HK1184741B - Method for purifying active polypeptides or immunocojugates - Google Patents

Description

Method for purifying active polypeptide or immunoconjugate

Background

Technical Field

The present invention provides methods for purifying a polypeptide or immunoconjugate from a solution comprising the active polypeptide or immunoconjugate and an acidic variant thereof, wherein the acidic variant is a deamidated species of said polypeptide or immunoconjugate. The invention also provides formulations comprising such purified polypeptides or immunoconjugates.

Background

Large-scale, economical protein purification is a factor in the biopharmaceutical industry. Therapeutic proteins are typically produced using prokaryotic or eukaryotic cell lines that are engineered to express the protein of interest from a recombinant plasmid containing the gene encoding the protein. The isolation of a desired protein from a mixture of components and cellular byproducts delivered to cells to a purity sufficient for use as a therapeutic agent (e.g., sufficient for use as a human therapeutic) presents a significant challenge to bioproduct manufacturers for a number of reasons.

Manufacturers of protein-based drug products must comply with strict regulatory standards, including extremely stringent purity requirements. To ensure safety, regulatory agencies, such as the U.S. Food and Drug Administration (FDA), require that protein-based pharmaceutical products be substantially free of impurities, including product-related contaminants (e.g., aggregates), fragments and variants of the recombinant protein, and process-related contaminants (e.g., host cell proteins, media components, viruses, DNA, and exotoxins). Although different protein purification schemes are available and widely used in the biopharmaceutical industry, they typically include an affinity purification step, such as protein a purification for antibodies, to achieve a pharmaceutically acceptable purity.

The creation of a purification scheme applicable to a particular biomolecule or different biomolecules, which is scalable, controllable, and strategically used with a particular resin or combination of resins, should allow its integration into the product formation process at a very early stage in the overall drug formation process. Designing a purification scheme approach can minimize costly changes to the manufacturing process that may otherwise be subsequently necessary in drug development or worse after approval. As the process becomes scaled and GMP production conditions are reached, additional inherent complications arise, including complications associated with resin packaging and buffer preparation. The manufacturing process and its capabilities can be improved by: simplifying the purification scheme, eliminating process steps, and maximizing yield and productivity while maintaining the integrity and purity of the molecules being purified. It is therefore desirable and advantageous to start with a simple and efficient process that can produce a drug substance of high quality and high safety.

One of the complications associated with the purification of pharmaceutical products is maintaining potency throughout the purification process. Many factors can contribute to the reduction or inhibition of efficacy, including modification of the drug product during development. Such modifications can occur at various stages of the process, for example when the protein is expressed in a cell, or when a protein that has been isolated from a cell is subjected to various conditions or buffers. The present invention provides a method for purifying the active polypeptide or immunoconjugate from a solution comprising a modified variant of the polypeptide or conjugate, wherein the presence of such modified variant results in an inhibition of the potency of the final drug product.

Summary of The Invention

The present invention provides a method for purifying a polypeptide of interest from a solution comprising the polypeptide and an acidic variant (e.g., a deamidated variant).

In particular, the invention provides a method for purifying an active immunoconjugate, wherein the immunoconjugate is deamidated at one or more residues, and wherein such deamidation results in inhibition of potency of said immunoconjugate, the method comprising: (a) contacting the immunoconjugate with an anion exchange AIEX chromatography matrix; and (b) eluting the bound immunoconjugate from the AIEX chromatography matrix with a high salt buffer, thereby separating the active conjugate from the deamidated variant.

The invention also provides a method for producing a purified polypeptide from a solution comprising the polypeptide and an acidic variant of the polypeptide, wherein the acidic variant of the polypeptide results in inhibition of the potency of the polypeptide, the method comprising: (a) contacting the polypeptide with an anion exchange (AIEX) chromatography matrix, and (b) eluting the bound polypeptide from the AIEX chromatography matrix with a high salt buffer, thereby separating said polypeptide from the acidic variant and producing a purified polypeptide.

The invention further provides a method of producing a purified polypeptide or immunoconjugate from a solution comprising the peptide and an acidic variant of the polypeptide, the method comprising: (a) producing the polypeptide or immunoconjugate in a bacterial cell expressing the polypeptide or immunoconjugate; (b) isolating inclusion bodies comprising the polypeptide or immunoconjugate from the bacterial cells; (c) refolding the polypeptide or immunoconjugate isolated from these inclusion bodies; (d) contacting a composition comprising the polypeptide or immunoconjugate with an AIEX chromatography matrix; and (e) eluting the bound polypeptide or immunoconjugate from the AIEX chromatography matrix with a high salt buffer, thereby purifying the polypeptide or immunoconjugate from the solution.

In certain embodiments, the acidic variant is a deamidated variant. In other embodiments, between about 75% to about 99% of the acidic or deamidated variant is removed during purification, particularly about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

The AIEX matrices of the invention comprise an ion exchange group of a quaternary or tertiary amine, a quaternary amino (Q) group, and in certain embodiments, the AIEX matrix is a Q sepharose.

The polypeptides or immunoconjugates of the invention are eluted with a linear or stepwise salt gradient. In certain embodiments, the linear salt gradient is from about Tris/HCl, 150mM NaCl to Tris/HCl in pH8.0, about 300mM NaCl in pH8.0, from Tris/HCl, about 175mM NaCl to Tris/HCl in pH8.0, about 275mM NaCl in pH8.0, or from Tris/HCl, about 192mM NaCl to Tris/HCl in pH8.0, about 245mM NaCl in pH 8.0.

In one embodiment, the polypeptide or immunoconjugate of the invention comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a Fab, Fab ', F (ab ')2, Fd, single chain Fv or scFv, disulfide linked Fv, V NAR binding domain, IgNar, internal antibody (intrabody), IgG Δ CH2, minibody, F (ab ')3, tetrabody, triabody, diabody (diabody), single domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2, or scFv-Fc. In some embodiments, the antibody or antigen binding fragment binds to a cell surface receptor, e.g., the cell surface receptor is CD 22. In other embodiments, the antibody or antigen-binding fragment thereof comprises a VH and VL sequence, wherein the VH sequence is selected from the group consisting of seq id nos: 6 to 11, and the VL sequence is selected from the group consisting of SEQ ID NOs: 2 and 12 to 15.

In another embodiment, the polypeptide or immunoconjugate comprises a toxin, wherein the toxin is selected from the group consisting of: pseudomonas exotoxin, ricin, abrin, diphtheria toxin, and subunits thereof, as well as botulinum toxins a through F or variants, or derivatives thereof. In some embodiments, the pseudomonas exotoxin or variant thereof has an amino acid sequence selected from the group consisting of seq id no:16 to 22 SEQ ID NO. In a particular embodiment, the immunoconjugate is a CAT-8015 immunotoxin comprising the VH-PE38 subunit of SEQ ID NO 1 and the VL subunit of SEQ ID NO 2.

The invention also provides a composition comprising a purified immunoconjugate having between less than about 25% and about 1% deamidated species, wherein said immunoconjugate is purified by any of the methods described above. The composition may be present at less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the deamidated species. In certain embodiments, the composition is a pharmaceutical composition comprising a purified polypeptide or immunoconjugate and a pharmaceutically acceptable carrier.

The invention also provides a formulation comprising 25mM sodium phosphate, 4% sucrose, 8% glycine, 0.02% polysorbate 80(PS80), 1mg/mL CAT-8015 in pH 7.4. In other embodiments, the formulation is freeze-dried.

The invention also provides a method for modifying the biological activity of a polypeptide solution comprising a polypeptide and a deamidated variant, the method comprising separating the polypeptide from the deamidated variant by linear elution AIEX chromatography; and combining the purified polypeptide with the deamidated variant in a fixed amount so as to obtain the desired biological activity of the polypeptide solution.

Brief description of the drawings

FIG. 1, a graph depicting the Ion Exchange Chromatography (IEC) curve of CAT-8015 reference standard. The pre-peak of CAT-8015 represents the majority of inactive deamidated, or isodeamidated (iso-deamidated) CAT-8015, while the main peak contains the majority of active intact CAT-8015 immunoconjugate.

FIG. 2 is a graph depicting the relative percent potency of CAT-8015 as a function of the percent of pre-peaks in the sample.

FIG. 3, is a graph depicting the elution profile of CAT-8015 laboratory scale purification by Q sepharose HP chromatography. CAT-8015 was purified using Q sepharose HP. Most of the active intact CAT-8015 remains in fractions (fractions) D5, D7, and D9 (spanning the main peak from D3 to D12, as shown by the above peaks).

FIG. 4, SDS-PAGE analysis (laboratory scale purification) of QHP loading and eluate pool samples. Lane 1 corresponds to the QHP loading cell; lane 2 corresponds to the QHP eluate pool; and lane 3 corresponds to the reference standard CAT-8015.

FIG. 5, Large Scale purification of CAT-8015 by Q Sepharose HP chromatography. CAT-8015 was purified using Q sepharose HP. As shown in this figure and Table 3, most of the active, intact CAT-8015 remained in fractions 5,6 and 7.

FIG. 6, SDS-PAGE analysis (Large Scale purification) of QHP loading and pool samples. Lane 1 corresponds to the QHP loading cell; and lane 2 corresponds to the QHP eluate pool.

Figure 7, a graph depicting the percentage of pre-peaks in the HA product as a function of dissolution pH, as illustrated in example 6.

Detailed description of the invention

The present invention provides a method for purifying the active polypeptide or immunoconjugate from a solution comprising the polypeptide or immunoconjugate and an acidic variant thereof. In one embodiment, the acidic variant comprises a deamidated form of the polypeptide or immunoconjugate. Most deamidated variants elute prior to the intact polypeptide under salt gradient elution conditions, compared to the expected elution behavior of anion exchange columns. Details of these methods are provided herein.

The terms "polypeptide", "peptide", "protein" and "protein fragment" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers as well as to non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring as well as synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those amino acids that are subsequently modified, such as hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carbonyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. These analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a chemical structure that is different from a typical amino acid, but that is functionally similar to a naturally occurring amino acid. Negatively charged amino acids include aspartic acid (or aspartate) and glutamic acid (or glutamate). Positively charged amino acids include arginine, histidine and lysine.

The "composition" to be purified herein includes the polypeptide of interest as well as one or more impurities. The composition may be "partially purified" (i.e., has been subjected to one or more purification steps, e.g., by non-affinity chromatography as described herein or may be obtained directly from the host cell or organism producing the polypeptide (e.g., the composition may comprise harvested cell culture fluid).

