RU2812161C2 - Antibody purification - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to a method of purifying antibodies from impurities, such as antibody degradation products. A method of purifying an anti-BCMA/anti-CD3 bispecific antibody is proposed. The antibody preparation is injected into the loading buffer onto the hydroxyapatite (HA) resin. The antibody preparation contains an intact bispecific antibody of interest with at least one type of impurity. Wash the resin with a wash buffer containing phosphate ions at a concentration of 10 to 50 mM. Elute the intact bispecific antibody of interest from the HA resin using an elution buffer containing phosphate ions at a concentration of 40 to 100 mM. The concentration of phosphate ions is increased compared to the wash buffer so that the bispecific antibody of interest is separated from at least one type of contaminant. The pH of the loading buffer, wash buffer and/or elution buffer is between pH 7.0 and 8.0. The resulting anti-BCMA/anti-CD3 bispecific antibody preparation contains not more than 1% of trimmed bispecific antibody as an impurity.

EFFECT: obtaining a method of antibody purification.

13 cl, 7 dwg, 5 tbl, 4 ex

Description

This application claims benefit to U.S. Provisional Application No. 62/635,943, filed February 27, 2018, the contents of which are hereby incorporated by reference in their entirety.

Technical field

The present invention relates to methods for purifying antibodies from impurities such as antibody degradation products. The purification methods of the present invention include the use of hydroxyapatite resins.

State of the art

Antibodies are important biological molecules for medical, diagnostic, industrial and other uses. Although many methods and reagents are available for recombinant antibody production, due to, for example, the size and molecular complexity of antibodies, efficient production and purification of the recombinant antibody of interest often remains difficult, particularly on a large/industrial scale.

For example, during the production of a recombinant antibody of interest, a degradation product related to the antibody of interest may sometimes be generated; the degradation product is an undesirable impurity. This degradation product may have some molecular properties very similar to those of the antibody of interest (eg, identical or nearly identical amino acid sequences or masses). Due to the molecular similarity between the intact antibody of interest and the degraded version of the antibody, efficient separation of the intact antibody from the degraded antibody can be very difficult.

Thus, there is a need for new and improved methods for purifying antibodies from impurities.

The essence of the invention

The present invention relates to methods for purifying an antibody of interest from one or more impurities.

In some embodiments, the present invention provides a method for purifying an antibody, comprising: A) introducing an antibody preparation into a loading buffer on a hydroxyapatite (HA) resin, wherein: the antibody preparation contains: I) an intact antibody of interest, and II) a clipped version of the antibody of interest, wherein the clipped the version of the antibody of interest is a degradation product of the intact antibody of interest and has a mass that differs by less than 10% from the mass of the intact antibody of interest; and B) eluting the intact antibody of interest from the HA resin using elution buffer.

In some embodiments, the present invention provides a method for purifying a bispecific antibody, comprising: A) introducing an antibody preparation into a hydroxyapatite (HA) resin loading buffer, wherein: the antibody preparation contains: I) an intact bispecific antibody of interest; and ii) at least one type of impurity, wherein the type of impurity is selected from the group consisting of: a) a clipped version of the bispecific antibody of interest, wherein the clipped version of the bispecific antibody of interest is a degradation product of the intact bispecific antibody of interest and has a mass difference of less than 10 % by weight of the intact bispecific antibody of interest; b) a first parent antibody, where the first parent antibody is a monospecific antibody having the same antigenic specificity as the first arm of the intact bispecific antibody; c) a second parent antibody, where the second parent antibody is a monospecific antibody having the same antigenic specificity as the second arm of the intact bispecific antibody; and d) high molecular weight compounds (HMMS); and B) eluting the intact bispecific antibody of interest from the HA resin using elution buffer.

In some embodiments, the present invention provides a method for purifying a bispecific antibody, comprising: A) introducing an antibody preparation into a hydroxyapatite (HA) resin loading buffer, wherein: I) the antibody preparation contains: a) an intact bispecific antibody of interest and b) a clipped version of the antibody of interest a bispecific antibody, wherein the clipped version of the antibody of interest is a degradation product of the intact bispecific antibody of interest and has a mass that differs by less than 10% from the weight of the intact bispecific antibody of interest; and ii) the ratio of clipped bispecific antibody molecules to intact bispecific antibody molecules in the antibody preparation is from at least 1:50 to no more than 1:5; B) eluting the intact bispecific antibody from the HA resin using elution buffer. In some embodiments, the method further includes the step C) of collecting a purified fraction eluted from the HA resin, wherein the purified fraction contains an intact bispecific antibody.

In some embodiments, the present invention provides a method for purifying an antibody, comprising: A) introducing an antibody preparation into a hydroxyapatite (HA) resin loading buffer, wherein: I) the antibody preparation contains: a) an intact antibody of interest, and b) a clipped version of the antibody of interest, wherein the clipped version of the antibody of interest is a degradation product of the intact antibody of interest and has a mass that differs by less than 10% from the mass of the intact antibody of interest; and ii) the clipped version of the antibody constitutes at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% of the antibody preparation by weight; B) eluting the intact antibody from the HA resin using an elution buffer, and C) collecting a purified fraction eluting from the HA resin, wherein the purified fraction contains the intact antibody and contains less than 1%, 2%, 3%, 4%, 5% , 6%, 7%, 8%, 9%, 10%, 15% or 20% wt. clipped antibody, where the purified fraction contains a lower wt. clipped antibody than the antibody preparation.

In some embodiments, in a method of the present invention comprising eluting an antibody of interest from an HA resin using an elution buffer, the elution buffer contains an ion. Optionally, the ion concentration in the buffer is increased during elution. Optionally, the ion concentration in the buffer around the HA resin is increased during elution.

In some embodiments, in a method of the present invention comprising an antibody, the antibody is a heterodimeric bispecific antibody.

In some embodiments, in a method of the present invention comprising collecting a purified fraction eluting from an HA resin, wherein the purified fraction contains an intact antibody of interest, the purified fraction contains at least 80%, 90%, 95%, 96%, 97% , 98% or 99% wt. intact antibody of interest.

In some embodiments, in the method of the present invention, including a purified fraction containing an intact bispecific antibody and a clipped bispecific antibody, the ratio of clipped bispecific antibody molecules to intact bispecific antibody molecules in the purified fraction is no more than 1:400, 1:200, 1:100 or 1:50.

In some embodiments, in a method of the present invention comprising an antibody of interest that is an anti-CD3 antibody or a bispecific antibody comprising an anti-CD3 arm, the antibody comprises at least one of the following: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO : 1; ii) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 2; iii) a VH region containing the amino acid sequence shown in SEQ ID NO: 1, and a VL region containing the amino acid sequence shown in SEQ ID NO: 3; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 2 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, in a method of the present invention comprising a bispecific antibody, the bispecific antibody is: i) an anti-BCMA/anti-CD3 bispecific antibody comprising an anti-BCMA arm and an anti-CD3 arm, or ii) an anti-FLT3/anti-CD3 bispecific antibody containing an arm anti-FLT3 and anti-CD3 arm.

In some embodiments, in a method of the present invention comprising a bispecific antibody comprising an anti-BCMA arm, the anti-BCMA arm comprises at least one of the following: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 5; ii) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 6; iii) a VH region containing the amino acid sequence shown in SEQ ID NO: 5, and a VL region containing the amino acid sequence shown in SEQ ID NO: 7; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 6 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 8.

In some embodiments, in a method of the present invention comprising a bispecific antibody comprising an anti-FLT3 arm, the anti-FLT3 arm comprises at least one of the following: i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 9; ii) a heavy chain containing the amino acid sequence shown in SEQ ID NO: 10; iii) a VH region containing the amino acid sequence shown in SEQ ID NO: 9, and a VL region containing the amino acid sequence shown in SEQ ID NO: 11; or iv) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10 and a light chain containing the amino acid sequence set forth in SEQ ID NO: 12.

In some embodiments, in a method of the present invention comprising incorporating an antibody preparation into an HA resin, the antibody preparation is incorporated into the HA resin to a density on the resin of 2, 3, 4, or 5 g/L to 8, 9, 10, 12. 15 or 20 g/l.

In some embodiments, in a method of the present invention comprising incorporating an antibody drug into an HA resin, at least 1, 5, 10, 50, 100, 500, 1000, or 5000 grams of the antibody drug are incorporated into the HA resin.

In some embodiments, in a method of the present invention involving an antibody preparation, the antibody preparation contains at least 50%, 60%, 70%, or 80%, but less than 90%, 95%, 97%, 98%, or 99% by weight. intact antibody of interest.

In some embodiments, in a method of the present invention involving a clipped antibody, the clipped antibody has a weight that differs by less than 0.1%, 0.5%, 1%, or 2% from the weight of the intact antibody. In some embodiments, in a method of the present invention involving a clipped antibody, the clipped antibody has a mass that is about 5 to 100 Da greater than the mass of the intact antibody. In some embodiments, in a method of the present invention involving a clipped antibody, the clipped antibody has a mass that is approximately 18 Da greater than the mass of the intact antibody.

In some embodiments, in a method of the present invention including a clipped antibody, the clipped antibody has a cleavage peptide bond in the polypeptide chain of the antibody, and wherein the cleavage peptide bond is in the heavy chain of the antibody. In some embodiments, in a method of the present invention including a clipped antibody, the clipped antibody has a cleavage peptide bond in the polypeptide chain of the antibody, and wherein the cleavage peptide bond is in the light chain of the antibody.

In some embodiments, in a method of the present invention involving a clipped antibody, the clipped antibody contains the same amounts of amino acids and the same amino acid sequences as the intact antibody. Alternatively, in some embodiments, the clipped antibody contains a different amount of amino acids than the intact antibody.

In some embodiments, in a method of the present invention comprising an antibody of interest comprising VH and VL domains that specifically bind to CD3, the corresponding clipped antibody contains a cleavage peptide bond in the VH domain that specifically binds to CD3.

In some embodiments, in the method of the present invention comprising an HA resin, the HA resin is a ceramic hydroxyapatite (cHA) resin.

In some embodiments, in the method of the present invention, the HA resin is washed with a wash buffer containing a phosphate ion after introducing the antibody preparation into the HA resin, but before eluting the intact bispecific antibody from the resin. Optionally, the wash buffer contains phosphate ions at a concentration of from about 5, 10, 15, 20 to 30, 40 or 50 mM.

In some embodiments, in the method of the present invention, including an elution buffer containing an ion, the ion is a phosphate. In some embodiments, the phosphate ion concentration during elution may increase from about 30, 40, or 50 mM to about 60, 70, 80, 100, 150, or 200 mM.

In some embodiments, in the method of the present invention, the pH of at least one of the loading buffer, wash buffer, and elution buffer is from 7.0 to 8.0, or about 7.0 to 8.0.

In some embodiments, in a method of the present invention comprising an antibody preparation, the antibody preparation contains proteins previously introduced and eluted from at least one of: i) a protein A resin and ii) an ion exchange resin. Optionally, the antibody preparation contains proteins previously loaded and eluted from both i) the protein A resin and ii) the ion exchange resin.

