Preparation of a composition containing two or more antibodies

Abstract

The present invention relates to means and a method for generating at least two antibodies. The method comprises the steps of: providing a nucleic acid encoding antibodies to a cell; culturing the cell; collecting antibodies from the culture; and The method includes the step of separating the generated antibody from half the antibody by ion exchange chromatography (IEX). In some embodiments, the antibody exhibits an IEX residence time that deviates by 10% or less from the average residence time of the individual antibodies under the IEX conditions used. The invention also relates to a composition of the antibody generated accordingly. In some embodiments, the invention relates to a composition comprising 2 to 10 recombinant antibodies, characterized in that at least two of the antibodies have an IEX residence time that deviates by 10% or less from the average residence time of the individual antibodies under the IEX conditions. The invention also relates to a composition comprising 2 to 10 recombinant antibodies, characterized in that at least two of the antibodies have a pI that differs by 0.4 units or less from the average pI of the at least two antibodies.

Description

Technology Field

[0001] The present invention relates to the field of antibodies, particularly to the field of therapeutic antibodies. Antibodies can be used for the treatment of humans. More specifically, the present invention relates to the generation and / or purification of a number of antibodies. A single host cell may generate a number of antibodies. Antibodies may also be generated by a mixture of host cells, each generating one of the antibodies. The present invention also relates to a method for generating a composition comprising these antibodies and purifying these antibodies.

[0002] Polyclonal antibodies are typically collected from the blood of subjects. The advantage of polyclonal antibodies is that pathogens are attacked through multiple targets and epitopes. The advantage of monoclonal or recombinant antibodies is their well-characterized specificity and function, which allows these antibodies to be used as precision agents with well-defined spectrums of action and toxicity.

[0003] The specificity of monoclonal antibodies can also be a disadvantage, particularly when multiple targets need to be addressed. While it is possible to mitigate this drawback by adding more antibodies to the drug, considering that even a single therapeutic antibody can be expensive, it is thought that the cost of such a polyclonal cocktail could quickly become high.

[0004] The development of bispecific, trispecific, and other multispecific antibodies has successfully introduced some forms of polyclonal antibodies into antibody therapeutics. In addition to an increase in the number of targets, this has also successfully introduced additional functions that could not previously be achieved with traditional monospecific mono- or polyclonal antibodies. Using multiple bispecific, trispecific, and other multispecific antibodies in the same treatment can provide further benefits.

[0005] The present invention provides an advancement in the art by describing a robust and economical method for purifying multiple antibody therapeutic agents produced from a single host cell or, alternatively, a mixture of host cells. The present invention is particularly useful for the economical production of a collection of two or more antibodies, preferably multispecific antibodies.

[0006] The present invention provides a method for generating at least two multispecific antibodies, wherein the method

[0007] - A step of providing a nucleic acid encoding a multispecific antibody to a cell;

[0008] - Step of culturing the above cells;

[0009] - A step of collecting multispecific antibodies from a culture; and

[0010] - The method comprises the step of separating the generated multispecific antibody from the half-antibody and optionally from the monospecific antibody and / or other unwanted antibody product-related impurities by ion exchange chromatography (IEX), and

[0011] The present method is characterized by exhibiting an IEX retention time that deviates by 10% or less from the average retention time of individual multispecific antibodies under IEX conditions in which multispecific antibodies are used. The retention times of each half antibody and optionally the monospecific antibody and / or other unwanted antibody product-related impurities are preferably outside the range over which the retention times of the multispecific antibodies span.

[0012] The present invention also provides a method for generating at least two multispecific antibodies, wherein the method

[0013] - A step of providing a nucleic acid encoding a multispecific antibody to a cell;

[0014] - Step of culturing the above cells;

[0015] - A step of collecting multispecific antibodies from a culture; and

[0016] - The method comprises the step of separating the generated multispecific antibody from the half antibody and optionally from the monospecific antibody and / or other unwanted antibody product-related impurities by ion exchange chromatography (IEX), and

[0017] The present method is characterized by exhibiting essentially the same IEX retention time under IEX conditions using a multispecific antibody. The retention times of each half antibody and optionally a monospecific antibody and / or other unwanted antibody product-related impurities are preferably outside the range over which the antibody retention time spans.

[0018] The present invention further provides a method for generating at least two antibodies, wherein the antibodies comprise monospecific and / or multispecific antibodies, and the method

[0019] - Step of providing nucleic acid encoding antibodies to cells;

[0020] - Step of culturing the above cells;

[0021] - A step of collecting antibodies from the culture; and

[0022] - Includes the step of separating the generated antibody from half the antibody by ion exchange chromatography (IEX), and

[0023] The present method is characterized by exhibiting essentially the same IEX retention time under IEX conditions in which the antibody is used. The retention times of each half antibody and optionally monospecific antibody are preferably outside the range over which the antibody retention times span.

[0024] The present invention also provides a composition comprising 2 to 10 recombinant antibodies that can be obtained by the method described herein.

[0025] A composition comprising 2 to 10 recombinant antibodies, such as a multispecific antibody, is also provided, characterized in that at least two of the antibodies have an IEX residence time that deviates by 10% or less from the average residence time of the individual antibodies under the IEX conditions used.

[0026] A composition comprising 2 to 10 recombinant antibodies is further provided, characterized in that at least two of the antibodies have essentially the same IEX residence time.

[0027] A composition comprising 2 to 10 recombinant antibodies is also provided, wherein at least two of the antibodies have isoelectric points (pI) that are preferably different from the average pI of the at least two antibodies by 0.4 units, 0.3, 0.2, and preferably 0.1 units or less. The pI of each of the at least two antibodies is preferably different from each other by 0.25 units or less. Specific details for implementing the invention

[0028] As used herein, the term “antibody” means a proteinaceous molecule of the class of immunoglobulins that contains one or more domains that bind to an epitope on an antigen, wherein such domains are variable regions of the antibody, are derived therefrom, or share sequence homology with them. Antibodies are typically composed of basic structural units, each having two heavy chains and two light chains. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the target to be treated (e.g., human antibodies in the case of human subjects). Antibodies having extended heavy chain and / or light chain variable regions are also incorporated herein. Antibodies according to the present invention are not limited to any particular format or method of production thereof.

[0029] Half antibodies are heavy and light chain combinations that do not associate with other heavy and light chain combinations and do not form an interface with another variable region or variable region-like polypeptide. Other unwanted antibody product-related impurities may be free light chains not associated with a heavy chain, free heavy chains not associated with a light chain or another heavy chain, or incompletely assembled antibodies lacking a light chain.

[0030] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells or PER-C6 cells, or various other cell lines known to those skilled in the art. Various organizations and companies have developed cell lines for the large-scale production of antibodies, for example, for clinical use. Non-limiting examples of such cell lines are particularly CHO cells, NS0 cells or PER-C6 cells or HEK293 cells. In certain preferred embodiments, the cell is a human cell. Preferably, it is a cell transformed by the adenovirus E1 region or its functional equivalent. In preferred embodiments, the cell is a CHO cell or a variant thereof. Preferably, it is a variant using a glutamine synthetase (GS) vector system for antibody expression. In one preferred embodiment, the cell is a CHO cell.

[0031] Cells may be provided with nucleic acids encoding antibodies. Cells will express the formed antibodies, assemble them, and excrete them into the supernatant of the cell culture. The introduction of a single heavy chain and a light chain (or rather, nucleic acids encoding them) leads to the production of a monoclonal antibody having two heavy chains each associated with a light chain.

[0032] The method of the present invention may be carried out with a mixture of cells comprising two or more types of cells that each generate different antibodies. The advantage of using such a mixture is that downstream processing of the collected antibodies can be simplified more efficiently. An additional advantage of the method is that the antibody products are collected and purified one by one. Furthermore, the mixture of two or more antibodies produced by the method of the present invention can reduce the number of tests required to obtain regulatory approval compared to the number of tests required individually for each antibody.

[0033] In a further embodiment, the cell is a homogeneous aggregate of cells essentially composed of replicas of a single cell provided with the nucleic acid encoding each antibody. The co-expression of several heavy chains in the cell enables various combinations of heavy chains. Combination with additional light chains increases the number of combinations. Various methods have been developed to favor the formation of specific combinations compared to other methods. Heavy chain variants have been generated that specifically promote the formation of heavy chain heterodimers to homodimers, or conversely, the formation of heavy chain homodimers to heterodimers. Heavy chains having specific homodimerization domains reduce the number of antibodies produced by these cells and / or increase the level of desirable antibodies compared to alternative combinations (e.g., higher production of heterodimers compared to homodimers).

