RU2795432C2 - Separation of antibodies with three light chains using cation exchange chromatography - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a method for separating H2L3 antibodies from an antibody composition containing H2L3 antibodies and H2L2 antibodies, and this method includes: (i) applying the antibody composition to a cation exchange resin so that the H2L3 antibodies and H2L2 antibodies bind to the resin, and the composition of antibodies has a pH of less than 6.0; (ii) applying the composition for elution with a salt concentration from 300 mm to 600 mm on a cation exchange resin; and (iii) collecting a composition containing H2L2 antibodies eluted from the resin, the said composition containing H2L2 antibodies containing one or more eluted column volumes selected from column volumes 1–9, as well as a method for separating anti-CD123 H2L3 antibodies from an anti-CD123 antibody composition comprising an anti-CD123 antibody H2L3 and an anti-CD123 antibody H2L2, wherein the anti-CD123 antibody H2L3 and anti-CD123 antibody H2L2 comprise the following: (i) heavy chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5–7, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of SEQ ID NO: 8–10, respectively, and (ii) an IgG heavy chain constant domain.

EFFECT: separation of H2L3 antibodies from compositions containing H2L3 antibodies and H2L2 antibodies using a cation exchange resin.

47 cl, 8 dwg, 4 tbl, 3 ex

Description

[1] This application claims priority to U.S. Provisional Application No. 62/562,188, filed September 22, 2017, which is incorporated herein by reference.

LINK TO ELECTRONIC SEQUENCE LISTING

[2] The contents of the electronically submitted sequence listing (name: 2921_097PC01_ST25; size: 15736 bytes; and date of creation: September 21, 2018) are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY

[3] The scope of the invention generally relates to methods for isolating antibodies with three light chains (H2L3) (for example, anti-CD123 H2L3 antibodies) or antigen-binding fragments thereof from an antibody composition containing H2L3 antibodies or antigen-binding fragments thereof and antibodies with two light chains. chains (H2L2) (for example, anti-CD123 H2L2) or their antigennegative fragments.

BACKGROUND OF THE INVENTION

[4] Recombinant antibodies engineered with reactive cysteine residues, ie "cysteine-engineered antibodies", can be covalently conjugated to drugs of interest to create targeted therapeutic agents. Studies have shown that mammalian cells stably transfected to express such cysteine-engineered antibodies also secrete a high molecular weight species known as the three light chain antibody (H2L3) (Gomez et al., Biotechnol Bioeng . 105(4): 748-60 ( 2010)). Cysteine-modified H2L3 antibodies are the product of a disulfide bond formation between the additional light chain and one of the engineered cysteine residues in the cysteine-modified H2L2 antibody. The level of cysteine-modified H2L3 antibodies in cell culture is related to the cell line and culture conditions. Although cell culture conditions can be modified to minimize H2L3 formation (eg, by using temperature shifts during cell culture), the result is highly cell line dependent (Gomez et al., Biotechnol Prof. 26(5): 1438-45 ( 2010)).

[5] Due to the similarity to the monomeric species, separation of H2L3 antibodies is a problem during the subsequent purification of monoclonal cysteine-modified H2L2 antibodies. In one specific study, hydrophobic interaction chromatography (HIC) was found to reduce H2L3 levels from approximately 3% to 0.5% in the purification of a cysteine-free engineered monoclonal antibody (Wollacott et al., mAbs 5(6): 925-935 ( 2013)). In the same study, cation exchange chromatography was used to remove H2L3 antibodies. However, even under modified conditions, this process was not enough to reduce the percentage of H2L3 to less than 1% in all tested cell lines. The authors concluded that the electrostatic effects in cation exchange chromatography were not strong enough to remove H2L3 antibodies. Thus, in order to obtain the most stable antibody product (eg, for therapeutic antibodies and immunoconjugates containing such antibodies), there is a need for more efficient separation of H2L3 species during antibody purification.

BRIEF DESCRIPTION OF THE INVENTION

[6] The present invention relates to the development of an efficient purification strategy for the separation of antibodies with three light chains (H2L3) from antibodies with two light chains (H2L2). These methods take advantage of the fact that cation exchange resins separate proteins primarily on the basis of charge. As stated herein, where the pH of the resin is lower than that of the antibody of interest (eg, 3.8 to 6.5), all kinds of antibodies, including H2L3 and H2L2, bind to the cation exchange resin. When antibodies are eluted from cation exchange resins using a high pH and/or low salt elution composition, H2L3 species are eluted not only in late fractions after the main peak of H2L2 species has been eluted, but also in earlier fractions containing the desired H2L2 species. However, as shown herein, optimizing cation exchange resins by using lower pH and higher salt concentrations can result in most or all of the H2L3 species being eluted in the late fractions after the main peak of the H2L2 species has been eluted. By using the optimized strong POROS™ XS cation exchange chromatography resin, the amount of H2L3 antibody in an antibody formulation can be reduced from 11% to less than 1%, and this level of recovery is reproducible in various cell lines.

[7] In some embodiments, a method for separating H2L3 antibodies or antigen-binding fragments thereof from an antibody composition comprising H2L3 antibodies or antigen-binding fragments thereof and H2L2 antibodies or antigen-binding fragments thereof comprises (i) applying the antibody composition to a cation exchange resin such that the H2L3 antibodies or their antigen-binding fragments and H2L2 antibodies or antigen-binding fragments bind to the resin; (ii) applying the eluent composition at a pH of about 3.8 to about 5.0 to a cation exchange resin; and (iii) collecting the H2L2 composition eluted from the resin.

[8] In some embodiments, a method for separating H2L3 antibodies or antigen-binding fragments thereof from an antibody composition comprising H2L3 antibodies or antigen-binding fragments and H2L2 antibodies or antigen-binding fragments thereof comprises (i) applying the antibody composition to a cation exchange resin such that the H2L3 antibodies or their antigen-binding fragments and H2L2 antibodies or antigen-binding fragments bind to the resin; (ii) applying the composition for elution with a salt concentration of from about 300 mm to about 600 mm on a cation exchange resin; and (iii) collecting the H2L2 composition eluted from the resin.

[9] In some embodiments, a method for separating anti-CD123 H2L3 antibodies or antigen-binding fragments thereof from an anti-CD123 antibody composition comprising anti-CD123 H2L3 antibodies or antigen-binding fragments thereof and anti-CD123 H2L2 antibodies or antigen-binding fragments thereof comprises (i) applying compositions of anti-CD123 antibodies on a cation exchange resin such that anti-CD123 antibodies H2L3 or antigen-binding fragments thereof and anti-CD123 antibodies H2L2 or antigen-binding fragments thereof bind to the resin; (ii) applying the eluent composition at a pH of about 3.8 to about 5.5 to a cation exchange resin; and (iii) collecting an anti-CD123 H2L2 composition eluted from the resin, wherein the anti-CD123 H2L3 antibodies or antigen-binding fragments thereof and the anti-CD123 H2L2 antibodies or antigen-binding fragments thereof comprise the heavy chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5-7, respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, respectively.

[10] In some embodiments, a method for separating anti-CD123 H2L3 antibodies or antigen-binding fragments thereof from an anti-CD123 antibody composition comprising anti-CD123 H2L3 antibodies or antigen-binding fragments thereof and anti-CD123 H2L2 antibodies or antigen-binding fragments thereof comprises (i) applying compositions of anti-CD123 antibodies on a cation exchange resin such that anti-CD123 antibodies H2L3 or antigen-binding fragments thereof and anti-CD123 antibodies H2L2 or antigen-binding fragments thereof bind to the resin; (ii) applying the composition for elution with a salt concentration of from about 300 mm to about 600 mm on a cation exchange resin; and (iii) collecting an anti-CD123 H2L2 composition eluted from the resin, wherein the anti-CD123 H2L3 antibodies or antigen-binding fragments thereof and the anti-CD123 H2L2 antibodies or antigen-binding fragments thereof comprise the heavy chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5-7, respectively, and the light chain variable region CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 8-10, respectively.

[11] In some embodiments, not more than 2% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, no more than 1% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, no more than 0.5% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L3 antibodies or antigen-binding fragments thereof.

[12] In some embodiments, at least 98%, at least 99%, or at least 99.5% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L2 antibodies or antigen-binding fragments thereof. In some embodiments, the H2L2 composition contains no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of H2L3 antibodies or antigen-binding fragments thereof in the antibody composition applied to the cation exchange resin.

[13] In some embodiments, the H2L2 composition contains one or more eluted column volumes selected from column volumes 1-9. In some embodiments, the H2L2 composition contains eluted column volumes 1-4. In some embodiments, the cation exchange resin comprises polystyrene crosslinked with divinylbenzene. In some embodiments, the implementation of the cation exchange resin contains surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -). In some embodiments, the particle size of the cation exchange resin is about 50 microns. In some embodiments, the cation exchange resin has a bimodal pore size distribution. In some embodiments, the bimodal pore size distribution includes approximately 500 nM diameter pores and approximately 22 nM diameter pores. In some embodiments, the cation exchange resin is a strong POROS™ XS cation exchange resin.

[14] In some embodiments, the elution composition contains a salt. In some embodiments, the salt in the elution composition is the chloride salt. In some embodiments, the chloride salt is sodium chloride, potassium chloride, calcium chloride, or magnesium chloride. In some embodiments, the salt concentration in the elution composition is from about 100 mM to about 600 mM, from about 300 mM to about 500 mM, or from about 350 mM to about 450 mM. In some embodiments, the salt concentration in the elution composition is from about 300 mM to about 500 mM, or from about 350 mM to about 450 mM. In some embodiments, the salt concentration in the elution composition is about 400 mM. In some embodiments, the elution composition has a pH of about 3.8 to about 6.5. In some embodiments, the elution composition has a pH of about 3.8 to about 5.0. In some embodiments, the elution composition has a pH of about 4.2.

