Optimization of elution conditions

Abstract

The present invention relates to a method for purifying antibodies or antibody fragments, comprising contacting a liquid sample with an affinity separation matrix to adsorb at least one antibody or antibody fragment onto the affinity separation matrix, the affinity matrix comprising a ligand based on Protein L or any variant thereof that binds to the κ light chain of an antibody or antibody fragment, and separating the at least one antibody or antibody fragment from the affinity separation matrix using an elution buffer having a concentration of 5 to 50 mM. The present invention also relates to a method for separating 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 methods for improving said conditions. [Background technology]

[0002] Immunoglobulins are some of the most widely used biopharmaceuticals in either production or development worldwide. Their high commercial demand for specific therapeutic markets, and therefore their value, has placed a premium on pharmaceutical companies to maximize the productivity of their respective mAb manufacturing processes while controlling associated costs.

[0003] In most cases, affinity chromatography is used as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal or polyclonal antibodies. A particularly interesting class of affinity reagents are proteins that can specifically bind to the constant part of the immunoglobulin molecule, such an interaction being unrelated to 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 feedstocks.

[0004] For immunoglobulins, immunoglobulin fragments, antibodies, or antibody fragments, such as Fab, single-chain variable fragments (scFv), bispecific T-cell engagers (BiTEs), domain antibodies lacking Fc chains but having kappa light chains of subclass 1, 3, or 4, a matrix containing protein L derived from Finegoldia magna (formerly Peptostreptococcus Magnus) (B Akerstrom, L Bjorck: 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, pp. 2234-2239, 1992 and U.S. Pat. No. 6,822,075) show great promise as a purification platform that provides the required high selectivity.

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

[0006] In most commercially available processes, targets bind to ligands on affinity matrices, typically requiring a pH of 2.5 to 3.5 for the target to dissociate from the affinity ligand and be eluted with an elution buffer. Such a low pH carries the risk of damaging the target protein, e.g., causing target protein aggregation or denaturation. This problem would result in lower productivity and yield of intact target protein. Therefore, regardless of whether the target is to be purified or separated by a chromatographic purification or separation process, it would be of great interest to be able to elute the target at a pH closer to neutral than previously possible, thereby minimizing the risks discussed above. U.S. Patent No. 10,844,112 discusses the use of specific buffers for elution. Previous attempts have been made to increase the elution pH by adding salt to the elution buffer, but such an approach requires the removal of high levels of salt from the eluate. Therefore, there remains significant interest in finding alternative methods for eluting target molecules at higher pHs than previously possible.

[0007] definition The terms "antibody" and "immunoglobulin" are used interchangeably herein and refer to an antigen-binding protein with a basic four-polypeptide chain structure consisting of two heavy (H) chains and two light (L) chains stabilized by inter- or intra-chain disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. In humans, there are two types of light chains: kappa chains and lambda chains. This term should be understood to include any antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, as well as antibody fragments, fusion proteins comprising antibodies or antibody fragments, and conjugates comprising antibodies or antibody fragments, such as antibody-drug conjugates (ADCs).

[0008] The term "mAb" refers to a monoclonal antibody.

[0009] The term "Fc region" refers to the C-terminal region of an IgG antibody, in particular the C-terminal region of the heavy chain of said IgG antibody. The term "Fc binding" refers to the ability to bind to said region.

[0010] The term "kappa light chain-containing protein" is used synonymously with "immunoglobulin kappa light chain-containing protein" and refers herein to a kappa light chain of subclass 1, 3, or 4 derived from an antibody. κI , V κIII and V κIV as in BHK Nilson et al.: J. Biol. Chem. 267, 10 pp. 2234-2239, 1992), and includes any intact antibody, antibody fragment, fusion protein, conjugate or recombinant protein containing a kappa light chain of subclass 1, 3 or 4.

[0011] The term "Fab" or "Fab fragment" refers to the antigen-binding region and contains both the constant and variable domains of both the heavy and light chains. Fab may contain a kappa and / or a lambda light chain.

[0012] The term "Fv fragment" refers to the fragment variable region and contains only two variable domains, VH and VL, which are held together in the Fv fragment by non-covalent interactions.

[0013] 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 three or more different types of antigens or three or more different epitopes on the same antigen. In contrast to mAbs, which are homodimers, bispecific or multispecific antibodies are heterodimers, having different variable regions, accounting for their bispecificity or multispecificity.