[0034] The method of the present invention is particularly suitable for generating two or more multispecific antibodies, including bispecific antibodies. Various methods for generating bispecific antibodies exist in the art. One approach uses a common light chain capable of forming a functionally variable domain having different heavy chains. One preferred method for generating bispecific antibodies involves using transgenic animals possessing a common chain in their genome, and immunizing these animals with an antigen generates a repertoire of antibodies specific to said antigen based on a non-common chain, wherein the repertoire consists of various antibodies comprising said common chain and a rearranged homologous chain. One animal may be immunized with different antigens, or different animals may be immunized individually with their respective antigens. Nucleic acids encoding the non-common chain or its variable region may be obtained from animal(s), e.g., B-cells, spleen, or lymphoid tissue. These may be used to generate nucleic acids capable of expressing each non-common chain, which can be introduced into the generating cells. The common chain may be introduced at the same or different times. The nucleic acid may be incorporated into the nucleus, and preferably into the genome of a host cell, so as to enable the host cell to produce a multispecific antibody or multimer that targets a number of antigens to which the transgenic animal(s) have been immunized (e.g., see WO2009 / 157771 incorporated herein by reference for the purpose of creating a variable region that can be combined with a common chain to create a functional variable domain specific to different targets and / or different epitopes).

[0035] Cells that produce a common light chain and two different heavy chains capable of forming a functional variable domain together with the common light chain produce bispecific antibodies having two different heavy and light chain combinations. Similarly, cells that produce a common light chain and three or more different heavy chains can produce multispecific antibodies capable of targeting three or more antigens, or combinations of two or more multispecific antibodies capable of targeting three or more antigens. Currently, a standard format of antibody (i.e., one constant portion and two variable domains) can be constructed and additional binding domains can be added. In this way, multispecific antibodies having one or more single-strand Fvs with additional binding specificity attached to the constant or one or more variable domains of the antibody can be produced. It is also possible to produce heavy chains having two or more variable regions. The additional heavy chain regions can advantageously be associated with different or common light chain variable regions. Refer to US 62 / 650467 incorporated herein by reference.

[0036] When cells produce two or more multispecific antibodies, this may also, in some cases, produce a certain amount of half antibody and an antibody having the same heavy chain or homodimer. The latter amount may be reduced by including modifications that promote heterodimer formation in the heavy chain. As indicated above, various methods exist for inducing heterodimerization of the heavy chain. Each domain having a modification is collectively referred to as a heterodimerization domain. A heavy chain having a heterodimerization domain favorable for interaction is referred to as having a compatible heterodimerization domain. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods for achieving such CH3 heterodimerization of the heavy chain have been described in the art.

[0037] One preferred method for generating a bispecific antibody is disclosed in US No. 9,248,181 and US No. 9,358,286. Specifically, a preferred modification for essentially generating only a bispecific full-length IgG molecule is an amino acid substitution L351K and T366K (EU numbering) ('KK-variant' heavy chain) in the first CH3 domain and an amino acid substitution L351D and L368E ('DE-variant' heavy chain) in the second domain, or vice versa. As previously described, the DE-variant and the KK-variant pair up to preferentially form a heterodimer (so-called 'DEKK' bispecific molecule). Homomerization of DE-mutated heavy chains (DEDE homomerization) or KK-mutated heavy chains (KKKK homomerization) rarely occurs due to reasons including strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0038] In the present invention, it is preferable that the cell be provided with a nucleic acid encoding a common light chain. Various methods for generating antibodies having different heavy chain variable regions and the same light chain variable regions are available to those skilled in the art. WO2004 / 106375 describes a phage library using a common light chain variable region. Phage selection yields variable domains having the same light chain variable region and different heavy chain variable regions. Additionally, non-human animals having a common chain and different homologous chains are described in WO2009 / 157771. Antibody selection in these animals yields variable domains having the same or similar common chain variable region and different homologous chain variable regions. WO2004 / 106375 and WO2009 / 157771 are incorporated herein by reference. The publications specifically mention the generation of antibodies having the same or similar common chain variable region and different homologous chain variable regions, preferably a common light chain variable region and a different heavy chain variable region, and nucleic acids encoding such antibodies.

[0039] In a preferred embodiment, the cell produces two or more heavy chains and one common light chain. Each heavy chain and light chain may have one or more variable regions connected to each chain. In a preferred embodiment, the cell produces three or more heavy chains. One of the three heavy chains preferably contains a portion of a compatible heterodimerization domain. Two or more other heavy chains preferably contain different portions of a compatible heterodimerization domain. When the first heavy chain is designated by the letter "A" and the two other heavy chains are designated by the symbols "B" and "C", the specific combination of heterodimerization domains leads primarily to the formation of combinations AB and AC. Combinations AA, BB, CC, and BC are disadvantageous due to the inclusion of heterodimerization domains. Such cells are effective in producing only two bispecific antibodies AB and AC (see FIG. 1). In the present invention, it is preferable for the cell to produce two antibodies by producing at least three heavy chains. In a preferred embodiment, one of the heavy chains comprises a portion of a compatible heterodimerization domain, and the two or more other heavy chains preferably comprise different portions of a compatible heterodimerization domain. A heavy chain shared by two or more bispecific or multispecific antibodies or multimers in the composition preferably has a CH3 DE portion of the heterodimerization domain. Another heavy chain in the bispecific or multispecific antibody preferably has a CH3 KK portion.

[0040] At least two antibodies are preferably multispecific antibodies, preferably bispecific antibodies. In a preferred embodiment, at least two of the antibodies share the same heavy chain. Cells can produce several series of bispecific antibodies by including different heterodimerization domains in the heavy chain. This implementation may lead to the predominant production of bispecific antibodies having heavy chain combinations AB and CD. Combination AB may be preferred by a DE / KK heterodimerization domain as described above in this specification, and CD may be preferred by a "knob in hole" heterodimerization domain, for example through charge engineering, or by the incorporation of other heterodimerization features known in the art. A shared heavy chain between different bispecific or multispecific multimer or antibody combinations with a shared heavy chain can be produced by providing a portion of the heterodimerization domain to the shared heavy chain and a complementary portion of the heterodimerization domain to the various combination chains. For example, the CH3 DE portion in the covalent heavy chain and the CH3 KK portion in the combination chain. One covalent heavy chain and two combination heavy chains will, in this environment, cause the production of antibodies with heavy chain combinations AB and AC (or AxBC and AxDE in the case of multispecific multimers) by double- or multispecific multimers or cells. Other possible combinations are AB, AE, CD, and CF; or AB, AE, AG, and CD, etc.

[0041] Antibodies generally have an isoelectric point (typically in the pH range of 6 to 10) that is distinct from other host cell proteins. Antibodies, e.g., multispecific antibodies and multispecific multimers, can be purified to a relatively high purity through the methods disclosed herein. The method may include several steps, such as culturing host cells, undergoing harvest clarification, and subsequently capturing proteins. IEX chromatography, such as anion exchange chromatography, may be used to remove host cell DNA, and cation exchange chromatography (CIEX), e.g., may be used to remove host cell proteins, leached protein A, and potential aggregates. Additional steps, such as viral filtration or hydrophobic interaction chromatography, may be included.

[0042] Antibodies are typically excreted by the producing cells. The harvesting of these antibodies typically involves the collection of the cell supernatant, followed by several purifications to remove unwanted cell debris or aggregates. Harvest clarification may include filtration, centrifugation, or a combination thereof of the culture supernatant of the antibody-producing cells. Antibody protein capture is typically performed by affinity purification. This can be carried out in several ways. This often involves purification using columns containing recombinant protein A, protein G, or protein L, which are bacterial proteins with known high specific binding ability to antibodies. Currently, various optimized mutations that bind more specifically to antibodies are available. For example, recombinant protein A with a non-essential domain removed is available, recombinant protein G with a deleted albumin binding site is available, and modified protein L is also available. The bound antibodies can be collected by elution on one or more of these columns. Anion- and cation-exchange chromatography may be used to further purify the formulation, for example, to purify bispecific or multispecific antibodies from half antibodies and / or homomeric antibodies and / or other unwanted antibody product-related impurities (if present). Hydrophobic interaction chromatography (HIC) is commonly used as an alternative polishing step in the antibody purification process. HIC provides orthogonal selectivity for ion exchange chromatography and can be an effective step for aggregate removal and host cell protein reduction. In the present invention, HIC may be used for analytical purposes to quantify two or more species having similar, preferably essentially identical, retention times and / or similar, preferably essentially identical pIs in IEX after the purification of two or more bispecific or multispecific antibodies or multimeric antibodies is completed. Thus, HIC is used to quantify the relative amounts of purified molecules.

