HK1249122B - Affinity chromatography purification with low conductivity wash buffer - Google Patents

Description

Affinity chromatography purification using low conductivity wash buffer

The present invention generally relates to the field of polypeptide purification and, more particularly, to improvements in multi-column purification methods for antibody purification.

Background Art

Proteins, particularly immunoglobulins, play a crucial role in today's serial medical products. For human application, each therapeutic protein must meet various E criteria. To ensure the safety of biopharmaceuticals for human use, byproducts accumulated during the production process must be specifically removed. To meet regulatory requirements, the manufacturing process must be followed by one or more purification steps. Purity, production capacity, and yield play a crucial role in determining the appropriate purification process.

Different methods are well established and widely used for protein purification, such as affinity chromatography (e.g., protein A or protein G affinity chromatography, single-chain Fv ligand affinity chromatography), ion exchange chromatography (e.g., cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (aminoethyl resins) and mixed-mode ion exchange), thiophilic adsorption (e.g., with β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-agarose, azaarenophilic resins or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis).

In order to purify the immunoglobulin produced by recombinant methods, a combination of different column chromatography steps is usually used. During the purification period, non-immunoglobulin contaminants such as host cell proteins and host cell DNA as well as endotoxins and viruses are exhausted. Therefore, affinity chromatography steps such as protein A affinity chromatography are usually followed by one or more additional separation steps. Usually, high conductivity buffers are described as washing steps for affinity chromatography. Due to, for example, high salt concentrations, further method steps may be needed when switching from one chromatography/separation step to another chromatography/separation step. High conductivity values in the eluent from a chromatography step may have a negative impact on subsequent chromatography steps.

US 6,127,526 describes a method for purifying a protein by protein A chromatography, comprising the steps of: (a) adsorbing the protein to protein A immobilized on a solid phase comprising silica or glass; (b) removing contaminants bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent; and (c) recovering the protein from the solid phase.

In WO 2011/038894 a Protein A chromatography method is reported in which host cell proteins and DNA are significantly depleted by specific washing steps before the immunoglobulins are recovered from the Protein A chromatography material.

Compositions and methods for isolating and purifying antibodies from sample matrices are reported in WO 2013/177118.

WO2013/033517 reports a method for isolating target polypeptides (such as antibodies) from viruses.

EP2583973 reports a method for purifying proteins, comprising one or more chromatography processes, wherein the buffer used in at least one chromatography process (equilibration buffer, wash buffer and elution buffer) contains amino acids; or dipeptides, oligopeptides or their polyamino acids, thereby purifying high-purity proteins with very low amounts of impurities (e.g., polymers or host cell proteins).

In EP 1561756 a method for purifying proteins is reported.

WO2015/024896 reports the use of hydroxyapatite chromatography to separate bispecific antibodies and bispecific antibody production by-products.

Ritzen et al. (J Chromatogr B Analyt Technol Biomed Life Sci, 2007, vol. 856, pp. 343-347) reported endotoxin reduction in monoclonal antibody formulations using arginine.

SUMMARY OF THE INVENTION

Herein is reported a method for producing bispecific antibodies by purifying the bispecific antibodies with an affinity chromatography step followed by one or more additional separation steps.

More specifically, it has been discovered that by using a low conductivity aqueous solution in the wash step of affinity chromatography prior to recovering the antibody from the chromatography material, the antibody purification process can be improved by avoiding the need for further processing steps before loading the eluate onto the next column/chromatography material and/or the content of specific host cell proteins can be reduced. For example, processing steps such as dilution or filtration of the Protein A affinity chromatography eluate can be omitted.

One aspect as reported herein is a method for producing a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, the method comprising the following steps

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution,

e) recovering the bispecific antibody from the affinity chromatography material,

f) Perform additional chromatography steps

This results in the generation of bispecific antibodies.

One aspect as reported herein is a method for purifying a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen from a sample, the method comprising the following steps:

a) providing a sample comprising the bispecific antibody,

b) applying the sample comprising the bispecific antibody to an affinity chromatography material,

c) washing the affinity chromatography material with a low conductivity aqueous solution,

d) recovering the bispecific antibody from the affinity chromatography material,

e) Perform additional chromatography steps

The bispecific antibody is thereby purified.

In one embodiment of all aspects the additional chromatography step following the affinity chromatography step is an ion exchange chromatography step or a multiplex ion exchange chromatography step.

In one embodiment of all aspects, the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single chain Fv ligand affinity chromatography. In a preferred embodiment of all aspects, the affinity chromatography is protein A affinity chromatography.

In one embodiment of all aspects the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less.

In one embodiment of all aspects the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.

In one embodiment of all aspects the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.

In one embodiment of all aspects the low conductivity aqueous solution has a pH of about 7 or greater.

In one embodiment of all aspects the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before or after washing the affinity chromatography material with the low conductivity aqueous solution.

In one embodiment, the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher. In one embodiment, the medium conductivity aqueous solution has a conductivity value of greater than 0.5 mS/cm to less than 20 mS/cm.

In one embodiment of all aspects the high or medium conductivity aqueous solution comprises histidine.

In one embodiment of all aspects the bispecific antibody is a bispecific antibody comprising,

a) the heavy and light chains of a first full-length antibody that specifically binds to a first antigen; and

b) a modified heavy chain and a modified light chain of a second full-length antibody that specifically binds to a second antigen, wherein the constant domains CL and CH1 are replaced with each other.

In one embodiment of all aspects the first antigen is human VEGF and the second antigen is human ANG-2.

In one embodiment of all aspects the first antigen is human ANG-2 and the second antigen is human VEGF.

In one embodiment of all aspects the first antigen is carcinoembryonic antigen (CEA) and the second antigen is CD3.

In one embodiment of all aspects the first antigen is CD3 and the second antigen is carcinoembryonic antigen (CEA).

In a preferred embodiment of all aspects, the first antigen binding site comprises as heavy chain variable domain (VH) SEQ ID NO: 1 and as light chain variable domain (VL) SEQ ID NO: 2; and the second antigen binding site comprises as heavy chain variable domain (VH) SEQ ID NO: 3 and as light chain variable domain (VL) SEQ ID NO: 4.

Detailed Description of the Invention

High-conductivity buffers are often used in wash steps for affinity chromatography. Switching from one chromatography/separation step to another may require further process steps, for example due to high salt concentrations. High conductivity values in the eluate from one chromatography step can have a negative impact on subsequent chromatography steps.

Herein is reported an improved purification method using affinity chromatography comprising washing the affinity chromatography material with a low conductivity aqueous solution followed by an additional separation step.

One aspect as reported herein is a method for producing a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, the method comprising the following steps

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody (a solution comprising the bispecific antibody) with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution, while at least 90% of the bispecific antibody remains bound to the affinity chromatography material,

e) recovering the bispecific antibody from the affinity chromatography material,

f) Perform additional chromatography steps

This results in the generation of bispecific antibodies.