[0014] The terms "Fc-binding polypeptide," "Fc binding agent," and "Fc-binding protein" refer to a polypeptide, molecule, or protein capable of binding to the crystallizable portion, Fc region, of an antibody, respectively, including, but not limited to, Protein A and Protein G, or any fragment or fusion protein thereof that maintains said binding properties.

[0015] The term "VH binding" refers to the ability to bind to the VH region of an antibody or antibody fragment.

[0016] As used herein, the term "liquid sample" refers to a liquid containing at least one target substance targeted for purification from other substances also present. A liquid sample can be, for example, an aqueous solution, an organic solvent system, or an aqueous / organic solvent mixture or solution. Source liquids are frequently complex mixtures or solutions containing many biomolecules (e.g., proteins, antibodies, hormones, and viruses), small molecules (e.g., salts, sugars, lipids, etc.), and even particulate matter. A typical source liquid of biological origin may begin as an aqueous solution or suspension but may also contain organic solvents used in prior separation steps, such as solvent precipitation, solvent extraction, etc. Examples of liquid samples that may contain valuable biological substances suitable for purification according to various embodiments of the present invention include, but are not limited to, culture supernatants from bioreactors, homogenized cell suspensions, plasma, plasma fractions, and milk. Alternatively, a liquid sample may be referred to as a "feed," "clarified cell culture feed," or "CCF."

[0017] A "buffer" is a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a change in pH by one unit. A buffer resists changes in pH through the action of its acid-base conjugate components. The term "physiological pH" refers to the pH of mammalian blood (i.e., 7.38 or approximately 7.4). The physiological pH range is therefore approximately 7.2 to 7.6. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases. Exemplary buffers used in the purification of biomolecules (e.g., protein molecules) include zwitterionic or "Good's" buffers; see, e.g., Good et al. (1966) Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol. 24:62. Acidic buffers commonly used in antibody processes are based on carboxylic acids.

[0018] "Wash solution" or "wash buffer" as used herein refers to a liquid used to remove impurities from a chromatography resin to which a target substance is bound. Two or more wash solutions designed to dissociate and remove different types of impurities nonspecifically associated with the chromatography resin may be utilized sequentially, e.g., using a series of wash solutions with different properties such as pH, conductivity, solvent concentration, etc.

[0019] "Binding buffer" refers to a buffer solution intended for loading a target molecule onto a chromatography column.

[0020] The term "equilibration buffer" as used in the present disclosure refers to a buffer solution used to prepare an affinity matrix with bound target proteins for elution. The equilibration buffer may also be used to wash the affinity matrix with bound target proteins.

[0021] As used interchangeably herein, "elution solution" or "elution buffer" refers herein to a liquid used to dissociate a target substance from a chromatography resin, thereby eluting the binding domain-containing protein from the immobilized binder, after washing with one or more wash solutions. The elution solution acts to dissociate the target substance without irreversibly denaturing it. Typical elution solutions are well known in the chromatography art and may have a different pH (typically a lower pH), a high salt concentration, a free affinity ligand or analog, or other substances that promote dissociation of the target substance from the chromatography resin. "Elution conditions" refer to process conditions imposed on a target substance-bound chromatography resin that dissociate the target substance from the chromatography resin, such as contacting the target substance-bound chromatography resin with an elution solution or elution buffer to produce such dissociation.

[0022] Preferably, the elution buffer has a low pH to disrupt the interaction between the separation matrix and the protein of interest. Typically, a low-pH elution buffer has a pH in the range of about 2 to about 5, for example, about 3 to about 4. Examples of buffers that control the pH within this range include glycine buffer, phosphate buffer, acetate buffer, and citrate buffer, as well as combinations thereof. Commonly used buffers are citrate buffer and acetate buffer, and most preferably sodium citrate buffer or sodium acetate buffer.

[0023] Ionic strength can be calculated using the following formula, which is well known within the art:

number

number

[0024] 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" are open-ended terms that take into account the presence of other recited items, but exclude prior art embodiments, so long as the presence of the other recited items does not change the basic or novel characteristics of the recited items.