The terms "polypeptide" or "polypeptide of interest" or "protein of interest" and "target protein" or "protein" are used interchangeably and refer to a protein or polypeptide, such as an antibody or immunoconjugate (as defined herein) to be purified from a mixture of proteins by a method of the invention, and other material such as an acidic variant of the polypeptide of interest.

An "acidic variant" is a variant of a polypeptide or immunoconjugate that is more acidic than the polypeptide of interest (e.g., as determined by cation exchange chromatography). An example of an acidic variant is a deamidated variant.

Deamidated proteins are proteins in which some or all of their free amide functional groups are hydroxylated to carboxylic acids, for example converting glutamine to glutamic acid. The rate of this reaction depends on the primary sequence, three-dimensional structure, pH, temperature, buffer type, ionic strength, and other solution properties. Importantly, deamidation introduces a negative charge to the molecule. As further explained below, this protein deamidation reaction may have a negative effect on protein activity.

As used herein, the terms "antibody" and "immunoconjugate" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), and antibody fragments described herein. The term "bispecific antibody" is intended to include any antibody which has two different binding specificities, i.e., which binds two different epitopes, which epitopes may be located on the same target antigen or, more commonly, on different target antigens.

Natural antibodies and immunoconjugates are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, and disulfidesThe number of linkages varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain at one end (V)_H) Followed by multiple constant domains. Each light chain has a variable domain at one end (V)_L) And a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light and heavy chain variable region domains (clothhia et al, j.mol.biol. [ journal of molecular biology ]]186,651-66,1985), Novotny and Haber, Proc. Natl. Acad. Sci. USA82,4592-4596 (1985)). Based on the composition of the heavy chains, five classes of human immunoglobulins are defined and designated IgG, IgM, IgA, IgE, and IgD. IgG class antibodies and IgA class antibodies are further divided into the following subclasses: namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2. The heavy chains in IgG, IgA, and IgD antibodies have three constant region domains, designated CH1, CH2, and CH3, and the heavy chains in IgM and IgE antibodies have four constant region domains CH1, CH2, CH3, and CH 4. Thus, a heavy chain has one variable region and three or four constant regions. Immunoglobulin structure and function are reviewed in the literature, e.g., Harlow et al eds, Antibodies: antibody Manual [ Antibodies: laboratory manual]Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).

The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to: fab, Fab ', F (ab')2, Fv and single chain Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies, which include antibodies other than those that may naturally occurAll other populations are identical except for mutations (which may be present in minor amounts). Monoclonal antibodies are highly specific and bind to a single antigen. Moreover, each monoclonal antibody is directed to a single determinant on the antigen, in contrast to polyclonal preparations which typically include different antibodies directed to different determinants (sites). An antibody "selectively binds" or "specifically binds" refers to the antibody reacting or associating with a site more frequently, more rapidly, for a longer duration, for a greater affinity, or a combination thereof, as compared to an alternative substance (including unrelated proteins). "selectively binds" or "specifically binds" refers to, for example, K to which an antibody binds to a protein_DIs at least about 0.1mM, but more often is at least about 1. mu.M. "selectively binds" or "specifically binds" often refers to K to which an antibody binds to a protein_DOften at least about 0.1 μ M or better, and at other times at least about 0.01 μ M or better. Due to sequence identity between homologous proteins of different species, specific binding energy can include antibodies that recognize tumor cell marker proteins of more than one species.

Antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to the sequence corresponding to an antibody derived from one particular species or belonging to a particular antibody type or subclass, while the remainder of the chain is identical to or homologous to the sequence corresponding to an antibody derived from another species or belonging to another antibody type or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. [ Proc. national academy of sciences ] USA81:6851-685 6855 (1984)).

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody comprising minimal sequences derived from non-human immunoglobulins. Typically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), e.g., mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some cases, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the recipient or donor antibody. These modifications were made in order to further improve antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally should also include a portion of at least one immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. For more details, see Jones et al, Nature [ Nature ]321:522-525 (1986); riechmann et al, Nature [ Nature ]332:323-329 (1988); and Presta, curr, Op, Structure, biol. [ structural biology ]2: 593-. See also the following review articles and references cited therein: vaswani and Hamilton, Ann. allergy, Asthma & Immunol. [ allergic Asthma and immunity ]1: 105-; harris, biochem. Soc. transactions [ Proc. Biochemical science ]23: 1035-; hurle and Gross, curr. Op. Biotech. [ Biotechnology ]5: 428-.

A "human antibody" is a substance that has an amino acid sequence that corresponds to the sequence of an antibody produced by a human and/or that has been made using any of the techniques disclosed herein for making human antibodies. The definition of human antibody specifically excludes humanized antibodies that include non-human antigen binding residues.

The term "immunoconjugate" or "conjugate" or "immunotoxin" as used herein refers to a compound or a derivative thereof that binds to a cell linking agent (e.g., an anti-CD 22 antibody or fragment thereof) and is defined by the following general formula: C-L-a, wherein C = cytotoxin, L = linker, and a = cell binding agent (e.g., anti-CD 22 antibody or antibody fragment). Immunoconjugates may also be defined by the formula in reverse order: A-L-C.

The term "cytotoxin" or "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioisotopes of Lu), chemotherapeutic agents such as methotrexate, doxorubicin, vinblastine (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes and fragments thereof (e.g., nucleolytic enzymes (nucleolytic enzymes)), antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof, as well as various anti-tumor or anti-cancer drugs disclosed below. Examples of cytotoxic agents include, but are not limited to: abrin, ricin, Pseudomonas Exotoxin (PE), Diphtheria Toxin (DT), botulinum toxin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically cause death through hepatotoxins. However, PE and DT can be modified to one form for use as an immunotoxin by: the native targeting component of the toxin (e.g., domain la of PE or B chain of DT) is removed and replaced with a different targeting moiety, such as an antibody.

In some embodiments, the toxin is a pseudomonas exotoxin. Pseudomonas aeruginosa exotoxin A (PE) is a very active monomeric protein (molecular weight 66 kD) secreted by Pseudomonas aeruginosa that inhibits protein synthesis (catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD to EF-2) in eukaryotic cells by catalyzing its ADP-ribosylation reaction to inactivate elongation factor 2 (EF-2).

The toxin contains three domains that act synergistically to cause cytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding. Domain II (amino acids 253-364) is responsible for translocation to the cytosol, while domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2, inactivating the protein and causing cell death. The function of domain Ib (amino acids 365-. See Siegel et al, J.biol.chem.264:14256-14261 (1989).

Pseudomonas Exotoxin (PE) as used in the present invention includes the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of the native PE and their cytotoxic fragments. Cytotoxic fragments of PE include fragments that are cytotoxic and that subsequently have or do not have proteolytic or other processing in the target cell (e.g., as a protein or precursor protein) include the cytotoxic fragments of PE including PE40, PE38, and PE 35. PE40 is a truncated derivative of PE, as previously described in the art. See Pai et al, Proc. Natl.Acad.Sci. [ Proc. Natl.Acad. ] USA,88:3358-62(1991); Kondo et al J.biol.chem. [ Biochemical journal ]263: 9470-. PE38 is a truncated PE consisting of amino acids 253-364 and 381-613 of the native PE. PE35 is a 35kD carboxy-terminal fragment of PE consisting of a Met at position 280 followed by amino acids 281-. In one embodiment, the cytotoxic fragment PE38 was used. PE38 is a precursor protein that can be activated to its cytotoxic form when processed in cells.

A "PE immunoconjugate" or "PE immunotoxin" is an immunoconjugate or immunotoxin comprising an antibody or antigen-binding fragment thereof and a PE toxin or variant thereof.

Reference to "purifying" a polypeptide or immunoconjugate from a composition comprising the polypeptide and one or more impurities refers to increasing the purity of the polypeptide in the composition by (completely or partially) removing at least one impurity from the composition. According to the invention, purification is carried out without using an affinity chromatography step.

The term "chromatography" refers to the process by which one solute of interest in a mixture is separated from other solutes in the mixture by the difference in the rate at which each solute of the mixture passes through a stationary medium under the influence of a mobile phase, or during binding and elution.

The terms "ion exchange" and "ion exchange chromatography" refer to chromatographic processes in which a solute of interest (e.g., a protein) in a mixture interacts with a charged compound attached (e.g., by covalent attachment) to a solid phase ion exchange material such that the solute of interest interacts with the charged compound non-specifically more or less than solute impurities or contaminants in the mixture. Contaminating solutes in the mixture elute from the column of ion exchange material faster or slower than the solute of interest, or bind to or are excluded from the resin relative to the solute of interest. "ion exchange chromatography" specifically includes cation exchange, anion exchange, and mixed mode chromatography.

The phrase "ion exchange material" refers to a solid phase that is negatively charged (i.e., cation exchange resin) or positively charged (i.e., anion exchange resin). Charge may be provided by attaching one or more charged ligands to the solid phase, for example by covalent attachment. Alternatively or in addition, the charge may be an inherent property of the solid phase (e.g., for silica gel, it bears an overall negative charge).

"anion exchange resin" refers to a solid phase that is positively charged, thereby having one or more positively charged ligands attached thereto. Any positively charged ligand, such as quaternary ammonium groups, attached to a solid phase suitable for forming an anion exchange resin may be used, commercially available anion resins including DEAEcellulose, Poros PI20, PI50, HQ10, HQ20, HQ50, D50 (from Applied Biosystems), Sartobind Q (from saikob Biosystems), etcDolich group (Sartorius)), MonoQ, MiniQ, Source15Q and 30Q, Q, DEAE, and ANXSepharose Fast Flow, Q Sepharose High Performance, QAE SEPHADEX^TMAnd FAST Q SEPHAROSE^TMGeneral medical company (GE Healthcare), WP PEI, WP DEAM, WP QUAT (from J.T. Baker), Hydrocell DEAE and Hydrocell QA (Biochrom Labs Inc.), UNOsphere Q, Macro-Prep DEAE and Macro-Prep High Q (from Burley (Biorad)), ceramic HyperD Q, ceramic HyperD DEAE, Trisacryl M and LS DEAE, SpheroexDEAE, QMA Spherosil LS, QMA Spherosil M and Mustang Q (from Pall Technologies), Millex Merge Base Type I and Type II anion resin and DOWEX MONOSHER E77, Weak Base anion (from Dow Liquid separation), Millex Melong Base Type III, Millex, and exchange ion TMAD 500, Mike, Michelle and 500 Michelle III), Weak Base anion resin and exchange ion TMAD, Michelle and Michelle ion E, DOWEX weak and strong anion exchange types I and II, Diaion weak and strong anion exchange types I and II, Duolite (from Sigma-Aldrich), TSK gel Q and DEAE5PW and 5PW-HR, toyopearSuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C (from Tosoh), QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q (from Wotman).

