CN113439090A - Preparation of a composition comprising two or more antibodies - Google Patents

Abstract

The present invention relates to a device and a method for producing at least two antibodies. The method can comprise the following steps: providing a cell with a nucleic acid encoding the antibody; culturing the cell; collecting the antibody from the culture; and separating the produced antibody from the half-antibody by ion exchange chromatography (IEX). In certain embodiments, such antibodies exhibit an IEX retention time that deviates from the mean value of the retention times of the individual antibodies by 10% or less under the IEX conditions used. The invention also relates to compositions of antibodies produced thereby. In certain aspects, the invention relates to compositions comprising 2-10 recombinant antibodies, characterized in that: under such IEX conditions, the IEX retention time of at least two of such antibodies deviates by 10% or less from the mean of the retention times of the individual antibodies. The invention also relates to a composition comprising 2 to 10 recombinant antibodies, characterized in that: the pI values of at least two of such antibodies differ from the average pI value of the at least two antibodies by 0.4 units or less.

Description

Preparation of a composition comprising two or more antibodies

The present invention relates to the field of antibodies, and more particularly to the field of therapeutic antibodies. Such antibodies can be used in the treatment of humans. More particularly, the invention relates to the production and/or purification of various antibodies. Such multiple antibodies can be produced by a single host cell. Such antibodies may also be produced by a mixture of host cells that each produce one of such antibodies. The invention also relates to methods for producing compositions comprising such antibodies and methods for purifying such antibodies.

Polyclonal antibodies are typically collected from the blood of an individual. One advantage (advantage) of polyclonal antibodies is: pathogens are attacked via multiple targets and epitopes. An advantage of monoclonal or recombinant antibodies is the well-characterized specificity and function, allowing such antibodies to be used as precise drugs with a well-defined spectrum of action and toxicity.

The specificity of a monoclonal antibody can also be a disadvantage, particularly when multiple targets need to be addressed. It is possible to reduce this disadvantage by adding more antibody to the drug, but considering that even a single therapeutic antibody may be expensive, it is expected that: the costs associated with such multi-plant mixtures can quickly become prohibitive.

The development of bi-, tri-, and other multispecific antibodies has successfully introduced certain aspects of a polyclonal antibody into antibody therapeutics. In addition to the increase in the number of targets, it also successfully introduces other functionalities previously unavailable using traditional monospecific monoclonal or polyclonal antibodies. The use of multiple di-, tri-, and other multispecific antibodies in the same treatment may provide even further benefits.

The present invention provides an advance in the art by describing a robust and economical method for the purification of multiple antibody therapeutics produced from a single host cell or, optionally, a mixture of host cells. The invention is particularly useful for the economical production of a collection of two or more antibodies, preferably multispecific antibodies.

Disclosure of Invention

The present invention provides a method for generating at least two multispecific antibodies, comprising:

-providing cells carrying nucleic acids encoding such multispecific antibodies;

-culturing such cells;

-collecting such multispecific antibodies from the culture; and

-separating the produced multispecific antibodies from the half-antibodies and optionally monospecific antibodies and/or other undesired antibody product-related impurities by ion exchange chromatography (IEX);

the method is characterized in that: such multispecific antibodies exhibit an IEX retention time that deviates from the mean value of the retention times of the individual multispecific antibodies by 10% or less under the IEX conditions used. The residence times of such corresponding half-antibodies and optionally monospecific antibodies and/or other impurities associated with the undesired antibody product preferably fall outside the range spanned by the residence times of such multispecific antibodies.

The present invention also provides a method for generating at least two multispecific antibodies, comprising:

-providing a cell with a nucleic acid encoding such a multispecific antibody;

-culturing the cell;

-collecting such multispecific antibodies from the culture; and

-separating the produced multispecific antibodies from the half-antibodies and optionally monospecific antibodies and/or other undesired antibody product-related impurities by ion exchange chromatography (IEX);

the method is characterized in that: such multispecific antibodies exhibit essentially the same IEX retention time under the IEX conditions used. The residence time of such corresponding half-antibodies and optionally monospecific antibodies and/or other impurities associated with the undesired antibody product preferably falls outside the range spanned by the residence times of such antibodies.

The present invention further provides a method for generating at least two antibodies, wherein such antibodies comprise a monospecific and/or a multispecific antibody, the method comprising:

-providing cells carrying nucleic acids encoding such antibodies;

-culturing such cells;

-collecting such antibodies from the culture; and

-separating the generated antibodies from the half-antibodies by ion exchange chromatography (IEX); the method is characterized in that: such antibodies exhibit essentially the same IEX retention time under the IEX conditions used. The residence time of such corresponding half-antibodies and optionally monospecific antibodies preferably falls outside the range spanned by the residence times of such antibodies.

The invention also provides a composition comprising 2-10 recombinant antibodies obtainable by a method as described herein.

Also provided is a composition comprising 2-10 recombinant antibodies (such as multispecific antibodies), characterized in that: under the IEX conditions used, the IEX retention time of at least two of such antibodies deviates by 10% or less from the mean of the retention times of the individual antibodies.

Further provided is a composition comprising 2-10 recombinant antibodies, characterized in that: the IEX retention times of at least two of such antibodies are substantially the same.

Also provided is a composition comprising 2-10 recombinant antibodies, characterized in that: the isoelectric Point (PI) of at least two of such antibodies preferably differs from the average PI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The pI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less.

Detailed Description

The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind an epitope on an antigen, wherein such domains are either derived from or share sequence homology with the variable region of an antibody. Antibodies are typically composed of basic building blocks each having two heavy chains and two light chains. The antibody for therapeutic use is preferably a natural antibody (e.g., a human antibody for a human subject) as close as possible to the subject to be treated. Antibodies with extended heavy and/or light chain variable regions are also included herein. An antibody according to the present invention is not limited to any particular format or method for producing it.

Half antibodies are heavy and light chain combinations that are not associated with another heavy chain and light chain combination and that do not form an interface with another variable region or variable region-like polypeptide. Other undesired antibody product-related impurities may be free light chains that are not associated with a heavy chain, free heavy chains that are not associated with a light chain or another heavy chain, or incompletely assembled antibodies that lack a light chain.