In some embodiments, the antibody produced by the method of the present invention is isolated and/or purified for use as pharmaceuticals or in the preparation of pharmaceuticals.

In some embodiments, the present invention provides an antibody purified by the methods of the present invention.

Brief description of drawings

In fig. 1 shows a flowchart of a method for producing a bispecific antibody that can be purified by the methods of the present invention.

In fig. 2 shows a diagram of an example of i) an intact bispecific antibody (left side panel) and ii) a clipped version of a bispecific antibody (right side panel), in which the clipped bispecific antibody is an impurity that may be present in an antibody preparation with an intact bispecific antibody.

In fig. 3 is a chromatogram depicting the separation of a bispecific anti-BCMA/anti-CD3 antibody of interest (“POI”) from a variety of different impurities by elution from an HA resin.

In fig. 4 is a graph showing the relative amounts of various proteins (including the antibody of interest and various impurities) in various fractions eluting from the HA resin in the HA chromatography experiment shown in the chromatogram of FIG. 3.

In fig. 5 is a chromatogram depicting the separation of a bispecific anti-BCMA/anti-CD3 antibody of interest (“POI”) from a variety of different impurities by elution from an HA resin.

In fig. 6 is a graph showing the relative amounts of various proteins (including the antibody of interest and various impurities) in various fractions eluting from the HA resin in the HA chromatography experiment shown in the chromatogram of FIG. 5, wherein the anti-BCMA/anti-CD3 bispecific antibody of interest is separated from a variety of different contaminants by elution from an HA resin.

In fig. 7 is a graph showing the relative amounts of various proteins (including the antibody of interest and various impurities) in various fractions eluting from an HA resin in an HA chromatography experiment in which an anti-FLT3/anti-CD3 bispecific antibody of interest is separated from a variety of different impurities by elution from HA resin.

Detailed description

The present invention relates to methods for purifying an antibody of interest from one or more impurities. The methods of the present invention involve the use of hydroxyapatite resin to separate the antibody of interest from impurities. In some embodiments, the antibody of interest is a bispecific antibody. In some embodiments, the impurity is an antibody related to the antibody of interest (i.e., having similar or the same amino acid sequences as the antibody of interest), but modified in one or more ways from the antibody of interest and having a different mass than the antibody of interest . Optionally, the mass of the antibody contaminant species is very similar to the mass of the antibody of interest. For example, in some embodiments, the weight of the antibody impurity species differs by less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, or 0. 01% by weight of the antibody of interest. Optionally, the methods of the present invention can be used to large-scale purify an antibody of interest from one or more impurities.

Definitions

Unless otherwise indicated, all terms of the art, notes and other scientific terms or terminology used in this description are intended to have the meanings commonly understood by those skilled in the art to which the invention belongs. In some cases, terms with a commonly understood meaning are defined herein for clarity and/or as a reference, and the inclusion of such definitions herein does not necessarily imply a material difference from the meaning generally understood in the art.

Unless otherwise specified, the following terms should be understood to have the following meanings:

An "antibody" is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain (ScFv) and domain antibodies (including, for example, shark antibodies and Camelids), diabodies and fusion proteins containing an antibody, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site. The term "antibody" includes monospecific, bispecific and polyspecific antibodies. An antibody includes an antibody of any class, such as IgG, IgA or IgM (or a subclass thereof), and the antibody may not belong to any particular class. Depending on the amino acid sequence of the constant region of the heavy chains of an antibody, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

Within the scope of the invention, the terms “heavy chain”, “light chain”, “variable region” or “variable domain”, “framework region”, “constant domain”, etc. have their meaning generally accepted in the field of immunology, and refer to domains in natural immunoglobulins and the corresponding domains of recombinant binding proteins (for example, humanized antibodies, bispecific antibodies, single chain antibodies, chimeric antibodies, etc.). The basic structural unit of natural immunoglobulins is a tetramer containing two light chains and two heavy chains, typically expressed as a glycoprotein of approximately 150,000 Daltons. The amino-terminal (N-terminal) portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C-terminal) portion of each chain defines the constant region. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region containing a CH1 domain, a hinge domain, CH2 and CH3 domains. The variable regions of the IgG molecule contain regions of hypervariability, referred to as complementarity determining regions (CDRs), containing antigen-contacting residues, and non-CDR segments, referred to as framework regions (FRs), typically supporting structure and determining the positioning of CDR loops (although some framework residues may also contact the antigen). Each VH and VL contains three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-c. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY) and class (eg, IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

A "bispecific" antibody or "dual specificity" antibody is a hybrid antibody containing two different antigen-binding sites. The two antigen-binding regions of a bispecific antibody bind to two different epitopes, which may be located on the same or different protein targets.

The term “intact” antibody refers to a recombinant antibody containing all of the expected peptide bonds and amino acids of a recombinant antibody (ie, expected given the nucleic acid sequences encoding the antibody polypeptides). Conversely, the term “clipped” antibody refers to a version of the corresponding “intact” antibody that lacks at least one peptide bond compared to the corresponding “intact” antibody. As used herein, references to “antibody of interest” generally refer to the intact antibody of interest unless the context clearly indicates otherwise.

As used herein, reference to an “approximate” value or parameter includes embodiments relating to that value or parameter as such, as well as values or parameters that may be 10% less or greater than the specified numerical value of that parameter. For example, a reference to "about 5 mg" includes 5 mg as well as any value between 4.5 mg and 5.5 mg.

Methods

The methods of the present invention can be used to purify an antibody of interest from one or more impurities. In the methods of the present invention, an antibody preparation (also referred to herein as a "parent sample") containing the antibody of interest and one or more impurity molecules is applied to a hydroxyapatite (HA) resin that binds to the antibody of interest and, optionally, one or more impurity molecules impurities. The HA resin is then washed to remove any loosely bound impurities (in some embodiments, all impurity molecules may leak through and not bind to the HA resin). The antibody of interest is then eluted from the HA resin using a phosphate elution buffer, which is typically introduced into the resin using a gradient of increasing concentration of phosphate ion. By eluting the antibody of interest from the HA resin, a purified sample is obtained containing the antibody of interest and fewer (or no) contaminants than was present in the original sample containing the antibody of interest. During the elution of the antibody of interest from the HA resin, any impurity molecules bound to the HA resin may also be eluted at some point along the gradient of increasing phosphate ion concentration. However, the impurity molecules elute from the HA resin under conditions sufficiently different from the elution conditions of the antibody of interest such that the antibody of interest can be effectively separated from the impurity molecules during elution. Below are additional details about the above process steps and the corresponding materials and steps.

Hydroxyapatite resin

Various hydroxyapatite resins are available commercially, and any available form of material can be used in the methods of the present invention. Optionally, hydroxyapatite is in crystalline form. Optionally, the hydroxyapatite is agglomerated to form particles and sintered at high temperatures into a stable porous ceramic mass.

In some embodiments, the HA resin of the present invention is a ceramic hydroxyapatite (cHA) resin. The terms "ceramic hydroxyapatite"/"cHA" refer to an insoluble hydroxylated calcium phosphate of the formula Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ sintered at high temperatures into a spherical, macroporous ceramic form. As used herein, the terms "ceramic hydroxyapatite"/"cHA" include, but are not limited to, type I and type II ceramic hydroxyapatite, as well as any suitable particle size unless otherwise specified. Typical cHA particle sizes that can be used in the methods of the present invention include, for example, a particle size (diameter) of 1-100 μm or 1-1000 μm, such as 20 μm, 40 μm or 80 μm. Examples of cHA resins that can be used in the methods of the present invention include CHT™ Type I and Type II resins (Bio-Rad). Any reference herein to "HA resin" or the like. includes cHA resin.

Typically, in the method of the present invention, the HA resin is provided in one or more chromatography columns. Column properties, such as diameter, length, and column packing density, can be selected based on various factors, including the needs of the specific purification project (i.e., the amount of protein to be purified) and factors related to the HA resin intended for column use, such as pore size, particle size, compressibility, loading capacity, and dynamic binding capacity. In addition, the methods of the present invention are often described in relation to the HA resin in a chromatography column; however, other suitable associated resin configurations are not excluded. In addition, reference in the present description to "HA column" or the like. refers to a chromatography column packed with HA resin.

Equilibration of the HA Column Before Protein Injection

In some embodiments, before introducing a sample containing the antibody of interest onto an HA column, the methods of the present invention may include the step of pre-equilibrating the column with one or more equilibration buffers. Equilibration buffers can be added to the column, for example, to ensure that the HA resin is pure at the start of the process (i.e., to ensure that the resin does not contain impurities already associated with the resin), and/or to ensure that ensure that the solution surrounding the HA resin is compatible with the sample intended to be injected into the resin.

In some embodiments, the equilibration buffer is a phosphate buffer containing, for example, sodium phosphate, where the concentration of phosphate ions in the buffer is from about 100 to 500 mM. Such equilibration buffers may also be referred to herein as "high phosphate equilibration buffers" or the like. For example, in an embodiment, the high phosphate equilibration buffer may contain from about 250 to 450 mM phosphate ions; in other embodiments, it may contain approximately 200, 250, 300, 350, 400, or 450 mM phosphate ions. This equilibration buffer has a relatively high concentration of phosphate ions in the elution buffer to elute any contaminants/impurities already present in the HA resin (i.e. present before the sample containing the protein of interest is introduced into the resin; such impurities may be present, e.g. The HA resin had previously been used for the purification process and had not been completely purified after previous use). The high phosphate equilibration buffer may have a pH of approximately 6.0 to 9.0. For example, in an embodiment, the high phosphate equilibration buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the high phosphate equilibration buffer contains approximately 400 mM phosphate ions and has a pH of approximately 7.5. In some embodiments, the high phosphate equilibration buffer may also be referred to herein as "equilibration buffer 1."

In some embodiments, the equilibration buffer is a phosphate buffer containing, for example, sodium phosphate, where the concentration of phosphate ions in the buffer is from about 1 to 20 mM. Such equilibration buffers may also be referred to herein as "low phosphate equilibration buffers" or the like. For example, in an embodiment, the low phosphate equilibration buffer may contain from about 1 to 10 mM phosphate ions; in other embodiments, it may contain approximately 1, 2, 3, 4, 5, or 10 mM phosphate ions. This equilibration buffer has a relatively low concentration of phosphate ions in the buffer to create conditions around the HA resin that are favorable for the protein of interest to bind to the resin. Optionally, the low phosphate equilibration buffer may further contain HEPES at a concentration of from about 1 to 50 mM. For example, in an embodiment, the low phosphate equilibration buffer may contain from about 2 to 30 mM HEPES; in other embodiments, it may contain about 5, 10, 15, 20 or 25 mM HEPES. The low phosphate equilibration buffer may have a pH of approximately 6.0 to 9.0. For example, in an embodiment, the low phosphate equilibration buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the low phosphate equilibration buffer contains approximately 2 mM phosphate ions, 20 mM HEPES, and has a pH of approximately 7.5. In some embodiments, the low phosphate equilibration buffer may also be referred to herein as "equilibration buffer 2." However, it is important that in the methods of the present invention, a low phosphate equilibration buffer can be used to pre-equilibrate the resin without prior use of a high phosphate equilibration buffer during the process.