One aspect as reported herein is a method for purifying a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen from a sample, the method comprising the following steps:

a) providing a (buffered aqueous) sample comprising the bispecific antibody,

b) applying the sample comprising the bispecific antibody to an affinity chromatography material,

c) washing the affinity chromatography material with a low conductivity aqueous solution, while at least 90% of the bispecific antibody remains bound to the affinity chromatography material,

d) recovering the bispecific antibody from the affinity chromatography material,

e) Perform additional chromatography steps

The bispecific antibody is thereby purified.

One aspect as reported herein is a method for producing a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, the method comprising the following steps

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody (a solution comprising the bispecific antibody) with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution, while at least 90% of the bispecific antibody remains bound to the affinity chromatography material, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less,

e) recovering the bispecific antibody from the affinity chromatography material,

f) Perform additional chromatography steps

This results in the generation of bispecific antibodies.

One aspect as reported herein is a method for purifying a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen from a sample, the method comprising the following steps:

a) providing a (buffered aqueous) sample comprising the bispecific antibody,

b) applying the sample comprising the bispecific antibody to an affinity chromatography material,

c) washing the affinity chromatography material with a low conductivity aqueous solution, wherein at least 90% of the bispecific antibody remains bound to the affinity chromatography material, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less,

d) recovering the bispecific antibody from the affinity chromatography material,

e) Perform additional chromatography steps

The bispecific antibody is thereby purified.

In one embodiment of all aspects, the additional chromatography step after the affinity chromatography step is an ion exchange chromatography step or a multivalent ion exchange chromatography step. In a preferred embodiment of all aspects, the additional chromatography step after the affinity chromatography step is a multivalent anion exchange chromatography step. In one embodiment of all aspects, the additional chromatography step after the affinity chromatography step is a multivalent cation exchange chromatography step. In one embodiment of all aspects, the additional chromatography step after the affinity chromatography step is a multivalent anion exchange chromatography material selected from CaptoAdhere ImpRes material (a multivalent anion exchange chromatography medium with a high flow agarose matrix, a multivalent strong anion exchanger as a ligand, an average particle size of 36-44 μm, and an ion capacity of 0.08-0.11 mmol Cl-/mL medium).

In general, certain additional process steps, such as pH adjustment, buffer exchange, dilution, diafiltration or conductivity adjustment, must be performed in the conversion from one column material to another. This is, for example, the case when a Protein A chromatography eluate is applied to a subsequent ion exchange chromatography material/resin. Here, if the chromatography resin is to function properly, the conductivity value of the applied sample must not exceed a certain low level. For example, the removal of certain impurities can be adversely affected. It has been found that further process steps can be avoided before the eluate is loaded onto the next chromatography column/chromatography material if a low-conductivity aqueous solution washing step is used in the previous affinity chromatography step. This washing step can have a significant impact on the final conductivity in the affinity chromatography (e.g. Protein A) eluate.

Table 1

It has been found that if the conductivity of the aqueous solution used in the washing step is low, i.e., a low conductivity aqueous solution is used for washing, the content of host cell protein can be reduced. In a preferred embodiment of all aspects, the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less. In one embodiment, the low conductivity aqueous solution has a conductivity value of from about 0.03 μS/cm to about 0.5 mS/cm. In one embodiment, the low conductivity aqueous solution has a conductivity value of from about 0.05 μS/cm to about 0.35 mS/cm. In one embodiment, the low conductivity aqueous solution is highly purified/deionized water.

It has been found that protein A affinity chromatography can be used for the purposes reported herein. In a preferred embodiment of all aspects, the affinity chromatography is protein A affinity chromatography. In one embodiment, the protein A affinity chromatography is selected from MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Mab Capture A affinity chromatography, ProSep UltraPlus affinity chromatography. In one embodiment, the affinity chromatography is protein G affinity chromatography. In one embodiment, the affinity chromatography is affinity chromatography using a recombinant protein as a ligand, which means that the affinity chromatography is a recombinant protein ligand affinity chromatography. In one embodiment, the affinity chromatography is affinity chromatography using a single-chain Fv as a ligand, which means that the affinity chromatography is a single-chain Fv ligand affinity chromatography. In one embodiment, the affinity chromatography comprises a mutant protein A coupled to a chromatography matrix or a fragment of protein A coupled to a chromatography matrix.

It has been found that the level of a (specific) host cell protein can be reduced. It has been found that in particular the level of phospholipase B-like 2 (PLBL2) can be reduced. In one embodiment, the (specific) host cell protein is a Chinese hamster ovary (CHO) host cell protein. In a preferred embodiment of all aspects, the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin. In one embodiment, the (specific) host cell protein is phospholipase B-like 2 (PLBL2).

It has been found that the low conductivity aqueous solution can contain a small amount of Tris or potassium phosphate. In one embodiment, the low conductivity aqueous solution contains tris (hydroxymethyl) aminomethane (Tris). In one embodiment, the low conductivity aqueous solution contains about 0.1mM to about 10mM Tris. In one embodiment, the low conductivity aqueous solution contains about 0.5mM to about 6.5mM Tris. In one embodiment, the low conductivity aqueous solution contains about 2mM Tris. In one embodiment, the low conductivity aqueous solution contains potassium phosphate. In one embodiment, the low conductivity aqueous solution contains about 0.05mM to about 5mM potassium phosphate. In one embodiment, the low conductivity aqueous solution contains about 0.05mM to about 2mM potassium phosphate. In one embodiment, the low conductivity aqueous solution contains about 0.5mM potassium phosphate.

It has been found that if the low conductivity aqueous solution has a certain pH, the effect of reducing the host cell protein content is significant. In one embodiment, the low conductivity aqueous solution has a pH of about 7 or higher. In one embodiment, the low conductivity aqueous solution has a pH of about 7.5 or higher. In one embodiment, the low conductivity aqueous solution has a pH of about 7.5 to about 8.5. In one embodiment, the low conductivity aqueous solution has a pH of about 8.

It has been found that the methods reported herein can reduce the level of host cell proteins, such as PLBL2, to a certain level, for example, when compared to the amount of PLBL2 loaded prior to a purification step, such as an affinity chromatography step. In one embodiment, the level of PLBL2 is reduced by at least 20-fold. In one embodiment, the level of PLBL2 is reduced by at least 40-fold. In one embodiment, the level of PLBL2 is reduced by at least 50-fold. In one embodiment, the level of PLBL2 is reduced by at least 90-fold. In one embodiment, the level of PLBL2 is reduced by at least 100-fold. In one embodiment, the level of PLBL2 is reduced by at least 50%. In one embodiment, the level of PLBL2 is reduced by at least 66%. In one embodiment, the level of PLBL2 is reduced by at least 80%. In one embodiment, the level of PLBL2 is reduced by at least 90%. In one embodiment, the level of PLBL2 is reduced by at least 95%. In some embodiments, the level of PLBL2 is reduced to less than 10 ng/mg antibody. In some embodiments, the level of PLBL2 is reduced to less than 5 ng/mg antibody. In some embodiments, the level of PLBL2 is reduced to less than 2 ng/mg antibody.