Suitable cells for antibody production are known in the art and include a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, a NS0 cell, or a PER-C6 cell, or a wide variety of other cell lines known to those of ordinary skill in the art having the present domain. Various organizations and companies have developed cell lines for large-scale production of antibodies, for example, for clinical applications. Non-limiting examples of such cell lines are CHO cells, NS0 cells or per.c6 cells or HEK293 cells, among others. In a particularly preferred embodiment, the cell is a human cell. Preferably, a cell transformed with an adenovirus E1 region or a functional equivalent thereof. In a preferred embodiment, the cell is a CHO cell or a variant thereof. Preferably, a variant for the expression of an antibody using a glutamine amide synthetase (GS) vector system. In a preferred embodiment, the cell is a CHO cell.

Such cells may be provided with nucleic acids encoding such antibodies. Such cells will express, assemble and drain the formed antibody into the supernatant of the cell culture. The introduction of a single heavy and light chain (more precisely, the nucleic acid encoding it) results in the production of a monoclonal antibody having two heavy chains each associated with a light chain.

A method of the invention may be performed using a cell mixture comprising two or more cells each producing a different antibody. The advantage of using such a mixture is: downstream processing of the collected antibodies can be more efficiently refined. Further advantages of a method are: the antibody product was collected and purified as one. Also, a mixture containing two or more antibodies generated by a method of the invention can reduce the number of tests required to obtain legal certification when compared to the number of tests required for each of such antibodies individually.

In a further embodiment, such cells are a homogeneous collection of cells consisting essentially of a copy of a single cell provided with nucleic acid encoding such a corresponding antibody. The common expression of several heavy chains in one cell allows for a variety of different heavy chain combinations. Combinations with additional light chains increase the number of combinations. Various methods have been developed to facilitate the formation of specific combinations over others. Heavy chain variants have been generated which specifically promote heavy chain heterodimer (heterodimers) over homodimer (homomodems) formation, or vice versa. Heavy chains with specific homo-or heterodimerization domains reduce the number of antibodies being produced by such cells and/or increase the level of preferred antibodies over alternative combinations (e.g., higher production of a heterodimer over a homodimer).

The method of the invention is particularly suitable for the production of two or more multispecific antibodies, including bispecific antibodies. Various methods for the generation of bispecific antibodies exist in the present domain. One approach uses a common light chain that can form a functional variable domain with different heavy chains. A preferred method for generating bispecific antibodies involves the use of a transgenic animal harboring a common chain within its genome such that immunization of such an animal with an antigen produces a repertoire of antibodies specific for the antigen on the basis of non-common chains, wherein the repertoire consists of various antibodies including the common chain and a rearranged cognate chain. An animal may be immunized with different antigens, or different animals may each be immunized with the corresponding antigen separately. The nucleic acid encoding the non-common strand or variable region thereof may be obtained from the animal(s), e.g., B cells, spleen or lymphoid tissue. These can be used to produce nucleic acids representing the corresponding non-common strands, which in turn can be introduced into the producer cell. The common strand may be introduced at the same or different times. The nucleic acid may be incorporated into the nucleus of a cell, and preferably the genome of the host cell, such that the host cell produces multispecific antibodies or multimers of target antigens for which the transgenic animal(s) have been immunized (for the purpose of production of variable regions that can be combined in a common chain to produce functional variable domains specific for different targets and/or different epitopes, see, for example, WO2009/157771, which is incorporated herein by reference).

A cell producing a common light chain and two different heavy chains, each forming a functional variable domain with the common light chain, among others, a bispecific antibody with two different heavy and light chain combinations. Likewise, a cell that produces a common light chain and three or more different heavy chains can form multispecific antibodies capable of targeting three or more antigens, or a combination of two or more multispecific antibodies capable of targeting three or more antigens. It is now possible to establish standard versions of antibodies (i.e. one constant part and two variable domains) and to add further binding domains. As such, multispecific antibodies having one or more single chain fvs with additional binding specificity attached to one or more of the constant portions or variable domains of an antibody can be made. It is also possible to generate heavy chains with two or more variable regions. The additional heavy chain regions may advantageously be combined with different or common light chain variable regions. See US 62/650467, which is hereby incorporated by reference.

When the cell produces two or more multispecific antibodies, it will also in some cases produce as dry quantities half antibodies as well as antibodies or homodimers with the same heavy chain. The number of the latter can be reduced by including heterodimers that promote modifications within the heavy chain. As mentioned previously, various methods exist for inducing heterodimerization of heavy chains. The corresponding domains with such modifications are collectively referred to as heterodimerization domains. Heavy chains with heterodimerization domains that facilitate interaction are said to have compatible heterodimerization domains. Such compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods are described in the art in which such CH3 heterodimerization of the heavy chain can be achieved.

A preferred method for generating bispecific antibodies is disclosed in US 9,248,181 and US 9,358,286. In particular, the favoured changes used to generate essentially bispecific only full length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain (the "KK-variant" heavy chain) and the amino acid substitutions L351D and L368E in the second domain (the "DE-variant" heavy chain), or vice versa. As mentioned previously, the DE-and KK-variants pair preferentially to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of a DE-variant heavy chain (DEDE homodimer) or of a KK-variant heavy chain (kkkkkk homodimer) hardly occurs due to strong repulsion between charged residues in the CH3-CH3 interface between identical heavy chains.

In the present invention, it is preferred that: such cells are provided with nucleic acids encoding a common light chain. There are a variety of different methods available to those skilled in the art for generating antibodies having different heavy chain variable regions but the same light chain variable region. WO 2004/106375 describes the use of a phage library of common light chain variable regions. Phage selection results in variable domains with the same light chain variable region but different heavy chain variable regions. Furthermore, non-human animals with a common strand as well as non-identical homologous strands are described in WO 2009/157771. Antibodies in such animals are selected to have variable domains with identical or similar common variable regions but non-identical homologous variable regions. WO 2004/106375 and WO2009/157771 are hereby incorporated by reference. Such patent publications (publications) are referenced particularly with respect to the production of antibodies (having identical or similar common variable regions and non-identical homologous variable regions, preferably a common light chain variable region and non-identical heavy chain variable regions) and nucleic acids encoding such antibodies.

In a preferred embodiment, such cells produce two or more heavy chains and a common light chain. Such corresponding heavy and light chains may have one or more variable regions associated with such corresponding chains. 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. The other two or more heavy chains preferably comprise another part of the compatible heterodimerization domain. If the first heavy chain is symbolically represented by the letter "a" and the other two by the letters "B" and "C", the particular combination of heterodimerization domains leads to the predominant formation of the combination AB and AC. Such combinations of AA, BB, CC and BC are not favored by the inclusion of heterodimerization domains. Such a cell produces only two bispecific antibodies AB and AC efficiently (see figure 1). In the present invention, it is preferred that: the cell produces the two antibodies by producing at least 3 heavy chains. In a preferred embodiment, one of such heavy chains contains one part of a compatible heterodimerization domain, while the other two or more heavy chains preferably include another part of the compatible heterodimerization domain. A heavy chain shared by two or more bi-or multispecific antibodies or multimers located in a composition preferably has the CH3 DE portion of the heterodimerization domain. The other heavy chains located in such bi-or multispecific antibodies preferably have a CH3KK moiety.

