Eluting conditions for protein l affinity chromatography

Abstract

The present invention relates to a method of purifying antibodies or antibody fragments: adsorbing at least one of said antibodies or antibody fragments onto an affinity separation matrix by contacting a liquid sample with the affinity separation matrix; wherein the affinity matrix is an affinity separation matrix comprising a ligand based on Protein L or any variant thereof binding to the kappa-light chain of the antibody or antibody fragment, and separating the at least one antibody or antibody fragment from the affinity separation matrix by using an elution buffer wherein the elution buffer has a concentration of 5-50 mM. The invention likewise relates to a method for isolating bispecific antibodies.

Description

Technical Field

[0001] The present invention relates to the field of chromatography, more particularly to affinity chromatography. More specifically, the present invention relates to elution conditions for protein L separation matrices and how to improve said conditions. Background Art

[0002] Immunoglobulins represent the world's most popular biopharmaceutical products in either manufacturing or development. The high commercial demand, and therefore the value, of this specific therapeutic market has led to a focus on pharmaceutical companies to maximize the productivity of their respective mAb manufacturing processes while controlling associated costs.

[0003] Affinity chromatography is often used as a key step in the purification of these immunoglobulin molecules, such as monoclonal or polyclonal antibodies. A particularly interesting class of affinity reagents are proteins that bind specifically to the constant portion of immunoglobulin molecules, an interaction that is independent of the antibody's antigen-binding specificity. Such reagents are widely used for affinity chromatography to recover immunoglobulins from a variety of samples, such as, but not limited to, serum or plasma preparations, or cell culture-derived materials.

[0004] For immunoglobulins, immunoglobulin fragments, antibodies or antibody fragments lacking Fc chains but having subclass 1, 3 or 4 kappa light chains, such as Fab, single-chain variable fragment (scFv), bispecific T cell engager (BiTE), domain antibodies, etc., a matrix (B) comprising protein L derived from Finegoldia magna (formerly known as Peptoscoccus Magnus) L J. Biol. Chem. 264, 19740-19746, 1989; W Kastem et al: J. Biol. Chem. 267, 12820-12825, 1992; B HK Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992 and US Pat. 6,822,075) show great promise as a purification platform providing the desired high selectivity.

[0005] Protein L matrices are commercially available, for example as ELISA from Cytiva TM Capto TM L and MabSelect TMVL and can be used to isolate proteins containing κ light chains, such as complete antibodies, Fab, scFv fragments, domain antibodies, etc. Approximately 75% of antibodies produced by healthy humans have κ light chains, and approximately 90% of therapeutic monoclonal antibodies and antibody fragments contain κ light chains (Carter, P., Lazar, G. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 17, 197–223 (2018). https: / / doi.org / 10.1038 / nrd.2017.227).

[0006] In most commercial methods, the target will be combined with the part of the affinity matrix so that the target is dissociated from the affinity ligand and eluted by elution buffer, and a pH of 2.5-3.5 is usually required. Such low pH has the risk of damaging the target protein, for example, causing target protein aggregation, target protein denaturation, etc. These problems will result in the lower productivity and output of the correct and undamaged target protein. Therefore, no matter how the target to be purified or separated by chromatography or separation method is, it is highly noteworthy that the target can be eluted at a pH closer to neutral pH than before, thereby minimizing the above-mentioned risk. US10,844,112B2 discusses the use of specific buffers for elution. In the past, attempts have been made to add salt to the elution buffer to improve the elution pH, but such methods need to remove high levels of salt from the eluate. Therefore, for finding an alternative method of eluting the target molecule at a higher pH than before, it is still very noteworthy.

[0007] definition

[0008] The terms "antibody" and "immunoglobulin" are used interchangeably herein and refer to antigen-binding proteins having a basic four-polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains, which are stabilized by interchain or intrachain disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. There are two types of light chains in humans, kappa chains and lambda chains. The term should be understood to include any antibody, including but not limited to monoclonal antibodies, bispecific antibodies, multispecific antibodies, and antibody fragments, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments, such as antibody-drug conjugates (ADCs).

[0009] The term "mAb" stands for monoclonal antibody.

[0010] The term "Fc region" refers to the C-terminal region of an IgG antibody, particularly the C-terminal region of the heavy chain of the IgG antibody. The term "Fc binding" refers to the ability to bind to the region.

[0011] The term "κ light chain-containing protein" is used as a synonym for "immunoglobulin κ light chain-containing protein" and refers herein to a protein comprising 1, 3, or 4 subclass κ light chains (also known as V κI 、V κIII and V κIV , as in BHK Nilsson et al: J. Biol. Chem. 267, 102234-2239, 1992), and includes any whole antibody, antibody fragment, fusion protein, conjugate, or recombinant protein containing 1, 3, or 4 subclass kappa light chains.

[0012] The term "Fab" or "Fab fragment" refers to the antigen binding region and includes the constant and variable domains of both the heavy and light chains. A Fab may contain a kappa light chain and / or a lambda light chain.