[15] In some embodiments, the method includes applying the equilibration composition to the cation exchange resin prior to applying the antibody composition to the cation exchange resin. In some embodiments, the balancing composition contains sodium acetate. In some embodiments, the concentration of sodium acetate in the balancing composition is from about 10 mM to 150 mM. In some embodiments, the concentration of sodium acetate in the balancing composition is about 50 mM. In some embodiments, the balancing composition has a pH of about 3.8 to about 6.5. In some embodiments, the balancing composition has a pH of about 4.2.

[16] In some embodiments, the antibody composition contains from about 10 to about 100 g/L of protein. In some embodiments, the antibody composition contains from about 30 g/L to about 50 g/L, or from about 35 g/L to about 45 g/L of protein. In some embodiments, the antibody composition contains approximately 40 g/L of protein. In some embodiments, the antibody composition has a pH of about 3.8 to about 6.5. In some embodiments, the antibody composition has a pH of about 4.2.

[17] In some embodiments, from about 1% to about 20% of the antibodies or antigen-binding fragments thereof in the antibody composition are H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, about 1% to about 15%, or about 5% to about 15%, or about 3% to about 12%, or about 10% to about 15% of the antibodies or antigen-binding fragments thereof in the antibody composition are H2L3 antibodies or their antigen-binding fragments. In some embodiments, the H2L2 composition contains at least 40%, at least 45%, at least 50%, or at least 55% of the H2L2 antibodies or antigen-binding fragments thereof in the antibody composition applied to the cation exchange resin.

[18] In some embodiments, the antibody composition comprises cysteine-engineered antibodies or antigen-binding fragments thereof. In some embodiments, cysteine-engineered antibodies, or antigen-binding fragments thereof, contain an engineered cysteine residue at position 442 according to EU/OU numbering. In some embodiments, the antibody composition comprises antibodies. In some embodiments, the antibody composition comprises antigen-binding antibody fragments. In some embodiments, the antibody composition comprises Fab, Fab', F(ab') ₂ , Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab') ₃ , tetrabody, triatelo, diabody, single domain antibody, DVD-Ig, Fcab, mAb ² , (scFv) ₂ or scFv-Fc. In some embodiments, the antibody composition comprises antibodies or antigen-binding fragments thereof derived from a CHO cell line.

[19] In some embodiments, the method further comprises conjugating the H2L2 antibodies or antigen-binding fragments thereof in the H2L2 composition with a cytotoxin to form an immunoconjugate composition. In some embodiments, the implementation of the composition of immunoconjugates receive in accordance with the methods described in this document. In some embodiments, the immunoconjugate composition contains no more than 2% H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, the immunoconjugate composition contains no more than 1% of H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, the immunoconjugate composition contains no more than 0.5% of H2L3 antibodies or antigen-binding fragments thereof.

[20] In some embodiments, the implementation of the composition H2L2 receive in accordance with the methods described in this document. H2L2 composition according to claim 46, containing not more than 2% of H2L3 antibodies or their antigen-binding fragments. In some embodiments, the H2L2 composition contains no more than 1% H2L3 antibodies or antigen-binding fragments thereof. In some embodiments, the H2L2 composition contains no more than 0.5% H2L3 antibodies or antigen-binding fragments thereof.

[21] In some embodiments, (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), and has a particle size of approximately 50 μm and a bimodal pore size distribution having pores with a diameter of approximately 500 nm, and pores with a diameter of approximately 22 nm; (ii) the elution composition contains from about 300 to 600 mm chloride salt and a pH from about 3.8 to about 5.0; (iii) the antibody composition contains from about 10 to about 100 g/L of protein, and from about 10% to about 15% of the antibodies or antigen-binding fragments thereof in the antibody composition are H2L3 antibodies or antigen-binding fragments thereof; (iv) the H2L2 composition contains one or more eluted column volumes selected from column volumes 1-9; and (v) not more than 2% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L3 antibodies or antigen-binding fragments thereof.

[22] In some embodiments, (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), and has a particle size of approximately 50 μm and a bimodal pore size distribution having pores with a diameter of approximately 500 nm, and pores with a diameter of approximately 22 nm; (ii) the elution composition contains from about 400 mm NaCl and has a pH of about 4.2; (iii) the antibody composition contains from about 30 to about 50 g/L of protein, and from about 10% to about 15% of the antibodies or antigen-binding fragments thereof in the antibody composition are H2L3 antibodies or antigen-binding fragments thereof; (iv) composition H2L2 contains eluted column volumes 1-4; and (v) not more than 1% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L3 antibodies or antigen-binding fragments thereof.

[23] In some embodiments, the composition comprises anti-CD123 antibodies or antigen-binding fragments thereof, wherein less than 1% of the anti-CD123 antibodies or antigen-binding fragments thereof are H2L3 antibodies or antigen-binding fragments thereof, and the anti-CD123 antibodies or antigen-binding fragments thereof contain the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of SEQ ID NO: 5-7, respectively, and the sequence of CDR1, CDR2 and CDR3 of the light chain variable region of SEQ ID NO: 8-10, respectively. In some embodiments, the anti-CD123 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 1. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof comprises the light chain variable region sequence of SEQ ID NO: 2. In some embodiments, the anti- The CD123 antibody, or antigen-binding fragment thereof, is constructed by introducing cysteine. In some embodiments, the anti-CD123 antibody comprises the heavy chain sequence of SEQ ID NO: 3. In some embodiments, the anti-CD123 antibody comprises the light chain sequence of SEQ ID NO: 4. In some embodiments, less than 0.5% of the anti-CD123 antibody, or their antigen-binding fragments are H2L3 antibodies or antigen-binding fragments thereof.

[24] In some embodiments, the composition comprises anti-CD123 immunoconjugates, wherein the immunoconjugates comprise anti-CD123 antibodies or antigen-binding fragments thereof bound to DGN549-C, wherein less than 1% of the anti-CD123 antibodies or antigen-binding fragments thereof are H2L3 antibodies or their antigen-binding fragments, wherein the anti-CD123 antibodies or antigen-binding fragments thereof comprise the heavy chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5-7, respectively, and the light chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO : 8-10, respectively. In some embodiments, the anti-CD123 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 1. In some embodiments, the anti-CD123 antibody or antigen-binding fragment thereof comprises the light chain variable region sequence of SEQ ID NO: 2. In some embodiments, the anti- The CD123 antibody, or antigen-binding fragment thereof, is constructed by introducing cysteine. In some embodiments, the anti-CD123 antibody comprises the heavy chain sequence of SEQ ID NO: 3. In some embodiments, the anti-CD123 antibody comprises the light chain sequence of SEQ ID NO: 4. In some embodiments, the immunoconjugate has the structure:

sulfonated-DGN549-C

[25] In some embodiments, less than 0.5% of the anti-CD123 antibodies or antigen-binding fragments thereof are H2L3 antibodies or antigen-binding fragments thereof.

BRIEF DESCRIPTION OF GRAPHICS/SHAPES

[26] FIG. 1 shows a Size Exclusion Ultra High Performance Liquid Chromatography (SEC-UPLC) chromatogram of a cysteine engineered monoclonal antibody (CysmAb) composition after protein A purification. . The y-axis of the chromatogram is a measure of the absorbance intensity (in EOP or absorbance units). The x-axis indicates the units of time (minutes) and is used to determine the retention time for each peak.

[27] FIG. 2 shows the percentage of aggregates (dark gray bars) and H2L3 antibodies (light gray bars) in different bioreactor production lots (A, B, C, D, E, F, G and H). H2L3 aggregates and antibodies in lots A and B were generated in cell line A. H2L3 aggregates and antibodies in lots C and D were generated in cell line B. H2L3 aggregates and antibodies in lots E, F, G and H were generated in the cell line C.

[28] FIG. 3 shows the percentage of H2L3 antibodies in the eluate after purification with ceramic hydroxyapatite (CHT) at various salt concentrations (100 mM, 95 mM, 90 mM or 85 mM potassium phosphate buffer). Load H2L3% (dark gray bars), eluate H2L3% (light gray bars).

[28] FIG. 4 shows the elution profile of cysteine-engineered mAb species superimposed on the elution profile of H2L3 aggregates and species. Fraction yield % (black line), aggregates % (light gray bars) and H2L3 % (dark gray bars) were measured for each fraction 1-10 (F1-F10) (x-axis).

[30] FIG. 5 shows the percentage of H2L3 in the elution pool at different pH values. High pH (solid line, black circle), medium pH (dashed line, open circle), low pH (dashed line, black circle), baseline H2L3 (straight black line).

[31] FIG. 6 shows the percentage of H2L3 in the eluate at various NaCl concentrations (420 mM, 410 mM, 400 mM, 390 mM and 380 mM) and collection volumes (CV1-3, CV1-4, CV1-5 and less than/equal to 7CV). Entry level H2L3 (straight black line).

[32] FIG. 7 shows the final elution conditions for POROS™ XS cation exchange chromatography based on a statistical analysis of the desirability of CysmAb purification.

[33] FIG. 8A shows the chemical structure of IMGN632. IMGN632 is a composition containing immunoconjugates containing the anti-CD123 antibody G4723 coupled to the cytotoxic payload DGN549-C in sodium bisulfite. Most of the immunoconjugate in the composition is in the sulfonated form shown in FIG. 8A.

