Antibody separation with a VH3 binding separation matrix

Abstract

The present invention relates to processes of isolation of VH3 comprising antibodies or antibody fragments using a Protein A affinity separation matrix capable of binding VH3, wherein the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less. It further relates to a process for separation of bispecific antibodies or antibody fragments comprising one VH3 chain from antibodies or antibody fragments comprising two or more VH3 chain fragments.

Description

[0001] ANTIBODY SEPARATION WITH A VH3 BINDING SEPARATION MATRIX

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of separation and isolation of antibodies and antibody fragments. More specifically it relates to improving elution conditions in methods using a VH3- bindning separation matrix.

[0004] BACKGROUND

[0005] Immunoglobulins and immunoglobulin fragments represent the most prevalent biopharmaceutical products in either manufacture or development worldwide. The high commercial demand for, and hence value of, this therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective manufacturing processes whilst controlling the associated costs.

[0006] Affinity chromatography is used in most cases, as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal or polyclonal antibodies, or fragments thereof. A particularly interesting class of affinity reagents is proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent of the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples, such as but not limited to serum or plasma preparations, or cell culture derived feed stocks. An example of such a protein is staphylococcal protein A (SpA), containing domains capable of binding to the Fc and Fab portions of IgG immunoglobulins from different species. These domains are commonly denoted as the E-, D-, A-, B- and C-domains.

[0007] SpA-based proteins have, due to their high affinity and selectivity, found a widespread use in the field of biotechnology, e.g. as ligands in affinity chromatography for capture and purification of antibodies as well as for detection or quantification. At present, SpA-based affinity medium is probably the most widely used affinity medium for isolation of monoclonal antibodies and their fragments from different samples including industrial cell culture supernatants. Accordingly, various matrices comprising protein A or protein A-derived ligands are commercially available, for example, in the form of MabSelect™ SuRe, MabSelect™ SuRe LX and MabSelect PrismA™ from Cytiva™, Uppsala, Sweden. Certain Protein A and Protein A-derived ligands have binding affinity for both the Fc part of an antibody and for certain VH domains of antibodies, in particular VH3. As a result, copurification of product-related impurities such as half-antibodies and truncated variants may occur and require elution schemes which are complex and / or not sufficiently mild. Additionally, the purification of bispecific antibodies, such as emicizumab, or fragments thereof requires complex elution schemes to ensure that only correctly paired bispecific antibodies or fragments thereof are obtained instead of a mixture of correctly and incorrectly paired antibodies and fragments. Thus, there is an unmet need in the field for simplified and reliable isolation methods for antibodies and fragments thereof.

[0008] More recently, ligands and separation matrices which selectively bind to a VH3 chain of an antibody or antibody fragment have been published and launched as commercial products. Publications towards such ligands and separation matrices include for instance W02023174900A1. An example of a commercial product is for instance MabSelect™ VH3 resin, launched by Cytiva™ in November 2023.

[0009] In most commercially available processes, the target will bind to the ligand of the affinity matrix such that normally a pH of 2.5-3.5 is necessary in order for the target to dissociate from the affinity ligand and be eluted with the elution buffer. Such low pH risks compromising the target protein, such as causing aggregation of the target protein, denaturation of the target protein, etc. These issues will lead to a lower productivity and yield of correct and uncompromised target protein. Thus, regardless of the target to be purified or separated by a chromatographic purification or separation process, it is of high interest to be able to elute the target at a pH closer to neutral pH than previously, thereby minimizing the risks discussed above. US 10,844,112 B2 discusses the use of a specific buffer for elution. Thus, there is still of great interest to find alternative ways of eluting target molecules at higher pH than previously.

[0010] SUMMARY OF THE INVENTION

[0011] It has been an objective for the present inventors to find a process wherein the elution conditions allow for a milder pH, i.e. higher than normally used, at the elution step for a target molecule from an affinity matrix. More specifically it has been an objective to achieve mild elution conditions for a VH3 binding separation matrix, such as the MabSelect™ VH3 resin.