[0013] The term "Fv fragment" refers to a fragment variable region and contains only two variable domains, VH and VL. VH and VL are held together in the Fv fragment by non-covalent interactions.

[0014] The term "bispecific antibody" refers to an antibody that can bind to two different types of antigens or two different epitopes on the same antigen. Similarly, a trispecific antibody refers to an antibody that can bind to three different types of antigens or three different epitopes on the same antigen. The term "multispecific antibody" refers to an antibody that can bind to more than two different types of antigens or more than two different epitopes on the same antigen. Bispecific or multispecific antibodies are heterodimers with different variable regions responsible for their bispecificity or multispecificity, in contrast to mAbs, which are homodimers.

[0015] The terms "Fc-binding polypeptide," "Fc-binding agent," and "Fc-binding protein" are intended to refer to a polypeptide, molecule, or protein, respectively, that is capable of binding to the crystallizable portion Fc region of an antibody, and include, but are not limited to, for example, Protein A and Protein G, or any fragment or fusion protein thereof that retains such binding properties.

[0016] The term "VH binding" refers to the ability of a VH region to bind to an antibody or antibody fragment.

[0017] As used herein, the term "liquid sample" refers to a liquid containing an attempt to purify at least one target substance from other substances that also exist. The liquid sample can be, for example, an aqueous solution, an organic solvent system, or an aqueous / organic solvent mixture or solution. Source liquid is normally a complex mixture or solution containing many biomolecules (such as proteins, antibodies, hormones, and viruses), small molecules (such as salts, sugars, lipids, etc.), and even particulate matter. Although the typical source liquid of biological origin can initially be used as an aqueous solution or suspension, it can also contain the organic solvent used in early separation steps, such as solvent precipitation, extraction, etc. The example of the liquid sample that can contain valuable biological substances that can be purified by various embodiments of the present invention includes, but is not limited to, culture supernatant, homogenized cell suspension, blood plasma, plasma fractions, and milk from a bioreactor. Alternatively, the liquid sample can be referred to as "feed," "clarified cell culture feed," or "CCF."

[0018] A "buffer" is a substance that, by being present in a solution, increases the amount of acid or base that must be added to cause a unit change in pH. Buffer solutions resist pH changes through the action of their acid-base conjugate components. The term "physiological pH" refers to the pH of mammalian blood (i.e., 7.38 or about 7.4). Therefore, the physiological pH range is 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 purifying biomolecules (e.g., protein molecules) include zwitterionic or "Good" buffers, see, for example, Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62. Acidic buffers commonly used in antibody technology are based on carboxylic acids.

[0019] "Washing liquid" or "washing buffer" as used herein refers to a liquid used to remove impurities from the chromatography resin bound to the target substance. More than one washing liquid can be used sequentially, for example, successive washing liquids having different properties, such as pH, conductivity, solvent concentration, etc., designed to dissociate and remove different types of impurities that are non-specifically bound to the chromatography resin.

[0020] "Binding buffer" refers to the buffered solution designed to load the target molecule onto the chromatography column.

[0021] The term "equilibration buffer" in the present disclosure refers to a buffer used to prepare the affinity matrix with bound target protein for elution. The equilibration buffer can also be used to wash the affinity matrix with bound target protein.

[0022] "Eluent" or "elution buffer" are used interchangeably herein and refer herein to a liquid used to dissociate the target substance from the chromatography resin after it has been washed with one or more washing liquids, thereby eluting the protein containing the binding region from the immobilized binding agent. The eluent is used to dissociate the target substance without irreversibly denaturing it. Typical eluents are well known in the field of chromatography and may have different pH (usually lower pH), higher salt concentration, free affinity ligands or the like, or other substances that promote the dissociation of the target substance from the chromatography resin. "Elution conditions" refer to the process conditions imposed on the chromatography resin bound to the target substance, which dissociate the target substance from the chromatography resin, for example, contacting the chromatography resin bound to the target substance with an eluent or elution buffer to produce such dissociation.

[0023] Elution buffer has low pH, thereby destroys the interaction between separation matrix and the interested protein.Usually, low pH elution buffer has the pH in the range of about 2 to about 5, for example, in the range of about 3 to about 4.The example of the buffer of control pH in this scope comprises glycine, phosphate, acetate and citrate buffer, and their combination.Common buffer is citrate and acetate buffer, most preferably sodium citrate or sodium acetate buffer.

[0024] The ionic strength was calculated according to the following formula well known in the art:

[0025]

[0026] in:

[0027] οI-ionic strength;

[0028] ο∑ - sum of values;

[0029] οc i - ion concentration; and

[0030] o -The square of the ionic charge.

[0031] As used herein, the terms "comprises," "comprising," "containing," "having," and the like may mean "includes," "including," and the like; "consisting essentially of" or "consists essentially of" is an open term that allows for more than what is described as long as the basic or novel characteristics of the described content are not changed by the presence of more than what is described, but excludes prior art embodiments.

