Improved methods for virus removal in protein purification

Abstract

This invention provides a method for improving the filtration capacity of a virus filter used in protein purification. [Solution] A method is provided for increasing the filtration capacity of a parvovirus filter during protein purification, comprising passing a composition containing the protein to be purified through a cation exchange step and an endotoxin removal step in any order, wherein the composition containing the protein to be purified is a cation exchange chromatography pool, the protein is a monoclonal antibody or a fragment thereof, and in the endotoxin removal step, the composition containing the protein to be purified is processed on a positively charged membrane surface.

Description

[Technical Field]

[0001] This invention originates from the field of protein purification. In particular, the invention relates to a method for increasing the filtration capacity of a virus filter by combining endotoxin removal in a prefiltration step with the use of a cation exchange medium. [Background technology]

[0002] Mammalian cell lines have become the first choice for the production of recombinant protein therapeutics due to their ability to properly fold proteins and perform post-translational modifications, such as glycosylation (Chu and Robinson Current Opinion in Biotechnology 12:180-187, 2001). However, these cell lines are also known to contain retrovirus-like particles (Lieber et al. Science 182:56-59, 1973; Lubiniecki et al. Dev Biol Stand 70:187-191, 1989) and carry the risk of potential adenovirus contamination (Garnick, Dev Biol Stand. Basel: Karger 93:21-29, 1998). While the biopharmaceutical industry, which manufactures recombinant protein drugs, has a good safety record, viral infections from blood and plasma-derived blood products have occurred in the past (Brown, Dev. Biol. Stand. 81, 1993; Thomas, Lancet 343:1583-1584, 1994). To mitigate the risk of viral contamination during recombinant protein production, downstream purification processes are designed to include stepping stones for the removal of endogenous and exogenous viruses. Adequate viral elimination can be achieved through a combination of several stepping stones that provide either viral inactivation or viral removal from the process feedstream. Viral inactivation is achieved using techniques such as low-pH incubation, heat treatment, and detergents, while viral removal is typically carried out using chromatography and filtration (Curtis et al., Biotechnology and Bioengineering 84(2):179-186, 2003).

[0003] Unlike chromatographic media that remove viruses based on physicochemical properties such as effective charge, viral filtration removes viruses by size exclusion and is therefore considered a more powerful technique. To date, the use of viral filtration in the downstream purification of mammalian cell culture-derived biopharmaceuticals has been limited to the removal of retroviruses (diameter 80-100 nm) due to the lack of high-capacity membranes with nominal pore sizes of less than 60 nm.

[0004] Recent advances in membrane technology have enabled the production of high-capacity membranes with a nominal pore size of 20 nm. These viral filters are resistant to parvovirus (18–26 nm in diameter) and allow the passage of proteins as small as 160 kD (approximately 8 nm), such as monoclonal antibodies (mAbs).

[0005] The high selectivity and high throughput associated with parvovirus filters are achieved by forming a thin retaining film on a microporous substrate. While the thin retaining layer allows for very precise separation of proteins and viruses, it is also susceptible to adhesion from impurities in the process feedstream, reducing filter capacity and flow rate. Contamination of virus filters is due to contaminants such as protein aggregates and denatured proteins. Bohonak and Zydney (Journal of Membrane Science 254(1-2):71-79, 2005) showed that the loss of filter capacity can be due to cake formation or pore blockage. Other recent reports (Bolton et al., Biotechnol. Appl. Biochem. 43:55-63, 2006; Levy et al., Filtration in the Biopharamaceutical Industry. (Meltzer, TH and Jornitz, MW, eds.) pp. 619-646, Marcel Dekker, New York, 1998) attributed filter fouling to the adsorption of impurities onto the pore walls. Several publications (Bolton et al., Biotechnology and Applied Biochemistry 42:133-142, 2005; Hirasaki et al., Polymer Journal 26(11):1244-1256, 1994; Omar and Kempf, Transfusion 42(8):1005-1010, 2002) have also demonstrated that reduced filter capacity or pore blockage can decrease virus retention by several orders of magnitude, affecting the robustness of unit operation.

[0006] Therefore, numerous recent studies have focused on identifying pre-filters to remove contaminants from the process feedflow, minimize viral filter contamination, and ensure high capacity, high yield, and robust virus retention. Bolton et al. (2006) conducted thorough research, including testing several membranes as pre-filters, demonstrating that using Viresolve® deep filters as pre-filters can increase the capacity of normal flowing parvovirus (NFP) membranes by almost an order of magnitude. Brown et al. (2008, Use of Charged Membranes to Identify Soluble Protein Foulants in order to Facilitate Parvovirus Filtration. IBC's 20 th Antibody Development and Production (San Diego, CA) evaluated a powerful cation exchange membrane adsorption device as a pre-filter for parvovirus-holding filters and showed that the virus filter's capacity can be increased several-fold for 11 different mAb flows. The authors hypothesized that the cation exchange membrane adsorption device removes large molecular weight (approximately 600-1500 kD) protein aggregates from the feed stream by competitive adsorption, preventing clogging of the virus filter. U.S. Patent No. 7,118,675 (Siwak et al.) describes a process using a charge-correcting membrane to remove protein aggregates from a protein solution to prevent contamination of a virus filter. [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention is at least in part based on experimental findings that contamination of parvovirus filters may be due to impurities other than those described in the literature, and that a more comprehensive prefiltration solution is needed to improve the virus filtration capacity. Therefore, this invention provides a novel prefiltration solution that performs significantly better than the best prefiltration approach described in the literature (cation exchange membrane adsorption device).

[0008] In one embodiment, the present invention relates to a method for improving the filtration capacity of a virus filter during protein purification, comprising passing a composition containing the protein to be purified through a cation exchange step and an endotoxin removal step, in any order, through the virus filter.

[0009] In one embodiment, the pore size of the virus filter is approximately 15 to 100 nm in diameter.

[0010] In another embodiment, the pore size of the virus filter is approximately 15 to 30 nm in diameter.

[0011] In yet another embodiment, the pore size of the virus filter is approximately 20 nm.

[0012] In a further embodiment, the virus to be removed is a parvovirus.

[0013] In further embodiments, the diameter of the parvovirus is approximately 18 to 26 nm.

[0014] In a different embodiment, the protein is an antibody or antibody fragment, for example, an antibody or fragment produced by recombinant DNA technology.

[0015] In another embodiment, the antibody is a therapeutic antibody.

[0016] In yet another embodiment, recombinant antibodies or antibody fragments are produced in mammalian host cells, such as Chinese hamster ovary (CHO) cells.

[0017] In a further embodiment, the composition containing the protein to be purified is first subjected to a cation exchange step and then, after being subjected to an endotoxin removal step, is virus filtered.

[0018] In a further embodiment, the composition containing the protein to be purified is first subjected to an endotoxin removal step and then, after being subjected to a cation exchange step, is virus filtered.

[0019] In another embodiment, the composition containing the protein to be purified is virus filtered after being simultaneously subjected to a cation exchange step and an endotoxin removal step by holding two media together in a single module.

[0020] In yet another embodiment, virus filtration is performed immediately after the endotoxin removal step.

[0021] In a further embodiment, virus filtration is performed immediately after the cation exchange step.

[0022] In a different embodiment, virus filtration is performed at a pH of about 4 to about 10.

[0023] In another embodiment, the protein concentration in the composition to be purified is about 1 to 40 g / L.