[0012] Additionally it has been an objective to design a process whereby the target compound is well separated from other molecules in a feed. Such a separation is particularly interesting for separating multispecific heteromeric antibodies, such as bi-specific heterodimeric antibodies, or trispecific heterotrimeric antibodies of interest from other antibodies that do not have a desired configuration of heavy chains and light chains, e.g. homodimers.

[0013] The objectives above have in a first aspect been attained through a process for isolation of an antibody or an antibody fragment comprising at least one VH3 chain, comprising the steps of: I. adsorbing at least one of the antibody or the antibody fragment onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;

[0014] II. washing the affinity separation matrix to remove impurities;

[0015] III. optionally equilibrating the affinity separation matrix with an equilibration buffer;

[0016] IV. separating the at least one antibody or antibody fragment from the affinity separation matrix by elution using an elution buffer; wherein the Protein A affinity separation matrix is a VH3 binding separation matrix, and wherein the equilibration buffer and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

[0017] According to a second aspect, the objectives above have been attained through a process for separation of bispecific antibodies or antibody fragments comprising one VH3 chain from variants of the antibody or antibody fragment comprising two or more VH3 chains or no VH3 chain comprising the steps of:

[0018] I. adsorbing a feed comprising at least the bispecific antibody onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;

[0019] II. washing the affinity separation matrix to remove impurities;

[0020] III. optionally equilibrating the affinity separation matrix with an equilibration buffer;

[0021] IV elution of the bispecific antibody from the affinity separation matrix using an elution buffer; wherein the Protein A affinity separation matrix is a VH3 binding separation matrix, and wherein the equilibration and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

[0022] The Protein A affinity separation matrix may be a VH3 binding separation matrix that does not bind to a Fc region of an antibody or antibody fragment.

[0023] The elution buffer may be chosen from the group consisting of acetate buffer, succinate buffer, propionate buffer, citrate buffer. Preferably the elution buffer is an acetate buffer, a propionate buffer or a succinate buffer. In one embodiment, the elution buffer is an acetate buffer. In one embodiment, the elution buffer is a propionate buffer. In one embodiment, the elution buffer is a succinate buffer.

[0024] The elution buffer may have a concentration of 20 mM or less, or 15 mM or less. The elution buffer may be substantially free of additional salts. Alternatively, the elution buffer may comprise an additional salt, whereby elution is achieved by lowering the salt content in the elution buffer in relation to previous buffers used in the process.

[0025] The elution is preferably performed with a decreasing pH gradient, optionally a step-wise elution, from pH 5-8 to about pH 3-4.

[0026] FIGURES

[0027] Figure 1: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3.4 using 50 mM Acetate.

[0028] Figure 2: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3.4 using 50 mM Acetate. Overlay of 50 mM Acetate with 0 mM NaCI, 25 mM NaCI, and 50 mM NaCI, respectively.

[0029] Figure 3: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3.2 using 50 mM Propionate.

[0030] Figure 4: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3.4 using 15 mM Succinate.

[0031] Figure 5: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3 using 20 mM Citrate.

[0032] Figure 6: Separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3 using 50 mM Citrate.

[0033] Figure 7: Comparison by overlay of chromatograms for separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3, between 50 mM Acetate, 50 mM Propionate, and 15 mM Succinate.

[0034] Figure 8: Comparison by overlay of chromatograms for separation of bispecific emicizumab (peak 1) and mispaired homodimer (peak 2) in a pH gradient from 5 to 3, between 50 mM Acetate, 20 mM Citrate, and 50 mM Citrate. DEFINITIONS

[0035] The terms "antibody" and "immunoglobulin" (abbreviated Ig) may be used interchangeably herein and refers to an antigen-binding protein having a basic four-polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains, said chains being stabilized by interchain or intrachain disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH). The CH region may comprise three different domains, CHI, CH2 and CH3. The VH region may comprise three different domains, VH1, VH2 and VH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. There are two types of light chain in humans, kappa chain and lambda chain. The term is to be understood to include any antibody, including but not limited to monoclonal antibodies, bi-specific antibodies, and multi-specific antibodies, as well as, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments, such as Antibody-Drug Conjugates (ADC).