Reference to a "solid phase" refers to a non-aqueous matrix to which one or more charged ligands can adhere. The solid phase may be a purification column, a discontinuous phase of discrete particles, a membrane, or a filter, etc. Examples of the material forming the solid phase include polysaccharides (e.g., agarose and cellulose); and other mechanically stable matrices such as silica gel (e.g., controlled pore glass), poly (styrene divinyl) benzene, polyacrylamide, ceramic particles, and derivatives of the foregoing.

The term "specific binding" as used herein to describe the interaction between a molecule of interest and a ligand bound to a solid phase matrix generally means that the protein of interest reversibly binds to a ligand at a binding site to which electrostatic forces, hydrogen bonds, hydrophobic forces, and/or van der Waals forces are bound, through a combination of steric complementarity of the protein and the structure of the ligand. The greater the spatial complementarity and the stronger the other forces at the binding site, the greater the binding specificity of the protein to its corresponding ligand. Non-limiting examples of specific binding include antibody-antigen binding, enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation, DNA binding protein-DNA binding, regulatory protein-protein interactions, and the like.

The term "non-specific binding" as used herein to describe an interaction between a molecule of interest and a ligand or other compound bound to a solid phase matrix means that the protein of interest binds to the ligand or compound on the solid phase matrix at the site of the interaction by electrostatic forces, hydrogen bonding, hydrophobic forces, and/or van der waals forces, but lacks structural complementarity that enhances the effects of non-structural forces. Examples of non-specific interactions include, but are not limited to: electrostatic forces, hydrophobic forces, and van der waals forces along with hydrogen bonding.

A "salt" is a compound formed by the interaction of an acid and a base. Salts useful in the present invention include, but are not limited to: an acetate salt (e.g., sodium acetate), a citrate salt (e.g., sodium citrate), a chloride salt (e.g., sodium chloride), a sulfate salt (e.g., sodium sulfate), or a potassium salt.

The term "detergent" refers to ionic and nonionic surfactants, such as polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; octyl glucoside sodium (sodium octyiglucoside); lauryl-, myristyl-, linoleyl-, or octadecyl-sulfobetaine; lauryl-, myristyl-, linoleyl-orOctadecyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl (lauroamidopropyl) -, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g. lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate, or sodium oleyl methyl taurate (sodium diodiummethyl oleyl-taurate); and MONAQUAT^TMIn a series (Monana industries, Inc) Parteson, N.J.), a useful detergent is a polysorbate, such as polysorbate 20 (TWEEN)) Or polysorbate 80 (TWEEN)）。

As used herein, a "buffer" is a solution that resists changes in pH by the addition of an acid or base through the action of its acid-base conjugate components. Various buffers can be used in the present invention, depending on the desired pH of the buffer during purification and the particular procedure [ see buffers. age for the Preparation and Use of buffers in Biological Systems, Gueffroy, D.ed.Calbiochem Corporation (1975) ]. Non-limiting examples of buffer components that can be used to control a desired pH range in a method of the invention include acetate, citrate, histidine, phosphate, ammonium buffers such as ammonium acetate, succinate, MES, CHAPS, MOPS, MOPSO, HEPES, Tris, and the like, as well as combinations of: TRIS-malic acid-NaOH, maleate, chloroacetate, formate, benzoate, propionate, pyridine, piperazine, ADA, PIPES, ACES, BES, TES, tricine, bicine, TAPS, ethanolamine, CHES, CAPS, methylamine, piperidine, O-boric acid, carbonic acid, lactic acid, succinic acid, diethylmalonic acid, diglucidin, HEPPS, HEPPSO, imidazole, phenol, POPSO, succinate, TAPS, amine-based substances, benzylamine, trimethyl or dimethyl or ethyl or aniline, ethylenediamine, or morpholine (mophiline). If desired, additional components (additives) may be present in the buffer, for example salts may be used to adjust the buffer ionic strength, such as sodium chloride, sodium sulfate and potassium chloride; and other additives such as amino acids (e.g., glycine and histidine), chaotropes (e.g., urea), alcohols (e.g., ethanol, mannitol, glycerol, and benzyl alcohol), detergents (see above), and sugars (e.g., sucrose, mannitol, maltose, trehalose, glucose, and fructose). The buffer components and additives used, as well as the concentrations, may vary depending on the type of chromatography being performed in accordance with the present invention.

A "loading buffer" is a substance used to load a composition comprising a polypeptide molecule of interest and one or more impurities onto an ion exchange resin. The loading buffer has a conductivity and/or pH such that the polypeptide molecule of interest (and typically one or more impurities) binds to the ion exchange resin, or such that the protein of interest flows through the column while the impurities bind to the resin.

The term "wash buffer" as used herein refers to a buffer used to wash or re-equilibrate the ion exchange resin prior to eluting the polypeptide molecule of interest. Conveniently, the wash buffer and loading buffer may be the same, but are not necessarily required.

An "elution buffer" is used to elute the polypeptide of interest from the solid phase. The conductivity and/or pH of the elution buffer is such that the polypeptide of interest is eluted from the ion exchange resin.

The "pI" or "isoelectric point" of a polypeptide refers to the pH at which the positive and negative charges of the polypeptide are in equilibrium. The pI can be calculated from the net charge of the amino acid residues or sialic acid residues of the carbohydrate to which the polypeptide is attached or can be determined by iso-focusing.

Reference to "binding" a molecule to an ion exchange material refers to exposing the molecule to the ion exchange material under appropriate conditions (pH/conductivity) such that the molecule is reversibly immobile in or on the ion exchange material due to ionic interactions between the molecule and one or more charged groups of the ion exchange material.

Reference to "washing" the ion exchange material means passing a suitable buffer through or over the ion exchange material.

"eluting" a molecule (e.g., a polypeptide or an impurity) from an ion exchange material refers to removing the molecule from the ion exchange material by changing the ionic strength of a buffer surrounding the ion exchange material such that the buffer competes with the molecule for charged sites on the ion exchange material.

As used in this disclosure and the claims, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise.

It should be understood that when the language "comprises" is used to describe embodiments, other similar embodiments are also provided as described with respect to "consisting of … …" and/or "consisting essentially of … …".

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B," a or B, "" a, "and" B. Similarly, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; a and B; b and C; a (alone); b (alone); and C (alone).

Pseudomonas aeruginosa exotoxin and other toxins

Toxins may be used with the antibodies of the present invention to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin, and subunits thereof, as well as botulinum toxins a through F. These toxins may be obtained from commercial sourcesAre readily available (e.g., Sigma Chemical Company, St. Louis, Mo.). Diphtheria toxin was isolated from corynebacterium diphtheriae. Ricin is the lectin RCA60 from castor (castor bean). The term also refers to toxic variants thereof. See, for example, U.S. patent nos. 5,079,163 and 4,689,401. Ricinus Communis Agglutinin (RCA) exists in two forms, and is named RCA according to their approximate molecular weights of 65kD and 120kD₆₀And RCA₁₂₀（Nicholson&Blaustein, j. biochem. biophysis. acta journal of biochemistry and biophysics]266:543(1972)). The a chain is responsible for inactivating protein synthesis and killing cells. The B chain links ricin to cell surface galactose residues and facilitates transfer of the A chain into the cytosol (Olsnes et al, Nature [ Nature)]249:627-631(1974) and U.S. Pat. No. 3,060,165).

Abrin includes toxic lectins from abrin. The toxic major components abrin a, B, c and d have molecular weights from about 63kD to 67k and consist of two disulfide-linked polypeptide chains a and B. The A chain inhibits protein synthesis; the B chain (abrin B) binds to the D-galactose residue (see Funatsu et al, Agr. biol. chem. [ Agrochemical ]52:1095 (1988); and Olsnes, Methods Enzymol [ Methods of enzymology ].50:330-335 (1978)).

In a preferred embodiment of the invention, the toxin is pseudomonas aeruginosa exotoxin (PE). Pseudomonas aeruginosa exotoxin (or exotoxin a) is an exotoxin produced by pseudomonas aeruginosa. The term "pseudomonas aeruginosa exotoxin" as used herein refers to a full-length native (naturally occurring) PE or a modified PE. Such modifications may include, but are not limited to: elimination of the various amino acids in domains Ia, removal of domains Ib, II and III, single amino acid substitutions and addition of one or more sequences at the carboxy terminus, such as KDEL (SEQ ID NO: 3) and REDL (SEQ ID NO: 4). See Siegel et al J.biol.chem. [ journal of biochemistry ]264: 14256-. In a preferred embodiment, the cytotoxic fragment of PE retains at least 50%, preferably 75%, more preferably at least 90%, and most preferably 95% of the cytotoxicity of the native PE. In a most preferred embodiment, the cytotoxic fragment is more toxic than native PE.

The natural pseudomonas aeruginosa exotoxin a (pe) is a very active monomeric protein (molecular weight 66 kD) secreted by pseudomonas aeruginosa that inhibits protein synthesis in eukaryotic cells. Such native PE sequences are provided by commonly assigned U.S. patent No. 5,602,095, which is hereby incorporated by reference. The ADP ribosylation reaction of elongation factor 2 (EF-2) is inactivated. The exotoxin contains three domains that act in combination to cause cytotoxicity. Domain Ia (amino acids 1-252) mediates cellular binding. Domain II (amino acids 253-364) is responsible for translocation into the cytosol, while domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2. The function of domain Ib (amino acids 365- & 399) remains undetermined, although a large part of it (amino acids 365- & 380) can be deleted without loss of cytotoxicity. See Siegal et al, (1989), supra.

PE employed in the present invention includes the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE, as well as cytotoxic fragments thereof. PE variants for use in the present invention are described in US7,355,012, and WO2007/016150 and WO 2009/032954. Cytotoxic fragments of PE include those moieties that are cytotoxic, with or without subsequent proteolytic or other processing in the target cell (e.g., as a protein or precursor protein). Cytotoxic fragments of PE include PE40, PE38, and PE 35.

In a preferred embodiment, PE has been modified to reduce or eliminate non-specific cell binding, often by removal of domain Ia as taught in U.S. patent No. 4,892,827, although this may also be accomplished by, for example, mutating certain residues of domain Ia. For example, U.S. patent No. 5,512,658 discloses a mutant PE in which domain Ia is present, but in which the basic residues of domain Ia at positions 57, 246, 247 and 249 are replaced with acidic residues (glutamic acid, or "E"), exhibiting significantly reduced non-specific cytotoxicity. The mutated form of PE is sometimes referred to as PE 4E.

