HK1239715A1 - Method to improve virus removal in protein purification - Google Patents

Description

Method for enhancing virus removal in protein purification

The present application is a divisional application of chinese patent application 201080044790.3 entitled "method for improving virus removal in protein purification" filed on 8/6/2010 by the applicant.

Background

Technical Field

The present invention is in the field of protein purification. In particular, the present invention relates to a method for increasing the filtration capacity of a virus filter by the combined use of an endotoxin removal and cation exchange matrix in a prefiltration process.

Description of the Related Art

Mammalian cell lines have become the primary choice for recombinant protein production technology due to their ability to perform the correct protein folding and post-translational modifications, such as glycosylation (Chu and RobinsonCurrent 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 to present a potential risk of foreign viral contamination (Garnick, Dev Biol stand.Basel: Karger 93: 21-29,1998). Although the biopharmaceutical industry produces recombinant protein drugs with good safety profiles, viral infections occur from blood and plasma-derived blood products (Brown, Dev. biol. Stand.81, 1993; Thomas, Lancet 343: 1583-. To reduce the risk of viral contamination during recombinant protein production, downstream purification processes were designed to include processing steps to remove endogenous and foreign viruses. Adequate viral clearance is obtained by a combination of several processing steps that provide for inactivation or removal of viruses from the feed stream (processfed stream). Virus inactivation is achieved using techniques such as incubation at low pH, heat and detergent treatment, while virus removal is typically performed using chromatography and filtration (Curtis et al, Biotechnology and Bioengineering 84(2): 179-186, 2003).

Unlike chromatographic matrices that remove viruses based on physicochemical properties (e.g., net charge), virus filtration removes viruses by size exclusion and is therefore considered to be a more powerful technique. To date, the use of viral filtration in downstream purification processes of biotherapeutics derived from mammalian cell cultures has been limited to the removal of retroviruses (80-100nm diameter) due to the lack of high-flux membranes with nominal pore sizes less than 60 nm.

Recent advances in membrane technology have enabled the production of high-throughput membranes with nominal pore sizes of 20 nm. These virus filters retain parvovirus (18-26nm diameter), allowing passage of proteins as large as 160kD (-8 nm), such as monoclonal antibodies (mAbs).

A parvovirus filter having high selectivity and high flux is obtained by coating a microporous substrate with a thin retention membrane layer. This thin retentate layer allows for very fine separation of proteins and viruses, but is also prone to fouling by impurities in the process feed stream, resulting in lower filtration capacity and flow. Viral filter fouling is due to contaminants such as protein aggregates and denatured proteins. Bohonak and Zydney (Bohonak and Zydney, Journal of Membrane Science 254(1-2):71-79,2005) showed that the loss of filter capacity could be due to filter cake formation or pore blocking. Other recent reports (Bolton et al, Biotechnol. appl. biochem.43:55-63,2006; Levy et al, Filtration in biomedical Industry. (Meltzer, T.H. and Jornitz, M.W. eds.) on page 619-. Several publications (Bolton et al, Biotechnology and Applied Biochemistry 42: 133-.

Thus, a great deal of recent research has focused on identifying prefilters (pre-filters) for removing fouling from process feed streams to minimize fouling of virus filters and to ensure high capacity, high throughput, and robust virus retention. Bolton et al (Bolton et al, 2006) conducted extensive research involving testing several membranes as prefilters and demonstrated the use of ViResolve^TMDepth filters act as prefilters and can increase the capacity of Normal Flux Parvovirus (NFP) membranes by almost an order of magnitude. Brown et al (Brown et al, 2008, Use of Charged Membranes to identified solution proteins in order to facility partial solutions filtration. IBC's20^thAntibody Development and Production, San Diego, CA) evaluated strong cation exchange membrane adsorbents as prefilters of parvovirus retention filters and demonstrated that virus filtration capacity could be increased several-fold for 11 different mAb streams. The authors hypothesized that the cation exchange membrane adsorbent removed large molecular weight (-600- & ltSUB & gt 1500kD) protein aggregates from the feed stream by competitive adsorption, preventing viral filter plugging. U.S. Pat. No. 7,118,675(Siwak et al) describes a process that utilizes charge-modified membranes to remove protein aggregates from protein solutions to prevent fouling of viral filters.

Summary of The Invention

The present invention is based, at least in part, on the experimental discovery that fouling of parvovirus filters can be caused by impurities other than those mentioned in the literature, requiring more comprehensive prefiltration solutions to improve virus filtration capacity. The present invention thus provides a novel prefiltration solution whose performance is significantly better than the best prefiltration process (cation exchange membrane adsorbent) mentioned in the literature.

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

In one embodiment, the pore size of the virus filter is between about 15 and about 100nm in diameter.

In another embodiment, the pore size of the virus filter is between about 15 and about 30nm in diameter.

In yet another embodiment, the pore size of the virus filter is about 20 nm.

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

In still further embodiments, the parvovirus has a diameter between about 18 and about 26 nm.

In various embodiments, the protein is an antibody or antibody fragment, such as an antibody produced by recombinant DNA techniques, or a fragment thereof.

In another embodiment, the antibody is a therapeutic antibody.

In yet another embodiment, the recombinant antibody or antibody fragment is produced in a mammalian host cell, e.g., a Chinese Hamster Ovary (CHO) cell.

In a further embodiment, the protein-containing composition to be purified is first subjected to a cation exchange step followed by an endotoxin removal step prior to virus filtration.

In yet a further embodiment, the composition comprising the protein to be purified is first subjected to an endotoxin removal step followed by a cation exchange step prior to virus filtration.

In another embodiment, the composition comprising the protein to be purified is subjected to a cation exchange step and an endotoxin removal step simultaneously prior to virus filtration, both media being held together in a single module.

In yet another embodiment, the endotoxin removal step is followed directly by virus filtration.

In a further embodiment, the virus filtration is performed directly after the cation exchange step.

In various embodiments, virus filtration is performed at a pH between about 4 and about 10.

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

In yet another embodiment, the antibody to be purified is directed 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.

In a further embodiment, the antibody is selected from the group consisting of an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-P53 antibody, an anti-cathepsin D antibody, an anti-Bcl-2 antibody, an anti-E-cadherin antibody, an anti-CA 125 antibody, an anti-CA 15-3 antibody, an anti-CA 19-9 antibody, an anti-c-erbB-2 antibody, an anti-P-glycoprotein antibody, an anti-CEA antibody, an anti-retinoblastoma protein antibody, an anti-ras oncoprotein antibody, an anti-Lewis X antibody, an anti-Ki-67 antibody, an anti-PCNA antibody, an anti-CD 3 antibody, an anti-CD 4 antibody, an anti-CD 5 antibody, an anti-CD 7 antibody, an anti-CD 8 antibody, an anti-CD 9/P24 antibody, an anti-CD 10 antibody, an anti-CD 3511 antibody, an anti-CD 13 antibody, an anti-CD 14 antibody, an anti-CD 15 antibody, an anti-CD 19 antibody, an anti-CD 23 antibody, an anti-CD 30 antibody, an anti-CD 31 antibody, an anti-CD 585 antibody, anti-CD 38 antibody, anti-CD 41 antibody, anti-LCA/CD 45 antibody, anti-CD 45RO antibody, anti-CD 45RA antibody, anti-CD 39 antibody, anti-CD 100 antibody, anti-CD 95/Fas antibody, anti-CD 99 antibody, anti-CD 106 antibody, anti-ubiquitin antibody, anti-CD 71 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 description of the drawings

FIG. 1: schematic of the experimental setup used for virus filtration studies.