[0036] The term "mAb" stands for monoclonal antibody.

[0037] The term "antibody fragment" is broadly used to refer to any fragments of antibodies with at least one retained binding to a target or an antigen. A non-exhaustive list of examples of antibody fragments are for instance Fab, Fab', Fab'-SH, F(ab')2, Fv, single-domain antibody (sdAb), single-chain Fv (scFv), diabodies, scFv dimers, tandem scFv (taFv), (scFv)2, single-chain diabodies (scDb), singlechain Fab (scFab), tandem scDb (TandAb), triabodies, tetrabodies, hexabodies, one-armed antibodies, and multispecific antibodies formed from antibody fragments such as Fab-scFv, scFv-Fc, Fab-scFv-Fc, scDb-Fc, and taFv-Fc, VHH fragments, etc.

[0038] The term "Fc region" refers to a Fragment crystallizable C-terminal region of an IgG antibody, in particular, the C-terminal region of the heavy chain(s) of said IgG antibody. The term "Fc binding" refers to the capability to bind to said region.

[0039] The term "Fab" or "Fab fragment" refers to the fragment antigen-binding region and includes both a constant domain and the variable domains of both the heavy and light chains. Fab are often monovalent having one antigen-binding site.

[0040] The term "bispecific antibody" stands for an antibody that can bind to two different types of antigen or two different epitopes on the same antigen. Likewise, a tri-specific antibody stands for an antibody that can bind to three different types of antigen or three different epitopes on the same antigen. The term "multi-specific antibody" stands for an antibody that can bind more than two different types of antigen or more than two different epitopes on the same antigen. A bi-specific or multi-specific antibody is heteromeric, with differing variable regions accounting for the bi- or multispecificity, as opposed to a mAb which is a homodimer. The bi-specificity or multi-specificity may be due to the variable light chains or the variable heavy chains.

[0041] The terms "VH-binding polypeptide", "VH-binding agent" and "VH-binding protein" mean a polypeptide, molecule, or protein, respectively, capable of binding to the variable heavy chain (VH) of an antibody, such as the Fab Portion. Such a polypeptide or protein includes, but is not limited to, e.g. Protein A and Protein G, or any fragment or fusion protein thereof that has maintained said binding property. The combination of the three types of VH chain disclosed above, is disclosed as i.e. VH3:VH3 for a homodimer of VH3, and i.e. VH1:VH3 or VH2:VH3 for heterodimer with one VH3 chain and one VH1 chain or one VH2 chain, respectively.

[0042] The term "liquid sample" or "sample" as used herein, refers to a liquid containing at least one target substance which is sought to be purified from other substances also present. Liquid samples can, for example, be aqueous solutions, organic solvent systems, or aqueous / organic solvent mixtures or solutions. The source liquids are often complex mixtures or solutions containing many biological molecules (such as proteins, antibodies, hormones, and viruses), small molecules (such as salts, sugars, lipids, etc.) and even particulate matter. While a typical source liquid of biological origin may begin as an aqueous solution or suspension, it may also contain organic solvents used in earlier separation steps such as solvent precipitations, extractions, and the like. Examples of liquid samples that may contain valuable biological substances amenable to the purification by various embodiments of the present invention include, but are not limited to, a culture supernatant from a bioreactor, a homogenized cell suspension, plasma, plasma fractions, and milk. The liquid sample or sample is often clarified before application to a chromatography resin.