Injection of a sample containing the antibody of interest into the HA column

When the HA column is ready for protein loading, the sample containing the antibody of interest and impurities is injected into the HA column. The buffer in which the sample is introduced into the HA column may be referred to herein as "loading buffer". In some embodiments, if a sample containing the antibody of interest is initially obtained for use in the method of the present invention, the sample is already in a suitable loading buffer for loading the sample onto the HA column. However, in other embodiments, before introducing the sample onto the HA column, it may be processed (eg, diluted, concentrated, or subjected to a buffer exchange) to modify the sample buffer conditions such that the sample is in a suitable buffer for introduction to the column. For example, in the methods of the present invention, the loading buffer cannot have a high concentration of phosphate ions that would interfere with binding of the antibody of interest to the HA resin. Thus, if the original sample containing the antibody of interest has a high concentration of phosphate ions, the sample will need to be, for example, diluted or subjected to buffer exchange until the phosphate ion concentration in the sample is reduced to a suitable low concentration to allow binding of the antibody of interest to the HA resin.

In some embodiments, the loading buffer contains no more than about 10 mM phosphate ions. For example, in some embodiments, the loading buffer contains less than about 10 mM, 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM phosphate ions. In some embodiments, the loading buffer contains 0 mM phosphate ions. Optionally, the loading buffer may contain various other salts or buffer components (eg Tris, glycine). The loading buffer may have a pH of approximately 6.0 to 9.0. For example, in an embodiment, the loading buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0.

In some embodiments, the sample containing the antibody of interest can be incorporated into the HA resin to a density on the resin of at least 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L. l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 12 g/l, 15 g/l, 20 g/l, 25 g/l or 30 g/l. In some embodiments, the sample containing the antibody of interest can be incorporated into the HA resin to a density on the resin of at least 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g /l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 12 g/l, 15 g/l, 20 g/l or 25 g/l up to no more than 2 g/l, 3 g/l, 4 g/l, 5 g/l, 6 g/l, 7 g/l, 8 g/l, 9 g/l, 10 g/l, 12 g/l, 15 g/l, 20 g/l, 25 g/l or 30 g/l, where the second value is greater than the first value.

Washing the HA Column

After introducing a sample containing the antibody of interest and impurities onto the HA column, but before eluting the antibody of interest, the methods of the present invention may optionally include the additional step of washing the loaded column with one or more wash buffers, for example, to remove nonspecifically immobilized impurities or other preparation or equilibrating the column for the elution step. One skilled in the art can determine the properties of any wash buffer. In one embodiment, the wash buffer is a phosphate buffer containing, for example, sodium phosphate, and the concentration of phosphate ions in the buffer is from about 5 to 50 mM. For example, in an embodiment, the wash buffer may contain from about 10 to 40 mM phosphate ions; in other embodiments, it may contain about 10, 20, 30, 40, or 50 mM phosphate ions. Optionally, the wash buffer may further contain HEPES at a concentration of from about 1 to 50 mM. For example, in an embodiment, the wash buffer may contain from about 2 to 30 mM HEPES; in other embodiments, it may contain about 5, 10, 15, 20 or 25 mM HEPES. The wash buffer may have a pH of approximately 6.0 to 9.0. For example, in an embodiment, the wash buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the wash buffer contains approximately 40 mM phosphate ions, 20 mM HEPES and has a pH of approximately 7.5.

Elution of the antibody of interest from the HA column

The methods of the present invention include the step of eluting the bound antibody of interest from the HA resin. The bound antibody of interest is eluted using one or more elution buffers. Typically, the elution buffer contains one or more salts or ions, and the concentration of the salts or ions is increased during elution.

In some embodiments, the elution buffer provided herein contains phosphate ions. Optionally, the concentration of phosphate ions in the elution buffer is increased from the initial concentration of about 20 mM to about 200 mM during elution. For example, in an embodiment, the concentration of phosphate ions in the elution buffer is increased from an initial concentration of about 40 mM to about 80 mM or from about 40 mM to about 100 mM during elution. The specific method and rate of increase in the concentration of phosphate ions in the elution buffer can be determined according to the antibody of interest, while taking into account the types of impurity molecules also associated with the HA resin. For example, the concentration of phosphate ions in the elution buffer can be increased using a smooth/flat linear gradient. The use of a gentle gradient may allow efficient separation of one or more molecules eluted from the HA resin under similar but different conditions. Alternatively, in some embodiments, the concentration of phosphate ions in the elution buffer can be increased using a steep gradient or it can be increased in a stepwise manner. Optionally, the elution buffer may further contain HEPES at a concentration of from about 1 to 50 mM. For example, in an embodiment, the elution buffer may contain from about 2 to 30 mM HEPES; in other embodiments, it may contain about 5, 10, 15, 20 or 25 mM HEPES. The elution buffer may have a pH of from about 6.0 to 9.0. For example, in an embodiment, the elution buffer may have a pH of about 7.0 to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In one embodiment, the elution buffer contains approximately 40-80 mM phosphate ions (raised by gradient), 20 mM HEPES and has a pH of approximately 7.5.

To optimize the elution of the antibody of interest from the HA column, as well as the separation of the antibody of interest from impurity molecules, elution conditions can be determined, including, but not limited to, the properties of the elution buffer suitable for use with the HA resin (such as buffer composition, pH, concentration , ionic strength, etc.); any necessary step or gradient change in the properties of the elution buffer; number of column volumes of elution buffer used; flow speed, etc.

Following elution, one or more peak fractions containing the antibody of interest are optionally collected individually or separately and optionally pooled, optionally pH adjusted, optionally filtered, and then optionally stored before further processing. Peak collection fractions can be identified by any suitable means, such as UV identification at A280 and initial collection when the UV signal exceeds the desired amount and/or is at the desired point corresponding to the elution conditions.

The material eluted from the HA resin and containing the antibody of interest may optionally be referred to herein as the "purified fraction" or the like. The purified fraction may contain material from a single HA resin eluting fraction, or it may be a combination of multiple combined HA resin eluting fractions. Typically, the purified fraction is prepared to balance the collection of a large amount of the antibody of interest with a small amount of impurity molecules. These competing goals often need to be balanced, e.g. there may be at least partial overlap between the conditions under which the antibody of interest is eluted from the HA resin and the conditions under which the impurity molecule species is eluted from the HA resin.

Any of the buffers for the methods of the present invention (for example, equilibration buffer, loading buffer, wash buffer or elution buffer) may also contain additional or alternative suitable components, such as acetate, succinate, MES, ACES, MOPSO, PIPES, BES, TAPSO, AMPSO, TRICINE, EPPS, bicine, DIPSO, HEPPSO, imidazole, Tris, bis-Tris, TAPS, arginine, glycine, acetonitrile, ethanol, methanol, 1% sodium dodecyl sulfate (SDS) or other surfactants, etc. P.

In some embodiments, any of the buffers provided herein may have a pH of from about 6.0 to about 9.0. In other embodiments, any of the buffers provided herein may have a pH of about 5.0 to 9.0, 5.5 to 9.0, 6.5 to 9.0, 7.0 to 9 ,0, from 7.5 to 9.0, from 7.0 to 8.0 or from 6.5 to 8.5.

In the buffers provided herein and described as containing "phosphate ions", phosphate ions can be formed in the buffer from any suitable phosphate salt, such as sodium phosphate or potassium phosphate. In addition, the solutions presented herein and described as being prepared using "sodium phosphate" can be prepared using any suitable sodium phosphate salt (eg, monobasic or dibasic).

After the antibody of interest has been eluted from the HA column, the HA column is optionally cleaned to remove impurities and other components that may degrade the resin in the column, and prepared for storage for later use. In one embodiment, the column is first regenerated using a buffer, such as a buffer containing sodium phosphate at a concentration of about 0.4 M and a pH of about 7.5; followed by an optional sanitation step using a cleaning solution such as about 1 M NaOH and about 0.5 M potassium phosphate, and then prepared for storage using a storage solution such as about 0.1 M NaOH.

Antibodies

The methods of the present invention can be used to purify an antibody of interest from one or more impurities. For example, the purified antibody can be used as pharmaceuticals or in the preparation of pharmaceuticals.

In some embodiments, the antibody purified by the method of the present invention is any type of antibody described herein. For example, the antibody purified by the method of the present invention may be a full-length antibody or an antibody fragment (eg, scFv or Fab) and may be monospecific or bispecific. Typically, the antibody of interest purified by the method of the present invention is a recombinant antibody.

IgG antibodies

In some embodiments, the antibody that can be purified by the method of the present invention is an immunoglobulin G (IgG) antibody. As is known in the art, an IgG antibody contains two heavy chains and two light chains and has an overall "Y" shape. In standard IgG molecules, the two heavy chains have the same amino acid sequence, and the two light chains have the same amino acid sequence. An IgG antibody can be described as having two "arms" (ie, a "first arm" and a "second arm"), where each arm contains one heavy chain and one light chain connected by a disulfide bond. In standard IgG molecules, the first arm of the antibody is identical to the second arm of the antibody (due to the fact that each arm contains a heavy chain and a light chain having the same amino acid sequence as the heavy chain and light chain in the other arm, respectively). The N-terminal region of the heavy chain contains the variable region of the heavy chain (VH), and the N-terminal region of the light chain contains the variable region of the light chain (VL). The VH and VL regions contain the portion of the antibody that specifically binds to the antigen. Thus, each arm of an IgG antibody can specifically bind to an antigen. In standard IgG molecules, both the first arm and the second arm of the IgG antibody bind to the same antigen (due to the fact that both arms contain heavy chains and light chains that have the same corresponding amino acid sequences). A standard IgG antibody may be referred to as "homodimeric" in that it contains 2 arms that are identical. The IgG antibody purified by the method of the present invention may be of the IgG1, IgG2, IgG3 or IgG4 subclass.

Bispecific IgG antibodies

In some embodiments, the antibody that can be purified by the method of the present invention is a bispecific IgG antibody. In a bispecific IgG antibody, each of the two arms of the antibody specifically binds to a different antigen. In addition, the heavy chain amino acid sequence in the first arm of an IgG bispecific antibody is different from the heavy chain amino acid sequence in the second arm of the same IgG bispecific antibody, and similarly, the light chain amino acid sequence in the first arm of an IgG bispecific antibody is generally different from the amino acid sequence light chain in the second arm of the same bispecific IgG antibody. Thus, a bispecific IgG antibody can be referred to as "heterodimeric" in view of the fact that it contains 2 arms that are different. The first arm of the bispecific IgG antibody may be described as being specific for a "first antigen" and the second arm of the bispecific IgG antibody may be described as being specific for a "second antigen". In some embodiments, the bispecific antibody is of an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecific antibody comprises an immunologically inert Fc region.