[0032] In case specific temperature data are not specified otherwise, all measurements and methods are carried out at room temperature (22 + / - 2°C). Summary of the Invention

[0033] One aim of the present inventors was to find a method wherein, during the step of eluting the target molecule from the affinity matrix, the elution conditions allow for a milder pH, ie a higher pH than normally used or a less acidic pH.

[0034] In addition, a goal is to design a method in which the target compound is well separated from other molecules in the feed. Such a separation is particularly meaningful for separating the bispecific heterodimeric antibody of interest from other antibodies that do not have the desired heavy and light chain configurations, such as homodimers.

[0035] The present inventors have surprisingly found that using an elution buffer that is less concentrated than usual and therefore has a lower ionic strength results in a less acidic elution pH of the target molecule in an antibody purification method.

[0036] Therefore, according to a first aspect, there is provided a method for purifying an antibody or antibody fragment, comprising the following steps:

[0037] adsorbing at least one of said antibodies or antibody fragments onto the affinity separation matrix by contacting the liquid sample with the affinity separation matrix,

[0038] washing the affinity separation matrix to remove impurities,

[0039] Equilibrating the affinity separation matrix with an equilibration buffer,

[0040] separating the at least one antibody or antibody fragment from the affinity separation matrix by using an elution buffer,

[0041] wherein the affinity separation matrix comprises a ligand based on protein L or any variant thereof that binds to the kappa-light chain of the antibody or antibody fragment, and wherein the elution buffer has a concentration of 5-50 mM.

[0042] According to a second aspect, the present invention provides a method for isolating a bispecific antibody, comprising the following steps:

[0043] by contacting a liquid sample with the affinity separation matrix, adsorbing a feed comprising at least the bispecific antibody onto the affinity separation matrix,

[0044] washing the affinity separation matrix to remove impurities,

[0045] Equilibrating the affinity separation matrix with an equilibration buffer,

[0046] Add elution buffer to the separation matrix to elute the bispecific antibody from the affinity separation matrix,

[0047] wherein the affinity separation matrix comprises a ligand based on protein L or any variant thereof that binds to the kappa-light chain of the bispecific antibody, and wherein the elution buffer has a concentration of 5-50 mM.

[0048] According to a third aspect, provided herein is a method for obtaining an elution pH above 4 from protein L or a protein L-derived chromatography separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by reducing the elution buffer concentration to below 50 mM, preferably to 10-50 mM.

[0049] For all aspects, the elution buffer may comprise a buffering dicarboxylic acid. The elution buffer may be a succinate buffer solution. The succinate buffer solution may have a concentration of 10-50 mM. The pH of the succinate buffer solution may be 5-3 or 5-2.8.

[0050] Alternatively, the elution buffer may comprise a buffering tricarboxylic acid. The elution buffer may be a citrate buffer solution. The citrate buffer solution may have a concentration of 5-45 mM. The pH of the citrate buffer solution may be 4.5-2.5.

[0051] Alternatively, the elution buffer may comprise a buffered monocarboxylic acid.The elution buffer may be a propionate buffer solution.

[0052] In all respects, elution is preferably performed in a pH range of about 5 to about 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Overlay of chromatograms from a trastuzumab run on a Protein L column using the indicated citrate elution buffer. The chromatograms zoom in on the elution peaks.

[0054] Figure 2 Overlay of chromatograms from a trastuzumab run on a Protein L column using 50 mM succinate elution buffer and the indicated equilibration buffer concentrations. The chromatogram zooms in on the elution peak.

[0055] Figure 3 is an overlay of chromatograms of bispecific antibodies (bsABs, as specified in the Examples) on a Protein L separation matrix, comparing elution using 10 mM citrate buffer and 10 mM equilibration / 50 mM succinate buffer. The chromatograms zoom in on the elution peaks.

[0056] Figure 4 involves Figure 1 and Figure 2 SEC analysis of the eluted fractions. Figure 4A Indicated are the positions where fractions were collected for the 10 mM / 50 mM succinate example. Figure 4B show Figure 4A SEC results of the indicated fractions.

[0057] Figure 5 shows a chromatogram of a bispecific antibody on a Protein L separation matrix using 15 mM succinate elution buffer. The scale on the right is pH. Figure 5A Shows an elution profile with a pH gradient, and Figure 5B An elution profile with step elution is shown.

[0058] Figure 6 shows a chromatogram of a bispecific antibody on a Protein L separation matrix using 50 mM propionate elution buffer. The scale on the right is pH. Figure 6A Shows an elution profile with a pH gradient, and Figure 6B An elution profile with step elution is shown.