FIG. 2: effect of sterile and depth filters on the capacity of Viresolve Pro parvovirus retention filters. The experiments were conducted at pH 5.5 and a conductance of 8.5 mS/cm. The mAb concentration was about 13 g/L.

Fig. 3(a) and (b): effect of cation exchange and endotoxin removal membrane adsorbent as prefilter on capacity of Viresolve Pro parvovirus filter. Data in 3(a) and 3(b) were generated with mAb1 at pH5.0 and 6.5, respectively.

Fig. 4(a) and (b): effect of novel prefiltration chain containing both cation exchange and endotoxin removal membrane sorbents on the capacity of Viresolve Pro parvovirus retention filter. The data in 4(a) and 4(b) were generated with MAb1 at pH5.0 and 6.5, respectively.

FIG. 5: the effect of the novel prefiltration train containing both cation exchange and endotoxin removal membrane adsorbents on the capacity of parvovirus retention filters using MAb2 compared to cation exchange prefiltration matrices.

Description of The Preferred Embodiment

I. Definition of

By "protein" is meant an amino acid sequence having a chain length sufficient to produce higher levels of tertiary and/or quaternary structure. Thus, proteins are distinguished from "peptides", which are also amino acid-based molecules, but do not have the structure. Typically, the proteins used herein have a molecular weight of at least about 15-20kD, preferably at least about 20 kD.

Examples of proteins encompassed within the definition herein include mammalian proteins such as CD4, integrins and subunits thereof such as beta7, growth hormones including human and bovine growth hormone, growth hormone releasing factor, parathyroid hormone, thyrotropin, lipoprotein, α -1-antitrypsin, insulin A-chain, insulin B-chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, clotting factors such as factor VIIIC, factor IX, tissue factor and von Willebrands factor (von Willebrands factor), anti-clotting factors such as protein C, atrial natriuretic factor, lung surfactant, plasminogen activator such as urokinase or tissue-type plasminogen activator (t-PA, e.g., urokinase or tissue-type plasminogen activator (t-PA)) Bmbozine, thrombin, tumor necrosis factors- α and- β, enkephalinase, RANTES (regulating expression and secretion of normally activated T cells), human macrophage inflammatory protein (MIP-1- α), serum albumins such as human serum albumin, Mullerian-inhibiting substance (mullerian-inhibiting substance), mouse gonadotropin-related peptides, DNase, inhibin, kinetin, Vascular Endothelial Growth Factor (VEGF), IgE, receptors for hormones or growth factors, integrins, protein A or D, rheumatoid factors, neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3, -4, -5 or-6 (NT-3, NT-4 NT, NT-5 or NT-6), or nerve growth factors such as NGF- β, platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, epidermal growth factor (FGF), transforming growth factor (TGF- β and TGF- β, TGF-35730, platelet-derived growth factor (TGF-derived growth factor), TGF-like TGF-3, TGF-364933, TGF-3623, TGF-3, TGF-364933, TGF-3, TGF-9, TGF-IGF-9, TGF-IGF-9, TGF-9-IGF-9, TGF-IGF-9-TGF-IGF-9, TGF-IGFSuch as CD3, CD8, CD19 and CD20, Erythropoietin (EPO), Thrombopoietin (TPO), osteoinductive factors, immunotoxins, Bone Morphogenic Proteins (BMP), interferons such as interferon- α, - β and- γ, Colony Stimulating Factors (CSF) such as M-CSF, GM-CSF and G-CSF, Interleukins (IL) such as IL-1 to IL-10, superoxide dismutase, T-cell receptors, surface membrane proteins, Decay Accelerating Factor (DAF), viral antigens such as part of the AIDS capsid, transporters, homing receptors, addressens, regulatory proteins, integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM, tumor associated antigens such as HER1(EGFR), HER2, HER3 or HER4 receptors, Apo2L/TRAIL, and fragments of any of the polypeptides listed above, as well as any of the immunoadhesins and any of the biological variants thereof;

as defined herein, the definition "protein" specifically includes therapeutic antibodies and immunoadhesins, including but not limited to antibodies to one or more of the following antigens: 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, and fragments thereof.

Other exemplary antibodies include, but are not limited to, antibodies selected from the group consisting of anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-P53 antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA 125 antibodies, anti-CA 15-3 antibodies, anti-CA 19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncogene protein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD 3 antibodies, anti-CD 4 antibodies, anti-CD 5 antibodies, anti-CD 7 antibodies, anti-CD 8 antibodies, anti-Ki-67 antibodies, anti-CD 9/p24 antibody, anti-CD 10 antibody, anti-CD 11c antibody, anti-CD 13 antibody, anti-CD 14 antibody, anti-CD 15 antibody, anti-CD 19 antibody, anti-CD 23 antibody, anti-CD 30 antibody, anti-CD 31 antibody, anti-CD 33 antibody, anti-CD 34 antibody, anti-CD 35 antibody, anti-CD 38 antibody, anti-CD 41 antibody, anti-LCA/CD 45 antibody, anti-CD 45RO antibody, anti-CD 45RA antibody, anti-CD 39 antibody, anti-CD 100 antibody, anti-CD 95/Fas antibody, anti-CD 99 antibody, anti-CD 106 antibody, anti-ubiquitin antibody, anti-CD 71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV-vimentin antibody, Anti-kappa light chain antibodies, anti-lambda light chain antibodies, anti-melanosome antibodies, anti-prostate specific antigen antibodies, anti-S-100 antibodies, anti-tau antigen antibodies, anti-fibrin antibodies, anti-keratin antibodies, and anti-Tn-antigen antibodies.

An "isolated" protein, such as an antibody, is one that has been identified, isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are materials that interfere with diagnostic or therapeutic uses of proteins (e.g., antibodies), which may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the protein (e.g., antibody) may be purified to: (1) to greater than 95% by weight, most preferably greater than 99% by weight, as determined by the Lowry method, (2) using a centrifugal cup sequencer to an extent sufficient to obtain at least 15 residues of the N-terminal or intermediate amino acid sequence; or (3) SDS-PAGE homogenization is achieved under reducing or non-reducing conditions using Coomassie Brilliant blue or, preferably, silver staining.

The protein is preferably substantially pure, ideally substantially homogeneous (i.e., free of contaminating proteins). By "substantially pure" protein is meant a composition comprising at least about 90% by weight protein, preferably at least about 95% by weight protein, based on the total weight of the composition.

By "substantially homogeneous" protein is meant a composition comprising at least about 99% by weight of protein, based on the total weight of the composition.

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length antibodies with immunoglobulin Fc regions), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies and single chain molecules, and antibodiesFragments (e.g., Fab, F (ab')₂And Fv).