The at least two antibodies are preferably multispecific, preferably bispecific antibodies. In a preferred embodiment, at least two of such antibodies share a common heavy chain. The cell can generate several series of bispecific antibodies by including different, non-identical heterodimerization domains in such heavy chains. Such an implementation may result in the predominant production of antibodies with heavy chain combinations AB and CD. The combination AB may be favoured by a DE/KK heterodimerization domain as mentioned herein before, whereas the CD is favoured by the incorporation of a "knob in hole" heterodimerization domain, or other heterodimerization features known in the art, e.g. via charge engineering. A shared heavy chain or combination of antibodies with the shared heavy chain between non-identical bi-or multispecific antibodies can be made by providing a shared heavy chain with a portion of a heterodimerization domain and various different combination chains with complementary portions of the heterodimerization domain. For example, the CH3 DE moiety located in the shared heavy chain and the CH3KK moiety located in such a combinatorial chain. One shared heavy chain and two combined heavy chains in this case will cause the cell to generate bi-or multispecific multimers with heavy chain combinations AB and AC (or AxBC and AxDE for multispecific multimers). Other possible combinations are AB, AE, CD and CF; or AB, AE, AG, and CD, such as these.

Antibodies typically have a unique isoelectric point (typically in the range of pH 6-10) compared to other host cell proteins. Such antibodies, such as multispecific antibodies, and in addition multispecific multimers, can be purified with a relatively high purity via the methods described herein. The method may include a number of steps, such as culturing the host cell, undergoing harvest clarification, followed by protein capture. IEX chromatography, such as anion exchange chromatography, may be used to remove host cell DNA and cation exchange Chromatography (CIEX) may be used, for example, to remove host cell proteins, gonolytic protein a, and potential aggregates. Additional steps such as virus filtration or hydrophobic interaction chromatography may be included.

The antibody is usually expelled by the producing cells. The harvesting of such antibodies typically involves the collection of a clarified solution of cells, followed by several purification steps to remove cell debris or aggregates (the presence of which is not required). Harvest clarification may involve filtration, centrifugation, or a combination of these of the supernatant of the culture of antibody-producing cells. Antibody protein capture is typically accomplished by affinity purification. This can be done in several ways. Typically this involves purification using columns with recombinant protein a, protein G or protein L, which are bacterial proteins with a known high specific binding capacity for antibodies. Today, a variety of different optimized mutants are available that bind antibodies more specifically. For example, a recombinant protein A from which its non-essential domains have been removed is available, a recombinant protein G from which its albumin binding site has been deleted is available, and in addition a modified protein L is available. The bound antibody may be collected by elution against one or more of such columns. Anion and cation exchange chromatography can be used to further purify the preparation, e.g., to purify bispecific or multispecific antibodies from half-antibodies and/or homodimeric antibodies and/or other impurities, if any, associated with undesired antibody products. Hydrophobic Interaction Chromatography (HIC) is commonly used as an alternative purification step in antibody purification. HIC offers an orthogonal selectivity for ion exchange chromatography and can be an efficient step for aggregate clearance and host cell protein reduction. In the present invention, HIC may be used for analytical purposes after purification of the two or more bi-or multispecific antibodies or multimers is completed to subsequently quantify the two or more species having similar (preferably substantially identical) residence times and/or similar (preferably substantially identical) pI values on IEX. Thus, HIC is used to quantify the relative amount of molecules purified.

A hydrophobic interaction resin is selected as the stationary phase, while the pH and/or conductivity of the mobile phase is modulated to achieve the desired selectivity. Antibodies generally attract positive charges at lower pH values, which affects their polarity as well as overall surface hydrophobicity. The pH conditions that allow for the isolation of the two or more antibodies located in the preparation can be selected.

In certain embodiments, the collected antibodies are 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 to collect such antibody located in the effluent fraction. The antibodies can then be run on one or more CIEX columns.

A preferred method for producing at least two antibodies is accomplished by the cells producing them as a single composition comprising the two or more antibodies.

A method for producing at least two antibodies preferably comprises culturing host cells that produce the two or more antibodies, collection of supernatant of such cells, processing the supernatant harvest in a harvest clarification process, which preferably comprises filtration using a pore size threshold that traps aggregates such as cells or cell debris and allows passage of antibody product. The antibody is eluted from the column after exposure to low pH by affinity chromatography using protein a, and the eluate is then neutralized using a suitable buffer.

As used herein, the term "isoelectric point (pI)" refers to the pH at which the average net charge of the protein surface (i.e., the potential of the electrical bilayer of the protein) is 0. In other words, the term refers to the point at which a group of the protein is dissociated such that the number of cations and anions are equal, and thus the net charge of the protein is 0.

As used herein, "pI" is calculated from the primary amino acids based on the ExPASy ProtParam tool using default parameters as of the earliest filing date (priority date) of this application. ProtParam is a tool that allows the computation of a variety of different physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or a user-entered protein sequence. The calculated parameters include the theoretical pI values. Gastiger e., Hoogland c., Gattiker a., Duvaud s., Wilkins m.r., Appel r.d., Bairoch a.; protein Identification and Analysis Tools on the ExPASy Server; (In) John M.Walker (ed): The Protocols Handbook, Humana Press (2005) pp.571-607.

The net surface charge of a protein varies with pH in a manner dependent on the pI value of the protein. At a pH equal to the pI value of a protein, the protein will not carry a net charge. At a pH below the pI, the protein will carry a net positive charge. If the buffer pH is raised above the pI of a protein, it will carry a net negative charge.

The pI value of a protein can be determined by its primary amino acid sequence and can therefore be calculated, and a buffer ensuring a known net charge of a protein of interest can then be selected. A negatively charged cation exchange resin can thus be selected when the protein of interest carries a net positive charge at the operating pH.