[0043] A "buffer" is a substance which, by its presence in solution, increases the amount of acid or alkali that must be added to cause unit change in pH. A buffered solution resists changes in pH by the action of its acid-base conjugate components. Buffered solutions for use with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions such that the pH of the solution is within a physiological range. The term "physiological pH" refers to the pH of mammalian blood (i.e., 7.38 or about 7.4). Thus, a physiologic pH range is from about 7.2 to 7.6. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases. Exemplary buffers for use in purification of biological molecules (e.g., protein molecules) include the zwitterionic or "Good" Buffers, see e.g., Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62. "Washing liquid", "wash buffer" or "column wash" as used herein all refer to the liquid used to carry away impurities from the chromatography resin to which is bound the target substance. More than one wash liquid can be employed sequentially, e.g., with the successive wash liquids having varying properties such as pH, conductivity, solvent concentration, etc., designed to dissociate and remove varying types of impurities that are non-specifically associated with the chromatography resin.

[0044] The term "equilibration buffer" refers in the present disclosure to a buffer used to prepare the affinity matrix, with bound target protein, for the elution, or for loading of the target molecule. Equilibration buffer may also be used for wash of the affinity matrix with bound target protein. The equilibration buffer is preferably the same kind of buffer as the elution buffer, differing only in pH.

[0045] "Elution liquid" or "elution buffer", which are used interchangeably herein, refers herein to the liquid that is used to dissociate the target substance from the chromatography resin, thereby eluting the binding region-containing protein from the immobilized binding agent, after it has been washed with one or more wash liquids. The elution liquid acts to dissociate the target substance without denaturing it irreversibly. Typical elution liquids are well known in the chromatography art and may have a different pH (typically lower pH), higher concentrations of salts, free affinity ligands or analogs, or other substances that promote dissociation of the target substance from the chromatography resin. "Elution conditions" refers to process conditions imposed on the target substance-bound chromatography resin that dissociate the target substance from the chromatography resin, such as the contacting of the target substance-bound chromatography resin with an elution liquid or elution buffer to produce such dissociation.

[0046] All buffers used herein are prepared by adding an acid at a preferred concentration and titrating with NaOH, KOH or another suitable base to achieve the desired pH.

[0047] Preferably the elution buffer has a low pH and thereby disrupts interactions between separation matrix and the protein of interest. Typically, the low pH elution buffer has a pH in the range from about 2 to about 5, such as in the range from about 3 to about 4. Examples of buffers that will control the pH within this range include glycine, phosphate, acetate, and citrate buffers, as well as combinations of these. Commonly used buffers are citrate and acetate buffers, most preferably sodium citrate or sodium acetate buffers.

[0048] DETAILED DESCRIPTION

[0049] As most targets, such as immunoglobulins or antibodies, and antibody fragments, will bind to the ligand of an affinity matrix such that normally a pH of 2.5-3.5 is necessary in order for the target to dissociate from the affinity ligand and be eluted with the elution buffer, it would be very advantageous to achieve a process which will allow for elution of the target at a milder, that is higher, pH. In particular, this is interesting for affinity matrices comprising ligands that are not specifically developed to provide a milder elution. Thus, the inventors wanted to provide a process that will lead to a milder elution even if the ligand is not specifically designed for a mild elution. The inventors decided to look at different types of elution buffers, and how that may impact the elution pH for a target.

[0050] In a first aspect of the present invention, a process for isolation of an antibody or an antibody fragment comprising at least one VH3 chain is provided, which allows for elution at a milder, or higher, pH than is generally obtained. The process comprises the steps of:

[0051] I. adsorbing at least one of the antibody or the antibody fragment onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;

[0052] II. washing the affinity separation matrix to remove impurities;

[0053] III. optionally equilibrating the affinity separation matrix with an equilibration buffer;

[0054] IV. separating the at least one antibody or antibody fragment from the affinity separation matrix by elution using an elution buffer.