[0025] In the absence of specific temperature data indicating otherwise, all measurements and methods are performed at room temperature (22 + / - 2°C). [Prior art documents] [Patent documents]

[0026] [Patent Document 1] U.S. Patent No. 6,822,075 [Patent Document 2] U.S. Patent No. 10,844,112 [Non-patent literature]

[0027] [Non-Patent Document 1] B Akerstrom, L Bjorck: J. Biol. Chem. 264, pp. 19740-19746, 1989 [Non-patent document 2] W Kastem et al.: J. Biol. Chem. 267, 12820-12825, 1992 [Non-patent document 3] B. H. K. Nilson et al.: J. Biol. Chem. 267, pp. 2234-2239, 1992 [Non-patent document 4] Carter, P., Lazar, G. Next generation antibody drugs: pursuit of the 'high-hanging fruit'. Nat Rev Drug Discov 17, pp. 197-223 (2018). https: / / doi.org / 10.1038 / nrd.2017.227 [Non-Patent Document 5] Good et al. (1966) Biochemistry 5:467 [Non-patent document 6] Good and Izawa (1972) Methods Enzymol. 24:62 Summary of the Invention [Problem to be solved by the invention]

[0028] It was our aim to find a process in which elution conditions allow for a milder pH, i.e. a higher pH than normally used, or a less acidic pH, in the elution step of the target molecule from the affinity matrix.

[0029] Furthermore, it was an objective to design a process in which the target compound would be conveniently separated from other molecules in the feed. Such separation is particularly interesting for separating the bispecific heterodimeric antibody of interest from other antibodies that do not have the desired configuration of heavy and light chains, e.g., homodimers. [Means for solving the problem]

[0030] The inventors have surprisingly found that the use of elution buffers at lower than usual concentrations, and therefore with lower ionic strength, results in a less acidic elution pH of target molecules in antibody purification processes.

[0031] Thus, according to a first aspect, there is provided herein a method for preparing a medicament for use in a pharmaceutical composition comprising the steps of: contacting the liquid sample with the affinity separation matrix, thereby adsorbing at least one of the antibody or antibody fragment onto 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 using an elution buffer; 1. A method for purifying an antibody or antibody fragment comprising: the affinity separation matrix comprises a ligand based on Protein L or any variant thereof that binds to the κ light chain of an antibody or antibody fragment, and the elution buffer has a concentration of 5 to 50 mM; A method is provided.

[0032] According to a second aspect, the present invention provides a method for producing a medicament for the treatment of a cancer, comprising the steps of: adsorbing the feed containing at least the bispecific antibody onto the affinity separation matrix by contacting the 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; 1. A method for isolating a bispecific antibody comprising: the affinity separation matrix comprises a ligand based on Protein L, or any variant thereof that binds to the κ light chain of the bispecific antibody, and the elution buffer has a concentration of 5 to 50 mM; A method is provided.

[0033] According to a third aspect, there is provided herein a method for obtaining an elution pH above 4 from Protein L or a Protein L-derived chromatographic separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by lowering the elution buffer concentration below 50 mM, preferably between 10 and 50 mM.

[0034] In all embodiments, the elution buffer may contain a buffered dicarboxylic acid. The elution buffer may be a succinate buffer. The succinate buffer may have a concentration of 10 to 50 mM. The pH of the succinate buffer may be 5 to 3, or 5 to 2.8.

[0035] Alternatively, the elution buffer may contain a buffered tricarboxylic acid. The elution buffer may be a citrate buffer. The citrate buffer may have a concentration of 5 to 45 mM. The pH of the citrate buffer may be 4.5 to 2.5.

[0036] Alternatively, the elution buffer may comprise a buffered monocarboxylic acid. The elution buffer may be a propionic acid buffer.