[0024] In yet another embodiment, the antibody to be purified is against one or more antigens selected from the group consisting of HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, VCAM, IL-17A and / or F, IgE, DR5, CD40, Apo2L / TRAIL, EGFL7, NRP1, mitogen-activated protein kinase (MAPK), and factor D.

[0025] In further embodiments, the antibodies include anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras tumor protein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD10 antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies, anti-CD19 antibodies, and anti-CD2 antibodies. The antibody is selected from the group consisting of 3 antibodies, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate-specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody. [Brief explanation of the drawing]

[0026] [Figure 1] This is a schematic diagram of the experimental preparation used for a virus filtration test. [Figure 2] This figure shows the effects of sterilization and deep filtration on the capacity of the Viresolve Pro parvovirus retention filter. Experiments were conducted at pH 5.5 and conductivity 8.5 mS / cm. The mAb concentration was approximately 13 g / L. [Figure 3a] This figure shows the effect of a cation exchange and endotoxin removal membrane adsorption device as a pre-filter on the performance of the Viresolve Pro parvovirus filter. Data 3(a) and 3(b) were obtained for MAb1 at pH 5.0 and 6.5, respectively. [Figure 3b]This figure shows the effect of a cation exchange and endotoxin removal membrane adsorption device as a pre-filter on the performance of the Viresolve Pro parvovirus filter. Data 3(a) and 3(b) were obtained for MAb1 at pH 5.0 and 6.5, respectively. [Figure 4a] This figure shows the effect of a novel pre-filtration train containing both cation exchange and endotoxin removal membrane adsorption devices on the ability of the Viresolve Pro parvovirus retention filter for MAb1. Data 4(a) and 4(b) were obtained at pH 5.0 and 6.5, respectively. [Figure 4b] This figure shows the effect of a novel pre-filtration train containing both cation exchange and endotoxin removal membrane adsorption devices on the ability of the Viresolve Pro parvovirus retention filter for MAb1. Data 4(a) and 4(b) were obtained at pH 5.0 and 6.5, respectively. [Figure 5] This figure shows the effect of a novel pre-filtration train containing both cation exchange and endotoxin removal membrane adsorption devices on the parvovirus retention capacity of MAb2, compared to a cation exchange pre-filtration medium. [Modes for carrying out the invention]

[0027] I. Definition A "protein" is defined as a sequence of amino acids whose chain length is sufficient to create a high level of tertiary and / or quaternary structure. Therefore, proteins are distinguished from "peptides," which are also amino acid-based molecules but do not possess such structures. Typically, proteins used herein have a molecular weight of at least about 15–20 kD, preferably at least about 20 kD.

[0028] Examples of proteins included in the definition herein include mammalian proteins, e.g., CD4, integrins and their subunits, e.g., beta-7; growth hormones, e.g., human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; α-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicular-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, e.g., factor VIIIC, factor IX, tissue factor and von Willebrand factor; anticoagulation factors, e.g., protein C; atrial natriuretic factor; pulmonary surfactants; plasminogen activators, e.g., urokinase or tissue-type plasminogen activators (t-PA, e.g., Activase®, TNKase®, Letebase®); bombazine; thrombin; tumor necrosis factor α and β; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted (regulated upon activation, expressed and secreted by normal T cells); human macrophage inflammatory protein (MIP-1-α); serum albumin, e.g., human serum albumin; Müller inhibitor; mouse gonadotropin-related peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); IgE, receptor for hormones or growth factors; integrins; protein A or D; rheumatoid factor; neurotrophic factors, e.g., bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor, e.g., NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor, e.g., aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF), e.g., TGF-α and TGF-β, e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor I and II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor-binding protein; other CD proteins, e.g., CD3, CD8, CD19 and CD20;Examples include erythropoietin (EPO); thrombopoietin (TPO); bone-inducing factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, e.g., interferon α, β, and γ; colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutases; T-cell receptors; surface membrane proteins; degeneration factors (DAFs); viral antigens, e.g., parts of the AIDS envelope; transport proteins; homing receptors; adlessins; regulatory proteins; integrins, e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; tumor-associated antigens, e.g., HER1 (EGFR), HER2, HER3, or HER4 receptors; Apo2L / TRAIL; and fragments of any of the above polypeptides; as well as immunoadhesins and antibodies that bind to any of the above proteins; and biologically active fragments or variants of any of the above proteins.

[0029] Specifically, the definition of "protein" as used herein includes therapeutic antibodies and immunoadhesins, such as antibodies against one or more of the following antigens, but is not limited to: HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4, VCAM, IL-17A and / or F, IgE, DR5, CD40, Apo2L / TRAIL, EGFL7, NRP1, mitosis-activated protein kinase (MAPK), and factor D, as well as fragments thereof.

[0030] Other antibodies include, but are not limited to, the following: anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras tumor protein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD10 antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti- CD15 antibody, anti-CD19 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate-specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody.

[0031] "Isolated" proteins, such as antibodies, are identified, isolated, and / or recovered from components of their natural environment. Contaminating components of the natural environment are substances that would interfere with the diagnostic or therapeutic use of proteins such as antibodies, and examples include enzymes, hormones, and other protein-like or non-protein-like solutes. In preferred embodiments, proteins, such as antibodies, are purified homogeneously by SDS-PAGE under reducing or non-reducing conditions, (1) to a value greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) using Coomassie blue, preferably silver staining.

[0032] The protein is preferably essentially pure and preferably essentially homogeneous (i.e., free from impurities). "Essentially pure" protein means a composition containing at least about 90% by weight, preferably at least about 95% by weight, of protein based on the total weight of the composition.

[0033] "Essentially homogeneous" protein means a composition that contains at least about 99% by weight of protein, based on the total weight of the composition.

[0034] The term "antibody" is used in its broadest sense and specifically includes monoclonal antibodies (e.g., full-length antibodies with an immunoglobulin Fc region), antibody compositions with multiepitope specificity, bispecific antibodies, diabodies and single-chain molecules, and antibody fragments (e.g., Fab, F(ab')2, and Fv).

[0035] A basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units accompanied by an additional polypeptide called a J chain, containing 10 antigen-binding sites, while IgA antibodies consist of 2 to 5 basic four-chain units, which can polymerize and combine with J chains to form multivalent aggregates. In the case of IgG, a four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Both H and L chains also have intrachain disulfide crosslinks at regular intervals. Each H chain has a variable domain (V) at its N-terminus. H ) and then in each of the α and γ chains there are three constant domains (C H ) and in the μ and ε isotypes, there are four C H It has a domain. Each L chain has a variable domain (V) at its N-terminus. L ) followed by a constant domain at the other end. V L is V Haligned together, and C L is aligned with the first constant domain of the heavy chain (C H 1). Certain amino acid residues are thought to form interfaces between the light and heavy chain variable domains. V H and V L pairings come together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0036] L chains from any vertebrate species can be assigned to one of two clearly different types called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to different classes or isotypes by the amino acid sequence of their heavy chain constant domains (CH). There are five classes of immunoglobulins having heavy chains called α, δ, ε, γ, and μ, namely IgA, IgD, IgE, IgG, and IgM. The γ and μ classes are further divided into subclasses based on relatively small differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0037] The term "variable" refers to the fact that a segment of the variable domain differs extensively in its sequence among antibodies. The V domain mediates antigen binding and limits the specificity of a particular antibody with respect to its specific antigen. However, variability is not uniformly distributed throughout the entire span of the variable domain. Instead, the V region consists of relatively invariant stretches called framework regions (FRs) of about 15-30 amino acid residues, separated by shorter regions called "hypervariable regions," each about 9-12 amino acid residues long, sometimes called "complementarity-determining regions" (CDRs), which have very high variability. The variable domains of the natural heavy and light chains each contain four FRs (mostly in a β-sheet configuration and linked by three hypervariable regions), which link the β-sheet structure and, in some cases, form loops that constitute part of it. The hypervariable regions within each chain are closely connected and held together by FRs, and together with hypervariable regions from other chains, they contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domain does not directly participate in antibody-antigen binding, but exhibits various effector functions, such as involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).