[34] FIG. 8B shows the non-sulfonated form of the immunoconjugate containing the anti-CD123 antibody G4723 coupled to the cytotoxic payload DGN549-C (monoimine structure), which may also be present in the IMGN632 formulation.

DETAILED DESCRIPTION OF THE INVENTION

[35] The present invention provides methods for separating H2L3 antibodies (e.g., anti-CD123 H2L3 antibodies) or antigen-binding fragments thereof from an antibody composition comprising H2L3 antibodies or antigen-binding fragments thereof and H2L2 antibodies (e.g., anti-CD123 antibodies) or antigen-binding fragments thereof. .

I. Definitions

[36] For ease of understanding of the present invention, a number of terms and phrases are provided below.

[37] "Cation exchange resin" refers to a solid phase that is negatively charged and which has free cations to exchange with cations in an aqueous solution passed through the solid phase. You can use any negatively charged ligand attached to the solid phase, suitable for the formation of cation exchange resin, such as carboxylate, sulfonate and others. Commercially available cation exchange resins include, but are not limited to, for example, those having a sulfonate-based group; a group based on sulfoethyl; a group based on sulfopropyl; a group based on sulfoisobutyl; a sulfoxyethyl group, a carboxymethyl group; groups based on sulfonic and carboxylic acids; a carboxylic acid group; a sulfonic acid group; and an orthophosphate-based group. As described herein, proteins (eg, antibodies or antigen-binding fragments thereof) can be separated based on the interaction between negatively charged groups on the cation exchange resin and positively charged groups on proteins (eg, antibodies or antigen-binding fragments thereof).

[38] The term "elute" and its grammatical variants refer to the removal of a molecule, e.g., a polypeptide of interest, from a resin (e.g., chromatographic material) using appropriate conditions, e.g., changing the ionic strength or pH of the buffer surrounding the resin (e.g., chromatographic material), by changing the hydrophobicity of the molecule, or by changing the chemical property of the ligand (eg charge) so that the protein of interest cannot bind the resin and is therefore eluted from the resin (eg chromatography column). The term "eluate" refers to the eluting component from the resin (eg, column) containing the polypeptide of interest when the solution is applied to the column. After elution of the polypeptide of interest, the resin (eg, column) can be regenerated, disinfected, and stored as needed.

[39] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. . In the context of this document, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecule, provided that the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavyweight constant domains. chains called alpha, delta, epsilon, gamma and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be unconjugated or conjugated to other molecules such as toxins, radioisotopes, etc.

[40] The term "antibody fragment" refers to a portion of an intact antibody with sufficient positive charge to bind to a cation exchange resin. "Antigen-binding fragment" refers to the portion of an intact antibody that binds to an antigen and has sufficient positive charge to bind to a cation exchange resin. The antigen-binding fragment may contain the antigenicity-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies.

[41] The term "antibody with three light chains" or "H2L3" or its antigennegative fragment refers to an antibody or its antigennegative fragment, which contains two heavy chains or fragments thereof and three light chains or fragments thereof.

[42] The term "antibody with two light chains" or "H2L2" or antigen-binding fragment refers to an antibody or antigen-binding fragment that contains two heavy chains or fragments thereof and two light chains or fragments thereof.

[43] The term "antibody composition" refers to a composition containing antibodies or antigen-binding fragments thereof. The antibody composition may contain antibodies and other components that have been obtained in cell culture (eg, from CHO cells), purified using a protein A column, and optionally further purified using an anion exchange column. In addition to antibodies or antigen-binding fragments thereof, the antibody composition may contain, for example, tris-acetic acid. The antibody composition may also contain aggregates.

[44] The "H2L2" composition refers to a composition eluted from a cation exchange resin that contains a higher proportion of H2L2 species than an antibody composition supported on a cation exchange resin.

[45] The "H2L3" composition refers to a composition eluted from a cation exchange resin that contains a higher proportion of H2L3 species than an antibody composition supported on a cation exchange resin.

[46] The term “cysteine-engineered” antibody or antigen-binding fragment thereof includes an antibody or antigen-binding fragment thereof with at least one cysteine (“Cys”) that is not normally present in a given light chain or heavy chain residue of an antibody or antigen-binding fragment thereof. Such a Cys, which may also be referred to as "engineered Cys", may be constructed using any standard molecular biology or recombinant technique (eg, by replacing the coding sequence for a non-Cys residue in the target residue with the coding sequence for Cys). For example, if the original residue is Ser with the coding sequence 5'-UCU-3', the coding sequence can be mutated (eg, by site-directed mutagenesis) to 5'-UGU-3', which encodes Cys. In certain embodiments, the Cys-engineered antibody, or antigen-binding fragment thereof, has a Cys-engineered heavy chain. In certain embodiments, the engineered Cys is in or near the heavy chain CH3 domain. In some embodiments, the engineered Cys is located at residue 442 of the heavy chain (EU/OU numbering; EU index, Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed., NIH publication No. 91-3242, 1991, the entire content of which is included to this document by reference). In some embodiments, the Fc region contains a cysteine at one or more positions 239, 282, 289, 297, 312, 324, 330, 335, 337, 339, 356, 359, 361, 383, 384, 398, 400, 440, 422 and 442, numbered with the EU index. In some embodiments, one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) in the light chain, A118 (EU numbering) in the heavy chain, and S400 (EU numbering) in the Fc region of the heavy chain. In some embodiments, the implementation of the variable domain of the light chain, for example, scFv, has a cysteine at position 100 according to Kabat. In certain embodiments, the heavy chain variable domain, eg scFv, has a cysteine at position 44 according to Kabat. Cysteine-engineered antibodies can be prepared as described, for example, in US Pat. No. 7,521,541, US Pat. No. 7,855,275, US Published Application No. 20110033378 and WO 2011/005481.

[47] A "monoclonal" antibody, or antigen-binding fragment thereof, refers to a homogeneous population of antibodies, or antigen-binding fragments thereof, involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. In this they differ from polyclonal antibodies, which usually include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof includes both intact and full-length monoclonal antibodies, as well as antibody fragments (for example, Fab, Fab', F(ab')2, Fv), single-stranded (ScFv) mutants, fusion proteins, containing part of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site. In addition, a "monoclonal" antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof, made by any number of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[48] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments thereof, which are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen binding fragments are human immunoglobulins in which residues from a complementarity determining region (CDR) are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and characteristics ("grafted CDR") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances, framework region (FR) residues of a human Fv immunoglobulin are replaced by corresponding residues of an antibody or fragment from a non-human species that has the desired specificity, affinity, and characteristics. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of additional residues either within the Fv framework region and/or within the replaced non-human residues to improve and optimize the specificity, affinity, and/or characteristics of the antibody or antigen-binding fragment thereof. In general, a humanized antibody or antigen-binding fragment thereof will contain substantially all of at least one, and typically two or three variable domains, containing all or substantially all of the CDR regions that correspond to a non-human immunoglobulin, and all or substantially all FR regions are human immunoglobulin consensus sequence regions. The humanized antibody, or antigen-binding fragment thereof, may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in US Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some embodiments, the "humanized antibody" is a surface-modified antibody.

[49] "Variable region" of an antibody refers to the variable region of the light chain of an antibody or the variable region of the heavy chain of an antibody, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FR, and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two ways to determine CDRs: (1) an approach based on interspecies sequence variability (i.e. Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md .), "Kabat"); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al, J. Molec. Biol. 273:927-948 (1997)). In addition, combinations of the two approaches are sometimes used in the art to determine CDR.

[50] The Kabat numbering system is generally used to designate a residue in a variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest. (5th Ed. , 1991, National Institutes of Health, Bethesda, Md.) ("Kabat").

[51] The numbering of amino acid positions as in Kabat refers to the numbering system used for heavy chain variable domains or light chain variable domains constituting antibodies in Kabat et al. (Sequences of Immunological Interest. (5th Ed., 1991, National Institutes of Health , Bethesda, Md.), "Kabat"). Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids, corresponding to a shortening or insertion of the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 in H2 and inserted amino acid residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after the FR amino acid residue 82 of the heavy chain. Kabat residue numbering can be determined for a given antibody by aligning regions of homology in the antibody sequence with a "standard" Kabat numbered sequence. Chothia, instead, refers to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The Chothia end of the CDR-H1 loop when numbering according to the Kabat numbering system varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places inserts in H35A and H35B; if neither 35A nor 35B is present , the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat's CDRs and Chothia's structural loops and are used in Oxford Molecular's AbM antibody modeling software.

A loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32..34 (Numbering according to Kabat) H1 H31-H35 H26-H35 H26-H32 (Numbering according to Chothia) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

[52] The term "human" antibody or antigen-binding fragment thereof means a human-produced antibody or antigen-negative fragment thereof, or an antibody or antigen-binding fragment thereof having an amino acid sequence corresponding to a human-produced antibody or antigen-negative fragment thereof, made using any method known to in this field of technology. This definition of a human antibody or antigen-binding fragment includes intact or full length antibodies and fragments thereof.

[53] the Term "chimeric" antibodies or antigennegative fragments thereof refers to antibodies or antigennegative fragments, and the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and characteristics at the time. how constant regions are homologous to sequences of antibodies or antigen-binding fragments obtained from another source (usually from a human) to avoid inducing an immune response in this species.

[54] The term "epitope" or "antigenic determinant" is used interchangeably herein and refers to that portion of an antigen that can be recognized and specifically binds to a particular antibody. When the antigen is a polypeptide, "epitopes" can be formed by either contiguous amino acids or non-contiguous amino acids formed by folding the protein's tertiary structure. Epitopes formed from contiguous amino acids are generally retained upon protein denaturation, while epitopes formed from tertiary structure are generally lost upon protein denaturation. An epitope usually contains at least 3 or more, usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[55] "Binding affinity" generally refers to the strength of the total number of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, in the context of this document, "binding affinity" refers to the actual binding affinity, which reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y as a whole can be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies usually bind antigen slowly and tend to dissociate easily, while high affinity antibodies usually bind antigen faster and tend to stay bound longer. A number of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this invention. Specific illustrative embodiments are described below.