PE40 is a truncated derivative of PE, deleted for domain Ia of the native PE molecule as previously described in the art. See Pai et al Proc.Nat' l Acad.Sci.USA88:3358-62(1991) and Kondo et al J.biol.chem. [ journal of biochemistry ]263: 9470-. PE35 is a 35kD carboxy-terminal fragment of PE in which amino acid residues 1-279 have been deleted and the molecule starts with Met at position 280, followed by amino acids 281-364 and 381-613 of the native PE. For example, PE35 and PE40 are disclosed in U.S. Pat. nos. 5,602,095 and 4,892,827. PE4E is a form of PE in which all of the domains of native PE are present, but in which the basic residues of domain Ia at positions 57, 246, 247 and 249 are replaced by acidic residues (glutamic acid or "E").

In some preferred embodiments, the cytotoxic fragment PE38 is employed. PE38 is a truncated PE precursor protein consisting of amino acids 253-, 364 and 381-613, which is activated to a cytotoxic form during intracellular processing (see, e.g., U.S. Pat. Nos. 5,608,039,7,355,012, and Pastan et al, Biochim. Biophys. acta, Biochem. Biophys. acta]1333:C₁-C₆(1997)）。

As noted above, some or all of domains Ib may be deleted and the remainder linked by a linker or directly by a peptide bond. Some of the amino moieties of domain II may be deleted. Also, the C-terminus may comprise the native sequence (REDLK) (SEQ ID NO: 5) of residue 609-613, or may comprise a variant found to maintain the ability of the construct to translocate into the cytosol, such as REDL (SEQ ID NO: 4) or KDEL (SEQ ID NO: 3), as well as repeats of these sequences. See U.S. patent No. 5,854,044; 5,821,238, respectively; and 5,602,095 and WO 99/51643. However, in preferred embodiments, the PE is PE4E, PE40, or PE38, any form of PE in which non-specific cytotoxicity has been eliminated or reduced to a level where significant toxicity to non-targeted cells does not occur, may be used in the immunoconjugate of the invention so long as it is still capable of translocation and EF-2 ribosylation in the target cell.

Conservatively modified variants of PE

Conservatively modified variants of PE, or cytotoxic fragments thereof, have at least 80% sequence similarity, preferably at least 85% sequence similarity, more preferably at least 90% sequence similarity, and more preferably at least 95% sequence similarity at the amino acid level to the PE of interest (e.g., PE 38).

The term "conservatively modified variants" applies to both amino acid sequences and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to amino acid sequences which encode identical or essentially identical amino acid sequences, or essentially identical nucleic acid sequences if the nucleic acid does not encode a nucleic acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is codon-specific, the codon can also be changed to any of the corresponding codons described without changing the encoded polypeptide. Such amino acid changes are "silent changes," which are a class of conservatively modified changes. Each nucleic acid sequence herein that encodes a polypeptide also accounts for every possible silent change in the nucleic acid. The skilled artisan will recognize that each codon in a nucleic acid (except AUG, which is typically the only codon for methionine) may be modified to produce a functionally identical molecule. Thus, each silent change in a nucleic acid encoding a polypeptide is inherent in each illustrated sequence.

With respect to amino acid sequences, the skilled artisan will recognize that a single substitution, deletion, or addition (which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence) to a nucleic acid, peptide, polypeptide, or protein sequence is a "conservatively modified variant" wherein the alteration results in the substitution of an amino acid with a chemically similar amino acid.

The pseudomonas exotoxin employed in the present invention can be assayed for a desired level of cytotoxicity by assays well known to those of ordinary skill in the art. Thus, cytotoxic fragments of PE, as well as conservatively modified variants of such fragments, can be readily assayed for cytotoxicity. Most candidate PE molecules can be assayed for cytotoxicity simultaneously by methods well known in the art. For example, the subunit of the candidate molecule can be assayed for cytotoxicity. The positively reactive subunits of these candidate molecules may continue to be reclassified and re-assayed until the desired cytotoxic fragment or fragments are identified. Such methods allow rapid screening of a large number of cytotoxic fragments or conservative variants of PE.

anti-CD 22/PE immunoconjugates

In one embodiment, the polypeptide of interest comprises an antibody that specifically binds CD 22. "CD 22" refers to a restricted linked B cell antigen belonging to the Ig superfamily. It is expressed in 60% to 70% of B cell lymphomas and leukemias and is not present on the cell surface or on stem cells at the early stages of B cell development. See, e.g., Vaickus et al, Crit. Rev. Oncol/Hematol.11: 267-. In another embodiment, the polypeptide of interest is an antibody fragment (e.g., Fab or scFv) that binds CD 22.

As used herein, reference to an antibody, the term "anti-CD 22" refers to an antibody that specifically binds to CD22 and includes reference to an antibody raised against CD 22. In some embodiments, the CD22 is a primate CD22, such as human CD 22. In one embodiment, the antibody is raised against human CD22, which is synthesized by a non-primate mammal after introduction into an animal that encodes cDNA for human CD 22. In another embodiment, the polypeptide of interest is a CD22 antibody immunoconjugate comprising PE38 exotoxin.

Method for preparing immunoconjugates of CD22/PE38One example is CAT-8015 described in International patent application publication Nos. WO98/41641 and WO2003/27135, U.S. Pat. Nos. 7,541,034, 7,355,012, and U.S. Pat. No. 2007/0189962, all of which are incorporated herein by reference. CAT-8015 is a recombinant immunotoxin protein, consisting of an antibody Fv fragment based on the murine anti-CD 22 antibody RFB4, RFB4 fused to a truncated form of the Pseudomonas aeruginosa exotoxin protein PE 38. The anti-CD 22Fv fragment consists of two domains V_LAnd V_H(ii) wherein the latter domain is modified so as to improve binding to the human CD22 target. CAT-8015 protein consists of two independent polypeptides V_LChain (SEQ ID NO: 2) and V_HChain (fused to PE38 Domain at C-terminus (V)_H-PE 38) (SEQ ID NO: 1)). Other V for the invention_LAnd V_HPE38 is described in U.S. Pat. Nos. 7,541,034, 7,355,012 and 2007/0189962. The two domains are designed to each contain engineered serine residues that allow for the formation of one intracellular disulfide bond. This feature increases the stability of the fusion protein.

V of CAT-8015_HThe amino acid sequence of the subunit P38 (SEQ ID NO: 1) is the following sequence:

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSAKASGG

PE38 sequences are shown in bold, V_HThe five amino acid linkers between the domains and the PE38 domain are underlined.

V of CAT-8015_LThe amino acid sequence of the subunit (SEQ ID NO: 2) is the following sequence:

MDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK(SEQ ID NO：2)

in other embodiments, the amino acid sequence of the VH domain of the immunoconjugate is one of:

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA(SEQ ID NO：6)

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGYNWGVLFAYWGQGTLVTVSA(SEQ ID NO：7)

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTTWGVLFAYWGQGTLVTVSA(SEQ ID NO：8)

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGSTYGVLFAYWGQGTLVTVSA(SEQ ID NO：9)

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA(SEQ ID NO：10)

MEVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSA(SEQ ID NO：11)

in another embodiment, the amino acid sequence of the VL domain of the immunoconjugate is one of:

MDIQMTQTTSSLSASLGDRVTISCRASQDIARYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK(SEQ ID NO：12)

MDIQMTQTTSSLSASLGDRVTISCRASQDIHGYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK(SEQ ID NO：13)

MDIQMTQTTSSLSASLGDRVTISCRASQDIGRYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK(SEQ ID NO：14)

MDIQMTQTTSSLSASLGDRVTISCRASQDIRGYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK(SEQ ID NO：15)

in certain other embodiments, the PE toxin of the immunoconjugate is a PE or variant thereof selected from the following:

natural PE

AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO：16)

PE40

GGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO：17)

PE38

GGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQID NO：18)

PE35

MWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO：19)

PE-LR

RHRQPRGWEQLPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO：20)

PE-LR-6X

RHRQPRGWEQLPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEEGGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWAGFYIAGDPALAYGYAQDQEPDAAGRIRNGALLRVYVPRSSLPGFYATSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEAGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDSEQAISALPDYASQPGKPPREDLK(SEQ ID NO：21)

PE-38(CAT-8015)

PEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK(SEQ ID NO：22)

The PE toxin of the immunoconjugate is fused or conjugated directly to a VH or VL domain or via a linker at the N-or C-terminus of the VH or VL domain. An example of a linker is described above for CAT-8015 and corresponds to the amino acid sequence KASGG (SEQ ID NO: 23). Additional linkers can be readily generated by techniques known in the art.

Expression of PE immunoconjugates

The PE immunoconjugates of the invention are expressed in cells, such as bacterial cells, and then isolated from inclusion bodies. Thereafter, the PE immunoconjugate isolated from the inclusion bodies was further purified using downstream purification steps.

Various host expression vector systems can be utilized to express the PE immunoconjugates of the invention. Such host expression systems represent vectors by which the coding sequence of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, express an antibody molecule of the invention in situ. These include, but are not limited to: microorganisms, such as bacteria (e.g., E.coli, Bacillus subtilis), plasmid DNA or cosmid DNA expression vectors transformed with recombinant phage DNA containing antibody coding sequences; yeast transformed with recombinant yeast expression vectors containing antibody coding sequences (e.g., saccharomyces, pichia); insect cell systems (e.g., baculoviruses) infected with recombinant viral expression vectors containing antibody coding sequences; plant cell systems infected with a recombinant viral expression vector containing antibody-encoding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) comprising recombinant expression constructs comprising promoters derived from the genome of a mammal (e.g., the metallothionein promoter) or from a mammalian virus (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

The DNA encoding the respective toxin (e.g., VH-PE 38) polypeptides of VL and VH-PE is introduced into an expression vector by techniques well known in the art.

"vector" refers to any vehicle used to clone and/or transfer a nucleic acid into a host cell. The vector may be a replicon to which it is possible to attach another DNA segment, such that replication of the attached segment occurs. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that acts as an autonomous unit of in vivo replication of DNA, i.e., is capable of replication under its own control. The term "vector" includes a vehicle for introducing the nucleic acid into a cell in vitro, or in vivo. A wide variety of vectors well known in the art can be used to manipulate the nucleic acid, integrate response elements and promoters (e.g., inducible promoters) into the gene, and the like. Possible vectors include, for example, plasmids, such as pBR322 or pUC plasmid derivatives, or Bluescript vectors. For example, insertion of a DNA fragment corresponding to a response element and a promoter into an appropriate vector can be accompanied by ligation of the appropriate DNA fragment into a selected vector having complementary binding ends. Alternatively, the ends of the DAN molecules may be enzymatically modified or an arbitrary site created by ligating nucleotide sequences (linkers) into the DNA ends. Such vectors may be engineered to contain selectable marker genes that provide for cell selection. Such markers allow for the identification and/or selection of host cells that express the protein encoded by the marker.