Bispecific IgG antibodies - methods of production

Methods for producing bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Conventionally, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-immunoglobulin light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

Recently, methods have been developed that use the following general steps to produce bispecific heterodimeric antibodies:

1) The first homodimer antibody (also referred to herein as the “first parent antibody”) and the second homodimer antibody (also referred to herein as the “second parent antibody”) are individually expressed and purified. The first homodimeric antibody is specific for a first target antigen of the resulting bispecific antibody, and the second homodimeric antibody is specific for a second target antigen of the resulting bispecific antibody. Thus, for example, if the goal is to produce a bispecific antibody having specificity for BCMA and CD3, an anti-BCMA monoclonal antibody (“first parent antibody”) and an anti-CD3 monoclonal antibody (“second parent antibody”) are separately expressed and purified.

2) The purified first homodimer/parent antibody and the purified second homodimer/parent antibody are then mixed and incubated together under conditions that promote the exchange of antibody arms such that heterodimeric bispecific antibodies are formed containing the first arm from the first parent antibody and the second arm from the second parent antibodies. These conditions typically involve a sequence of reducing conditions followed by oxidizing conditions. Reducing conditions promote the cleavage of disulfide bonds holding the two heavy chains of homodimeric antibodies together and thus allow switching of antibody arms between the first parent antibody and the second parent antibody. Then, under subsequent oxidative conditions, new disulfide bridges are formed, stabilizing the newly formed bispecific antibodies. This general approach for producing bispecific antibodies is shown in FIG. 1. In FIG. 1 shows a first parent antibody ("Parent Antibody A"; gray) and a second parent antibody ("Parent Antibody B"; black), each of which is a monospecific homodimer and contains a first arm and a second arm. Typically, the first parent antibody and the second parent antibody are specific for different antigens. The first parent antibody and the second parent antibody are then mixed and subjected to reduction and oxidation steps, resulting in the formation of a bispecific antibody of interest comprising a first arm from parent antibody A and a second arm from parent antibody B and having the corresponding specificities of both arms.

Optionally, the amino acid sequence of the antibody heavy chain can be modified in one or more ways to promote the formation of bispecific antibodies. For example, the heavy chain of one arm of a bispecific antibody may contain a modification of the amino acids in the first hinge region such that the replaced amino acid in the first hinge region has the opposite charge to the corresponding amino acid in the hinge region of the other arm of the resulting bispecific antibody. This is described, for example, in international patent application No. PCT/US2011/036419 (WO2011/143545). In another approach, the formation of a desired heteromultimeric or heterodimeric protein (eg, a bispecific antibody) is enhanced by altering or engineering the contact region between the first and second immunoglobulin-like Fc region (eg, the hinge region and/or the CH3 region). In this approach, the bispecific antibody may comprise a CH3 region, wherein the CH3 region contains a first CH3 polypeptide and a second CH3 polypeptide interacting with each other to form a CH3 contact region, wherein one or more amino acids in the CH3 contact region destabilize homodimer formation and are not electrostatically unfavorable to homodimer formation. This approach is also described in international patent application No. PCT/US2011/036419 (WO2011/143545).

The above method and other methods for producing bispecific antibodies are further described, for example, in international patent applications No. PCT/IB2011/054899 (WO2012/059882), PCT/US2011/036419 (WO2011/143545) and Giese et al, Biotechnology Progress, " Bispecific Antibody Process Development: Assembly and Purification of Knob and Hole Bispecific Antibodies,” 17 Jan 2018, and the references cited therein, each of which is incorporated herein by reference for all purposes. The antibody purification methods of the present invention can be used to purify bispecific antibodies produced by any suitable method.

Bispecific IgG antibodies - specificity

In some embodiments, an antibody that can be purified by the method of the present invention is a full-length human IgG bispecific antibody, wherein a first variable domain of a first arm of the bispecific antibody can bind to a first antigen, and a second variable domain of a second arm of the bispecific antibody can bind to a second antigen. The first antigen and the second antigen may have any of the characteristics of an antigen presented herein. In some embodiments, the first antigen is on a first cell type and the second antigen is on a second cell type.

In some embodiments, the antibody that can be purified by the method of the present invention is a full-length bispecific human IgG antibody, wherein the first variable domain of the antibody can recruit the activity of a human immune effector cell by specifically binding to an effector antigen located on the human immune effector cell, and where the second the variable domain of an antibody can specifically bind to a target antigen.

The human immune effector cell that can be bound by an antibody provided herein can be any of a variety of immune effector cells known in the art. For example, the immune effector cell may be a member of a human lymphoid cell lineage, including, but not limited to, a T cell (eg, a cytotoxic T cell), a B cell, and a natural killer (NK) cell. The immune effector cell may also be, for example, a member of the human myeloid lineage, including, but not limited to, a monocyte, a neutrophil granulocyte, and a dendritic cell. Such immune effector cells may have a cytotoxic or apoptotic effect on a target cell or any desired effect upon activation by binding of the effector antigen. An effector antigen is an antigen (eg, a protein or polypeptide) expressed on a human immune effector cell. Examples of effector antigens that may be bound by an antibody provided herein include, but are not limited to, human CD3 (or CD3 complex (cluster of differentiation)), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

A target antigen is expressed on a target cell in a disease (eg, inflammatory disease, proliferative disease (eg, cancer), immunological disorder, neurological disease, neurodegenerative disease, autoimmune disease, infectious disease (eg, viral infection or parasitic infection), allergic reaction, graft-versus-host disease, or host-versus-graft disease). The target antigen is not an effector antigen. Non-limiting examples of target antigens include BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine receptor type 5), CD19, HER (human epidermal growth factor receptor)-2/neu, HER-3, HER-4, EGFR ( epidermal growth factor), FLT3 (Fms-like tyrosine kinase 3), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis antigen Y, CD20, CD33, CD30, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl-GM1, poly-SA, GD2, carbonic anhydrase IX (MN/CA IX), CD44v6, Shh (Sonic Hedgehog), Wue-1, plasma cell antigen, (membrane-bound) IgE, MCSP (melanoma-specific chondroitin sulfate proteoglycan), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; desmoglein 4, neoepitope E-cadherin, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and MIS (Mullerian inhibitory substance) receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen ), endosialin, EGFRvIII, LG, SAS and CD63.

In some embodiments, the antibody purified by the method of the present invention may be any antibody described in US Application No. 15/085644, filed March 30, 2016 (Publication No. US20160297885), or US Application No. 15/993874, filed May 31, 2018 (Publication No. US20180346601), hereby incorporated herein by reference in its entirety for all purposes.

In some embodiments, the antibody purified by the method of the present invention may be a bispecific IgG antibody in which one arm of the antibody specifically binds to cluster of differentiation 3 (CD3). Information about CD3 is given, for example, in UniProtKB ID #P07766.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the heavy chain VH region of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQG TTVTVSS (SEQ ID NO: 1). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds CD3, the heavy chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTV SSastkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkcrvrcprcpappvagpsvflfppkpkdtlmisrtpevtcvvvavshedpevqfnwyvdgvevhnaktkpree (SEQ ID NO: 2). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the heavy chain VH region of the CD3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 1.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEI K (SEQ ID NO: 3). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the light chain of the CD3-binding arm has an amino acid sequence comprising the amino acid sequence: DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIKrt vaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec (SEQ ID NO: 4). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 3.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the heavy chain VH region of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 1 and the CD3 light chain VL region. binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to CD3, the heavy chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2, and the light chain of the CD3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, in a bispecific IgG antibody, wherein one arm of the antibody specifically binds CD3 , the VH region of the heavy chain of the CD3 binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 of the VH sequences shown in SEQ ID NO: 1, and the VL region of the light chain of the CD3 binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 sequences VL given in SEQ ID NO: 3.

In some embodiments, the antibody purified by the method of the present invention may be a bispecific IgG antibody in which one arm of the antibody specifically binds to B-cell maturation antigen (BCMA). Information about BCMA is given, for example, in UniProtKB ID #Q02223.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds BCMA, the heavy chain VH region of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLV TVSS (SEQ ID NO: 5). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the heavy chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSas tkgpsvfplapcsrstsestaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssnfgtqtytcnvdhkpsntkvdktverkcevecpecpappvagpsvflfppkpkdtlmisrtpevtcvvvavshedpevqfnwyvdgvevhnaktkpreeqf (SEQ ID NO: 6). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds BCMA, the heavy chain VH region of the BCMA-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 5.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEI K (SEQ ID NO: 7). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKrt vaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec (SEQ ID NO: 8). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 7.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the heavy chain VH region of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 5 and the light chain VL region of the BCMA-binding arm. binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the heavy chain of the BCMA binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 6, and the BCMA-binding arm light chain has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, in a bispecific IgG antibody, in which one arm of the antibody specifically binds to BCMA , the VH region of the heavy chain of the BCMA binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 of the VH sequences shown in SEQ ID NO: 5, and the VL region of the light chain of the BCMA binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 sequences VL given in SEQ ID NO: 7.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds BCMA, the heavy chain VH region of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFFSSYPMSWVRQAPGKGLEWVSAIGgSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLV TVSS (SEQ ID NO: 13). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to BCMA, the VL region of the light chain of the BCMA-binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPGTLLSLPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQEWPLTFGQGTKVEI K (SEQ ID NO: 14). In some embodiments, in any reference herein to an antibody comprising a VH region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 5, the antibody may alternatively comprise a VH region containing the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, in any reference herein to an antibody comprising a VL region having an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 7, the antibody may alternatively comprise a VL region comprising the amino acid sequence set forth in SEQ ID NO: 14. Likewise, the heavy and light chains of the anti-BCMA antibody containing the VH and VL sequences of SEQ ID NO: 13 and SEQ ID NO: 14, respectively, are also included herein.

In some embodiments, the antibody purified by the method of the present invention may be a bispecific IgG antibody in which one arm of the antibody specifically binds fms-like tyrosine kinase 3 (FLT3). Information about FLT3 is given, for example, in UniProtKB ID #P36888.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VH region of the heavy chain of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGF DIWGQGTLVTVSS (SEQ ID NO: 9). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds FLT3, the heavy chain of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the heavy chain VH region of the FLT3-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VH sequence set forth in SEQ ID NO: 9.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the VL region of the light chain of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIK (SEQ ID NO: 11). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the light chain of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12). In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds FLT3, the VL region of the light chain of the FLT3-binding arm has an amino acid sequence comprising CDR1, CDR2, and CDR3 of the VL sequence set forth in SEQ ID NO: 11.

In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the heavy chain VH region of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 9 and the VL region of the FLT3 light chain - binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, in a bispecific IgG antibody in which one arm of the antibody specifically binds to FLT3, the heavy chain of the FLT3 binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 10, and the light chain of the FLT3-binding arm has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, in a bispecific IgG antibody, wherein one arm of the antibody specifically binds to FLT3 , the VH region of the heavy chain of the FLT3 binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 of the VH sequences shown in SEQ ID NO: 9, and the VL region of the light chain of the FLT3 binding arm has an amino acid sequence comprising the CDR1, CDR2 and CDR3 sequences VL given in SEQ ID NO: 11.