The basic 4-chain antibody unit is a heterotetrameric glycoprotein, comprising two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 such basic heterotetrameric units, with an additional polypeptide called the J chain, containing 10 antigen binding sites, while IgA antibodies comprise 2-5 such basic 4 chain units, which can be combined with the J chain to polymerize into multivalent assemblies (assembly). In the case of IgG, the 4 chain units are typically about 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly arranged intrachain disulfide bonds. The N-terminus of each H chain has a variable domain (V)_H) For the case of α and gamma chains, each variable domain is followed by 3 constant domains (C)_H) And for the case of μ and isotypes, four C's after the variable domain_HA domain. The N-terminus of each L chain has a variable domain (V)_L) The other end of the variable domain is followed by a constant domain. V_LAnd V_HPairing, C_LTo the first constant domain (C) of the heavy chain_H1) And (6) pairing. Specific amino acid residues are thought to form the interface between the light and heavy chain variable domains. Paired V_HAnd V_LTogether forming a single antigen binding site. For the structure and properties of different types of antibodies see, e.g., Basic and clinical immunology, 8 th edition, Daniel p.sties, Abba i.terr and Tristram g.parsolw (eds.), Appleton&Lange, Norwalk, CT,1994, page 71 and chapter 6.

Based on the amino acid sequence of the constant domains, the L chain of any vertebrate species can be assigned to one of two well-distinguished types, termed κ and λ. Amino acid sequence based on the constant domain of its heavy chain (C)_H) Immunoglobulins can be divided into different classes or subclasses there are five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, with heavy chains called α, γ and μ, respectively, based on C_HSequence and workWith relatively subtle differences in energy, the γ and μ classes are further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

The term "variable" refers to the fact that certain fragment sequences of variable domains differ widely between antibodies. The V domain mediates antigen binding, defining the specificity of a particular antibody for its particular antigen. However, the variable and non-uniform distribution is throughout the variable domains. In contrast, the V regions consist of relatively invariant segments (stretches) called Framework Regions (FR) separated by about 15-30 amino acid residues, separated by short regions of high variation called "hypervariable regions" or sometimes called "complementarity determining regions" (CDRs) of about 9-12 amino acid residues in length, respectively. The variable domains of native heavy and light chains each comprise 4 FRs, largely adopting a β -sheet conformation, connected by 3 hypervariable regions, which form circular junctions, in some cases forming in part a β -sheet structure. The hypervariable regions of each chain are tightly linked together by FRs and are responsible for forming the antigen-binding site of antibodies with the hypervariable regions of the other chain (see Kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term "hypervariable region" (also known as "complementarity determining region" or CDR) refers to the amino acid residues (typically 3 or 4 short regions of high sequence variation) of an antibody within the V region domain 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) a cross-species sequence variability-based approach (i.e., Kabat et al, Sequences of Proteins of immunological interest (National Institute of Health, Bethesda, MS 1991)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia, c. et al, j.mol.biol.196:901-917(1987)). However, if two residues are identifiedOther techniques define overlapping rather than identical regions, which may be combined to define hybrid (hybrid) CDRs.

The term "monoclonal antibody" refers herein to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the population comprises individual antibodies that are identical except for potential naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes). In addition to specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The phrase "monoclonal" means that the characteristics of an antibody are obtained from a substantially homogeneous population of antibodies and is not considered to require production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be made by the hybridoma method described for the first time in Kohler et al, Nature,256495(1975), or may be made by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be monoclonal antibodies using, for example, Clackson et al, Nature,352624-,222581-597(1991), isolated from phage antibody libraries.

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical with or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the fragments exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al, proc.natl.acad.sci.usa,81:6851-6855(1984))。

"intact" antibodies are suchAn antibody comprising an antigen binding site and a CL, and comprising at least a heavy chain domain C_H1、C_H2 and C_H3。

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; Zapata et al, Protein Eng.8(10):1057-1062[1995]) (ii) a Single chain antibody molecules and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a remaining "Fc" fragment, the name reflecting the ability to crystallize readily. The Fab fragment consists of the entire variable domains (V) of the L and H chains_H) And a first constant domain of a heavy chain (C)_H1) And (4) forming. Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen binding site. Pepsin treatment of antibodies produced a single large F (ab')₂Fragment of, said F (ab')₂The fragments correspond approximately to two disulfide-linked Fab fragments with different antigen binding activity, yet are capable of crosslinking with antigen. Fab' fragments differ from Fab fragments in C_H1 domain has several additional residues at the carboxy terminus, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domains have a free thiol group. F (ab')₂Antibody fragments were originally produced as paired Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxy-terminal portions of the two H chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which are also recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment, containing the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain in close, non-covalent association. The folding of these two domains gives rise to 6 hypervariable loops (3 loops each for the H and L chains) providing amino acid residues for antigen binding and the antigen-binding specificity of the resulting antibody. However, even a single variable domain (or half of an Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains joined into a single polypeptide chain. Preferably, the sFv polypeptide is further comprised in V_HAnd V_LA polypeptide linker between the domains, which enables the sFv to form a desirable structure for antigen binding. For an overview of sFv, see Pluckthun in the pharmaceutical of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994).

The term "bivalent body" refers to a small antibody fragment prepared by reaction at V_HAnd V_LsFv fragments (see above) were constructed with short linkers (about 5-10 residues) between domains to achieve V domains that pair inter-chain rather than intra-chain, resulting in bivalent fragments, i.e., fragments with 2 antigen binding sites. Bispecific diabodies are heterodimers of two "cross over" sFv fragments, of which 2 antibodies have a V_HAnd V_LThe domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; hollinger et al, Proc. Natl. Acad. Sci. USA906444-.

An antibody that "binds" to a molecular target or antigen of interest is one that is capable of binding the antigen with sufficient affinity to allow the antibody to be effectively targeted to cells expressing the antigen.

An antibody that "specifically binds" or is "specifically directed to" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to the particular polypeptide or an epitope on the particular polypeptide under conditions that do not substantially bind to any other polypeptide or polypeptide epitope. In this embodiment, the antibody binds to less than 10% of the other polypeptide or polypeptide epitope as determined by Fluorescence Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA) as described above.

A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab ', F (ab') that is mostly human in sequence₂Or other antigen binding sequence of an antibody) contains minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region (also the CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some instances, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, "humanized antibody" herein also encompasses residues not found in either the recipient antibody or the donor antibody. These modifications are made to further improve and optimize the performance of the antibody. The most preferred humanized antibodies also comprise at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. For more details, see Jones et al, Nature,321522-525 (1986); reichmann et al, Nature,332323-329 (1988); and Presta, curr, op, struct, biol,2:593-596(1992)。

"Effector function" of an antibody refers to those biological activities attributed to the Fc region of an antibody (either the Fc region of a native sequence or the Fc region of an amino acid sequence variant) 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; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a type of cytotoxicity,wherein secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are required to kill target cells by this mechanism. The primary cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. In ravatch and Kinet, annu.9Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of 457-92 (1991). To assess ADCC activity of a target molecule, an ACDD assay in vitro is performed, for example, as described in U.S. Pat. nos. 5,500,362 or 5,821,337. Effective effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be assessed in vivo, for example, in Clynes et al, PNAS USA95652-656 (1998).