[0059] Figure 7 shows a chromatogram of trastuzumab run on a Protein L column. The chromatogram zooms in on the elution peak. The scale on the right is pH. Figure 7A Step elution using 15 mM succinate elution buffer is shown. Figure 7B Step elution using 50 mM propionate elution buffer is shown. Figure 7C Step elution using 20 mM citrate elution buffer is shown. DETAILED DESCRIPTION

[0060] The inventors aimed to find a method in which, during the step of eluting the target molecule from the affinity matrix, the elution conditions allow for a milder pH, ie a higher pH than normally used.

[0061] The inventors have surprisingly found that in an antibody purification method a low concentration of elution buffer is advantageous in obtaining a milder elution pH, ie a less acidic elution pH, for the target molecule compared to a higher concentration of the same buffer.

[0062] Therefore, in a first aspect, the present invention relates to a method for purifying an antibody or antibody fragment, comprising the following steps:

[0063] adsorbing at least one of said antibodies or antibody fragments onto the affinity separation matrix by contacting the liquid sample with the affinity separation matrix,

[0064] washing the affinity separation matrix to remove impurities,

[0065] Equilibrating the affinity separation matrix with an equilibration buffer,

[0066] separating the at least one antibody or antibody fragment from the affinity separation matrix by using an elution buffer,

[0067] The elution buffer has a concentration of 5-50 mM.

[0068] The affinity separation matrix comprises a ligand based on protein L or any variant thereof that binds to the kappa light chain of an antibody or antibody fragment. In other words, the separation matrix is ​​protein L or a separation matrix derived from protein L. In the foregoing, the separation matrix is ​​referred to as a protein L separation matrix for convenience.

[0069] Any buffer solution used in the equilibration buffer and elution buffer in the methods disclosed herein must be a material that functions well as a buffer. That is, the material has good buffering properties (see "Definition" above). A skilled artisan will know which materials have good buffering properties and which materials do not, and which such materials are suitable for use in the present methods.

[0070] The elution buffer may preferably be a monocarboxylic acid, a dicarboxylic acid, or a tricarboxylic acid. A dicarboxylic acid will result in two carboxyl groups per acid molecule in solution. A tricarboxylic acid will result in three carboxyl groups per acid molecule in solution. Therefore, the more carboxyl groups present, the more counterions will be required to achieve the desired pH, resulting in a higher ionic strength.

[0071] The monocarboxylic acid used in the methods disclosed herein can be, for example, formic acid, acetic acid, and propionic acid, and is preferably formic acid or propionic acid. Preferably, propionic acid is used as the elution buffer. The elution buffer can therefore preferably be a 50 mM or lower propionate solution. However, compared to the commonly used citrate buffer, a higher concentration of propionate buffer, such as 75 mM or 100 mM, will also result in a higher elution pH.

[0072] The dicarboxylic acid used in the method may be, for example, oxalic acid, malonic acid and succinic acid. Preferably, succinic acid is used as the elution buffer. The elution buffer may therefore preferably be a 10-50 mM, for example, 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM succinate solution.

[0073] Elution of the target molecule is typically performed by lowering the pH. This can be done using a decreasing pH gradient or in a stepwise manner. The succinate elution buffer may have a pH of 5-3 or 5-2.8. By gradually increasing the amount of elution buffer relative to the equilibration buffer, a decreasing pH is achieved, and elution will occur. The equilibration buffer should preferably have a pH above 5, for example 5.5 or about 5.8-6. Elution occurs within a pH range of about 5 to about 3.

[0074] In the examples herein, an equilibrium buffer comprising the same buffer salt or acid as the elution buffer is used. This is to ensure a linear gradient in the examples, and in order to better show the advantages of the elution conditions. However, it will be appreciated by those skilled in the art that any conventional equilibrium buffer can be used, such as phosphate buffered saline (PBS), Tris buffered saline (TBS) with different concentrations of NaCl, etc. For the present invention, elution buffer is important in affecting the increase elution pH of the target molecule. However, it should be noted that the present invention focuses on the buffer solution having the required impact on the elution pH. According to the method disclosed herein, no other salt is used to increase or affect the elution pH. Therefore, according to the present disclosure, increasing the elution pH is achieved in the absence of other salts.

[0075] like Figure 2 As can be seen, for the Protein L separation matrix, elution with a succinate solution as disclosed above results in an elution pH of approximately 4.5. This is significantly higher than previously achieved with, for example, acetate buffer or citrate buffer having a concentration equal to or greater than 50 mM. For example, when using citrate at a concentration equal to or greater than 50 mM, a decrease in elution pH was previously observed, which is undesirable.

[0076] The tricarboxylic acid used in the method may preferably be a citrate buffer solution. The elution buffer may be a 5-45mM, such as 5mM, or 10mM, or 15mM, or 20mM, or 25mM, or 30mM, or 35mM, or 40mM, or 45mM citrate solution.