In the method as reported herein, further washing steps may be carried out using medium and/or high conductivity aqueous solutions. In one embodiment, the low conductivity aqueous solution washing step is carried out before or after the high conductivity aqueous solution washing step. In one embodiment, the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or more. In one embodiment, the high conductivity aqueous solution has a conductivity value from about 20 mS/cm to about 100 mS/cm. In one embodiment, an intermediate washing step is carried out with a medium conductivity aqueous solution between the low conductivity aqueous solution washing step and the high conductivity aqueous solution washing step. In one embodiment, the medium conductivity aqueous solution has a conductivity value of greater than 0.5 mS/cm to less than 20 mS/cm.

It has been found that when the high or medium conductivity aqueous solution further comprises an amino acid, the host cell protein reduction effect can be improved. In one embodiment, the high or medium conductivity aqueous solution comprises an amino acid. In one embodiment, the high or medium conductivity aqueous solution comprises histidine. In one embodiment, the high or medium conductivity aqueous solution comprises histidine and Tris.

It has been found that the use of a hydrophobic interaction chromatography step can be omitted. In one embodiment, the use or method is free of a hydrophobic interaction chromatography method/step.

One aspect as reported herein is a method for producing a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, the method comprising

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody with a protein A affinity chromatography material,

d) washing the protein A affinity chromatography material with a low conductivity aqueous solution,

e) recovering the bispecific antibody from the protein A affinity chromatography material,

f) Perform additional chromatography steps

Thus, bispecific antibodies are generated.

wherein the additional chromatography step following the affinity chromatography step is a multiplexed anion exchange chromatography step, and wherein the low conductivity aqueous solution is water, and wherein the first antigen-binding site comprises as heavy chain variable domain (VH) SEQ ID NO: 1, and as light chain variable domain (VL) SEQ ID NO: 2; and the second antigen-binding site comprises as heavy chain variable domain (VH) SEQ ID NO: 3 and as light chain variable domain (VL) SEQ ID NO: 4.

One aspect as reported herein is a method for purifying a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen from a sample, the method comprising the following steps:

a) providing a sample comprising the bispecific antibody,

b) applying the sample containing the bispecific antibody to a protein A affinity chromatography material,

c) washing the protein A affinity chromatography material with a low conductivity aqueous solution,

d) recovering the bispecific antibody from the protein A affinity chromatography material,

e) performing an additional chromatography step,

thereby purifying the bispecific antibody,

wherein the additional chromatography step after the affinity chromatography step is a multiplexed anion exchange chromatography step, and wherein the low conductivity aqueous solution is water, and wherein the first antigen binding site comprises SEQ ID NO: 1 as the heavy chain variable domain (VH), and SEQ ID NO: 2 as the light chain variable domain (VL); and the second antigen binding site comprises SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL).

One aspect as reported herein is a method for producing a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, the method comprising the following steps

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less,

e) recovering the bispecific antibody from the affinity chromatography material,

f) performing an additional chromatography step,

This results in the generation of bispecific antibodies.

One aspect as reported herein is a method for purifying a bispecific antibody comprising a first antigen binding site that specifically binds to a first antigen and a second antigen binding site that specifically binds to a second antigen, said method comprising the following steps

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less,

e) recovering the bispecific antibody from the affinity chromatography material,

f) performing an additional chromatography step,

This results in the generation of bispecific antibodies.

The term "process" as used herein also includes the use of individual process steps for avoiding a conductivity adjustment step (directly) before a subsequent further chromatography step. In particular, the conductivity adjustment step is avoided after the viral inactivation step and before an ion exchange chromatography step (directly) performed thereafter.

One aspect as reported herein is to use the steps of the method as reported herein to avoid a conductivity adjustment step after recovery of the bispecific antibody from the affinity chromatography material and before performing further chromatography steps.

The terms "anti-Ang2/VEGF antibody" and "bispecific antibody that binds to Ang2 and VEGF" or "bispecific antibody to Ang2 and VEGF" refer to antibodies that bind to Ang2 and VEGF with sufficient affinity to allow the antibody to be used as a diagnostic and/or therapeutic agent targeting Ang2 and VEGF. In one embodiment, the extent of binding of the anti-Ang2/VEGF antibody to unrelated non-Ang2 or VEGF proteins is less than about 10% of the binding of the antibody to Ang2 and VEGF, as measured, for example, by ELISA or surface plasmon resonance. In certain embodiments, the anti-Ang2/VEGF antibody binds to an epitope of Ang2 and an epitope of VEGF that is conserved among Ang2 or VEGF from different species. The foregoing also applies to the terms "bispecific antibody to Factor IXa and Factor X" or "bispecific antibody to Her3 and EGFR," etc.

The specific antibodies used in the methods as reported herein are bispecific antibodies against Factor IXa and Factor X (anti-FIXa/X antibodies; IgG4 isotype) as described in WO2012/067176, bispecific antibodies against Angiopoietin 2 (Ang2) and Vascular Endothelial Growth Factor A (VEGF-A) (anti-Ang2/VEGF-A antibodies; vanucizumab; IgG1 isotype) as described in WO2011/117329 or SEQ ID NOs: 01 to 04, or bispecific antibodies against Her3 and EGFR (anti-Her3/EGFR antibodies; IgG1 isotype). The terms VEGF or VEGF-A may be used interchangeably herein.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an antigen epitope in an in vitro assay, preferably a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden). The affinity of binding is defined by the terms ka (rate constant for antibody binding from the antibody/antigen complex), kd (dissociation constant), and _KD ( _kd / _ka ). Binding or specific binding refers to a binding affinity ( _KD ) of ^10-7 mol/L or less.

The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

"Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Fab fragments are antibody fragments obtained by papain digestion of (full-length/intact) antibodies.

A "bispecific antibody" is an antibody with two different antigen-binding specificities. As used herein, the term "bispecific" antibody refers to an antibody with at least two binding sites, each binding site binding a different epitope.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA _2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "Fc-region" herein is used to define the C-terminal region of the immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or C-terminal glycyl-lysine dipeptide (Gly446Lys447) in the Fc region may or may not exist. Unless otherwise specified herein, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acids have been introduced, including the offspring of these cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the initially transformed cells are included herein. The term "cell" includes cells for expressing nucleic acids. In one embodiment, the host cell is a CHO cell (e.g., CHO K1, CHO DG44) or a BHK cell or a NS0 cell or a SP2/0 cell or a HEK 293 cell or a HEK 293EBNA cell or a COS cell. In another embodiment, the cell is a CHO cell or a BHK cell or a cell. As used herein, the expression "cell" includes subject cells and their offspring.

The term "washing" refers to the application of a solution to an affinity chromatography material to remove non-specifically bound polypeptides and non-polypeptide compounds from the chromatography material, in particular to remove host cell proteins and host cell DNA. The term "washing" does not include eluting bound material from the affinity chromatography material.