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is a receptor that binds IgG antibodies (gamma receptors), a receptor comprising the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants and alternative splicing forms of these receptors, and an Fc γ RII receptor comprising Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), having similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains in its cytoplasmic domain an Immunoreceptor Tyrosine Activation Motif (ITAM). The inhibitory receptor Fc γ RIIB contains an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in its cytoplasmic domain (see M).Annu.Rev.Immunol.15:203-234(1997)). Ravatch and Kinet, annu.9457-92 (1991); capel et al, immunolmethods425-34 (1994); and deHaas et al, j.lab.clin.med.126330-41(1995)) have reviewed FcRs. The other end-to-end (FcR),including future-identified fcrs, are encompassed by the term "FcR" herein. The term also encompasses the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG into the fetus (Guyer et al, j.117587(1976) and Kim et al, J.24:249(1994))。

A "human effector cell" is a leukocyte that expresses one or more fcrs and performs effector functions. Preferably, the cells express at least Fc γ RIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMCs and MNK cells being preferred. Effector cells can be isolated from natural sources (e.g., blood).

"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 their respective antigens. To assess complement activation, CDC assays can be performed, e.g., Gazzano-Santoro et al, j202163 (1996).

The terms "conjugation," "conjugated," and "conjugation" refer to any and all forms of covalent or non-covalent attachment, including, but not limited to, direct genetic or chemical fusion, coupling through linkers or cross-linking agents, and non-covalent association, such as with a leucine zipper. Antibody conjugates have another entity, such as a cytotoxic compound, drug, composition, compound, radioactive element, or detectable label, attached to the antibody or antibody fragment.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with dysfunction and those in need of prevention of dysfunction.

"mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, non-human higher primates, livestock and farm animals, as well as zoo, stadium or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, mice, cats, and the like. Preferably, the mammal is a human.

"dysfunction" is any indication that would benefit from treatment with a protein. Including chronic and acute dysfunctions or diseases, including pathological indications that predispose the mammal to the dysfunction in question.

A "therapeutically effective amount" is at least the minimum concentration required to produce a measurable improvement or prevention of a particular disorder. Knowing that a therapeutically effective amount of a protein is generally known in the art, a therapeutically effective amount of a protein found below can be determined using standard techniques within the skill of those in the art (e.g., a physician of ordinary skill).

Mode for carrying out the invention

A. Protein production

According to the present invention, the production of proteins by recombinant DNA techniques is generally known in the art, i.e., by culturing cells transformed or transfected with a vector containing a nucleic acid encoding a protein.

The production of proteins by recombinant means can be achieved by transfecting or transforming appropriate host cells with expression or cloning vectors and culturing in conventional nutrient media suitable for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences. One skilled in the art can select culture conditions, such as substrate, temperature, pH, etc., without undue experimentation. In general, the principles, procedures and Practical techniques for maximizing Cell culture productivity can be found in Mammalian Cell Biotechnology, A Practical Approach, M.Butler, eds (IRLPress,1991) and Sambrook et al, Molecular Cloning, A Laboratory Manual, New York, Cold spring Harbor Press. Methods of transfection are known to those of ordinary skill in the art and include, for example, CaPO₄And CaCl₂Transfection, electroporation, microinjection, and the like. Suitable techniques are also described in Sambrook et al, supra. Other transfectionsDescription of the technology is given in Shaw et al, Gene23315 (1983); WO 89/05859; graham et al, Virology52456-457(1978) and U.S. P.4,399, 216.

Nucleic acids encoding the desired protein can be inserted into replicable vectors for cloning or expression. Suitable vectors are publicly available and may take the form of plasmids, cosmids, viral particles or phages. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. Generally, DNA is inserted into the appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes and enhancer elements, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

The protein form may be recovered from the culture medium or host cell lysate. If membrane bound, it may be released from the membrane using a suitable detergent or by enzymatic cleavage. The cells used for expression may also be disrupted by a variety of physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

The protein may be purified by any suitable technique known in the art, for example, fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or a cation exchange resin (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, use of a protein A agarose column (e.g., DEAE)G-75) gel filtration to remove contaminants such as IgG, and forms of metal chelating column binding epitope tags.

B. Antibody preparation

In some embodiments of the invention, the selected protein is an antibody. Techniques for producing antibodies (including polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies) are as follows.

(i) Polyclonal antibodies

Polyclonal antibodies are typically generated by multiple Subcutaneous (SC) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. It is useful to conjugate the relevant antigen to a protein that is immunogenic to the species to be immunized, such as keyhole limpet hemocyanin (limpet), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl A, synthetic trehalose dicorynomycolate). One skilled in the art can select an immunization protocol without undue experimentation.

One month later, boosts were performed by subcutaneous injections at multiple sites with an initial amount of peptide or conjugate from 1/5 to 1/10 in freund's complete adjuvant. After 7 to 14 days, the animals were bled and the antibody titer in the serum was determined. Animals were boosted until titer plateaus. Preferably, the animal is boosted with conjugates of the same antigen but conjugated to different proteins and/or conjugated via different cross-linking agents. Conjugates are also made in recombinant cell culture as protein fusions. In addition, a coagulant such as alum is suitable for enhancing the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., a population comprising a single antibody is identical, except for potential naturally occurring mutations that may be present in minor amounts. Thus, the phrase "monoclonal" means that the antibody is not characterized as a mixture of different antibodies.

For example, monoclonal antibodies can be made using the hybridoma method, which is first described in Kohler et al, Nature,256495(1975), or may be produced by a recombinant DNA method (U.S. Pat. No. 4,816,567).

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

The immunizing agent typically comprises the protein to be formulated. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if cells of non-human mammalian origin are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells. Goding, Monoclonal antibodies: Principles and Practice, academic Press (1986), pages 59-103. Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium, which preferably contains one or more agents that inhibit the growth or survival of the unfused, parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium (e.g., HAT medium). Among the preferred myeloma Cell lines are mouse myeloma lines, such as those derived from 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 cells are also describedAre used for the production of human monoclonal antibodies (Kozbor, j. immunol.,1333001 (1984); brodeur et al, Monoclonal Antibody Production Techniques and Applications, pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

Monoclonal antibodies to the antigen produced in the medium in which the hybridoma cells are grown are assayed. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Can be prepared, for example, by Munson et al, anal. biochem,107220(1980) to determine the binding affinity of the monoclonal antibody.

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and grown using standard methods (Goding, supra). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo in animals as ascites tumors.

The immunological agent typically comprises an epitope protein to which the antibody binds. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian cell sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells. Goding, Monoclonal antibodies: Principles and Practice, Academic Press (1986), pages 59-103.

Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are cells that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a culture medium (e.g., HAT medium). Preferred immortalized Cell lines are mouse myeloma lines, which are available, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American type culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human monoclonal antibodies. Kozbor, J.Immunol.,1333001 (1984); brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp 51-63.

The culture medium in which the hybridoma cells were cultured was assayed for the presence of monoclonal antibodies against the formulated protein. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such assays and techniques are known in the art. Can be prepared by, for example, Munson and Pollard, anal. biochem,107220(1980) to determine the binding affinity of the monoclonal antibody.