[0077] The citrate elution buffer may have a pH of 4.5-2.5. By gradually increasing the amount of elution buffer relative to the equilibration buffer, a decreasing pH is achieved and elution will occur. The equilibration buffer should have a pH of about or above 5, such as 5.5 or 6.0. Elution occurs in the pH range of about 5 to about 3. Figure 1 As can be seen, when using a Protein L separation matrix, an elution pH of at most 3.7 can be achieved with a citrate buffer solution having the lowest concentration. This is in contrast to the disclosure of US Pat. No. 10,844,112 B2, in which citrate buffer is disclosed as being generally more unfavorable for Protein L affinity matrices and an elution pH of less than 3.0 is shown for a 100 mM citrate elution buffer. By using a lower concentration of citrate buffer according to the present disclosure, a higher and more favorable elution pH is achieved, such as Figure 1 In addition, the lowest concentration of citrate buffer shown herein results in a higher elution pH than the highest elution pH (pH 3.6) shown using acetate buffer in US 10,844,112 B2.

[0078] Therefore, an advantage of using low concentrations and thereby low ionic strengths for the elution buffer according to the present invention is that the pH at which elution occurs can thereby be increased, as can be seen in the examples herein.

[0079] In a second aspect, the present invention relates to a method for isolating a bispecific antibody, comprising the following steps:

[0080] by contacting a liquid sample with the affinity separation matrix, adsorbing a feed comprising at least the bispecific antibody onto the affinity separation matrix,

[0081] washing the affinity separation matrix to remove impurities,

[0082] Equilibrating the affinity separation matrix with an equilibration buffer,

[0083] Add elution buffer to the separation matrix to elute the bispecific antibody from the affinity separation matrix,

[0084] The elution buffer has a concentration of 5-50 mM.

[0085] The elution buffer in this second aspect is as disclosed above for the first aspect.

[0086] The pH range for elution is as disclosed in the first aspect.

[0087] The separation matrix in this second aspect is as disclosed above for the first aspect.

[0088] Elution buffer is added with a pH gradient of about pH 6-5.8 to about pH 2.5-2.8. For example, when using a succinate buffer, the pH of the succinate equilibration buffer is preferably about 5.8-6.0, and the pH of the succinate elution buffer is about 5 to about 3 or about 5 to about 2.8. For example, when using a citrate buffer, the pH of the citrate equilibration buffer is preferably about 5.5, and the pH of the citrate elution buffer is about 4.5 to about 2.5 or about 4.5 to about 2.8. For example, when using a propionate buffer, the pH of the propionate equilibration buffer is preferably about 5.8-6.0, and the pH of the propionate elution buffer is about 5 to about 3 or about 5 to about 3.5.

[0089] like Figure 3As seen, the elution conditions using succinate buffer and citrate buffer provide a clear separation of the homodimer and heterodimer of bispecific antibodies on a protein L separation matrix. Peak 1 under the highest elution pH can correspond to very weakly bound unwanted species of a separation matrix (e.g., non-specific binding). Peak 2 under the second high elution pH corresponds to a heterodimer bsAb with 1 κ VL chain and 1 λ VL chain, which is a bsAb as a target for purification and separation. This has been confirmed (not shown) by LC-MS data. Peak 3 corresponds to a homodimer species with 2 κ VL chains, also confirmed (not shown) by LC-MS data. As in the case of the first aspect, the elution pH when using succinate buffer is higher than the elution pH when using citrate buffer. However, both types of buffers can achieve an elution pH of 3.6 or higher than 3.6, or even an elution pH of about 4 for succinate, and therefore both provide good separation under favorable pH.

[0090] in addition, Figure 6A and 6B It is clearly shown that the use of propionate buffer provides a clear separation of homodimers and heterodimers of the bispecific antibody on the Protein L separation matrix, similar to that described above. Figure 6B For step elution, the separation is particularly clear.

[0091] According to a third aspect, provided herein is a method for obtaining an elution pH above 4 from protein L or a protein L-derived chromatography separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by reducing the elution buffer concentration to below 50 mM, preferably to 10-50 mM. The composition of the elution buffer and the pH and more specifically the concentration of the elution buffer are as disclosed in the previous two aspects.

[0092] Thus, the methods of the present disclosure allow for elution of antibodies or antibody fragments from a Protein L separation matrix at a higher pH than is typically used. This would be advantageous for antibodies or antibody fragments because the elution conditions are closer to neutral pH than previously demonstrated methods, resulting in a lower risk of denaturation of the antibodies or antibody fragments. Additionally, acidic pH can promote aggregation, which is mitigated by the methods of the present disclosure.

[0093] The present invention is further disclosed in the following examples. These examples should not be construed as limiting the scope of the invention to the exact configuration or conditions shown therein, but are included merely to illustrate the advantages of the present invention. The scope of the invention is defined by the appended claims, and any embodiments falling within these definitions, even if not explicitly disclosed, constitute part of the present invention.