[0038] The term “hypervariable region” (also known as “complementarity-determining region” or CDR), as used herein, refers to the amino acid residues of an antibody located within the V-region domain of an immunoglobulin (usually three or four short regions with very high sequence variability) that form the antigen-binding site and are the primary determinants of antigen specificity. There are at least two methods for identifying CDR residues: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, MS 1991); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al., J. Mol. Biol.). 196 : 901-917 (1987). However, unless two residue identification techniques limit the overlapping regions but not identical regions, they can be combined to identify hybrid CDRs.

[0039] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for any spontaneous mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically contain multiple different antibodies directed to multiple different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and is not intended to require antibody production by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention are those described in Kohler et al., Nature, 256They can be produced by the hybridoma method first described in 495 (1975), or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" are described, for example, by Clackson et al., Nature, 352 :624-628 (1991) and Marks et al., J. Mol. Biol., 222 It can also be isolated from phage antibody libraries using the technique described in :581-597 (1991).

[0040] The monoclonal antibodies described herein specifically refer to "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass, as well as (insofar as they exhibit the desired biological activity) fragments of such antibodies (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984).

[0041] "Undamaged" antibodies have an antigen-binding site, as well as CL and at least the long-chain domain C. H 1. C H 2 and C H It includes 3.

[0042] An "antibody fragment" is a portion of an intact antibody, preferably containing the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; and linear antibodies (U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng.). 8(10) Examples include single-chain antibody molecules and multispecific antibodies formed from antibody fragments (see 1057-1062

[1995] ).

[0043] Papain digestion of the antibody produced two identical antigen-binding fragments called "Fab" fragments, as well as a residual "Fc" fragment (this name reflects its ability to easily crystallize). The Fab fragment contains a variable region domain (V) of the H chain. H ) and the first constant domain of one heavy chain (C H It consists of a complete L chain with 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-bonded Fab fragments with different antigen-binding activities and can still crosslink to the antigen. The Fab' fragment contains one or more cysteine ​​from the antibody hinge region. H It differs from the Fab fragment by having 2-3 additional residues at the carboxyl terminus of one domain. Fab'-SH is the herein designation for Fab' fragments in which one or more cysteine ​​residues in the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair with Fab' fragments that have a hinged cysteine ​​between them. Other chemical couplings of the antibody fragments are also known.

[0044] The Fc fragment contains the carboxyl-terminal portions of both H chains held together by a disulfide. The effector function of an antibody is determined by the sequence within the Fc region, which is also recognized by an Fc receptor (FcR) found on certain types of cells.

[0045] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a rigid, non-covalently associated dimer of one heavy chain and one light chain variable region domain. The folding of these two domains gives rise to six hypervariable loops (three from the H and L chains, respectively) that contribute amino acid residues for antigen binding and confer antigen specificity to the antibody. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to antigens, albeit with lower affinity than the complete binding site.

[0046] A "single-stranded Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further contains V H and V L The sFv contains polypeptide linkers between its domains, which allow it to form a desirable structure for antigen binding. For further information on sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0047] The term "diabody" refers to the fact that interchain pairing of the V domain is achieved rather than intrachain pairing, thereby producing a bivalent fragment, i.e., a fragment having two antigen-binding sites. H and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) that have short linkers (approximately 5-10 residues) between domains. A bispecific diabody is a V of two antibodies. H and V L Diabodies are heterodimers of two "cross-linked" sFv fragments located on polypeptide chains with different domains. Diabodies are, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 This is described in more detail in 6444-6448 (1993).

[0048] An antibody that "binds" to the molecular target or antigen is one that can bind to the antigen with sufficient affinity so that the antibody is useful in targeting cells that express the antigen.

[0049] An antibody that "specifically binds to" or "is specific to" a particular polypeptide or epitope on a particular polypeptide is one that binds to the particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In such embodiments, the degree of antibody binding to these other polypeptides or polypeptide epitopes is less than 10% compared to binding to the target polypeptide or epitope, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0050] Humanized non-human (e.g., mouse) antibodies are mostly chimeric immunoglobulins, immunoglobulin chains, or fragments of human sequences (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) that contain minimal sequences derived from non-human immunoglobulins. For the most part, the humanized antibody is a human immunoglobulin (recipient antibody), in which case residues from the recipient's hypervariable region (including the CDR) are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, or rabbit, with the desired specificity, affinity, and capability. In some cases, Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibody" may also include residues not found in either the recipient or donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also includes at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin. For further details, see Jones et al., Nature. 321 :522-525 (1986); Reichmann et al., Nature, 332 :323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2 See 593-596 (1992).

[0051] Antibody "effector function" refers to the biological activity that can be attributed to the antibody's Fc region (native sequence Fc region or amino acid sequence variant Fc region), and varies with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0052] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) causes these cytotoxic effector cells to specifically bind to antigen-carrying target cells, which are then killed by cytotoxins. Antibodies "arm" the cytotoxic cells and are necessary for the killing of target cells by this mechanism. NK cells, the main cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression in hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol. 9 This is summarized in Table 3 on page 464 of 457-92 (1991). To assess the ADCC activity of the molecule, an in vitro ACDD test, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, can be performed. Useful effector cells for such tests include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule can be assessed in vivo, for example, in Clynes et al., PNAS USA. 95 It can be assessed using an animal model such as the one disclosed in :652-656 (1998).

[0053] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. The preferred FcR is the naturally occurring human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and encompass the FcγRI, FcγRII, and FcγRIII subclasses, e.g., allele variant receptors, and alternatively, spliced ​​forms of these receptors. The FcγRII receptor encompasses FcγRIIA ("activating receptor") and FcγRIIB ("suppressive receptor"), which have similar amino acid sequences differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The suppressive receptor FcγRIIB contains an immunoreceptor tyrosine-based suppressive motif (ITIM) in its cytoplasmic domain (M. Daeron, Annu. Rev. Immunol. 15 See 203-234 (1997). FcR is Ravetch and Kinet, Annu. Rev. Immunol. 9 : 457-92 (1991);Capel et al., Immunomethods 4 : 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126 This is discussed in 330-41 (1995). Other FcRs, such as those to be identified in the future, are included in the term "FcR" as used herein. This term also includes FcRn, a neonatal receptor involved in the transport of maternal IgG to the fetus (Guyer et al., J. Immunol.). 117 : 587 (1976) and Kim et al., J. Immunol. 24 : 249 (1994).

[0054] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, but PBMCs and MNK cells are preferred. Effector cells can be isolated from native sources, such as blood.

[0055] Complement-dependent cytotoxicity, or CDC, refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind to their cognitive antigens. For assessing complement activation, see, for example, Gazzano-Santoro et al., J. Immunol. Methods. 202 A CDC test, such as the one described in 163 (1996), may be performed.