[0037] For all embodiments, elution is preferably carried out in the pH range of about 5 to about 3. [Brief explanation of the drawings]

[0038] [Figure 1] An overlay of chromatograms of trastuzumab on a Protein L column run with the indicated citrate elution buffers. The chromatogram is zoomed in on the elution peak. [Figure 2] Chromatogram overlay of trastuzumab on a Protein L column run with 50 mM succinate elution buffer and the indicated equilibration buffer concentrations. The chromatogram is zoomed in on the elution peak. [Figure 3] Chromatogram overlay of bispecific antibodies (bsAB, as specified in the Examples) on Protein L separation matrix comparing the use of 10 mM citrate elution buffer and 10 mM equilibration / 50 mM elution succinate buffer. The chromatogram is zoomed in on the elution peak. [Figure 4A] SEC analysis of elution fractions from Figures 1 and 2. For the 10 mM / 50 mM succinate example, the locations where fractions were collected are indicated. [Figure 4B]Regarding SEC analysis of elution fractions from Figures 1 and 2, the SEC results for the fractions shown in Figure 4A are shown. [Figure 5A] Chromatogram of bispecific antibody on Protein L separation matrix using 15 mM succinate elution buffer. Right axis is pH. pH gradient elution profile is shown. [Figure 5B] Chromatogram of bispecific antibody on Protein L separation matrix using 15 mM succinate elution buffer. Right axis is pH. Elution profile with step elution is shown. [Figure 6A] Chromatogram of bispecific antibody on Protein L separation matrix using 50 mM propionic acid elution buffer. Right axis is pH. pH gradient elution profile is shown. [Figure 6B] Chromatogram of bispecific antibody on Protein L separation matrix using 50 mM propionic acid elution buffer. Right axis is pH. Elution profile with step elution is shown. [Figure 7A] Chromatogram of trastuzumab run on a Protein L column. The chromatogram is zoomed in on the elution peak. The right axis is pH. Step elution using 15 mM succinate elution buffer is shown. [Figure 7B] Chromatogram of trastuzumab run on a Protein L column. The chromatogram is zoomed in on the elution peak. The right axis is pH. Step elution using 50 mM propionic acid elution buffer is shown. [Figure 7C] Chromatogram of trastuzumab run on a Protein L column. The chromatogram is zoomed in on the elution peak. The right axis is pH. Step elution using 20 mM citrate elution buffer is shown. DETAILED DESCRIPTION OF THE INVENTION

[0039] The inventors had the objective of finding a process in which elution conditions would allow for a milder pH, ie a higher pH than is normally used, in the elution step of the target molecule from the affinity matrix.

[0040] The inventors have surprisingly found that in antibody purification processes, a low concentration of an elution buffer is advantageous compared to a high concentration of the same buffer in achieving a less acidic elution pH, which is a milder elution pH for the target molecule.

[0041] Thus, in a first aspect, the present invention provides a method for producing a soluble polymer comprising the steps of: contacting the liquid sample with the affinity separation matrix, thereby adsorbing at least one of the antibody or antibody fragment onto 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 using an elution buffer; 1. A method for purifying an antibody or antibody fragment comprising: The elution buffer has a concentration of 5 to 50 mM. Regarding the method.

[0042] 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. Alternatively expressed, the separation matrix is ​​a Protein L or Protein L-derived separation matrix. Here, the separation matrix is ​​conveniently referred to as a Protein L separation matrix.

[0043] Any buffer used as an equilibration buffer or elution buffer in the methods disclosed herein should be a material that functions favorably as a buffer, i.e., has good buffering properties (see "Definitions" above). Those skilled in the art will recognize which materials have good buffering properties and which do not, and which such materials are applicable to the present methods.

[0044] The elution buffer may preferably be a monocarboxylic acid, dicarboxylic acid, or tricarboxylic acid. A dicarboxylic acid in solution provides two carboxyl groups per acid molecule present in the solution. A tricarboxylic acid in solution provides three carboxyl groups per acid molecule present in the solution. Therefore, the more carboxyl groups present, the more counterions are required to achieve the desired pH, resulting in a higher ionic strength.

[0045] The monocarboxylic acid used in the method disclosed herein may be, for example, formic acid, acetic acid, or propionic acid, preferably formic acid or propionic acid. Preferably, propionic acid is used as the elution buffer. Therefore, the elution buffer may preferably be a propionic acid solution of 50 mM or less. However, for propionic acid buffers, higher concentrations, for example, 75 mM or 100 mM, also result in a higher elution pH than commonly used citrate buffers.