[0055] The equilibration buffer and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

[0056] The affinity separation matrix is a VH3 binding separation matrix. An example of such a separation matrix is MabSelect™ VH3 from Cytiva™. However, any Protein A affinity separation matrix that binds to the VH3 chain of an antibody or an antibody fragment could be used in the process disclosed.

[0057] In a second aspect of the present invention, a process for separation of multispecific antibodies or antibody fragments comprising one VH3 chain from variants of the antibody or antibody fragment comprising two or more VH3 chains or no VH3 chain is provided. The process comprises the steps of:

[0058] I. adsorbing a feed comprising at least the bispecific antibody onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;

[0059] II. washing the affinity separation matrix to remove impurities;

[0060] III. optionally equilibrating the affinity separation matrix with an equilibration buffer;

[0061] IV elution of the bispecific antibody from the affinity separation matrix using an elution buffer. The equilibration buffer and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

[0062] The affinity separation matrix is a VH3 binding separation matrix. An example of such a separation matrix is MabSelect™ VH3 from Cytiva™. However, any Protein A affinity separation matrix that binds to the VH3 chain of an antibody or an antibody fragment could be used in the process disclosed.

[0063] The MabSelect™ VH3 separation matrix is devoid of an Fc interaction. Hence, a homodimer consisting of either VH1:VH1 or VH2:VH2 should not bind and therefore pass through the resin without binding to the same, while the other homodimer VH3:VH3 binds with two interactions, thus creating avidity effect and therefore binding more stringently compared to the correctly paired heterodimer VH1:VH3 or VH2:VH3. Consequently, the heterodimer VH1 / VH2:VH3 will elute at a milder pH than the VH3:VH3 homodimer. By eluting in a gradient or two step elution with different pH's, a separation between VH1 / VH2:VH3 and VH3:VH3 is feasible.

[0064] The monocarboxylic acid for the equilibration buffer and the elution buffer is preferably chosen from acetic acid or propionic acid. The dicarboxylic acid for equilibration buffer and the elution buffer is preferably succinic acid. The tricarboxylic acid is typically citric acid. Preferably equilibration buffer and the elution buffer is an acetate buffer, a propionate buffer or a succinate buffer.

[0065] The buffer solutions may comprise counter-ions, depending on the composition of the buffer solution. Alternative counter-ions for the buffer solutions that may be used are sodium, potassium, ammonium, Tris or other bases. As an example, a succinate buffer might be prepared by titrating the acid with NaOH (rendering a sodium succinate buffer), KOH (rendering a potassium succinate buffer) etc. The skilled person is well aware of suitable counter-ions that may be used for the buffer solutions within the technical field.

[0066] The elution buffer may have a concentration of 20 mM or less, or 15 mM or less. In particular for succinate buffer and citrate buffer, it is preferable that the concentration of the elution buffer is 20 mM or 15 mM.

[0067] The present inventors have unexpectedly discovered that use of the herein mentioned buffers at the herein mentioned concentrations will break any VH3 interaction between a target molecule and the Protein A affinity separation matrix used. In addition, the breaking of the VH3 interaction will occur at a higher, and consequently a milder, pH compared to the conventional processes used within the technical field. In one embodiment, the elution buffer is substantially free of additional salts. That is to say that apart from any salt comprising a counter-ion being used for producing an elution buffer, such as for instance Na-citrate to obtain a citrate buffer, there is no additional salt, such as additional NaCI, added to the elution buffer. In another embodiment, the elution buffer comprises additional salt.

[0068] The presence of additional salt may improve the resolution between two peaks when separating bispecific heterodimeric antibodies from homodimeric antibodies. However, this may also lead to the elution pH being somewhat decreased, as shown below in the Examples. Thus, the skilled person needs to determine, with regards to the specific antibody or antibody fragment being separated and possible elution pH, whether to include an additional salt in the elution buffer to improve resolution, or whether to exclude an additional salt so as to increase the elution pH.