[0056] The terms “conjugate,” “conjugated,” and “conjugated” refer to any and all types of covalent or non-covalent bonding, including, but not limited to, direct genetic or chemical fusion, coupling via linkers or crosslinkers, and non-covalent associations, such as those using leucine zippers. An antibody conjugate has another entity bound to an antibody or antibody fragment, such as a cytotoxic compound, drug, composition, compound, radioactive element, or detectable label.

[0057] "Treatment" refers to both therapeutic treatment and preventive or deterrent measures. Those requiring treatment include those who already have a disability, as well as those for whom disability should be prevented.

[0058] For the purposes of this treatment, “mammal” refers to any animal classified as a mammal, such as humans, non-human higher primates, livestock and farm animals, as well as zoo, sport, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, mice, cattle, etc. Preferably, the mammal is human.

[0059] A “disorder” is any condition that would benefit from treatment using proteins. Examples include chronic and acute disorders or diseases, such as pathological conditions that make mammals susceptible to the disorder.

[0060] A "therapeutic effective dose" is the minimum concentration required to produce a measurable improvement or prevention of a particular disorder. While the therapeutic effective doses of known proteins are well known in the art, the effective doses of the following proteins to be discovered in the future can be determined by standard techniques that are well within the scope of the skills of those skilled in the art, e.g., ordinary physicians.

[0061] II. Method of carrying out the present invention A. Protein preparation According to the present invention, proteins are produced by recombinant DNA technology, that is, by culturing cells that have been transformed or transfected with a vector containing nucleic acids encoding proteins, as is well known in the art.

[0062] The preparation of proteins by recombinant means can be achieved by transfecting or transforming suitable host cells with expression or cloning vectors, inducing promoters, selecting transformants, or culturing them in modified conventional nutrient media where appropriate for amplifying genes encoding desired sequences. Culture conditions, such as medium, temperature, and pH, can be selected by those skilled in the art without excessive experimentation. Generally, principles, protocols, and techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, Ed. (IRL Press, 1991) and Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press. Transfection methods are known to those skilled in the art, and examples include CaPO4 and CaCl2 transfection, electroporation, and microinjection. Appropriate techniques are also described in Sambrook et al. (above). Additional transfection techniques are described in Shaw et al., Gene 23 : 315 (1983);WO 89 / 05859;Graham et al., Virology 52 This is described in : 456-457 (1978) and U.S. Patent No. 4,399,216.

[0063] Nucleic acids encoding a desired protein can be inserted into a vector that can be replicated for cloning or expression. Suitable vectors are publicly available and can take the form of plasmids, cosmids, viral particles, or phages. Suitable nucleic acid sequences can be inserted into vectors by various methods. Generally, DNA is inserted into a suitable restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, signal sequences, origins of replication, one or more marker genes, and one or more enhancer elements, promoters, and termination sequences. Construction of a suitable vector containing one or more of these components is done using standard ligation techniques known to those skilled in the art.

[0064] The protein morphology can be recovered from the culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable surfactant or by enzymatic cleavage. Cells used for expression can also be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonic treatment, mechanical disruption, or cell lysants.

[0065] Protein purification can be achieved by any suitable technique known in the art, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation exchange resin (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using a Protein A Sepharose column (e.g., Sephadex® G-75) to remove impurities such as IgG, and a metal chelate column to bind epitope-tagged forms.

[0066] B. Antibody preparation In one embodiment of the present invention, the preferred protein is an antibody. Techniques for producing antibodies, such as polyclonal, monoclonal, humanized, bispecific, and heterozygous antibodies, are described below.

[0067] (i) Polyclonal antibodies Polyclonal antibodies are generally produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen with an immunogenic protein in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvants (monophosphoryl lipid A, synthetic trehalose dicolinomicolate). Those skilled in the art can select an immunization protocol without excessive experimentation.

[0068] One month later, animals are boosted by subcutaneous injection at multiple sites with peptides or conjugates in Freund's complete adjuvant at 1 / 5 to 1 / 10 of the original amount. After 7 to 14 days, animals are blood drawn and serum is tested for antibody titer. Animals are boosted until titer plateaus. Preferably, animals are boosted with conjugates of the same antigen but coupled to different proteins and / or via different crosslinking reagents. Conjugates can be prepared in recombinant cell culture as protein fusions. Furthermore, agglutinants, such as alum, are appropriately used to enhance the immune response.

[0069] (ii) Monoclonal antibody Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical, except for any naturally occurring mutations that may exist in small amounts. Therefore, the modifier "monoclonal" indicates a characteristic of the antibody that it is not a mixture of distinct antibodies.

[0070] For example, monoclonal antibodies are described in Kohler et al., Nature. 256 They can be produced using the hybridoma method first described in 495 (1975), or by the recombinant DNA method (U.S. Patent No. 4,816,567).

[0071] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0072] The immunizing agent typically contains the protein to be prescribed. Generally, peripheral blood lymphocytes ("PBLs") are used when human-derived cells are desired, or spleen cells or lymph node cells are used when non-human mammalian-derived cells are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). The immortalized cell line is usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are preferably seeded and grown in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fused parent myeloma cells. For example, if parent myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the proliferation of HGPRT-deficient cells.

[0073] Preferred myeloma cells are those that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, preferred myeloma cell lines include mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, California, USA), and those derived from SP-2 cells available from the American Type Culture Collection (Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also been described for human monoclonal antibody production (Kozbor, J. Immunol., 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0074] The culture medium in which hybridoma cells are proliferating is tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0075] The binding affinity of monoclonal antibodies is, for example, as described in Munson et al., Anal. Biochem. 107 This can be confirmed by the Skatchard analysis in :220 (1980).

[0076] After hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones can be subcloned using a limited dilution procedure and proliferated by standard methods (Goding, above). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be proliferated in vivo as ascites tumors in animals.

[0077] Immunotherapeutic agents typically contain epitope proteins to which antibodies bind. Generally, when human-derived cells are desired, peripheral blood lymphocytes ("PBLs") are used, or when non-human mammalian-derived cells are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0078] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), where these substances prevent the proliferation of HGPRT-deficient cells.

[0079] Preferred immortalized cell lines are those that efficiently fuse, support stable high-level antibody expression by selected antibody-producing cells, and are sensitive to media such as HAT medium. Even more preferred are mouse myeloma cell lines, which are available, for example, from the Salk Institute Cell Distribution Center (San Diego, California, USA) and the American Type Culture Collection (Rockville, Maryland). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0080] The culture medium in which hybridoma cells are cultured can then be tested for the presence of monoclonal antibodies directed against the protein to be formulated. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be found, for example, in Munson et al., Anal. Biochem. 107 This can be confirmed by the Skatchard analysis in :220 (1980).

[0081] After the desired hybridoma cells are identified, the clones can be subcloned using a limited dilution procedure and proliferated by standard methods (Goding, above). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be proliferated in vivo as ascites tumors in animals.

[0082] Monoclonal antibodies secreted by subclones can be appropriately separated from culture media, ascites fluid, or serum using conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0083] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector, which is then transfected into host cells that would otherwise not produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize monoclonal antibodies in recombinant host cells. For a discussion of recombinant expression of antibody-encoding DNA in bacteria, see Skerra et al., Curr. Opinion in Immunol., 5 :256-262 (1993) and Pluckthun, Immunol. Revs. 130 :151-188 (1992) is one example.