[0046] The dicarboxylic acid used in this method may be, for example, oxalic acid, malonic acid, or succinic acid. Preferably, succinic acid is used as the elution buffer. Therefore, the elution buffer may be a succinic acid solution of preferably 10 to 50 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

[0047] Elution of target molecules is typically achieved by lowering the pH. This can be done using a decreasing pH gradient or stepwise. The succinic acid elution buffer can have a pH of 5 to 3, or 5 to 2.8. The pH is lowered and elution occurs by gradually increasing the amount of elution buffer relative to the equilibration buffer. The equilibration buffer preferably has a pH greater than 5, e.g., 5.5, or about 5.8 to 6. Elution occurs within a pH range of about 5 to about 3.

[0048] In the examples herein, an equilibration buffer containing the same buffer salt or acid as the elution buffer is used. This is to ensure a linear gradient in the examples and better visualize the benefits of the elution conditions. However, those skilled in the art will recognize that any commonly used equilibration buffer, such as phosphate-buffered saline (PBS) containing various concentrations of NaCl, Tris-buffered saline, etc., can be used. For the present invention, it is the elution buffer that is important in influencing the increase in elution pH of the target molecule. However, it is noted that the present invention focuses on the buffer with respect to the desired effect on elution pH. No additional salts are used to increase or affect the elution pH according to the methods disclosed herein. Thus, the increase in elution pH is achieved according to the present disclosure without additional salts.

[0049] As can be seen in Figure 2, elution with the succinic acid solution disclosed above results in an elution pH of approximately 4.5 for the Protein L separation matrix. This is significantly higher than the elution pH previously achieved using, for example, acetate buffers or citrate buffers at concentrations of 50 mM or greater. For example, it has previously been observed that the use of citrate at concentrations of 50 mM or greater undesirably reduces the elution pH.

[0050] The tricarboxylic acid used in this method may preferably be a citrate buffer. The elution buffer may be a 5 to 45 mM citrate solution, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or 45 mM.

[0051] The citrate elution buffer may have a pH between 4.5 and 2.5. By gradually increasing the amount of elution buffer relative to the equilibration buffer, a decrease in pH is achieved, and elution occurs. The equilibration buffer should have a pH of about 5 or greater, e.g., 5.5 or 6.0. Elution occurs within a pH range of about 5 to about 3. As seen in Figure 1, when using a Protein L separation matrix, an elution pH of up to 3.7 can be achieved with the lowest citrate buffer concentration. This contrasts with the disclosure of U.S. Pat. No. 10,844,112, which discloses that citrate buffers are less advantageous for the Protein L affinity matrix overall and shows an elution pH of less than 3.0 for a 100 mM citrate elution buffer. Thus, as shown in Figure 1, a higher and more advantageous elution pH is achieved by using a lower concentration citrate buffer according to the present disclosure. Furthermore, the lowest concentration of citrate buffer shown herein results in a higher elution pH than the highest elution pH using acetate buffer (pH 3.6) shown in US Pat. No. 10,844,112.

[0052] Thus, an advantage of using a low concentration for the elution buffer according to the present invention, thereby achieving a low ionic strength, is that the pH at which elution occurs can then be increased, as can be seen in the examples herein.

[0053] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: adsorbing the feed containing at least the bispecific antibody onto the affinity separation matrix by contacting the 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; 1. A method for isolating a bispecific antibody comprising: The elution buffer has a concentration of 5 to 50 mM. Regarding the method.

[0054] The elution buffer in the second aspect is as disclosed in relation to the first aspect above.

[0055] The pH range for elution is as disclosed in relation to the first aspect.

[0056] The separation matrix in this second embodiment is as disclosed in connection with the first embodiment above.

[0057] The elution buffer is added in a pH gradient from about pH 6-5.8 to about pH 2.5-2.8. For example, when a succinate buffer is used, the pH of the succinate equilibration buffer is preferably about 5.8-6.0, and the pH of the succinate elution buffer is about 5-3, or about 5-2.8. For example, when a citrate buffer is used, the pH of the citrate equilibration buffer is preferably about 5.5, and the pH of the citrate elution buffer is about 4.5-2.5, or about 4.5-2.8. For example, when a propionic acid buffer is used, the pH of the propionic acid equilibration buffer is preferably about 5.8-6.0, and the pH of the propionic acid elution buffer is about 5-3, or about 5-3.5.