[56] The phrase "or more" when used herein to denote binding affinity refers to a stronger binding between a molecule and its binding partner. The phrase "or more", when used herein, refers to a stronger binding, represented by a smaller numerical value of Kd. For example, an antibody that has an affinity for an antigen of "0.6 nM or more" has an affinity of the antibody for the antigen of <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM, etc. or any value less than 0.6 nM.

[57] The term "specifically binds" usually means that the antibody binds to the epitope through its antigen-binding domain, and that the binding results in some complementarity between the antigen-binding domain and the epitope. By this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity by which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" with higher specificity than to the corresponding epitope "D". ".

[58] By "predominantly binds" is meant that an antibody specifically binds to an epitope more easily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that "predominantly binds" to a given epitope is more likely to bind to that epitope than to the corresponding epitope, even though such an antibody may cross-react with a related epitope.

[59] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be separated by non-amino acids. In addition, these terms include an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation to a labeled moiety. The term also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that since the polypeptides of this invention are based on antibodies, in some embodiments, the implementation of the polypeptides may occur as single chains or linked chains.

[60] The term "immunoconjugate" or "conjugate" in the context of this document refers to a compound or derivative thereof that is associated with a cell-binding agent (i.e., an anti-CD123 antibody or fragment thereof) and is defined by the general formula: C-A, where C = cytotoxin (e.g., a maytansinoid, a benzodiazepine compound, including pyrrolobenzodiazepines (PBDs) and tetracyclic benzodiazepines such as indolinobenzodiazepines) and A = an antibody or antigen-binding fragment thereof, e.g., an anti-CD123 antibody or antibody fragment. The immunoconjugate may optionally contain a linker and be defined by the general formula C-L-A, where C = cytotoxin, L = linker and A = antibody or antigen-binding fragment, for example, an anti-CD123 antibody or antibody fragment. Immunoconjugates can also be determined using the general formula in reverse order: C-A or A-L-C. Immunoconjugates may also contain multiple cytotoxins (C) per antibody or antigen-binding fragment (A) or multiple cytotoxins (C) and linkers (L) per antibody or antigen-binding fragment (A).

[61] A "linker" is any chemical moiety that is capable of linking a compound, typically a drug (such as a maytansinoid, a benzodiazepine compound, including pyrrolobenzodiazepines (PBD) and tetracyclic benzodiazepines such as indolinobenzodiazepines), to a cell-binding agent (such as anti-CD123 antibody or fragment thereof) in a stable covalent manner. Linkers may be susceptible to, or substantially resistant to, for example, disulfide bond cleavage, under the conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups and thioether groups.

[62] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients constituting the formulation and/or the mammal being treated with them.

[63] The term "pharmaceutical formulation" refers to a formulation that is in such a form as to provide the biological activity of the active ingredient to be effective, and which does not contain any additional ingredients that are unacceptably toxic to the subject to whom the formulation will be administered. . The composition may be sterile.

[64] The terms "(human) IL-3Rα", "interleukin-3 receptor alpha", or "CD123", used interchangeably herein, refer to any native (human) IL-3Rα or CD123 unless otherwise indicated. The CD123 protein is an interleukin-3-specific subunit of a heterodimeric cytokine receptor (IL-3 receptor or IL-3R). The terms encompass "full length" full-length CD123 polypeptides as well as any form of a CD123 polypeptide resulting from intracellular processing. The term also includes naturally occurring variants of CD123, such as those encoded by splice variants and allelic variants. The CD123 polypeptides described herein may be isolated from a variety of sources, such as different types of human tissue or other source, or produced by recombinant or synthetic methods. When specified specifically, "CD123" may be used to refer to a nucleic acid that encodes a CD123 polypeptide. Human CD123 sequences are known and include, for example, those associated with NCBI identification numbers NP_002174 and NM_002183 (protein and nucleic acid sequences for human CD123 variant 1), and NP_001254642 and NM_001267713 (protein and nucleic acid sequences for human CD123 variant 2). In the context of this document, the term "human CD123" refers to CD123 containing the sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

1 MVLLWLTLLL IALPCLLQTK EDPNPPITNL RMKAKAQQLT WDLNRNVTDI ECVKDADYSM

61 PAVNNSYCQF GAISLCEVTN YTVRVANPPF STWILFPENS GKPWAGAENL TCWIHDVDFL

121 SCSWAVGPGA PADVQYDLYL NVANRRQQYE CLHYKTDAQG TRIGCRFDDI SRLSSGSQSS

181 HILVRGRSAA FGIPCTDKFV VFSQIEILTP PNMTAKCNKT HSFMHWKMRS HFNRKFRYEL

241 QIQKRMQPVI TEQVRDRTSF QLLNPGTYTV QIRARERVYE FLSAWSTPQR FECDQEEGAN

301 TRAWRTSLLI ALGTLLALVC VFVICRRYLV MQRLFPRIPH MKDPIGDSFQ NDKLVVWEAG

361 KAGLEECLVT EVQVVQKT (SEQ ID NO:11)

1 MVLLWLTLLL IALPCLLQTK EGGKPWAGAE NLTCWIHDVD FLSCSWAVGP GAPADVQYDL

61 YLNVANRRQQ YECLHYKTDA QGTRIGCRFD DISRLSSGSQ SSHILVRGRS AAFGIPCTDK

121 FVVFSQIEIL TPPNMTAKCN KTHSFMHWKM RSHFNRKFRY ELQIQKRMQP VITEQVRDRT

181 SFQLLNPGTY TVQIRARERV YEFLSAWSTP QRFECDQEEG ANTRAWRTSL LIALGTLLAL

241 VCVFVICRRY LVMQRLFPRI PHMKDPIGDS FQNDKLVVWE AGKAGLEECL VTEVQVVQKT

(SEQ ID NO:12)

[65] The term "anti-CD123 antibody" or "antibody that binds to CD123" means an antibody that is capable of binding to CD123 with sufficient affinity such that the antibody becomes useful as a diagnostic and/or therapeutic agent for targeting CD123 (e.g. , huMov19 antibody (M9346A)). The degree of binding of an anti-CD123 antibody to an unrelated non-CD123 protein is less than about 10% of the binding of the antibody to CD123 as measured, for example, by radioimmunoassay (RIA).

[66] The term "IMGN632" refers to the immunoconjugate composition shown in FIG. 8. The immunoconjugate composition contains immunoconjugates containing an average of 1.5 to 2.1 DGN549-C cytotoxic agents per huCD123-6Gv4.7 antibody ("G4723A") in the sulfated version (FIG. 8A). The immunoconjugate composition may also contain a non-sulfonated immunoconjugate (monoimine structure shown in Fig. 8B).

[67] As used in this specification and claims, the singular forms include the plural forms unless the context clearly dictates otherwise.

[68] It should be understood that where embodiments are described herein with the wording "comprising", otherwise similar embodiments described in terms of "consisting of" and/or "consisting primarily of" are also provided. .

[69] The term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A" and "B". Likewise, the term "and/or" used in a phrase such as "A, B and/or C" is intended to cover 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; B and C; A (separately); B (separately); and C (separately).

II. Cation exchange resins

[70] In accordance with the methods provided herein, cation exchange resins can be used to separate antibodies with three light chains (H2L3) and their antigen-binding fragments from a composition containing antibodies H2L3 and antibodies with two light chains (H2L2) and their antigen-binding fragments. fragments.

[71] One exemplary cation exchange resin used in the methods presented herein is POROS™ XS optimized strong cation exchange resin (Thermos Fisher, formerly Life Technologies Corporation, Carlsbad, CA; 10,000 ml = Cat. No. 440334; 5000 ml 4404335, 1000 ml = 4404336, 250 ml = 4404337, 10 ml = 82071, and 50 ml = 82072).

[72] The cation exchange resin may contain, for example, polystyrene crosslinked with divinylbenzene. The cation exchange resin may have surface functional groups of sulfopropyl (-CH2CH2CH2SO3-). The cation exchange resin may contain polystyrene crosslinked with divinylbenzene and have sulfopropyl surface functional groups (-CH2CH2CH2SO3-).

[73] In some embodiments, the cation exchange resin is not a Fractogel SE HiCap (EMD Millipore) column. In some embodiments, the implementation of the cation exchange resin is not based on methacrylate.

[74] The cation exchange resin may have a particle size of approximately 50 microns. The cation exchange resin may have a bimodal pore size distribution, for example, with a pore diameter of about 500 nM and a pore diameter of about 22 nM. The cation exchange resin may have a particle size of approximately 50 μm and a bimodal pore size distribution, for example with a pore diameter of approximately 500 nM and a pore diameter of approximately 22 nM.

[75] The cation exchange resin may contain polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH2CH2CH2SO3-), have a particle size of approximately 50 μm, and have a bimodal pore size distribution having a pore diameter of 500 nM and a pore diameter of approximately 22 Nm.

[76] The volume of the cation exchange resin may be of a certain size. For example, the volume of the cation exchange resin may be from about 10 to about 15,000 ml. The volume of the cation exchange resin may be from about 20 to about 25 ml. The volume of the cation exchange resin may be from about 100 to about 150 ml. The volume of the cation exchange resin may be from about 10,000 to about 15,000 ml. The volume of the cation exchange resin may be approximately 13,800 ml. The volume of the cation exchange resin may be 32 L or more.