[0069] By lowering the concentration of the buffer solution used for elution, the conductivity is generally decreased. Lowering of conductivity has previously been shown to cause elution. However, the lower conductivity for elution has not previously been tied to elution at a higher pH, in particular for a Protein A affinity separation matrix, and even more particularly for a Protein A affinity separation matrix capable of binding to the VH3 chain, where the VH3 interaction between the target molecule and the Protein A affinity separation matrix is dissociated at the herein disclosed elution pH ranges. The elution pH as shown in the Examples below, at which pH the above-mentioned dissociation occurs, is at least 4.0.

[0070] The elution is preferably performed with the use of a gradient with decreasing pH. Normally two solutions of the elution buffer are used with different pH. The first elution buffer solution will have a pH of about 5-8, and the second elution buffer solution a pH of about 3-4. The change during elution from the first elution buffer to the second elution buffer may be gradual or stepwise.

[0071] In one embodiment, the equilibration buffer and / or any washing buffers used before the equilibration and elution comprises salt, and the target is eluted using a decreasing salt gradient. Thus, elution is achieved by lowering the salt content in the elution buffer. The salt may for instance be, but is not limited to, NaCI, KCI, Na2SO4, NH4CI and (NH4)2SO4. By decreasing the salt concentration during the elution step, the VH3 interaction between the target molecule and the Protein A affinity separation matrix is dissociated. In this embodiment, the elution buffer may comprise salt, as long as the salt concentration in the elution buffer is decreased in relation to the washing buffers and / or equilibration buffer.

[0072] As mentioned above, by using the herein mentioned elution buffers, at the herein mentioned concentrations, the present inventors have shown that the pH at which the target elutes in the processes disclosed above, will be higher, as compared to the conventional processes known within the technical field. The inventors have also shown that upon separation between a correctly paired bi-specific antibody, and a mis-paired homodimeric antibody, the elution buffers used above will lead to a milder elution pH for both peaks. However, the elution pH for the first elution peak, comprising the correctly paired bi-specific antibody, is the most important elution pH. For all tested elution buffers, with or without additional salt, the elution peak of the correctly paired bi-specific antibody is at 4.0 or higher. This is important in that the integrity of the target is more likely maintained with the processes as disclosed herein.

[0073] Thus, by using low concentrations of the specified elution buffers, with or without any additional salt added, the inventors have shown that the elution pH may be increased.

[0074] The washing of the affinity separation matrix to remove impurities is normally conducted with at least one washing step, more commonly at least two or more washing steps, in order to remove different kinds of impurities in the liquid sample from the separation matrix. The skilled person is familiar with how to wash a separation matrix efficiently and properly before elution of the target.

[0075] EXAMPLES

[0076] In the examples below, the antibody Emicizumab (Hemlibra™) was used. An Emicizumab comprising feed from GeneArt™ (Thermo Fisher Scientific Inc.) expressed in HEK293 cells was used. The feed was first purified on a MabSelect™ VL resin from Cytiva™.

[0077] Emicizumab is an asymmetric bispecific antibody with two different heavy chains, of which one with a variable region of class VH1 and the other one with a variable region of class VH3 (VH1:VH3). The light chain is the same on both arms of the antibody. Mutations in heavy chains (CH3) facilitate heavy chains hetero-dimerization through electric repulsion and attractions. Two possible mispairings of the antibody includes homodimers of VH1:VH1 and VH3:VH3.

[0078] The MabSelect™ VH3 separation matrix was packed in Tricorn™ 5 / 50 (1 ml CV) for the experiments below.

[0079] The experiments were performed on an AKTA™ Pure from Cytiva™. The method used on the AKTA™ is shown in Table 1. TABLE 1. MabSelect™ VH3 Purification method

[0080] 4 min RT = 0,25ml / min; 5 min RT = 0,2ml / min

[0081] For the pH 5 elution buffer A, and the pH 3 elution buffer B used in the experiments, as well as the pH gradient used for each buffer, see Table 2 below. TABLE 2. Elution buffers

[0082] The resulting chromatograms are shown in Figures 1-6. The resulting pH for the elution peaks are summarized in Table 3.