In some embodiments, the present invention provides an anti-BCMA/anti-CD3 bispecific antibody, wherein the anti-BCMA antibody arm has any of the characteristics described above for the anti-BCMA arm, and the anti-CD3 antibody arm has any of the characteristics described above for the anti-CD3 arm . In some embodiments, the present invention provides an anti-FLT3/anti-CD3 bispecific antibody, wherein the anti-FLT3 antibody arm has any of the characteristics described above for the anti-FLT3 arm, and the anti-CD3 antibody arm has any of the characteristics described above for the anti-CD3 arm .

The present invention also relates to methods for purifying a monospecific antibody having affinity for any of the above antigens and/or containing any of the amino acid sequences described above. For example, the present invention also relates to the purification of a monospecific, homodimeric anti-CD3 antibody containing the amino acid sequence VH set forth in SEQ ID NO: 1.

Impurities

The methods of the present invention can be used to purify an antibody of interest from one or more impurities.

Impurities include, for example, clipped versions of the antibody of interest, protein aggregates, and, in the case of a bispecific antibody of interest, parental monospecific antibodies associated with the formation of the bispecific antibody of interest. These different impurities may also be referred to herein as different "types of impurities", "impurity molecules" or the like.

Clipped versions of the antibody of interest

The terms "clipped versions of the antibody of interest", "clipped antibodies" or the like. refer to a recombinant antibody in which one or more polypeptide bonds in the antibody are cleaved from the corresponding intact antibody of interest. In contrast, the term "intact" antibody refers to a recombinant antibody containing all of the expected peptide bonds and amino acids of a recombinant antibody (i.e., expected given the nucleic acid sequences encoding the antibody polypeptides)

In this regard, clipped antibodies can be considered degradation products related to the antibody of interest. Cleavage of a peptide bond in an antibody may occur, for example, by enzymatic (eg, protease-mediated) or non-enzymatic activity.

In some embodiments, if a peptide bond in an antibody polypeptide is cleaved, after cleavage, the cleaved portion of the polypeptide chain may no longer be covalently associated with the rest of the antibody; in this case, the cleaved portion of the polypeptide chain may dissociate from the rest of the antibody. This most often occurs when cleavage occurs at a peptide bond near the N- or C-terminus of a polypeptide chain, and results in the formation of a clipped antibody that is missing one or more amino acids compared to the corresponding intact antibody. These clipped antibodies have a lower mass than the corresponding intact antibody due to the loss of one or more amino acids in the antibody.

Alternatively, in some other embodiments, if a peptide bond in an antibody polypeptide is cleaved, after cleavage, the cleaved portion of the polypeptide chain may remain covalently linked to the rest of the antibody (eg, through an intrachain or interchain disulfide bond). In this case, even if cleavage of the peptide bond in the antibody occurs, the cleaved portion of the polypeptide chain will remain associated with the rest of the antibody through the remaining intact covalent bonds connecting the cleaved portion of the polypeptide chain to the rest of the antibody. In this case, the clipped antibody will still have the same number of amino acids and amino acid sequences as the intact antibody. Additionally, in at least some embodiments, this type of clipped antibody may have a slightly larger mass than the corresponding intact antibody. This increase in mass may result, for example, from one or more chemical reactions occurring after cleavage of the peptide bond. During such reactions, one or more atoms (eg, H, O) may react with atoms of the antibody polypeptide chain and become covalently associated with the antibody chain, resulting in an increase in the mass of the clipped antibody compared to the corresponding intact antibody.

Because A "clipped" antibody is formed from the corresponding "intact" antibody, the "clipped" version of the antibody has the same amino acid sequence (in the case of no loss of amino acids in the antibody as a result of cleavage of the peptide bond) or almost the same amino acid sequence (in the case of loss of one or more amino acids sequences in the antibody as a result of peptide bond cleavage) as the corresponding “intact” version of the antibody.

Typically, the clipped version of an antibody has a mass similar to that of the corresponding intact antibody. As described above, in some embodiments, a clipped antibody may have a smaller mass than the corresponding intact antibody (eg, if the clipping results in the loss of one or more amino acids in the antibody). Alternatively, in some embodiments, the clipped antibody may have a greater mass than the corresponding intact antibody (in the case where the clipping does not result in the loss of any amino acids in the antibody and, instead, results in the addition of at least an atom in the antibody through one or more reactions occurring as a result of cleavage of the peptide bond).

In some embodiments, the clipped version of the antibody has a mass that differs by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% , 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% based on the weight of the corresponding intact antibody of interest. That is, in some embodiments, the clipped version of the antibody of interest has a mass that differs from the mass of the corresponding intact antibody of interest by no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4 %, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03% , 0.02% or 0.01%.

As described above, in some embodiments, the clipped version of the antibody of interest has a lower mass than the corresponding intact antibody of interest. For example, in some embodiments, the clipped version of the antibody has a mass of no more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% , 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01% less than the mass of the corresponding intact antibody of interest. In other words, if the clipped version of an antibody has a mass that is no more than 10% less than the mass of the corresponding intact antibody of interest, then, for example, if the intact antibody of interest has a mass of 100,000 Da, then the clipped version of the antibody has a mass that is no more than 10,000 Yes, less than the mass of the intact antibody (10% of 100,000 is 10,000), i.e. it has a mass of 90,000 to 100,000 Da.

As described above, in some embodiments, the clipped version of the antibody of interest has a greater mass than the corresponding intact antibody of interest. For example, in some embodiments, the clipped version of the antibody has a mass of no more than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2% , 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% greater than the mass of the corresponding intact antibody of interest. In other words, if the clipped version of an antibody has a mass no more than 1% greater than the mass of the corresponding intact antibody of interest, then, for example, if the intact antibody of interest has a mass of 100,000 Da, then the clipped version of the antibody has a mass of no more than 1000 Yes, greater than the mass of the intact antibody (1% of 100,000 is 1000), i.e. it has a mass of 100,000 to 101,000 Da.

High molecular weight compounds (HMMS)/protein aggregates

In some embodiments, impurities associated with the process of the present invention are referred to as "high molecular weight compounds" (HMMS). The term "HMMS" refers to any high molecular weight contaminants or impurities, but generally represents an association of at least two proteins forming an aggregate. By way of example, HMMS may include a plurality of aggregated molecules of the antibody of interest and/or aggregates of host cell proteins used to produce the antibody of interest. Aggregates can be formed by any means, including, for example, covalent or non-covalent binding of molecules.

Parental antibodies

In some embodiments, the impurity relevant to the method of the present invention is a "parent antibody" or the like. This type of impurity molecule is important in the case of methods of the present invention in which the antibody of interest is a bispecific antibody derived from two different parent antibodies, for example, as shown in FIG. 1. These parent antibodies are monospecific homodimers. Parental antibodies may be present in an antibody preparation with a bispecific antibody of interest due to a variety of possible mechanisms, for example: i) in some cases, some parental antibody molecules are not separated into a first arm and a second arm during the reconstitution step to separate the parent antibodies into separate first arms and second arms (and thus the antibodies remain as monospecific homodimers); or ii) in some cases, a separate first arm and second arm from the same type of parent antibody combine, thereby forming a monospecific homodimer (rather than participating in the formation of a heterodimeric bispecific antibody). As used herein, the term “parent antibody” refers to homodimeric molecules resulting from any of the above mechanisms. In addition, the antibody preparation provided herein may contain the parent antibody of one or both parent antibodies as an impurity.

Purification of the antibody of interest from impurities

The present invention relates to methods for purifying an antibody of interest from one or more impurities.

In some embodiments, in the method of the present invention, the antibody of interest is contained in an antibody preparation (also referred to herein as a "parent sample") containing the antibody of interest as well as one or more types of impurity molecules. In this starting material, in the case of the method of the present invention, the antibody of interest may contain, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85% , 90% or 95% wt. protein in the antibody preparation. Additionally, in some embodiments of the method of the present invention, the purified fraction (also referred to herein as a “purified sample”) is collected as an eluate from the HA resin. This purified fraction contains the antibody of interest and, in some embodiments, still contains one or more impurity molecules. In some embodiments, the purified fraction provided herein may contain, for example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98% or 99% wt. protein in the purified fraction.

In some embodiments, in the method of the present invention, the starting sample contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95 % wt. of the antibody of interest, and a subsequent purified sample in the same method contains at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96 %, 97%, 98% or 99% wt. antibody of interest, where the second value is greater than the first value.

In some embodiments, in the method of the present invention, the starting sample contains at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% , 9%, 10%, 15%, 20% or 25% wt. a clipped version of the antibody of interest. In some embodiments, in the method of the present invention, the purified sample contains no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% wt. a clipped version of the antibody of interest. In some embodiments, in the method of the present invention, the starting sample contains at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25% wt. clipped version of the antibody of interest, and a subsequent purified sample in the same method contains no more than approximately 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5 %, 6%, 7%, 8%, 9%, 10%, 15% or 20% wt. a clipped version of the antibody of interest, where the second value is less than the first value.

In some embodiments, in the method of the present invention, the original sample contains an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody is at least about 1:100, 1:50, 1:25, 1:20, 1 :10, 1:5, 1:4 or 1:3. In some embodiments, in the method of the present invention, the purified sample contains an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody is no more than about 1:100, 1:50, 1:25, 1:20, or 1: 10. In some embodiments, in the method of the present invention, the original sample contains an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody in the original sample is at least about 1:100, 1:50, 1:25, 1: 20, 1:10, 1:5, 1:4 or 1:3, and wherein the ratio of clipped antibody to intact antibody in a subsequent purified sample in the same method is no more than about 1:200, 1:100, 1:50, 1:25, 1:20 or 1:10, where the second ratio is less than the first ratio. In some embodiments, in the method of the present invention, the parent sample contains an intact antibody of interest and a clipped version of the antibody of interest, wherein the ratio of clipped antibody to intact antibody in the parent sample is between approximately one of the ratios in group A (group A ratios: 1:100, 1 :50, 1:25, 1:20, 1:10, 1:5 or 1:4) to one of the ratios in group B (group B ratios: 1:50, 1:25, 1:20, 1:10 , 1:5, 1:4 or 1:3), and where the ratio of clipped antibody to intact antibody in a subsequent purified sample in the same method is no more than about 1:200, 1:100, 1:50, 1:25, 1:20 or 1:10, where the ratio in the purified sample is less than the ratio in the original sample.

In some embodiments, the initial sample produced by the method of the present invention contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest. In some embodiments, the purified sample obtained by the method of the present invention contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest. In some embodiments, the initial sample produced by the method of the present invention contains at least 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 grams of intact antibody of interest, and subsequent purified a sample in the same method contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000 or 5000 grams of intact antibody of interest, where the first value is greater than the second value.

