome and a human light-chain transchromosome, referred to as "TC mice", can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727. In addition, cows that carry human light and heavy chain chromosomes have been described in the art (for example, Kuroiwa et al. (2002) Nature Bio-technology 20: 889-894 and PCT application No. WO 2002/092812 ) and can be used to construct the anti-IL-13 antibodies of this description.
[0052] The recombinant human antibodies of the invention, which include, but are not limited to, anti-IL-13 antibodies, an antigen binding part of the same, or antibodies related to anti-IL-13 presented herein can be isolated by screening a recombinant combinatorial antibody library, for example, a scFv phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from lymphocytes humans. Methodologies for the preparation and screening of such libraries are known in the art. In addition to the commercially available kits for the generation of phage display libraries (for example, the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the phage display kit (phage display) Stratagene Sur- fZAPTM, catalog No. 240612, whose full instructions are incorporated here), examples of particularly accessible methods and reagents for use in generating and screening antibody exposure libraries can be found, for example, in Ladner et al., US patent no. 5,223,409; Kang et al. PCT publication no. WO 92/18619; Dower et al. PCT publication no. WO 91/17271; Winter et al. PCT publication no. WO 92/20791; Markland et al. PCT publication no. WO 92/15679; Breitling et al. PCT publication no. WO 93/01288; McCafferty et al. PCT publication no. WO 92/01047; Garrard et al. PCT publication no. WO 92/09690; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; McCafferty et al., Nature (1990) 348: 552-554; Griffiths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. 1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrard et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982; whose full instructions are hereby incorporated.
[0053] The human monoclonal antibodies of this description can also be prepared using SCID mice, in which human immune cells have been reconstructed in such a way that a human antibody response can be generated under immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
[0054] In certain embodiments, the methods of the invention include anti-IL-13 antibodies and part of antibodies, antibodies related to anti-IL-13 and part of antibodies, and human antibodies and part of antibodies with equivalent properties anti-IL-13 antibodies, such as high affinity binding to hIL-13 with low dissociation kinetics and high neutralization capacity. In one aspect, the invention provides treatment with an isolated human antibody, or antigen binding part thereof, which dissociates from hIL-13 with a Kd of about 1 x 10-8 M or less and a dissociation rate constant of 1 x 10-3 s- 1 or less, both determined by surface plasmon resonance. In specific non-limiting modalities, a purified anti-IL-13 antibody according to the invention competitively inhibits the binding of ABT-308 to IL-13 under physiological conditions.
[0055] In yet another embodiment of the invention, the antibodies or fragments thereof, such as, but not limited to, anti-IL-13 antibodies or fragments thereof, may be altered, wherein the antibody constant region is modi - stayed to reduce at least one biological effector function mediated by constant region in relation to an unmodified antibody. To modify an antibody of the invention in such a way as to exhibit reduced binding to the Fc receptor, the immunoglobulin constant region segment of the antibody can be subjected to mutation in particular regions necessary for Fc receptor (FcR) interactions (see, for example, example, Canfield and Morrison (1991) J. Exp. Med. 173: 1483-1491; and Lund et al. (1991) J. of Immunol. 147: 2657-2662, whose full instructions are incorporated here). The reduction in the antibody's FcR binding capacity may also reduce other effector functions that depend on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cell cytotoxicity. 3. Antibody production 3.1 General production strategies
[0056] To express an antibody of the invention, the DNAs encoding full and partial length heavy and light chains are inserted into one or more expression vectors, such that the genes are operatively linked to control sequences transcriptional and translational. (See, for example, US Patent No. 6,914,128, the full instruction of which is hereby incorporated by reference.) In this context, the term "operably linked" is intended to mean that an antibody gene is linked in a vector such that the transcriptional and translational control sequences within the vector satisfy their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into an expression vector using standard methods (for example, binding complementary restriction sites in the vector and fragment of the antibody gene, or blunt-ended binding if restriction sites are not present). Prior to insertion of the antibody or antibody-related heavy or light chain sequences, the expression vector can now load the antibody constant region sequences. For example, an approach to convert the anti-IL-13 antibody or VH and VL sequences related to the anti-IL-13 antibody to full-length antibody genes is to insert them into expression vectors that already encode the constants - light chain and heavy chain tests, respectively, such that the VH segment is operatively linked to the CH segment (s) within the vector and the VL segment is operatively linked to the CL segment within of the vector. In addition or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector in such a way that the signal peptide is linked in structure to the terminal amino of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain genes, a recombinant expression vector of the invention can carry one or more regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, optimizers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), whose full instruction is hereby incorporated by reference. Those skilled in the art will note that the design of the expression vector, which includes the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that target high levels of protein expression in mammalian cells, such as promoters and / or optimizers derived from cytomegalovirus (CMV) (such as the promoter / optimizer) CMV virus), simian virus 40 (SV40) (such as the SV40 promoter / optimizer), adenovirus, (for example, the adenovirus late major promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, for example, U.S. patent no. 5,168,062 by Stinski, U.S. patent no. 4,510,245 by Bell et al. And U.S. patent no. 4,968,615 by Schaffner et al., Whose full instructions are hereby incorporated by reference.
[0058] In addition to the antibody chain genes and regulatory sequences, a recombinant expression vector of the invention can carry one or more additional sequences, such as a sequence that regulates the replication of the vector in host cells (e.g., origins replication) and / or a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US patents Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al., Whose instructions are incorporated herein by reference). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, in a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate ductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).
[0059] An antibody, or antibody part, of the invention can be prepared by recombinant expression of immunoglobulin heavy and light chain genes in a host cell. To express an antibody in a combining manner, a host cell is transfected with one or more recombinant expression vectors that carry fragments of DNA encoding the antibody's immunoglobulin heavy and light chains, such that the heavy and light are expressed in the host cell and secreted in the medium in which the host cells are cultured, from such medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody light and heavy chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; The Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. patents nos. 4,816,397 & 6,914,128, whose full instructions are incorporated here.
[0060] For the expression of heavy and light chains, the expression vector (s) encoding the light and heavy chains is (are) transfected into a host cell using standard techniques. The various forms of the term "transfection" are intended to include a wide variety of techniques commonly used for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, such as mammalian host cells, is adequate due to the fact that such eukaryotic cells, and in particular mammary cells - beasts, are prone than prokaryotic cells to gather and secrete an immunologically active and properly folded antibody. The prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibody (Boss and Wood (1985) Immunology Today 6: 12-13, whose full instruction is incorporated herein by reference).
[0061] The host cells suitable for cloning and expressing DNA in the vectors in this document consist of the higher eukaryotic cells, yeasts or prokaryotes described above. Prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis r B. licheniformis (for example, B. licheniformis 41 P presented in DD 266,710, published on April 12, 1989), Pseudomonas, such as P. aeruginosis, and Streptomyces. A suitable E. coli cloning host consists of E. coli 294 (ATCC 31,446), although other strains, such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are suitable . These examples are illustrative rather than limiting.
[0062] In addition to prokaryotes, eukaryotic microbes such as yeast or filamentous fungi are suitable cloning or expression hosts for polypeptide encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species and strains are commonly available and are useful in this document, such as Schizocaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum ( ATCC 36.906), K. thermotolans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium, and hosts of Aspergillus, such as A. nidulans and A. niger.
[0063] Host cells suitable for the expression of glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculo-viral strains, variants and permissive insect host cells corresponding to hosts, such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa caliphalic NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus at present. document, in accordance with the present invention, particularly for the transfection of Spodoptera frugiperda cells. Cultures of cotton, corn, potato, soy, petunia, tomato and tobacco plant cells can also be used as hosts.
Mammalian host cells suitable for expression of the recombinant antibodies of the invention include Chinese hamister ovary (CHO cells) (which include CHO dhfr cells, described in Urlaub and Chasin, (1980) PNAS USA 77: 4216-4220 , used with a selectable DHFR marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601-621, whose full instructions are incorporated by reference), NS0 myeloma cells , COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion. of the antibody in the culture medium in which host cells are growing. Other examples of useful mammalian host cell line consist of monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol: 36:59 m (1977)); newborn hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a line of human hepatoma (Hep G2), whose full instructions are hereby incorporated by reference.
[0065] Host cells are transformed with the hi cloning expression vectors described above for antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify the genes encoding the desired strings.
[0066] Host cells used to produce an antibody can be grown in a variety of media. Commercially available media such as Ham's F10 ™ (Sigma), Minimal Essential Medium ™ ((MEM), (Sigma), RPMI-1640 (Sigma) and Modified Dulbecco's Eagle's Medium ™ ((DMEM), Sigma) are suitable for host cell culture In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al, Anal. Bio-chem. 102: 255 (1980), US patents 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469; WO 90/03430; WO 87/00195; or US patent No. Re 30,985, can be used as a culture medium for cells host, whose full instructions are hereby incorporated by reference. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as, HEPES), nucleotides (such as, adenosine and thymidine), antibiotics (tai s such as gentamicin drug), residual elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements can also be included in suitable concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, H, and the like, consist of those previously used with the host cell selected for expression, and will be evident to those skilled in the art.
[0067] Host cells can also be used to produce parts of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are included in the scope of the present invention. For example, in certain embodiments, it may be desirable to transfect a host cell with DNA encoding the light chain or the heavy chain (but not both) of an antibody of this invention. Combinant DNA technology can also be used to remove some or all of the DNA encoding each or both of the heavy and light chains that is not necessary for binding to IL-13, specifically, hIL-13. The expressed molecules from such truncated DNA molecules they are also encompassed by the antibodies of the invention. In addition, bifational antibodies can be produced in which a light chain and a heavy chain consist of an antibody of the invention and the other light and heavy chains are specific for an antigen other than IL-13 by cross-linking an antibody from invention to a second antibody using standard chemical cross-linking methods.
[0068] In a system suitable for the recombinant expression of an antibody, or antigen binding part thereof, of the invention, a recombinant expression vector that encodes both the antibody heavy chain and the antibody light chain it is introduced into dhfr-CHO cells through calcium phosphate-mediated transfection. Within the recombinant expression vector, each of the antibody's heavy and light chain genes is operatively linked to the CMV optimizer / AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows for the selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transforming host cells are cultured to allow expression of the antibody light and heavy chains and the intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select for transformants, cultivate host cells and recover the antibody from the culture medium.
[0069] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted in the medium. In one aspect, if the antibody is produced intracellularly, as a first step, fragments of particulate, host cells or lysed cells (for example, which result from homogenization), can be removed, for example, by means of centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems can first be concentrated using a commercially available protein concentration filter, for example, an Amicon ™ ultrafiltration unit or Millipore Pellicon ™.
[0070] Prior to the process of the invention, procedures for purifying antibodies from cell fragments initially depend on the site of expression of the antibody. Some antibodies can be secreted directly from the cell in the surrounding growth medium; others are done intracellularly. For the latter antibodies, the first stage of a purification process typically involves: cell lysis, which can be done by a variety of methods, including mechanical shear, osmotic shock or enzymatic treatments. Such disruption releases all cell contents in the homogenate, and, in addition, produces subcellular fragments that are difficult to remove due to their small size. These are usually removed by differential centrifugation or by means of filtration. Where the antibody is secreted, supernatants from such expression systems are generally concentrated first using a commercially available protein concentration filter, for example, an Amicon ™ or Millipore Pellicon ™ ultrafiltration unit. Where the antibody is secreted in the medium, recombinant host cells can also be separated from the cell culture medium, for example, by tangential flow filtration. The antibodies can be further recovered from the culture medium using the antibody purification methods of the invention. 3.2. Exemplary production strategy
[0071] In certain embodiments, the initial production step of the anti-IL-13 antibody involves using centrifuge bottle and Biowave pouch operations to expand CHO cells that express the anti-IL-13 antibody from from a single frozen vial to the desired biomass for inoculation of a 110 L seed bioreactor. A frozen vial of CHO cells from the primary cell bank is melted and placed in the growth medium (SR-512) and centrifuged. The cells are resuspended in the growth medium and expanded at 37 ° C and 5% CO2 in disposable centrifuge bottles, shaker bottles and / or Biowave bags of increasing volume. Duplicate 20 L Biowave bags are used to maximize final cell expansion prior to inoculation into the seed bioreactor. When the cell density reaches> 2.0 x 106 viable cells / mL from both 20 L Biowave bags in approximately 15 to 17 days, the culture is transferred to a 110 L seed bioreactor loaded with the growth medium - SR-520 cement for further expansion. After inoculation, the target temperature is 37 ° C and the pH is adjusted to a target of 7.1 and controlled by adding NaOH and sparging CO2. Dissolved oxygen (DO) in the bioreactor is controlled at a target value of 40% by spraying with air and oxygen. Once the cell density reaches> 2.6 x 106 viable cells / mL after approximately 2 to 4 days, the culture is transferred to a 3000 L production bioreactor.
[0072] In certain embodiments, a partial load from a 3000 L production bioreactor is used to further expand the cell culture. Initially, the reactor is loaded with the growth medium (SR-520) and inoculated with the batch from the 110 L seed bioreactor. During this short filling stage, the temperature, dissolved oxygen and pH are controlled at 37 ° C, 40% and 7.1, respectively. The pH of the culture is controlled by spraying CO2 and adding NaOH. Typically, cells were grown for 2 to 4 days before reaching the production stage density of> 1.6 x 106 viable cells / mL.
[0073] The SR-521 production medium (1950 L) is added to the cell culture in the 3000 L bioreactor to start the production stage. Defoamer C is added to reduce foaming. The pH of the culture is controlled to a target value of 6.9 with intermittent spray of CO2 and addition of NaOH. The temperature and dissolved oxygen are controlled at target values of 35 ° C and 40%, respectively. The DO in the bioreactor is initially controlled to the desired value by spraying the air and supplemented with pure oxygen if necessary. In certain embodiments, the temperature is reduced to a target value of 33 ° C, when the viable cell density reaches> 3.0 x 106 cells / mL, and the pH and DO are maintained at target values of 6.9 and 40%, respectively, while in other modes the target value of 35 ° C is maintained. Glucose (SR-334) is added as needed. Cultures are collected and purified as described below, when cell viability drops to <50%. 4. Purification of antibody 4.1 Purification of antibody in general
[0074] The invention provides methods for producing a purified (or "reduced HCP") antibody preparation from a mixture comprising an antibody and at least one HCP. The present invention also provides methods in which the final purified preparation is reduced to leached protein A. The purification process of the invention begins at the separation step when the antibody has been produced using the methods described above and methods conventional in the art. Table 1 summarizes a modification of a purification scheme. Variations in this scheme, which include, but are not limited to, variations where the protein A affinity chromatography step is omitted or the order of the ion exchange steps are reversed, are considered and are included in the scope of this invention. Table 1 Purification steps with their associated purpose

[0075] Once the clarified solution or mixture that comprises the antibody has been obtained, the separation of the antibody from the other proteins produced by the cell, such as HCPs, is performed using a combination of different purification techniques, which include ion exchange separation step (s) and hydrophobic interaction separation step (s). The separation steps separate protein mixtures based on their load, degree of hydrophobicity or size. In one aspect of the invention, the separation is performed using chromatography, which includes cationic, anionic and hydrophobic interaction. Several different chromatography resins are available for each of these techniques, allowing for the precise adaptation of the purification scheme to the particular protein involved. The essence of each of the separation methods is that proteins can be caused to cross at a different rate down a column, achieving physical separation that increases as they pass further down the column, or selectively adhere to the separation medium, being then eluted differently by different solvents. In some cases, the antibody is separated from impurities, when the impurities specifically adhere to the column and the antibody does not, that is, the antibody is present in the flow through.