Proteins with different pI values will have different degrees of charge at a given pH and thus different affinities for positively charged surface groups located on the particles of the anion exchange medium; thus, different proteins will bind to the resin in different amounts, facilitating their separation. Thus, by producing heterodimeric polypeptides having unique pI values relative to such homodimeric and hapten and/or other antibody product-associated impurities, such heterodimeric polypeptides can be readily purified using standard elution techniques, e.g., by applying a pH gradient, or by applying a salt or conductive gradient at a fixed pH, or a combination of a pH and a conductive gradient. In embodiments of the invention, the constant portion and the light chain located in such antibodies have substantially the same sequence in different antibodies. Such antibodies typically differ substantially only in the amino acid sequence of the heavy chain variable region. For such antibodies, it is generally sufficient to calculate the pI value of the heavy chain variable region as shown herein. The calculation and the standard are then set by using the pI value of the heavy chain variable region instead of the pI value of the (half) antibody. The average pI value of the heavy chain variable region of the antibody represents the residence time of the antibody in a CIEX-column. Such antibodies, if any, may have the same or different heterodimerization domains. They preferably have the same heterodimerization domain. Such antibodies may further differ in the correct amino acid sequence of such constant portions.

In connection with the ion exchange chromatography (IEX) step(s), this is the process of separating ions and polar molecules according to their affinity for the ion exchanger. It acts on a variety of charged molecules-including large proteins, small nucleotides, and amino acids. IEX is typically used for protein purification. The water soluble and charged protein forms ionic bonds with the insoluble stationary phase. The bound molecules may then be eluted and collected using an eluant having a higher concentration of ions and/or a different pH. The salt concentration or pH may be varied in a stepwise manner; by gradually changing the mobile phase of the chromatographic run, or a combination of these. 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 is generally in the range from 5 to 9, preferably from 6 to 8.

When the molecule of interest is positively charged at the pH used for chromatography, CIEX is typically used. The molecule is positively charged because the pH used for chromatography is lower than the pI value of the molecule. In this version, the stationary phase is negatively charged, while positively charged molecules are loaded to be attracted to the stationary phase. Anion exchange chromatography is when the stationary phase is positively charged, and negatively charged molecules (meaning that the pH used for chromatography is higher than pI value) are loaded to be attracted to the stationary phase.

An antibody (such as a multispecific antibody) is typically bound to the IEX column in a binding phase under conditions that promote binding of the antibody (such as a multispecific antibody) to the substrate. The IEX column is typically subsequently washed to remove unbound material. Elution from the column is completed in an elution phase. The residence time of an antibody (such as a multispecific antibody) is typically calculated from the beginning of the elution phase. It is the amount of time the antibody spends on the column when the elution phase is initiated. If a sample contains several compounds, each compound in the sample will typically spend a different amount of time on the column depending on its chemical composition, i.e., each will have a different residence time. Residence times are usually quoted in units of seconds or minutes.

In a method of the invention, the residence time of such antibodies (such as multispecific antibodies) is preferably substantially the same. Different antibodies may have different residence times in 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 co-purification of two or more antibodies (such as two or more multispecific antibodies) in a single IEX chromatography run. Residence times that deviate from the mean of the residence times of such individual antibodies by 10% or less are typically sufficiently close to allow co-purification of two or more antibodies (such as two or more multispecific antibodies) in a single IEX chromatography run.

A suitable CIEX HPLC method for antibody purification and/or analysis according to the invention uses an ion exchange column of the TSKgel SP-STAT (7 μm particle size, 4.6mM i.d. × 10cmL, Tosoh 21964) series. Such columns are packed with non-porous resin particles that are additionally separated for velocity and high resolution analysis of biomolecules. The particles in the TSKgel STAT column contain an open access network of multiple layers of ion exchange groups for loading capacity, and the particle size makes these columns suitable for HPLC and FPLC systems.

One suitable method involves equilibration of TSKgel SP-STAT (7 μm particle size, 4.6mM I.D.times.10 cmL, Tosoh 21964) using buffer A (sodium phosphate buffer, 25mM, pH 6.0), after which the antibody is drained from the column by increasing the salt concentration and running a gradient of buffer B (25mM sodium phosphate, 1mM NaCl, pH 6.0). The flow rate was set at 0.5 mL/min. The injected sample mass for the test sample as well as the control group was 10. mu.g, while the injected volume was 10-100. mu.L. The chromatogram was analyzed with respect to peak patterns (peak patterns), retention time and peak area of the main peak observed as a result of 220 nm. For larger amounts of antibody, the method may be proportional.

A typical plot of a CIEX chromatographic run of antibody preparations is depicted in fig. 2. Such antibodies for this run were collected from transfected cells as shown in the example, and were purified from many other proteins located in the culture medium by using a protein a column. As shown in the demonstration example, the antibody was eluted by acid elution, followed by neutralization and buffer exchange to PBS pH 7.4. A sample of the antibody preparation was then loaded onto the CIEX column. After washing, the associated proteins are eluted by applying a salt gradient. Such CIEX conditions are the same for the samples in fig. 2A and 2B. The residence time was calculated for the top of the peak of the bispecific antibody. The retention time of two or more antibodies (such as multispecific antibodies) preferably deviates from the average of the retention times of the two or more antibodies by 10% or less. A deviation of greater than 10% often results in inefficient separation of such antibodies from the half-antibodies and, optionally, for multispecific antibodies, from homodimers and/or other antibody product-related impurities. In a preferred embodiment, the retention time of the two or more antibodies deviates from the average of the retention times of the two antibodies by 9% or less. Preferably with a mean deviation from the retention time of the two or more antibodies of 8%, 7%, 6% or 5% or less. In a preferred embodiment, the retention time of the two or more antibodies deviates from the average of the retention times of the two or more antibodies by 4% or less. Preferably 3% or less, preferably 2% or less. Increasingly similar residence times generally allow for increasingly efficient separation of such multispecific antibodies from half-antibodies and optionally homodimers and/or other antibody product-related impurities, and thus allow for cleaner collection of the two antibodies located in fractions of the IEX column.

In the means and methods of the invention, the average residence time of the two or more antibodies (such as the two or more multispecific antibodies) is calculated as to such antibodies to be co-purified or collected. Thus, in embodiments where such antibodies to be purified are multispecific antibodies, the average residence time of the two or more antibodies is calculated accordingly. The retention time of the antibody not collected (such as homodimeric antibody) was not used for the calculation of the mean.

If this item is selected to be an antibody (such as a multispecific antibody) having substantially the same IEX retention time under the conditions of IEX, an antibody such as a multispecific antibody may be selected for co-purification in a method of the invention. Such antibodies (such as multispecific antibodies) may also be tailored by appropriate modification of one or more variable regions to have substantially the same IEX retention time under the same or similar conditions used for IEX.