[0043] A hydrophobic interaction resin is selected as the stationary phase, and the pH and / or conductivity of the mobile phase are adjusted to achieve the required selectivity. Antibodies typically attract a positive charge at lower pH, which affects polarity and overall surface hydrophobicity. pH conditions that allow for the separation of two or more antibodies in the formulation may be selected.

[0044] In some embodiments, the collected antibody is first separated from other proteins by affinity purification, preferably by protein A extraction. Subsequently, the affinity-purified antibody is run on an anion exchange column under conditions for collecting antibodies from the distribution fraction. Subsequently, the antibody may be run on one or more CIEX columns.

[0045] A preferred method for generating at least two antibodies is carried out by generating them by a cell as a single composition comprising two or more antibodies.

[0046] A method for generating at least two antibodies preferably comprises the steps of culturing host cells that generate two or more antibodies, collecting the supernatant of the cells, and preferably processing the supernatant harvest in a harvest clarification process comprising filtration using a pore size cut-off that captures aggregates such as cells or cell residues and allows the passage of antibody products. The antibodies are collected from the cell culture medium by affinity chromatography using protein A. The antibodies bound to the protein resin are eluted from the chromatography column after exposure to a low pH, and the eluent is subsequently neutralized using a suitable buffer.

[0047] As used herein, the term “isoelectric point (pI)” refers to the pH at which the average net charge of the protein surface, that is, the potential of the protein’s electric double layer, is zero. In other words, this term refers to the point at which the protein group dissociates so that the number of cations and anions is equal, and the net charge of the protein becomes zero.

[0048] As used herein, “pI” is calculated based on primary amino acids according to the ExPASy, ProtParam tool using default parameters of the earliest filing date (priority date) of this application. ProtParam is a tool that enables the calculation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL, or for a user-input protein sequence. The calculated parameters include theoretical pIs. Reference [Gasteiger E., Hoogland C., Gattiker A., ​​Duvaud S., Wilkins MR, Appel RD, Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005) pp. 571-607].

[0049] The net surface charge of a protein changes with pH in a manner indicated by the protein's pI. At a pH equal to the protein's pI, the protein will not have a net charge. At a pH below the pI, the protein will have a net positive charge. If the buffer pH rises above the protein's pI, it will have a net negative charge.

[0050] The pI of a protein can be determined by its primary amino acid sequence and calculated accordingly, and then a buffer that ensures a known net charge for the protein of interest can be selected. Thus, when the protein of interest has a net positive charge at the operating pH, a negatively charged cation exchange resin is selected.

[0051] Proteins having different pI values ​​will have varying degrees of charge at a given pH and thereby have different affinities for positively charged surface groups on the particles of the anion exchange medium; thus, different proteins will bind to the resin with different strengths, facilitating its separation. Thus, by generating heterodimeric polypeptides having distinct pIs for homodimeric and half-antibody contaminants and / or other antibody product-related impurities, the heterodimeric polypeptides can be easily purified using standard elution techniques, for example by applying a pH gradient, or by applying a salt or conductivity gradient or a combination of pH and conductivity gradients at a fixed pH. In embodiments of the present invention, the constant portion and light chain within the antibody essentially have the same sequence in different antibodies. These antibodies typically differ essentially only in the amino acid sequence of the heavy chain variable region. For these antibodies, this is typically sufficient to calculate the pI of the heavy chain variable region as indicated herein. Subsequently, calculations and standards are established by using the pI of the heavy chain variable region instead of the pI of the (half)-antibody. The average pI of the variable region of the antibody's heavy chain represents the retention time of the antibody in the CIEX column. The antibody may have the same or different heterodimerization domains, where present. It preferably has the same heterodimerization domain. Additionally, the antibody may differ slightly in the exact amino acid sequence in the constant region.

[0052] In the case of ion-exchange chromatography (IEX) step(s), this is a process that separates ions and polar molecules based on their affinity for ion exchangers. This applies to various charged molecules, including large proteins, small nucleotides, and amino acids. IEX is often used for protein purification. Soluble and charged proteins form ionic bonds with an insoluble stationary phase. Subsequently, the bonded molecules can be eluted and collected using an eluent having a higher ion concentration and / or a different pH. The salt concentration or pH can be changed in a stepwise manner; by progressively changing the mobile phase of the chromatography run; or by a combination thereof. Two types of ion chromatography are anion-exchange and cation-exchange. Cation-exchange chromatography (CIEX) is preferred in the present invention. Antibody CIEX is preferably performed at a physiological pH. The pH typically ranges from 5 to 9, preferably from 6 to 8.

[0053] CIEX is typically used when the molecule of interest is positively charged under the pH used for chromatography. The molecule is positively charged because the pH for chromatography is lower than the molecule's pI. In this type of chromatography, the stationary phase is negatively charged and loaded so that positively charged molecules are attracted to it. In anion-exchange chromatography, the stationary phase is positively charged and loaded so that negatively charged molecules (meaning the pH for chromatography is higher than the pI) are attracted to it.

[0054] Antibodies, such as multispecific antibodies, are typically bound to the IEX column during the binding phase under conditions that promote binding to the substrate. The IEX column is typically subsequently washed to remove unbound material. Elution from the column is performed during the elution phase. The retention time of the antibody, such as a multispecific antibody, is typically calculated from the start of the elution phase. It is the time the antibody spends on the column when the elution phase begins. If the sample contains several compounds, each compound within the sample will typically spend a different amount of time on the column depending on its chemical composition; that is, each will have a different retention time. Retention times are typically expressed in seconds or minutes.

[0055] The retention times of antibodies, such as multispecific antibodies, are preferably essentially the same in the method of the present invention. Different antibodies may have different retention times under the same column and conditions. In the present invention, it has been found that antibodies, such as multispecific antibodies, may be selected or designed to have IEX retention times that are sufficiently close to allow for the simultaneous purification of two or more antibodies, such as two or more multispecific antibodies, in a single IEX chromatography run. A retention time that deviates by 10% or less from the average of the retention times of individual antibodies is typically sufficiently close to allow for the simultaneous purification of two or more antibodies, such as two or more multispecific antibodies, in a single IEX chromatography run.

[0056] The CIEX HPLC method suitable for antibody purification and / or analysis according to the present invention uses an ion exchange column of the TSKgel SP-STAT series (7 μm particle size, 4.6 mM ID × 10 cm L, Tosoh 21964). The column is packed with non-porous resin particles for rapid, high-resolution analysis as well as the isolation of biomolecules. While the particles within the TSKgel STAT column contain an open access network of multilayer ion-exchange groups for loading capacity, the particle size makes these columns suitable for HPLC and FPLC systems.

[0057] A suitable method involves equilibrating TSKgel SP-STAT (7 μm particle size, 4.6 mM ID × 10 cm L, Tosoh 21964) with buffer A (sodium phosphate buffer, 25 mM, pH 6.0)—subsequently, removing antibodies from the column by increasing the salt concentration and running a gradient of buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate is set to 0.5 mL / min. The injection sample mass for the test sample and control is 10 μg, and the injection volume is 10 to 100 μl. The chromatogram is analyzed for the peak pattern, retention time, and peak area for the observed main peak based on the 220 nm results. This method can be scaled for larger amounts of antibodies.

[0058] A typical graph of a CIEX chromatography run of an antibody preparation is shown in Fig. 2. The antibodies for this run were collected from transfected cells as shown in the examples and purified from many other proteins in the medium by a Protein A column. The antibodies were eluted by neutralization following acid elution and buffer exchange with PBS pH 7.4 as shown in the examples. Subsequently, samples of the antibody preparation were loaded onto a CIEX column. After washing, a salt gradient was applied to elute the associated proteins. CIEX conditions were the same for the samples in Figs. 2a and 2b. Retention times are calculated relative to the top of the peak of the bispecific antibody. The retention times of two or more antibodies, such as a multispecific antibody, preferably deviate by no more than 10% from the average of the retention times of the two or more antibodies. A deviation of more than 10% typically results in inefficient separation of the antibodies from half antibodies and, optionally, from homodimers and / or other antibody product-related impurities in the case of a multispecific antibody. In a preferred embodiment, the retention time of two or more antibodies deviates from the average retention time of the two antibodies by 9% or less. Preferably, it deviates from the average retention time of two or more antibodies by 8%, 7%, 6%, or 5% or less. In a preferred embodiment, the retention time of two or more antibodies deviates from the average retention time of two or more antibodies by 4% or less. Preferably 3% or less, preferably 2% or less. Increasingly similar retention times typically enable increasingly efficient separation of multispecific antibodies from half antibodies and optionally homodimers and / or other antibody product-related impurities, thereby enabling a cleaner collection of two antibodies from the fraction of the IEX column.