Different methods for protein recovery and purification are well established and widely used, such as affinity chromatography using microproteins (e.g. protein A or protein G affinity chromatography), affinity chromatography using recombinant proteins as ligands (e.g. single-chain Fv as ligand, e.g. kappa selection), ion exchange chromatography (e.g. cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g. with β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-agarose, aza-arenophilic resin or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoretic methods (e.g. gel electrophoresis, capillary electrophoresis). These methods can be independently combined in the different embodiments reported herein.

The term "Protein A" refers to a Protein A polypeptide obtained from a natural source or produced synthetically.

The term "Protein A chromatography material" refers to an inert solid phase to which Protein A is covalently attached.

In one embodiment, the Protein A chromatography material is selected from Mab Select Sure, ProSep vA, Mab Capture A, ProSep Ultra Plus, Mab Select, Mab Select Xtra, Poros A, or ProSep A.

The term "high conductivity aqueous solution" refers to an aqueous solution having a high conductivity value. The conductivity value may be about 20 mS/cm or higher.

The term "medium conductivity aqueous solution" refers to an aqueous solution having a medium conductivity value. The conductivity value may be greater than 0.5 mS/cm to less than 20 mS/cm.

The term "low conductivity aqueous solution" refers to an aqueous solution having a low conductivity value. The conductivity value may be about 0.5 mS/cm or less. If the pH is about 8.5 or higher, the conductivity value may be about 1.2 mS/cm or less.

Specific embodiments of the present invention

1. A method for producing a bispecific antibody comprising a first antigen-binding site that specifically binds to a first antigen and a second antigen-binding site that specifically binds to a second antigen, the method comprising the following steps:

a) culturing cells comprising a nucleic acid encoding a bispecific antibody,

b) recovering the bispecific antibody from the cells or culture medium,

c) contacting the bispecific antibody with an affinity chromatography material,

d) washing the affinity chromatography material with a low conductivity aqueous solution,

e) recovering the bispecific antibody from the affinity chromatography material,

f) performing an additional chromatography step,

This results in the generation of bispecific antibodies.

2. The method according to embodiment 1, wherein the additional chromatography step after the affinity chromatography step is an ion exchange chromatography step or a multicomponent ion exchange chromatography step.

3. The method according to any one of embodiments 1 to 2, wherein the further chromatography step following the affinity chromatography step is a multiplex anion exchange chromatography step.

4. The method according to any one of embodiments 1 to 3, wherein the multivalent anion exchange chromatography material is selected from CaptoAdhere ImpRes material (a multivalent anion exchange chromatography medium with a high-flow agarose matrix, a multivalent strong anion exchanger as a ligand, an average particle size of 36-44 μm, and an ion capacity of 0.08-0.11 mmol Cl-/mL culture medium).

5. The method according to any one of embodiments 1 to 4, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less.

6. The method according to any one of embodiments 1 to 5, wherein the low conductivity aqueous solution has a conductivity value of from about 0.03 μS/cm to about 0.5 mS/cm.

7. The method according to any one of embodiments 1 to 6, wherein the low conductivity aqueous solution has a conductivity value of from about 0.05 μS/cm to about 0.35 mS/cm.

8. The method according to any one of embodiments 1 to 5, wherein the low conductivity aqueous solution is highly purified/deionized water.

9. The method according to any one of embodiments 1 to 8, wherein the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single-chain Fv ligand (KappaSelect) affinity chromatography.

10. The method according to any one of embodiments 1 to 9, wherein the affinity chromatography is protein A affinity chromatography.

11. The method according to any one of embodiments 1 to 10, wherein the Protein A affinity chromatography is selected from MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography.

12. The method according to any one of embodiments 1 to 11, wherein the content of (specific) host cell proteins is reduced.

13. The method according to any one of embodiments 1 to 12, wherein the (specific) host cell protein is a Chinese Hamster Ovary (CHO) host cell protein.

14. The method according to any one of embodiments 1 to 13, wherein the (specific) host cell protein is a phospholipase.

15. The method according to any one of embodiments 1 to 14, wherein the (specific) host cell protein is phospholipase A, phospholipase B, phospholipase C or phospholipase D.

16. The method according to any one of embodiments 1 to 15, wherein the (specific) host cell protein is phospholipase B-like 2 (PLBL2).

17. The method according to any one of embodiments 1 to 16, wherein the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

18. The method according to any one of embodiments 1 to 17, wherein the low conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).

19. The method according to any one of embodiments 1 to 18, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 10 mM Tris.

20. The method according to any one of embodiments 1 to 19, wherein the low conductivity aqueous solution comprises from about 0.1 mM to about 8 mM Tris.

21. The method according to any one of embodiments 1 to 20, wherein the low conductivity aqueous solution comprises from about 0.5 mM to about 6.5 mM Tris.

22. The method according to any one of embodiments 1 to 21, wherein the low conductivity aqueous solution comprises about 2 mM Tris.

23. The method according to any one of embodiments 1 to 17, wherein the low conductivity aqueous solution contains potassium phosphate.

24. The method according to any one of embodiments 1 to 17, wherein the low conductivity aqueous solution comprises from about 0.2 mM to about 5 mM potassium phosphate.

25. The method according to any one of embodiments 1 to 17, wherein the low conductivity aqueous solution comprises from about 0.05 mM to about 2 mM potassium phosphate.

26. The method according to any one of embodiments 1 to 17, wherein the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.

27. The method according to any one of embodiments 1 to 26, wherein the low conductivity aqueous solution has a pH of about 7 or higher.

28. The method according to any one of embodiment 27, wherein the low conductivity aqueous solution has a pH of about 7.5 or higher.

29. The method according to any one of embodiments 1 to 26, wherein the low conductivity aqueous solution has a pH of about 7 to about 9.5.

30. The method according to any one of embodiments 1 to 27, wherein the low conductivity aqueous solution has a pH of about 7.5 to about 8.5.

31. The method according to any one of embodiments 1 to 27, wherein the low conductivity aqueous solution has a pH of about 8.

32. The method according to any one of embodiments 1 to 31, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

33. The method according to any one of embodiments 1 to 32, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

34. The method according to any one of embodiments 1 to 33, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

35. The method according to any one of embodiments 1 to 34, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

36. The method according to any one of embodiments 32 to 35, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher.

37. The method according to any one of embodiments 32 to 36, wherein the high conductivity aqueous solution has a conductivity value of from about 20 mS/cm to about 100 mS/cm.

38. The method according to any one of embodiments 32 to 37, wherein the moderate conductivity aqueous solution has a conductivity value of greater than 0.5 mS/cm to less than 20 mS/cm.

39. The method according to any one of embodiments 32 to 38, wherein the high or medium conductivity aqueous solution comprises an amino acid.

40. The method according to any one of embodiments 32 to 39, wherein the high or medium conductivity aqueous solution comprises histidine.

41. The method according to any one of embodiments 32 to 40, wherein the high or medium conductivity aqueous solution comprises histidine and Tris.

42. The method according to any one of embodiments 1 to 41, wherein the method is free of a hydrophobic interaction chromatography method/step.