In one embodiment, the generated antibodies (such as bi-and/or multispecific antibodies) sought to be co-purified have similar pI values. The isoelectric point (pI) of at least two of such antibodies preferably differs from the average pI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The pI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less.

A slight to no difference in pI values of such antibodies generally allows a good co-purification. Advantageously, the pI values of the corresponding half antibodies within an antibody differ more from the mean value. This difference promotes a good separation of such half-antibodies from "co" -migrating intact antibodies in the CIEX chromatography step.

In embodiments where such antibodies sought to be co-purified are bi-or multispecific antibodies, preference is given to: the average difference in pI values of the variable domains located in each of such antibodies sought to be co-purified from the pI values of the variable domains of the other antibody(s) to be co-purified is greater than 0.2 (preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably greater than 1.8 or 2.0) units. In this embodiment, the pI values of the variable domains located in an antibody preferably differ by at least 0.2 units greater than the difference between "x" and "y", preferably it is 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, 5, 0, 1.2, 1.5, 02.0 or 2.5) greater than the difference between "x" and "y", where "x" is the average of the pI values of the two variable domains of a first one of such antibodies and "y" is the average of the pI values of the two variable domains of a second one of such antibodies. A difference as mentioned in the pI values of variable domains located in an antibody generally indicates a good separation of such antibody product-related impurities from such antibodies sought to be co-purified and/or from monospecific antibodies.

Certain compositions comprising two or more bi-or multispecific antibodies have constant regions and light chains with amino acid sequences that are similar or have a substantially identical amino acid sequence. Such two or more bi-and/or multispecific antibodies are typically not identical to each other substantially only in the amino acid sequence of such variable domains or substantially only in the amino acid sequence of such heavy chain variable regions. In such cases, it is often necessary to determine the pI value of the whole antibody. Conversely, the pI values of such variable domains and/or the pI values of such heavy chain variable regions may be determined. This provides a means to assess whether such antibodies can migrate close together in a CIEX chromatography step (in other words whether such antibodies have a residence time that is sufficiently the same to allow co-purification).

In one embodiment of a method or composition as disclosed herein, the two or more bi-or multispecific antibodies have constant regions and light chains with the same amino acid sequence or with substantially the same amino acid sequence. The two or more bi-or multispecific antibodies may be co-purified in one CIEX chromatography step when the average pI value of the variable domains located in each antibody differs by 0.7 units or less from the average pI value of the variable domains of the respective antibody sought to be co-purified. In a preferred embodiment, the average "x" of the pI values of the two variable domains of a first of such antibodies and the average "y" of the pI values of the two variable domains of a second of such antibodies differ by 0.6 units or less, preferably by 0.5 units or less from the average "x" and "y" of the first and second antibodies sought to be co-purified. The difference between "x" and "y" and the mean value of "x" and "y" of the first and second antibodies sought to be co-purified is preferably 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such bi-and/or multispecific antibodies typically have substantially the same residence time. In this embodiment, the constant regions of such antibodies are substantially identical. The pI values and in particular the average values "x" and "y" of such antibodies as a whole represent the pI values of the corresponding antibodies. In this embodiment, preferred are: the average difference in pI values of the variable domains located in each of such antibodies sought to be co-purified from the pI values of the variable domains located in such antibodies is greater than 0.2 (preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably greater than 1.8 or 2.0 units, in this embodiment, the difference in pI values of the variable domains located in an antibody is preferably at least 0.2 units greater than the difference between "x" and "y", preferably it is 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". a difference as mentioned in the pI values of the variable domains located in an antibody generally indicates a good separation of the half-antibody from such antibodies sought to be co-purified and/or from mono-specific antibodies.

In one embodiment of a method or composition as disclosed herein, the two or more bi-or multispecific antibodies have constant regions and light chains with the same amino acid sequence or with substantially the same amino acid sequence. The two or more bi-or multispecific antibodies may be co-purified in one CIEX chromatography step when the average pI value of the heavy chain variable region located in each antibody differs from the average pI value of the heavy chain variable region of the corresponding antibody sought to be co-purified by 0.7 units or less. In a preferred embodiment, the average "m" of the pI values of the two heavy chain variable regions of a first one of such antibodies and the average "n" of the pI values of the two heavy chain variable regions of a second one of such antibodies differ by 0.6 units or less, preferably by 0.5 units or less from the average of the "m" and "n" of the first and second antibodies sought to be co-purified. The difference between "m" and "n" and the mean value of "m" and "n" of the first and second antibodies sought to be co-purified is preferably 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such bi-and/or multispecific antibodies typically have substantially the same residence time. In this embodiment, the constant regions of such antibodies are substantially identical. The pI values and in particular the average values "m" and "n" of such antibodies as a whole represent the pI values of the corresponding antibodies. In this embodiment, preferred are: the pI values of the heavy chain variable regions located in each of such antibodies sought to be co-purified differ from the average pI values of the heavy chain variable regions located in such 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 more than 1.8 or 2.0) units. In this embodiment, the difference in pI values of the heavy chain variable regions located in an antibody is preferably at least 0.2 units greater than the difference between "m" and "n", preferably it is at least 0.3, 0.4, 0.5 (preferably at least 0.6, 0.7, 7, 0)0.8, 0.9, 1.0, 1.2, 1.5, 2.0 or 2.5 greater than the difference between "m" and "n". A difference as mentioned in the pI values of the heavy chain variable regions located in an antibody generally indicates a good separation of the half-antibody from such antibody which is sought to be co-purified and/or from the monospecific antibody.

Such antibodies, such as multispecific antibodies, may have or be selected to have heavy and light chain combinations (half antibodies) or homodimers (e.g., monospecific antibodies) or other antibody product-related impurities that have retention times under the IEX conditions used that are significantly different from the retention times of such intact antibodies or desired antibodies, such as multispecific antibodies. In one embodiment, where the cells exhibit a common light chain, the selection typically falls on the heavy chain. The heavy chain may be modified such that such half-antibodies or homodimers have very different retention times. In a preferred embodiment, the residence time of such half-antibodies and/or homodimers differs by greater than 10% from the average residence time of such corresponding antibodies or multispecific antibodies. In a preferred embodiment, the average of the pI values of the individual heavy and light chain combinations of an antibody sought to be not co-purified differs from the average of the pI values of the heavy and light chains of the at least two antibodies to be co-purified by more than 0.5 units.

The invention further provides a composition comprising 2 to 10 recombinant antibodies obtainable by a method as described herein. Also provided is a composition comprising 2-10 recombinant antibodies, characterized in that: the IEX retention times of at least two of such antibodies are substantially the same.

The invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that: the pI values of at least two of such antibodies differ from the average pI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The pI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less.

The invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that: the average "x" of the pI values of the two variable domains of the first antibody and the average "y" of the pI values of the two variable domains of the second antibody differ by 0.7, 0.6 and preferably 0.5 units or less from the average "x" and "y" of the first and second antibodies sought to be co-purified. The difference between "x" and "y" and the mean value of "x" and "y" of the first and second antibodies sought to be co-purified is preferably 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such antibodies (such as multispecific antibodies) typically have substantially the same retention time. In this embodiment, the constant regions of such antibodies are substantially identical. The pI values of the different variable domains of the antibody, each comprising a heavy chain variable region and a light chain variable region, and in particular the average of such pI values, represent the pI value of the antibody as a whole.

The invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that: the average of the pI values of the two heavy chain variable regions of the two variable domains of the first antibody "m" and the average of the pI values of the two heavy chain variable regions of the two variable domains of the second antibody "n" differs by 0.7, 0.6 and preferably 0.5 units or less from the average of the "m" and "n" of the first and second antibodies sought to be co-purified. The difference between "m" and "n" and the mean value of "m" and "n" of the first and second antibodies sought to be co-purified is preferably 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such antibodies (such as multispecific antibodies) typically have substantially the same retention time. In this embodiment, the constant region and the light chain variable region of such antibodies are substantially identical. The pI values of the different heavy chain variable regions of the antibody and in particular the average of such pI values represent the pI values of the antibody as a whole.

In a preferred embodiment, such IEX residence times and/or such pI values are preferably substantially the same for all such antibodies to be collected in the composition. In a preferred embodiment, at least two of such antibodies are bispecific antibodies. Preferably, at least two of such antibodies share one and the same heavy chain.

In some embodiments, one or two V_H/V_LThe common light chain variable region of the binding region includes a germline IgV kappa 1-39 x 01 variable region V-segment. In a particular embodiment, one or two V_H/V_LThe light chain variable region of the binding region includes a kappa light chain V-segment IgV kappa 1-39 x 01. IgV kappa 1-39 is a shorthand for the immunoglobulin variable kappa 1-39 gene. The genes are also called immunoglobulin kappa variable 1-39, IGKV139, IGKV 1-39. The external identifiers for this gene are: HGNC: 5740; entrez Gene: 28930, respectively; ensembl: ENGG 00000242371. The amino acid sequence for this V-region is provided in the sequence identification number: 25 in (b). The V-region may also be combined with one of the 5J-regions. The preferred J-regions are jk1 and jk5, and the linked sequences are denoted IGKV1-39/jk1 and IGKV1-39/jk5, with alternative names IgV κ 1-39 x 01/IGJ κ 1 x 01 or IgV κ 1-39 x 01/IGJ κ 5 x 01 (according to the nomenclature of the IMGT database global website located at IMGT. In some embodiments, one or two V_H/V_LThe light chain variable region of the binding domain includes kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 1X 05 (SEQ ID NO: 26 and SEQ ID NO: 27, respectively).

In certain embodiments, one or both V of a bispecific antibody_H/V_LThe light chain variable region of the binding domain comprisesComprises the following steps: an LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 22), an LCDR2 comprising the amino acid sequence AAS and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 24) (i.e. such CDRs according to IMGT, IGKV 1-39). In certain embodiments, one or both V of a bispecific antibody_H/V_LThe light chain variable region of the binding region comprises: an LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 22), an LCDR2 comprising the amino acid sequence AASLQS (SEQ ID NO: 23) and an LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 24).

In certain embodiments, one or both V of a bispecific antibody_H/V_LThe binding region comprises 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%) identical or 100% identical to the amino acid sequence set forth in sequence identification number: 26. In certain embodiments, one or both V of a bispecific antibody_H/V_LThe binding region comprises 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%) identical or 100% identical to the sequence identification number set forth: 27.

For example, in some embodiments, relative to the sequence identification number: 26 or sequence identification number: 27, one or two of a bispecific antibody_VH/VLThe variable light chain of the binding region may have from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. In certain embodiments, one or both V's of a bispecific antibody are relative to the amino acid sequence specified_H/V_LThe variable region of the light chain of the binding domain comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 20 to 1 and preferably 0 amino acid insertions, deletions, substitutions, additions or a combination of these.

In other embodiments, one or both V of a bispecific antibody_H/V_LThe light chain variable region of the binding region comprises the sequence identification number: 26 or sequence identification number: 27. In certain embodiments, two V's of a bispecific antibody_H/V_LThe binding regions comprise the same V_LAnd (4) a zone. In one embodiment, two V's of a bispecific antibody_H/V_LV of the binding region_LIncluding the recited sequence identification numbers: 26. In one embodiment, two V's of a bispecific antibody_H/V_LV of the binding region_LIncluding the recited sequence identification numbers: 27.

Bispecific antibodies, such as those disclosed in the methods herein, can be provided in a number of formats. Many different types of bispecific antibodies are known in the art. For example, not with two VH/V_LBispecific antibody versions of the exemplary antibodies in combination have at least one variable domain comprising a heavy chain variable region and a light chain variable region. This variable domain may be linked to a single chain Fv-fragment, monomer, a VH and a Fab-fragment providing a second binding activity.

Bispecific antibodies, such as those disclosed in the methods provided herein, are generally of the human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4, for example). In certain embodiments, such antibodies are of the human IgG1 subtype. Full-length IgG antibodies are preferred because of their favorable half-life and for low immunogenicity. Thus, in certain embodiments, such bispecific antibodies are full length IgG molecules. In one embodiment, such bispecific antibodies are full length IgG1 molecules.

In certain embodiments, the antibody comprises a crystallizable fragment (Fc). The Fc region of bispecific antibodies is preferably composed of a human constant region. A constant region or Fc of a bispecific antibody may contain one or more (preferably no more than 10, preferably no more than 5) amino acid differences to the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab-arm of such bispecific antibodies can further comprise an Fc-region comprising modifications that facilitate formation of the bispecific antibody, modifications that affect Fc-mediated effector function, and/or other features described herein.

In one aspect, provided is a pharmaceutical composition comprising two or more antibodies as defined herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a governmental regulatory agency or listed in the united states pharmacopeia or another generally recognized pharmacopeia for use in animals (particularly humans), and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, such as these, that are physiologically compatible. The term "carrier" means a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or aqueous common salt solutions as well as aqueous dextrose and glycerol solutions may be employed 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 such as these), the active compound may be encapsulated within a substance to protect the compound from acids and other natural conditions that may inactivate the compound.

Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier, or are suitable for reconstitution into a liquid solution or suspension for intravenous administration. The compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

Also included are solid preparations which are intended to be converted, shortly before use, to liquid preparations for oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions.

A "bispecific antibody" is an antibody as described herein, wherein one domain of the antibody binds to a first antigen and a second domain of the antibody binds to a second antigen, wherein the first and second antigens are not the same. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region/light chain variable region (V)_H/V_L) Combining a first epitope on an antigen, and a second V_H/V_LThe combination incorporates a second tabulation. The term further includes antibodies in which a VH is capable of specifically recognizing a first antigen, paired with the V in an immunoglobulin variable region_HV of_LCapable of specifically recognizing a second antigen. Formed V_H/V_LThe pair (pair) will bind either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO 2010/108127 and Schaefer et al (Cancer Cell 20, 472-. A bispecific antibody according to the invention is not limited to any particular format or method for generating it.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as: after aligning the sequences for optimal comparison purposes, the residues in a candidate sequence are the same percentage as the residues in a selected sequence. Comparison of percent sequence identity of nucleic acid sequences was determined using default values using the AlignX application of Vector NTI Programmabovance 10.5.2 software, such default values being determined using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J (1994) Nuc.acid Res.22: 4673. sup. 4680), swgapdna score matrix (swgapdpnarnt score matrix), a gap opening penalty of 15 (gapopen penalty) and a gap extension penalty of 6.66 (gapextension penalty). Amino acid sequences were aligned using default values using the AlignX application of Vector NTI Program Advance 11.5.2 software using a modified ClustalW algorithm (Thompson, j.d., Higgins, d.g., and Gibson t.j., 1994), a blosum62mt2 scoring matrix (blosum62mt2 score matrix), a gap open penalty of 10, and a gap extension penalty of 0.1.

The term "common light chain" as used herein means two light chains (or their V's) located in a bispecific antibody_LPortion). The two light chains (or VL portions thereof) may be identical or have some amino acid sequence differences while the binding specificity of the full-length antibody is unaffected. The terms "common light chain", "common V_L"," Single light chain "," Single V_L", with or without the addition of the term" rearranged ", are used interchangeably herein. "common" also means that the amino acid sequences of the light chains are not identical functional equivalents. Many variants of such light chains exist in which mutations (deletions, substitutions, insertions and/or additions) are present which do not affect the formation of functional binding regions. The light chain of the invention may also be a light chain as indicated herein above, having from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination of these. For example, light chains that are not identical but are still functionally equivalent are made or found to fall within the scope of the definition of a common light chain as used herein, e.g., by introducing and testing conservative amino acid changes, changes in amino acids located within regions that do not or only partially contribute to binding specificity when paired with the heavy chain, and the like. The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include an essentially intact IgG, but it does not necessarily have all the functions of an intact IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain containing a constant region(C) And variable regions (V) which can be broken down into regions designated CH1, CH2, CH3, V_HAnd C_L、V_LThe domain of (a). An IgG antibody binds to antigen via a variable region domain included in the Fab portion and can, upon binding, interact with molecules and cells of the immune system via such constant domains (mostly based on the Fc portion). Full length antibodies according to the invention encompass IgG molecules in which mutations providing the desired characteristics may be present. A full-length IgG should not have a deletion of a substantial portion of any of such regions. However, IgG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule are encompassed by the term "full-length IgG". For example, such an IgG molecule may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR regions, wherein such deleted amino acids are not necessary for the antigen or epitope binding specificity of the IgG.

Since an antibody typically recognizes an epitope of an antigen, and such an epitope may also be present in other compounds, an antibody according to the invention that "specifically recognizes" an antigen may also recognize other compounds if such other compounds contain the same epitope. Thus, the term "specifically recognizes", in the sense that an antigen interacts with an antibody, does not exclude the binding of such an antibody to other compounds containing an epitope of the same species.

The term "epitope" or "antigenic determinant" means an address on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear or conformational epitopes). Epitopes formed from linked linear amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally disorganized upon treatment with denaturing solvents. An epitope may typically comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an Epitope are known to those having ordinary skill in the art and include techniques in the art such as X-ray crystallography, deuterium-exchange mass spectrometry (HDX-MS), and two-dimensional nuclear magnetic resonance (2-dimensional nuclear magnetic resonance), peptide scanning (pepscan), and alanine scanning (alanine scan) according to the nature of the Epitope (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, vol.66, g.e.morris, Ed. (1996)).

For purposes of clarity and a concise description of the invention, features are described herein as part of the same or separate embodiments, however, it will be understood that: the scope of the present invention may include embodiments having all or some of the described combinations of features.

In order that the invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The term "including" is used without limitation to the use of other forms such as "include" of the verb prototype, "include" of the third-party's singular verb, and "include" of the verb's past form.

Drawings

FIG. 1 shows a schematic view of a

In one embodiment, the composition comprises two bispecific antibodies sharing a common arm. The figure depicts an antibody with heavy chain (1) and light chain (4). The 4 heavy chains have 3 non-identical variable regions (5, 6 and 7). The heavy chain with the shared variable region (5) has a portion of a heterodimerization domain (3). Such heavy chains with variable regions (6) and (7) have compatible portions of the heterodimerization domain (2). The preferred pairing of heterodimerization regions (2) and (3) can direct the formation of bispecific antibodies.

FIG. 2

Picture a: the bispecific antibody PB4516 generated the CIEX-plot at 220nm for number 8(p 08); picture B: the bispecific antibody PB6892 generated a CIEX-plot of number 4(p04) at 220 nm.

FIG. 3

FIG. 3 a: the bispecific antibody PB4516 generated the CIEX-plot at 220nm, No. 10(p 10);

FIG. 3 b: bispecific antibody PB11244 generated the CIEX-plot at 220nm for code 1(p 01);

FIG. 3 c: two bispecific antibodies were recognized in straight PB (pbxxxx (x)) per box. The heavy chain variable regions for PB11244 and PB4516 are indicated in straight target 1 and target 2. The sequence of the light chain is the same for all antibodies and has the serial identification number: 26, common light chain IgKV1 × 39/jk 1. For each heavy chain variable region, pI values calculated with the ExPASy ProtParam tool are indicated in the straight-line pI. The difference in pI values between the two heavy chain variable regions is shown in the last straight line demonstrating the average VH between PB11244 and PB4516_pIThe difference in value was 0.08.