[0083] TABLE 3. pH of elution peaks

[0084] * Resolution measured in elution volume in ml between the highest points of the two elution peaks.

[0085] The results above clearly show that all of the tested buffers lead to a higher elution pH as compared to the reference of 50 mM Citrate. Even the citrate buffer with a lower concentration of 20 mM leads to a higher elution pH, as compared to the reference citrate buffer with a higher concentration of 50 mM. See Fig. 8 for a comparative overlay of 50 mM Acetate, 20 mM Citrate and 50 mM Citrate. The lower concentration of citrate, as well as acetate clearly leads to a higher elution pH.

[0086] In particular, acetate without additional salt, as well as the propionate and succinate elution buffers lead to an elution pH for both peaks above 4.0. Additionally, the resolution between the two peaks is clear. See Fig. 7 for a comparison between these buffers, clearly showing how similarly the bispecific antibody elutes, and at about the same pH.

[0087] While the acetate buffer with additional salt leads to a higher resolution, the elution pH is decreased compared to acetate buffer without additional salt, in particular for peak 2. This is clear from the comparative overlay of Fig. 2.

Claims

CLAIMS1. A process for isolation of an antibody or an antibody fragment comprising at least one VH3 chain, comprising the steps of:I. adsorbing at least one of the antibody or the antibody fragment onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;II. washing the affinity separation matrix to remove impurities;III. optionally equilibrating the affinity separation matrix with an equilibration buffer;IV. separating the at least one antibody or antibody fragment from the affinity separation matrix by elution using an elution buffer; wherein the Protein A affinity separation matrix is a VH3 binding separation matrix, and wherein the equilibration buffer and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

2. A process for separation of bispecific antibodies or antibody fragments comprising one VH3 chain from variants of the antibody or antibody fragment comprising two or more VH3 chains or no VH3 chain comprising the steps of:I. adsorbing a feed comprising at least the bispecific antibody onto a Protein A affinity separation matrix by contacting a liquid sample with the affinity separation matrix;II. washing the affinity separation matrix to remove impurities;III. optionally equilibrating the affinity separation matrix with an equilibration buffer;IV elution of the bispecific antibody from the affinity separation matrix using an elution buffer; wherein the Protein A affinity separation matrix is a VH3 binding separation matrix, and wherein the equilibration and the elution buffer is a mono-, di- or tricarboxylic acid at a concentration of 50 mM or less.

3. The process according to any one of claim 1 or claim 2, wherein the Protein A affinity separation matrix is a VH3 binding separation matrix that does not bind to a Fc region of an antibody or antibody fragment.

4. The process of any of the preceding claims, wherein the elution buffer is chosen from the group consisting of acetate buffer, succinate buffer, propionate buffer, citrate buffer.

5. The process of claim 4, wherein the elution buffer is an acetate buffer, a propionate buffer or a succinate buffer.

6. The process of claim 5, wherein the buffer is an acetate buffer.

7. The process of claim 5, wherein the buffer is a propionate buffer.

8. The process of claim 5, wherein the buffer is a succinate buffer.

9. The process of any of the preceding claims, wherein the elution buffer has a concentration of 20 mM or less, or 15 mM or less.

10. The process of any of the preceding claims, wherein the elution buffer is substantially free of additional salts.

11. The process of any of the preceding claims, wherein the elution buffer comprises an additional salt, whereby elution is achieved by lowering the salt content in the elution buffer in relation to previous buffers used in the process.

12. The process of any of the preceding claims, wherein the elution is performed with a decreasing pH gradient, optionally a step-wise elution, from pH 5-8 to about pH 3-4.

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