43. A method according to any one of embodiments 1 to 42, wherein the bispecific antibody is a bispecific antibody comprising a) the heavy chain and light chain of a first full-length antibody that specifically binds to a first antigen; and b) a modified heavy chain and a modified light chain of a second full-length antibody that specifically binds to a second antigen, wherein the constant domains CL and CH1 are replaced with each other.

44. The method according to any one of embodiments 1 to 43, wherein the bispecific antibody is a bispecific antibody against Factor IXa and Factor X or a bispecific antibody against HER3 and EGFR or a bispecific antibody against Ang2 and VEGF.

45. The method according to any one of embodiments 1 to 44, wherein the first antigen is human VEGF and the second antigen is human ANG-2 or the first antigen is human ANG-2 and the second antigen is human VEGF.

46. A method according to any one of embodiments 44 to 45, wherein the first antigen binding site comprises SEQ ID NO: 1 as the heavy chain variable domain (VH), and SEQ ID NO: 2 as the light chain variable domain (VL); and the second antigen binding site comprises SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL).

47. A method for purifying a bispecific antibody from a sample, wherein the bispecific antibody comprises a first antigen-binding site that specifically binds a first antigen and a second antigen-binding site that specifically binds a second antigen, the method comprising the steps of:

a) providing a sample comprising the bispecific antibody,

b) applying the sample comprising the bispecific antibody to an affinity chromatography material,

c) washing the affinity chromatography material with a low conductivity aqueous solution,

d) recovering the bispecific antibody from the affinity chromatography material,

e) Perform additional chromatography steps

The bispecific antibody is thereby purified.

48. The method according to embodiment 47, wherein the further chromatography step following the affinity chromatography step is an ion exchange chromatography step or a multiplex ion exchange chromatography step.

49. The method according to any one of embodiments 47 to 48, wherein the further chromatography step following the affinity chromatography step is a multiplex anion exchange chromatography step.

50. The method according to embodiment 49, wherein the multinary anion exchange chromatography material is selected from Capto Adhere Imp Res material (multinary anion exchange chromatography with a high-flow agarose matrix, multinary strong anion exchangers as ligands, an average particle size of 36-44 μm, and an ion capacity of 0.08-0.11 mmol Cl-/mL medium).

51. The method according to any one of embodiments 47 to 50, wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less.

52. The method according to any one of embodiments 47 to 51, wherein the low conductivity aqueous solution has a conductivity value of from about 0.03 μS/cm to about 0.5 mS/cm.

53. The method according to any one of embodiments 47 to 52, wherein the low conductivity aqueous solution has a conductivity value of from about 0.05 μS/cm to about 0.35 mS/cm.

54. The method according to any one of embodiments 47 to 51, wherein the low conductivity aqueous solution is highly purified/deionized water.

55. The method according to any one of embodiments 47 to 54, wherein the affinity chromatography is protein A affinity chromatography or protein G affinity chromatography or single-chain Fv ligand (KappaSelect) affinity chromatography.

56. The method according to any one of embodiments 47 to 55, wherein the affinity chromatography is protein A affinity chromatography.

57. The method according to any one of embodiments 47 to 56, wherein the Protein A affinity chromatography is selected from the group comprising MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Mab Capture A affinity chromatography, ProSep UltraPlus affinity chromatography.

58. The method according to any one of embodiments 47 to 57, wherein the content of a (specific) host cell protein is reduced.

59. The method according to any one of embodiments 47 to 58, wherein the (specific) host cell protein is a Chinese Hamster Ovary (CHO) host cell protein.

60. The method according to any one of embodiments 47 to 59, wherein the (specific) host cell protein is a phospholipase.

61. The method according to any one of embodiments 47 to 60, wherein the (specific) host cell protein is phospholipase A, phospholipase B, phospholipase C or phospholipase D.

62. The method according to any one of embodiments 47 to 61, wherein the (specific) host cell protein is phospholipase B-like 2 (PLBL2).

63. The method according to any one of embodiments 47 to 62, wherein the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or clusterin.

64. The method according to any one of embodiments 47 to 63, wherein the low conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).

65. The method according to any one of embodiments 47 to 64, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 10 mM Tris.

66. The method according to any one of embodiments 47 to 65, wherein the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.

67. The method according to any one of embodiments 47 to 66, wherein the low conductivity aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.

68. The method according to any one of embodiments 47 to 67, wherein the low conductivity aqueous solution comprises about 2 mM Tris.

69. The method according to any one of embodiments 47 to 68, wherein the low conductivity aqueous solution contains potassium phosphate.

70. The method according to any one of embodiments 47 to 69, wherein the low conductivity aqueous solution comprises about 0.2 mM to about 5 mM potassium phosphate.

71. The method according to any one of embodiments 47 to 69, wherein the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.

72. The method according to any one of embodiments 47 to 71, wherein the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.

73. The method according to any one of embodiments 47 to 72, wherein the low conductivity aqueous solution has a pH of about 7 or higher.

74. The method according to any one of embodiments 47 to 73, wherein the low conductivity aqueous solution has a pH of about 7.5 or higher.

75. The method according to any one of embodiments 47 to 73, wherein the low conductivity aqueous solution has a pH of about 7 to about 9.5.

76. The method according to any one of embodiments 47 to 74, wherein the low conductivity aqueous solution has a pH of about 7.5 to about 8.5.

77. The method according to any one of embodiments 47 to 54, wherein the low conductivity aqueous solution has a pH of about 8.

78. The method according to any one of embodiments 47 to 77, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

79. The method according to any one of embodiments 47 to 77, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

80. The method according to any one of embodiments 47 to 77, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before or after washing the affinity chromatography material with a low conductivity aqueous solution.

81. The method according to any one of embodiments 47 to 77, wherein the method further comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before washing the affinity chromatography material with a low conductivity aqueous solution.

82. The method according to any one of embodiments 78 to 81, wherein the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher.

83. The method according to any one of embodiments 78 to 82, wherein the high conductivity aqueous solution has a conductivity value of from about 20 mS/cm to about 100 mS/cm.

84. The method according to any one of embodiments 78 to 83, wherein the moderate conductivity aqueous solution has a conductivity value from greater than 0.5 mS/cm to less than 20 mS/cm.

85. The method according to any one of embodiments 78 to 84, wherein the high or medium conductivity aqueous solution comprises an amino acid.

86. The method according to any one of embodiments 78 to 85, wherein the high or medium conductivity aqueous solution comprises histidine.

87. The method according to any one of embodiments 78 to 86, wherein the high or medium conductivity aqueous solution comprises histidine and Tris.

88. The method according to any one of embodiments 47 to 87, wherein the method is devoid of a hydrophobic interaction chromatography method/step.

89. A method according to embodiments 47 to 88, wherein the bispecific antibody is a bispecific antibody comprising a) the heavy chain and light chain of a first full-length antibody that specifically binds to a first antigen; and b) a modified heavy chain and a modified light chain of a second full-length antibody that specifically binds to a second antigen, wherein the constant domains CL and CH1 are replaced with each other.