[77] The cation exchange resin may be in the form of a column.

III. Antibodies and their antigen-binding fragments

[78] Antibodies and antigen-binding fragments thereof (eg, therapeutically useful antibodies and antigen-binding fragments thereof) typically contain two heavy chains or fragments thereof and two light chains or fragments thereof. However, species with three light chains (H2L3) have also been found containing two heavy chains or fragments thereof and three light chains or fragments thereof. This H2L3 species may occur with higher probability in cysteine-engineered antibodies and their antigen-binding fragments, for example, when the H2L3 species results from a disulfide bond formed between the additional light chain and one of the engineered cysteines on the antibody or antigen-binding fragments thereof. Accordingly, the antibody composition used herein may comprise cysteine-engineered antibodies or antigen-binding fragments thereof. Similarly, an H2L2 or H2L3 antibody or antigen-binding fragment thereof may be a cysteine engineered H2L2 or H2L3 antibody or antigen-binding fragment thereof. A cysteine-engineered antibody or antigen-binding fragment thereof may, for example, contain an engineered cysteine residue at position 442 according to EU/OU numbering.

[79] In some embodiments, the antibodies or antigen-binding fragments thereof are humanized antibodies or antigen-binding fragments thereof. In some embodiments, the humanized antibody or fragment thereof is a surface-altered antibody or antigen-binding fragment thereof. In other embodiments, the antibodies or antigen-binding fragments thereof are fully human antibodies or antigen-binding fragments thereof.

[80] For example, an anti-CD123 antibody or antigen-binding fragment thereof can be used in these methods. The anti-CD123 antibody, or antigen-binding fragment thereof, may comprise the huCD123-6Gv4.7 antibody sequences shown in Tables 1-3 below. For example, an anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise the heavy chain variable region CDR-1, CDR-2, and CDR-3 sequences of SEQ ID NOs: 5, 6, and 7, respectively, the light chain variable region CDR-1, CDR-2 and CDR-3 sequences of SEQ ID NOs: 8, 9 and 10, respectively. An anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise a heavy chain variable domain comprising the sequence recited in SEQ ID NO: 1. An anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise a light chain variable domain comprising the sequence shown in SEQ ID NO: 2. An anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 1 and a light chain variable domain comprising the sequence set forth in SEQ ID NO: 2. An anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise a heavy chain variable domain, containing the sequence shown in SEQ ID NO: 3. An anti-CD123 antibody or antigen-binding fragment for use in the methods presented herein may contain a light chain containing the sequence shown in SEQ ID NO: 4. An anti-CD123 antibody or its antigen-binding fragment for use in the methods provided herein may contain a heavy chain variable domain containing the sequence shown in SEQ ID NO: 3 and a light chain containing the sequence shown in SEQ ID NO: 4.

[81] In one example, an anti-CD123 antibody or antigen-binding fragment thereof for use in the methods provided herein may comprise a heavy chain variable domain and a light chain variable domain containing the sequences shown in Table 1. In another example, anti-CD123 an antibody or antigennegative fragment thereof for use in the methods provided herein may comprise a heavy chain and a light chain containing the sequences shown in Table 2. In another example, an anti-CD123 antibody or antigennegative fragment thereof for use in the methods provided in herein may contain the heavy and light chain variable region complementarity-determining regions containing the sequences shown in Table 3.

[82] An anti-CD123 antibody or antigen-binding fragment thereof can bind to an epitope at amino acids 205 to 346 of human CD123.

[83] An antibody or antigen-binding fragment thereof (e.g., a cysteine-engineered antibody or antigen-binding fragment thereof, an anti-CD123 antibody or antigen-binding fragment thereof, or a cysteine-engineered antibody or antigen-binding fragment thereof) for use in these methods can be produced by a recombinant method. For example, an antibody or antigen-binding fragment thereof (e.g., a cysteine-engineered antibody or antigen-binding fragment thereof, an anti-CD123 antibody or antigen-binding fragment thereof, or a cysteine-engineered antibody or antigen-binding fragment thereof) for use in these methods can be produced in a mammalian cell line. , for example, in a CHO cell.

IV. Antibody compositions

[84] In accordance with the methods proposed herein, antibody compositions containing both antibodies with three light chains (H2L3) and their antigen-binding fragments, and antibodies with two light chains (H2L2) and their antigen-binding fragments can be applied to a cation exchange column for separating H2L3 and H2L2 species.

[85] Antibody compositions for use in the methods provided herein can be compositions in which from about 1% to about 20% of the antibodies or antigen-binding fragments thereof are H2L3 antibodies or antigen-binding fragments thereof. Antibody compositions for use in the methods provided herein may be compositions in which from about 1% to about 15%, or from about 5% to about 15%, or from about 3% to about 12%, or from about 10% to about 15% of the antibodies or antigen-binding fragments thereof in an antibody composition are H2L3 antibodies or antigen-binding fragments thereof.

[86] An antibody composition for use in the methods provided herein may contain a specific concentration of protein such that a specific loading density is applied to the cation exchange resin. The protein concentration (load density) may be, for example, from about 10 g/l to about 100 g/l. The protein concentration (load density) can be from about 30 g/L to about 50 g/L. The protein concentration (load density) can be from about 30 g/L to about 45 g/L. The protein concentration (load density) can be from about 30 g/L to about 40 g/L. The protein concentration (load density) may be approximately 40 g/l.

[87] In addition to the H2L2 and H2L3 species, the antibody composition may contain aggregates. For example, an antibody composition may contain from about 1% to about 10% aggregates. The antibody composition may contain from about 1% to about 5% aggregates. The antibody composition may contain from about 2% to about 5% aggregates.

[88] The composition of antibodies may have a specific pH, for example, from about 3.8 to about 6.5. The antibody composition may have a pH of about 3.8 to about 5.5. The antibody composition may have a pH from about 3.8 to about 5.0. The antibody composition may have a pH from about 3.8 to about 4.7. The antibody composition may have a pH of from about 3.8 to about 4.4. The antibody composition may have a pH from about 3.8 to about 4.2. The antibody composition may have a pH of from about 4.0 to about 5.0. The antibody composition may have a pH of from about 4.0 to about 4.7. The antibody composition may have a pH of from about 4.0 to about 4.4. The antibody composition may have a pH of from about 4.0 to about 4.2. The antibody composition may have a pH of about 4.2.

[89] the pH of the antibody composition can be, for example, the same as the pH of the balancing composition (binding composition), which, as described in more detail below, can be applied to the cation exchange resin before applying the antibody composition to the cation exchange resin. The pH of the antibody composition can be, for example, the same as the pH of the elution composition, which, as described in more detail below, can be applied to the cation exchange resin after the antibody composition to elute the H2L2 composition. The pH of the antibody composition may be the same as the pH of the balancing composition (binding composition) and the elution composition.

[90] In some embodiments, the antibody composition does not have a pH of 6.0. In some embodiments, the antibody composition has a pH less than 6.0.

[91] The antibody composition may contain protein A-purified antibodies or antigen-binding fragments thereof. The antibody composition may contain antibodies, or antigen-binding fragments thereof, that have been purified with Protein A and purified in an anion exchange column. Thus, an antibody composition may contain components such as buffers (eg, tris-acetic acid) and/or antibody aggregates, in addition to soluble H2L2 and H2L3 antibodies or antigen-binding fragments thereof.

V. Elution Solutions and Elution Methods for the Preparation of Compositions H2L2 and H2L3

[92] In accordance with the methods provided herein, antibodies with three light chains, (H2L3) and their antigen-binding fragments and antibodies with two light chains (H2L2) and their antigen-binding fragments can be separately eluted from the cation exchange column.

[93] In particular, the eluent composition may be applied to a cation exchange resin (eg, column) for preferential elution of the H2L2 species, and then the H2L2 composition may be collected from the resin. Elution compositions are provided herein for use in such methods.

[94] The elution composition for use in the methods provided herein may contain a salt. The salt may be a chloride salt, such as sodium chloride, potassium chloride, calcium chloride or magnesium chloride. In one case, the salt is sodium chloride. The concentration of salt (eg, sodium chloride) in the elution composition may be, for example, from about 100 mm to about 600 mm. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 200 mM to about 600 mM. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 300 mm to about 600 mm. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 400 mm to about 600 mm. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 200 mm to about 500 mm. The concentration of salt (eg, sodium chloride) in the eluent composition may be from about 300 mM to about 500 mM. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 400 mm to about 500 mm. The concentration of salt (eg, sodium chloride) in the elution composition may be from about 380 mm to about 420 mm. The concentration of salt (eg, sodium chloride) in the elution composition may be approximately 400 mm.

[95] In some embodiments, the elution composition does not have a salt concentration of 100 mM. In some embodiments, the elution composition has a salt concentration greater than 100 mM.

[96] The elution composition for use in the methods provided herein may have a specific pH. The pH may be, for example, from about 3.8 to about 6.5. The elution composition may have a pH of from about 3.8 to about 5.5. The elution composition may have a pH of from about 3.8 to about 5.0. The elution composition may have a pH of from about 3.8 to about 4.7. The elution composition may have a pH of from about 3.8 to about 4.4. The elution composition may have a pH of from about 3.8 to about 4.2. The elution composition may have a pH of from about 4.0 to about 5.0. The elution composition may have a pH of from about 4.0 to about 4.7. The elution composition may have a pH of from about 4.0 to about 4.4. The elution composition may have a pH of from about 4.0 to about 4.2. The elution composition may have a pH of about 4.2.