[0084] In further embodiments, the antibody is described in McCafferty et al., Nature, 348 It can be isolated from an antibody phage library generated using the technique described in :552-554 (1990). Clackson et al., Nature,352 :624-628 (1991) and Marks et al., J. Mol. Biol., 222 581-597 (1991) describes the isolation of mouse and human antibodies, respectively, using a phage library. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10 :779-783 (1992)), as well as combined infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21 :2265-2266 (1993)) is described. Therefore, these techniques are a viable alternative to the traditional monoclonal antibody hybridoma technique for the isolation of monoclonal antibodies.

[0085] The DNA can also be further modified by substituting the coding sequence in the constant domains of the human heavy and light chains, for example, instead of the homologous mouse sequence (U.S. Patent No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81 Modification can be achieved by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence, as described in :6851 (1984).

[0086] Typically, such non-immunoglobulin polypeptides are substituted in place of the constant domain of an antibody, or they are substituted in place of the variable domain of one antigen-binding site of the antibody, to create a chimeric bivalent antibody containing one binding site specific to a different antigen and another antigen-binding site specific to a different antigen.

[0087] Chimeric or hybrid antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including crosslinking agents. For example, immunotoxins can be constructed by disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimidates.

[0088] (iii) Humanization and human antibodies The antibodies used in this formulation method may further include humanized or human antibodies. Humanized non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of the antibody), which contain the minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies), in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, Fv framework residues of human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of a human immunoglobulin consensus sequence. Humanized antibodies also optimally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin (Jones et al., Nature, 321 :522-525 (1986); Reichmann et al., Nature, 332 :323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0089] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often called "implant" residues, and are typically obtained from an "implant" variable domain. Humanization is essentially described in Winter and co-workers, Jones et al., Nature. 321 :522-525 (1986);Riechmann et al., Nature 332 :323-327 (1988);Verhoeyen et al., Science 239 This can be carried out according to the method of :1534-1536 (1988), or by substituting rodent CDRs or CDR sequences with corresponding sequences in a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially fewer than intact human variable domains are substituted with corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies in which several CDR residues and possibly several FR residues are substituted with residues from similar sites in rodent antibodies.

[0090] The selection of human variable domains to be used in the production of humanized antibodies is crucial for reducing antigenicity, both for light and heavy chains. According to the so-called "best fit" method, the variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence most closely resembling the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol.). 151 :2296 (1993);Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a specific framework derived from the consensus sequences of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89 :4285 (1992);Presta et al., J. Immnol., 151 :2623 (1993)).

[0091] Furthermore, it is important that antibodies are humanized while retaining high affinity for antigens and other favorable biological properties. To achieve this goal, according to preferred methods, humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. Scrutiny of these representations allows for the analysis of possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and transfer sequences so that desired antibody properties, such as increased affinity for the target antigen(s), are achieved. Generally, CDR residues are involved in directly and almost substantially influencing antigen binding.

[0092] Alternatively, it is possible here to create transgenic animals (e.g., mice) that can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production during immunization. For example, the antibody heavy chain linkage region (J) in chimeric and germline mutant mice. HIt has been described that isozygous deletion of the gene results in complete suppression of endogenous antibody production. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice induces the production of human antibodies upon antigen administration. For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90 :2551 (1993);Jakobovits et al., Nature, 362 :255-258 (1993);Bruggermann et al., Year in Immuno., 7 See 33 (1993). Human antibodies can also be obtained from phage-represented libraries (Hoogenboom et al., J. Mol. Biol., 227 :381 (1991);Marks et al., J. Mol. Biol., 222 :581-597 (1991).

[0093] Human antibodies can also be produced using various techniques known in the art, such as phage-represented libraries (Hoogenboom and Winter, J. Mol. Biol.). 227 : 381 (1991);Marks et al., J. Mol. Biol. 222 : 581 (1991)). Techniques by Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1): 86-95 (1991). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice, in which case the endogenous immunoglobulin genes are partially or completely inactivated. Upon antigen administration, human antibody production is observed, which is very similar in all respects to that observed in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is documented, for example, in U.S. Patents No. 5,545,807; No. 5,545,806, No. 5,569,825, No. 5,625,126, No. 5,633,425, and No. 5,661,016, as well as in the following scientific publication: Marks et al., Bio / Technology 10 : 779-783 (1992);Lonberg et al., Nature 368 : 856-859 (1994);Morrison, Nature 368 : 812-13 (1994);Fishwild et al., Nature Biotechnology 14 : 845-51 (1996);Neuberger, Nature Biotechnology 14 : 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13 This is described in 65-93 (1995).

[0094] (iv) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT) The antibodies of the present invention can also be used in ADEPT by conjugating them with a prodrug activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81 / 01145) into an active anticancer drug (see, for example, WO88 / 07378 and U.S. Patent No. 4,975,278).

[0095] Enzymatic components of immune complexes useful for ADEPT include any enzyme capable of acting on the prodrug in a manner that converts it to its more active cytotoxic form.

[0096] Enzymes useful in the method of the present invention include glycosidase, glucose oxidase, human lysozyme, human glucuronidase, alkaline phosphatase (useful for converting phosphate-containing prodrugs into free drugs); aryl sulfatase (useful for converting sulfate-containing prodrugs into free drugs); cytosine deaminase (useful for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil); proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidase (e.g., carboxypeptidase G2 and carboxypeptidase A); and cathepsins (e.g., cathepsin B and L) (peptide-containing). Examples of enzymes useful for converting prodrugs into free agents include, but are not limited to, D-alanyl carboxypeptidase (useful for converting prodrugs containing D-amino acid substituents); carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase (useful for converting glycosylated prodrugs into free agents); β-lactamase (useful for converting drugs derivatized with β-lactams into free agents); and penicillin amidases, such as penicillin V amidase or penicillin G amidase (useful for converting drugs derivatized with their amine nitrogen using phenoxyacetyl or phenylacetyl groups, respectively, into free agents). Alternatively, antibodies having enzymatic activity known in the art, also known as "abzymes," can be used to convert the prodrugs of the present invention into free active agents (e.g., Massey, Nature). 328 See 457-458 (1987). Antibody-abzyme conjugates may be prepared as described herein for the delivery of abszymes to tumor cell populations.

[0097] The enzyme of this invention can be covalently bound to an anti-IL-17 or anti-LIF antibody by techniques well known in the art, such as the use of the heterobifunctional crosslinking agent described above. Alternatively, a fusion protein comprising at least the antigen-binding region of the antibody of the present invention, which is linked to at least the functionally active portion of the enzyme of the present invention, can be constructed using recombinant DNA techniques well known in the art (e.g., Neuberger et al., Nature). 312 (See 604-608 (1984)).

[0098] (iv) Bispecific and multispecific antibodies A bispecific antibody (BsAb) is an antibody that has binding specificity to at least two different epitopes. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0099] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the simultaneous expression of two immunoglobulin heavy-light chain pairs, in which case the two chains have different specificities (Millstein et al., Nature, 305 :537-539 (1983). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of 10 possible antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule (which is usually performed by affinity chromatography) is quite cumbersome, and the product yield is low. A similar method is described in WO 93 / 08829, as well as in Traunecker et al., EMBO J. 10 Disclosed in :3655-3659 (1991).

[0100] An antibody variable domain (antibody-antigen binding site) with desired binding specificity can be fused with an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain and includes at least a portion of the hinge, CH2, and CH3 regions. Preferably, it has a first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. The immunoglobulin heavy chain fusion, and optionally the DNA encoding the immunoglobulin light chain, are inserted into a separate expression vector and co-transfected into a suitable host organism. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology. 121 See 210 (1986).