[0059] In the means and method of the present invention, the average retention time of two or more antibodies, such as two or more multispecific antibodies, is calculated for the antibodies to be co-purified or collected. Accordingly, in an embodiment where the antibody to be purified is a multispecific antibody, the average retention time of two or more antibodies is calculated based on the multispecific antibody. The retention time of antibodies not to be collected, such as homodimeric antibodies, is not used for the calculation of the average.

[0060] In the method of the present invention, an antibody such as a multispecific antibody may be selected for co-purification by selecting an antibody such as a multispecific antibody that has essentially the same IEX retention time under the conditions used for IEX. The antibody such as a multispecific antibody may also be fitted through appropriate modification of one or more variable regions to have essentially the same IEX retention time under the same or similar conditions used for IEX.

[0061] In one embodiment, the generated antibodies to be co-purified, such as dual- and / or multispecific antibodies, have similar pIs. At least two of the antibodies have isoelectric points (pIs) that are preferably different from the average pI of the at least two antibodies by 0.4 units, 0.3, 0.2, and preferably 0.1 units or less. The pIs of each of the at least two antibodies are preferably different from each other by 0.25 units or less.

[0062] If the difference in the pI of the antibodies is small or non-existent, it typically enables good co-purification. Advantageously, the pI of each half-antibody within the antibody differs more from the average. This difference facilitates good separation of the half-antibodies from the "co-transfer" complete antibody during the CIEX chromatography step.

[0063] In an embodiment where the antibody to be co-purified is a bispecific or multispecific antibody, it is preferable that the pI of the variable domain in each antibody to be co-purified differs from the average of the pIs of the variable domains of the other antibody(s) to be co-purified by more than 0.2, preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably 1.8 or 2.0 units. In this embodiment, the difference in pI of the variable domain within the antibody is preferably at least 0.2 units greater than the difference between "x" and "y", where "x" is the average of the pIs of two variable domains of the first antibody among the antibodies and "y" is the average of the pIs of two variable domains of the second antibody among the antibodies, preferably at least 0.3, 0.4, 0.5, preferably at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0, or 2.5 greater than the difference between "x" and "y". Such a difference in the pI of the variable domain within the antibody typically indicates good separation from the antibody to be co-purified of antibody product-related impurities and / or good separation from the single-specific antibody.

[0064] Some compositions comprising two or more bispecific or multispecific antibodies have invariant regions and light chains with similar or essentially identical amino acid sequences. These two or more bispecific and / or multispecific antibodies typically differ from one another only in the amino acid sequence of the essentially variable domain or only in the essentially heavy chain variable region. In such cases, it is often not necessary to determine the pI of the entire antibody. Instead, the pI of the variable domain and / or the pI of the heavy chain variable region may be determined. This provides a means to evaluate whether the antibodies can move close to each other during the CIEX chromatography step, that is, whether the antibodies have sufficiently identical retention times to allow for co-purification.

[0065] In one embodiment of the method or composition as disclosed herein, two or more bispecific or multispecific antibodies have a constant region and a light chain having the same amino acid sequence or essentially the same amino acid sequence. Two or more bispecific or multispecific antibodies may be co-purified in a CIEX chromatography step when the average pI of the variable domain within each antibody differs by 0.7 units or less from the average pI of the variable domain of each antibody to be co-purified. In a preferred embodiment, the average "x" of the pI of the two variable domains of the first antibody among the antibodies and the average "y" of the two variable domains of the second antibody among the antibodies differ by 0.6 units or less, preferably 0.5 units or less, from the average of "x" and "y" of the first and second antibodies to be co-purified. "x" and "y" are preferably 0.4 units from the average of "x" and "y" of the first and second antibodies to be co-purified; Preferably different by 0.3; preferably 0.2; and preferably 0.1 units or less. These dual- and / or multispecific antibodies typically have essentially the same retention time. In the present embodiment, the constant regions of the antibodies are essentially identical. The pI of these antibodies, and in particular the mean "x" and "y", represent the pI of each antibody as a whole. In the present embodiment, the pI of the variable domain within each antibody to be co-purified is preferably different from the mean of the pIs of the variable domains of the antibodies by more than 0.2, preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably 1.8 or 2.0 units. In these embodiments, the difference in pI of the variable domain within the antibody is preferably at least 0.2 units greater than the difference between "x" and "y", preferably at least 0.3, 0.4, 0.5, preferably at least 0.6, 0.0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0, or 2.5 are larger. Such differences in the pI of the variable domain within the antibody typically indicate good separation of the half antibody from the antibody to be co-purified and / or good separation from the single-specific antibody.

[0066] In one embodiment of the method or composition as disclosed herein, two or more bispecific or multispecific antibodies have constant regions and light chains having the same amino acid sequence or essentially the same amino acid sequence. Two or more bispecific or multispecific antibodies may be co-purified in a CIEX chromatography step when the average pI of the heavy chain variable region within each antibody differs by 0.7 units or less from the average pI of the heavy chain variable region of each antibody to be co-purified. In a preferred embodiment, the average "m" of the pI of the two heavy chain variable regions of the first antibody among the antibodies and the average "n" of the pI of the two heavy chain variable regions of the second antibody among the antibodies differ by 0.6 units or less, preferably 0.5 units or less, from the average of the "m" and "n" of the first and second antibodies to be co-purified. "m" and "n" are preferably 0.4 units from the average of the "m" and "n" of the first and second antibodies to be co-purified; Preferably different by 0.3; preferably 0.2; and preferably 0.1 units or less. These dual- and / or multispecific antibodies typically have essentially the same retention time. In the present embodiment, the constant regions of the antibodies are essentially identical. The pI of these antibodies, and in particular the mean "m" and "n", represent the pI of each antibody as a whole. In the present embodiment, the pI of the heavy chain variable region within each antibody to be co-purified is preferably different from the mean of the pIs of the heavy chain variable regions of the antibodies by more than 0.2, preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably 1.8 or 2.0 units. In this embodiment, the difference in pI of the heavy chain variable region within the antibody is preferably at least 0.2 units greater than the difference between "m" and "n", and preferably at least 0.3, 0.4, 0.0.5, preferably at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0, or 2.5 greater. Such a difference in the pI of the heavy chain variable region within the antibody typically indicates good separation of the half antibody from the antibody to be co-purified and / or good separation from the single-specific antibody.

[0067] Antibodies, such as multispecific antibodies, may have, or may be selected to have, heavy chain and light chain combinations (half antibodies) or homodimers (e.g., monospecific antibodies) or other antibody product-related impurities having a retention time significantly different from that of the whole antibody or the desired antibody, e.g., multispecific antibody, under the IEX conditions used. In embodiments where the cells express a common light chain, the choice is typically in the heavy chain. The heavy chain may be modified so that the half antibody or homodimer has very different retention times. In a preferred embodiment, the retention times of the half antibody and / or homodimer differ by more than 10% of the average retention times of each antibody or multispecific antibody. In a preferred embodiment, the average pI of the individual heavy chain and light chain combination of the antibody not to be co-purified differs by more than 0.5 units from the average pI of the heavy chains and light chains of at least two antibodies to be co-purified.

[0068] The present invention further provides a composition comprising 2 to 10 recombinant antibodies obtainable by the method described herein. A composition comprising 2 to 10 recombinant antibodies is also provided, characterized in that at least two of the antibodies have essentially the same IEX residence time.

[0069] The present invention further provides a composition comprising 2 to 10 recombinant antibodies, wherein at least two of the antibodies differ from the average pI of the at least two antibodies by 0.4 units, 0.3, 0.2, and preferably 0.1 units or less. The pI of each of the at least two antibodies preferably differs from each other by 0.25 units or less.