[97] In some embodiments, the elution composition does not have a pH of 6.0. In some embodiments, the composition has an eluting pH of less than 6.0.

[98] The elution composition for use in the methods provided herein may have a particular combination of salt concentration and pH. For example, the salt (eg, sodium chloride) concentration may be from about 300 mM to about 600 mM, and the pH may be from about 3.8 to about 5.5. The salt (eg, sodium chloride) concentration may be from about 300 mM to about 500 mM, and the pH may be from about 3.8 to about 5.0. The salt (eg, sodium chloride) concentration may be from about 380 mM to about 420 mM, and the pH may be from about 4.0 to about 4.4. The salt concentration (eg sodium chloride) may be about 400 mM and the pH may be about 4.2.

[99] In some embodiments, the elution composition does not have 100 mM sodium chloride at pH 6.0.

[100] As demonstrated herein, applying the elution composition provided herein (e.g., low pH, high salt concentration) to a cation exchange resin provided herein containing an antibody composition provided herein with antibodies H2L2 and H2L3, or antigen-binding fragments thereof, can elute the H2L2 composition with little or no H2L3 contamination. This is because the methods provided herein can cause a continuous H2L3 elution (after the H2L2 elution peak) instead of both early (along with the H2L2 elution peak) and late (after the H2L2 elution peak) elution.

[101] Thus, the H2L2 composition provided herein may contain one or more eluted column volumes. For example, the H2L2 composition may contain one eluted column volume selected from column volumes 1-9. The H2L2 composition may contain two eluted column volumes selected from column volumes 1-9 (eg, column volumes 1 and 2 or column volumes 3 and 4). The H2L2 composition may contain three, four, five, six, seven, eight or nine eluted column volumes selected from column volumes 1-9. The H2L2 composition may also contain eluted column volumes 1-9 (ie, a pool of the first nine column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-8 (ie, a pool of the first four column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-7 (ie, a pool of the first four column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-6 (ie, a pool of the first four column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-5 (ie, a pool of the first four column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-4 (ie, a pool of the first four column volumes). The H2L2 composition provided herein may contain eluted column volumes 1-3 (ie, a pool of the first four column volumes).

[102] Using the methods provided herein, the H2L3 species can be efficiently separated from the H2L2 species in an antibody composition. For example, the methods used herein can result in an H2L2 composition containing no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the H2L3 species that was present in the antibody composition coated on cation exchange resin. On the other hand, the methods used herein can result in an H2L3 composition containing at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of H2L3 species that were present in the composition of the antibodies applied to the cation exchange resin.

[103] Using the methods described herein, an H2L2 composition can be prepared in which no more than 2%, no more than 1%, or no more than 0.5% of the antibodies or antigen-binding fragments are H2L3 species. Using the methods described herein, an H2L2 composition can be prepared in which at least 98%, at least 99%, or at least 99.5% of the antibodies or antigen-binding fragments thereof in the H2L2 composition are H2L2 antibodies or antigen-binding fragments thereof.

[104] Using the methods described herein, it is also possible to obtain an H2L2 composition containing fewer aggregates than an antibody composition coated on a cation exchange resin. For example, using the methods described in this document, it is possible to obtain an H2L2 composition containing no more than 1% of aggregates or no more than 0.5% of aggregates. Using the methods provided herein, an H2L2 composition containing approximately 0.3% aggregates, approximately 0.2% aggregates, or approximately 0.1% aggregates can be obtained.

[105] Advantageously, the methods provided herein also provide a high yield. For example, using the methods provided herein, an H2L2 composition can be prepared that contains at least 40%, at least 45%, at least 50%, or at least 55% of the H2L2 antibodies or antigen-binding fragments thereof in the antibody composition. applied to the cation exchange resin.

[106] To balance the cation exchange resin and promote binding of the H2L2 and H2L3 antibodies to the resin, the balancing composition (or binding composition) can be applied to the resin before the antibody composition is applied to the resin. An equilibrating composition (binding composition) can be used to maintain the pH and/or conductivity of the resin. Suitable buffers that can be used for this purpose are well known in the art and include any pH buffer that is compatible with the selected resin used in the chromatography step to separate the H2L3 and H2L2 species. The balancing composition (binding composition) may contain, for example, sodium acetate, at a concentration that is high enough to maintain pH, but not too high to prevent binding of antibodies and their antigen-binding fragments in the composition of antibodies to the cation exchange resin.

[107] Composition for balancing (composition for binding) may contain, for example, from 10 mm to 150 mm sodium acetate. Composition for balancing (composition for binding) may contain, for example, from 25 mm to 150 mm sodium acetate. Composition for balancing (composition for binding) may contain 50 mm sodium acetate.

[108] In some embodiments, the balancing composition (binding composition) does not contain 20 mM sodium acetate. In some embodiments, the composition for balancing (composition for binding) contains more than 20 mm sodium acetate.

[109] Composition for balancing (composition for binding) can also have a certain pH, for example, from about 3.8 to about 6.5. Composition for balancing (composition for binding) may have a pH of from about 3.8 to about 5.5. Composition for balancing (composition for binding) may have a pH of from about 3.8 to about 5.0. Composition for balancing (composition for binding) may have a pH of from about 3.8 to about 4.7. Composition for balancing (composition for binding) may have a pH of from about 3.8 to about 4.4. Composition for balancing (composition for binding) may have a pH of from about 3.8 to about 4.2. Composition for balancing (composition for binding) may have a pH of from about 4.0 to about 5.0. Composition for balancing (composition for binding) may have a pH of from about 4.0 to about 4.7. Composition for balancing (composition for binding) may have a pH of from about 4.0 to about 4.4. Composition for balancing (composition for binding) may have a pH of from about 4.0 to about 4.2. The balancing composition (binding composition) may have a pH of about 4.2.

[110] In some embodiments, the balancing composition (binding composition) does not have a pH of 6.0. In some embodiments, the balancing composition (binding composition) has a pH less than 6.0.

[111] As demonstrated herein, a method for separating H2L3 antibodies, or antigen-binding fragments thereof, from an antibody composition comprising H2L3 antibodies, or antigen-binding fragments thereof, and H2L2 antibodies, or antigen-binding fragments thereof, may include (i) applying the antibody composition to a cation exchange resin such that the antibodies H2L3 or antigen-binding fragments thereof and H2L2 antibodies or antigen-binding fragments thereof bind to the resin; (ii) applying the eluent composition to a cation exchange resin; and (iii) collecting the H2L2 composition eluted from the resin. The method may optionally include applying the equilibration composition (binding composition) to the cation exchange resin before the antibody composition is applied to the cation exchange resin. As demonstrated herein, the choice of cation exchange resin and the pH and salt concentration of the eluent composition can advantageously result in all H2L3 species being eluted after the H2L2 peak is reached and allows collection of H2L2 compositions with little (e.g., less than 1%) or lack of H2L3 species.

VI. Application of H2L2 compositions

[112] In accordance with the methods proposed herein, antibodies with three light chains (H2L3) and their antigen-binding fragments can be separated from antibodies with two light chains (H2L2) and their antigen-binding fragments to obtain compositions of H2L2. Such H2L2 compositions are useful, for example, for therapeutic purposes. For example, H2L2 compositions can be used to prepare pharmaceutical compositions containing highly pure H2L2 antibodies or antigen-binding fragments thereof, eg compositions containing no more than, for example, 1% or 0.5% H2L3 species.

[113] Compositions of H2L2, obtained in accordance with the methods described herein, can also be used to obtain immunoconjugates. Advantageously, immunoconjugates prepared from the H2L2 compositions provided herein will contain few or no H2L3 species. Such immunoconjugates can be prepared using a linker group to link a drug or prodrug to an antibody or antigen-binding fragment thereof. Suitable linker groups are well known in the art and include, for example, disulfide groups, thioether groups, acid groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. A single immunoconjugate may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs or prodrugs per antibody or antigen-binding fragment thereof. A composition containing such immunoconjugates may have an average of about 1 to about 10, about 1 to about 5, about 1 to about 3, or about 1.5 to about 2.1 drugs or prodrugs per antibody or antigen-binding agent thereof. fragment.

[114] The composition of immunoconjugates obtained in accordance with the methods proposed herein may contain no more than 2%, no more than 1%, or no more than 0.5% of H2L3 species. An immunoconjugate composition prepared according to the methods provided herein may contain at least 98%, at least 99%, or at least 99.5% H2L2 species.

[115] For example, an H2L2 composition containing anti-CD123 H2L2 antibodies or antigen-binding fragments thereof (eg, huCD123-6Gv4.7; G4723A) can be conjugated to a cytotoxic agent to form an immunoconjugate. The cytotoxic agent can be, for example, an indoline benzodiazepine cancer cell killing agent such as DGN549-C. Methods for preparing such immunoconjugates are presented, for example, in WO 2017/004025 and WO 2017/004026; the contents of which are incorporated herein by reference in their entirety.

[116] In some embodiments, an H2L2 composition comprising anti-CD123 H2L2 antibodies or antigen-binding fragments thereof (eg, huCD123-6Gv4.7; G4723A) may be conjugated to DGN549-C. The resulting immunoconjugate composition may contain an average of 1.5 to 2.1 cytotoxins (DGN549-C) per antibody (huCD123-6Gv4.7; G4723A). The immunoconjugates can be formulated in sodium bisulfate to form IMGN632 as shown in FIG. 8A (and 8B).

EXAMPLES

[117] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of this application.