The term "expression vector" refers to a vector, plasmid or vehicle designed to express an inserted nucleic acid sequence after translocation into a host. The cloned gene (i.e., the inserted nucleic acid sequence), e.g., encoding anti-CD 22VH, anti-CH 22VL, or anti-CD 22VH or VL, fused to a PE toxin, is typically placed under the control of control elements (e.g., a promoter, minimal promoter, enhancer, or the like). Initiation control regions or promoters, which are used to drive expression of a nucleic acid in a desired host cell, are numerous and familiar to those of ordinary skill in the art. Essentially any promoter capable of driving expression of these genes can be used in the expression vector, including but not limited to: viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue-specific promoters, pathogenesis-or disease-related promoters, development-specific promoters, inducible promoters, light-regulated promoters; including but not limited to: SV40 early (SV 40) promoter region, promoters contained in the 3' Long Terminal Repeat (LTR) of Rous Sarcoma (RSV), E1A or Major Late Promoter (MLP) of adenovirus (Ad), Human Cytomegalovirus (HCMV) major immediate early promoter, Herpes Simplex Virus (HSV) Thymidine Kinase (TK) promoter, IE1 promoter, elongation factor 1 alpha (EF1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, pantothenic acid C (Ube) promoter, globulin promoter, regulatory sequences of mouse metallothionein-L promoter and transcriptional control region, broad-spectrum expression promoters (HPRT, vimentin, beta-actin, tubulin, and the like), promoters of intermediate fibers (desmin, neurofilament protein, keratin, GFAP, and the like), Promoters for therapeutic agents (MDR, CFTR or factor VIII types and the like), promoters associated with pathogenesis or disease-furthermore, these expression sequences can be modified by the addition of enhancers or regulatory sequences and the like.

The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases, a DNA sequence (including a coding sequence) is transcribed to form messenger rna (mrna). The messenger RNA is then translated to form a polypeptide product having the relevant biological activity. Moreover, the expression process may involve further processing steps to the transcription of the RNA product, such as splicing to remove introns, and/or post-transcriptional processing of a polypeptide product.

The VL and VH-PE38 polypeptides are expressed in a cell, for example a bacterial cell, such as E.coli. These polypeptides are expressed, for example, in E.coli and isolated from inclusion bodies. In certain embodiments, the VL and VH-PE38 subunits are expressed in different cells. For example, VL is expressed on a first vector in one cell, while VH-PE38 is expressed on a second vector in a different cell. The inclusion bodies from each cell line were recovered and lysed. In certain embodiments, the inclusion bodies are solubilized at a pH range of about 9.0 to about 10.5. In other embodiments, the inclusion bodies are solubilized at ph9.0, ph10.0, or ph 10.5. The solubilized VL and VH-PE38 inclusion bodies were combined to form an immunoconjugate comprising the VL and VH-PE38 subunits.

In other embodiments, the VL and VH-PE38 subunits are expressed on different vectors in the same cell, e.g., VL is expressed on a first vector in a cell and VH-PE38 is expressed on a different vector in the same cell. Inclusion bodies from the cells were recovered, lysed, and the VL and VH-PE38 subunits were combined to form an immunoconjugate. In certain other embodiments, the VL and VH-PE38 subunits are expressed on the same vector in the same cell.

Downstream chromatography steps were used to further purify this immunoconjugate.

Chromatographic conditions

As understood in the art, the loading, washing and elution conditions for chromatography of the present invention should depend on the particular chromatography media/ligand used. Of course, the process of the present invention incorporates the use of other protein purification methodologies such as salt precipitation, affinity chromatography, hydroxyapatite chromatography, reverse phase liquid chromatography, or other conventionally used protein purification techniques. However, it is contemplated that the process of the present invention should eliminate or significantly reduce the need for additional purification steps.

Anion exchange chromatography is also performed during the chromatographic separation of the polypeptide of interest. As is well known in the art, the anion exchanger may be based on different materials, taking into account the matrix and the attached charged groups. For example, the following matrices may be used, wherein the mentioned materials may be more or less cross-linked: agarose based (e.g. SEPHAROSE Fast)(e.g., Q-SEPHAROSE FF), and SEPHAROSE HighBased on cellulose (e.g. DEAE)) (ii) a Silica-based and synthetic polymer-based, or resins such as SuperQ-650 (from TOSOHBIOP) and Macro High Q (from BIO-RAD). For anion exchange resins, the charged groups covalently attached to the matrix can be, for example, Diethylaminoethyl (DEAE), Quaternary Aminoethyl (QAE), and/or quaternary ammonium (Q). In certain embodiments, the resin is selected from the group including, but not limited to: q Sepharose High Performance, QSepharofast Flow, DEAE Sepharose Fast Flow, Capto Q, Capto DEAE, Toyopearls SuperQ650(M), Toyopearls SuperQ650(S), Toyopearls DEAE650(M), Toyopearls DEAE650(S), TSKgel SuperQ-5PW (30), TSKgel SuperQ-5PW (20), TSKgel DEAE-5PW (30), TSKgel DEAE-5PW (20), EMDChemics: fractogel EMD DEAE (S), Fractogel EMD DEAE (M), Fractogel EMD DDMAE (S), Fractogel EMD DMAE (M), Fractogel EMD TMAE (S), Fractogel EMD TMAE (M), and Baker Bond XWP500PolyQuat-35, SPE. In one embodiment of the process, the anion exchange resin employed is Q-Sepharose

Although any of these resins can be used for small scale purification of antibodies, certain sizes of resins and lower cost facilitate production scale separations. If the size of the resin is too small, considerable back pressure is generated in the system. Furthermore, the amount of polypeptide that can be purified is limited. If the resin is too costly to make or purchase, it is not economically viable or operational for large scale purification applications.

Therefore, the resin used in the present invention must be of a certain size in order to provide effective scale-up without excessive cost. By "purification at the manufacturing level" is meant the purification of an antibody from a recombinant preparation on a scale that is satisfactory for commercial scale production. The resin used in the predetermined step should be the same as that used in the final protocol at the manufacturing level, since one may not easily predict the change in conditions necessary to separate these aggregates if the resin is changed. Certain resins used for small-scale or laboratory-scale purification may not be amenable to large-scale purification. Such resins for use in the present invention include: such as Q-SEPHAROSE HP. However, the skilled artisan will recognize other anion exchange resins for commercial scale production.

The resin column used when packing an anion exchange chromatography column is reflected by the dimensions of the column, i.e. the diameter of the column and the height of the resin, and varies depending on factors such as the amount of antibody in the applied solution and the binding capacity of the resin used.

The exchange resin may be equilibrated with a buffer prior to anion exchange chromatography. Any of a variety of buffers are suitable for equilibration with the exchange resin, such as sodium acetate, sodium phosphate, TRIS, phosphate, bis-TRIS, and L-histidine. One skilled in the art will appreciate that many other buffers may be used for equilibration so long as the pH and conductivity of the antibody solution used are about the same. When the "bind-wash" (bind-wash) procedure is performed, the equilibration buffer and wash buffer are the same. When performing the "bind-elute" process, the elution buffer may be made of one or more buffer substances in order to control the pH. The salt used is, for example, a high solubility salt, such as sodium chloride or potassium phosphate, but any salt that maintains the functionality of the antibody and allows the antibody monomer to be removed from the resin may be used.

In performing the "bind-elute" process, elution of the antibody monomer from the resin is performed with a substantially non-denaturing buffer having a pH and ionic strength sufficient to effectively elute the monomeric antibody, thereby recovering the antibody-containing eluate while leaving aggregates bound to the resin. In this context, effectively eluted means that at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% of the antibody loaded onto the resin is recovered. After ion exchange, only about 1.0%, preferably only 0.5%, most preferably less than 0.1% of the aggregates remain in the antibody preparation.

In one embodiment, the elution is performed as a gradient step elution. In another embodiment, the elution is performed according to a linear gradient.

Surprisingly, deamidated variants of these immunoconjugate proteins elute at higher salt concentrations despite a significant net increase in negative charge due to deamidation of one asparagine residue. Thus, these reduced potency variants are separated from the more active protein by ion exchange chromatography as described herein.

In certain embodiments of the invention, about 75% to about 99% of the acidic or deamidated variants present in the starting sample of the polypeptide or the immunoconjugate are removed during purification. In other embodiments, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the amidated variant is removed. Thus, a composition comprising the active polypeptide or immunoconjugate has at least between about 25% to about 1% deamidated species, e.g., less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

Deamidated variants of the invention include immunoconjugates comprising a PE toxin or variant thereof, wherein deamidation occurs at one or more residues in the immunoconjugate, e.g., at one or more residues in the PE toxin or variant thereof. In certain embodiments, deamidation occurs at 1,2, 3, 4, or 5 residues in the immunoconjugate. In other embodiments, the immunoconjugate comprising a PE toxin or variant thereof is deamidated at 1,2, 3, 4, or 5 residues in the PE toxin or variant thereof, e.g., at position 358 of SEQ ID NO:1, at position 495 of SEQ ID NO:16, at position 243 of SEQ ID NO:17, at position 227 of SEQ ID NO:18, at position 200 of SEQ ID NO:19, at position 212 of SEQ ID NO:20, at position 212 of SEQ ID NO:21, or at position 229 of SEQ ID NO: 22.

In one embodiment, the salt concentration of the elution buffer is high enough to displace antibody monomers from the resin without displacing aggregates. However, it will be appreciated that an increase in pH and lower salt concentration may be used in order to elute antibody monomers from the resin.

Any or all of the chromatographic steps of the present invention may be performed by any of the robot segments. For example, chromatography can be performed in a column. The column may be run from top to bottom or from bottom to top with or without pressure. During this chromatography, the direction of liquid flow in the column can be reversed. Chromatography may also be carried out using a batch process in which the solid medium is separated from the liquid used, so as to be loaded, washed and eluted by any suitable means, including gravity, centrifugation or filtration. Chromatography is also performed by contacting the sample with a filter that absorbs or retains certain molecules in the sample more strongly than others. In the following description, various embodiments of the present invention are described in the context of chromatography performed in a column. However, it should be understood that the use of a column is only one of several chromatography models that can be used, and the illustration of the invention using a column is not limited to the application of the invention to column chromatography, as those skilled in the art can also readily apply these teachings to other models, such as models using batch processes or filters.