After identification of the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and cultured using standard methods (Goding, supra). Suitable media for this purpose include, for example, Dulbecco's ModifiedEagle's medium or RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in an animal.

Monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA encoding the monoclonal antibody can be optionally isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells function as a preferred source of such DNA. Once isolated, the DNA may be placed in an expression vector and then transfected into a host cell, such as an e.coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cell. For recombinant expression of antibody-encoding DNA in bacteria, a review of the literature includes Skerra et al, curr. opinion in immunol,5256, 262(1993) and Pl ü ckthun, Immunol.130:151-188(1992)。

In other embodiments, the methods may be selected from the group consisting of methods using McCafferty et al, Nature,348552 (1990) the antibody was isolated from a phage library of antibodies generated by the technique described in. Clackson et al, Nature,352624-,222581-597(1991) describes the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology,10779-783(1992)), and combinatorial infection and in vivo recombination as strategies to construct very large phage libraries (Waterhouse et al, Nuc. acids. Res.,21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

The DNA may also be modified, for example, by replacing the homologous mouse sequences with the coding sequences for the human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl Acad. Sci. USA,816851(1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide.

Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or for one antigen binding site of a variable domain of an antibody, to produce a chimeric bivalent antibody comprising one antigen binding site with specificity for one antigen, and another antigen binding site with specificity for a different antigen.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include thiolimine (iminothiolate) and methyl-4-mercaptobutyrate (mercaptobutyramide).

(iii) Humanized and human antibodies

The antibodies used in the formulation methods may further comprise humanized or human antibodies. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')₂Or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies comprise human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework region residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies also contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. In general, a humanized antibody comprises substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. Jones et al, Nature321:522-525(1986)(ii) a Riechmann et al, Nature332323-329(1988) and Presta, curr, Opin, Structure, biol.2:593-596(1992)。

Methods for humanizing non-human antibodies are generally known in the art. In general, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically from an "import" variable domain. Humanization can be based essentially on Winter and colleagues Jones et al, Nature321522-525 (1986); riechmann et al, Nature332323-327 (1988); verhoeyen et al, Science2391534-1536(1988) or by replacing rodent CDR or CDR sequences with corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an entire human variable domain is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The selection of human variable domains for making the light and heavy chains in the humanized antibody is very important for reducing antigenicity. According to the so-called "best-fit" approach, the variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human Framework (FR) of the humanized antibody. Sims et al, j.immunol.,1512296 (1993); chothia et al, J.mol.biol.,196:901(1987). Another approach uses specific frameworks derived from the common sequence of all human antibodies of a particular subclass of light or heavy chains. The same framework can be used for several different humanized antibodies. Carter et al, proc.natl.acad.sci.usa,894285 (1992); presta et al, J.Immunol.,151:2623(1993)。

more importantly, the humanized antibodies retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and familiar to those skilled in the art. Computer programs are available which suggest and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Review of these displays allows analysis of the likely role of a residue in the function of a candidate immunoglobulin sequence, i.e., the ability of the residue to affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the receptor and input into the sequence such that the desired antibody characteristics, such as increased affinity for the target antigen, are obtained. In general, CDR residues are directly and for the most part substantially involved in affecting antigen binding.

Alternatively, transgenic animals can be produced that are capable of producing a comprehensive repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region (J) in chimeric and germline mutant mice has been described_H) Gene, to obtain complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice will result in antigen-based stimulated human antibody production. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa,902551 (1993); jakobovits et al, Nature,362255-258 (1993); bruggermann et al, Yeast in Immuno,7:33(1993). Human antibodies can also be derived from phage display libraries (Hoogenboom et al, j.mol.biol.,227381 (1991); marks et al, j.mol.biol.,222:581-597(1991))。

human antibodies, including phage display libraries, can also be produced using a variety of techniques known in the art. Hoogenboom and Winter, j.227381 (1991); marks et al, j.mol.biol.222:581(1991). The technique of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985) and Boerner et alEt al, j.147(1):86-95(1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. Based on the stimulus, human antibody production is observed, which closely mimics in all respects that observed in humans, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. patent nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and in the following scientific publications: marks et al, Bio/Technology10779 and 783 (1992); lonberg et al, Nature368:856-859(1994)；Morrison，Nature368812-13 (1994); fishwild et al, Nature Biotechnology14:845-51(1996)；Neuberger,Nature Biotechnology14826(1996) and Lonberg and Huszar, Intern.Rev.Immunol.13:65-93(1995)。

(iv) Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anticancer drug. See, for example, WO 88/07378 and U.S. patent No. 4,975,278.

The enzyme component of the immunoconjugate for ADEPT comprises any enzyme capable of acting on the prodrug in a manner that converts the prodrug to a more active cytotoxic form.

Enzymes that may be used in the method of the invention include, but are not limited to, glycosidases, glucose oxidase, human lysozyme, human glucuronidase, alkaline phosphatase for converting phosphate-containing prodrugs to free drugs; arylsulfatase for converting the sulfuric acid-containing prodrug into a free drug; cytosine deaminase for converting non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; proteases, e.g.Serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g.carboxypeptidase G2 and carboxypeptidase A) and cathepsins (e.g.cathepsins)B and L) for converting a peptide-containing prodrug to a free drug, D-alanylcarboxypeptidase for converting a prodrug containing a D-amino acid substitution, carbohydrate-cleaving enzymes such as β galactosidase and neuraminidase for converting a glycosylated prodrug to a free drug, β lactamase for converting a β lactam-derived drug to a free drug, and penicillin amidases such as penicillin V amidase or penicillin G amidase for converting an amine nitrogen-derived phenoxyacetyl or phenylacetyl drug to a free drug, respectively328:457-458(1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of abzymes to a tumor cell population.

The enzymes of the invention can be covalently bound to anti-IL-17 or anti-LIF antibodies by techniques generally known in the art, for example using the heterobifunctional cross-linkers discussed above. Alternatively, recombinant DNA techniques generally known in the art may be used (see, e.g., Neuberger et al, Nature312604-608(1984)) a fusion protein comprising at least the antigen-binding region of an antibody of the invention linked to at least a functionally active part of an enzyme of the invention.

(v) Bispecific and multispecific antibodies

Bispecific antibodies (BsAb) are antibodies that have binding specificity for at least two different epitopes. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods of making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities. Millstein et al, Nature,305:537-539(1983). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a mixture of perhaps 10 different antibody moleculesOf which only one has the correct bispecific structure. Purification of the correct molecule is rather cumbersome, usually by affinity chromatography steps, and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al, EMBO j,103655-3659 (1991).

Antibody variable domains (antibody-antigen binding sites) with the desired binding specificity can be fused to immunoglobulin constant domain sequences. The fusion is preferably to an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. Preferably, a first heavy chain constant region (CH1) is present in at least one of the fusions, this region containing the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain (and, if desired, immunoglobulin light chain) fusions is inserted into a different expression vector and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology121:210(1986)。

According to different methods, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. The fusion is preferably of an immunoglobulin heavy chain constant region, including at least a portion of the hinge, the CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments in an embodiment, when unequal ratios of the three polypeptide chains are used in the construction to provide optimal yields. However, when expressing at least two polypeptides in equal ratios results in high yields, or when the ratios are not particularly important, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

According to another approach described in WO 96/27011, the interface between antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains in the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), the same or similar sized "holes" are created at the interface of the second antibody molecule that compensate for the large side chains. This provides a mechanism for increasing the yield of heterodimers relative to other unwanted side products (e.g., homodimers).