[0076] As noted above, the precise suitability of a purification scheme depends on the consideration of the protein to be purified. In certain embodiments, the separation steps of the present invention are employed to separate an antibody from one or more HCPs. Antibodies that can be purified successfully using the methods described herein include, but are not limited to, human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM antibodies. In certain embodiments, the purification strategies of the present invention exclude the use of protein A affinity chromatography, for example, in the context of the purification of IgG3 antibodies, as IgG3 antibodies bind to protein A ineffectively. Other factors that allow the specific suitability of a purification scheme include, but are not limited to: the presence or absence of an Fc region (for example, in the context of the full-length antibody, as compared to a Fab fragment of the same ) due to the fact that protein A binds to the Fc region; the particular germline sequences employed in generating the antibody of interest; and the amino acid composition of the antibody (for example, the primary sequence of the antibody, as well as the hydrophobicity / general charge of the molecule). Antibodies that share one or more characteristics can be purified using appropriate purification strategies to take advantage of these characteristics. 4.2 Primary recovery
[0077] The initial steps of the purification methods of the present invention involve the first phase of clarification and primary recovery of antibody from a sample matrix. In addition, the primary recovery process may also consist of a point at which viruses that may be present in the sample matrix are reduced or inactivated. For example, any one or more of a variety of viral reduction / inactivation methods can be used during the primary purification recovery phase that includes heat inactivation (pasteurization), pH inactivation, solvent / detergent treatment , gamma and UV irradiation and the addition of certain chemical inactivating agents, such as β-propiolactone or, for example, copper phenanthroline, according to US patent no. 4,534,972, whose full instruction is hereby incorporated by reference. In certain modalities of the present invention, the sample matrix is exposed to viral pH reduction / inactivation during the primary recovery phase.
[0078] Viral pH reduction / inactivation methods include, but are not limited to, incubating the mixture for a period of time at low pH and subsequently neutralizing the pH and removing particulates by means of filtration. In certain modalities, the mixture will be incubated at a pH between about 2 and 5, at a pH between about 3 and 4, which includes, but is not limited to, at a pH of about 3.5. The pH of the sample mixture can be reduced by any suitable acid that includes, but is not limited to, citric acid, acetic acid, caprylic acid or other suitable acids. The choice of pH level depends largely on the stability profile of the antibody product and buffer components. It is known that the quality of the target antibody during the reduction / inactivation of low pH viruses is affected by the pH and the duration of the low pH incubation. In certain embodiments, the low pH incubation duration will be from 0.5 h to 2 h, which includes, but is not limited to, 0.5 h to 1.5 h, and which includes, but is not limited to, durations of about 1 hour. The reduction / inactivation of viruses is dependent on these same parameters in addition to the concentration of protein, which can limit the reduction / inactivation in high concentrations. In this way, the appropriate parameters of protein concentration, pH and duration of reduction / inactivation can be selected to achieve the desired level of viral reduction / inactivation.
[0079] In certain modalities, viral reduction / inactivation can be achieved through the use of appropriate filters. A non-limiting example of a suitable filter is the Ultipor DV50 ™ filter available from Pall Corporation. Although certain embodiments of the present invention employ such filtration during the primary recovery phase, in other embodiments, it is employed in other stages of the purification process, which include as the penultimate or final stage of purification. In certain modalities, alternative filters are used for viral reduction / inactivation, such as, but not limited to, Viresolve ™ filters (Millipore, Billerica, Mass.); Zeta Plus VR ™ filters (CUNO; Meriden, Conn.); and Planova ™ filters (Asahi Kasei Pharma, Planova Division, Buffalo Grove, 111.).
[0080] In those modalities where viral reduction / inactivation is employed, the sample mixture can be adjusted, as needed, for additional purification steps. For example, after low pH viral reduction / inactivation, the pH of the sample mixture is typically adjusted to a more neutral pH, for example, from about 4.5 to about 8.5, and that includes, but it is not limited to, about 4.9, before continuing the purification process. In addition, the mixture can be washed with water for injection (WFI) to obtain the desired conductivity.
[0081] In certain embodiments, primary recovery will include one or more centrifugation steps to further clarify the sample matrix and thus assist in the purification of anti-IL-13 antibodies. Centrifugation of the sample can be performed, for example, but not as a limitation, at 7,000 x g to approximately 12,750 x g. In the context of large-scale purification, such centrifugation can occur in line with a flow rate adjusted to achieve, for example, but not as a limitation, a turbidity level of 150 NTU in the resulting supernatant. Such a supernatant can then be collected for further purification.
[0082] In certain embodiments, primary recovery will include the use of one or more depth filtration steps to further clarify the sample matrix and thus assist in purifying the antibodies of the present invention. Depth filters contain medium filtration media that have a graduated density. Such graduated density allows larger particles to be captured close to the filter surface, while smaller particles that penetrate the larger open areas on the filter surface, are only captured in the smaller openings closer to the center of the filter. In certain modalities, the depth filtration step may consist of a delipid depth filtration step. Although certain modalities employ the depth filtration steps only during the primary recovery phase, other modalities employ depth filters, which include delipid depth filters, during one or more additional purification phases. Non-limiting examples of depth filters that can be used in the context of the present invention include Cuno ™ depth filters model 30 / 60ZA (3M Corp.), and 0.45 / 0.2μm bilayer filter cartridges Sartopore ™. 4.3 Affinity chromatography
[0083] In certain embodiments, the primary recovery sample is subjected to affinity chromatography to further purify the antibody of interest away from HCPs. In certain embodiments, the chromatographic material is able to bind selectively or specifically to the antibody of interest. Non-limiting examples of such a chromatographic material include: protein A, protein G, chromatographic material which comprises the antigen bound by the antibody of interest, and the chromatographic material which comprises an Fc-binding protein. In specific embodiments, the affinity chromatography step involves submitting the primary recovery sample to a column comprising a suitable protein A resin. Protein A resin is useful for the affinity purification and isolation of a variety of antibody isotypes, particularly IgG1 IgG2 and IgG4. Protein A is a bacterial cell wall protein that binds to mammalian IgGs mainly through its Fc regions. In its native state, protein A has five IgG binding domains, as well as other domains of unknown function.
[0084] There are several commercial sources for protein A resin. Suitable reins include, but are not limited to, MabSelect ™ available from GE Healthcare and ProSep Ultra Plus ™ from Millipore. A non-limiting example of a suitable column packed with MabSelect ™ consists of a column about 1.0 cm in diameter x about 21.6 cm in length (~ 17 mL of bed volume). This column size can be used for small scale purifications and can be compared with other columns used for larger scales. For example, a 20 cm x 21 cm column whose bed volume is about 6.6 L can be used for larger purifications. Regardless of the column, the column can be packaged using a suitable resin, such as MabSelect ™ or ProSep Ultra Plus ™.
[0085] In certain modalities, it will be advantageous to identify the dynamic binding capacity (DBC) of the protein A resin, in order to adapt the purification to the antibody of particular interest. For example, but not as a limitation, the DBC of a MabSelect ™ or ProSept Ultra Plus ™ column can be determined by a single flow rate load or dual flow load strategy. The single flow rate load can be evaluated at a speed of about 300 cm / h for the entire loading period. The dual flow rate loading strategy can be determined by loading the column to about 35 mg of protein / mL of resin at a linear speed of about 300 cm / h, then reducing the linear speed in half to allow for longer residence time for the last part of the load.
[0086] In certain embodiments, the protein A column can be balanced with a suitable buffer before loading the sample. A non-limiting example of a suitable buffer is a Tris / NaCl buffer, pH about 7.2. A non-limiting example of suitable equilibrium conditions is 25 mM Tris, 100 mM NaCl, pH of about 7.2. After this equilibration, the sample can be loaded onto the column. After loading the column, the column can be washed once or multiple times using, for example, the equilibration buffer. Other washes, which include washes using different buffers, can be used to elute the column. For example, the column can be washed using one or more column volumes of 20 mM citric acid / sodium citrate, 0.5 M NaCl at a pH of about 6.0. This wash can optionally be followed by one or more washes using the equilibration buffer. The protein A column can then be eluted using a suitable elution buffer. A non-limiting example of a suitable elution buffer is an acetic acid / NaCl buffer, pH of about 3.5. Suitable conditions consist, for example, of 0.1 M acetic acid, pH of about 3.5. The eluate can be monitored using techniques well known to those skilled in the art. For example, the absorbance in OD280 can be followed. The column eluate can be collected starting with an initial deflection of about 0.5 AU to a reading of about 0.5 AU at the rear edge of the elution peak. The elution fraction (s) of interest can then be prepared for further processing. For example, the collected sample can be titrated to a pH of about 5.0 with the use of Tris (for example, 1.0 M) at a pH of about 10. Optionally, this title sample can be filtered and additionally processed. 4.4 Ion exchange chromatography
[0087] In certain embodiments, the present invention provides methods for producing a reduced HCP antibody preparation from a mixture comprising an antibody and at least one HCP by subjecting the mixture to at least one exchange separation step ionic in such a way that an eluate comprising the antibody is obtained. Ion exchange separation includes any method by which two substances are separated based on the difference in their respective ion charges and can employ cation exchange material or anion exchange material.
[0088] The use of a cation exchange material versus an anion exchange material is based on the total protein charge. Therefore, it is included in the scope of this invention to employ an anion exchange step before using a cation exchange step, or a cation exchange step before using an anion exchange step. In addition, it is included in the scope of this invention to employ only one cation exchange step, only one anion exchange step or any serial combination of the two.
[0089] In the execution of the separation, the initial antibody mixture can be placed in contact with the ion exchange material using any of a variety of techniques, for example, using a purification technique. batch or a chromatographic technique.
[0090] For example, in the context of batch purification, the ion exchange material is prepared, or equilibrated to, in the desired starting buffer. Under preparation, or equilibration, a slurry of ion exchange material is obtained. The antibody solution is placed in contact with the slurry to absorb the antibody to be separated into the ion exchange material. The solution comprising the HCP (s) that do not bind to the ion exchange material is separated from the slurry, for example, by letting the slurry settle and removing the supernatant. The slurry can be subjected to one or more washing steps. If desired, the slurry can be placed in contact with a solution of greater conductivity to desorb the HCPs that have bound to the ion exchange material. In order to elute bound polypeptides, the salt concentration of the buffer can be increased.
[0091] Ion exchange chromatography can also be used as an ion exchange separation technique. Ion exchange chromatography separates molecules based on differences between the total charge of the molecules. For the purification of an antibody, the antibody must have a charge opposite to that of the functional group attached to the ion exchange material, for example, resin, in order to bind. For example, antibodies, which generally have a total positive charge at the buffer pH below their pI, will bind well to the cation exchange material, which contains negatively charged functional groups.
[0092] In ion exchange chromatography, the charged layers on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided that the ionic intensity of the surrounding buffer is low. Elution is generally achieved by increasing the ionic intensity (ie, conductivity) of the buffer to compete with the solute for the charged locations of the ion exchange matrix. Changing the pH and thus changing the solute charge is another way to achieve solute elution. The change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution).
[0093] Anionic or cationic substituents can be attached to matrices in order to form anionic or cationic supports for chromatography. Non-limiting examples of anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulphonate (S). Cellulose ion exchange resins, such as DE23 ™, DE32 ™, DE52 ™, CM-23 ™, CM-32 ™ and CM-52 ™, are available from Whatman Ltd. Maidstone, Kent, UK Cross-linked ion exchangers and SEPHADEX® based are also known. For example, DEAE-, QAE-, CM- and SP- SEPHADEX® and DEAE-, Q-, CM and S-SEPHAROSE® and SEPHAROSE® fast flow are all available from Pharmacia AB. In addition, both DEAE and CM-derived ethylene glycol copolymer, such as TOYOPEARL ™ DEAE-650S or M and TOYOPEARL ™ CM-650S or M are available from Toso Haas Co., Philadelphia, Pa. , an anion exchange step is completed using a Pall Mustang Q membrane cartridge.
[0094] A mixture comprising an antibody and impurities, for example, HCP (s), is loaded onto an ion exchange column, such as a cation exchange column. For example, but not as a limitation, the mixture can be loaded in a load of about 80 g protein / L of resin, depending on the column used. An example of a suitable cation exchange column consists of a column 80 cm in diameter x 23 cm long, whose bed volume is about 116 L. The mixture loaded in this column cation can be subsequently washed with washing (equilibration buffer). The antibody is then eluted from the column and a first elute is obtained.
[0095] This ion exchange step facilitates the capture of the antibody of interest, while reducing impurities, such as HCPs. In certain respects, the ion exchange column consists of a cation exchange column. For example, but not by way of limitation, a suitable resin for such a cation exchange column consists of CM HyperDF ™ resin. These resins are available from commercial sources, such as Pall Corporation. This cation exchange procedure can be performed at or around room temperature. 4.5 Ultrafiltration / Diafiltration
[0096] Certain embodiments of the present invention employ the steps of ultrafiltration and / or diafiltration to further purify and concentrate the antibody sample. Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.I., 1996); and one: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). A filtration process consists of tangential flow filtration as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalog" pp. 177-202 (Bedford, Mass., 1995/96). Ultrafiltration is generally considered to be filtration using filters with a pore size less than 0.1 μm. By using filters with such a small pore size, the sample volume can be reduced by permeating the sample buffer through the filter, while the antibodies are retained behind the filter.
[0097] Diafiltration is a method of using ultrafilters to remove and exchange non-aqueous salts, sugars and solvents, to separate free species from bound species, to remove low molecular weight material and / or to cause rapid change in ionic environments and / or pH. Micro-solutes are removed most effectively by adding solvent to the solution which is ultrafiltered at a rate approximately equal to the rate of ultratfiltration. This washes the micro-species from the solution in constant volume, effectively purifying the retained antibody. In certain embodiments of the present invention, a diafiltration step is employed to change the various buffers used in connection with the present invention, optionally, before additional chromatography or other purification steps, as well as to remove impurities from the preparations of anti-body. 4.6 Hydrophobic interaction chromatography
[0098] The present invention also provides methods for producing a reduced HCP antibody preparation from a mixture comprising an antibody and at least one HCP which further comprises a hydrophobic interaction separation step. For example, a first eluate obtained from an ion exchange column can be subjected to a hydrophobic interaction material in such a way that a second eluate that has a reduced level of HCP is obtained. Hydrophobic interaction chromatography steps, such as those presented in this document, are generally performed to remove protein aggregates, such as antibody aggregates and process-related impurities.
[0099] In the execution of the separation, the sample mixture is put in contact with the HIC material, for example, with the use of a batch purification technique or with the use of a column. Before HIC purification, it may be desirable to remove any chaotropic agents or very hydrophobic substances, for example, by passing the mixture through a pre-column.