A variety of different loading, washing and elution conditions may be used, as desired. In some embodiments, the initial loading conditions are altered such that the proteins (e.g., antibodies) eluted from the initial non-HT are applied directly to the HT column.

Elution can be achieved, for example, by changing the salt conditions in the liquid phase. For example, the salt and/or conductivity of the liquid phase is increased (linearly or distributed) to the point where the antibody elutes. Exemplary wash conditions include, for example, 10mM phosphate, ph6.7, where elution is achieved by increasing the salt concentration (e.g., to 10mM phosphate, 1.5M NaCl, ph 6.7) (distributed or in a linear fashion). All of the various embodiments or options described herein may be combined in any or all variations.

Prior to applying the sample to the column, the column may be equilibrated with the buffer or salt to be used for chromatographic analysis of the protein. As discussed below, chromatography (and loading of the protein to be purified) may occur in a variety of buffers or salts, including sodium, potassium, ammonium, magnesium, calcium, chloride, fluoride, acetate, phosphate, and/or citrate and/or Tris buffers. Citrate buffers and salts are preferred by those skilled in the art for ease of handling. Such buffers or salts may have a pH of at least about 5.5. In some embodiments, equilibration may occur in a solution comprising Tris or a sodium phosphate buffer. In some embodiments, equilibration occurs at a pH of at least about 5.5. Equilibration may occur at a pH between about 6.0 and about 8.6, preferably at a pH between about 6.5 and 7.5. Most preferably, the solution includes sodium phosphate at a concentration of about 25 millimolar and a pH of about 6.8.

The protein purification process of the present invention may be applied to remove an acidic variant from any protein. Some proteins specifically contemplated for use in the present invention include antibodies or variants thereof. Other proteins include, but are not limited to: recombinant fusion proteins comprising one or more constant antibody immunoglobulin domains, optionally an Fc portion of an antibody, and a protein of interest.

Formulations

Formulations of purified polypeptides or immunoconjugates are prepared by combining a purified polypeptide or immunoconjugate of The invention with a pharmaceutically acceptable carrier (e.g., carrier, excipient) for storage and use (Remington, The Science and Practice of pharmacy [ pharmaceutical Science and Practice ] version 20 mack publishing, 2000). Suitable pharmaceutically acceptable carriers include, but are not limited to: non-toxic buffers such as phosphate, citrate and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (for example, octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, for example methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, n-aspartic acid, histidine, arginine or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and nonionic surface activity, such as TWEEN or polyethylene glycol (PEG).

The antibody and/or immunoconjugate compositions of the invention (i.e., PE attached to the antibody) are particularly useful for parenteral administration, such as, for example, by intravenous administration or administration to the lumen of a body cavity or organ. Compositions for administration will generally comprise a solution of the antibody and/or immunoconjugate dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional, well known sterilization techniques. These compositions may contain pharmaceutically acceptable auxiliary substances as required under substantially physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium greening, calcium chloride, sodium lactate and the like. The concentration of the fusion protein in these formulations can vary widely and should be selected based primarily on the following conditions: fluid volume, viscosity, body weight, and the like to suit the particular mode of administration selected and the needs of the patient.

Thus, a typical pharmaceutical immunoconjugate composition for intravenous administration would be about 0.3 to about 50 μ g/kg per day, especially 20-50 μ g/kg per day of total treatment, with the dose preferably administered continuously or dispensed (allocate) three times per day. The actual method for preparing the administrable composition will be known or apparent to those of ordinary skill in the art and is described in more detail in publications such as Remington's Pharmaceutical Science 19the, Mack publishing company, Easton, Pa. (1995).

Compositions comprising the immunoconjugate of the invention may be administered for therapeutic treatment. In therapeutic applications, the composition is administered to a patient suffering from a disease in an amount sufficient to treat or at least partially arrest the disease and its complications. An amount sufficient to achieve the above is defined as a "therapeutically effective dose". The amount effective for this purpose should depend on the severity of the disease and the general health of the patient.

Single or multiple administrations of these compositions may be carried out, depending on the dosage and frequency as required and tolerated by the patient. In any case, the combination should provide a sufficient amount of the protein of the invention to effectively treat the patient. The dose may be administered three times every other day or continuously every other day for a period (e.g., 21 days), but may be applied periodically until a therapeutic effect is achieved or until side effects interrupt treatment. Generally, the dose should be sufficient to treat or ameliorate the symptoms or signs of the disease without unacceptable toxicity to the patient. An effective amount of the compound is one that provides subjective relief of one or more symptoms or an objectively identifiable improvement, as indicated by a clinician or other qualified observer.

In one embodiment, the immunoconjugate is formulated as a pharmaceutical composition comprising at least one acceptable excipient. A pharmaceutically acceptable CAT-8015 immunoconjugate formulation comprises 0.5mg/mL to 2.5mg/mL CAT-8015, typically 1.0mg/mL, 1.1mg/mL, 1.2 mg/mL, 1.3mg/mL, 1.4mg/mL, or 1.5mg/mL, in 25mM sodium phosphate, 4% sucrose, 8% glycine, 0.02% polysorbate 80(PS80), pH 7.4. In additional embodiments, the sodium phosphate can be in the range of 20mM to 100mM, 25mM to 50mM, or 25mM to 35 mM; sucrose may be at 2%, 3%, 4%, 5% or 6%; glycine may be in the range of 5% -10%, typically 5%, 6% or 7%; polysorbate 80 may be in the range from about 0.01% to about 1%, typically 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%; wherein the pH is in the range of 6.5 to 8.0, typically at pH7.2, 7.3, 7.4, 7.5 or 7.6. Other buffers known to those of ordinary skill in the art may also be utilized.

In certain embodiments of the invention, the formulation is freeze-dried. The term "freeze-drying" refers to any composition or formulation prepared in a frozen state under high vacuum conditions by rapid freezing and dehydration of the dried form. "lyophilization" or "freeze-drying" refers to the process of freezing and drying a solution. Lyophilized formulations or compositions are often ready for use as a preparation or reconstituted by the addition of sterile distilled water. In certain embodiments, the freeze-dried formulations of the present invention are reconstituted into vials.

For intravenous administration, the formulation of the invention, e.g., a liquid formulation or a formulation reconstituted from a lyophilized formulation, is placed in a vial with the immunoconjugate in the formulation present at the concentrations specified above. The formulation is removed from the vial and added to an Intravenous (IV) bag solution, wherein the IV bag contains from about 30mL to about 100mL of solution, typically 50mL, 60mL, 70mL, or 80 mL. A separate IV bag "protection solution" may also be added to the total volume of the IV bag, wherein the protection solution comprises an amount of polysorbate 80 such that the polysorbate 80 present in the final IV bag solution is in the range of 0.001% to about 3% polysorbate 80, typically in the range of about 0.01% to about 0.1%, and more typically 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%. The protective solution may be pre-formulated in a vial such that the concentration of polysorbate 80 is about 0.5% to about 5%, and may be 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%. The protective solution prevents the immunoconjugate or drug (e.g., CAT-8015) from being absorbed onto the contact surface of the IV bag, thereby preventing or inhibiting the immunoconjugate or drug from being affixed to the IV bag during administration and allowing the patient to receive an appropriate dose of the immunoconjugate or drug. The IV bag solution may be administered by infusion to a patient for varying durations, typically 30 minutes to 1 hour, typically 30 minutes.

The immunoconjugates and different uses of the formulations of the invention are encompassed in various disease states caused by specific human cells, which can be eliminated by the toxic action of the protein. One application of the immunoconjugates of the invention is for the treatment of a B cell malignancy or malignant B cells that express CD 22. Exemplary B cell malignancies include chronic B-lymphocytic cell (B-CLL), childhood acute lymphoblastic leukemia (pALL), Follicular Lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), non-hodgkin's lymphoma (NHL), and Hairy Cell Leukemia (HCL).

All documents, patents, journal articles and other materials cited in this application are hereby incorporated by reference.

Although the present invention has been fully described in connection with several embodiments thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims unless they depart therefrom.

Examples of the invention

Example 1 expression, recovery and Inclusion body isolation of CAT-8015

Fermentation of different cell lines containing the CAT-8015VL and CAT-8015VH-PE38 expression vectors was performed. The fermentate was harvested by continuous centrifugation. The fermentation harvest was passed through a continuous centrifuge at a rate of 0.5 to 0.8L per minute at 2-8 ℃ and centrifuged at a speed of about 15,000 rpm. After centrifugation, the cell pellet (paste) was frozen at 70 ℃ or less.

Following this treatment, the VH-PE38 and VL cell mass were thawed at 2 ℃ to 8 ℃ for 12 to 24 hours. The cells were lysed, releasing inclusion bodies comprising VL and VH-PE38 products. Subsequently, the resulting inclusion bodies are solubilized and V is obtained_HPE38 and V_LAnd (3) obtaining the product.

The product was concentrated to approximately 1mg/mL (as determined by Coomassie Brilliant blue Total protein assay) using a 30kDA ultrafiltration hollow fiber cartridge. The retentate is then diafiltered with 5 to 6 volumes of 20mM Tris, 100mM urea pH7.4 to achieve a conductivity of 2.5 to 3.0 mS/cm. The product was stored at 2 ℃ to 8 ℃ for up to 72 hours.

EXAMPLE 2 analytical Scale purification of active CAT-8015 by anion exchange chromatography on high Performance resin

V_HExpression of the P38 subunit results in the formation of a deamidated variant of the subunit. This amidation was found to occur in the PE38 portion of the immunoconjugate. V_HDeamidation of the-P38 subunit resulted in a decrease in the potency of the CAT-8015 protein. Surprisingly, the chromatographic conditions described below successfully removed the deamidated variant, thereby providing the ability to remove inactive species during purification. Because the deamidation occurs at the PE38 portion of the fusion construct, the chromatographic conditions can be used to remove any deamidated variant of a PE conjugate.

CAT-8015 was renatured from the isolated inclusion bodies and subsequently purified by a 4-column procedure. Table 1 provides a summary of the renaturation and purification unit operations.