[0070] The present invention further provides a composition comprising 2 to 10 recombinant antibodies, characterized in that the average pI "x" of the two variable domains of the first antibody and the average pI "y" of the two variable domains of the second antibody differ from the average of "x" and "y" of the first and second antibodies to be co-purified by no more than 0.7, 0.6, and preferably 0.5 units. "x" and "y" differ from the average of "x" and "y" of the first and second antibodies to be co-purified by preferably 0.4; preferably 0.3; preferably 0.2; and preferably 0.1 units or less. Such antibodies, such as multispecific antibodies, typically have essentially the same retention time. In the present embodiment, the constant regions of the antibodies are essentially the same. The average pI of the different variable domains of the antibodies, including the pI, and in particular the heavy chain variable region and the light chain variable region, respectively, represents the pI of the antibody as a whole.

[0071] The present invention further provides a composition comprising 2 to 10 recombinant antibodies, wherein the average pI "m" of the two heavy chain variable regions of the two variable domains of the first antibody and the average pI "n" of the two heavy chain variable regions of the two variable domains of the second antibody differ from the average of the "m" and "n" of the first and second antibodies to be co-purified by no more than 0.7, 0.6, and preferably 0.5 units. The "m" and "n" differ from the average of the "m" and "n" of the first and second antibodies to be co-purified by preferably 0.4; preferably 0.3; preferably 0.2; and preferably 0.1 units or less. Such antibodies, such as multispecific antibodies, typically have essentially the same retention time. In the present embodiment, the constant region and the light chain variable region of the antibody are essentially the same. The pI, and in particular the average of the pIs of the different heavy chain variable regions of the antibody, represents the pI of the antibody as a whole.

[0072] In a preferred embodiment, the IEX retention time and / or pI are preferably essentially the same for all antibodies to be collected in the composition. In a preferred embodiment, at least two of the antibodies are bispecific antibodies. Preferably, at least two of the antibodies share the same heavy chain.

[0073] In some embodiments, the common light chain variable region of one or both of the VH / VL binding regions comprises the germline IgVκ1-39*01 variable region V-segment. In some embodiments, the light chain variable region of one or both of the VH / VL binding regions comprises the kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39. The external ID of the gene is HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence for the V-region is provided in SEQ ID NO. 25. The V-region may be combined with one of five J-regions. Preferred J-regions are jk1 and jk5, and the combined sequences are denoted as IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (nomenclature according to the IMGT database World Wide Web at imgt.org). In a given embodiment, the light chain variable region of one or both of the VH / VL binding regions comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (sequence number 26 and sequence number 27, respectively).

[0074] In some embodiments, the light chain variable regions of one or both of the VH / VL binding regions of the bispecific antibody comprise LCDR1 containing the amino acid sequence QSISSY (SEQ No. 22), LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence QQSYSTP (SEQ No. 24) (i.e., the CDR of IGKV1-39 according to IMGT). In some embodiments, the light chain variable regions of one or both of the VH / VL binding regions of the bispecific antibody comprise LCDR1 containing the amino acid sequence QSISSY (SEQ No. 22), LCDR2 containing the amino acid sequence AASLQS (SEQ No. 23), and LCDR3 containing the amino acid sequence QQSYSTP (SEQ No. 24).

[0075] In some embodiments, one or both of the VH / VL binding regions of the bispecific antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO. 26. In some embodiments, one or both of the VH / VL binding regions of the bispecific antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO. 27.

[0076] For example, in some embodiments, the variable light chain of one or both of the VH / VL binding regions of the bispecific antibody may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to SEQ ID NO. 26 or SEQ ID NO. 27. In some embodiments, the light chain variable region of one or both of the VH / VL binding regions of the bispecific antibody comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 or 1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence.

[0077] In another embodiment, the light chain variable region of one or both of the VH / VL binding regions of the bispecific antibody comprises the amino acid sequence of SEQ ID NO. 26 or SEQ ID NO. 27. In a given embodiment, both VH / VL binding regions of the bispecific antibody comprise the same VL region. In one embodiment, the VL of both VH / VL binding regions of the bispecific antibody comprises the amino acid sequence presented in SEQ ID NO. 26. In one embodiment, the VL of both VH / VL binding regions of the bispecific antibody comprises the amino acid sequence presented in SEQ ID NO. 27.

[0078] Bispecific antibodies such as those disclosed in the method of this specification may be provided in a number of formats. Bispecific antibodies of a number of different formats are known in the art. For example, a bispecific antibody format other than a classical antibody having two VH / VL combinations has at least a variable domain comprising a heavy chain variable region and a light chain variable region. Such variable domains may be linked to a single-chain Fv fragment, a monobody, VH, and a Fab fragment that provide a second binding activity.

[0079] Bispecific antibodies such as those disclosed in the method provided herein are generally of human IgG subclasses (e.g., IgG1, IgG2, IgG3, IgG4). In a given embodiment, the antibody is of the human IgG1 subclass. Full-length IgG antibodies are preferred due to their advantageous half-life and low immunogenicity. Accordingly, in a given embodiment, the bispecific antibody is a full-length IgG molecule. In one embodiment, the bispecific antibody is a full-length IgG1 molecule.

[0080] In a given embodiment, the antibody comprises a crystallizable fragment (Fc). The Fc of the bispecific antibody preferably consists of a human constant region. The constant region or Fc of the bispecific antibody may contain one or more, preferably 10 or fewer, amino acid differences from the constant region of a naturally occurring human antibody, preferably 5 or fewer. For example, in a given embodiment, each Fab-arm of the bispecific antibody may further comprise an Fc-region comprising a modification that promotes the formation of the bispecific antibody, a modification that affects Fc-mediated effector function, and / or other features described herein.

[0081] In one aspect, a pharmaceutical composition is provided comprising two or more antibodies as defined herein and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” means approved by government regulatory agencies for use in animals, particularly in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias, and includes any and all physiologically suitable solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption retardants, etc. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle administered with the compound. Such pharmaceutical carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, etc. Water or saline solutions, and aqueous solutions of dextrose and glycerol, may be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration may be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the active compound may be coated with a specific material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0082] Pharmaceutical compositions suitable for administration to human patients are typically formulated in a liquid carrier for parenteral administration, for example, or are suitable for reconstitution as a liquid solution or suspension for intravenous administration. The composition may be formulated in the form of a dosing unit for ease of administration and uniformity of dosage.

[0083] In addition, solid formulations intended for conversion into liquid formulations for oral or parenteral administration immediately before use are included. Such liquid forms include solutions, suspensions, and emulsions.

[0084] "Bispecific antibody" is an antibody as described herein, wherein one domain of the antibody binds to a first antigen and, meanwhile, a second domain of the antibody binds to a second antigen, and said first antigen and second antigen are not identical. The term "bispecific antibody" also includes an antibody in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope on the antigen and a second VH / VL combination binds to a second epitope. The term further includes an antibody in which VH can specifically recognize the first antigen and VL paired with VH within the immunoglobulin variable region can specifically recognize the second antigen. The resulting VH / VL pair will bind to antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008 / 027236, WO 2010 / 108127 and in the literature [Schaefer et al (Cancer Cell 20, 472-486, October 2011)]. The bispecific antibody according to the present invention is not limited to any specific bispecific format or method of production thereof.