[00100] For example, in the context of batch purification, the HIC material is prepared or balanced to the desired equilibration buffer. A slurry of the HIC material is obtained. The antibody solution is placed in contact with the slurry to absorb the antibody to be separated into the HIC material. The solution comprising HCPs that do not bind to the HIC material is separated from the slurry, for example, by allowing the slurry to settle and removing the supernatant. The slurry can be subjected to one or more washing steps. If desired, the slurry can be brought into contact with a solution of less conductivity to desorb the antibodies that have bound to the HIC material. In order to elute bound antibodies, the salt concentration can be decreased.
[00101] While ion exchange chromatography depends on the antibody loads to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of antibodies. The hydrophobic groups in the antibody interact with the hydrophobic groups in the column. The more hydrophobic a protein is, the stronger it will interact with the spine. In this way, the HIC step removes impurities derived from the host cell (for example, DNA and other species related to low and high molecular weight products).
[00102] Hydrophobic interactions are strongest at high ionic intensity, therefore, this form of separation is conveniently performed after salt precipitations or ion exchange procedures. The absorption of the antibody in an HIC column is favored by high concentrations of salt, but the actual concentrations may vary over a wide range depending on the nature of the antibody and the particular HIC ligand chosen. Several ions can be arranged in a so-called solufobic series depending on whether hydrophobic interactions are promoted (salt precipitation effects (salting-out)) or disrupt the water structure (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are classified in terms of increasing salt precipitation effect as Ba ++; Ca ++; Mg ++; Li +; Cs +; Na +; K +; Rb +; NH4 +, while anions can be classified in terms of increasing chaotropic effect as P0 ---; S04--; CH3C03 -; CI-; Br-; N03-; ClO4-; I-; SCN-.
[00103] In general, sulfates of Na, K or NH4 effectively promote the interaction of ligand-protein in HIC. Salts that influence the intensity of the interaction can be formulated, as given by the following relationship: (NH4) 2SO4> Na2SO4> NaCl> NH4Cl> NaBr> NaSCN. In general, salt concentrations between about 0.75 and about 2 M ammonium sulfate or between about 1 and 4 M NaCl are useful.
[00104] The HIC columns generally comprise a base matrix (for example, cross-linked agarose or synthetic copolymer material) to which hydrophobic binders (for example, alkyl and aryl groups) are coupled. A suitable HIC column comprises an agarose resin substituted by phenyl groups (for example, a Phenyl Sepharose ™ column). Many HIC columns are commercially available. Examples include, but are not limited to, a fast-flowing, low- or high-substituted Phenyl Sepharose ™ 6 column (Pharmacia LKB Bio-technology, AB, Sweden); high performance Phenyl Sepharose ™ column (Pharmabel LKB Biotechnology, AB, Sweden); high-performance Octil Sepharose ™ column (Pharmacia LKB Biotechnology, AB, Sweden); Fractogel ™ EMD Propil or Fractogel ™ EMD Phenyl columns (E. Merck, Germany); Macro-Prep ™ Meil or Macro-Prep ™ t-Butyl holders (Bio-Rad, California); WP Hl-Propyl (C3) ™ column (J. T. Baker, New Jersey); and Toyopearl ™ ether, phenyl or butyl columns (TosoHaas, PA). 4.7 Exemplifying purification strategies
[00105] In certain modalities, primary recovery proceeds through the initial use of centrifugation and filtration steps to remove cells and cell fragments (which include HCPs) from the collection of the production bioreactor. For example, but not by way of limitation, a culture that comprises antibodies, media and cells can be subjected to centrifugation at approximately 7000 x g to approximately 11,000 x g. In certain modalities, the resulting sample supernatant is then passed through a filter set comprising multiple depth filters. In certain instances, the filter set comprises around twelve 40.64 cm (16 inch) depth filters Cuno ™ model 30 / 60ZA (3M Corp.) and around three round filter housings equipped with three 76.2 cm (30 inch) 0.45 / 0.2 μm Sartopore ™ 2 filter cartridges (Sartorius). The clarified supernatant is collected in a vessel, such as a pre-sterilized collection vessel, and maintained at approximately 8 ° C. This temperature is then adjusted to approximately 20 ° C before the capture chromatography step or steps described below. It should be noted that an element skilled in the art may vary from the conditions mentioned above and will still be included in the scope of the present invention.
[00106] In certain embodiments, primary recovery will be followed by affinity chromatography using protein A resin. There are several commercial sources for protein A resin. A suitable resin consists of MabSelect ™ available from GE Healthcare. An example of a suitable column packed with MabSelect ™ consists of a column about 1.0 cm in diameter x about 21.6 cm in length (~ 17 mL of bed volume). This column size can be used for bench scale. This can be compared with other columns used for extended scales. For example, a 20 cm x 21 cm column whose bed volume is about 6.6 L can be used for commercial production. Regardless of the column, the column can be packaged using a suitable resin, such as MabSelect ™.
[00107] In certain respects, the protein A column can be balanced with a suitable buffer before loading the sample. An example of a suitable buffer is a Tris / NaCl buffer, pH about 6 to 8, which includes, but is not limited to, about 7.2. A specific example of suitable conditions consists of 25 mM Tris, 100 mM NaCl, pH 7.2. After this equilibration, the sample can be loaded onto the column. After loading the column, the column can be washed once or multiple times using, for example, the equilibration buffer. Other washes that include washes using different buffers can be used before column elution. For example, the column can be washed using one or more column volumes of 20 mM citric acid / sodium citrate, 0.5 M NaCl at a pH of about 6.0. This wash can optionally be followed by one or more washes using the equilibration buffer. The protein A column can then be eluted using a suitable elution buffer. An example of a suitable elution buffer is an acetic acid / NaCl buffer, pH around 3.5. Suitable conditions are, for example, 0.1 M acetic acid, pH 3.5. The eluate can be monitored using techniques well known to those skilled in the art. For example, the absorbance at OD280 can be followed. The column eluate can be collected starting with an initial deflection of about 0.5 AU to a reading of about 0.5 AU at the rear edge of the elution peak. The elution fraction (s) of interest can then be prepared for further processing. For example, the collected sample can be titrated to a pH of about 5.0 with the use of Tris (for example, 1.0 M) at a pH of about 10. Optionally, this title sample can be filtered and further processed.
[00108] The dynamic bonding capacity (DBC) of the MabSelect ™ column can be determined by a single flow rate load or dual flow load strategy. The single flow rate load can be evaluated at a speed of about 300 cm / h for the entire loading period. The dual flow rate loading strategy can be determined by loading the column to about 35 mg of protein / mL of resin at a linear speed of about 300 cm / h, then reducing the linear speed in half to allow time longer stay for the last part of the load.
[00109] The protein eluate can then be further purified by employing a pH-mediated virus reduction / inactivation step. In certain modalities, this step will involve adjusting the eluate pH to between about 3 and about 5, which includes, but is not limited to, about 3.5, for approximately 1 hour. The reduction of pH can be facilitated with the use of known acid preparations, such as citric acid, for example, 3 M citric acid. Exposure to acidic pH reduces, if not completely eliminates, pH-sensitive viral contaminants and precipitates some contaminants from the medium / cell. After this viral reduction / inactivation step, the pH is adjusted to about 4.9 or 5.0 using a base, such as sodium hydroxide, for example, 3 M sodium hydroxide, for about twenty about forty minutes. This adjustment can occur at around 20 ° C.
[00110] In certain modalities, the pH-adjusted culture is further purified using an anion exchange column. A non-limiting example of a column suitable for this step consists of a column 60 cm in diameter x 30 cm in length, whose bed volume is about 85 L. The column is packed with an anion exchange resin, such as as Q Sepharose ™ Quick-flow available from GE Healthcare. The column can be balanced using about seven column volumes of a suitable buffer, such as Tris / sodium chloride. An example of suitable conditions consists of 25 mM Tris, 50 mM sodium chloride at pH 8.0. An element skilled in the art may vary the conditions, but they remain within the scope of the present invention. The column is loaded with the sample collected from the protein A purification step described above. In another aspect, the column is loaded from the eluate collected during the cation exchange. After loading the column, the column is washed with the equilibration buffer (for example, Tris / sodium chloride buffer). The continuous flow comprising the antibodies can be monitored using a UV spectrophotometer at OD280nm. This anion exchange step reduces process-related impurities, such as nucleic acids such as DNA and host cell proteins. The separation occurs due to the fact that the antibodies of interest do not interact substantially with or bind to the solid phase of the column, for example, Q Sepharose ™, but many impurities interact with and bind to the solid phase of the column. The anion exchange can be performed at about 12 ° C.
[00111] In certain embodiments, the pH-adjusted culture is then further purified using a cation exchange column. In certain embodiments, the equilibrium buffer used in the cation exchange column consists of a buffer that has a pH of about 5.0. An example of a suitable buffer consists of about 210 mM sodium acetate, pH 5.0. After equilibration, the column is loaded with the sample prepared from the primary recovery step above. The column is packed with a cation-exchange resin, such as CM Sepharose ™ Fast-flowing available from GE Healthcare. The column is then washed using the equilibration buffer. The column is then subjected to an elution step with the use of a buffer that has a higher ionic intensity, as compared to the wash or equilibration buffer. For example, a suitable elution buffer can be about 790 mM sodium acetate, pH 5.0. The antibodies will be eluted and can be monitored using a UV spectrophotometer adjusted to OD280nm. In a particular example, the elution collection can be from the top 3 OD280nm to the bottom 8 OD280nm. It should be understood that an element skilled in the art can vary conditions and still fall within the scope of the invention.
[00112] In certain embodiments, the pH-adjusted culture, the cation exchange eluate or the anion exchange eluate, is filtered using, for example, a 40.64 cm (16 inch) Cuno ™ delipid filter. This filtration, using the delipid filter, can be accompanied, for example, by a 76.2 cm (30 inch) 0.45 / 0.2 μm Sartopore ™ bilayer filter cartridge. The ion exchange elution buffer can be used to wash the residual volume remaining in the filters and prepared for ultrafiltration / diafiltration.
[00113] In order to complete the ultratfiltration / diafiltration step, the filtration medium is prepared in a suitable buffer, for example, 20 mM sodium phosphate, pH 7.0. A salt, such as sodium chloride, can be added to increase ionic strength, for example, 100 mM sodium chloride. This ultratfiltration / diafiltration step serves to concentrate anti-IL-13 antibodies, remove sodium acetate and adjust the pH. Commercial filters are available to carry out this step. For example, Millipore manufactures a 30 kD cut molecular weight (MWCO) cellulose ultrafilter membrane cassette. This filtration procedure can be carried out at or around room temperature.
[00114] In certain modalities, the sample from the capture filtration step above is subjected to a second ion exchange separation step. This second ion exchange separation will, in certain embodiments, involve separation based on the opposite charge of the first ion exchange separation. For example, if an anion exchange step is employed after primary recovery, the second chromatographic ion exchange step may consist of a cation exchange step. Conversely, if the primary recovery step is followed by a cation exchange step, this step will be followed by an anion exchange step. In certain modalities, the first ion exchange eluate can be directly subjected to the second ion exchange chromatographic step where the first ion exchange eluate is adjusted to the appropriate buffer conditions. Suitable anionic and cationic separation conditions and materials are described above.
[00115] In certain embodiments of the present invention, the sample containing antibodies will be further processed using a hydrophobic interaction step of separation. A non-limiting example of a column suitable for such a step consists of a column 80 cm in diameter x 15 cm in length, whose bed volume is about 75 L, which is packed with a suitable resin used for HIC , such as, but not limited to, Phenyl HP Sepharose ™ available from Amersham Biosciences, Upsala, Sweden. The continuous flow preparation obtained from the previous anion exchange chromatographic step that comprises the antibodies of interest can be diluted with an equal volume around 1.7 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. This can then be subjected to filtration using a 0.45 / 0.2 μm Sartopore ™ 2 bilayer filter or its equivalent. In certain modalities, the hydrophobic chromatography procedure involves two or more cycles.
[00116] In certain modalities, the HIC column is first balanced with the use of a suitable buffer. A non-limiting example of a suitable buffer is 0.85 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. An element skilled in the art can vary the equilibrium buffer and remain within the scope of the present invention, by changing the concentrations of buffering agents and / or by replacing equivalent buffers. In certain embodiments, the column is then loaded with a continuous anion exchange flow sample and washed multiple times, for example, three times, with a suitable buffer system, such as ammonium sulfate / sodium phosphate. An example of a suitable buffer system includes the 1.1 M ammonium sulphate buffer, 50 mM sodium phosphate with a pH of around 7.0. Optionally, the column can go through additional washing cycles. For example, a second wash cycle can include multiple column washes, for example, one to seven times, using a suitable buffer system. A non-limiting example of a suitable buffer system includes 0.85 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0. In one aspect, the loaded column still undergoes a third wash using a suitable buffer system. The column can be washed multiple times, for example, one to three times, using a buffer system, such as 1.1 M ammonium sulfate, 50 mM sodium phosphate at a pH around 7, 0. Again, an element skilled in the art can vary buffering conditions and remain within the scope of the present invention.
[00117] The column is eluted using a suitable elution buffer. A suitable example of such an elution buffer consists of 0.5 M ammonium sulfate, 15 mM sodium phosphate at a pH around 7.0. The antibodies of interest can be detected and collected using a conventional spectrophotometer from the top side at 3 OD280nm to the bottom side of the peak at 3 OD280nm.
[00118] In certain aspects of the invention, the elute from the hydrophobic chromatography step is subjected to filtration to remove viral particles, which include intact viruses, if present. A non-limiting example of a suitable filter is the Ultipor DV50 ™ filter available from Pall Corporation. Other viral filters can be used in this filtration step and are well known for elements skilled in the art. The HIC elute is passed through a pre-moistened filter of about 0.1 μm and a 5.08 x 76.2 centimeter (2 x 30 inch) filter set around the Ultipor DV50 ™ 34 psig. In certain embodiments, after the filtration process, the filter is washed using, for example, the HIC elution buffer to remove any antibodies retained in the filter housing. The filtrate can be stored in a pre-sterilized container at around 12 ° C.
[00119] In certain modalities, the above filtrate is again subjected to ultrafiltration / diafiltration. This step is important if a professional's purpose is to use the antibody, for example, in a pharmaceutical formulation. This process, if employed, can facilitate the concentration of the antibody, remove previously used buffer salts and replace them with a particular formulation buffer. In certain embodiments, continuous diafiltration with multiple volumes, for example, two volumes, of a formulation buffer is performed. A non-limiting example of a suitable formulation buffer is the 5 mM methionine buffer, 2% mannitol, 0.5% sucrose, pH 5.9 (without Tween). Upon completion of this diavolume exchange, antibodies are concentrated. Once a predetermined antibody concentration has been achieved, then a professional can calculate the amount of 10% Tween that should be added to reach a final Tween concentration of about 0.005% (by volume) ).
[00120] Certain embodiments of the present invention will include additional purification steps. Examples of additional purification procedures that can be performed before, during or after the ion exchange chromatography method include ethanol precipitation, isoelectric focusing, reverse phase HPLC, silica chromatography, Sepharose ™ heparin chromatography, chromatography on additional anion exchange and / or additional cation exchange chromatography, chromato-focusing, SDS-PAGE, ammonium sulphate precipitation, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography (for example, using G protein, an antibody, substrate, ligand or specific antigen as capture reagent).