90. The method according to any one of embodiments 47 to 89, wherein the bispecific antibody is a bispecific antibody against Factor IXa and Factor X or a bispecific antibody against HER3 and EGFR or a bispecific antibody against Ang2 and VEGF.

91. The method according to embodiments 47 to 54, wherein the first antigen is human VEGF and the second antigen is human ANG-2 or the first antigen is human ANG-2 and the second antigen is human VEGF.

92. A method according to embodiments 90 to 91, wherein the first antigen binding site comprises SEQ ID NO: 1 as the heavy chain variable domain (VH) and SEQ ID NO: 2 as the light chain variable domain (VL); and the second antigen binding site comprises SEQ ID NO: 3 as the heavy chain variable domain (VH) and SEQ ID NO: 4 as the light chain variable domain (VL).

The following examples and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be understood that modifications can be made to the procedures described without departing from the spirit of the invention.

Sequence Listing Description

SEQ ID NO: 01 Variable heavy chain domain VH of <VEGF>

SEQ ID NO: 02 Variable light chain domain VL of <VEGF>

SEQ ID NO: 03 Variable heavy chain domain VH of <ANG-2>

SEQ ID NO: 04 Variable light chain domain VL of <ANG-2>

Example 1

Materials and Methods

Antibody

The present invention is exemplified by bispecific antibodies against Factor IXa and Factor X (anti-FIXa/X antibodies; IgG4 isotype) as described in WO2012/067176, bispecific antibodies against Ang2 and VEGF-A (anti-Ang2/VEGF-A antibodies; vanucizumab; IgG1 isotype) as described in WO2011/117329 or SEQ ID NOs: 01 to 04, or bispecific antibodies against Her3 and EGFR (anti-Her3/EGFR antibodies; IgG1 isotype).

Detection methods for total host cell protein (HCP), phospholipase B-like protein 2 (PLBL2), and clusterin

a) CHO HCP assay

Methods The residual CHO HCP content in the samples was determined by electrochemiluminescence immunoassay (ECLIA) on a cobas e 411 immunoassay analyzer (Roche Diagnostics).

The assay is based on the sandwich principle using polyclonal anti-CHO HCP antibodies from sheep.

First incubation: Chinese hamster ovary host cell protein (CHO HCP) from 15 μL sample (neat and/or diluted) and biotin-conjugated polyclonal CHO HCP-specific antibody formed a sandwich complex, which became bound to streptavidin-coated microparticles through the interaction of biotin with streptavidin.

Second incubation: After adding a polyclonal CHOHCP-specific antibody labeled with a ruthenium complex (tris(2,2'-bipyridyl)ruthenium(II) complex), a ternary sandwich complex is formed on the microparticles.

The reaction mixture is drawn into a measurement chamber, where the microparticles are magnetically captured on an electrode surface. Unbound material is then removed in a wash step. A voltage is then applied to the electrodes, inducing chemiluminescent emission, which is measured by a photomultiplier tube.

Finally, the concentration of CHO HCP in the test sample was calculated from the standard curve of CHO HCP with known concentrations.

b) CHO PLBL2 assay

Methods The residual Chinese hamster ovary (CHO) phospholipase B-like 2 protein (PLBL2) content in samples was determined by electrochemiluminescence immunoassay (ECLIA) on a cobas e411 immunoassay analyzer (Roche Diagnostics).

The assay is based on the sandwich principle and uses a monoclonal anti-CHO PLBL2 antibody derived from mouse.

In the first incubation step, CHO PLBL2 from 30 μL of sample (pure and/or diluted), biotin-labeled monoclonal CHO PLBL2-specific antibody and ruthenium complex (tris(2,2'-bipyridyl)ruthenium(II)-complex)-labeled monoclonal CHO PLBL2-specific antibody formed a sandwich complex.

In a second step following the addition of streptavidin-coated microparticles, the ternary complex is bound to the solid phase via the interaction of biotin and streptavidin.

The reaction mixture is drawn into a measurement chamber, where the microparticles are magnetically captured on an electrode surface. Unbound material is then removed in a wash step. Applying a voltage to the electrodes induces chemiluminescence, which is measured by a photomultiplier tube.

Finally, the concentration of CHO PLBL2 in the test samples was calculated from the standard curve of CHO PLBL2 with known concentrations.

c) Clusterin assay

Methods The residual clusterin content in the samples was determined by a commercial assay from Merck Millipore (GyroMark HT Kit GYRCLU-37K) according to the manufacturer's instructions.

Briefly, the assay is based on a sandwich ELISA, following the steps:

1) Bind the rat clusterin biotinylated capture antibody to a streptavidin-coated affinity column of Bioaffy 1000nL CD.

2) Capture rat clusterin molecules from the sample to anti-clusterin antibodies,

3) Binding of a second dye-labeled anti-clusterin detection antibody to the captured molecules,

4) Rat clusterin was quantified using Gyrolab Evaluator.

Example 2

Purification of bispecific anti-Ang2/VEGF-A antibody (IgG1 isotype) by Protein A chromatography

Antibody: Anti-Ang2/VEGF-A antibody

General chromatography conditions:

Column resin: Protein A material "Mab Select SuRe" (GE-Healthcare) Height: 20.1 cm, CV: 15.79 ml

Equipment: Avant 150

Flow rate: 300 cm/h in all steps

After equilibration of the column (step 1), the solution containing the anti-Ang2/VEGF-A antibody was applied to the Protein A affinity column. The initial amount of PLBL2 loaded in the solution containing the anti-Ang2/VEGF-A antibody was 919.7 ng PLBL2/mg antibody. The initial amount of CHOP loaded in the solution containing the anti-Ang2/VEGF-A antibody was 682,304 ng CHOP/mg antibody.

The chromatography steps were performed according to the following general scheme:

Step 1: Balance:

Step 2: Loading the solution containing the antibody

Step 3: Washing I

Step 4: Washing II

Step 5: Washing III

Step 6: Wash IV (Additional Wash)

Step 7: Elution

After elution from the protein A affinity column, the protein was determined by size exclusion chromatography (SEC) and spectrophotometric (OD) analysis.

SEC:

Resin: TSK 3000 (Tosoh)

Column: 300 x 7,8 mm

Flow rate: 0.5 ml/min

Buffer: 200 mM potassium phosphate containing 250 mM potassium chloride, adjusted to pH 7.0

Wavelength: 280nm

OD:

Ratio coefficient: 1,54

Wavelength: 280nm minus 320nm

Specific buffer conditions for Protein A chromatography

a) Control (wash with equilibration buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 5: Washing III: ---

Step 6: Wash IV: ---

Step 7: Elution: 50 mM acetic acid, pH 4,0

b) Low conductivity wash (using Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 6: Wash IV: 2 mM Tris, pH 8,0

Step 7: Elution: 50 mM acetic acid, pH 4,0

c) High conductivity wash (using Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 700 mM Tris, pH 7,2

Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 6: Wash IV: ---

Step 7: Elution: 50 mM acetic acid, pH 4,0

d) Low conductivity Tris + high conductivity histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7,0

Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4,0

Example 3

Purification of bispecific anti-FIXa/X antibody (IgG4 isotype) by Protein A chromatography

Purification of anti-FIXa/X antibodies was tested in two different chromatographic setups:

Setup 1

General conditions were according to those described in Example 2.