[0058] As can be seen in Figure 3, both elution conditions using succinate and citrate buffers provide clear separation of the bispecific antibody homodimer and heterodimer on the Protein L separation matrix. Peak 1 at the highest elution pH may correspond to an undesired species that binds very easily to the separation matrix, such as nonspecific binding. Peak 2 at the second highest elution pH corresponds to a heterodimeric bsAb with one kappa VL chain and one lambda VL chain, which is the bsAb targeted for purification and separation. This was confirmed by LC-MS data (not shown). Peak 3 corresponds to a homodimeric species with two kappa VL chains, also confirmed by LC-MS data (not shown). As in the first embodiment, the elution pH using succinate buffer is higher than that using citrate buffer. However, both types of buffer allow for an elution pH of 3.6 or higher, or even approximately 4 for succinate, and therefore both provide good separation at favorable pHs.

[0059] Furthermore, Figures 6A and 6B clearly show that the use of a propionic acid buffer, as before, provides a clear separation of bispecific antibody homodimers and heterodimers on a Protein L separation matrix. The separation is particularly clear during the step elution in Figure 6B.

[0060] According to a third aspect, there is provided herein a method for obtaining an elution pH above 4 from Protein L or a Protein L-derived chromatographic separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by lowering the elution buffer concentration below 50 mM, preferably between 10 and 50 mM. The composition of the elution buffer, as well as the pH and more specific concentration of the elution buffer, are as disclosed for the previous two aspects.

[0061] Thus, the disclosed method allows for elution of antibodies or antibody fragments from a Protein L separation matrix at a higher pH than commonly used. This may 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 denaturing the antibody or antibody fragment. Furthermore, acidic pH can contribute to aggregation, which is mitigated by the disclosed method.

[0062] 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 settings or conditions set forth in those examples, but are included merely to illustrate the advantages of the present invention. The scope of the present invention is defined by the appended claims, and any embodiment that falls within those definitions forms part of the present invention, even if not explicitly disclosed. [Example]

[0063] material Sodium dihydrogen phosphate and disodium 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)aminomethane were purchased from Merck (Darmstadt, Germany). Formic acid 98-100% EMSURE® from Merck and acetonitrile LC / MS grade (OPTIMA®) were purchased from Fisher Scientific. Propionic acid was purchased from Fluka Chemical, and sodium propionate was purchased from Sigma-Aldrich.

[0064] device An AKTA Pure 25 system (Cytiva™, Uppsala, Sweden) controlled by Unicorn 7.7 was used for all chromatographic runs. An AKTA Explorer 10 XT (Cytiva, Uppsala, Sweden) was used for SEC analysis. A BioAccord LC-MS instrument (Waters), an Acquity I-Class UPLC system coupled with an Acquity RDa detector.

[0065] method Protein L chromatography MabSelect VL (Protein L affinity resin, Cytiva™, Uppsala, Sweden) was packed into a 0.5 cm diameter column with a 10 cm bed height. The column volume (CV) was approximately 2 mL. Detailed information about MabSelect VL chromatography is summarized in Table 1. The column load was either purified mAb, purified cell culture harvest of mAb, or purified cell culture harvest of bispecific Ab (bsAb). The mAb used in this experiment was trastuzumab. The bsAb used is a bsAb containing the kappa class 1 light chain from trastuzumab, the lambda class 2 light chain from avelumab, and the trastuzumab heavy chain, available from ThermoFisher. For mAb purification runs, all runs were loaded with 10 mg of mAb per mL of resin, and for bsAb purification runs, all runs were loaded with 2.5 mg of bsAb per mL of resin. The column was loaded and run in bind-elute mode. The target protein was eluted with elution buffer over 20 CV with a gradient of 0-100%. For all chromatography runs, the system was run at a flow rate corresponding to a 6-minute residence time. All chromatograms were recorded by monitoring UV absorbance at 280 nm. Elution from selected runs was collected in fractions and analyzed by SEC for monomer purity and / or identity using LC-MS.

[0066] [Table 1]

[0067] [Table 2]

[0068] Size exclusion chromatography SEC analysis was performed using a Superdex 200 Increase column (10 x 300 mm, Cytiva™). 50 μl of sample was injected per run. The mobile phase consisted of 200 mM phosphate at pH 6.8. Sample elution was isocratic for 30 min at a flow rate of 0.8 mL / min. UV absorbance at 280 nm was recorded for all chromatograms.