Example 1: Assessing H2L3 levels

[118] It has previously been shown that mammalian cells stably transfected to express such cysteine-engineered antibodies also secrete a high molecular weight species known as a tri-light chain (H2L3) antibody (Gomez et al., Biotechnol Bioeng. 2010 Mar 1;105 (4):748-60). These antibodies contain an additional (third) light chain linked to an engineered cysteine on the antibody. Separation of this new high molecular weight (HMW) species poses a problem during subsequent purification of cysteine engineered monoclonal antibodies (CysmAb) due to the similarity to the monomeric species. In order to develop a robust and efficient purification strategy for the removal of H2L3 species, the H2L3 content of an exemplary antibody was evaluated, and various methods for removing H2L3 were evaluated.

[119] 0 The illustrative monoclonal antibody tested was the cysteine engineered huCD123-6Gv4.7 (G4723A) antibody (see WO 2017/004025 and WO 2017/004026, the contents of which are incorporated herein by reference in their entirety; see also Tables 1-3 higher). The antibody was expressed in CHO cells and then purified from purified cell culture supernatants using protein A chromatography. After protein A chromatography, the purity (including percentage of monomer, aggregates, H2L3 and small molecular weight species) of the antibody was assessed using size exclusion ultra performance liquid chromatography (SEC-UPLC). SEC-UPLC analysis showed the H2L3 species (FIG. 1). The SEC-UPLC chromatogram shows the retention time for each peak of the CysmAb composition species: monomer (17.686 minutes), aggregates (15.336 minutes), H2L3 (16.638 minutes) and small molecular weight species (19.008 and 22.445 minutes) (Fig. 1).

[120] Experiments were analyzed using eight different production batches of the bioreactor of the same antibody (A, B, C, D, E, F, G and H). The amount of H2L3 depended both on the cell line and on the cultivation condition and ranged from 2 to 11% (Fig. 2).

Example 2 Ceramic hydroxyapatite chromatography is ineffective for separating H2L3

[121] Removal of the H2L3 species has not been effectively achieved by ceramic hydroxyapatite (CHT) chromatography. (Fig. 3). In these experiments, the CHT column was equilibrated with a buffer containing 20 mM potassium phosphate at pH 6.7. CysmAb with 4.7% H2L3 species was loaded onto CHT™ Resin (BioRad Laboratories, Hercules, CA) and eluted by stepwise elution using 100 mM, 95 mM, 90 mM, or 85 mM potassium phosphate buffer at pH 6.7. The elution peak was collected at mAU above 50 in 1CV fractions. Single step yield of antibody and H2L3 % of each fraction was analyzed and used for calculation and comparison with single step yield of antibody and H2L3 % virtual pool with 6 column volumes (CV). It was expected that high molecular weight species (ie, aggregates and H2L3) would bind more strongly to the CHT resin due to their larger size and would elute in subsequent fractions. In addition, it was thought that elution with a lower concentration of phosphate would result in broader elution peaks and a lower yield in one virtual pool step while improving separation. However, in FIG. 3 shows that H2L3 species removal did not improve within the tested phosphate range and remained at 2.5%. Additional experiments to change the salt concentration also failed to effectively separate the H2L3 species, and there was little opportunity to change the pH in the CHT column. Thus, CHT chromatography is unable to efficiently separate H2L3.

Example 3 Optimized Cation Exchange Chromatography Efficiently Separates H2L3

[122] Removal of H2L3 species was verified using POROS™ XS strong cation exchange chromatography. POROS™ XS strong cation exchange resin (Life Technologies Corporation, Carlsbad, CA) was equilibrated with a buffer containing 50 mM sodium acetate at pH 5.5. A CysmAb formulation with 3% aggregates and 4% H2L3 species was loaded onto a POROS™ column XS and eluted using a gradient elution using 0-200 mm NaCl for 20 CV. The elution peak was collected at mAU above 50 in fractions of 0.5 CV. Antibody yield at one stage, aggregate % and H2L3% of each fraction were analyzed and plotted against the number of fractions. On FIG. 4 shows that aggregated species were eluted in late fractions (F8-F10) and effectively separated from the main peak of the eluted antibody. H2L3 species were also eluted in different fractions from the main peak of the eluted antibody. A significant portion of the H2L3 species eluted in late fractions and effectively separated from the main H2L2 antibody peak (FIG. 4). However, a subset of H2L3 species co-eluted with earlier antibody peak fractions (FIG. 4). The early eluting H2L3 species is believed to result from the fact that the free cysteine in the H2L3 antibodies is capped with glutathione. The glutathione-capped species is more acidic (lower PI) and hence eluted in an earlier fraction. The late-eluting H2L3 species is thought to result from free cysteine in H2L3 antibodies being capped with cysteine. The cysteine capped species is less acidic (higher PI) and hence eluted in a later fraction.

[123] Based on these results, further optimization of the binding and elution conditions from the POROS™ XS column was done to cause the early eluting H2L3 species to be eluted in a later fraction for more efficient separation from the main H2L2 peak. Lowering the binding and elution pH in Poros XS chromatography was found to significantly improve the removal of H2L3 particles (FIG. 5).

[124] NaCl gradient elution was performed using POROS™ XS chromatography at pH 5.0, 4.7 and 4.4 (at pH 5.0, 150-300 mM NaCl for 20CV; at pH 4.7 and 4, 4, 0-500 mM NaCl for 25 CV). The POROS™ XS resin coating material (antibody formulation) contained approximately 8.4% H2L3 species. The elution peak was collected at mAU above 100 in fractions of 0.5 CV. Analyzed the yield of antibodies at one stage and H2L3% of each fraction. H2L3 % vpool with different volume collected was plotted against the output of each vpool. The number of co-eluted H2L3 species in earlier antibody peak fractions decreased with decreasing pH. On FIG. 5 shows that lowering the binding and elution pH from the POROS™ XS column significantly improved the removal of H2L3 species such that lower pH elution gave a product with lower levels of H2L3 species. At pH 4.4, H2L3 was efficiently separated from the antibody and eluted only in later fractions. Elution at a lower pH produces a product (composition H2L2) with a lower content of H2L3 species.

[125] Salt concentration and peak elution volume collected have also been shown to influence H2L3 removal (FIG. 6). In these experiments, the starting material (antibody composition) with approximately 4.1% H2L3 species was loaded onto a POROS™ XS column equilibrated with 50 mM sodium acetate at pH 4.2. Bound antibody was eluted using 380-420 mM NaCl in 50 mM sodium acetate buffer at pH 4.2. The elution peak was collected at mAU above 100 in fractions of 1 CV. H2L3% virtual pools were eluted at various NaCl concentrations and collection volumes. On FIG. 6 shows that the lower the NaCl concentration and the lower the volume collected, the lower the H2L3% achieved in the elution pool. Over the entire range of NaCl H2L3 concentrations tested, % of virtual pools was reduced from 4.1% to <1%.

[126] The results of the experiment in FIG. 6 were analyzed using the JMP® Profile Prediction Tool. A mathematical model was built to predict H2L3% yield and total macromolecular species (HMW)% at different salt concentrations and volumes collected. On FIG. 7 shows that the salt concentration significantly affected all responses, while the collected volume significantly affected only the yield at one stage. The desirability of each response was determined based on product quality requirements and practical considerations. For the final process condition, combinations of salt concentration and collection volume with the highest overall desirability were considered.

[127] By optimizing binding and elution conditions, including pH, salt concentration, loading density, and collection volume, H2L3 levels were consistently reduced to < 1% in the final product (Table 4; experiments were performed under the same POROS conditions as described above, at pH 4.2, with 400 mM NaCl, collecting 4CV (i.e. column volume 1-4)).

[128] Based on the experiments described above, it was determined that the ideal range for pH, salt, load density and volume collected is as follows: pH from about 3.8 to 6.5, salt concentration from about 100 mM to 600 mM, a loading density of about 10-100 g/l, and a collection volume of about 1 to 9 CV.

[129] It should be appreciated that the "Detailed Description" section, and not the "Summary" and "Abstract" sections, is for interpretation of the claims. The "Summary" and "Abstract" sections identify one or more, but not all, illustrative embodiments of the present invention as intended by the inventor(s), and thus are not intended to limit the present invention and the appended claims in any way.

[130] The present invention has been described above with functional building blocks illustrating the implementation of these functions and their relationships. The boundaries of these functional building blocks have been arbitrarily defined herein for convenience of description. Alternative boundaries can be defined as long as the specified functions and their relationships are properly performed.

[131] The foregoing description of specific embodiments fully reflects the general nature of the invention, which others can, applying knowledge within the prior art, easily modify and / or adapt for various applications of such specific embodiments, without undue experimentation, without departing from the general concept of this invention. . Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the instructions and guidance provided herein. It should be understood that the language or terminology in this document is intended to be descriptive and not restrictive, so that the terminology or language in this description should be interpreted by the skilled artisan in light of the instructions and guidance.

[132] The scope and scope of this invention should not be limited by any of the above illustrative embodiments, but should only be determined in accordance with the following claims and their equivalents.

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SEQUENCE LIST

<110> ImmunoGen, Inc.