[0085] When referring to nucleic acid or amino acid sequences in this specification, “percent (%) identity” is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after aligning the selected sequence and the candidate sequence for the purpose of optimal comparison. The sequence identity percentage for comparing nucleic acid sequences is determined using the AlignX application of the Vector NTI Program Advance 10.5.2 software using default settings, which use a modified ClustalW algorithm (reference [Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22: 4673-4680]), a swgapdnarnt score matrix, a gap-opening penalty of 15, and a gap-extending penalty of 6.66. Amino acid sequences are aligned using the AlignX application of the Vector NTI Program Advance 11.5.2 software with default settings, which use the modified ClustalW algorithm (reference [Thompson, JD, Higgins, DG, and Gibson TJ, 1994]), the blosum62mt2 score matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0086] As used herein, the term "common light chain" refers to two light chains (or their VL portions) within a bispecific antibody. The two light chains (or their VL portions) may be identical or may have some amino acid sequence differences that do not affect the binding specificity of the full-length antibody. The terms "common light chain," "common VL," "single light chain," and "single VL," with or without the addition of the term "rearranged," are all used interchangeably herein. "Common" also refers to functional equivalents of light chains having non-identical amino acid sequences. Many variants of said light chains exist, and these variants contain mutations (deletions, substitutions, insertions, and / or additions) that do not affect the formation of the functional binding region. The light chain of the present invention may also be a light chain as specified above, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, light chains that are not identical but are still functionally equivalent may be prepared or found by introducing and testing, for example, conservative amino acid changes, changes in amino acids within a region that do not contribute to or only partially contribute to binding specificity when paired with a heavy chain, as defined in the definition of a common light chain as used herein. The terms 'whole-length IgG' or 'whole-length antibody' according to the present invention are defined as comprising an essentially complete IgG, but this does not necessarily possess all the functions of a whole IgG. To avoid misunderstanding, the whole-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be degraded into domains designated as CH1, CH2, CH3, VH, and CL, VL.IgG antibodies bind to antigens through variable domains contained within the Fab portion and, after binding, can interact with molecules and cells of the immune system through constant domains, usually through the Fc portion. The full-length antibody according to the present invention comprises an IgG molecule in which mutations providing desired characteristics may be present. The full-length IgG must not have a substantial portion of any of these regions. However, an IgG molecule with one or several amino acid residues deleted without substantially altering the binding characteristics of the resulting IgG molecule is included within the term "full-length IgG." For example, such an IgG molecule may preferably have 1 to 10 amino acid residues deleted within a non-CDR region, wherein the deleted amino acids are not essential for the binding specificity of IgG.

[0087] Since antibodies typically recognize epitopes of antigens, and since such epitopes may likewise be present in other compounds, an antibody according to the present invention that “specifically recognizes” an antigen may likewise recognize other compounds if those other compounds contain the same type of epitope. Accordingly, regarding antigen-antibody interactions, the term “specifically recognizes” does not exclude the binding of the antibody to other compounds containing the same type of epitope.

[0088] The term "epitope" or "antigenic determinant" refers to a region on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by the tertiary folding of a protein (so-called linear and stereochemical epitopes). Epitopes formed from continuous linear amino acids are typically retained upon exposure to a denaturing solvent, whereas epitopes formed by the tertiary folding stereochemical structure are typically lost upon treatment with a denaturing solvent. Epitopes can typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids within a specific spatial stereochemical structure. Methods for determining the spatial conformation of an epitope are known to those skilled in the art and include techniques in the art, for example, X-ray crystallography, HDX-MS and two-dimensional nuclear magnetic resonance, Pepscan, and alanine scans depending on the properties of the epitope (see, for example, [Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)]).

[0089] For clarity and brevity, features are described herein as part of identical or distinct embodiments, but it will be understood that the scope of the invention may include embodiments having all or some of the disclosed features.

[0090] To facilitate a better understanding of this description, certain terms are defined first. Additional definitions are provided throughout the detailed description. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art, and the conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used.

[0091] As used herein, the singular forms (“a,” “an,” and “the”) include plural objects unless the context clearly indicates otherwise. The use of the term “including,” as well as other forms such as “include,” “include,” and “included,” is not restrictive.

[0092] Brief explanation of the drawing

[0093] Fig. 1

[0094] A schematic diagram of an embodiment comprising two bispecific antibodies that share a common arm. This diagram illustrates an antibody having a heavy chain (1) and a light chain (4). The four heavy chains have three different variable regions (5, 6, 7). The heavy chain having the shared variable region (5) has one part (3) of a heterodimerization domain. The heavy chain having the variable region (6) and the variable region (7) has compatible parts of the heterodimerization domain (2). A preferred pair of heterodimerization regions (2) and heterodimerization regions (3) may indicate the formation of a bispecific antibody.

[0095] Fig. 2

[0096] Panel A: CIEX profile at 220 nm of bispecific antibody PB4516 generation number 8 (p08). Panel B: CIEX profile at 220 nm of bispecific antibody PB6892 generation number 4 (p04).

[0097] Fig. 3

[0098] Fig. 3a: CIEX profile at 220 nm of bispecific antibody PB4516 generation number 10 (p10).

[0099] Fig. 3b: CIEX profile at 220 nm of bispecific antibody PB11244 generation number 1 (p01).

[0100] Fig. 3c: Two bispecific antibodies per box are identified in the PB(PBXXXX(X)) column. The heavy chain variable regions for PB11244 and PB4516 are shown in the columns for Target 1 and Target 2. The light chain sequences are identical for all antibodies and have the amino acid sequence of the common light chain IgKV1*39 / jk1 of sequence number 26. The pI calculated using the ExPASy, ProtParam tool is displayed for each heavy chain variable region in the pI column. The pI difference between the two heavy chain variable regions is shown in the last column, which demonstrates that the average VH pI difference between PB11244 and PB4516 is 0.08.

[0101] Fig. 4

[0102] CIEX profile at 220 nm of the antibody formulation from individual colony cp12 of pool FST2. The CIEX profile shows sharp peaks for co-exhausting antibodies PB4516 and PB11244. The profile indicates that the sample contains a limited amount of product-related impurities. This also demonstrates good separation between the individually migrating product-related impurities and the co-exhausting bispecific antibodies.

[0103] Fig. 5

[0104] CIEX profile of the antibody formulation of colony CP07 at 220 nm. Colonies were selected from a set of individual colonies of the single colony FST2cp09. Second subcloning was performed to confirm that the FST2cp09-cp07 cell line is a clonal cell line. Bispecific antibody-specific ELISA showed the presence of 743 µg / ml of the EGFR / HER2 bispecific antibody PB11244 and 1134 µg / ml of the EGFR / HER3 bispecific antibody PB4516.

[0105] Fig. 6

[0106] Retention time of antibody homomers (PGXXXX) having two identical variable domains. The amino acid sequence of the heavy chain variable domain has the sequence shown for the common light chain IgKV1*39 / jk1 of sequence no. 26 and the MF of Fig. 8.

[0107] Fig. 7. Two bispecific antibodies are identified per box in the PB (PBXXXX(X)) column. The heavy chain variable region (MFXXXX) of each bispecific antibody is shown in the columns for Target 1 and Target 2. The light chain sequence is identical for all antibodies and has the amino acid sequence of the common light chain IgKV1*39 / jk1 of Sequence No. 26. The pI calculated using the ExPASy and ProtParam tools is displayed for each heavy chain variable region in the pI column. The pI difference between the two heavy chain variable regions and the measured retention times are shown in the last two columns. The measured retention times, calculated pIs, and average pIs indicate that the couple of bispecific antibodies can be efficiently co-eluted in CIEX chromatography. The antibodies possess an IgG1 constant region and a common light chain. Heavy chains with a shared heavy chain variable region (identical MF) containing either a CH3 DE heavy chain or a KK heavy chain are shown. Retention times are indicated for CIEX chromatography performed for each bispecificity, and exemplary conditions for CIEX chromatography are described in the Materials and Methods section. Many different CIEX chromatography conditions will result in suitable retention times between the listed bispecific antibody pairs with the provided pI values.

[0108] Fig. 8

[0109] Amino acid sequences of the CDRs of the heavy chain variable region (MFXXXX) and light chain variable region of each antibody, and the amino acid sequence of the common light chain variable region.

[0110] Examples

[0111] Example 1

[0112] Materials and Methods

[0113] cell line

[0114] HEK293 and CHO-K1 were maintained in the growth medium.

[0115] Production of bispecific antibodies

[0116] To ensure the efficient heteromerization and formation of bispecific antibodies, bispecific antibodies were generated using proprietary CH3 technology. CH3 technology enables the efficient pairing of two different heavy chain molecules as previously described by using charge-based point mutations in the CH3 region (PCT / NL2013 / 050294; published in WO 2013 / 157954 A1).

[0117] The VH gene was cloned into one of two different backbone IgG1 vectors. Depending on the binding partner, VH was cloned into an IgG1 backbone containing a CH3 variant with the heterodimer variant "DE" or into an IgG1 backbone containing the complementary CH3 heterodimer variant "KK". In the case of a bispecific or multispecific antibody in which two or more antibodies share a heavy chain. The covalent chain preferably has the CH3 heterodimer variant "DE" (also referred to as the DE-heavy chain), and two or more specific heavy chains have the CH3 heterodimer variant "KK" (also referred to as the KK-heavy chain).