[0069] Liquid chromatography-mass spectrometry (data not shown) LC-MS analysis was performed using a BioResolve RP mAb polyphenyl column (450 Å, 2.7 μm, 2.1 × 50 mm, Waters). Samples diluted to 0.1 g / L were reduced with DTT and digested with FabRICATOR® (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 mAbs or bsAbs were eluted in a gradient using a mobile phase containing 0.1% formic acid in acetonitrile at a flow rate of 0.5 mL / min over a total of 15 minutes. The column was maintained at 60°C throughout the analysis.

[0070] result Example 1 Purification of mAb using citrate buffer and Protein L separation matrix In this example, Protein L chromatography as described above was performed using experimental settings 3, 4, and 5 in Table 2. The resulting chromatograms are shown in Figure 1. It is clear that the lower the concentration of both Buffer A and B, and therefore the lower the ionic strength, as shown in Table 3, the higher the elution pH that can be achieved.

[0071] [Table 3]

[0072] Example 2 Purification of mAb using succinate buffer and protein L separation matrix In this example, Protein L chromatography as described above was performed using experimental settings 1 and 2 in Table 2. The resulting chromatogram is shown in Figure 2. It is clear that a higher elution pH can be achieved with lower concentrations of Buffer A and lower ionic strength, as shown in Table 4, using the indicated concentrations of Buffer B.

[0073] [Table 4]

[0074] In this case, it can be seen in FIG. 2 that the elution pH is significantly increased by about 1 pH unit compared to the citrate buffer in FIG.

[0075] Example 3 SEC analysis Fractions were collected from the above chromatography run as shown in Figure 4A. The results of the SEC analysis are summarized in Table 5. An exemplary SEC curve from the 10 mM / 50 mM succinate run is shown in Figure 4B.

[0076] [Table 5]

[0077] For the succinate elution experiments, it is clear that the first fraction from the main peak in the chromatogram, with an elution pH of 4.5, contains 100% or nearly 100% of the monomeric mAb to be purified. When eluted with citrate buffer, it is clear that the lowest concentration of citrate buffer, 10 mM / 10 mM, yields 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 does not show as good a separation of the monomeric mAb from other molecules present in the feed.

[0078] In the 10 mM / 50 mM succinate and 20 mM / 50 mM succinate cases, the second fraction still contains a significant amount of target monomer, but also contains a small amount of low molecular weight (LMW) species. Without being bound by any theory, this LMW is believed to contain a single light chain. In addition, the second fraction also contains high molecular weight (HMW) species. Without being bound by any theory, this is believed to contain aggregates. The amount of LMW and HMW then increases in the third and fourth fractions.

[0079] For 20 mM / 20 mM citrate and 50 mM / 50 mM citrate, the first fraction from the main peak also contains LMW, and the LMW increases with the following two fractions.

[0080] Again, this indicates that the low succinate buffer concentrations specified above, and even the lowest citrate buffer concentrations, result in low ionic strength and provide good separation of monomeric mAb from other species, e.g., LMW and HMW, contained in the feed.

[0081] Example 4 Separation of bispecific antibodies on Protein L separation matrix 4A. Comparison of 10 mM citrate buffer with 10 mM succinate buffer In this example, the bsAb defined above was run using experimental settings 1 and 5 in Table 2 and on a Protein L separation matrix as described above. The chromatogram (Figure 3) shows three peaks in the pH gradient. In 10 mM / 50 mM succinate buffer, the three peaks elute at pH 4.8, 4.1, and 3.8, respectively. In 10 mM / 10 mM citrate buffer, the three peaks elute at pH 4.8, 3.9, and 3.6, respectively.

[0082] LC-MS analysis (data not shown) indicates that the second peak contains a heterodimeric bispecific antibody containing one kappa VL chain and one lambda VL chain. The third peak contains a monomeric Ab containing two kappa VL chains (homodimer) (data not shown). The first peak contains species or antibody variants that bind nonspecifically and / or weakly to the separation matrix and contains a monomeric Ab containing two lambda VL chains (homodimer).

[0083] 4B. Separation using 15mM succinate buffer In this example, the bsAb defined above was run on a Protein L separation matrix using experimental setup 7 in Table 2 and as described above. Elution was performed using a pH gradient from 6 to 3.4 (Figure 5A) or step elution (Figure 5B). In step elution, pH 4.8 was used to elute lambda homodimers, pH 4.3 was used to elute lambda-kappa heterodimers, and pH 3.4 was used to elute kappa homodimers.