In addition, any of the above descriptions regarding a) the amount or b) the purity of the antibody of interest in the original sample or purified sample can be considered together with respect to a single sample. For example, as indicated above, the original sample may contain at least approximately 80% of the mass. antibody of interest; further, as noted above, the original sample may contain at least about 10 grams of the antibody of interest. Thus, the present invention also relates to a starting sample containing at least about 80% by weight. of the antibody of interest and at least 10 grams of the antibody of interest, etc.

General methods

Unless otherwise indicated, the practice of the present invention uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology known to those skilled in the art. Such methods are fully described in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995), and, where appropriate , subsequent editions and the corresponding websites of the above links.

The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, various modifications of the invention in addition to those presented and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the claims.

Examples

Example 1: Purification of Anti-BCMA/Anti-CD3 Bispecific Antibody by cHA Resin Chromatography

Target:

This example examined methods for separating full-length human bispecific IgG of interest from various impurities. The antibody of interest was a heterodimeric anti-BCMA/anti-CD3 bispecific antibody (ie, one arm of the bispecific antibody was specific for BCMA and the other arm was specific for CD3). Impurities present with the bispecific antibody of interest at the start of purification included: i) a clipped version of the intact anti-BCMA/anti-CD3 bispecific antibody of interest; ii) homodimeric, monospecific parental anti-BCMA antibodies; iii) homodimeric, monospecific anti-CD3 antibodies; and iv) protein aggregates/high molecular weight compounds (HMMS).

Purification of an intact bispecific antibody of interest from a clipped version of the bispecific antibody presents a special challenge because the clipped version of the bispecific antibody contained the same number of amino acids and the same amino acid sequences as the intact bispecific antibody of interest and differed in mass from the intact bispecific antibody by only 18 Da. Specifically, the mass of the intact anti-BCMA/CD3 bispecific antibody is 148,095.5 Da, while the mass of the corresponding clipped version of the bispecific antibody is 148,113.5 Da (as determined by mass spectrometry). Thus, the clipped bispecific antibody has a mass difference of less than 0.1% (even less than 0.02%) compared to the mass of the intact bispecific antibody (i.e., 0.1% of 148,095 Da is 148 Da; 0.02% from 148095 Yes is 29.6 Yes). In other words, the mass of the clipped bispecific antibody is approximately 100.01% of the mass of the intact bispecific antibody of interest. Thus, the mass of the clipped bispecific antibody is very similar to the mass of the intact bispecific antibody of interest.

The clipped version of the bispecific antibody contains a cleavage of the anti-CD3 heavy chain peptide bond between the 56th and 57th amino acids of the heavy chain. The anti-CD3 heavy chain has the amino acid sequence set forth in SEQ ID NO: 2, and thus cleavage occurs between amino acids R and G in the sequence "RG" of SEQ ID NO: 2 ("RG" appears in SEQ ID NO: 2 only once). This cleavage is believed to result in the addition of 1 oxygen atom and 2 hydrogen atoms in the clipped bispecific antibody (corresponding to an 18 Da increase in mass) compared to the intact bispecific antibody. In addition, although the anti-CD3 heavy chain contains a cleaved peptide bond, the cleaved 56 amino acid portion of the heavy chain (ie, the first 56 amino acids of the chain) remains linked to the rest of the antibody via an intact, remaining intrachain disulfide bond. The intrachain disulfide bond is between C22 and C98 of the anti-CD3 heavy chain (ie, C22 and C98 of the sequence shown in SEQ ID NO: 2). The continued binding of the cleaved 56-amino acid moiety to the rest of the antibody via an intrachain peptide bond is shown schematically in. 2.

In fig. 2, the diagram on the left shows an intact bispecific antibody of interest containing intact heavy and light chains for the anti-CD3 arm and the anti-BCMA arm of the bispecific antibody (in the figure, the longer chains correspond to the heavy chains of the antibody and the shorter chains correspond to the light chains). In addition, in FIG. 2 also shows the various disulfide bonds within an antibody, including intrachain disulfide bonds (eg, linking different amino acids in the same light chain or heavy chain), as well as interchain disulfide bonds (eg, linking the heavy chain of the anti-BCMA arm to the heavy chain of the anti-BCMA arm). CD3 or linking the light chain of the anti-CD3 arm to the heavy chain of the anti-CD3 arm).

Another challenge in developing a method for purifying an intact bispecific antibody of interest from various impurities has been to develop a method that will efficiently separate the bispecific antibody of interest from the impurities when relatively large quantities of the bispecific antibody of interest need to be purified. For example, one of the goals in this example was to develop a method for purifying a bispecific antibody that would be effective for methods in which at least 1 gram of bispecific antibody needs to be purified. As is known in the art, the large-scale purification of proteins often presents many challenges that do not arise (or are much less problematic) when purifying the same proteins on a small scale, resulting, for example, from difficulties in achieving clear chromatographic resolution between different proteins when performing chromatography. on a large scale.

Materials and methods:

The starting material for this work was an antibody preparation containing a bispecific anti-BCMA/CD3 IgG antibody of interest, as well as various impurities such as those noted above. The amino acid sequences of the bispecific antibody polypeptides are given in the following SEQ ID NO: anti-BCMA heavy chain: SEQ ID NO: 6; anti-BCMA light chain: SEQ ID NO: 8; anti-CD3 heavy chain: SEQ ID NO: 2; anti-CD3 light chain: SEQ ID NO: 4. The bispecific antibody of interest is prepared from two separate parent antibodies as described above herein. More specifically, the parental anti-CD3 and anti-BCMA monospecific antibodies are purified by protein A chromatography, and these purified parental anti-CD3 and anti-BCMA monospecific antibodies are used to prepare the anti-BCMA/CD3 bispecific antibody of interest. The resulting anti-BCMA/CD3 bispecific antibody is then purified by ion exchange chromatography. The antibody preparation used as purification starting material in this example was eluted from the ion exchange column and contained the bispecific antibody of interest and various remaining impurities, as well as buffers/salts at approximately 50 mM Tris and 60 mM glycine, pH 7.5 . The antibody preparation contained more than 85% wt. an intact bispecific antibody of interest; it also contained approximately 8% clipped bispecific antibody, approximately 1% monospecific parent anti-BCMA antibody, approximately 1% monospecific parent anti-CD3 antibody, and approximately 2% protein aggregates/high molecular weight compounds (HMMS). Thus, although the antibody preparation used as starting material in this method contained an intact bispecific antibody of interest that was already relatively pure, the goal was to develop a method for increasing the purity of the intact bispecific antibody.

Results:

A variety of different chromatographic resins and conditions were tested in an attempt to identify the appropriate resin and buffer conditions that would allow efficient purification of intact anti-BCMA/CD3 bispecific antibody of interest from clipped bispecific antibody, monospecific parent antibodies, and protein aggregates. During this process, for example, many different ion exchange resins and hydrophobic interaction resins were tested, as well as different buffer conditions and pH. After extensive testing, hydroxyapatite resin was the only resin identified that could effectively purify intact anti-BCMA/CD3 bispecific antibody from various contaminants, including clipped bispecific antibody.

In fig. 3 shows the chromatographic profile of the elution of a bispecific antibody of interest from a ceramic hydroxyapatite (“cHA”) resin column and the competitive separation of the anti-BCMA/CD3 bispecific antibody of interest from a variety of different contaminants, including a clipped version of the bispecific antibody, both parent antibodies, and high molecular weight proteins. In the chromatographic experiment shown in FIG. 3, additional parental anti-BCMA antibodies and parental anti-CD3 antibodies were added to the antibody preparation loaded into the cHA resin to more clearly identify the elution position of these molecules from the cHA column (however, no additional intact or clipped bispecific antibodies were added). In fig. 3, the X-axis from left to right shows the elution sequence from the cHA column (ie, the material on the left elutes from the cHA column earlier/at a lower salt concentration than the material on the right). Typically, the eluate is collected from the column in successive fractions; thus, the X-axis can also be considered to reflect the sequence of eluate fractions from the cHA resin column. The Y-axis shows UV absorbance (at 280 nM) and conductivity, which are separately indicated in the graph. UV absorbance corresponds to the presence of protein being eluted, and conductivity corresponds to the salt concentration of the eluted material. Thus, in FIG. Figure 3 shows the elution profile of various proteins from a cHA resin column, because the salt concentration in the elution buffer flowing through the cHA resin increases. Looking at the UV graph in FIG. 3 from left to right, it shows various peaks and shoulders corresponding to the intact anti-BCMA/CD3 bispecific antibody of interest or various impurities. Specifically, from left to right, the first UV peak corresponds to the elution of the first parent antibody ("parent antibody 1"; monospecific anti-BCMA homodimer). The early part of the next large peak (and the largest peak in the graph) corresponds to the protein of the intact bispecific antibody of interest ("POI"). The last/tail portion of the same large peak corresponds to a clipped version of the bispecific antibody (“clipped version”). Although there is some overlap between the elution profile of the intact bispecific antibody and the clipped bispecific antibody, these two types of antibodies elute from the cHA column in significantly different salt conditions and fractions to significantly separate the intact bispecific antibody from the clipped version of the bispecific antibody. Finally, after elution of the clipped version of the bispecific antibody, the last large peak/shoulder from the cHA column corresponds to the elution of the second parent antibody (anti-CD3 monospecific homodimer) as well as high molecular weight compounds ("HMMS") (also referred to as protein aggregates) from the cHA column . Thus, in the graph in FIG. 3 shows that intact anti-BCMA/CD3 antibody can be effectively purified from a variety of contaminants, including the corresponding clipped and parent antibody, by cHA resin chromatography.

In fig. 4 is a graph that provides more detailed information about the different molecules present in the sequentially eluted fractions from a cHA resin column, according to the cHA elution profile shown in FIG. 3. In particular, in FIG. 4 provides details of the relative amounts of: i) intact anti-BCMA/CD3 bispecific antibody of interest ("POI"); ii) a clipped version of the bispecific antibody; iii) the first parental antibody/monospecific antibody against BCMA; iv) a second parental antibody/monospecific anti-CD3 antibody; and v) high molecular weight compounds ("HMMS") in various fractions eluted from the cHA column. On the X axis in Fig. 4, from left to right, shows the sequence of eluate from a cHA column (i.e., from low salt concentration to high salt concentration; each data point corresponds to the fraction eluted from the column). On the Y axis in Fig. 4 on the left shows the percentage of each of i) the protein of the intact anti-BCMA/CD3 bispecific antibody of interest ("POI"); ii) a clipped version of the bispecific antibody; iii) the first parental antibody/monospecific antibody against BCMA; and iv) a second parental antibody/anti-CD3 monospecific antibody in each fraction. On the Y axis of Fig. Figure 4 on the right shows the % HMMS in each fraction.