[00121] In certain embodiments of the present invention, the anti-IL-13 antibody consists of an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM isotype antibody comprising the variable region sequences of heavy and light chain described in Figure 1. In certain embodiments, the anti-IL-13 antibody consists of an IgG1, IgG2, IgG3 or IgG4 isotype antibody comprising the heavy and light chain variable region sequences described in Figure 1. 5. Test methods for sample purity 5.1 Host cell protein assay
[00122] The present invention also provides methods for determining residual levels of host cell protein (HCP) concentration in the isolated / purified antibody composition. As described above, HCPs are desirably excluded from the final target substance product, for example, the anti-IL-13 antibody. Exemplary HCPs include proteins that originate from the source of antibody production. Failure to identify and sufficiently remove HCPs from the target antibody can lead to reduced efficacy and / or particular adverse reactions.
[00123] For use in the present invention, the term "HCP ELISA" refers to an ELISA where the second antibody used in the assay is specific for HCPs produced from cells, for example, CHO cells, used to generate the antibody (for example, anti-IL-13 antibody). The second antibody can be produced according to conventional methods known to those skilled in the art. For example, the second antibody can be produced using HCPs obtained by simulated production and purification procedures, that is, the same cell line used to produce the antibody of interest is used, but the cell line is not used. transfected with the antibody's DNA. In an exemplary embodiment, the second antibody is produced using HPCs similar to those expressed in the cell expression system of choice, that is, the cell expression system used to produce the target antibody.
[00124] Generally, the HCP ELISA comprises placing a liquid sample comprising HCPs between two layers of antibodies, that is, a first antibody and a second antibody. The sample is incubated during the time that the HCPs in the sample are captured by the first antibody, for example, but is not limited to, goat anti-CHO, affinity purified (Cygmis). A second labeled antibody, or mixture of antibodies, specific for the HCPs produced from the cells used to generate the antibody, for example, biotinylated anti-CHO HCP, is added and binds to the HCPs within the sample. In certain modalities, the first and second antibodies consist of polyclonal antibodies. In certain respects, the first and second antibodies consist of mixtures of polyclonal antibodies raised against HCPs, for example, but are not limited to, a mix of biotinylated goat host cell anti-protein 599/626/748. The amount of HCP contained in the sample is determined using the appropriate test based on the label of the second antibody.
[00125] The HCP ELISA can be used to determine the level of HCPs in an antibody composition, such as an eluate or continuous flow obtained using the process described above. The present invention also provides a composition comprising an antibody, in which the composition has no detectable level of HCPs, as determined by an immunoabsorbent assay linked to the HCP enzyme ("ELISA"). 5.2 Testing of affinity chromatographic material
[00126] In certain embodiments, the present invention also provides methods for determining residual levels of affinity chromatographic material in the isolated / purified antibody composition. In certain contexts, such material leaches into the antibody composition during the purification process. In certain embodiments, an assay for identifying the protein A concentration in the isolated / purified antibody composition is employed. For use in the present invention, the term "protein A ELISA" refers to an ELISA where the second antibody used in the assay is specific to protein A used to purify the antibody of interest, for example, an anti-IL- 13. The second antibody can be produced according to conventional methods known to those skilled in the art. For example, the second antibody can be produced using recombinant or naturally occurring protein A, in the context of conventional methods for antibody generation and production.
[00127] Generally, the protein A ELISA comprises placing a liquid sample comprising protein A (or possibly containing protein A) between two layers of anti-protein A antibodies, that is, a first anti-protein A antibody and a second anti-protein A antibody. The sample is exposed to a first layer of anti-protein A antibody, for example, but is not limited to, polyclonal antibodies or mixtures of polyclonal antibodies, and incubated for a period enough that protein A in the sample is captured by the first antibody. A second labeled antibody, for example, but not limited to, polyclonal antibodies or mixtures of polyclonal antibodies, specific to protein A, is then added and binds to protein A captured within the sample. Additional non-limiting examples of anti-protein A antibodies useful in the context of the present invention include antibodies to chicken protein A and biotinylated anti protein A. The amount of protein A contained in the sample is determined using the appropriate test based on the label of the second antibody. Similar tests can be used to identify the concentration of alternative affinity chromatographic materials.
[00128] The protein A ELISA can be used to determine the level of protein A in an antibody composition, such as an eluate or continuous flow obtained using the process described above. The present invention also provides a composition comprising an antibody, wherein the composition has no detectable level of protein A, as determined by an immunosorbent assay linked to the protein A enzyme ("ELISA"). 6. Additional modifications
[00129] The antibodies of the present invention can be modified. In some embodiments, the antibodies or antigen binding fragments of the same are chemically modified to provide a desired effect. For example, the pegylation of antibodies or antibody fragments of the invention can be performed using any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3: 4-10 (1992); EP 0 154 316; and EP 0 401 384, each of which is incorporated by reference in its entirety. In one aspect, pegylation is carried out through an acrylic reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A water-soluble polymer suitable for pegylating the antibodies and antibody fragments of the invention consists of polyethylene glycol (PEG). For use in the present invention, "polyethylene glycol" is intended to include any of the forms of PEG that have been used to derive other proteins, such as mono (C1-C10) alkoxy- or aryloxy polyethylene glycol.
[00130] The methods for preparing the pegylated antibody and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as an aldehyde derivative or reactive ester of PEG, under suitable conditions, so that the antibody or antibody fragment is attached to one or more PEG groups, and (b) obtain the reaction products. It will be evident for an element skilled in the art to select the optimal reaction conditions or the acrylic reactions based on the known parameters and the desired result.
[00131] The antibodies and pegylated antibody fragments specific for IL-13 can generally be used to treat IL-13 related disorders of the invention by administering the anti-IL-13 antibodies and antibody fragments described herein. . Generally, pegylated antibodies and antibody fragments have an increased half-life, as compared to un pegylated antibodies and antibody fragments. Un pegylated antibodies and antibody fragments can be used alone, together or in combination with other pharmaceutical compositions.
[00132] An antibody or antibody part of the invention can be derived from or linked to another functional molecule (for example, another peptide or protein). Accordingly, the antibodies and part of antibodies of the invention are intended to include the derived and otherwise modified forms of the anti-human hIL-13 antibodies described herein, which include the immunoadhesion molecules. For example, an antibody or antibody part of the invention can be functionally linked (through chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody (for example , a bispecific antibody or a diabody, a detectable agent, a cytotoxic agent, a pharmaceutical agent and / or a protein or peptide that can mediate the association of the antibody or antibody part with another molecule (such as a streptavidin core region or a polyhistidine tag).
[00133] A type of derived antibody is produced by cross-linking two or more antibodies (of the same or different types, for example, to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, which have two distinctly reactive groups separated by a suitable (for example, m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (for example, disuccinimidyl suberate). Such binders are available from Pierce Chemical Company, Rockford, IL.
[00134] Useful detectable agents with which an antibody or antibody part of the invention can be derived include fluorescent compounds. Suitable fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin and the like. An antibody can also be derived with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When an antibody is derived with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent of horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a color reaction product, which is detectable. An antibody can also be derived with biotin and detected by indirect measurement of avidin or streptavidin binding. 7. Pharmaceutical compositions
[00135] The antibodies and antibody parts of the invention can be incorporated into pharmaceutical compositions suitable for administration to an individual. Typically, the pharmaceutical composition comprises an antibody or antibody part of the invention and a pharmaceutically acceptable carrier. For use in the present invention, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delay and isotonic agents, and the like, which are physiologically compatible . Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable vehicles may additionally comprise small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which optimize the useful life or effectiveness of the antibody or antibody part.
[00136] The antibodies and antibody parts of the invention can be incorporated into a pharmaceutical composition suitable for parenteral administration. The antibody or antibody parts can be prepared as a solution for injection that contains, for example, 0.1 to 250 mg / ml of antibody. The solution for injection may be composed of a liquid or lyophilized dosage form in an amber or flint bottle, ampoule or pre-filled syringe. The buffer may consist of approximately 1 to 50 mM L-histidine, (optimally, 5 to 10 mM), at pH 5.0 to 7.0 (optimally, pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution in a concentration of 0 to 300 mM (optimally, 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, mainly 0 to 10% sucrose (optimally, 0.5 to 1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, mainly 1 to 10% mannitol (optimally, 24%). Stabilizers can be used in both lyophilized and liquid dosage forms, mainly 1 to 50 mM L-methionine (optimally, 5 to 10 mM). Other suitable bulking agents include glycine, argin, which can be included as 0 to 0.05% polysorbate-80 (optimally, 0.005 to 0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.
[00137] In one aspect, the pharmaceutical composition includes the antibody in a dosage of about 0.01 mg / kg to 10 mg / kg. In another aspect, antibody dosages include approximately 1 mg / kg administered every other week, or approximately 0.3 mg / kg administered weekly. A person skilled in the art can assess the dosage and regimen suitable for administration to an individual.
[00138] The compositions of this invention can be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (for example, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The form depends, for example, on the intended mode of administration and the therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. One mode of administration is parenteral (for example, intravenous, subcutaneous, intraperitoneal, intramuscular). In one aspect, the antibody is administered by intravenous injection or infusion. In another aspect, the antibody is administered by intramuscular or subcutaneous injection.
[00139] Therapeutic compositions need to be typically sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for the high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody or antibody part) in the required amount in a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtrate sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile lyophilized powders for the preparation of sterile injectable solutions, the preparation methods consist of vacuum drying and spray drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution of the same. The proper fluidity of a solution can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. The prolonged absorption of injectable compounds can be caused by the inclusion in the composition of an agent that delays absorption, for example, monostearate salts and gelatin.
[00140] The antibodies and antibody parts of the present invention can be administered by a variety of methods known in the art, a route / mode of administration consisting of subcutaneous injection, intravenous injection or infusion. As will be seen by the person skilled in the art, the route and / or method of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a vehicle that will protect the compound against rapid release, such as a controlled release formulation, which includes implants, transdermal patches and microencapsulated delivery systems. Biocompatible and biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortesters and polylactic acid. Many methods for the preparation of such formulations are either patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, whose full instruction is hereby incorporated by reference.
[00141] In certain respects, an antibody or antibody part of the invention can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be included in a hard or soft gelatin capsule, compressed into tablets, or directly incorporated into the individual's diet. For oral therapeutic administration, the compounds can be incorporated with excipients and used in the form of ingestible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, discs, and the like. To administer a compound of the invention by means of administration other than parenteral, it may be necessary to coat the compound with or co-administer the compound with a material to prevent its inactivation.
[00142] Supplementary active compounds can also be incorporated into the compositions. In certain aspects, an antibody or antibody part of the invention is co-formulated with and / or co-administered with one or more additional therapeutic agents that are useful for the treatment of disorders, in which the activity of IL-13 is harmful. For example, an anti-hIL-13 antibody or antibody part of the invention can be co-formulated and / or co-administered with one or more additional antibodies that bind other targets (for example, antibodies that bind other cytokines or that bind cell surface molecules). In addition, one or more antibodies of the invention can be used in combination with two or more of the therapeutic agents mentioned above. Such combination therapies can advantageously use lower dosages of the therapeutic agents administered, thus avoiding possible toxicities or complications associated with the various monotherapies. The skilled person will note that when the antibodies of the invention are used as part of a combination therapy, a lower dosage of the antibody may be desirable than when the antibody alone is administered to an individual (e.g., a synergistic therapeutic effect. can be achieved through the use of combination therapy which, in turn, allows the use of a lower dose of the antibody to achieve the desired therapeutic effect).
[00143] It should be understood that the antibodies of the invention or part of antigen binding thereof can be used alone or in combination with an additional agent, for example, a therapeutic agent, said additional agent being selected by the element versed in the technique for its intended purpose. For example, the additional agent may consist of a therapeutic agent recognized in the art as being useful for treating the disease or condition that is treated by the antibody of the present invention. The additional agent can also consist of an agent that imparts a beneficial attribute to the therapeutic composition, for example, an agent that affects the viscosity of the composition.
[00144] It should also be understood that the combinations that are to be included in this invention are those combinations useful for their intended purpose. The agents presented below are illustrative and are not intended to be limiting. The combinations that are part of this invention can consist of the antibodies of the present invention and at least one additional agent selected from the lists below. The combination may also include more than one additional agent, for example, two or three additional agents, if the combination is such that the formed composition can perform its intended function.
[00145] Some combinations consist of non-steroidal anti-inflammatory drug (s) also mentioned as NSAIDS, which include drugs like ibuprofen. Other combinations consist of corticosteroids that include prednisolone; the well-known side effects of steroid use can be reduced or even eliminated by reducing the required steroid dose when treating patients in combination with the antibodies of this invention. Non-limiting examples of therapeutic agents for rheumatoid arthritis with which an antibody, or part of an antibody, of the invention can be combined include the following: cytokine suppressant anti-inflammatory drug (s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF and PDGF. The antibodies of the invention, or antigen binding part thereof, can be combined with the antibodies to cell surface molecules, such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their ligands, which include CD 154 (gp39 or CD40L).
[00146] Some combinations of therapeutic agents can interfere at different points in the subsequent inflammatory and autoimmune cascade; examples include TNF antagonists such as chimeric, humanized or human TNF antibodies, D2E7, (US order no. serial 08 / 599.226, filed February 9, 1996, whose full instruction is hereby incorporated by reference), cA2 ( Re- micade ™), CDP 571, anti-TNF antibody fragments (for example, CDP870), and soluble TNF p55 or p75 receptors, derived from them, (P75TNFRIgG (Enbrel ™) or p55TNFRl gG (Lenercept), soluble IL-13 receptor (sIL-13), and also TNFcc-converting enzyme (TACE) inhibitors; similarly, IL-1 inhibitors (eg, interleukin-1 converting enzyme inhibitors, such as like Vx740, or IL-1RA, etc.) can be effective for the same reason. Other combinations include interleukin 11, anti-P7s and p-selectin glycoprotein ligand (PSGL). More other combinations involve other essential elements of the response. autoimmune disease that can act parallel to, dependent on or according to the function of IL-13. combination includes anti-CD4 depletion inhibitors. Further other combinations include antagonists of the co-stimulating pathway CD80 (B7.1) or CD86 (B7.2) which include antibodies, soluble receptors or antagonist ligands.
[00147] The antibodies of the invention, or antigen-binding part thereof, can also be combined with agents, such as methotrexate, 6- MP, azathioprine sulfasalazine, mesalazine, chloroquine / hydroxychloroquine olsalazine, pencilamine, aurothiomalate (intramuscular and oral) , azathioprine, colchicine, corticosteroids (oral, inhaled and local injection), β-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifin, ipratropium and cyclopyrope FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroids, such as prednisolone, phosphodiesterase inhibitors, adensosin agonists, antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with signaling by pro-inflammatory cytokines, such as TNFα or IL-1 (for example, IRAK, NIK, IKK, p38 or MAP kinase inhibitors), converting enzyme inhibitors are IL-1β (eg Vx740), anti-P7s, p-selectin glycoprotein ligand (PSGL), TNFα converting enzyme (TACE) inhibitors, T cell signaling inhibitors, such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (for example, soluble p55 or p75 TNF receptors and p75TNFRIgG derivatives (Enbrel. TM.) And p55TNFRIgG (Lenercept), sIL-1 RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-1)) and anti-inflammatory cytokines (for example, IL-4, IL-10 , IL-11, IL-13 and TGFβ). Some combinations include methotrexate or leflunomide and in cases of severe or moderate rheumatoid arthritis, cyclosporine.