[0094] Example

[0095] Material

[0096] Sodium dihydrogen phosphate and disodium hydrogen phosphate, sodium chloride, sodium hydroxide, acetic acid, citric acid, trisodium citrate, succinic acid, disodium succinate hexahydrate, DL-dithiothreitol (DTT), tris(hydroxymethyl)aminomethane HCl and tris(hydroxymethyl)alkylmethane were purchased from Merck (Darmstadt, Germany). Formic acid 98-100% Purchased from Merck, and acetonitrile LC / MS GRADE Purchased from Fisher Scientific. Propionic acid was purchased from Fluka Chemical, and sodium propionate was purchased from Sigma-Aldrich.

[0097] equipment

[0098] Controlled by Unicorn 7.7 Pure 25 System (Cytiva TM , Uppsala, Sweden) was used for all chromatographic runs. Explorer 10XT (Cytiva, Uppsala, Sweden) was used for SEC analysis, and the LC-MS instrument was BioAccord (Waters), Acquity I-Class UPLC system coupled to Acquity RDa detector.

[0099] method

[0100] Protein L chromatography

[0101] MabSelect VL (Protein L affinity resin, Cytiva TM, Uppsala, Sweden) was packed in a 0.5 cm diameter column with a bed height of 10 cm. The column volume (CV) was approximately 2 mL. The detailed information of the MabSelect VL chromatogram is summarized in Table 1. The column load was purified mAb, clarified cell culture harvest of mAb, or clarified cell culture harvest of bispecific Ab (bsAb). The mAb used in this experiment was trastuzumab. The bsAb used was a bsAb comprising a κ1 class light chain from trastuzumab, a λ2 class light chain from avelumab, and a heavy chain of trastuzumab, available from ThermoFisher. For mAb purification runs, all runs were loaded with 10 mg mAb / mL resin, and for bsAb purification runs, all runs were loaded with 2.5 mg bsAb / mL resin. The column was loaded and operated in bind-elute mode. The target protein was eluted with a gradient of elution buffer from 0 to 100% over 20 CVs. For all chromatographic runs, the system was operated at a flow rate corresponding to a 6-minute residence time. All chromatograms were recorded by monitoring UV absorbance at 280 nm. The eluate from selected runs was collected in fractions and analyzed for monomer purity by SEC and / or identity by LC-MS.

[0102] Table 1: MabSelect VL chromatogram

[0103] step Volume (CV) Buffer composition Binding buffer 3 20 mM phosphate 150 mM NaCl pH 7.2 Loading mAb or bsAb, as specified above Salt washing 7 20 mM phosphate 500 mM NaCl pH 7.2 Washing / Balancing 3-5 Buffer A, see Table 2 Elution* 5 0-100% Buffer B, 20 CV, see Table 2 peeling 3 0.5 M acetic acid balance 2 20 mM phosphate, 150 mM NaCl pH 7.2 CIP (cleaning in place) 3 0.1M NaOH Rebalancing 1.5 20 mM phosphate 500 mM NaCl pH 7.2 Rebalancing 3 20 mM phosphate 150 mM NaCl pH 7.2

[0104] Table 2: Equilibration buffer (buffer A) and elution buffer (buffer B)

[0105] Experimental setup Buffer A: Equilibration buffer Buffer B: elution buffer 1 10 mM succinate, pH 5.8 50 mM succinate, pH 2.8 2 20 mM succinate, pH 6 50 mM succinate, pH 2.8 3 50 mM citrate, pH 5.5 50 mM citrate, pH 2.5 4 20 mM citrate, pH 5.5 20 mM citrate, pH 2.5 5 10 mM citrate, pH 5.5 10 mM citrate, pH 2.8 6 50 mM propionate, pH 6 50 mM propionate, pH 3.2 7 15 mM succinate, pH 6 15 mM succinate, pH 3.4

[0106] Size exclusion chromatography

[0107] Use Superdex 200 Increase column (10x300mm, Cytiva TM SEC analysis was performed using a flow cytometer (100 nm, 50 μl) per run. A 50 μl sample volume was injected for each run. The mobile phase consisted of 200 mM phosphate, pH 6.8. The sample was eluted isocratically at a flow rate of 0.8 mL / min for 30 minutes. UV absorbance at 280 nm was recorded for all chromatograms.

[0108] Liquid chromatography-mass spectrometry (data not shown)

[0109] Using BioResolve RP mAb Polyphenyl columns ( 2.7 μm, 2.1x50 mm, Waters) for LC-MS analysis. The sample diluted to 0.1 g / L was reduced with DTT and The samples were digested with a 50 μl column (Genovis) at 37°C for 3 hours before injection. 1 μl of sample was injected per run. The mobile phase consisted of 0.1% formic acid, and the mAb or bsAb was eluted using a gradient of 0.1% formic acid / acetonitrile at a flow rate of 0.5 mL / min for a total of 15 minutes. The column was maintained at 60°C during analysis.