[0084] [Table 6]

[0085] As can be seen in Figures 5A and 5B, a clear separation of the heterodimer from the two homodimers is achieved.

[0086] 4C. Separation using 50 mM propionic acid buffer In this example, the bsAbs defined above were run on a Protein L separation matrix using experimental setup 6 in Table 2 and as described above. Elution was performed in a gradient (FIG. 6A) or step elution (FIG. 6B). In the step elution, pH 4.8 was used to elute lambda homodimers, pH 4.4 was used to elute lambda-kappa heterodimers, and pH 3.5 was used to elute kappa homodimers.

[0087] [Table 7]

[0088] As can be seen in Figures 6A and 6B, a clear separation of the heterodimer from the two homodimers is achieved.

[0089] Example 5 mAb Purification: Comparison of 15 mM Succinate, 50 mM Propionate, and 20 mM Citrate In this example, Protein L chromatography as described above was performed using experimental settings 4, 6, and 7 in Table 2. The results of the experiments are summarized in Table 8 below, and the resulting chromatograms are shown in Figure 7A (15 mM succinate), Figure 7B (50 mM propionate), and Figure 7C (20 mM citrate).

[0090] [Table 8]

[0091] Thus, the use of lower concentrations of elution buffer than commonly used has been shown to increase the elution pH that can be achieved. Furthermore, separation of heterodimeric antibodies from homodimeric antibodies can be achieved while still achieving an increased elution pH.

[0092] Furthermore, succinate and propionate buffers have been shown to be advantageous alternatives to the commonly used citrate buffer. In particular, low succinate concentrations and lower than commonly used citrate concentrations have been shown to be advantageous in achieving higher elution pH on Protein L separation matrices. Without being bound by any theory, the lower ionic strength of the buffer is believed to be the reason for the higher elution pH. Therefore, buffers composed of monocarboxylic or dicarboxylic acids may be advantageous compared to tricarboxylic acids because the ionic strength at a given pH is lower for monocarboxylic and dicarboxylic acids. This is due to the fact that fewer sodium ions (from NaOH titration) are required to achieve the same pH.

Claims

1. The following steps: contacting the liquid sample with an affinity separation matrix, thereby adsorbing at least one antibody or antibody fragment onto 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 using an elution buffer; 1. A method for purifying an antibody or antibody fragment comprising: the affinity separation matrix comprises a ligand based on Protein L, or any variant thereof, which binds to the κ light chain of the antibody or antibody fragment, and the elution buffer has a concentration of 5 to 50 mM; method.

2. The following steps: contacting a liquid sample with an affinity separation matrix, thereby adsorbing a feed comprising at least the bispecific antibody onto 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; 1. A method for isolating a bispecific antibody comprising: the affinity separation matrix comprises a ligand based on Protein L, or any variant thereof, that binds to the κ light chain of the bispecific antibody, and the elution buffer has a concentration of 5 to 50 mM. method.

3. 1. A method for obtaining an elution pH of greater than 4 from Protein L or a Protein L-derived chromatographic separation matrix using a carboxylic acid-based elution buffer, wherein the elution pH is obtained by reducing the elution buffer concentration below 50 mM, preferably between 10 and 50 mM.

4. 4. The method of claim 1, wherein the elution buffer comprises a buffered dicarboxylic acid.

5. 5. The method of claim 4, wherein the elution buffer is a succinate buffer.

6. 6. The method of claim 5, wherein the succinate buffer has a concentration of 10 to 50 mM.

7. 7. The method according to claim 5 or 6, wherein the succinate buffer has a concentration of 10 to 20 mM, preferably 15 mM.

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

9. 4. The method of claim 1, wherein the elution buffer comprises a buffered tricarboxylic acid.

10. 10. The method of claim 9, wherein the elution buffer is a citrate buffer.

11. 11. The method of claim 9, wherein the citrate buffer has a concentration of 5 to 45 mM.

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

5.

13. 4. The method of claim 1, wherein the elution buffer comprises a buffered monocarboxylic acid.

14. 14. The method of claim 13, wherein the elution buffer is a propionic acid buffer.

15. 15. The method of claim 14, wherein the propionic acid buffer has a concentration of 50 mM.

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

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