[0118] HEK293 cells were transiently transfected with a DNA-FUGENE mixture and cultured further. Seven days after transfection, the supernatant was harvested and the medium was refilled. Fourteen days after transfection, the supernatants were mixed and filtered through 0.22 μM. The sterile supernatant was stored at 4°C. Suspension-adapted 293F cells were cultured in T125 flasks on a shaker stand until a density of 3.0 × 10⁶ cells / ml was reached. Cells were seeded into individual wells of a 24-deep-well plate at a density of 0.3 to 0.5 × 10⁶ viable cells / ml. Cells were transiently transfected with individual sterile DNA:PEl-MIX and cultured further. Seven days after transfection, the supernatant was harvested and filtered through 0.22 μM. The sterile supernatant was stored at 4°C.

[0119] Generation of a stable cell line pool co-expressing two bispecific antibodies

[0120] CHO cells were transfected with three heavy chain constructs and a common light chain construct in a molar ratio of common light chain construct (cLC):EGFR heavy chain:HER2 heavy chain:HER3 heavy chain = 2.5:2:1:1. Ten pools of stably transfected cells were obtained (AJ). ELISA analysis of anti-EGFR, anti-HER2, and anti-HER3 antibodies was performed on the supernatants of the ten pools on days 3 and 6. All three antibodies were detected in all pools.

[0121] Generation of a stable cell line clone co-expressing two bispecific antibodies.

[0122] The pool was plated on a semi-solid medium and allowed to grow for 7 to 10 days. A single colony was selected and seeded into a 24-well culture plate. The colony was re-seeded before collecting antibodies from the supernatant of the culture.

[0123] Determination of antibody titer

[0124] The anti-HER2 antibody titer of samples containing a single bispecific antibody was determined by ELISA against Erbb-2 Fc protein (R&D System). The anti-HER3 titer of samples containing a single bispecific antibody was determined by ELISA against human Erbb-3 Fc protein (R&D System). The anti-EGFR antibody titer of samples containing a single bispecific antibody was determined by ELISA against human EGFR ECD-Fc protein (R&D System). Starting at 5 µg / ml, the wells of the ELISA plates were coated using serial twofold dilutions of the antigen.

[0125] An ELISA assay to quantify EFGR × HER2 and EGFR × HER3 bispecific antibodies in a composition containing a mixture of two bispecific antibodies was performed by coating an ELISA plate with EGFR-Fc (R&D System). After washing, the plate was incubated with the sample. After washing, the presence of a conjugated bispecific antibody having one EFGR arm and one HER2 arm was detected by incubating with labeled HER2-Fc. The presence of a conjugated bispecific antibody having one EFGR arm and one HER3 arm was detected by incubating with labeled HER3-Fc.

[0126] IgG purifiers

[0127] Purification of IgG was performed using affinity chromatography. Purification was carried out under sterile conditions using vacuum filtration. First, the pH of the medium was adjusted to pH 8.0, and subsequently, the product was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) on a shaking platform at 600 rpm for 2 hours at 25°C. Next, the beads were collected by vacuum filtration. The beads were washed twice with PBS (pH 7.4). Subsequently, IgG was eluted at pH 3.0 using 0.1 M citric acid buffer, and the IgG fraction was immediately neutralized using Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). The sample was finished with a final buffer of PBS pH 7.4.

[0128] Cation-exchange chromatography (CIEX)

[0129] CIEX-HPLC chromatography was performed using ion exchange columns of the TSKgel SP-STAT series (7 μm particle size, 4.6 mM ID × 10 cm L, Tosoh 21964). The columns are packed with non-porous resin particles for rapid, high-resolution analysis as well as the isolation of biomolecules. While the particles within the TSKgel STAT columns contain an open access network of multilayer ion-exchange groups for loading capacity, the particle size makes these columns suitable for HPLC and FPLC systems.

[0130] TSKgel SP-STAT (7 μm particle size, 4.6 mM ID × 10 cm L, Tosoh 21964) was equilibrated using buffer A (sodium phosphate buffer, 25 mM, pH 6.0), and then antibodies were removed from the column by increasing the salt concentration and running a gradient of buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate was set to 0.5 mL / min. The injection sample mass for all test samples and controls (in PBS) was 10 μg, and the injection volume was 10 to 100 μl. Chromatograms were analyzed for the peak pattern, retention time, and peak area for the main peak observed based on the 220 nm results.

[0131] result

[0132] The CIEX profiles of the bispecific antibodies PB4516p08 and PB6892p04 were compared (see Fig. 2). It was observed that the production of PB6892 contained a significant amount of impurities. Additionally, the retention time of the PB6892 bispecific antibody fraction was significantly lower than that of the PB4516 bispecific antibody fraction. In the case of co-production by the same cells and subsequent co-purification using CIEX, the retention times of the two bispecific antibodies are preferably closer to each other. For this reason, the variable region of the HER2 arm for PB6892 was replaced with a different variable region. The EGFR × HER2 bispecific antibody PB11244 was produced using a heavy chain having the variable region MF2032. The CIEX profiles of PB4516p10 and PB11244p01 are shown in Fig. 3. The retention times of the bispecific antibody fractions are 16.310 and 16.950, respectively. These retention times are sufficiently similar to enable co-purification using CIEX under the indicated conditions. Additionally, this figure shows that the retention times of impurities are sufficiently different to allow for efficient separation in the analysis and preparative columns. The bispecific antibody preparations were produced in HEK293 cells.

[0133] CHO-K1 cells were used for co-generation. CHO cells were transfected with a construct containing three heavy chains with variable regions of MF3755 (EGFR), M2032 (HER2), and MF3178 (HER3), respectively, and transfected into CHO-K1 cells with a construct expressing the light chain variable region of SEQ No. 26. Vector-positive cells were selected and pooled. Ten individual pools of transfected CHO-K1 cells (identified as AJ) were generated.

[0134] Table 1 shows the amounts of the bispecific antibodies PB4516 (EGFR × HER3) and PB11244 (EGFR × HER2) produced by each pool. In addition, the total amount of IgG produced as well as the ratios of the amounts are shown. Pools F and J were selected for subcloning.

[0135] Table 2 shows the amounts of bispecific antibodies PB4516 (EGFR × HER3) and PB11244 (EGFR × HER2) produced by each clone.

[0136] CIEX profiles were analyzed using antibodies generated by clone FST2cp12 (see Fig. 4). It is evident that two bispecific antibodies are efficiently co-eluted in the same CIEX elution fraction.

[0137] Clone FST2cp09 was further subcloned to confirm that the cell line was cloned and to determine the additional CIEX profile of the generated antibodies. Figure 5 shows the CIEX profile. It is evident that two bispecific antibodies efficiently co-elute in the same CIEX elution fraction. The relative contribution of the two bispecific antibodies in co-elution is analyzed by ELISA and / or by a hydrophobic interaction column. Bispecific antibody-specific ELISA revealed the presence of 743 µg / ml of the EGFR / HER2 bispecific antibody PB11244 and 1134 µg / ml of the EGFR / HER3 bispecific antibody PB4516.

[0138] Example 2

[0139] Generation of a stable cell line pool co-expressing two bispecific antibodies

[0140] Cell lines expressing the 2 × 2 bispecific antibodies listed in FIG. 7 are generated as follows. CHO cells are transfected with three heavy chain constructs and a common light chain construct. The three heavy chains are identified by the heavy chain variable regions (MFXXXX) indicated in the box. The light chain contains the light chain variable region sequence of IgVk1*39 / jk1 of SEQ ID NO. 26. The two bispecific antibodies each have one heavy chain in common and one different heavy chain. For example, the first couple specified in FIG. 7 shares a common heavy chain containing the same HER3 binding arm, which includes a heavy chain variable region (MF3178) and a different second binding arm. PB4528 has an EGFR binding arm with a heavy chain variable region (MF4003), and PB4188 has a HER2 binding arm with a heavy chain variable region (MF3958). The shared heavy chain has a KK CH3 region of a compatible DE / KK heteromerization domain. The shared heavy chain arm may also have a DE CH3 region. For example, in FIGS. 3 to 5, the two bispecific antibodies are co-purified PB11244 and PB4516. As shown in FIG. 3c, PB11244 and PB4516 share the same EGFR binding arm having a heavy chain variable region (MF3755), PB11244 has a HER2 binding arm having a heavy chain variable region (MF2032), and PB4516 has a HER3 binding arm having a heavy chain variable region (MF3178). While the shared heavy chain arm within this pair of bispecific antibodies has a DE CH3 region, the different HER2 and HER3 binding arms have a KK CH3 region.