[00148] The pharmaceutical compositions of the invention can include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody part of the invention. A "therapeutically effective amount" refers to an effective amount, in dosages and for the period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody or part of the antibody can vary according to factors, such as the condition of the disease, age, sex and weight of the individual, and the ability of the antibody or part of the antibody to elicit a response desired in the individual. A therapeutically effective amount also consists of one in which any toxic or harmful effects of the antibody or part of the antibody are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[00149] Dosage regimens can be adjusted to provide the optimal desired response (for example, a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. In certain embodiments, it is especially advantageous to formulate parenteral compositions in the form of a dosage unit for ease of administration and uniformity of dosage. The dosage unit form, for use in the present invention, refers to physically distinct units suitable as unitary dosages for mammalian individuals to be treated; each unit comprising a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is stipulated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the composition technique of such an active compound for the treatment of sensitivity in individuals.
[00150] A non-limiting and exemplary range for a prophylactic or therapeutically effective amount of an antibody or antibody part of the invention is 0.01 to 20 mg / kg, 1 to 10 mg / kg or 0.3 to 1 mg / kg. It should be noted that the dosage values may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular individual, the specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the individual who administers or supervises the administration of the compositions, and that the dosage ranges presented in this document are exemplary only and are not intended to limit the scope or practice of the claimed composition. 8. Uses of the antibodies of the invention 8.1 Uses of the anti-IL-13 antibody in general
[00151] Given their ability to bind IL-13, the anti-IL-13 antibodies, or antigen binding parts thereof, of the invention can be used to detect IL-13, in one aspect, hIL- 13 (for example, in a sample matrix, in one aspect, a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA ) or tissue immunohistochemistry. The invention provides a method for the detection of IL-13 in a biological sample which comprises contacting a sample with an antibody, or part of antibody, of the invention and detecting the antibody bound to IL-13 or unbound antibody, to detect, thus, the IL-13 in the sample. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I, 131I, 35S or 3H. Detection of IL-13 in a sample can be useful in a diagnostic context, for example, in diagnosing a condition associated with increased IL-13 and / or it can be useful in identifying an individual who may benefit from treatment with an anti-IL-13 antibody.
[00152] As an alternative to detection assays that involve labeled anti-IL-13 antibody, IL-13 can be detected in a sample by a competition immunoassay using, for example, rhIL-13 standards labeled with a detectable substance and an unlabeled anti-IL-13 antibody, such as an anti-hIL-13 antibody. In this assay, the sample, the labeled rhIL-13 standards and the anti-hIL-13 antibody are combined and the amount of labeled rhIL-13 standard bound to the unlabeled antibody is determined. The amount of hIL-13 in the sample is inversely proportional to the amount of labeled rhIL-13 standard bound to the anti-hIL-13 antibody.
[00153] The antibodies and part of antibodies of the invention are able to neutralize the activity of IL-13 in vitro and in vivo, in one aspect, an activity of hIL-13. Consequently, the antibodies and part of antibodies of the invention can be used to inhibit IL-13 activity, for example, in a cell culture containing IL-13, in human subjects or in other mammalian species that have IL-13 with which an antibody of the invention is cross-reacted (for example, primates, such as baboon, crab monkey and rhesus). In one aspect, the invention provides an isolated human antibody, or antigen binding part of it, that neutralizes human IL-13 activity, and at least one additional IL-13 primate selected from the group consisting of Baboon IL-13, Marmoset IL-13, Chimpanzee IL-13, Crab Monkey IL-13 and Rhesus IL-13, but does not neutralize mouse IL-13 activity. In one aspect, IL-13 is human IL-13. For example, in a cell culture that contains, or is suspected to contain, hIL-13, an antibody or antibody part of the invention can be added to the culture medium to inhibit hIL-13 activity in the culture.
[00154] In another aspect, the invention provides a method for inhibiting IL-13 activity in an individual suffering from a disorder in which IL-13 activity is harmful. For use in the present invention, the phrase "a disorder in which IL-13 activity is harmful" is intended to include diseases and other disorders in which the presence of IL-13 in an individual suffering from the disorder has been shown to be or is suspected of being responsible for the pathophysiology of the disorder or a factor that contributes to the worsening of the disorder. Consequently, a disorder in which IL-13 activity is harmful is a disorder in which inhibition of IL-13 activity is expected to alleviate the symptoms and / or progress of the disorder. Such disorders can be evidenced, for example, by an increase in the concentration of IL-13 in a biological fluid of an individual suffering from the disorder (for example, an increase in the concentration of IL-13 in serum, plasma, synovial fluid , etc. of the individual), which can be detected, for example, with the use of an anti-IL-13 antibody, as described above. In one aspect, the antibodies or antigen binding part thereof can be used in therapy to treat the diseases or disorders described in this document. In another aspect, the antibodies or antigen-binding part thereof can be used to manufacture a remedy for the treatment of the diseases or disorders described in this document. There are countless examples of disorders in which IL-13 activity is harmful. For example, IL-13 plays a critical role in the pathology associated with a variety of diseases involving immune and inflammatory elements, which include, but are not limited to, respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Additional IL-13-related disorders include, but are not limited to: atopic disorders (eg, atopic dermatitis and allergic rhinitis); inflammatory and / or autoimmune conditions of the skin, gastrointestinal organs (for example, inflammatory bowel diseases (IBD), such as ulcerative colitis and / or Crohn's disease), and liver (for example, cirrhosis, fibrosis); scleroderma; tumors or cancers, for example, Hodgkin's lymphoma.
Consequently, anti-IL-13 antibodies, antigen-binding parts of the same or vectors that express the same in vivo are indicated for the treatment of diseases, such as asthma or other inflammatory and / or autoimmune conditions where there is an abnormal expression of IL-13, leading to an excess of IL-13 or in cases of complications due to exogenously administered IL-13. 8.2 Use of anti-IL-13 antibody in respiratory disorders
[00156] In certain embodiments of the present invention, an anti-IL-13 antibody, or antigen binding part thereof, is employed in the treatment of one or more disorders associated with IL-13, which include, but are not limited to , respiratory disorders (for example, asthma (for example, allergic or non-allergic asthma (for example, asthma due to infection with, for example, respiratory syncytial virus (RSV), for example, in younger children )), chronic obstructive pulmonary disease (COPD) and other conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production, for example, cystic fibrosis and pulmonary fibrosis.
[00157] In certain embodiments, this application provides methods of treatment (for example, reduction, improvement) or prevention of one or more symptoms associated with a respiratory disorder, for example, asthma (for example, allergic or non-allergic asthma) ; allergies; chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation, eosinophilia, fibrosis and excess mucus production, for example, cystic fibrosis and pulmonary fibrosis. For example, asthma symptoms include, but are not limited to, wheezing, shortness of breath, bronchoconstriction, airway hyperreactivity, reduced lung capacity, fibrosis, airway inflammation and mucus production. The method comprises administering to the individual an IL-13 antibody, or a fragment thereof, in an amount sufficient to treat (for example, reduce, ameliorate) or prevent one or more symptoms. The IL-13 antibody can be administered therapeutically or prophylactically, or both. The IL-13 antagonist, for example, the anti-IL-13 antibody, or fragment thereof, can be administered to the individual, alone or in combination with other therapeutic modalities, as described herein. In certain embodiments, the individual is a mammal, for example, a human suffering from a disorder associated with IL-13, as described in the present document.
[00158] As noted above, IL-13 has been involved in an essential role in causing pathological responses associated with asthma. However, other mediators of different immune pathways are also involved in the pathogenesis of asthma, and blocking these mediators, in addition to IL-13, may offer additional therapeutic benefits. Thus, the binding proteins of the invention can be incorporated into a bispecific antibody, in which the bispecific antibody is able to bind target pairs that include, but are not limited to, IL-13 and a pro-cytokine -inflammatory, such as tumor necrosis factor α (TNF-α). TNF-α can amplify the inflammatory response in asthma and can be linked to disease severity (McDonnell et al, Progress in Respiratory Research (2001), 31 (New Drags for Asthma, Allergy and COPD), 247-250. ). This suggests that blocking both IL-13 and TNF-α may have beneficial effects, particularly in severe airway disease. In a non-limiting embodiment, the bispecific antibody of the invention binds the targets IL-13 and TNF-α and is used for the treatment of asthma.
[00159] In another embodiment, the binding proteins of the invention can be used to generate bispecific antibody molecules that bind IL-13 and IL-1beta, IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and DL-25; IL-13 and TARC; EL-13 and MDC; IL-13 and M1F; IL-13 and TGF-β; EL-13 and LHR agonist; DL-13 and CL25; IL-13 and SPRR2a; EL-13 and SPRR2b; and DL-13 and ADAM8. The present invention also provides bispecific antibodies capable of binding IL-13 and one or more targets involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1; IFNG, histamine and histamine receptors, EL1A, DL1B, BL2, IL3, EL4, IL5, IL6, IL7, IL8, IL9, IL10, ELI 1, IL12A, IL12B, IL14, IL15, IL16, IL17, IL18, EL19 , IL-20, IL-21, IL-22, EL-23, EL-24, EL-25, IL-26, IL-27, EL-28, IL-30, EL-31, EL-32, IL -33, tTLG, PDGFB, IL2RA, EL4R, IL5RA, IL8RA, DL8RB, IL12RB1, IL12RB2, EL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL13 , CCL20, CCL22, CCL24.CX3CL1, CXCLl, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6 , TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR and chitinase. EXAMPLES 1. The production of anti-IL-13 antibody
[00160] A drug substance production batch consists of an ABT-308 monoclonal antibody solution obtained from the germinal series, production, primary recovery and capture, and fine purification of the drug substance derived from a single cycle of the production reactor. 1.1 Preparation of medium
[00161] The solutions are prepared according to GMP Solution Records with purified water that meets USP / EP / JP standards. The formulated medium solution consists of 0.1 μm filtered in a suitably sized bag, bioreactor or pre-sterilized container. The 0.1 μm filter is tested for integrity after use. The compositions of the production and growth medium are given in Table 2. Table 2. Composition of the cell culture medium

1.2 Expansion of inoculum
[00162] The Biowave vial and pouch operations serve to expand CHO cells from a single frozen vial of MCB to the desired biomass for inoculation of a 110 L seed bioreactor. A vial frozen from the cell bank primary is defrosted and placed in the growth medium (SR-512) and centrifuged. The cells are resuspended in the growth medium and expanded at 37 ° C and 5% CO2 in disposable centrifuge bottles or Biowave bags of increasing volume. Duplicate 20 L Biowave bags are used to maximize the expansion of final cell mass prior to inoculation into the seed bioreactor. When cell density reaches> 2.0 x 106 viable cells / mL from both 20 L Biowave bags in approximately 15 to 17 days, the culture is transferred to a 110 L seed bioreactor loaded with the medium SR-520 for additional expansion. After inoculation, the target temperature is 37 ° C and the pH is adjusted to a target of 7.1 and controlled by adding NaOH and spraying CO2. Dissolved oxygen (DO) in the bioreactor is controlled at a target value of 40% by spraying with air and oxygen. Once the cell density reaches> 2.6 x 106 viable cells / mL after approximately 2 to 4 days, the culture is transferred to a 3000 L production bioreactor. 1.3 Short fill bioreactor
[00163] A partial fill of the 3000 L production bioreactor is used to further expand the cell culture. Initially, the reactor is loaded with the growth medium (SR-520) and inoculated with the batch from the 110 L seed bioreactor.
[00164] During this short filling stage, the temperature, dissolved oxygen and pH are controlled at 37 ° C, 40% and 7.1, respectively. The pH of the culture is controlled by spraying CO2 and adding NaOH. Typically, cells grow for 2 to 4 days before reaching the required density of> 1.6 x 10 6 viable cells / mL. 1.4 Production bioreactor
[00165] The SR-521 production medium (1950 L) is added to the cell culture in the 3000 L bioreactor to start the production stage. Defoamer C is added to reduce foaming. The pH of the culture is controlled to a target value of 6.9 by spraying CO2 and adding intermittent NaOH. The temperature and dissolved oxygen are controlled at target values of 35 ° C and 40%, respectively. The DO in the bioreactor is initially controlled to the desired value by spraying air and supplemented with pure oxygen if necessary. In certain embodiments, the temperature is reduced to a target value of 33 ° C, when the viable cell density reaches> 3.0 x 106 cells / mL, and the pH and DO are maintained at target values of 6.9 and 40%, respectively. Glucose (SR-334) is added as needed. Cultures are collected and purified as described below, when cell viability drops to <50%. 1.5 Process performance
[00166] Process performance and test results during the process are given in Table 3 and Table 4, respectively. Table 3. Performance of the cell culture process for manufacturing ABT-308

Table 4. Test results during the cell culture process

The. Specification 2. The isolation and purification of the anti-IL-13 antibody
[00167] The primary recovery and capture operations include clarification of the collection by means of filtration, capture of the antibody by protein A affinity chromatography and low pH viral inactivation followed by depth filtration. Fine purification operations include anion exchange chromatography, hydrophobic interaction chromatography, viral filtration, ultrafiltration / diafiltration and final filtration, bottling and freezing. 2.1 Preparation of solutions
[00168] The solutions are prepared according to GMP Solution Records with purified water USP (USP-PW) or water for injection (WFI). The main solutions consist of 0.2 μm filtered in irradiated bags, containers vaporized in place or subjected to autoclave. 2.2 Primary recovery and clarification
[00169] The purpose of primary filtration recovery is to remove cells and cell fragments from the collection of the production bioreactor. The unprocessed collection is passed through a filter set consisting of depth filters, delipid depth filters and membrane filters. The clarified supernatant is collected in the collection tank and kept at 2 to 8 ° C. Controls during the process for clarified collection include ABT-308 concentration by Poros A chromatography, biocharge and endotoxin testing. 2.3 Protein A affinity chromatography
[00170] The purpose of protein A affinity chromatography is to capture ABT-308 from the clarified collection and reduce the impurities related to the process. Three chromatography cycles are typically performed to process the entire collection. The product concentrations from the three cycles are combined for further processing.
[00171] A column of 45 cm in diameter x 22 cm in length (35 L) is packed with MabSelect® protein resin (GE Healthcare) or ProSep Ultra Plus ™ (Millipore) and qualified for use. The storage buffer is removed from the column by purified water USP (USP-PW) followed by 0.2 M acetic acid and finally by rinsing with USP PW. The column is equilibrated with 25 mM Tris, 100 mM NaCl, pH 7.2, then loaded with clarified collection to a maximum of 32 g of protein / L of resin for MabSelect® protein A resin (GE Healthcare ) or 45 g of protein / L of resin for the ProSep Ultra Plus ™ kingdom (Millipore). The column is washed with 25 mM Tris, 100 mM NaCl, pH 7.2, then with 20 mM sodium citrate, 0.5 M NaCl, pH 6.0 and, finally, washed again with 25 mM Tris, 100 mM NaCl, pH 7.2. The antibody is eluted from the column with 0.1 M acetic acid, pH 3.5. After each cycle, the pH of the eluate concentration is adjusted to a target of 4.1, if required. Controls during the process include the determination of protein concentration by A280, SE-HPLC, biocarrier and endotoxin test.