TABLE 1-CAT-8015 product scheme

Step unit operation

FIG. 1 shows an analytical Ion Exchange Chromatography (IEC) curve for a reference standard sample of CAT-8015. As shown in the curve, the pre-peak occurs before the main peak elutes. The biological activity of CAT-8015 relative to a reference standard was tested on each fraction eluted in IEC using an apoptosis bioassay that measures the ability of test samples to induce dose-dependent apoptotic death of Daudi cell lines expressing the CD22 receptor. Once bound to CD22 and internalized, CAT-8015 induced apoptosis of Daudi cells via Caspase3/7, which can be measured by the Caspase-GloTM3/7 assay system. The potency of the assay sample is determined by dividing the EC50 of the assay sample by the 50% effective concentration of the reference standard (EC 50). The results of the apoptosis bioassay showed that the relative potency of CAT-8015 correlated with the percentage of pre-peaks of CAT-8015, as shown in FIG. 1. Figure 2 shows the correlation between this relative potency and the percentage of pre-peaks.

The pre-peak fraction and the main peak fraction from multiple IEC analyses were collected, pooled and subjected to peptide profile analysis and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. The results were compared to those obtained from peptide mapping and LC-MS/MS experiments with purified CAT-8015. Analysis of the purified CAT-8015 drug substance showed that Asn-358 was partially deamidated to Asp-358 and homo-Asp-358 (Table 2). Asp-358 and homo-Asp-358 were found to be significantly enriched in the pre-peak fraction, whereas the main peak fraction was enriched in intact CAT-8015 (Table 2). Taken together, these results show that deamidation at Asn-358 results in a loss of relative potency for a cell-based bioassay. The Asp-358 residue is present in the PE toxin portion of the immunoconjugate, thereby demonstrating that an immunoconjugate comprising a PE toxin or variant thereof, in which Asp-358 is present, should likely undergo deamidation and undergo a reduction in potency or viability.

Table 2:

amino acid 358 based on peptide mapping analysis and LC-MS/MS assignment of CAT-8015 drug substrate, pre-peak fraction and main peak fraction

Amino acids	Drug substance (%)	Front Peak (%)	Main Peak (%)
				N358	78.1	3.1	88.9
D358	11.9	44.0	2.2
				Same as-D358	10.0	52.9	8.9

D358= deamidated Asn-358; homo-D358 = homo-deamidated Asn-358; n358= Asn-358.

The isolation of deamidated CAT-8015 from intact CAT-8015 was achieved by anion exchange chromatography using strong ion exchange groups, such as Q (quaternary amino) coupled to small diameter resins such as Source15 (particle diameter: 15 μm; GE Healthcare) and Sepharose highPerformance (particle diameter: 34 μm; GE Healthcare). The use of small diameter chromatography resins in biological manufacturing processes is complicated by the significant back pressure generated under typical operating conditions, as defined by column geometry, flow rate, and buffer composition. Based on these considerations and the requirements of a High resolution chromatography step, Q Sepharose High Performance was selected for the separation of deamidated CAT-8015 from intact CAT-8015. Chromatographic conditions were developed that achieved high resolution while maintaining operability on different fabrication scales.

Example 3 laboratory Scale purification of CAT-8015

The column was first pre-equilibrated with 5 Column Volumes (CV) of buffer C (pre-equilibration/strip (strip) buffer: 10mM Tris/HCl, pH8.0, 1.0M NaCl) and then equilibrated with 5 CV of buffer A (equilibration buffer: 10mM Tris/HCl, pH 8.0) at a linear flow rate of 100 cm/hr. The chromatography resin was Q Sepharose HighPerform (QHP, GEHealthcare), in a Millipore Vantage column, 2.2cm 19.5cm, and run on AKTAExplorer. An intermediate purified product pool was prepared for loading onto the high performance anion exchange column using a 10kDa MWCO membrane by diafiltration with 10 volumes of buffer a. The diafiltration pool of hydrophobic interaction product was loaded onto a QHP column at a linear flow rate of 100cm/hr, followed by a 2-fold CV re-equilibration step with buffer a at the same flow rate. CAT-8015 was eluted with a linear gradient of 10 CV (from 35% buffer B (elution buffer: 10mM Tris/HCl, pH8.0, 500mM NaCl) to 55% buffer B) at a linear flow rate of 100 cm/hr. The eluted product was monitored at 280 nm. Fractions were collected and analyzed for% pre-peak by analytical Ion Exchange Chromatography (IEC). Fractions containing less than 25% of the pre-peak were pooled. The QHP cell was analyzed for% pre-peak by analyzing IEC on a strong anion exchange column. Relative potency was measured by apoptosis bioassay, as explained above.

At pH8.0, CAT-8015 binds strongly to the anion exchange resin, with no protein being detected by absorption at A280 in the fractions flowing through. After the first washing step with Tris/HCl, 175mM NaCl in pH8.0, CAT-8015 is eluted from the column with a linear salt gradient from 35% B (175 mM NaCl in Tris/HCl, pH 8.0) to 55% B (275 mM NaCl in Tris/HCl, pH 8.0). CAT-8015 eluted from the column between 39% B (192 mM NaCl in Tris/HCl, pH 8.0) and 49% B (245 mM NaCl in Tris/HCl, pH 8.0). FIG. 3 shows a QHP chromatographic curve of CAT-8015.

Fractions were analyzed by analyzing IEC. Table 3 shows the results of fractions eluting between 44.5% (223 mM NaCl) and 47.2% (236 mM NaCl).

Table 3:

IEC analysis of the collected QHP fractions

Fraction numbering	% front peaka	% main peak
			C12	41.0	59
D12	34.5	65.5
			D11	27.0	73
D9	17.6	82.4
			D7	15.9	84.1
D5	18.7	81.3
			D3	21.9	78.1

^aThe pre-peak contained greater than 90% deamidated CAT-8015.

Table 3 shows that anion exchange chromatography operating in a salt gradient elution mode can separate deamidated CAT-8015 from intact CAT8015 in an efficient manner.

Surprisingly, intact CAT-8015 eluted at a higher salt concentration, although the negative charge was clearly increased due to deamidation of an asparagine residue. The results are consistent with the chromatographic curves observed by IEC. Samples containing CAT-8015 were injected into an analytical anion exchange column (PL-SAX, Varian) equilibrated at pH8.0 with a Tris/HCl buffer system and eluted by one step and in combination with gradient elution steps (FIG. 1).

Fractions were pooled according to a pooling (pooling) standard of less than 25% of the pre-peak content. The QHP pool was analyzed for% pre-peak and relative potency by SDS-PAGE, analytical IEC and apoptosis bioassay. As shown in FIG. 4, SDS-PAGE analysis showed that the QHP load cell and eluted sample contained highly purified CAT-8015. However, QHP loading pools do not meet the target specification for purity by IEC and bioactivity. Purity and potency measurements of the QHP loading cell, as compared to the QHP eluate cell, as shown in table 4 below, show that the anion exchange chromatography step using QHP results in a significant increase in purity of IEC and relative potency by CAT-8015. The QHP loading pool was generated from the intermediate purification step II.

Table 4:

CAT-8015 purity as measured by IEC and bioactivity

Step (ii) of	% front peak	% main peak	Relative potency (%)
				QHP sample loading pool	53.8	46.2	52
QHP eluate pool	16.5	83.8	80

The QHP product pool was then diafiltered into formulation buffer to yield CAT-8015 drug substance.

Thus, manufacture of CAT-8015 drug substance requires isolation of deamidated CAT-8015 from active CAT-8015. Anion exchange chromatography using high performance resins (e.g., QHP) to isolate deamidated CAT-8015 from intact CAT-8015 and to increase bioactivity to target specifications are prerequisites for successful CAT-8015 drug substance performance.

EXAMPLE 4 Large Scale purification of CAT-8015

The column was first pre-equilibrated with 5 CV of buffer C (equilibration/stripping buffer: 10mM Tris/HCl, pH8.0, 1.0M NaCl) at a linear flow rate of 66cm/hr, and then equilibrated with 5 CV of buffer A at a linear flow rate of 76 cm/hr. The chromatography resin was Q Sepharose HighPerformance (QHP, GE Healthcare) run on a K Prime instrument in a BP300, 30cm x22cm bed. An intermediate purified product pool was prepared for loading onto the high performance anion exchange column by diafiltration with 10 volumes of buffer A (equilibration buffer: 10mM Tris/HCl, pH 8.0) using a 10kDa MWCO membrane. The diafiltration product pool was loaded onto a QHP column at a linear flow rate of 64cm/hr, followed by a 2-fold CV reequilibration step using buffer A at 76 cm/hr. CAT-8015 was eluted with a 10-fold CV linear gradient (from 35% buffer B (elution buffer: 10mM Tris/HCl, pH8.0, 500mM NaCl) to 55% buffer B) at a linear flow rate of 76 cm/hr. The elution of the product was monitored at 280 nm. Fractions were collected and analyzed for% pre-peak by analytical Ion Exchange Chromatography (IEC). Fractions containing less than 25% of the pre-peak were pooled. The QHP cell was analyzed for% pre-peak by analyzing IEC on a strong anion exchange column. Relative potency was measured by apoptosis bioassay.

Large scale anion exchange chromatography of CAT-8015 was performed as described above. Due to equipment limitations, QHP purification was performed at reduced flow rates. CAT-8015 is eluted from the column at a conductivity between 22.3mS/cm and 26.4 mS/cm. FIG. 5 shows a QHP chromatographic curve of CAT-8015 purified according to the method described above.

Fractions were analyzed by analysis of IEC. Table 5 shows the results of fractions eluting at a conductivity between 23.8 and 25.4 mS/cm. Table 5 shows that anion exchange chromatography performed according to a linear salt gradient elution mode can separate deamidated CAT-8015 from intact CAT-8015. Isolation of deamidated CAT-8015 from intact CAT-8015 occurs in a conductivity range of less than 2 mS/cm.

TABLE 5

IEC analysis of% Pre-Peak purity of collected fractions

Fraction (b) of	% front peak	% main peak
			1	55.1	44.9
2	39.0	61
			3	30.1	69.9
4	25.1	74.9
			5	18.7	81.3
6	14.7	85.3
			7	16.4	83.6

Fractions 4 through 7 were combined according to a pre-peak content convergence index of less than 25%. QHP pools were analyzed for% pre-peak and relative potency by SDS-PAGE and SEC, analytical IEC, and apoptosis bioassay. SDS-PAGE analysis, as shown in FIG. 6, shows QHP loading wells and highly purified CAT-8015 containing eluted sample. However, this QHP loading pool fails to meet the target specification by SEC, IEC, and relative potency versus purity. Purity and potency measurements of the QHP loading pool compared to the QHP eluate pool, as shown in tables 6 and 7 below, show that the anion exchange chromatography step using QHP results in a significant increase in purity of SEC, IEC and relative potency by CAT-8015. The QHP loading pool was generated from the intermediate purification step II.