[0101] According to a different approach, an antibody variable domain (antibody-antigen binding site) with desired binding specificity can be fused with an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain and includes at least a portion of the hinge, CH2, and CH3 regions. Preferably, it has a first heavy chain constant region (CH1) containing the site necessary for light chain binding, which is present in at least one of the fusions. The immunoglobulin heavy chain fusion, and optionally the DNA encoding the immunoglobulin light chain, are inserted into a separate expression vector and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative ratios of the three polypeptide fragments in embodiments where the unequal ratios of three polypeptide chains used in construction provide the optimal yield. However, if the expression of at least two polypeptide chains in equal ratios yields a high yield, or if the ratio is not particularly significant, it is possible to insert coding sequences for two or all three polypeptide chains into a single expression vector.

[0102] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface includes at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" having the same or similar size as the large side chain(s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers against other undesirable end products such as homodimers.

[0103] In a preferred embodiment of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having first-binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing second-binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from undesirable immunoglobulin chain combinations, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy method of separation. This approach is disclosed in WO94 / 04690 (published March 3, 1994). Further details on the generation of bispecific antibodies can be found, for example, in Suresh et al., Methods in Enzymology, 121 See 210 (1986).

[0104] Bispecific antibodies include crosslinked or "heterozygous" antibodies. For example, one antibody in a heterozygous antibody may be bound to avidin and the other to biotin. Such antibodies have been proposed, for example, to direct immune system cells to target undesirable cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO91 / 00360, WO92 / 200373). Heterozygous antibodies can be produced using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with numerous crosslinking techniques.

[0105] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For the production of bivalent antibody fragments that are not necessarily bispecific, the following techniques may also be used. For example, Fab' fragments recovered from E. coli can be chemically bound in vitro to form bivalent antibodies (Shalaby et al., J. Exp. Med., 175 See pages 217-225 (1992).

[0106] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical linking. (Brennan et al., Science) 229 : 81 (1985) describes a method in which intact antibodies are proteolytically cleaved to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-TNB derivative to form a bispecific antibody. The produced bispecific antibody can be used as an active agent for selective enzyme immobilization.

[0107] The Fab' fragment can be directly recovered from E. coli and chemically bound to form a bispecific antibody. (Shalaby et al., J. Exp. Med.) 175 217-225 (1992) describes the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was secreted separately from Escherichia coli and attached to a supported chemical bond in vitro to form a bispecific antibody. This bispecific antibody bound to cells overexpressing the ErbB2 receptor and normal human T cells, and was able to induce lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0108] Various techniques for directly producing and isolating bivalent antibody fragments from recombinant cell cultures are also described. For example, bivalent heterodimers have been produced using a leucine zipper (Kostelny et al., J. Immunol., 148 (5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' moieties of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, and then reoxidized to form an antibody heterodimer. Hollinger et al., Proc. Natl. Acad. Sci. USA, 90 The “diabody” technique described in 6444-6448 (1993) provides an alternative mechanism for producing bispecific / bivalent antibody fragments. The fragments are formed by linkers that are too short to allow pairing between two domains on the same chain, thereby separating the light chain variable domain (V L ) and the heavy chain variable domain (V H ) includes. Therefore, V of one fragment H and V L The domain is complementary to another fragment V L and V HIt is paired with a domain, thereby forming two antigen-binding sites. Another strategy for producing bispecific / bivalent antibody fragments using single-chain Fv(sFv) dimers has also been reported (Gruber et al., J. Immunol., 152 See :5368 (1994).

[0109] Antibodies with a valency greater than 2 are intended. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol.). 147 : 60 (1991).

[0110] Exemplary bispecific antibodies can bind to two different epitopes on a given molecule. Alternatively, to concentrate cellular defense mechanisms on cells expressing a specific protein, an anti-protein arm may be combined with an arm that binds to a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or to an Fc receptor for IgG (FcγR), such as FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing a specific protein. Such antibodies possess a protein-binding arm, as well as an arm that binds to a cytotoxic agent or radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another such bispecific antibody binds to the protein and also to tissue factor (TF).

[0111] (v) Heterozygous antibody Heterozygous conjugated antibodies are also within the scope of the present invention. Heterozygous conjugated antibodies consist of two covalently bound antibodies. Such antibodies have been proposed, for example, to direct immune system cells to target undesirable cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO91 / 00360, WO92 / 200373 and EP 03089). It is intended that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, such as methods involving crosslinking agents. For example, immunotoxins can be constructed by using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutylimide, as disclosed, for example, in U.S. Patent No. 4,676,980.

[0112] C. Purification of proteins, including antibodies When a target polypeptide is expressed in recombinant cells other than human-derived cells, the target polypeptide will not contain any human-derived proteins or polypeptides. However, to obtain a preparation that is substantially homogeneous with respect to the target polypeptide, it is necessary to purify the target polypeptide from the recombinant cell proteins or polypeptides. As a first step, the culture medium or lysate is typically centrifuged to remove granular cell debris. The membrane and soluble protein fractions are then separated. The target polypeptide can then be purified from the soluble protein fraction and from the membrane fraction of the culture lysate, depending on whether the target polypeptide is membrane-bound or not. The following steps are examples of appropriate purification procedures: fractionation on an immunoaffinity or ion-exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on silica or cation-exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE, ammonium sulfate precipitation; gel filtration using, e.g., Sephadex® G-75; and a Protein A Sepharose column to remove contaminants such as IgG.

[0113] Most companies currently manufacturing monoclonal antibodies (MAbs) employ a three-column platform approach that includes protein A affinity chromatography for product capture, followed by flow-through anion exchange chromatography to extract load contaminants such as host cell proteins (HCPs), endotoxins, host DNA, and leached protein A, and then retention-type cation exchange chromatography or hydrophobic interaction chromatography (HIC) to remove positively charged contaminants, including residual HCPs and product aggregates.

[0114] Viruses that can be present in a protein solution are larger than the proteins themselves. Therefore, it is presumed that viruses can be removed from the proteins by filtration, according to their size.

[0115] Viral filtration can remove larger viruses, such as retroviruses (80-100 nm in diameter), typically using high-capacity membranes with a nominal pore size of approximately 60 nm. High-capacity membranes with a nominal pore size of 20 nm are also commercially available, allowing filtration to remove smaller viruses, such as parvoviruses (18-26 nm in diameter), while allowing the passage of proteins as large as 160 kD (approximately 8 nm), such as monoclonal antibodies. The present invention is primarily intended to address the problems typically associated with filtering such small viruses by using virus removal filters with smaller pore sizes.

[0116] Typically, the viral filtration step can be performed at one of several points in a given downstream process. For example, in a typical monoclonal antibody purification process, viral filtration may be performed after the low-pH viral inactivation step, or after the intermediate column chromatography step, or after the final column chromatography step.

[0117] According to the present invention, the virus filtration unit operation can be performed at any stage of the downstream process. Virus filtration during the downstream processing of monoclonal antibodies is typically performed after the affinity chromatography (capture) and ion exchange purification (final purification) steps.

[0118] An example of the experimental setup used in the experiments disclosed herein is shown in Figure 1. However, it should be emphasized that the present invention is not limited in this way. Other configurations well known in the art are also suitable and may be used in the methods of the present invention.

[0119] In parallel flow viral filtration, the protein solution is typically extruded outwards at a constant flow rate onto the retaining sides. The differential pressure across the virus removal filter causes the protein solution to permeate through the filter, while the viruses are retained on the retaining sides.