The method used to obtain the data shown in FIG. 3 was also used on an antibody preparation to which no additional antibodies were added; the data from this chromatographic experiment are shown in FIG. 5. Thus, the data in FIG. 5 depicts the cHA resin purification of a typical antibody preparation eluted from an ion exchange column during the production of anti-BCMA/CD3 bispecific antibodies, containing primarily the intact anti-BCMA/CD3 bispecific antibody of interest, as well as various impurities, including a clipped version of the bispecific antibody . In fig. 5, the X-axis from left to right shows the sequence of eluting material from the cHA column. The Y-axis shows UV absorbance (at 280 nM) and conductivity, which are separately indicated in the graph. The peaks of parent antibody 1 and parent antibody 2 were smaller in Fig. 5 than in Fig. 3, because into the sample injected into the cHA column in FIG. 5 did not add additional parent antibody 1 and parent antibody 2 (while in Fig. 3, additional parent antibody 1 and parent antibody 2 were added to the sample).

In fig. 6 is a graph that provides additional information on the yield of intact anti-BCMA/CD3 bispecific antibody of interest from the cHA resin, as well as the relative amount of clipped bispecific antibody in the various fractions eluting from the cHA resin. The X-axis from left to right shows the sequence of eluate from the cHA column (i.e., from low salt concentration to high salt concentration; each data point corresponds to the fraction eluted from the column). Along the vertical/Y axis are three different variables for each fraction: Variable 1) (diamonds): total yield of intact bispecific antibody ("%POI"), which is the total amount of intact bispecific antibody against BCMA/CD3 (i.e., the antibody of interest) protein) isolated from the cHA column up to and including this fraction in the chromatographic experiment (in other words, as if all the material eluting from the cHA column during the experiment up to and including this fraction were collected and pooled, and then the total amount of intact protein was measured) anti-BCMA/CD3 bispecific antibody in this pooled material); in addition, the "% POI" value is reported as the allocated percentage of the total amount of intact anti-BCMA/CD3 bispecific antibody/protein of interest loaded onto the cHA column (ie, not a gram value). Variable 2) (squares): % of protein in each respective fraction that is clipped bispecific antibody ("% clipped antibody"). Variable 3) (triangles): total yield of clipped bispecific antibody ("total clipped antibody"), representing the total amount of clipped bispecific antibody recovered from the cHA column prior to that fraction in the chromatography experiment. Moreover, in the graph in FIG. 6, the Y-axis on the left shows the %POI values and the Y-axis on the right shows the % clipped antibody values.

Fig. 6 also contains information about the correspondence of some of the different fractions in FIG. 6 different points in the UV absorption on the chromatographic profile shown in FIG. 5. Thus, for example, in FIG. Figure 5 shows that the highest UV absorption/protein elution peak from the cHA resin corresponds to the protein of interest/intact anti-BCMA/CD3 bispecific antibody. The top of this large UV absorption/protein elution peak from the cHA column is also referred to in the experiment as the “top”; the chromatographic peak fraction corresponding to this apex point is indicated in FIG. 6. Then various points after the top of the peak are also indicated in Fig. 6. These points are "90%", "80%", "70%", "60%" and "50%" and are calculated as follows. The UV absorption value at the vertex is set as the starting point for additional calculations. A UV value representing 90% of the UV value of the vertex is then determined. The 90% UV peak value occurring after the UV peak value achieved during the chromatographic experiment (ie, towards the tail of the peak) is reported as the “90%” fraction; it may also be referred to herein as the "90% post-apex" fraction or the like. The same is repeated for the values 80%, 70%, 60% and 50% (i.e. each of these values is located further towards the tail of the peak and thus corresponds to an increasingly larger amount of collected material). As shown in FIG. 6, with a decrease in the % vertex value, the % of clipped antibody in the fractions increases. This is consistent with the clipped bispecific antibody eluting from the cHA column later/at a higher salt concentration than the intact anti-BCMA/CD3 bispecific antibody. Thus, if a larger fraction of the peak apex is collected (ie, with a lower % fraction after the peak apex), then more of the clipped bispecific antibody will also be collected due to the partial overlap between the elution profiles of the intact bispecific antibody and the clipped bispecific antibody.

Various values in Fig. 6 are also presented below in Table 1. As shown in Table 1, in the cHA purification method of the present invention, for example, if the 90% post-peak eluate from the cHA column is collected (i.e., % POI yield to the "90" fraction % after peak apex"), then 54% of the intact bispecific antibody/protein of interest loaded onto the cHA column is isolated, and this isolated protein pool contains 0% of the clipped bispecific antibody. In another example, if the 50% post-peak eluate from a cHA column is collected (i.e., the % POI yield to the "50% post-peak" fraction), then 74% of the intact bispecific antibody/protein of interest introduced into the cHA column is recovered. column, and this isolated protein aggregate contains 0.2% clipped bispecific antibody. Thus, as shown in FIG. 6 and Table 1, the cHA resin can effectively achieve reliable purification of the intact bispecific antibody of interest from the clipped bispecific antibody.

Table 1

Peak aggregate %POI % of total clipped antibody Top of the peak 34 0 90% after top of peak 54 0 80% after top of peak 61 0.1 70% after top of peak 67 0.1 60% after top of peak 70 0.2 50% after top of peak 74 0.2

To obtain the cHA chromatography results shown in FIG. 3-6, cHA chromatography was carried out as follows. An antibody preparation containing the anti-BCMA/CD3 bispecific antibody of interest and various impurities was applied to a chromatography column containing cHA resin. The cHA resin was cHA type 1, 40 μm particle size (Bio-Rad). In the case of the chromatographic experiments shown in FIG. 3 and 4, the sample was injected into the cHA resin until the protein density on the cHA resin was 30 g/L. In the case of the chromatographic experiments shown in FIG. 5 and 6, the sample was injected into the cHA resin until the protein density on the cHA resin was 10 g/L. Before introducing the sample into the cHA resin, the resin was pre-equilibrated with 5 column volumes of equilibration buffer 1 and then 5 column volumes of equilibration buffer 2. The composition of the various buffers in this method is shown below in Table 2. Antibody preparation containing partially purified anti-BCMA bispecific antibody of interest CD3 was loaded onto a cHA resin column in a loading buffer containing approximately 50 mM Tris and 60 mM glycine, pH 7.5. After injecting the antibody preparation into the cHA resin column, the column was washed with 3 column volumes of wash buffer. The bispecific antibody of interest (ie, the intact bispecific antibody) was then eluted from the cHA resin using 20 column volumes of elution buffer in which the sodium phosphate concentration was increased from 40 mM to 80 mM during the elution. As shown in FIG. 3-6, various impurities present in the antibody preparation with the bispecific antibody of interest are also eluted from the cHA column by the elution buffer, but this is done at significantly different salt concentrations compared to the intact anti-BCMA/CD3 bispecific antibody of interest, thus that the intact bispecific antibody can be effectively separated from various impurities, including the clipped version of the intact bispecific antibody.

After eluting the protein of interest from the cHA resin column using the elution buffer in the manner described above, the cHA resin can be desorbed with 5 column volumes of desorption buffer, followed by sanitation with 5 column volumes of sanitation buffer, followed by treatment with 5 column volumes of storage buffer.

For each of the above analyzes of different material eluting from the cHA column, the type and amount of different species of molecules in different fractions/pools were determined by analytical cation exchange (CEX) chromatography.

table 2

Buffer name Composition Column volumes Balancing buffer 1 400 mM sodium phosphate, pH 7.5 5 Balancing buffer 2 20 mM HEPES, 2 mM sodium phosphate, pH 7.5 5 Boot buffer Protein aggregate in approximately 50 mM Tris, 60 mM glycine, adjusted to pH 7.5 N/A Wash buffer 20 mM HEPES, 40 mM sodium phosphate 3 Elution buffer Gradient 20 column volumes from 40 to 100 mM sodium phosphate 20 Desorption buffer 400 mM sodium phosphate, pH 7.5 5 Buffer for sanitation 500 mM potassium phosphate, 1 M sodium hydroxide 5 Storage Buffer 100 mM sodium hydroxide 5

Example 2: Purification of Anti-BCMA/Anti-CD3 Bispecific Antibody by cHA Resin Chromatography - Bulk Load Samples

Target:

In this example, the goal was to determine whether the bispecific antibody purification method described in Example 1 could be effectively used with different cHA resin column mass loading samples. For example, a specific goal was to determine, for various mass loading samples on a cHA resin column, whether more than 50% of the input bispecific antibody could be consistently recovered while yielding no more than 1% of the clipped bispecific antibody as an impurity in the isolated purified bispecific antibody product.

Materials and methods:

The materials and methods in this example were the same as in example 1, except that a sample of the antibody preparation was introduced into the cHA resin until the protein density on the cHA resin (in different chromatographic experiments) was 8 g/l, 10 g/l l or 12 g/l.

Results:

The results of these chromatography experiments are summarized below in Table 3. In all 3 chromatography experiments, 90% of the post-peak material was collected and analyzed (as described in Example 1). As shown in Table 3, for each of the bulk samples of 8 g/L, 10 g/L and 12 g/L, more than 50% of the input intact bispecific antibody was isolated as purified bispecific antibody, and the isolated purified bispecific antibody product contained less than 1% clipped bispecific antibody as an impurity.

Thus, these experiments demonstrated that the intact bispecific antibody of interest can be consistently and efficiently separated from the clipped bispecific antibody by cHA resin chromatography under different cHA resin mass loading assays.

Table 3

Mass load Total output POI Total clipped antibody 8 grams/liter 56% 0.4 10 grams/liter 58% 0.5 12 grams/liter 58% 0.8

Example 3: Purification of anti-BCMA/anti-CD3 bispecific antibody by cHA resin chromatography - different pH samples

Target:

In this example, the objective was to determine whether the bispecific antibody purification method described in Example 1 could be effectively carried out under different pH conditions.

Materials and methods:

The materials and methods in this example were the same as in example 1, except that the pH of the buffers used throughout the method was pH 7.0, pH 7.5 or pH 8.0 (in different chromatographic experiments). In these chromatographic experiments, a sample of the antibody preparation was injected into the cHA resin to a protein density of 30 g/L on the cHA resin.

Results:

The results of these chromatography experiments are summarized below in Table 4. In all 3 chromatography experiments, peak apex material was collected and analyzed (as described in Example 1). As shown in Table 4, intact bispecific antibody protein of interest was successfully isolated under each of the different pH conditions tested, and at pH 7.5 the highest yield of protein of interest was achieved (in these chromatographic experiments, the amount of clipped bispecific antibody in the purified material was not separately determined).

Thus, these experiments demonstrated that the intact bispecific antibody of interest can be efficiently purified by cHA resin chromatography under different pH conditions.

Table 4

pH buffer Total output POI 7.0 thirty% 7.5 40% 8.0 20%

Example 4: Purification of Anti-FLT3/Anti-CD3 Bispecific Antibody by cHA Resin Chromatography

Target:

In this example, the objective was to determine whether the bispecific antibody purification method described in Example 1 could be effectively carried out using a different bispecific antibody than Example 1, which also required separation of the intact bispecific antibody of interest from various contaminants, including the corresponding clipped bispecific antibody with a similar mass. In this example, the antibody of interest was a heterodimeric anti-FLT3/anti-CD3 bispecific antibody.