LIU Fang

LI Xinfang

<120> Separation of antibodies with three light chains using a cation exchange

chromatography

<130> 2921.097PC01/EKS/CLD/BMB

<150> 62/562 188

<151> 09/22/2017

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<170> PatentIn version 3.5

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<---

Claims (74)

1. A method for separating H2L3 antibodies from an antibody composition containing H2L3 antibodies and H2L2 antibodies, said method comprising: (i) applying the antibody composition to a cation exchange resin such that the H2L3 antibodies and H2L2 antibodies bind to the resin, wherein the antibody composition has a pH of less than 6.0; (ii) applying the composition for elution with a salt concentration of from 300 mm to 600 mm on a cation exchange resin; And (iii) collecting a composition containing H2L2 antibodies eluted from the resin, said composition containing H2L2 antibodies containing one or more eluted column volumes selected from column volumes 1-9; wherein said H2L3 antibodies and H2L2 antibodies comprise an IgG heavy chain constant domain. 2. The method according to claim 1, characterized in that no more than 2% of the antibodies in the composition containing H2L2 antibodies are H2L3 antibodies. 3. The method according to any one of paragraphs. 1, 2, characterized in that at least 98% of the antibodies in the composition containing H2L2 antibodies are H2L2 antibodies. 4. The method according to any one of paragraphs. 1, 2, characterized in that the composition containing H2L2 antibodies contains no more than 25% of H2L3 antibodies in the antibody composition applied to the cation exchange resin. 5. The method according to any one of paragraphs. 1-4, characterized in that the composition containing H2L2 antibodies contains eluted column volumes 1-4. 6. The method according to any one of paragraphs. 1-5, characterized in that said cation exchange resin contains polystyrene crosslinked with divinylbenzene and/or surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -). 7. The method according to any one of paragraphs. 1-6, characterized in that the particle size of the cation exchange resin is 50 microns. 8. The method according to any one of paragraphs. 1-7, characterized in that said cation exchange resin has a bimodal pore size distribution, wherein the bimodal pore size distribution includes pores with a diameter of 500 nM and pores with a diameter of 22 nM. 9. The method according to any one of paragraphs. 1-8, characterized in that the salt in said composition for elution contains a chloride salt. 10. The method according to claim 9, wherein said chloride salt is sodium chloride, potassium chloride, calcium chloride or magnesium chloride. 11. The method according to any one of paragraphs. 1-9, characterized in that the composition for elution has a pH of less than 6.0. 12. The method according to any one of paragraphs. 1-11, characterized in that said method includes applying the composition with a pH of less than 6.0 to the cation exchange resin before applying the antibody composition to the cation exchange resin. 13. The method of claim 11, wherein said composition, which is applied to the cation exchange resin prior to application of the antibody composition, contains sodium acetate. 14. The method according to any one of paragraphs. 1-13, characterized in that the antibody composition contains from 10 to 100 g/l of protein. 15. The method according to any one of paragraphs. 1-14, characterized in that from 1% to 20% in the antibody composition are H2L3 antibodies. 16. The method according to any one of paragraphs. 1-15, characterized in that the composition containing the H2L2 antibodies contains at least 40% of the H2L2 antibodies in the antibody composition applied to the cation exchange resin. 17. The method according to any one of paragraphs. 1-16, characterized in that the antibody composition contains cysteine-engineered antibodies. 18. The method of claim 17 wherein said cysteine-engineered antibodies contain an engineered cysteine residue at position 442 according to EU/OU numbering. 19. The method according to any one of paragraphs. 1-18, characterized in that the antibody composition contains antibodies produced by the CHO cell line. 20. The method according to p. 1, characterized in that (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), has a particle size of 50 μm and a bimodal pore size distribution, including a pore diameter of 500 nM and a pore diameter of 22 nM ; (ii) the composition for elution contains from 300 mm to 600 mm chloride salt and has a pH of less than 6.0; (iii) the antibody composition contains from 10 to 100 g/l of protein and from 10% to 15% of the antibodies in the antibody composition are H2L3 antibodies; And (iv) not more than 2% of the antibodies in the composition containing H2L2 antibodies are H2L3 antibodies. 21. The method according to p. 1, characterized in that (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), has a particle size of 50 μm and a bimodal pore size distribution, including a pore diameter of 500 nM and a pore diameter of 22 nM ; (ii) the composition for elution contains 400 mm NaCl and has a pH of 4.2; (iii) the antibody composition contains 30 to 50 g/L of protein and 10% to 15% of the antibodies in the antibody composition are H2L3 antibodies; (iv) a composition containing H2L2 antibodies contains eluted column volumes 1-4; And (v) not more than 1% of the antibodies in the composition containing the H2L2 antibodies are H2L3 antibodies. 22. The method according to any one of paragraphs. 1-21, characterized in that said IgG heavy chain constant domain is an IgG1 heavy chain constant domain. 23. The method according to any one of paragraphs. 1-21, characterized in that said IgG heavy chain constant domain is an IgG2 heavy chain constant domain. 24. The method according to any one of paragraphs. 1-21, characterized in that said IgG heavy chain constant domain is an IgG4 heavy chain constant domain. 25. A method for separating anti-CD123 H2L3 antibodies from an anti-CD123 antibody composition comprising anti-CD123 H2L3 antibody and anti-CD123 H2L2 antibody, said method comprising: (i) applying the anti-CD123 antibody composition to a cation exchange resin such that the anti-CD123 antibody H2L3 and anti-CD123 antibody H2L2 bind to the resin, wherein the pH of said antibody composition is less than 6.0; (ii) applying the composition for elution with a pH of less than 6.0 and with a salt concentration of from 300 mm to 600 mm on a cation exchange resin; And (iii) collecting a composition containing anti-CD123 H2L2 antibodies eluted from the resin, said composition containing anti-CD123 H2L2 antibodies containing one or more eluted column volumes selected from column volumes 1-9, wherein the H2L3 anti-CD123 antibody and the H2L2 anti-CD123 antibody comprise: (i) the heavy chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 5-7, respectively, and the light chain variable region CDR1, CDR2 and CDR3 sequences of SEQ ID NO : 8-10, respectively, and (ii) an IgG heavy chain constant domain. 26. The method of claim 25, wherein the anti-CD123 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 2. 27. The method according to claim 25 or 26, characterized in that the anti-CD123 antibody is constructed by introducing cysteine. 28. The method according to any one of paragraphs. 25-27, characterized in that no more than 2% of the antibodies in the composition containing H2L2 antibodies are H2L3 antibodies. 29. The method according to any one of paragraphs. 25-28, characterized in that at least 98% of the antibodies in the composition containing H2L2 antibodies are H2L2 antibodies. 30. The method according to any one of paragraphs. 25-29, characterized in that the composition containing H2L2 antibodies contains no more than 25% of H2L3 antibodies in the antibody composition applied to the cation exchange resin. 31. The method according to any one of paragraphs. 25-27, characterized in that the composition containing the H2L2 antibodies contains the eluted column volumes 1-4. 32. The method according to any one of paragraphs. 25-31, characterized in that said cation exchange resin contains polystyrene crosslinked with divinylbenzene and/or surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -). 33. The method according to any one of paragraphs. 25-32, characterized in that the particle size of the cation exchange resin is 50 microns. 34. The method according to any one of paragraphs. 25-33, characterized in that said cation exchange resin has a bimodal pore size distribution, wherein the bimodal pore size distribution includes pores with a diameter of 500 nM and pores with a diameter of 22 nM. 35. The method according to any one of paragraphs. 25-33, characterized in that the salt in said composition for elution is a chloride salt. 36. The method according to claim 35, wherein said chloride salt is sodium chloride, potassium chloride, calcium chloride or magnesium chloride. 37. The method according to any one of paragraphs. 25-35, characterized in that said method includes applying the composition with a pH of less than 6.0 to the cation exchange resin before applying the antibody composition to the cation exchange resin. 38. The method of claim 37, wherein said composition, which is applied to the cation exchange resin prior to application of the antibody composition, contains sodium acetate. 39. The method according to any one of paragraphs. 25-38, characterized in that the antibody composition contains from 10 to 100 g/l of protein. 40. The method according to any one of paragraphs. 25-39, characterized in that from 1% to 20% of the antibodies in the antibody composition are H2L3 antibodies. 41. The method according to any one of paragraphs. 25-40, characterized in that the composition containing the H2L2 antibodies contains at least 40% of the H2L2 antibodies in the antibody composition applied to the cation exchange resin. 42. The method according to any one of paragraphs. 25-41, characterized in that the antibody composition contains cysteine-engineered antibodies. 43. The method according to any one of paragraphs. 25-42, characterized in that the antibody composition contains antibodies produced by the CHO cell line. 44. The method according to any one of paragraphs. 25-43 further comprising conjugating H2L2 antibodies in a composition containing H2L2 antibodies with DGN549-C to form a composition containing an immunoconjugate, wherein said immunoconjugate has the following structure:

45. The method according to p. 25, characterized in that (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), has a particle size of 50 μm and a bimodal pore size distribution, including a pore diameter of 500 nM and a pore diameter of 22 nM ; (ii) the composition for elution contains from 300 mm to 600 mm chloride salt and has a pH of less than 6.0; (iii) the antibody composition contains from 10 to 100 g/l of protein, and from 10% to 15% of the antibodies in the antibody composition are H2L3 antibodies; And (iv) not more than 2% of the antibodies in the composition containing the H2L2 antibodies are H2L3 antibodies. 46. The method according to claim 25, characterized in that (i) the cation exchange resin contains polystyrene crosslinked with divinylbenzene, surface functional groups of sulfopropyl (-CH ₂ CH ₂ CH ₂ SO ₃ -), has a particle size of 50 μm and a bimodal pore size distribution, including a pore diameter of 500 nM and a pore diameter of 22 nM ; (ii) the composition for elution contains 400 mm NaCl and has a pH of 4.2; (iii) the antibody composition contains from 30 to 50 g/l of protein, and from 10% to 15% of the antibodies in the antibody composition are H2L3 antibodies; (iv) a composition containing H2L2 antibodies contains eluted column volumes 1-4; And (v) not more than 1% of the antibodies in the composition containing the H2L2 antibodies are H2L3 antibodies. 47. The method according to any one of paragraphs. 25-46, characterized in that said IgG heavy chain constant domain is an IgG1 heavy chain constant domain.

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