[0120] In so-called "vertical flow" or "dead-end" viral filtration, the same viral filters used in parallel viral filtration may be used, but the peripheral equipment and operating procedures are considerably simpler and less expensive than those in parallel flow viral filtration. Therefore, in principle, "vertical flow" filtration involves placing a polymer-containing solution in a pressure vessel before filtration, and then pressurizing the solution with the help of a pressure source, preferably nitrogen (gas) or air, to pass it through a viral removal filter. Alternatively, a pump can be used on the holding side to filter the liquid through the viral removal filter at a predetermined flow rate.

[0121] The fineness of a filter is generally expressed as the pore size, or as the approximate molecular weight (relative molecular weight) at which the filter stops the molecule (the so-called cutoff).

[0122] Virus filters are well known in the art and are supplied, in particular, by Millipore (Massachusetts, USA) and Asahi Chemical Industry Co., Ltd. (Japan). A suitable parvovirus-retaining filter is Viresolve® Pro (Millipore Corp., Billerica, MA). The Viresolve® Pro membrane has an asymmetric bilayer structure and is made of polyethersulfone (PES). The membrane structure is designed to retain viruses larger than 20 nm while allowing proteins with a molecular weight of less than 180 kDa to pass through the membrane. Other filters suitable for removing small viruses, such as parvovirus, from protein solutions include Novasip® DV20 and DV50 virus removal filter capsules (Pall Corp., East Hills, NY), Virosart® CPV, Planova 20N (Asahi Kasei), and BioEX (Asahi Kasei). Novasip DV20 capsule filters utilize Ultipor VF DV20 ruffled membrane cartridges to remove parvoviruses and other viruses as small as 20 nm from protein solutions of 5-10 liters. Novasip DV50 capsule filters incorporate Ultipor VF DV50 Ultipleat® membrane cartridges to remove viruses larger than 40-50 nm. Novasip Ultipor VF capsule filters are supplied non-sterile and can be gamma-irradiated. Virosart® CPV utilizes a bilayer polyethersulfone chiral membrane to retain parvoviruses larger than 4 log and retroviruses of 6 log.

[0123] Pre-filtration of the supply solution can have a dramatic impact on filter performance. Pre-filtration typically aims to remove impurities and contaminants, such as protein aggregates, DNA, and other trace substances, which can cause contamination of the viral filter.

[0124] According to the present invention, a significant enhancement of the effectiveness of the virus filter can be achieved by a pre-filtration step that includes the use of both cation exchange and endotoxin removal media. In this context, the terms “media (singular)” or “media (plural)” are used to encompass any means for carrying out the cation exchange and endotoxin removal steps, respectively. Therefore, the term “cation exchange media” specifically includes, but is not limited to, cation exchange resins, matrices, absorbers, etc. The term “endotoxin removal media” includes, but is not limited to, any positively charged membrane surface, such as chromatographic endotoxin removal media, endotoxin affinity removal media, etc.

[0125] Suitable cation exchange media for use in the prefiltration stage of the present invention include, but are not limited to, Mustang® S, Sartobind® S, Viresolve® Shield, SPFF, SPXL, Capto® S, Poros® 50HS, Fractogel® S, Hypercel® D, etc. (these are commercially available).

[0126] Suitable endotoxin removal media for use in the prefiltration stage of the present invention include, but are not limited to, Mustang® E, Mustang® Q, Sartobind® Q, Chromasorb®, Possidyne®, Capto® Q, QSFF, Poros® Q, Fractogel® Q, etc. (these are commercially available).

[0127] The prefiltration step can be carried out, for example, by employing a manufacturing process chromatography pool and processing the pool on a filtration train containing an endotoxin removal and cation exchange medium and a parvovirus filter. The endotoxin removal and cation exchange medium act as prefiltration steps, and the capacity of the parvovirus filter is independent of the order of the two steps in the filtration train. The filtration train can operate sequentially as a single step, or it can be operated as different unit operations. For example, in one embodiment, the chromatography pool is first processed on the endotoxin removal medium, the collected pool is then processed on the cation exchange medium, and the subsequent pool is filtered with a parvovirus filter. As described above, the order in which the cation exchange medium and endotoxin removal medium are applied in the process sequence does not affect the parvovirus filtration capacity. The process can be operated over a wide pH range, for example, in the range of pH 4 to 10, and the optimal filtration capacity depends on the target impurity profile and product attributes. Similarly, the protein concentration can vary over a wide range, for example, from 1 to 40 g / L, but does not limit the mass processing capacity of the parvovirus filter.

[0128] The present invention will be further understood by reference to the following embodiments, however, they do not limit the scope of the invention. All references throughout this disclosure are incorporated herein by reference. [Examples]

[0129] material and method 1. Protein solution Since viral filtration during the downstream processing of monoclonal antibodies is performed after affinity chromatography (capture stage) and ion exchange stage (final purification stage), all filtration experiments were carried out using ion exchange (cation or anion exchange) chromatography pools in commercially viable manufacturing processes. The mAb concentrations and pool conductivity of the cation exchange and anion exchange pools were 10 mg / ml and 10 mS / cm, and 8 mg / ml and 4 ms / cm, respectively. Filtration experiments were performed using fresh feedstock (within 24 hours of manufacture) or feedstock that had been frozen at -70°C after manufacture and thawed at 4-8°C before use. No significant differences were observed between the results obtained using fresh feedstock and freeze-thawed feedstock. Protein concentrations were determined using a UV-vis spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE), and absorbance was measured at 280 nm.

[0130] 2. Membrane Filtration experiments were conducted using Viresolve® Pro (Millipore Corp., Billerica, MA) parvovirus-retaining filters. The Viresolve® Pro membrane has an asymmetric bilayer structure and is made of polyethersulfone (PES). The membrane structure is designed to retain viruses larger than 20 nm, while allowing proteins with molecular weights less than 180 kDa to pass through. Pre-filters evaluated for Viresolve® Pro in this test included Viresolve® Optiscale 40 deep filter (Millipore Corp., Billerica, MA), Fluorodyne EX Mini 0.2 μm sterile filter (Pall Corp., East Hills, NY), and membrane absorbers from the Mustang® family (Pall Corp., East Hills, NY). The membrane absorbers were obtained from the vendor in fully enclosed Acrodisc® units. Table 1 summarizes the key characteristics (functional groups, bed volume, pore size, etc.) of all pre-filters used in this test. [Table 1]

[0131] 3. Experiment Setup Filtration experiments were conducted using the specially designed apparatus shown in Figure 1. The load substance, i.e., the manufacturing process mAb pool, was placed in the load reservoir and filtered through a filtration train consisting of different combinations of pre-filters and commercially available parvovirus filters. In all filtration experiments, a constant filtration flow rate (P) was maintained. max The following method was used: A pressure transducer was placed upstream of each filter and connected to a Millidaq or Netdaq system to record differential pressure data as a function of time or mass processing rate. The filtrate from the parvovirus filter was collected in a reservoir and held on a load cell to record the mass processing rate as a function of time.

[0132] Results and Discussion Downstream purification of mAbs expressed in mammalian cell cultures typically involves a first step of centrifugation and deep filtration to remove cells and cell fragments, followed by affinity chromatography for mAb capture and host cell protein (HCP) removal, and then cation exchange chromatography, viral filtration, and anion exchange chromatography to further remove impurities such as aggregates, viruses, leached protein A, and HCP. Most experiments in this study were performed using a cation exchange pool, with cation exchange chromatography being the second chromatography step.