[00172] After the first cycle, the column is regenerated with 0.2 M acetic acid and rinsed with USP-PW. After the second cycle, the column is regenerated with 0.2 M acetic acid, rinsed with USP-PW, then disinfected with 0.1 M acetic acid, 20% ethanol followed by washing and short term storage in 50 mM sodium acetate, 20% ethanol, pH 5. After the third cycle, the column is regenerated with 0.2 M acetic acid and rinsed with USP-PW. It is then cleaned with 0.4 M acetic acid, 0.5 M NaCl, 0.1% Tween 80, followed by USP-PW, followed by 50 mM NaOH, 1.0 M NaCl , then, USP-PW. Finally, it is disinfected with 0.1 M acetic acid, 20% ethanol, followed by washing and storage in 50 mM acetic acid, 20% ethanol, pH 5.0. 2.4 Low pH Incubation and Filtration
[00173] Low pH incubation is a dedicated viral reduction step that provides additional assurance of viral safety by inactivating unexpected enveloped viruses that could be present in protein eluate A. The purpose of filtration after low pH incubation is to remove any precipitates that may form during the low pH treatment.
[00174] The pH of the combined protein A chromatography eluates is adjusted to a target value of 3.5 with 0.5 M phosphoric acid and maintained at 18 to 25 ° C for 60 to 70 minutes. The mixture is then adjusted to pH 5 with 1 M Tris, pH 10, clarified by a combination of depth filters and membrane filters, then cooled to 10 to 14 ° C. In-process controls for the low pH treatment and filtration step include determination of protein concentration by A280, SE-HPLC, biocharge and endotoxin testing. 2.5 Performance of the primary recovery and capture process
[00175] The performance of the process for the primary recovery and capture operations is given in Table 5 and the results of the controls during the process are given in Table 6. Table 5. Performance of the primary recovery and capture process
a Combined yield due to sampling error after low pH inactivation.
[00176] B The depth filter area was three times larger in batch 56136BI than in batches 57058BI and 58207BI. The larger filter area in batch 56136BI resulted in decreased yield. Table 6. Test results during the primary recovery and capture process
a Samples of biocharge and endotoxin taken at the start of the next unit operation. b Sample not taken. 2.6 Strong anion exchange chromatography
[00177] The purpose of the strong anion exchange chromatography step is to reduce impurities related to the process, such as host cell proteins, DNA and endotoxins. It can also serve as a viral elimination step. In certain embodiments, a Q Sepharose ™ FF resin column is operated in a continuous flow mode in which the antibody flows through the column and the impurities remain attached to the resin, in alternative modalities, a Mustang Q ™ membrane (Pall Corp. ) is used in place of the Q Sepha- rose FF resin column. Operations are performed at 10 to 14 ° C.
[00178] A column of 45 cm in diameter x 22 cm in length (35 L) is packed with Q Sepharose ™ FF resin (GE Healthcare) and qualified for use. The column is equilibrated with 25 mM Tris, 50 mM NaCl, pH 8.0. The pH of the filtered and neutralized inactivation solution is adjusted to 8.0 with 1 M Tris, pH 10, the conductivity is adjusted to 5.0 to 6.5, and the solution is filtered through membrane and delipid filters. The Q Sepharose charge is pumped through the column at a maximum charge of 80 g of protein / L of resin. After loading, the column is washed with 25 mM Tris, 50 mM NaCl, pH 8.0 and the continuous flow and wash are combined. This consists of Q Sepharose ™ continuous flow concentration and washing (QFTW). In-process controls for the Q Sepharo-se ™ step include A280 concentration, SE-HPLC, biocharge and endotoxin testing.
[00179] The column is regenerated with 25 mM sodium phosphate, 1.0 M NaCl, pH 7.0, followed by a rinse with WFI, and disinfected with 1.0 M NaOH followed by a rinse with WFI. The column is then neutralized with 25 mM sodium phosphate, 1.0 M NaCl, pH 7.0 and stored in 25 mM sodium phosphate, 20% isopropanol, pH 7.0. 2.7 Hydrophobic interaction chromatography
[00180] The purpose of the Phenyl Sepharose ™ step is to remove aggregates, ABT-308 fragments and process-related impurities. The operations are carried out at 10 to 14 ° C.
[00181] A column of 60 cm in diameter x 15 cm in length (42 L) is packed with Phenyl Sepharose ™ HP resin (GE Healthcare) and qualified for use. The column is equilibrated with WFI, then, 20 mM sodium phosphate, 1.1 M ammonium sulfate, pH 7.0.
[00182] The continuous flow of Q Sepharose ™ and the wash is diluted 1: 1 (by volume) with 40 mM sodium phosphate, 2.2 M ammonium sulfate, pH 7.0. This solution, the Fenil Sepharose ™ filler, is filtered through a 0.2 μm filter and loaded onto the column at a maximum load of 64 g of protein / L of resin. The column is washed with 25 mM sodium phosphate, 1.4 M ammonium sulfate, pH 7.0 and ABT-308 is eluted from the column with 11 mM sodium phosphate, 0.625 M south - ammonium fact, pH 7.0. In-process controls for the Phenyl Sepharose ™ step include A280 concentration, SE-HPLC, biocharge and endoxin test.
[00183] The column is regenerated with WFI, then disinfected with 1 M NaOH, rinsed with WFI and stored in 25 mM sodium phosphate, 20% isopropanol, pH 7. 2.8 Nanofiltration
[00184] Nanofiltration consists of a dedicated viral elimination step that provides an additional guarantee of viral safety by physically removing unexpected viruses> 20 nm in diameter that could be present in the phenyl sepharose HP column eluate. Operations are performed at 10 to 14 ° C.
[00185] The Phenyl Sepharose ™ HP column eluate is passed through a 0.1 μm filter and a Ultipor DV20 filter set pre-moistened with 15 mM histidine, pH 5.6. After filtration, the filter set is washed with 15 mM of histidine, pH 5.6, to recover any retained ABT-308. After use, an integrity test is performed on the DV20 filter and the filter is discarded. If the filter does not pass the integrity test, the solution can be refiltered as described above. In-process controls for the nanofiltration step include A280 protein concentration, SE-HPLC, biocharge and endotoxin testing. 2.9 ABT-308 drug substance formulation by Ultrafiltration / Diafiltration
[00186] The purpose of the UF / DF step is to diafiltrate the drug substance in the final formulation buffer, 15 mM histidine, pH 5.6 and ABT-308 concentration. These operations are performed in 10 to 14 ° C.
[00187] The nanofiltrate is concentrated to approximately 50 g / L with the use of MWCO 30 kDa polyether sulfone membranes, diafiltered with the formulation buffer, then concentrated to approximately 180 g / L. The UF system is drained from the product and rinsed with the diafiltration buffer to recover any remaining product in the system. The concentrate and wash are combined to produce ABT-308 diafiltered at a concentration of approximately 120 to 160 g / L. ABT-308 concentrate is filtered through membrane filters. In-process controls for the ultratfiltration / diafiltration step include A280 concentration, SE-HPLC, biocharge and endotoxin testing.
[00188] After each procedure, the ultrafiltration system is washed with WFI and cleaned with a 250 ppm sodium hypochlorite solution, then disinfected and stored in 0.1 M sodium hydroxide. 2.10 Final filtration, bottling and freezing
[00189] The filtration and bottling operations are performed in a Class 100 area at 2 to 8 ° C in a Class 100 laminar flow hood. The formulated ABT-308 is filtered through a 0.2 μm filter in bottles of Pyrogen-free and pre-sterilized PETG. The labeled bottles are placed in an empty freezing unit at - 80 ° C (nominal) until freezing and then transferred to storage freezers kept at -80 ° C (nominal). In-process controls for the final filtration and bottling step include A280, biocharge and endotoxin testing (drug substance test results). 2.11 Performance of the fine purification process
[00190] The performance of the process for the primary recovery and capture operations is given in Table 7 and the results of the controls during the process are given in Table 8. Table 7. Process performance, fine purification

Table 8. Test results during the fine purification process


[00191] a. Biocharge and endotoxin samples taken at the beginning of the next unit operation.
[00192] b. The 6 EU / mL endotoxin result is not considered to be significant due to the fact that the subsequent drug substance meets the <0.2 EU / mg specification. In addition, lot 56003BF consisted of an engineering process and was not released for human use.
[00193] c. Specification of drug substance release.
[00194] d. Result of drug substance release. 3. Determination of host cell protein concentration in antibody compositions
[00195] This procedure describes the test methodology for determining the concentration of residual host cell protein in antibody samples. The enzyme linked immunosorbent assay (ELISA) is used to place the host cell protein (Antigens) between two layers of specific antibodies. This is followed by blocking non-specific sites with casein. The host cell proteins are then incubated during the time that the antigen molecules are captured by the first antibody (Coating antibody). A second antibody (anti-biotinylated host cell protein) is then added, which attaches to the antigen (host cell proteins). HRP-conjugated neutravidine is added, which binds to biotinylated host cell anti-protein. This is followed by the addition of blue substrate. The chromogenic substrate is hydrolyzed by the antibody conjugated to the bound enzyme, producing a blue color. The reaction is stopped with 2M H3PO4, changing the color to yellow. The color intensity is directly proportional to the amount of antigen bound in the well.
[00196] Preparation of 50 mM Sodium Bicarbonate (Coating Buffer), pH 9.4. To a 1 L beaker add: 900 mL of Milli-Q water; 4.20 g ± 0.01 g of sodium bicarbonate. Stir until completely dissolved. Adjust pH to 9.4 with 1 N NaOH. Transfer to a 1 L volumetric flask and add Milli-Q water until the volume of the flask is reached. Mix by inversion until homogeneous. Filter through a 0.22 μm sterile filter unit. Store at nominal 4 ° C for up to 7 days from the date of preparation.
[00197] Preparation of 0.104 M Na2HPO4 * 7H2O, 1.37 M NaCl, 0.027 M KCl, 0.0176 M KH2PO4, pH = 6.8 to 6.9 (10X PBS). Add approximately 400 mL of Milli-Q water to a glass beaker. Add 13.94 g ± 0.01 g of Na2HPO4 x 7H2O. Add 40.0 g ± 0.1 g of NaCl. Add 1.00 g ± 0.01 g of KCl. Add 1.20 g ± 0.01 g of KH2PO4. Shake until smooth. Transfer to a 500 mL volumetric flask. QS to 500 mL volume with Milli-Q water. Mix by inversion. Filter through a 0.2 μm sterile filter unit. Store at room temperature for up to 7 days.
[00198] Preparation of 1X PBS + 0.1% Triton X-100, pH 7.40: (Plate wash buffer). In a 4 L graduated cylinder, mix 400 mL of 10 X PBS (step 5.2) with 3500 mL of Milli-Q water. Check the pH and adjust if necessary to 7.40 ± 0.05 with 1 N HCl or 1 N NaOH. Add Milli-Q water until the volume of the bottle is reached. Cover the cylinder tightly with parafilm and mix by inversion until smooth. Transfer to a 4 L bottle. Remove 4 mL of 1 X PBS and discard. Add 4 ml of X-100 triton to 3996 ml of 1 X PBS. Place on the stirring plate and shake until completely dissolved. Filter the amount of plate wash buffer needed for the preparation of dilution buffer through a 0.22 μm sterile filter unit. Store at room temperature for up to 7 days.
[00199] Preparation of coating antibody mixture: goat anti CHO 599/626/748 (batch no. G11201 @ 1.534 mg / mL), affinity purified: NOTE: Stocks stored at - 80 ° C nominal in vials. Prepare rates. Take one rate per plate at the time of use.
[00200] Immediately before use: Dilute the antibody mixture to have a final concentration of 4 μg / mL in 50 mM cold sodium bicarbonate, as shown below. For example: add 31 μLs of coating antibody mixture to 11969 μLs of cold coating buffer. Mix by inversion delicately.
[00201] Preparation of a mixture of anti-biotinylated host cell protein, 599/626/748 (lot no. G11202 @ 0.822 mg / mL): NOTE: Stocks stored at nominal -80 ° C in vials. Prepare rates. Take one rate per plate at the time of use. Immediately before use: dilute the biotinylated antibody mixture to have a final concentration of 1 μg / mL in casein at 37 ° C ± 2 ° C, as shown below. For example: add 14.6 μLs of biotinylated antibody mixture to 11985 μLs of casein at 37 ° C ± 2 ° C. Mix by inversion delicately.
[00202] Preparation of Neutravidine-HRP. Reconstitute new batches (2 mg vial) to 1 mg / mL, as shown below: Add 400 μL of Milli-Q water to the vial, then add 1600 μL of 1X PBS, for a total of 2 mL. Vortex gently. Store at - 20 ° C nominal. Prepare aliquots with the desired volume so that 1 aliquot per plate is used. Prepare in polypropylene tube. Qualify new batches to determine the work concentration. Designate expiration of 6 months from the date of preparation. For example, if the concentration of work was determined to be 0.2 μg / mL, then prepare as explained below. Immediately before use: defrost a Neutravidin-HRP aliquot at room temperature. Dilute the Neutravidine solution from 1 mg / mL to 0.1 mg / mL (100 μg / mL) with Casein at 37 ° C ± 2 ° C. For example: Dilute X10, add 50 μL of neutravidine to 450 μL of Casein. Vortex gently. Additionally dilute the solution from 100 μg / mL to 0.2 μg / mL with Casein at 37 ° C ± 2 ° C. For example: Dilute X500, add 24 μL of neutravidine (100 μg / mL) to 11976 μL of Casein. Vortex gently.
[00203] Preparation of 5.7 2M phosphoric acid (Stop solution). Prepare a 2 M phosphoric acid solution from concentrated phosphoric acid as explained below. From the% of phosphoric acid indicated on the label, the density (1.685g / ml) and the weight of the formula (98 g / mol), calculate the volume of concentrated phosphoric acid needed to prepare 500 ml of 2M of phosphoric acid. Add the volume of concentrated phosphoric acid calculated above to the flask. Add Milli-Q water until the volume of the bottle is reached and mix by inversion until it is homogeneous. Store at room temperature for up to 6 months from the date of preparation.
[00204] Preparation of dilution buffer (X100 diluted in casein in 1X PBS + 0.1% Triton X100, pH 7.4). Dilute Casein X100 at 37 ° C ± 2 ° C in 1X PBS + 0.1% sterile Triton X100 by 0.22 μm filtration, pH 7.4 (from above). For example: Add 1 mL of Casein at 37 ° C ± 2 ° C to 99 mL of 1X PBS + 0.1% Triton X100 sterilized by 0.22 μm filtration, pH 7.4. Mix well. Prepare new for each use.
[00205] Preparation of Standards. Host cell protein standards (Antigen standards) (lot no. G11203 @ 1,218 mg / mL): NOTE: Stocks stored at nominal -80 ° C in 70 aliquots. Defrost an aliquot at room temperature. Perform serial dilutions in polypropylene tubes using the dilution buffer.
[00206] Sample Preparation. In polypropylene tubes, dilute final bulk samples to 24 mg / mL in dilution buffer. Record the concentration. NOTE: use the solutions below to prepare reinforced samples and to prepare the 12 mg / mL solutions mentioned below. In polypropylene microtubes, further dilute the solutions from 24 mg / mL to 12 mg / mL in dilution buffer. Load triplicate wells for each of the 12 mg / mL solutions on the plate for a total of 6 wells.