Materials and methods:

The heterodimeric anti-FLT3/anti-CD3 bispecific antibody used in this example contained the same anti-CD3 heavy chain and light chain as in Example 1. The amino acid sequences of the polypeptides in the FLT3-binding arm of the bispecific antibody are given in the following SEQ ID NO: heavy chain anti-FLT3: SEQ ID NO: 10; anti-FLT3 light chain: SEQ ID NO: 12.

The clipped version of the bispecific antibody in the example included cleavage at the same position of the anti-CD3 heavy chain as described in Example 1.

The materials and methods in this example were the same as in Example 1, except that, as described above, the sample antibody preparation contained an anti-FLT3/anti-CD3 bispecific antibody. A sample of the antibody preparation was injected into the cHA resin until the protein density on the cHA resin was 10 g/L.

Results:

In fig. 7 is a graph that provides information on the yield of intact anti-FLT3/CD3 bispecific antibody of interest from the cHA resin, as well as the relative amount of clipped bispecific antibody in the various cHA resin eluting fractions. The X-axis from left to right shows the sequence of eluate from the cHA column (i.e., from low salt concentration to high salt concentration; each data point corresponds to the fraction eluted from the column). The vertical/Y axis shows three different variables for each fraction: variable 1) (diamonds): % POI; variable 2) (squares): % clipped antibody; and variable 3) (triangles), each of which was determined as described in example 1 and shown in Fig. 6. Moreover, in the graph of FIG. 7, the Y-axis on the left shows the %POI values and the Y-axis on the right shows the % clipped antibody values. Fig. 7 also includes information regarding the correspondence of some of the fractions shown in FIG. 7, different UV absorption points on the corresponding chromatographic profile (not shown). The points indicated as "top", "85%" and "60%" were determined in the same way as for FIG. 6.

Different values from fig. 7 are also shown below in Table 5. As shown in Table 5, the cHA resin purification method of the present invention makes it possible, for example, to recover more than 80% of the intact anti-FLT3/CD3 bispecific antibody of interest while recovering less than 1% of the clipped bispecific antibody in the purified antibody product.

Thus, as shown in FIG. 7 and Table 5, purification of the intact anti-FLT3/CD3 bispecific antibody from the clipped bispecific antibody can be effectively achieved using the cHA resin.

Table 5

Peak aggregate %POI % of total clipped antibody Top of the peak 55 0.2 85% after top of peak 72 0.4 60% after top of peak 82 0.7

Although the presented ideas are described with reference to various uses, methods, kits and compositions, it should be understood that various changes and modifications can be made without deviating from the ideas presented in the present description and the claims below. The examples described above are provided to better illustrate the ideas described and not to limit the scope of the ideas presented herein. Although the presented ideas are described in terms of these example embodiments, those skilled in the art will appreciate that numerous variations and modifications of these example embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the teachings of the present invention.

All references cited herein, including patents, patent applications, articles, manuals, etc., and references cited therein are therefore incorporated herein by reference in their entirety. To the extent that one or more of the included literature and similar materials differ from or conflict with this application, including, by way of non-limiting examples, certain terms, usage of terms, methods described, or the like, this application shall control.

The foregoing description and examples describe in detail some specific embodiments of the invention and the best mode of implementation provided by the inventors of the present invention. However, it should be understood that, despite how detailed the above text may be, the present invention can be practiced in a variety of ways, and the present invention should be considered in light of the claims and any equivalents thereof.

It should be understood that while embodiments are described herein using the term “comprising,” otherwise similar embodiments described using the terms “consisting of” and/or “consisting essentially of” are also provided.

Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention includes not only the entire group as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group in which there is no one or more group members. The present invention also provides for the specific exclusion of one or more of any group members from the claimed invention.

Unless otherwise specified, all technical and scientific terms used in this specification have the meaning commonly understood by one skilled in the art to which the invention belongs. In the event of a conflict, this description, including definitions, will control. Throughout this specification and claims, the term “comprise” or variations thereof such as “comprises” or “comprising” will be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers . Unless the context otherwise requires, terms in the singular shall include the plural, and terms in the plural shall include the singular. Any examples following the term “for example” are not intended to be exhaustive or limiting. The term “or” when used to list multiple options (eg, “A, B, or C”) should be interpreted to include any one or more of the options unless the context clearly indicates otherwise.

The present description provides examples of methods and materials, although methods and materials similar or equivalent to those presented herein may also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative and not limiting only.

--->

LIST OF SEQUENCES

<110> PFIZER INC.

<120> ANTIBODY PURIFICATION

<130>PC72423A

<150> US 62/635,943

<151> 2018-02-27

<160> 12

<170> Patent In version 3.5

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Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

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Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

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Gln Gly Thr Thr Val Thr Val Ser Ser

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Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

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Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

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Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

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Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

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Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

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Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

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Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

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Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

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Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

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Val Arg Cys Pro Arg Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

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Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

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Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu

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Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

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Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

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Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

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Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

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Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

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Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

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Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

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Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

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Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

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Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

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Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Gly Ser Gly Gly Ser Leu Pro Tyr Ala Asp Ile Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Tyr Trp Pro Met Asp Ile Trp Gly Gln Gly Thr Leu Val Thr

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Val Ser Ser

115

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Gly Ser Gly Gly Ser Leu Pro Tyr Ala Asp Ile Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Trp Pro Met Asp Ile Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly

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Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

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Val Asp Lys Thr Val Glu Arg Lys Cys Glu Val Glu Cys Pro Glu Cys

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Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

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Val Val Ala Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

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Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

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Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

340 345 350

Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly Phe Tyr Pro

355 360 365

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

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Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

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Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

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Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

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Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

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Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

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Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

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Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

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Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

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Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

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Ser Phe Asn Arg Gly Glu Cys

210 215

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ser Ala Ile Ser Gly Gly Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp Leu Ser Pro Ser Asp Val Gly Trp Gly Tyr Gly Phe Asp

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Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Gly Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp Leu Ser Pro Ser Asp Val Gly Trp Gly Tyr Gly Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

210 215 220

Lys Cys Glu Val Glu Cys Pro Glu Cys Pro Ala Pro Pro Val Ala Gly

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Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

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Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

290 295 300

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

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Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

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Tyr Asp Thr Phe Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

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20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Thr Phe Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (45)

1. A method for purifying an anti-BCMA/anti-CD3 bispecific antibody, comprising: A) introducing the antibody preparation into a loading buffer onto a hydroxyapatite (HA) resin, where: The antibody preparation contains: I) an intact bispecific antibody of interest; And ii) at least one type of impurity, where the type of impurities is selected from the group consisting of: a) a truncated version of the bispecific antibody of interest, wherein the truncated version of the bispecific antibody of interest is a degradation product of the intact bispecific antibody of interest and has a mass that differs by less than 1% from the mass of the intact bispecific antibody of interest; b) a first parent antibody, where the first parent antibody is a monospecific antibody having the same antigenic specificity as the first arm of the intact bispecific antibody; c) a second parent antibody, where the second parent antibody is a monospecific antibody having the same antigenic specificity as the second arm of the intact bispecific antibody; And d) high molecular weight compounds (HMMS); B) washing the resin with a wash buffer containing phosphate ions in a concentration of 10 to 50 mM; And C) eluting the intact bispecific antibody of interest from the HA resin using an elution buffer containing phosphate ions at a concentration of 40 to 100 mM, wherein the concentration of phosphate ions is increased relative to the wash buffer such that the bispecific antibody of interest is separated from at least one type of impurity; wherein the pH of at least one of the loading buffer, wash buffer and elution buffer is at a pH level of from 7.0 to 8.0 or between these values; And wherein the anti-BCMA/anti-CD3 bispecific antibody comprises an anti-BCMA arm and an anti-CD3 arm, wherein (a) anti-CD3 arm contains (i) a VH region containing the amino acid sequence of SEQ ID NO:1 and a VL region containing the amino acid sequence of SEQ ID NO:3, or (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:2, and a light chain containing the amino acid sequence of SEQ ID NO:4; And (b) the anti-BCMA arm contains (i) a VH region containing the amino acid sequence of SEQ ID NO:5 and a VL region containing the amino acid sequence of SEQ ID NO:7, or (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:6, and a light chain containing the amino acid sequence of SEQ ID NO:8. 2. The method according to claim 1, where the ratio of truncated bispecific antibody molecules and intact bispecific antibody molecules in the antibody preparation is at least from 1:50 to no more than 1:5; optionally, where the method includes collecting the purified fraction eluted from the HA resin, wherein the purified fraction contains the intact bispecific antibody. 3. The method according to claim 1, further comprising collecting the purified fraction eluted from the HA resin, wherein the purified fraction contains the intact antibody of interest, and where the purified fraction contains at least 95 wt.%, 96 wt.%, 97 wt.%, 98 wt.%. or 99% wt. intact antibody of interest. 4. The method according to claim 2 or 3, where the purified fraction contains an intact bispecific antibody and a truncated bispecific antibody, where the ratio of truncated bispecific antibody molecules and intact bispecific antibody molecules in the purified fraction is no more than 1:100. 5. The method according to any one of claims 1-4, wherein the truncated antibody has a mass that is approximately 5-100 Da greater than the mass of the intact antibody. 6. The method according to any one of claims 1 to 5, wherein the truncated antibody contains a cleaved peptide bond in the polypeptide chain of the antibody, and wherein the cleaved peptide bond is in the heavy chain of the antibody. 7. The method according to any one of claims 1 to 6, wherein the truncated antibody contains the same number of amino acids and the same amino acid sequences as the intact antibody. 8. The method according to any one of claims 1-6, where the truncated antibody contains a different number of amino acids than the intact antibody. 9. The method according to any one of claims 1 to 8, wherein the HA resin is a ceramic hydroxyapatite (cHA) resin. 10. Method according to any one of claims 1 to 9, wherein the phosphate ion concentration is increased during elution from approximately 40 to 100 mM. 11. The method according to any one of claims 1 to 10, wherein the antibody preparation contains proteins previously loaded onto or eluted from at least: i) Protein A resins and ii) ion exchange resin. 12. Method according to any one of claims 1 to 11, wherein the antibody is isolated and/or purified for use as pharmaceuticals or for the production of pharmaceuticals. 13. An antibody preparation comprising an anti-BCMA/anti-CD3 bispecific antibody comprising an anti-BCMA arm and an anti-CD3 arm, wherein (a) anti-CD3 arm contains (i) a VH region containing the amino acid sequence of SEQ ID NO:1 and a VL region containing the amino acid sequence of SEQ ID NO:3, or (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:2, and a light chain containing the amino acid sequence of SEQ ID NO:4; And (b) the anti-BCMA arm contains (i) a VH region containing the amino acid sequence of SEQ ID NO:5 and a VL region containing the amino acid sequence of SEQ ID NO:7, or (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:6, and a light chain containing the amino acid sequence of SEQ ID NO:8; And where the preparation contains no more than 1% of the trimmed bispecific antibody as an impurity.