In a preferred embodiment of the method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. This asymmetric structure was found to be advantageous for the isolation of ideal bispecific compounds from unwanted immunoglobulin chain combinations, since the presence of immunoglobulin light chains in only half of the bispecific molecules provides an easy to implement way of isolation. This method is disclosed in WO 94/04690 published 3.3.1994. For additional details on the generation of bispecific antibodies, see, e.g., Suresh et al, Methods in Enzymology,121:210(1986)。

bispecific antibodies comprise cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO 91/00360, WO 92/200373). Heteroconjugated antibodies can be made using any convenient crosslinking method. Suitable crosslinking agents are generally known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a variety of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. The following techniques may also be usedBivalent antibody fragments that are not necessarily bispecific are produced. For example, Fab' fragments recovered from e. See Shalaby et al, j.exp.med,175:217-225(1992)。

bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science22981(1985) describes procedures in which intact antibodies are proteolytically cleaved to yield F (ab')₂And (3) fragment. These fragments are reduced in the presence of dimercaptoalcohol complexing reagent sodium arsenite to stabilize the vicinal dimercapto and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. Then, one Fab '-TNB derivative is converted into another Fab' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes.

Coli can be directly recovered and chemically coupled to form bispecific antibodies. Shalaby et al, j.exp.med,175217-225(1992) describes fully humanized bispecific antibodies F (ab')₂And (3) producing molecules. Coli, and direct chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques have also been described for the production and isolation of bivalent antibody fragments directly from recombinant cell cultures. For example, a leucine zipper has been used to produce bivalent heterodimers. Kostelny et al, j. immunol.,148(5):1547-1553(1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Reducing the antibody homodimers in the hinge region to form monomers, thenAnd then oxidized to form antibody heterodimers. Hollinger et al, Proc. Natl. Acad. Sci. USA,906444-. The fragments comprise a light chain variable domain (V) joined by a linker_L) Linked heavy chain variable domains (V)_H) The linker is too short to allow pairing between the two domains of the same strand. Thus, V of a segment_HAnd V_LThe domains are forced to complement the V of another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific/bivalent antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, j.immunol.,152:5368(1994)。

antibodies with more than two titers are contemplated. For example, trispecific antibodies may be prepared. Tutt et al, j.147:60(1991)。

An exemplary bispecific antibody can bind to two different epitopes on a given molecule. Alternatively, an anti-protein arm may be combined with an arm that binds a trigger molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or an Fc receptor of IgG (fcyr), such as fcyri (CD64), fcyrii (CD32), and fcyriii (CD16), such that the cellular defense mechanisms are focused on cells expressing a particular protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing a particular protein. Such antibodies have a protein-binding arm, and an arm that binds a cytotoxic agent or radionuclide chelator (e.g., EOTUBE, DPTA, DOTA, or TETA). Another bispecific antibody of interest binds to a protein of interest and further binds to Tissue Factor (TF).

(vi) Heteroconjugate antibodies

Heteroconjugated antibodies also fall within the scope of the invention. Heteroconjugated antibodies comprise two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection. WO 91/00360, WO 92/200373 and EP 03089. It is contemplated that the antibodies, including those involving cross-linking agents, may be prepared in vitro using any method known in synthetic protein chemistry. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include thiolimine (iminothiolate) and methyl-4-mercaptobenzimidate, and are disclosed, for example, in U.S. Pat. No. 4,676,980.

C. Protein purification, including antibodies

When the target polypeptide is expressed in a recombinant cell derived from a source other than human, the target polypeptide is completely free of a protein or polypeptide derived from human. However, the target polypeptide must be purified from the recombinant cellular protein or polypeptide to obtain a substantially homogeneous preparation of the target polypeptide. As a first step, the culture medium or lysate is typically centrifuged to remove particulate cell debris. The membrane and soluble protein fraction are then separated. The target polypeptide is then 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 procedures are examples of suitable purification procedures: fractionation based on immunoaffinity or ion exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on cation exchange resins (e.g. DEAE); carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and a protein a sepharose column to remove contaminants, such as IgG.

Most companies currently produce monoclonal antibodies (MAb) using a three-column platform method comprising protein a affinity chromatography for product capture, anion exchange chromatography followed by flow-through (flow-through) mode, extraction of negatively charged contaminants such as Host Cell Proteins (HCP), endotoxins, host DNA and leaked protein a, followed by cation exchange chromatography or Hydrophobic Interaction Chromatography (HIC) in retention mode to remove positively charged contaminant types, including residual HCP and product aggregates.

Viruses that may be present in the protein solution are larger than the protein itself. It is thus assumed that viruses can be removed from proteins by filtration, depending on size.

Viral filtration can remove larger viruses, such as retroviruses (80-100nm diameter), typically using high-throughput membranes with a nominal pore size of about 60 nm. Since high-throughput membranes with nominal pore size of 20nm are also commercially available, it is possible to remove smaller viruses, such as parvovirus (18-26nm diameter), by filtration while allowing passage of proteins as large as 160kD (. about.8 nm), such as monoclonal antibodies. The present invention is primarily intended to solve the problems associated with the use of smaller pore size virus removal filters, and with such smaller virus filtration.

Typically, the virus filtration step may be performed at any of several nodes in a given downstream process. For example, in a typical monoclonal antibody purification process, virus filtration may be performed after a low pH virus inactivation step, or after an intermediate column chromatography step, or after a final column chromatography step.

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

The experimental setup used in the experiments disclosed herein is illustrated in fig. 1. However, it should be emphasized that the invention is not so limited. Other devices commonly known in the art are also suitable and may be used in the methods of the present invention.

In tangential flow viral filtration, the protein solution is typically pumped to the retentate side at about a constant flow rate. The pressure differential created across the virus removal filter causes the protein solution to permeate through the filter while the virus remains on the retentate side.

In the case of so-called "normal-flow" or "dead-end" viral filtration, the same viral filter used in tangential viral filtration can be used, but the surrounding instrumentation and operating procedures are simpler and less expensive than in the case of tangential flow viral filtration. Thus, in principle, "normal flow" filtration involves placing the macromolecule-containing solution in a pressure vessel prior to filtration and forcing the solution through a virus removal filter with the aid of a pressure source (suitably nitrogen or air). Alternatively, a pump can be used on the retentate side to filter the liquid through the virus removal filter at a predetermined flow rate.

The degree of sensitivity (fineness) of a filter is generally expressed in terms of pore size or approximate molecular weight (relative molecular weight) of the molecules retained by the filter, and thus the molecular weight is referred to as trapped.