[00207] Reinforcement Preparation. In a polypropylene microtube, prepare a 10 ng / mL host cell protein booster from the 20 ng / mL standard prepared above, by diluting 2X with dilution buffer. Load three wells for the 10 ng / mL reinforcement solution onto the plate. Use the standard 20 ng / mL solution from step 6.1 to reinforce the samples.
[00208] Preparation of reinforced samples. In polypropylene microtubes, reinforce 300 μL of each solution in a final mass of 24 mg / mL with 300 μL of the 20 ng / mL reinforcement solution (6.1). Load triplicate wells for each sample solution reinforced by a total of 6 wells.
[00209] Preparation of Control. A control range needs to be adjusted for each new control stock solution, prior to use in routine testing. Control stage: Prepare 150 μL aliquots of a batch of ABT-308 drug substance concentrate and store frozen at -80 ° C nominal for up to three years.
[00210] Work Control Preparation. Defrost a control aliquot at room temperature. In polypropylene tubes, dilute the control to 24 mg / mL with dilution buffer. In polypropylene microtubes, additionally dilute the 24 mg / mL control solution with 12 mg / mL dilution buffer. Prepare a single control of loading and dilution in 3 wells of the plate.
[00211] ELISA procedures. Fill the plate wash bottle with plate wash buffer (referring to step 5.3, 1X PBS + 0.1% Triton X-100). Start the plate washer. Check the following parameters: Parameters should be adjusted for: Plate type: 1 For each cycle (a total of 5 cycles): Volume: 400 μLs; Rinse time: 10 seconds; Asp .: 4 seconds.
[00212] Test procedure. Coat plates with 100 μL / 4 μg / mL well of goat coating antibody mixture in 50 mM cold sodium bicarbonate. Tap the side of the plate until the coating solution covers the bottom of the wells evenly, cover with electrical tape and incubate at 4 ° C nominal, while shaking on a plate shaker (or equivalent) at speed 3 by 18 hours ± 1 hour. After incubating overnight, remove the plate from the refrigerator and allow it to equilibrate to room temperature. Shake the coating. Dry the plate with paper towels. Block with 300 μL / well of Casein at 37 ° C ± 2 ° C, cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 1 hour. Prepare standard, sample, control, reinforcement and reinforced samples during the block incubation. Wash the plate 5 times with Wash Buffer. Dry the plate with paper towels. Using an 8-channel pipette, pipette 100 μL / well of standards, samples, reinforcements, reinforced samples and control in triplicate wells of the plate. Pipette 100 μL / well of dilution buffer into all empty wells of the plate to serve as blank solutions. Cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 1 hour. Fill out a template to use as a guide when loading the card.
[00213] Configuration of card reader. Configure model, insert concentrations for patterns. Do not enter dilution factors for samples, control, reinforcement or reinforced samples. Designate the wells containing diluents as blank solutions to be subtracted from the total wells. Wash the plate 5 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of biotinylated goat antibody. Cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 1 hour. Wash the plate 5 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of Neutravidine-HRP conjugate solution. Cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 1 hour. Wash the plate 5 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of cold blue K Substrate, cover with electrical tape and incubate at room temperature for 10 minutes (start the timer as soon as the substrate is added to the first row), while shaking at speed 3 on the stirrer. Lab-line titration plate (or equivalent). Stop the reaction by adding 100 μL / well of 2M phosphoric acid (Step 5.7). Place the plate on a plate shaker at speed 3 for 3 to 5 minutes. Read the plate at 450 nm.
[00214] Data analysis and calculations. NOTE: only samples, reinforcements, reinforced samples and control, with optimal densities that fall within the practical quantification limit (2.5 ng / mL of standard) of the standard curve and that meets% CV or% difference criteria indicated below are accepted. If the OD's in the sample fall below the 2.5 ng / mL standard, the result should be reported as less than 2.5 ng / mL. This value should then be divided by the concentration of diluted sample (12 mg / mL) to report the value in ng / mg. If the sample is high in host cell concentration causing the unreinforced and / or reinforced sample to be above the standard curve, report the value as> 100 ng / mL. This value should then be divided by the diluted sample concentration (12 mg / mL) to report the value in ng / mg. Consider the zero value of the sample for reinforcement recovery calculations when the sample is below the standard of 2.5 ng / mL.
[00215] Standard curve. Standard concentrations should be entered in the protocol model. A quadratic curve fit is used. The determination coefficient must be = 0.99 and the CV% between triplicate wells must be = 20%. If these criteria have not been met: A standard (1 level, 3 wells) can be downloaded. If 1.25 ng / mL is lowered, only samples and samples reinforced with optimal densities that fall within the optimal densities of 2.5 ng / mL and 100 ng / mL (the remaining standard curve points) are acceptable. Additionally, for the triplicates of each standard level, if a single well is clearly contaminated or shows low connection, it can be lowered. If a well is lowered from a standard level, the remaining copies must have a% difference = 20%. The% CV for the lowest standard, which shows OD values close to the bottom (blank solutions) of the plate, should be = 30%. If a well is lowered, the% difference for the remaining copies must be = 35%. If the lowest standard is lowered, only samples and samples reinforced with optimal densities that are included in the optimal densities of the remaining standard curve level are acceptable.
[00216] Samples. CV% should be = 20% between triplicate wells. Report% CV between triplicate wells. One well from each sample dilution can be lowered. The remaining copies must have a% difference = 20%. Note: if the OD of the unreinforced sample is below the standard OD of 2.5 ng / mL, the% of difference criteria does not apply to the results of the unreinforced. Refer to the calculation above.
[00217] Calculate actual host cell concentration in ng / mg from the average value (ng / mL), as shown below: CHO host cell protein (ng / mg) = "Unreinforced sample result (ng / mL) ) "average _ diluted sample concentration (12 mg / mL).
[00218] Reinforcements. CV% should be = 20% between triplicate wells. Record% of CV. A well from the reinforcement can be lowered. The remaining points must have a difference of 20% = 20%. Refer to the calculation above. Report the host cell concentration in ng / mL. This result will be used in reinforcement recovery calculations. The resulting concentration for the reinforcement (ng / mL) needs to be ± 20% of the theoretical reinforcement concentration. Record the result and indicate approved or disapproved. If the reinforcement result is not within 20% of the theoretical, the test must be repeated. The mean reinforcement concentration (ng / mL) x 100 = needs to be 100% ± 20% 10 ng / mL.
[00219] Reinforced samples. % CV should be = 20% "between triplicate wells. Record% CV between triplicate wells. One well from each reinforced sample dilution can be downloaded. The remaining copies must have a% difference = 20% Refer to the calculation above Report "Reinforced sample result" for each dilution in ng / mL Record the% difference between duplicate dilutions The% difference between dilutions should be = 25% These results will be used in reinforcement recovery calculations.
[00220] Calculate% of reinforcement recovery for each dilution set using the formula below:% of reinforcement recovery = reinforced sample value - non-reinforced sample value X 100 reinforcement value. NOTE: (1) If the value of the OD's of non-reinforced sample falls below the standard of 2.5 ng / mL, consider the value as zero when calculating the% of reinforcement recovery. The% recovery of reinforcement needs to be 100% ± 50% (50% to 150%) for each dilution, for each sample. Record the results and pass / fail.
[00221] Control. CV% should be = 20% between triplicate wells. Record% of CV result. A well from the control can be lowered. The remaining copies must have a% difference = 20%. Refer to the calculation above. Report the host cell concentration in the control in ng / mL. Calculate the host cell concentration in ng / mg as shown below: Host cell protein (ng / mg) = results of control host cell protein in ng / mL. 4. Determination of protein A concentration in antibody compositions
[00222] In this ELISA, the plates are coated with Chicken Anti-protein A and incubated. Non-specific sites are blocked with casein in PBS. The plates are washed in 1X PBS + 0.1% Triton X-100 to remove unbound material. The sample standards and Cys-protein A are diluted in 1X PBS + 4.1% Triton X + 10% Casein. The solutions are denatured by heating to 95 ° C ± 2 ° C, separating protein A from the antibody. In certain modalities, for example, if (GE Healthcare), the solutions are then added to the plate and incubated. In alternative modalities, for example, if the protein affinity stage includes the use of ProSep Ultra Plus ™ (Milipore), the solutions are cooled and 0.85% NaCl + 12.5% 1 N acid acetic + 0.1% Tween 20, are added to each tube (1: 1) to further assist in the separation of protein A from the sample protein. The tubes are vortexed, incubated and centrifuged. Supernatants are removed and further processed. Unbound material is washed with 1X PBS + 0.1% Triton X-100. Biotinylated chicken anti-protein A is added to the microtiter plate and incubated. The plate is washed to remove unbound material and the Neutravidine - Peroxidase conjugate is added.
[00223] Neutravidine will bind to biotinylated chicken Anti-protein A that has been bound to wells. The plate is washed again to remove unbound Neutravidine and K blue substrate (tetramethylbenzidine (TMB)) is added to the plate. The substrate is hydrolyzed by the bound Neutravidine producing a blue color. The reaction is stopped with phosphoric acid, changing the color to yellow. The intensity of the yellow color in the wells is directly proportional to the concentration of protein A present in the wells.
[00224] Preparation of Reagents and Solutions. Casein bottles need to be heated to 37 ° C ± 2 ° C; sonicated for 2 minutes and separated in aliquots. The aliquots must be stored at a nominal 4 ° C. When the test is to be carried out, the number of casein aliquots required should be set at 37 ° C ± 2 ° C. The coating buffer and substrate are used cold (taken at a nominal 4 ° C just before use).
[00225] 50 mM sodium bicarbonate (Coating buffer), pH 9.4. To a 1 L beaker, 900 ml of Milli-Q water, 4.20 g ± 0.01 g of sodium bicarbonate are added. Stir until completely dissolved. Adjust pH to 9.4 with 1 N NaOH. Transfer to a 1 L volumetric flask and add Milli-Q water to complete the volume. Mix by inversion until smooth. Filter through a sterile 0.22 CA μm filter unit. Store at nominal 4 ° C for up to 7 days from the date of preparation.
[00226] 104 M Na2HPO4 * 7H20, 1.37 M NaCl, 0.027 M KCl, 0.0176 M KH2PO4, pH = 6.8 to 6.9. (10 X PBS): Add approximately 400 mL of Milli-Q water to a glass beaker. Add 13.94 g ± 0.01 g of Na2HPO4 x 7H20. Add 40.0 g ± 0.1 g of NaCl. Add 1.00 g ± 0.01 g of KCl. Add 1.20 g ± 0.01 g of KH2PO4. Shake until smooth. Transfer to a 500 mL volumetric flask. QS at 500 mL volume with Milli-Q water. Mix by inversion. Filter through a 0.2 CA μm sterile filter unit. Store at room temperature for up to 7 days.
[00227] 1X PBS + 0.1% Triton X-100, pH 7.40: (Plate wash buffer). In a 4 L graduated cylinder, mix 400 mL of 10 X PBS (see above) with 3500 mL of Milli-Q water. Check the pH and adjust if necessary to 7.40 ± 0.05 with 1 N HCl or 1 N NaOH. Add Milli-Q water until the volume of the bottle is reached. Cover the cylinder tightly with parafilm and mix by inversion until smooth. Transfer to a 4 L bottle. Remove 4 mL of 1 X PBS and discard. Add 4 ml of X-100 triton to 3996 ml of 1 X PBS. Place on the stirring plate and shake until completely dissolved. Store at room temperature for up to 7 days.
[00228] Chicken Anti-protein A Coating Antibody. Obtain an aliquot of antibody per plate at the time of use. To qualify new batches of Chicken Anti-protein A, it may be necessary to use and qualify Conjugated Chicken Anti-protein A (prepared from the same coating batch) together. Immediately before use: Dilute the antibody mixture in 50 mM cold sodium bicarbonate to the concentration determined during the coating qualification. For example: if during the qualification the coating concentration to be loaded on was determined to be 6 μg / mL and if the stock concentration is 3000 g / mL, then add 24 μLs of coating antibody to 11976 μLs of buffer cold coating. Mix by inversion gently.
[00229] Biotinylated chicken anti protein A. Obtain an aliquot of antibody per plate at the time of use. To qualify new batches of conjugated chicken Anti-protein A-Biotin, it may be necessary to use and qualify with the same batch of chicken Anti-protein A that was prepared. Immediately before use: Dilute the biotinylated antibody in Casein at 37 ° C ± 2 ° C to the concentration determined during the qualification of biotinylated antibody. For example: if during qualification the concentration of biotinylated antibody to be loaded onto the plate was determined to be 4 μg / mL and the stock concentration is 1000 μg / mL, then add 48 μLs of biotinylated antibody to 11952 μLs of Casein at 37 ° C ± 2 ° C. Mix by inversion gently. Neutravidine-HRP. Reconstitute new batches (2 mg / vial) at 1 mg / mL as shown below: Add 400 μL of Milli-Q water to the vial, then add 1600 μL of 1 X PBS, for a total of 2 mL. Vortex gently. Store at -80 ° C nominal. Prepare the aliquots with the desired volume so that 1 aliquot per plate is used. Prepare in polypropylene tube. Designate the 6 month expiration date from the preparation date. For example, if the concentration of work was determined to be 0.1 μg / mL, then prepare as explained below. Immediately before use, defrost a Neutravidine-HRP aliquot at room temperature. Dilute the Neutravidine solution from 1 mg / mL to 0.01 mg / mL (10 μg / mL) with Casein at 37 ° C ± 2 ° C. For example: Dilute X10, add 50 μL of neutravidine to 450 μL of Casein. Vortex gently, X10 again, add 100 μL of X10 neutralravidine to 900 μL of Casein. Vortex gently. Additionally dilute the solution from 10 μg / mL to 0.1 μg / mL, with Casein at 37 ° C ± 2 ° C. For example: Dilute X100, add 120 μL of neutralravidine (10 μg / mL) to 11880 μL of Casein. Invert several times gently to mix. Stop solution (1 N of acquired phosphoric acid is used). Store at room temperature for up to 1 year from the date of receipt. Dilution buffer (1X PBS + 4.1% Triton X100 + 10% Casein, pH 7.4). Add 86 mL of 1X PBS + 0.1% Triton X100, pH 7.4 (from Step 5.3) to a beaker or flask, add 4 mL of Triton X-100 and 10 mL of Blocking Casein in PBS, and shake to mix / dissolve. It may take 20 to 30 minutes to dissolve the triton. This equals a solution of 1X PBS + 4.1% Triton X100 + 10% Casein, pH 7.4. Filter through a sterile 0.22 CA μm filter unit. Prepare new for each use. This is sufficient for 1 plate. Protein A patterns (Antigen patterns). NOTE: Stocks stored at -20 ° C nominal in 70 μL aliquots. Defrost an aliquot on ice. Perform serial dilutions according to the examples on the polypropylene tubes in the table below using dilution buffer (see above) using the concentration indicated in the manufacturers' COA: For example, if the COA indicates that the stock concentration is 2.1 mg / mL (2100000 ng / mL), so: Defrost samples on ice. In polypropylene microcentrifuge tubes, dilute the final bulk samples to 20 mg / mL in dilution buffer (above). Perform 2 separate dilutions. Record the concentration. Use the solutions below to prepare the reinforced samples and to prepare the 10 mg / mL solutions. In polypropylene microcentrifuge tubes, additionally dilute the 20 mg / mL to 10 mg / mL solutions in dilution buffer. Reinforcement Preparation. In a polypropylene microcentrifuge tube, prepare a 0.296 ng / mL protein A boost from the 0.593 ng / mL standard prepared above in Step 6.1, by diluting it 2X with dilution buffer. Perform a single dilution. The triplicate wells for the 0.296 ng / mL reinforcement solution will be loaded onto the plate. Use the standard 0.593 ng / mL solution from Step 6.1 to reinforce the samples.