[0110] result

[0111] Example 1 - Purification of mAb using citrate buffer and protein L separation matrix

[0112] In this example, the protein L chromatography above was performed using experimental settings 3, 4, and 5 of Table 2. The resulting chromatogram is shown in Figure 1 Obviously, the lower the concentration of both buffers A and B, and therefore the lower the ionic strength (as shown in Table 3), the higher the elution pH that can be achieved.

[0113] Table 3: Buffer composition and ionic strength of investigated citrate buffers

[0114] Buffer A: Ionic strength (M) Buffer B Ionic strength (M) 50mM pH 5.5 0.1961 50mM pH 2.5 0.01046 20mM pH 5.5 0.0725 20mM pH 2.5 0.00404 10mM pH 5.5 0.03454 10mM pH 2.8 0.00341

[0115] Example 2 - Purification of mAb using succinate buffer and protein L separation matrix

[0116] In this example, the protein L chromatography was performed according to the experimental settings 1 and 2 of Table 2. The resulting chromatogram is shown in Figure 2 It is clear that the lower the concentration of buffer A, and by using a specified concentration of buffer B, the lower the ionic strength (as shown in Table 4), and the higher the elution pH that can be achieved.

[0117] Table 4: Buffer composition and ionic strength of investigated succinate buffers

[0118] Buffer A: Ionic strength (M) Buffer B: Ionic strength (M) 10mM pH 5.8 0.0225 50mM pH 2.8 0.00198 20mM pH 6 0.0506 50mM pH 2.8 0.00198

[0119] Here Figure 2 It can be observed that Figure 1 The elution pH increased significantly by approximately one (1) pH unit compared to the citrate buffer.

[0120] Example 3 - SEC analysis

[0121] Fractions were collected from the above chromatography experiments, as Figure 4A The results of the SEC analysis are summarized in Table 5. An exemplary SEC curve from a 10 mM / 50 mM succinate experiment is shown in Figure 4B middle.

[0122] Table 5: SEC analysis of collected fractions

[0123]

[0124]

[0125] Clearly, for the succinate elution experiment, the first fraction from the main peak of the chromatogram, which was eluted at a pH of 4.5, contained 100% or nearly 100% of the monomeric mAb to be purified. When eluted with citrate buffer, it was clear that the lowest concentration of citrate buffer, 10 mM / 10 mM, resulted in the highest amount of monomeric mAb in the first fraction from the main peak (10 mM, elution pH 3.75, 100% target monomer), while 50 mM citrate did not show the same good separation of the monomeric mAb from other molecules present in the feed.

[0126] In the case of 10mM / 50mM succinate and 20mM / 50mM succinate, the second fraction still contains a considerable amount of target monomer, but also contains a small amount of low molecular weight (LMW) species. Without being bound by any theory, it is believed that this LMW contains a single light chain. In addition, the second fraction also contains high molecular weight (HMW) species. Without being bound by any theory, it is believed that it contains aggregates. The amount of LMW and HMW then increases in the third and fourth fractions.

[0127] In the case of 20 mM / 20 mM citrate and 50 mM / 50 mM citrate, the first fraction from the main peak also contained LMW, which increased in the following two fractions.

[0128] Again, this shows that low concentrations of succinate buffer as specified above, and minimal concentrations of citrate buffer, and thus low ionic strength, lead to good separation of monomeric mAb from other species contained in the feed, such as LMW and HMW.

[0129] Example 4 - Separation of bispecific antibodies on a protein L separation matrix

[0130] 4A. Comparison of 10 mM Citrate and 10 mM Succinate Buffers

[0131] In this example, the bsAbs defined above were run according to experimental setups 1 and 5 of Table 2 and on a Protein L separation matrix according to above. Figure 3 ) shows three peaks according to the pH gradient. For 10mM / 50mM succinate buffer, the three peaks elute at pH 4.8, 4.1, and 3.8, respectively. For 10mM / 10mM citrate buffer, the three peaks elute at pH 4.8, 3.9, and 3.6, respectively.

[0132] LC-MS analysis (data not shown) showed that the second peak contained heterodimeric bispecific antibodies comprising one κ VL chain and one λ VL chain. The third peak contained monomeric Abs comprising two κ VL chains (homodimers) (data not shown). The first peak contained species or antibody variants that were nonspecifically and / or weakly bound to the separation matrix, including monomeric Abs comprising two λ VL chains (homodimers).

[0133] 4B. Separation using 15 mM succinate buffer

[0134] For this example, the bsAbs defined above were run according to experimental setup 7 of Table 2 and on a Protein L separation matrix according to above. Figure 5A ) or by step elution ( Figure 5B In the step elution, pH 4.8 was used to elute the lambda homodimer, pH 4.3 was used to elute the lambda-κ heterodimer, and pH 3.4 was used to elute the kappa homodimer.

[0135] Table 6: Elution pH of entities in the feed using 15 mM succinate

[0136] peak entity Elution pH A λ homodimer 4.9 B bsAb (heterodimer) 4.4 C κ homodimer 4.0

[0137] like Figure 5A and Figure 5B As can be seen in Figure 3, a clear separation of the heterodimer from the two homodimers was achieved.