Antibody: Anti-FIXa/X

Initial loading amount of PLBL2 determined in a solution containing anti-FIXa/X antibodies: 557 ng PLBL2/mg antibody. Initial loading amount of CHOP determined in a solution containing anti-FIXa/X antibodies: 387377 ng CHOP/mg antibody.

Specific buffer conditions for Protein A chromatography

a) High conductivity wash (Tris buffer only)

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 700 mM Tris, pH 7,2

Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 6: Wash IV: ---

Step 7: Elution: 50 mM acetic acid, pH 4,0

b) Low conductivity Tris + high conductivity histidine (His)/Tris

Step 1: Equilibration: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 2: Sample loading

Step 3: Wash 1: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 4: Wash II: 200 mM His/1000 mM Tris, pH 7,0

Step 5: Wash III: 25 mM Tris, 25 mM NaCl, pH 7,0

Step 6: Wash IV: 2 mM Tris, pH 8.0

Step 7: Elution: 50 mM acetic acid, pH 4,0

Setup 2

General chromatography conditions

Column resin: Protein A material "Mab Select SuRe" (GE-Healthcare) Height: 20.1 cm, CV: 15.79 ml

Equipment: Avant 150

Flow rate: 300 cm/h in all steps

After equilibration of the column (step 1), a solution containing anti-FIXa/X antibodies is applied to the Protein A affinity column.

Initial loading amount of PLBL2 determined in a solution containing anti-FIXa/X antibodies: 557 ng PLBL2/mg antibody. Initial loading amount of CHOP determined in a solution containing anti-FIXa/X antibodies: 387,377 ng CHOP/mg antibody.

The chromatography steps were performed according to the following general scheme:

Step 1: Balance:

Step 2: Loading of solution containing antibody

Step 3: Washing I

Step 4: Wash II

Step 5: Wash III (Additional Wash)

Step 6: Elution

Specific buffer conditions for Protein A chromatography

a) High conductivity wash (using only NaSO4 buffer)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Washing III: ---

Step 6: Elution: 35 mM acetic acid, pH 4,0

b) Low conductivity wash (Tris 1mM) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 1 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

c) Low conductivity wash (Tris 2mM) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 35 mM acetic acid, pH 4,0

d) Low conductivity wash (Tris 4mM) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 4 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

e) Low conductivity wash (Tris 6mM) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 6 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

f) Low conductivity wash (Tris 4mM, pH 7.8) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 4 mM Tris, pH 7.8

Step 6: Elution: 50 mM acetic acid, pH 4,0

g) Low conductivity wash (Tris 4mM, pH 8.2) + high conductivity wash (with NaSO4)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 4 mM Tris, pH 8.2

Step 6: Elution: 50 mM acetic acid, pH 4,0

h) Low conductivity wash (Tris 2mM) + high conductivity wash (with histidine (His)/Tris 1M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash 1: 200 mM His/1000 mM Tris, pH 7,0

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 35 mM acetic acid, pH 4,0

i) Low conductivity wash (Tris 2mM) + high conductivity wash (histidine (His)/Tris 0.85M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash 1: 200 mM His/850 mM Tris, pH 7,0

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

j) Low conductivity wash (Tris 2 mM) + high conductivity wash (histidine (His)/Tris 0.7 M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash 1: 200 mM His/700 mM Tris, pH 7,0

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

k) Low conductivity wash (Tris 2mM) + high conductivity wash (histidine (His)/Tris 0.55M)

Step 1: Equilibration: 20 mM NaPO4, pH 7.5

Step 2: Sample loading

Step 3: Wash 1: 200 mM His/550 mM Tris, pH 7,0

Step 4: Wash II: 20 mM NaPO4, pH 7.5

Step 5: Wash III: 2 mM Tris, pH 8.0

Step 6: Elution: 50 mM acetic acid, pH 4,0

Example 4

General procedures/conditions:

Simulated cell culture medium

Blank harvested cell culture fluid was generated using untransfected CHO-DP12 cells cultured in serum-free medium. Fermentation was performed at a 2 L scale using a representative cell culture method. At the end of 14 days of fermentation, the cell culture fluid was harvested by centrifugation and sterile filtration. The harvested cell culture fluid (HCCF) was then stored at -70°C until the experiment.

Purified PLBL2

Recombinant CHO PLBL2 with a C-terminal hexahistidine tag was expressed in a 35 L scale transient transfection and purified from harvested cell culture fluid as previously described (Vanderlaan et al., 2015). Purified PLBL2 was then formulated in PBS solution and stored at -70°C until experimentation.

Purified antibodies

Recombinant humanized antibodies (bispecific antibodies against Her3 and EGFR (anti-Her3/EGFR antibody; IgG1 isotype)) were expressed in CHO cells and purified using column chromatography to ensure that the PLBL2 concentration was below 20 ng/mg. Before starting each study, each antibody buffer was exchanged into PBS using a PD-10 desalting column (GE Healthcare).

Preparation of sample material for Protein A chromatography

For packed bed column chromatography, to normalize the population and abundance of host cell proteins in the Protein A load between antibodies, purified antibodies were diluted to the same concentration using PBS and spiked into HCCF from a non-producing cell line to yield a final antibody titer of 5 g/L. A control was also prepared in which PBS was added instead of purified antibody to assess nonspecific host cell protein binding to the Protein A resin in the absence of antibody.

For each high-throughput Protein A batch purification experiment, loading was prepared in 96-well plates. In each well, purified antibody was mixed with different ratios of PLBL2 and PBS to achieve target relative PLBL2 concentrations of 0-20,000 ng/mg and an antibody titer of 5 g/L.

Packed bed column chromatography

All packed bed column chromatography experiments were performed using a 0.66 cm inner diameter × 20 cm bed height MabSelect SuRe (GE Healthcare) Protein A resin column. For each purification, the column was first equilibrated with 25 mM tris, 25 mM NaCl, pH 7.7 (equilibration buffer) for 3 column volumes (CV). Protein A loading was then applied to a target loading density of 30 μg antibody/resin, followed by washing the column with equilibration buffer for 3 CVs, washing with different types of wash buffer for 3 CVs, and then washing with equilibration buffer for another 3 CVs. Subsequently, the antibody was eluted at low pH with 0.1 M acetic acid (pH 2.8), and the eluate pool was collected starting at 0.5 OD from the beginning of the elution peak; the pooling was terminated after 2.8 CVs. To control the run with empty HCCF doped with PBS, a 2.8 CV simulated elution pool was generated starting from 1 CV to 3.8 CV after the start of the elution phase. At the end of each run, each Protein A eluate was titrated to pH 5.0 using 1.5 M tris base. The column was then cleaned with 0.1 M sodium hydroxide solution. The volumetric flow rate for all stages was 20 CV/h, except for the loading, first equilibration wash, and elution stages (flow rate was 15 CV/h).