Virus filters are known in the art and are supplied by Millipore of Massachusetts, USA and asahi chemical Industry co. Suitable parvovirus retention filters includePro(Millipore Corp.,Billerica,MA)。Pro membranes have asymmetric bilayer structures and are made from Polyethersulfone (PES). The membrane structure is designed to retain viruses with a size greater than 20nm while allowing proteins with a molecular weight of less than 180kDa to permeate through the membrane. Other suitable filters for removing parvoviruses (including parvoviruses) from protein solutions include Novasip^TMDV20 and DV50 virus removal filters (Filter capsules) (Pall Corp., East Hills, NY),CPV, PLANOVA 20N (Asahi Kasei), and BioEX (Asahi Kasei). The Novasip DV20 gradient membrane filter removes parvovirus and other viruses as small as 20nm from up to 5-10 liters of protein solution using an Ultipor VF-grade DV 20-grade folded membrane cartridge (multiplexed membrane cartridge). The Novasip DV50 stage membrane filter integrates an Ultipor VF DV50 stageMembrane sleeve for removal of viruses 40-50nm and larger. Supplied byThe novasippultipor VF membrane filter is sterile and may also be gamma-irradiated.CPV utilizes a double layer of polyethersulfone asymmetric membrane, retaining more than 4 logs of parvovirus and 6 logs of retrovirus.

The prefiltering of the feed solution has a surprising effect on the filtration performance. The pre-filtration is typically aimed at removing impurities and contaminants, such as protein aggregates, DNA and other trace materials, that may cause fouling of the virus filter.

According to the present invention, a significant enhancement of the efficiency of the virus filter can be achieved by a pre-filtration step, including the use of cation exchange and endotoxin removal matrices. In this context, the term "medium" or "matrix" is used to cover any means for performing the cation exchange and endotoxin removal steps, respectively. Thus, the term "cation exchange media" specifically includes, but is not limited to, cation exchange resins, matrices, adsorbents, and the like. The term "endotoxin removal media" includes, but is not limited to, any positively charged membrane surface, including, for example, chromatographic endotoxin removal matrices, endotoxin affinity removal matrices, and the like.

Suitable cation exchange media for use in the prefiltration step of the present invention include, but are not limited toS、S、Shield、SPFF、SPXL、S、50 HS、S、D, etc., are commercially available.

Endotoxin removal matrices suitable for use in the prefiltration step of the present invention include, but are not limited toE、Q、Q、Q、QSFF、Q、Q, etc., are all commercially available.

The prefiltration step may be carried out, for example, by using the chromatography liquid (pool) to be treated and treating said liquid on a filtration train (filtrationtrain) comprising an endotoxin removal and cation exchange matrix and a parvovirus filter. The endotoxin removal and cation exchange matrix functions as a prefiltration step, the capacity of the parvovirus filter being independent of the order of the two steps in the filter train. The filter chain may work continuously as a single step or may be operated as different units of operation. For example, in one embodiment, the chromatography liquid is first passed through an endotoxin removal matrix, then the collected liquid is passed through a cation exchange matrix, and then the liquid is filtered with a parvovirus filter. As mentioned above, the order of application of the cation exchange matrix and endotoxin removal matrix in the treatment sequence does not affect the parvovirus filtration capacity. The treatment can be operated over a wide pH range, for example in the pH range of 4-10, with the optimum filtration capacity depending on the impurity composition and product properties aimed at. Similarly, the protein concentration can be varied over a wide range, e.g., 1-40g/L, without limiting the mass flux of the parvovirus filter.

The present invention will be more fully understood by reference to the following examples. The described embodiments should not be viewed as limiting the scope of the invention. All citations throughout the disclosure are expressly incorporated herein by reference.

Examples

Materials and methods

1. Protein solution

Since virus filtration during downstream processing of monoclonal antibodies is performed after affinity chromatography (capture step) and ion exchange (refining) step, all filtration experiments are performed with commercially relevant ion exchange (cation or anion exchange) chromatography liquids to be processed. The mAb concentration and the conductance of the cation exchange solution and the conductance of the anion exchange solution were 10mg/ml and 10mS/cm, and 8mg/ml and 4mS/cm, respectively. Filtration experiments were performed with either fresh feedstock (used within 24 hours after production), or with feedstock frozen at-70 ℃ after production and thawed at 4-8 ℃ before use. No significant differences were found in the results obtained with fresh or freeze-thawed material. The protein concentration was determined by measuring the absorbance at 280nm using a UV-vis spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE).

2. Film

By usingPro (Millipore Corp., Billerica, MA) parvovirus retention filter filtration experiments were performed.Pro membranes have an asymmetric bilayer structure and are made from Polyethersulfone (PES). The membrane structure is designed to leave viruses larger than 20nm in size while allowing proteins with molecular weights less than 180kDa to permeate through the membrane. Evaluated in this studyPrefiltration of Pro comprisingOptiscale 40 depth filters (Millipore Corp., Billerica, MA), Fluorodyne EX Mini 0.2 μm sterile filters (Pall Corp., East Hills, NY) andfamily of membrane adsorbents (Pall corp., East Hills, NY). The membrane adsorbent is completely encapsulatedUnits, obtained from a vendor. Table 1 summarizes the key properties (functional groups, bed volume, pore size, etc.) of all prefilters used in this study.

Table 1: key Properties of the Pre-Filter

3. Experimental setup

The filtration experiment was carried out using a custom-made device shown in fig. 1. The loading material (i.e., the mAb fluid to be processed) was placed in the loading reservoir and filtered through a filter train consisting of a combination of different prefilters and commercially available parvovirus filters. In all filtration experiments, a constant filtration flow rate (P) was used_max) Square blockThe method is carried out. Pressure sensors were placed upstream of each filter and coupled to Millidaq or Netdaq systems to record different pressure data as a function of time or mass flux. The filtrate from the parvovirus filter was collected in a reservoir that was kept on the loading chamber to record mass flux as a function of time.

Results and discussion

Downstream purification of mabs expressed in mammalian cell cultures typically utilizes centrifugation and depth filtration as the first step to remove cells and cell debris, followed by affinity chromatography for mAb capture and removal of Host Cell Proteins (HCPs), followed by cation exchange chromatography, virus filtration, and anion exchange chromatography to further remove impurities such as aggregates, viruses, leaked protein a, and HCPs. Most of the experiments in this study were performed with cation exchange solution, with cation exchange chromatography as the second chromatography step.

FIG. 2 shows the use of a therapeutic mAb feed stream and different prefilters at 200L/m²Experimental data at constant flow,/hr, different pressures across Viresolve Pro. The X-axis represents the mass of mAb loaded per square meter of virus filter. The Y-axis represents the differential pressure across the virus filter as a function of mass flux. The data clearly show that the depth filter provides several orders of magnitude increase in virus filtration capacity compared to the sterile filter. Bolton et al (Bolton et al, appl. biochem.43:55-63, 2006) made similar observations as they evaluated ViresolvePrefilter^TMA depth filter membrane matrix as a prefilter of NFP parvovirus retention filters (Millipore Corp.) for polyclonal IgG solutions. The authors attribute the increase in capacity to the selective adsorption of the fouling agent, denatured protein, due to hydrophobic interactions.

Although deep filtration membranes have traditionally been used successfully to clarify cell culture fluids, there are considerable limitations worth additional consideration when used in downstream capture steps, such as prefilters as parvovirus retention filters.