[00230] Preparation of reinforced samples. In polypropylene microcentrifuge tubes, reinforce 500 μL of each solution in a final mass of 20 mg / mL with 500 μL of the 0.593 ng / mL reinforcement solution. Save for denaturation. The triplicate wells for each reinforced sample solution will be loaded onto the plate for a total of 6 wells.
[00231] Preparation of Control. Obtain a batch of ABT-308 drug substance. Prepare 150 μL aliquots and store frozen at - 80 ° C nominal for three years from the date of aliquoting.
[00232] Work control: Defrost a control rate on ice. In a polypropylene microcentrifuge tube, dilute the control to 10 mg / mL with dilution buffer to have a final volume of 1000 μLs. Prepare a single dilution. Save for denaturation. The triplicate control wells will be loaded onto the plate.
[00233] Denaturation. For blank plate solutions, add 1000 μLs of dilution buffer to the microcentrifuge tubes equal to the number of blank solutions that will be run on the plate. The tube caps can be covered with parafilm to prevent them from bursting during heating or a second shelf can be placed on top of the tubes to keep the caps closed. Heat standards, non-reinforced samples, reinforced samples, reinforcement, blank solutions and control at 95 ° C ± 2 ° C for 15 minutes. Remove the parafilm from the tubes during cooling, if used. Allow to cool for 15 minutes and centrifuge for 5 minutes at approximately 10,000 rpm. Transfer 700 μLs of the supernatant into the microtubes to load on the plate. Be careful not to affect the triton / protein pellet.
[00234] Plate washer and bath setup instructions. Fill a plate wash bottle with plate wash buffer (referring to Step 5.3, 1X PBS + 0.1% Triton X-100). Start the plate washer. Check the following parameters: Parameters should be adjusted for: Plate type: 1 For each cycle (a total of 4 cycles): Asp speed: 10 mm / s; Volume: 400 μLs; Rinse time: 5 seconds; Asp time: 6 seconds. Activate the water bath and adjust to 95 ° C. Allow the water bath temperature to equilibrate to 95 ° C ± 2 ° C for at least 30 minutes.
[00235] Test procedure: A checklist can be used as a guide by checking the steps as they are completed. Additionally, record all equipment used during the test. The amount of Casein aliquots to be used for each day the test will be performed must be set at 37 ° C ± 2 ° C. The coating buffer and substrate are used cold. Prepare the standard, sample, control, reinforcement and reinforced samples before and during the block incubation. It may take longer than the 1-hour block incubation to prepare dilutions, transfer to eppendorf tubes, denature for 15 minutes, cool for 15 minutes, centrifuge for 5 minutes and transfer to microtubes. Leave at least 40 minutes before blocking the plates. Samples, Reinforced Samples, Standards, Control, Test Booster and blank solutions are loaded onto the plate horizontally from rows B to G using a 12-channel pipette. The patterns are loaded from high to low concentration. The plate coating, addition of biotin, addition of neutralravidine, addition of substrate and addition of stop solution are made vertically from columns 2 to 11.
[00236] Coat the plates with 100 μL / well of coating antibody in 50 mM cold sodium bicarbonate. Tap the side of the plate until a coating solution covers the bottom of the wells evenly, cover with electrical tape and incubate at 4 ° C nominal, while shaking on a plate shaker (or equivalent) at speed 3.
[00237] After incubating overnight, remove the plate from the refrigerator and allow it to equilibrate to room temperature. Shake the coating. Dry the plate with paper towels. Block with 300 μL / well of Casein at 37 ° C ± 2 ° C, cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker ( or equivalent) at 80 rpm ± 5 rpm for 1 hour ± 10 minutes.
[00238] Prepare the pattern, sample, control, reinforcement and reinforced samples before and during the blocking incubation. Wash the plate 4 times with washing buffer. Dry the plate with paper towels. Using an 8-channel pipette, piping 100 μL / well of standards, samples, reinforcements, reinforced samples, blank solutions and denatured controls in triplicate wells of the plate. Wells outside the plate are not used, add untreated dilution buffer to these wells. Cover with electrical tape and incubate at 37 ° C ± 2 C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 2 hours. Fill out a template to use as a guide when loading the card.
[00239] Card reader configuration. Wash the plate 4 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of biotinylated antibody. Cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 1 hour.
[00240] Wash the plate 4 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of Neutravidine-HRP conjugate solution. Start the timer as soon as neutravidine is added to the last row. Cover with electrical tape and incubate at 37 ° C ± 2 ° C, while shaking on a Lab-line Environ plate shaker (or equivalent) at 80 rpm ± 5 rpm for 30 minutes. Wash the plate 4 times with washing buffer. Dry the plate with paper towels. Add 100 μL / well of blue K substrate, cover with electrical tape and incubate at room temperature for 10 minutes (start the timer as soon as the substrate is added to the first row), while shaking at speed 3 on the Lab titration plate shaker -line (or equivalent). Stop the reaction by adding 100 μL / well of 1 N phosphoric acid. Place the plate on a plate shaker at speed 3 for 3 minutes. Read the plate at 450 nm.
[00241] Data analysis and calculations. NOTE: Only samples, reinforcements, reinforced samples and control with optimal densities that fall within the practical quantification limit of the standard curve and that meet the% CV or% of difference criteria indicated below, are accepted. If the OD's in the sample fall below the standard curve, the result should be reported as less than 0.18 ng / mL (LOQ test). This value should then be divided by the diluted sample concentration (10 mg / mL) to report the value in ng / mg. If the sample is high in protein A concentration causing the sample not reinforced and / or reinforced to be above the standard curve (2 ng / mL), then dilute additionally so that it is within the standard curve. This value should then be divided by the diluted sample concentration to report the value in ng / mg. For reinforcement recovery calculations, subtract the non-reinforced sample value (ng / mL) from the reinforced sample value (ng / mL), even when the non-reinforced sample value (ng / mL) is below the curve. If the value is negative or the 'range' is obtained, then consider the non-reinforced sample as zero for the reinforcement recovery calculations.
[00242] Standard curve. Standard concentrations should be inserted into the protocol model. A quadratic curve fit is used. The determination coefficient must be = 0.99 and the% CV between the triplicate wells must be = 20%. If these criteria have not been met: A standard (1 level, 3 wells) can be downloaded. If 0.18 ng / mL is lowered, only samples and samples reinforced with optimal densities that fall within the optimal densities of 0.26 ng / mL and 2 ng / mL (the remaining standard curve points) are acceptable. Additionally, for the triplicates of each standard level, if a single well is clearly contaminated or shows low connection, it can be lowered. If a well is lowered from a standard level, the remaining copies must have a% difference = 20%. The% CV for the lowest standard, which shows OD values close to the bottom (blank solution) of the plate, should be = 30%. If a well is lowered, the% difference for the remaining copies must be = 35%. If the lowest standard is lowered, only samples and samples reinforced with optimal densities that are included in the optimal densities of the remaining standard curve level are acceptable.
[00243] Calculate the% difference as shown below:% difference = (Abs. (Dilution of result 1 - dilution of result 2) / average value) X 100%. The test needs to be repeated if the standards do not meet the above criteria. Report% CV and / or% difference values and standard curve determination coefficient results.
[00244] Samples. CV% should be = 20% between triplicate wells. Report% CV between triplicate wells. One well from each sample dilution can be lowered. The remaining copies must have a% difference = 20%. Note: If the OD of the unreinforced sample is below the lowest standard OD, the% of difference criteria does not apply to the results of the non-reinforced: Refer to the calculation above.
[00245] Report "Result of unreinforced sample" for each dilution in ng / mL. These values will be used in reinforcement recovery calculations. Calculate the mean "Unreinforced sample result (ng / mL)" and the% difference between dilutions. Report the results. The% difference between dilutions must be = 25%. Calculate the concentration of real protein A in ng / mg from the average value (ng / mL), as shown below: protein A (ng / mg) = "Average non-reinforced sample result (ng / mL)" Concentration of diluted sample (10 mg / mL). Record the result.
[00246] Reinforcements. The CV% should be = 20% between the triplicate wells. Record the CV%. A well from the reinforcement can be lowered. The remaining points must have a% difference = 20%. Refer to the calculation above. Report the protein A concentration in ng / mL. This result will be used in reinforcement recovery calculations. The resulting reinforcement concentration (ng / mL) must be ± 20% of the theoretical reinforcement concentration. Record the result and indicate pass or fail. If the reinforcement result is not within 20% of the theory, the test needs to be repeated. The mean reinforcement concentration (ng / mL) x 100 = needs to be 100% ± 20% 0.296 ng / mL
[00247] Reinforced samples. The CV% should be = 20% between the triplicate wells. Record the% CV between the triple wells. One well from each reinforced sample dilution can be lowered. The remaining copies must have a difference of 20% = Refer to the calculation above. Report "enhanced sample result" for each ng / mL dilution. Record the% difference between duplicated dilutions. The% difference between dilutions should be = 25%. These results will be used in the reinforcement recovery calculations. Calculate the% reinforcement recovery for each dilution set using the formula below:% reinforcement recovery = reinforced sample value - non-reinforced sample value X 100. Reinforcement value. NOTE: For reinforcement recovery calculations, subtract the non-reinforced sample value (ng / mL) from the reinforced sample value (ng / mL), even when the non-reinforced sample value (ng / mL) is below the curve. If the value is negative or the 'range' is obtained, then consider the unreinforced sample as zero for the reinforcement recovery calculations. Reinforcement recovery% must be 100% ± 50% (50% to 150% o) for each dilution for each sample. Record the results and pass / fail.
[00248] Control. The CV% should be = 20% between the triplicate wells. Record the% CV result. A well from the control can be lowered. The remaining copies must have a% difference = 20%. Table 9. Results of protein A and residual host cell protein assay
Table 10. Results of protein A and residual host cell protein assay: In process samples

[00249] Several publications are mentioned in this document, the contents of which are incorporated herein by reference, in their entirety.

权利要求:
Claims (8)
[0001]
1. Method for purifying an anti-IL-13 antibody from a sample mixture comprising an anti-IL-13 antibody and at least one host cell protein (HCP), said method CHARACTERIZED by the fact that it includes: (a) depth filtration of said first pH-adjusted sample and collection of a depth-filtered sample; (b) contacting said filtered sample in depth with a Protein A affinity chromatography resin, washing said affinity chromatography resin with a buffer comprising 25 mM Tissue, 100 mM NaCl, pH 7.2, followed by a wash with a buffer comprising 20 mM sodium citrate / citric acid, 0.5 M NaCl, pH 6, and then with a buffer comprising 25 mM Tris, 100 mM NaCl, pH 7.2 , and collect an affinity chromatography sample; (c) subjecting said affinity chromatography sample to a reduction in pH thereby forming a sample of reduced pH, wherein said reduction in pH is for a pH of about 3 to about 4; (d) adjust said reduced pH sample to a pH of about 4.5 to about 8.5 and put said adjusted pH sample in contact with an anion exchange material and collect an anion exchange sample ; (e) contacting said anion exchange sample with a hydrophobic interactive chromatography (HIC) material and collecting a HIC sample, in which said anti-IL-13 antibody comprises six complementarity determining regions (CRDs): CDR -H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, where: CDR-H1 comprises amino acids 33 to 38 of SEQ ID NO: 1, CDR-H2 comprises amino acids 52 to 67 of SEQ ID NO: 1, CDR-H3 comprises amino acids 100 to 112 of SEQ ID NO: 1, CDR-L1 comprises amino acids 24 to 34 of SEQ ID NO: 2, CDR-L2 comprises amino acids 50 to 55 of SEQ ID NO: 2, and CDR-L3 comprises amino acids 89 to 97 of SEQ ID NO: 2.
[0002]
2. Method, according to claim 1, CHARACTERIZED by the fact that said reduction in pH is accomplished by mixing a suitable acid with said sample mixture, and in which said suitable acid is selected from the group that consists of citric acid, acetic acid and caprylic acid.
[0003]
3. Method, according to claim 1, CHARACTERIZED by the fact that said Protein A resin is MabSelect ™ resin.
[0004]
4. Method according to any one of claims 1 to 3, CHARACTERIZED by the fact that said anion exchange material is: (i) an anion exchange resin; or (ii) an anion exchange material which is an anion exchange resin comprising an anionic substituent; or (iii) an anion exchange material which is an anion exchange resin comprising a diethylaminoethyl (DEAE) anionic substituent; or (iv) an anion exchange material which is an anion exchange resin comprising an anionic quaternary aminoethyl (QAE) substituent; or (v) an anion exchange material which is an anion exchange resin comprising an anionic quaternary amine substituent (Q); or (vi) an anion exchange material that is a Q-sepharose anion exchange resin; or (vii) an anion exchange material that is a Mustang® Q membrane.
[0005]
5. Method, according to claim 1, CHARACTERIZED by the fact that said anion exchange step comprises a first anion exchange step and a second anion exchange step.
[0006]
6. Method, according to claim 1, CHARACTERIZED by the fact that said HIC material is: (i) a HIC column whose stationary phase comprises hydrophobic groups, or (ii) a HIC column whose stationary phase it comprises hydrophobic groups selected from the group consisting of alkyl groups, aryl groups and a combination thereof; or (iii) a HIC column selected from the group consisting of phenyl sepharose (such as Fenyl Sepharose ™ 6 Fast Flow column, Fenyl Sepharose ™ High Performance column), Octil Sepharose ™ High Performance column , Fractogel ™ EMD Propyl, Fractogel ™ EMD Phenyl columns, Macro-Prep ™ Methyl, Macro-Prep ™ t-Butyl supports, WP HI-Propyl (C3) ™ column and Toyopearl ™ ether, phenyl or butyl columns ; or (iv) an agarose resin substituted with phenyl groups.
[0007]
7. Method, according to claim 1, CHARACTERIZED by the fact that it also comprises a filtration step, in which said HIC sample is subjected to filtration to remove viral particles and to facilitate the change of buffer.
[0008]
8. Method according to claim 1, CHARACTERIZED by the fact that said anti-IL-13 antibody or antigen binding portion thereof is a humanized antibody.

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法律状态:
2017-12-05| B25A| Requested transfer of rights approved|Owner name: ABBVIE INC. (US) |

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2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-06-02| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-10-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-05| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 05/01/2021, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/10/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US25341109P| true| 2009-10-20|2009-10-20|

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US61/253.411|2009-10-20|

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PCT/US2010/053388|WO2011050071A2|2009-10-20|2010-10-20|Isolation and purification of anti-il-13 antibodies using protein a affinity chromatography|

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