[0133] Figure 2 shows the therapeutic mAb supply flow rate of 200 L / min using different pre-filters. 2 This graph shows experimental data on differential pressure when passing through Viresolve Pro at a constant flow rate per hour. The X-axis represents the mass of loaded mAbs per square meter of virus filter. The Y-axis represents the differential pressure through the virus filter as a function of mass processing capacity. The data clearly shows that the deep filter provides an increase in viral filtration capacity by several orders of magnitude compared to the sterile filter. A similar observation was made by Bolton et al. (Appl. Biochem. 43:55-63, 2006) when evaluating the effect of Viresolve Prefilter™ (deep filter medium) as a prefilter for NFP parvovirus retention filters (Millipore Corp.) using a polyclonal IgG solution. The authors believe that the increase in capacity is due to the selective adsorption of contaminants (denatured proteins) due to hydrophobic interactions.

[0134] Deep filters have traditionally been successfully used for clarifying cell culture fluids; however, there are a number of limitations that must be considered, especially when used downstream of the capture stage, such as as a prefilter for parvovirus retention filters. (a) Deep filters do not improve substrate stability, hinder hygiene in the process sequence after installation, and create the possibility of open-type processing and the growth of contaminating microorganisms. (b) The composition of the deep filter includes diatomaceous earth as a key component, which is typically food grade and presents a quality issue. (c) Diatomaceous earth is generally supplied from nature (lacking well-defined chemical processes) and therefore may exhibit lot-to-lot variability. (d) Deep filters also tend to filter out metals, β-glycans and other impurities, and this removal needs to be demonstrated and confirmed in downstream operations.

[0135] Since the downstream unit operations of deep filters must be designed to provide adequate filtration, these limitations pose a significant burden on process development. However, even when the requirements for filtration filtration are met, there is reason for concern that certain lots of deep filters may have significantly higher filtration than those that the process can clean, because the main components are supplied from nature, i.e., they lack well-defined chemical synthesis processes.

[0136] Therefore, there has been considerable interest in developing pre-filters that do not exhibit these limitations. As mentioned above, Brown et al. (Brown et al. IBC's 20 th A recent study by Antibody Development and Production (San Diego, CA, 2008) showed that Mustang S, a potent load ion exchanger, can several times increase the capacity of parvovirus retention filters when used as a prefilter. Therefore, experiments were conducted to evaluate the effects of different prefiltration media on Viresolve® Pro. Experimental data at pH 5.0 and 6.5 are shown in Figures 3(a) and (b). The data showed that the cation exchange media had a slight advantage over the endotoxin removal adsorbent at pH 5.0, but this advantage disappeared at pH 6.5. The overall capacity of both media was higher than that using sterile filters (Figure 2); however, it was significantly insufficient to successfully perform unit operations at a production scale.

[0137] Based on the hypothesis that both cation exchange and endotoxin removal media can remove two different contaminants and that both can filter contaminants, we designed experiments using a novel pre-filtration train containing both cation exchange and endotoxin removal media. The experimental results are shown in Figures 4(a) and (b) (pH 5.0 and pH 6.5, respectively). The data clearly show that the combination of the two media is significantly better than each of the media alone. For example, at pH 5.0, the combination of cation exchange and endotoxin removal media provides a capacity improvement of more than an order of magnitude at a differential pressure of 20 psa. A similar trend was observed at pH 6.5, however, the overall capacity was lower than that obtained at pH 5.0. This is likely because impurities are removed more robustly at lower pH.

[0138] The experimental results in MAb2 are shown in Figure 5. Consistent with the data in Figure 4, the novel pre-filtration train containing both the endotoxin removal medium and the cation exchange medium showed a significant increase in capacity, suggesting that the endotoxin removal medium and the cation exchange medium work synergistically to remove two different types of contaminants.

[0139] conclusion Previous studies have largely focused on using deep filters or cation exchange membrane adsorbents as pre-filters to increase the capacity of parvovirus-retaining filters. Deep filters offer a robust mechanism for increasing viral filtration capacity, but limitations associated with them, such as filtered-out material, restrict their application to specific stages in downstream purification streams. Cation exchange membrane adsorbents can increase parvovirus filtering capacity for some monoclonal antibody (mAb) feed streams, but they are not universally applicable, as the data from this study show, suggesting that numerous contaminants may exist that need to be addressed to further improve the performance of parvovirus removal filters.

[0140] As demonstrated by the experimental results above, the present invention highlights two aspects: (1) the endotoxin removal medium itself, when used for prefiltration, can effectively increase the capacity of the parvovirus filter; and (2) when the endotoxin removal and cation exchange medium are connected in a prefiltration train, the parvovirus filtration capacity can be increased several times, reducing raw material costs and facilitating successful virus filtration operations on a production scale.

Claims

1. A method for improving the filtration capacity of a virus filter during protein purification, comprising passing a composition containing the protein to be purified through a cation exchange step and an endotoxin removal step, either simultaneously or in any order, through the virus filter.

2. The method according to claim 1, wherein the pore size of the virus filter is approximately 15 to approximately 100 nm in diameter.

3. The method according to claim 2, wherein the pore size of the virus filter is approximately 15 to approximately 30 nm in diameter.

4. The method according to claim 3, wherein the pore size of the virus filter is approximately 20 nm.

5. The method according to claim 3 or 4, wherein the virus to be removed is a parvovirus.

6. The method according to claim 5, wherein the diameter of the parvovirus is approximately 18 to approximately 26 nm.

7. The method according to claim 1, wherein the protein is an antibody or an antibody fragment.

8. The method according to claim 7, wherein the protein is a recombinant antibody or antibody fragment.

9. The method according to claim 8, wherein the recombinant antibody or antibody fragment is produced in a mammalian host cell.

10. The method according to claim 9, wherein the mammalian host cells are Chinese hamster ovary (CHO) cells.

11. The method according to claim 1, wherein the composition containing the protein to be purified is first subjected to a cation exchange step, then to an endotoxin removal step, and finally filtered for virus.

12. The method according to claim 1, wherein the composition containing the protein to be purified is first subjected to an endotoxin removal step, then to a cation exchange step, and then subjected to viral filtration.

13. The method according to claim 1, wherein the composition containing the protein to be purified is subjected to an endotoxin removal step and a cation exchange step simultaneously, and then filtered for virus.

14. The method according to claim 11, wherein the virus is filtered immediately after the endotoxin removal step.

15. The method according to claim 12, wherein the virus is filtered immediately after the cation exchange step.

16. The method according to claim 13, wherein the virus is filtered immediately after the simultaneous endotoxin removal and cation exchange step.

17. The method according to claim 1, wherein the virus filtration is performed at a pH of approximately 4 to approximately 10.

18. The method according to claim 1, wherein the protein concentration in the composition is about 1 to 40 g / L.

19. The method according to claim 8, wherein the antibody is against one or more antigens selected from the group consisting of HER1 (EGFR), HER2, HER3, HER4, VEGF, CD20, CD22, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, VCAM, IL-17A and / or F, IgE, DR5, CD40, Apo2L / TRAIL, EGFL7, NRP1, mitosis-activated protein kinase (MAPK), and factor D.

20. The aforementioned antibodies include anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras tumor protein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD23 antibody, and anti-CD30 antibody. The method according to claim 8, wherein the antibody is selected from the group consisting of antibodies, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA / CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-kappa light chain antibody, anti-lambda light chain antibody, anti-melanosome antibody, anti-prostate-specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody, and anti-Tn-antigen antibody.

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