[0138] 4C. Separation using 50 mM propionate buffer

[0139] For this example, the bsAbs defined above were run according to experimental setup 6 of Table 2 and on a Protein L separation matrix according to the above. Figure 6A ) or by step elution ( Figure 6B In the step elution, λ homodimers were eluted using pH 4.8, λ-κ heterodimers were eluted using pH 4.4, and κ homodimers were eluted using pH 3.5.

[0140] Table 7: Elution pH of entities in the feed using 50 mM propionate

[0141]

[0142]

[0143] like Figure 6A and Figure 6B As can be seen in Figure 3, a clear separation of the heterodimer from the two homodimers was achieved.

[0144] Example 5. mAb purification: comparison of 15 mM succinate, 50 mM propionate, and 20 mM citrate

[0145] In this example, the protein L chromatography according to the above was performed according to the experimental settings 4, 6 and 7 of Table 2. The experimental results are summarized in Table 8 below, and the resulting chromatograms are shown in Table 8. Figure 7A (15 mM succinate), Figure 7B (50 mM propionate) and Figure 7C (20 mM citrate).

[0146] Table 8

[0147]

[0148] Therefore, it has been shown that using a lower concentration of elution buffer than is commonly used will increase the achievable elution pH. Additionally, separation of heterodimeric antibodies from homodimeric antibodies can be achieved while still achieving an increased elution pH.

[0149] In addition, it has been shown that succinate and propionate buffers are advantageous alternatives to conventional citrate buffers. Specifically, it has been shown that low concentrations of succinate and concentrations lower than conventional citrate concentrations are advantageous for achieving higher elution pH on a Protein L separation matrix. Without being bound by any theory, it is believed that the lower ionic strength of the buffer solution is the reason for the higher elution pH. Therefore, compared with tricarboxylic acids, buffers consisting of monocarboxylic acids or dicarboxylic acids may be advantageous because the ionic strength at a given pH is lower for monocarboxylic acids and dicarboxylic acids. This is due to the need for less sodium ions (from NaOH titration) to achieve the same pH.

Claims

1. A method for purifying an antibody or antibody fragment, comprising the following steps: adsorbing at least one of said antibodies or antibody fragments onto an affinity separation matrix by contacting a liquid sample with the affinity separation matrix, washing the affinity separation matrix to remove impurities, Equilibrating the affinity separation matrix with an equilibration buffer, separating the at least one antibody or antibody fragment from the affinity separation matrix by using an elution buffer, wherein the affinity separation matrix comprises a ligand based on protein L or any variant thereof that binds to the kappa-light chain of the antibody or antibody fragment, and wherein the elution buffer has a concentration of 5-50 mM.

2. A method for isolating a bispecific antibody, comprising the following steps: adsorbing a feed comprising at least the bispecific antibody onto an affinity separation matrix by contacting a liquid sample with the affinity separation matrix, washing the affinity separation matrix to remove impurities, Equilibrating the affinity separation matrix with an equilibration buffer, adding an elution buffer to the separation matrix to elute the bispecific antibody from the affinity separation matrix, wherein the affinity separation matrix comprises a ligand based on protein L or any variant thereof that binds to the kappa-light chain of the bispecific antibody, and wherein the elution buffer has a concentration of 5-50 mM.

3. A method for obtaining an elution pH above 4 from protein L or a protein L-derived chromatography separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by reducing the elution buffer concentration to below 50 mM, preferably to 10-50 mM.

4. The method of any one of claims 1 to 3, wherein the elution buffer comprises a buffered dicarboxylic acid. The method according to claim 4 , wherein the elution buffer is a succinate buffer solution. The method according to claim 5 , wherein the succinate buffer solution has a concentration of 10-50 mM.

7. The method according to any one of claims 5 or 6, wherein the succinate buffer solution has a concentration of 10-20 mM, preferably 15 mM.

8. The method according to any one of claims 5-7, wherein the pH of the succinate buffer solution is 3-5 or 2.8-5.

9. The method of any one of claims 1 to 3, wherein the elution buffer comprises a buffered tricarboxylic acid.

10. The method according to claim 9, wherein the elution buffer is a citrate buffer solution.

11. The method according to any one of claims 9 or 10, wherein the citrate buffer solution has a concentration of 5-45 mM.

12. The method according to any one of claims 9 to 11, wherein the pH of the citrate buffer solution is 4.5 to 2.

5.

13. The method of any one of claims 1-3, wherein the elution buffer comprises a buffered monocarboxylic acid. The method according to claim 13 , wherein the elution buffer is a propionate buffer solution.

15. The method according to claim 14, wherein the propionate buffer solution has a concentration of 50 mM.

16. The method of any one of claims 1 to 15, wherein the elution is performed at a pH range of about 5 to about 3.

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