(a) The depth filter is not alkaline stable, preventing the disinfection of the processing chain after installation, resulting in open processing and potential bioburden growth.

(b) The composition of the depth filtration membrane comprises diatomaceous earth as a key component, which is generally food grade and presents quality concerns.

(c) Diatomaceous earth is generally of natural origin-lacking well-defined chemistry-and thus can have batch variations.

(d) Depth filters also tend to leak metals, beta-glycans, and other impurities, the removal of which needs to be confirmed and validated using downstream operations.

The above limitations place additional burdens on the process and therefore require the design of operating units downstream of the depth filtration membrane to provide proper removal of the leachable species. However, even if the desired leak clearance is met, consideration is given to the fact that a particular batch of depth filtration membranes may have leaks that are significantly higher than the process is capable of clearing, since the key components are naturally derived, in other words, they lack a well-defined chemical synthesis process.

Thus, there is significant interest in developing prefilters that do not suffer from these limitations. As described above, Brown et al (Brown et al, IBC's 20)^thAntibody Development and Production, San Diego, CA, 2008) recently showed that a strongly negatively charged ion exchanger Mustang S can increase the capacity of a parvovirus retention filter several times when used as a prefilter. Thus, experiments were conducted to evaluate different pairs of prefiltered mediaEffect of Pro. The experimental data at pH5.0 and 6.5 are shown in FIGS. 3(a) and (b). The data show that although at pH5.0, the cation exchange matrix exhibits a slight advantage over the endotoxin removal adsorbent, which advantage is lost at pH 6.5. Although the overall capacity was higher with both matrices than with the sterile filter (fig. 2); but still understandThere is a significant shortage of capacity required to successfully perform a unit operation on a production scale.

Based on the hypothesis that both cation exchange and endotoxin removal matrices can remove two different foulants; both of these foulants can cause filter fouling, and experiments were designed with a new prefiltering chain that included both cation exchange and endotoxin removal matrices. The experimental results show that in fig. 4(a) and (b), the completion was at pH5.0 and pH 6.5, respectively. The data clearly show that the combination of the two matrices is clearly superior to each matrix by itself. For example, at pH5.0, the combination of cation exchange and endotoxin removal matrix provides a capacity improvement of more than an order of magnitude at a pressure differential of 20 psi. A similar trend was seen at pH 6.5, but the overall capacity was lower than that obtained at pH 5.0. This may be due to more efficient removal of impurities at lower pH.

The results of the experiment using mAb2 are shown in figure 5. Consistent with the data in fig. 4, the novel prefiltration chain containing both an endotoxin removal matrix and a cation exchange matrix substantially increased capacity, suggesting that the endotoxin removal matrix and the cation exchange matrix act synergistically to remove two different types of contaminants.

Conclusion

Most of the previous work focused on using depth filtration membranes or cation exchange membrane adsorbents as prefilters to increase the capacity of parvovirus retention filters. While depth filters provide a powerful mechanism for increasing virus filtration capacity, limitations associated therewith (e.g., leakage) limit their application to specific stages in downstream purification sequences. Cation exchange membrane adsorbents can increase the parvovirus filter capacity of some monoclonal antibody (mAb) feed streams, but as can be seen from the data in this study, they are not universally applicable, suggesting that multiple foulants may be present that need to be addressed to further improve parvovirus removal filter performance.

As demonstrated by the above experimental results, the present invention emphasizes two aspects-1) the endotoxin removal matrix itself can effectively increase the capacity of the parvovirus filter when used in prefiltering, and (2) the coupling of endotoxin removal and cation exchange matrices in the prefiltering chain can provide several times increased parvovirus filtration capacity, reduce raw material consumption and facilitate successful virus filtration operations at the production scale.

Claims (15)

1. A method for improving the filtration capacity of a virus filter in a downstream process of protein purification, consisting of subjecting a composition comprising a recombinant protein or antibody produced by a mammalian host cell to a cation exchange step and an endotoxin removal step simultaneously or in any order before passing through said virus filter, wherein the virus to be removed is a parvovirus, wherein the composition comprising the recombinant protein or antibody has been subjected to at least the following steps of a capturing step comprising affinity chromatography, and a refining step comprising cation chromatography and anion chromatography.

2. The method of claim 1, wherein the pore size of the virus filter is between about 15 and about 100nm in diameter.

3. The method of claim 2, wherein the pore size of the virus filter is between about 15 and about 30nm in diameter.

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

5. The method of any one of the preceding claims, wherein the parvovirus has a diameter of between about 18 and about 26 nm.

6. The method of claim 1, wherein the protein is an antibody or antibody fragment.

7. The method of any one of the preceding claims, wherein the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

8. A method for improving the filtration capacity of a viral filter during protein purification comprising subjecting a composition comprising a recombinant protein or antibody produced by a mammalian host cell to an endotoxin removal step prior to viral filtration followed by a cation exchange step.

9. A method for improving the filtration capacity of a viral filter during protein purification comprising subjecting a composition comprising a recombinant protein or antibody produced by a mammalian host cell to a simultaneous endotoxin removal step and cation exchange step prior to viral filtration.

10. The method of claim 8, wherein the endotoxin removal step is directly followed by viral filtration.

11. The method of claim 9, wherein the simultaneous endotoxin removal and cation exchange steps are followed directly by virus filtration.

12. The method of claim 1, wherein the virus filtration is performed at a pH between about 4 and about 10.

13. The method of claim 1, wherein the concentration of protein in the composition is about 1-40 g/L.

14. The method of any one of the preceding claims, wherein the antibody is directed 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.

15. The method according to any one of the preceding claims, wherein the antibody is selected from the group consisting of an anti-estrogen receptor antibody, an anti-progesterone receptor antibody, an anti-P53 antibody, an anti-cathepsin D antibody, an anti-Bcl-2 antibody, an anti-E-cadherin antibody, an anti-CA 125 antibody, an anti-CA 15-3 antibody, an anti-CA 19-9 antibody, an anti-c-erbB-2 antibody, an anti-P-glycoprotein antibody, an anti-CEA antibody, an anti-retinoblastoma protein antibody, an anti-ras oncoprotein antibody, an anti-Lewis X antibody, an anti-Ki-67 antibody, an anti-PCNA antibody, an anti-CD 3 antibody, an anti-CD 4 antibody, an anti-CD 5 antibody, an anti-CD 7 antibody, an anti-CD 8 antibody, an anti-CD 9/P24 antibody, an anti-CD 10 antibody, an anti-CD 11c antibody, an anti-CD 13 antibody, an anti-CD 14 antibody, an anti-CD 15 antibody, an anti-CD 19 antibody, an anti-CD 23 antibody, an anti-CD 30 antibody, an anti-CD 39, anti-CD 35 antibody, anti-CD 38 antibody, anti-CD 41 antibody, anti-LCA/CD 45 antibody, anti-CD 45RO antibody, anti-CD 45RA antibody, anti-CD 39 antibody, anti-CD 100 antibody, anti-CD 95/Fas antibody, anti-CD 99 antibody, anti-CD 106 antibody, anti-ubiquitin antibody, anti-CD 71 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.