TABLE 6

CAT-8015 purity as determined by SEC

Step (ii) of	% monomer	% aggregates	% others
				QHP sample loading pool	92.7	0.7	6.6
QHP eluate pool	99.0	1.0	0

TABLE 7

CAT-8015 purity as determined by IEC and bioactivity

Step (ii) of	% front peak	% main peak	Relative potency (%)
				QHP sample loading pool	50.3	49.7	51
QHP eluate pool	17	83	75

Subsequently, the QHP product pool was diafiltered into formulation buffer to produce CAT-8015 drug substance.

Examples 2 to 4 show the ability of deamidated CAT-8015 to be isolated from intact CAT-8015 using anion exchange chromatography on a resin (e.g., Q Sepharose High performance) and to increase its relative potency to meet the target specification (see tables 4 and 6). Deamidated CAT-8015 is distinguished from intact CAT-8015 by an additional negative charge. Most of deamidated CAT-8015 eluted prior to intact CAT-8015 under salt gradient elution conditions compared to the expected elution behavior of the anion exchange column (see tables 3 and 5). This unexpected elution pattern was observed under analytical scale, laboratory scale, and large scale anion exchange chromatography conditions. This elution pattern was unexpected because a linear high salt elution buffer would be expected to cause a higher negative charge for the variant. Thus, examples 2 to 4 show that using a linear elution buffer, a deamidated species can be removed from an active immunoconjugate using anion exchange chromatography. Separation of deamidated CAT-8015 from the completed CAT-8015 occurs within a specific conductivity range, enhancing the need for high resolution anion exchange resins, careful control of elution conditions, and testing of collected fractions during processing.

Example 5 modification of the biological Activity of CAT-8015 formulation

The efficacy of the CAT-8015 composition was calibrated by mixing a specific amount of the deamidated pre-peak with the main peak of activity. To obtain a composition containing a particular CAT-8015 potency, aliquots of the pre-peak product of CAT-8015 were combined with aliquots of the main peak product of CAT-8015 to arrive at a composition with a particular CAT-8015 potency. By controlling the level of efficacy of CAT-8015 in the composition, a CAT-8015 formulation was generated for administration of a desired dose of a particular volume of recombinant CAT-8015.

Example 6 adjustment of pH during solubilization results in a reduction of deamidated species, as measured after capture and intermediate purification steps

Although deamidated species can be removed from active immunoconjugates using a purification step as described above, the level of CAT-8015 deamidated species can also be effectively reduced by adjusting the pH at an early step in the purification process (i.e., the refolding step (step 4 in table 1 above)). Refolding operations to achieve lower levels of CAT-8015 deamidated species include the following sub-steps:

refolding substep 1: dissolution, clarification and concentration:VH-PE38 and VL inclusion bodies were thawed at room temperature (15-30 ℃) for 12 to 24 hours. VH-PE38 and VL inclusion bodies at 1:1 molarThe ratios were combined and adjusted to 15% (w/v) solids by adding 50mM Tris, 20mM EDTA, pH 7.4. These inclusion bodies were solubilized by adding 5kg of inclusion body solubilization buffer (50 mM ethanolamine, 8M urea, 0.5M arginine, 2mM EDTA, 10mM DTE) per kg of 15% (w/v) solid inclusion body suspension. The pH of the inclusion body solubilization buffer was varied between pH9.0 and 10.5, increasing in 0.5pH units. Dissolution was carried out at room temperature (15-30 ℃) for 2 hours with continuous stirring. Solubilized inclusion bodies were clarified by depth filtration through a series of filters. The clear filtrate was filtered by tangential flow to 5-6g/L and concentrated using a 5kDa Molecular Weight Cut Off (MWCO) ultrafiltration membrane.

Refolding substep 2: and (3) refolding:CAT-8015 refolding was initiated by 10-fold dilution of the clarified and concentrated inclusion body filtrate into a pre-cooled (2-8 ℃) refolding buffer (50 mM ethanolamine, 1M arginine, 2mM EDTA, 0.91mM oxidized glutathione, pH 9.5). The refolding solution was maintained at 2-8 ℃ for 48 to 72 hours with continued mixing. Refolding solution by concentrated and dialysis filtration before placed at room temperature (15-30C) and termination. The refold solution was concentrated by tangential flow filtration using a 10kDa MWCO membrane and diafiltered with 10 volumes of 20mM sodium phosphate, ph 7.4. The concentrated and diafiltered refold solution was filtered through a 0.2 μm filter (TMAE loading).

As part of the capture step (step 5 of Table 1 above), the CAT-8015 formulation obtained from the above refolding operation was loaded onto a Fractogel TMAE column (EMD Biosciences or equivalent) equilibrated with 20mM potassium phosphate, pH 7.4. After loading, the column was washed first with 20mM potassium phosphate, pH7.4, and then with 20mM potassium phosphate, 0.1% (w/w) Triton X100, pH7.4, followed by a subsequent wash with 20mM potassium phosphate, 100mM sodium chloride, pH 7.4. The product is eluted from the column in a reverse phase flow with 20mM potassium phosphate, 200mM sodium chloride, pH 7.4. The column was extracted with 2M sodium chloride (strip), sterilised with 1N sodium hydroxide and stored at room temperature in 0.1N sodium hydroxide.

As part of intermediate purification step 1, hydroxyapatite chromatography was performed. The hydroxyapatite chromatography step operates as a flow-through chromatography step. The product obtained from the above capture step was directly loaded onto a ceramic hydroxyapatite column (Bio-Rad Laboratories or equivalent) equilibrated with 400mM potassium phosphate, 200mM sodium chloride, pH7.4, followed by 20mM potassium phosphate, 200mM sodium chloride, pH 7.4. Under the chromatographic conditions, the product (HA product) was collected in the flow-through fraction. The column was extracted with 400mM potassium phosphate, 200mM sodium chloride, pH7.4, sterilized with 1N sodium hydroxide and stored at room temperature in 0.1N sodium hydroxide.

The percentage of the pre-peak in the HA product from above was analyzed by high performance anion exchange chromatography. Table 8 and figure 7 show the percentage of the pre-peak in the HA product as a function of the dissolution pH. As shown in table 8 and fig. 7, solubilizing VH-PE38 and VL inclusions at lower pH resulted in less deamidated CAT-8015 product. The ability to control the deamidation of CAT-8015 in early steps in the renaturation and purification process can increase the overall process yield while maintaining the quality of the final purified drug substance.

TABLE 8

Percentage of pre-peak in HA product as a function of dissolution pH

Dissolution pH	9.0	9.5	10.0	10.5
					Front Peak (%)	9.8	14.5	22.1	31.8

Claims (23)

1. A method for producing a purified polypeptide from a solution comprising a polypeptide and a deamidated species of the polypeptide, wherein said deamidated species of the polypeptide results in an inhibition of the potency of said polypeptide, the method comprising: (a) contacting the polypeptide with an anion exchange chromatography matrix, and (b) eluting bound polypeptide from the anion exchange chromatography matrix with a linear salt gradient from Tris/HCl, 150mM NaCl at pH8.0 to Tris/HCl, 300mM NaCl at pH8.0, thereby separating said polypeptide from deacylated species and producing a purified polypeptide, wherein 75% to 99% of said deamidated species are removed; the polypeptides include an anti-CD 22 antibody or antigen-binding fragment thereof and a pseudomonas aeruginosa exotoxin or cytotoxic fragment thereof, and conservatively modified variants thereof.

2. The method of claim 1, wherein the anion exchange chromatography matrix comprises quaternary ammonium and tertiary ammonium ion exchange groups.

3. The method of claim 2, wherein the anion exchange chromatography matrix comprises a quaternary ammonium ion exchange group.

4. The method of claim 3, wherein the anion exchange chromatography matrix is Q sepharose.

5. The method of claim 1, wherein the linear salt gradient is from 175mM NaCl in Tris/HCl, pH8.0 to 275mM NaCl in Tris/HCl, pH 8.0.

6. The method of claim 1, wherein the linear salt gradient is from 192mM NaCl in Tris/HCl, pH8.0 to 245mM NaCl in Tris/HCl, pH 8.0.

7. The method of claim 1, wherein about 80% of the deamidated species is removed.

8. The method of claim 1, wherein about 85% of the deacylated species are removed.

9. The method of claim 1, wherein about 90% of the deacylated species are removed.

10. The method of claim 1, wherein about 95% of the deacylated species are removed.

11. The method of claim 1, wherein about 96%, 97%, 98% or 99% of the deacylated species are removed.

12. The method of any one of claims 1-11, wherein the antibody or antigen-binding fragment comprises a Fab, Fab ', F (ab')₂Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, internal antibody, IgG △ CH₂Mini antibody, F (ab')₃Tetrad, tred, diabody, single domain antibody, DVD-Ig, Fcab, mAb²、a(scFv)₂Or scFv-Fc.

13. The method of any one of claims 1-11, wherein said pseudomonas exotoxin or cytotoxic fragment thereof, and conservatively modified variants thereof, has an amino acid sequence selected from the group consisting of seq id no:16 to 22 SEQ ID NO.

14. The method of any one of claims 1-11, wherein the pseudomonas exotoxin or cytotoxic fragment thereof, and conservatively modified variants thereof, has the amino acid sequence of SEQ ID No. 22.

15. The method of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof comprises a VH and VL sequence.

16. The method of claim 15, wherein said VH sequence is selected from the group consisting of seq id nos: SEQ ID NOS: 6 to 11.

17. The method of claim 15, wherein said VL sequence is selected from the group consisting of seq id nos: SEQ ID NO 2, and 12 to 15.

18. The method of claim 1, wherein the polypeptide is V comprising SEQ ID NO 1_HSubunit PE38 and V of SEQ ID NO. 2_LSubunit CAT-8015 immunotoxin.

19. A composition comprising a purified polypeptide having less than 25%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% deamidated species, wherein the polypeptide is purified by the method of claim 1 or 4.

20. The composition of claim 19, wherein the polypeptide comprises V of SEQ ID NO 1_HSubunit PE38 and V of SEQ ID NO 2_LA subunit.

21. A pharmaceutical composition comprising the composition of any one of claims 19 or 20 and a pharmaceutically acceptable carrier.

22. A formulation comprising the composition of any one of claims 19 or 20 and at least one excipient selected from the group consisting of: sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium hydroxide, and water.

23. A formulation comprising the composition of any one of claims 19 or 20, comprising 1mg/mL CAT-8015 comprising the VH-PE38 subunit of SEQ ID NO:1 and the VL subunit of SEQ ID NO:2 in 25mM sodium phosphate, 4% sucrose, 8% glycine, 0.02% polysorbate 80, pH 7.4.