[0141] The molar ratio of common light chain construct (cLC):common heavy chain construct:different heavy chain construct 1:different heavy chain construct 2 is 2.5:2:1:1. A pool of stably transfected cells is obtained. ELISA analysis of the antigen is performed on the supernatant collected from the pool. All three antigen-binding species are detected in the pool. The CIEX retention times of the bispecific antibodies in each couple of Figure 7 were determined under similar CIEX conditions and are shown in the 8th column. The deviation from the average retention time is calculated using the formula 100x((AB) / (A+B)), where A is the retention time of the bispecific antibody with the longest retention time. For example, the deviation for the first couple is 100x((16.46-16.24) / (16.46+16.24)) = 0.67 or 0.7%.

[0142] The antibodies in the collected supernatant are first separated from other proteins in the supernatant by acid elution and rapid neutralization following protein A extraction. Subsequently, the buffer of the collected antibodies is exchanged with PBS. Subsequently, the sample is loaded onto a CIEX column, washed, and eluted by applying an increasing salt gradient. The absorption of the eluent is measured at 220 nm, and the retention time is calculated from the onset of the salt gradient and the observation of peak(s) for the bispecific antibodies. The bispecific antibodies are collected, and each bispecific antibody in the collected eluent is verified by ELISA. The retention time for each bispecific antibody is shown in the final column. It is evident that multiple pairs have retention times that allow for efficient co-elution in the CIEX column. It is also evident that CIEX chromatography provides good separation of the co-eluting bispecific antibodies and their respective homodimers (if present). FIG. 6 lists the retention times of various antibodies having heavy chain homodimers containing a heavy chain variable region present in co-administered bispecific antibodies. It is evident that the retention times of the homodimers are sufficiently different from the retention times of each bispecific antibody. For example, in the first box of FIG. 7, homodimers PG3178, PG3958, and PG4003 may be present in the product. The retention times of each homodimer are approximately 22, 12, and 13 (rows 1 to 3 of FIG. 6), whereas the retention times of the bispecific antibodies containing a heavy chain variable region are approximately 19 and 19.5 (rows 1 and 2 of FIG. 7).

[0143] [Table 1]

[0144]

[0145] Table 1: Quantification of EGFRxHER2 and EGFRxHER3 bispecific antibody production in cell pools. Culture supernatants from 10 pools (AJ) were evaluated. ELISA assays were based on EGFR-Fc coating, binding of the generated antibodies, and detection using labeled HER2-Fc or labeled HER3-Fc. Pools A through J were analyzed using two ELISA assays. The bispecific antibodies PB4516 and PB11244 are IgG1 heavy-chain antibodies possessing compatible DE / KK heteromerization domains. The heavy chains are combined with a common light chain. The bispecific antibodies share the MF3755 heavy-chain variable region on one heavy chain, and each possesses a different heavy-chain variable region on the other IgG1 heavy chain, namely MF3178 for PB4516 and MF2032 for PB11244.

[0146] [Table 2]

[0147]

[0148] Table 2: Selected pools were used for single-cell cloning. Eighteen colonies were selected from three pools. Two independent pools F (FST1 and FST2) and one pool J (JST1) were used for single-cell cloning. The designation "cp" followed by a number identifies individual colonies within a pool. The selected colonies were grown and used for antibody collection. Individual colonies from the same pool produced different amounts and different ratios of their respective bispecific antibodies.

Claims

Claim 1 A method for generating at least two types of antibodies, wherein at least one of the antibodies is a multispecific antibody, and comprises: - a step of providing a nucleic acid encoding the at least two types of antibodies to cells; - a step of culturing the cells; - a step of collecting the at least two types of antibodies from the culture of the culturing step; and - a step of separating the generated antibodies from half antibodies by ion exchange chromatography (IEX), wherein, under the IEX conditions used, the IEX retention time of the at least two types of antibodies deviates from the average of the retention times of the at least two individual antibodies by 10% or less. Claim 2 A method according to claim 1, wherein the step of collecting at least two types of antibodies from the culture comprises purifying the antibody from another protein by antibody affinity purification. Claim 3 A method according to claim 2, comprising further applying size-exclusion chromatography, gel-filtration chromatography, and / or anion-exchange chromatography to at least two antibodies purified by the antibody affinity purification. Claim 4 A method according to claim 1, wherein, after the step of separating the antibodies generated by the IEX, the relative expression levels of the at least two types of collected antibodies are quantitatively analyzed by hydrophobic interaction chromatography (HIC). Claim 5 A method according to claim 4, wherein the specificity of at least two types of collected antibodies is verified by ELISA. Claim 6 A method according to claim 1, wherein the retention time of each of the above half antibodies is outside the range over which the retention times of the above at least two types of antibodies overlap. Claim 7 In claim 6, the method wherein the cells produce three heavy chains. Claim 8 In claim 7, the method wherein the heavy chains comprise domains for efficient heteromerization of the heavy chains. Claim 9 A method according to claim 1, wherein at least two of the antibodies are bispecific antibodies. Claim 10 A method according to claim 1, wherein at least two of the antibodies share a single identical heavy chain. Claim 11 A method according to claim 1, wherein the antibodies have a pI that differs by 0.4 units or less from the average isoelectric point (pI) of the at least two types of antibodies. Claim 12 A method according to claim 1, wherein the at least two types of antibodies are selected to have heavy chain and light chain combinations having different retention times exceeding 10% of the average retention times of all antibodies under the IEX conditions used. Claim 13 In claim 12, the method wherein the pI of the heavy chain and light chain combinations of each of the at least two types of antibodies differs by more than 0.4 units from the average pI of the at least two types of antibodies. Claim 14 In claim 12, the method wherein the heavy chains comprise one CH3 domain for the preferential heteromerization of the heavy chains. Claim 15 In claim 1, the heavy chains of the antibodies are IgG heavy chains. Claim 16 The method of claim 1, wherein the cells generate three or more heavy chains, one heavy chain comprises amino acid substitutions L351K and T366K (EU numbering) within the CH3 region, and another heavy chain comprises amino acid substitutions L351D and L368E within the CH3 region. Claim 17 A method for generating at least two types of antibodies, wherein at least one of the antibodies is a multispecific antibody, and comprises: - a step of providing a nucleic acid encoding the at least two types of antibodies to cells; - a step of culturing the cells; - a step of collecting the at least two types of antibodies from the culture of the culturing step; and - a step of separating the generated antibodies from half of the antibodies by ion exchange chromatography (IEX), wherein the at least two types of antibodies exhibit IEX retention times that deviate from the average of the retention times of the individual antibodies by 10% or less under the IEX conditions used, and after the step of separating the generated antibodies by IEX, the at least two types of collected antibodies are quantitatively analyzed for relative expression levels by hydrophobic interaction chromatography (HIC), and the specificity of the at least two types of collected antibodies is verified by ELISA. Claim 18 delete Claim 19 A composition comprising 2 to 10 types of recombinant antibodies, wherein at least 2 of the antibodies have IEX retention times that deviate by 10% or less from the average of the retention times of the individual antibodies under the IEX conditions used, and at least 1 of the antibodies is a multispecific antibody. Claim 20 A composition comprising 2 to 10 types of recombinant antibodies, wherein at least 2 of the antibodies have a pI that differs from the average pI of the at least 2 antibodies by 0.4 units or less, and at least 1 of the antibodies is a multispecific antibody. Claim 21 A composition according to claim 19, characterized in that the IEX retention times of all said antibodies deviate by no more than 10% from the average retention times of said individual antibodies under the IEX conditions used; and / or the pI of all said antibodies differs by no more than 0.4 units from the average pI of said at least two types of antibodies. Claim 22 A composition according to claim 19, wherein at least two of the above antibodies are bispecific antibodies. Claim 23 A composition according to claim 22, wherein at least two of the antibodies share a single identical heavy chain. Claim 24 The method of claim 1 further comprises selecting at least two antibodies having IEX retention times that deviate by 10% or less from the average of the retention times of at least two individual antibodies having the same IEX retention times under the IEX conditions used. Claim 25 A method according to claim 1, wherein at least one of the antibodies is a monospecific antibody. Claim 26 A method according to claim 1, wherein at least two of the antibodies are multispecific antibodies. Claim 27 A composition according to claim 19, wherein at least one of the above antibodies is a monospecific antibody. Claim 28 A composition according to claim 19, wherein at least two of the above antibodies are multispecific antibodies.

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