A) Purification of anti-Her3/EGFR antibody (IgG1 isotype) by Protein A chromatography

Specific wash buffer conditions for purification of anti-Her3/EGFR antibodies (IgG1 isotype) using the general procedure of Example 4:

a) Deionized water

Example 5

The bispecific anti-Ang2/VEGF-A antibody was purified by protein A chromatography and washed with high-purity deionized water.

The cell culture fluid (HCCF) from the harvest of CHO expression culture was processed by MabSelect SuRe affinity chromatography in bind-elute mode. After HCCF was loaded onto the column to reach a maximum loading density of 38 g _mAb /l _resin , the column was washed with 25 mM Tris, 25 mM NaCl, pH 7.2 for 5 column volumes. Then, an additional wash of 5 column volumes was performed with 0.7 M Tris/HCl, pH 7.2. The third wash step was performed with highly purified water for five column volumes. The column-bound antibody was eluted with 50 mM acetate, pH 3.4. The elution pool was collected in a maximum of three column volumes based on an OD ₂₈₀ of 500 to 250 mAU (path length 1 cm).

The affinity elution pool was adjusted to pH 3.5 with acetic acid and held for 30 minutes. The pool was then adjusted to pH 5.0 with 1.5 M Tris base and clarified by depth filtration. The depth filtered pool was adjusted to pH 7.0 with 1.5 M Tris base. The conductivity value of the adjusted pool was determined to be less than 6 mS/cm. No dilution step with water was required before loading the material onto the next chromatography column, CaptoAdhere ImpRes.

result:

Example 6

Purification of bispecific anti-Ang2/VEGF-A antibodies in Protein A chromatography without additional washing with high-purity deionized water (Comparative Example)

Harvested cell culture fluid (HCCF) from CHO expression cultures was processed by MabSelect SuRe affinity chromatography in bind-elute mode. After HCCF was loaded onto the column to reach a maximum loading density of 38 g _mAb /l _resin , the column was washed for 5 column volumes with 25 mM Tris, 25 mM NaCl, pH 7.2. Then, an additional wash of 5 column volumes was performed with 0.7 M Tris/HCl, pH 7.2. A third wash step was performed again using 25 mM Tris, 25 mM NaCl, pH 7.2. The column-bound antibody was eluted with 50 mM acetate, pH 3.4. The eluted pool was collected over a maximum of three column volumes based on an OD ₂₈₀ of 500 to 250 mAU (path length 1 cm).

The affinity elution pool was adjusted to pH 3.5 with acetic acid and held for 30 minutes. The pool was then adjusted to pH 5.0 with 1.5 M Tris base and clarified by depth filtration. The depth filtered pool was adjusted to pH 7.0 with 1.5 M Tris base. The conductivity value of the adjusted pool was determined to be 6 mS/cm. An additional dilution step with water was required before loading the material onto the next chromatography column, Capto Adhere Imp Res.

result:

Claims (13)

1. A method for producing a bispecific antibody, said bispecific antibody comprising a first antigen-binding site that specifically binds to a first antigen and a second antigen-binding site that specifically binds to a second antigen, said method comprising the following steps: a) Culture cells containing nucleic acids encoding the bispecific antibodies. b) Recover the bispecific antibody from the cells or culture medium. c) Contact the bispecific antibody with the affinity chromatography material. d) Washing the affinity chromatography material with a low-conductivity aqueous solution having a conductivity value from 0.03 μS/cm to 0.5 mS/cm, and additionally, before or after washing the affinity chromatography material with the low-conductivity aqueous solution, washing the affinity chromatography material with a medium-conductivity aqueous solution having a conductivity value from greater than 0.5 mS/cm to less than 20 mS/cm. e) Recover bispecific antibodies from affinity chromatography materials. f) Perform additional chromatography steps. This generates the bispecific antibody. 2. A method for purifying a bispecific antibody from a sample, said bispecific antibody comprising a first antigen-binding site that specifically binds to a first antigen and a second antigen-binding site that specifically binds to a second antigen, said method comprising the following steps: a) Provide a sample containing the bispecific antibody, b) Apply the sample containing the bispecific antibody to the affinity chromatography material. c) Washing the affinity chromatography material with a low-conductivity aqueous solution having a conductivity value from 0.03 μS/cm to 0.5 mS/cm, and additionally, before or after washing the affinity chromatography material with the low-conductivity aqueous solution, washing the affinity chromatography material with a medium-conductivity aqueous solution having a conductivity value greater than 0.5 mS/cm to less than 20 mS/cm. d) Recover the bispecific antibody from the affinity chromatography material. e) Perform additional chromatography steps. This allows for the purification of the bispecific antibody. 3. The method according to claim 1 or 2, wherein the additional chromatography step following the affinity chromatography step is an ion exchange chromatography step or a multi-component ion exchange chromatography step. 4. The method according to claim 1 or 2, wherein the affinity chromatography is protein A affinity chromatography, protein G affinity chromatography, or single-chain Fv ligand affinity chromatography. 5. The method according to claim 1 or 2, wherein the affinity chromatography is protein A affinity chromatography. 6. The method of claim 1 or 2, wherein the low conductivity aqueous solution contains 0.1 mM to 8 mM Tris. 7. The method of claim 1 or 2, wherein the low conductivity aqueous solution comprises 0.05 mM to 2 mM potassium phosphate. 8. The method of claim 1 or 2, wherein the low conductivity aqueous solution has a pH of 7 or higher. 9. The method of claim 1 or 2, wherein the method further comprises washing the affinity chromatography material with a high-conductivity aqueous solution and a medium-conductivity aqueous solution before or after washing with a low-conductivity aqueous solution, wherein the high-conductivity aqueous solution has a conductivity value of 20 mS/cm or higher, and wherein the medium-conductivity aqueous solution has a conductivity value of greater than 0.5 mS/cm and less than 20 mS/cm. 10. The method of claim 9, wherein the aqueous solution with high or medium conductivity comprises histidine. 11. The method of claim 1 or 2, wherein the bispecific antibody is a bispecific antibody comprising a) a heavy chain and a light chain of a first full-length antibody that specifically binds to a first antigen; and b) a heavy chain and a light chain of a second full-length antibody that specifically binds to a second antigen, wherein the constant structural domains CL and CH1 are replaced by each other. 12. The method of claim 1 or 2, wherein the first antigen is human VEGF and the second antigen is human ANG-2 or the first antigen is human ANG-2 and the second antigen is human VEGF. 13. The method according to claim 1 or 2, wherein the first antigen binding site comprises SEQ ID NO: 1 as a heavy chain variable domain (VH) and SEQ ID NO: 2 as a light chain variable domain (VL); and the second antigen binding site comprises SEQ ID NO: 3 as a heavy chain variable domain (VH) and SEQ ID NO: 4 as a light chain variable domain (VL).