FIG. 4

CIEX-plot of one antibody preparation of individual colonies cp12 of pool FST2 at 220 nm. The CIEX-plot shows a sharp peak for the co-eluting antibodies PB4516 and PB 11244. The graph shows that: the sample contains a limited amount of product related impurities. It also shows a good separation between the co-eluting bispecific antibody and the respective migrated product related impurities.

FIG. 5

CIEX-plot of an antibody preparation of community CP07 at 220 nm. The community is selected from a collection of individual communities of the single community FST2cp 09. A second sub-selection was performed to ensure that the FST2cp09-cp07 cell line was a single source cell line. Bispecific antibody specific ELISA indicated the presence of the EGFR/HER2 bispecific antibody PB11244 at 743. mu.g/mL and the EGFR/HER3 bispecific antibody PB4516 at 1134. mu.g/mL.

FIG. 6

Retention time of antibody homodimers (PGXXXX) with two identical variable domains. The amino acid sequence of the heavy chain variable region has the sequence shown in figure 8 for MF and one is the serial identification number: common light chain of 26 IgKV1 × 39/jk 1.

FIG. 7

Two bispecific antibodies were recognized in straight PB (pbxxxx (x)) per box. The heavy chain variable regions (MFXXXX) of each of such bispecific antibodies are indicated in direct targets 1 and 2. The sequence of the light chain is the same for all antibodies and has the serial identification number: 26, common light chain IgKV1 × 39/jk 1. For each heavy chain variable region, pI values calculated with the ExPASy ProtParam tool are indicated in the straight-line pI. The pI value difference between the two heavy chain variable regions and the measured residence time are indicated in the last two straight rows. The measured residence time and the calculated and average pI values indicate: bispecific antibodies of a pair can be co-eluted efficiently in CIEX chromatography. Such antibodies have an IgG1 constant region and a common light chain. Heavy chains with CH3 DE heavy chain or KK heavy chain with shared heavy chain variable regions (same MF) are indicated. The retention time for the CIEX chromatography performed on each specific antibody is indicated, and one exemplary condition for CIEX chromatography is described in the materials and methods section. Many other CIEX chromatography conditions will result in suitable residence times between the aligned bispecific antibody pairings with the provided pI values.

FIG. 8

The amino acid sequences of the CDRs and the amino acid sequence of the common light chain variable region of the corresponding antibody heavy chain variable region (MFXXXX).

Examples

Example 1

Materials and methods

Cell line

HEK293 and CHO-K1 were maintained in growth medium.

Production of bispecific antibodies

Bispecific antibodies were generated using proprietary CH3 technology (to ensure efficient heterodimerization and formation of a bispecific antibody as previously described (PCT/NL 2013/050294; published as WO 2013/157954 a1), the CH3 technology exploits charge-based point mutations in the CH3 region to allow efficient pairing of two non-identical heavy chain molecules.

One VH gene was cloned into one of two IgG1 vectors of non-identical architecture. Depending on the binding partner, the VH was cloned into an IgG1 framework comprising a CH3 variant with heterodimerization variant "DE" or an IgG1 framework comprising the complementary CH3 heterodimerization variant "KK". In the case of bi-or multispecific antibodies in which two or more antibodies share a heavy chain, the shared chain preferably has the CH3 heterodimerization variant "DE" (also referred to as the DE-heavy chain), while the two or more distinct heavy chains have the CH3 heterodimerization variant "KK" (also referred to as the KK-heavy chain).

HEK293 cells were transiently transfected with DNA-FUGENE mixture and further cultured. At 7 days after transfection, the supernatant was harvested and the medium was refreshed. At 14 days after transfection, the supernatant was combined and filtered through 0.22. mu.M. The sterile supernatant was stored at 4 ℃. Suspension adapted 293F cells were cultured in T125 flasks on an oscillator plate to a 3.0X 10⁶Density of individual cells/mL. The cells are in a range of 0.3-0.5X 10⁶Viable cells/mL were seeded into each well of a 24 deep well culture dish. Such cells were transiently transfected with individual sterile DNA: PEl-MIX and further cultured. At 7 days after transfection, the supernatant was harvested and filtered through 0.22. mu.M. The sterile supernatant was stored at 4 ℃.

Generation of a pool of stable cell lines co-expressing two bispecific antibodies

CHO cells were transfected with 3 heavy chain constructs and a common light chain construct in a common light chain construct (cLC): EGFR heavy chain: HER2 heavy chain: HER3 heavy chain at a molar ratio of 2.5:2: 1:1. 10 pools (A-J) of stably transfected cells were obtained. ELISA assays for anti-EGFR, anti-HER 2, and anti-HER 3 antibodies were performed on the day 3 and day 6 of the 10 pools, supernatant. All 3 specificities could be determined in all pools.

Generation of stable clones of cell lines co-expressing two bispecific antibodies

Such pools were plated in semi-solid medium and allowed to grow for 7-10 days. The single colonies were picked and seeded into 24-well culture plates. The colonies were reseeded before antibody collection from the supernatant of the culture.

Determination of antibody titers (antibodies)

The anti-HER 2 antibody titers of samples containing one single bispecific antibody were determined by ELISA against the Erbb-2Fc protein (R & D systems). The anti-HER 3 titer of samples containing one single bispecific antibody was determined by ELISA against human Erbb-3-Fc protein (R & Dsystems). The anti-EGFR antibody titers of samples containing one single bispecific antibody were determined by ELISA against human EGFR ECD-Fc protein (R & D systems). Serial 2-fold dilutions of such antigens were used to coat the wells of one ELISA plate, starting at 5 μ g/mL.

ELISA assays for quantifying EFGRxHER2 and EGFR x HER3 bispecific antibodies in compositions comprising the two bispecific antibodies were performed by coating ELISA plates with EGFR-Fc (R & Dsystems). After washing, such discs are incubated with the sample. After washing, the presence of bound bispecific antibody with an EFGR arm at grade one HER2 arm was detected by incubation with the labeled HER 2-Fc. The presence of bound bispecific antibodies with an EFGR arm at grade one HER3 arm was detected by incubation with the labeled HER 3-Fc.

IgG purification

Purification of IgG was performed using affinity chromatography. Purification was performed under sterile conditions using vacuum filtration. First, the pH of the medium was adjusted to pH 8.0 and the resultant was then incubated with protein ASepharose CL-4B beads (50% v/v) (Pierce) at 25 ℃ for 2 hours on a plate shaker set at 600 rpm. Second, such beads are harvested by vacuum filtration. The beads were washed 2 times with PBS pH 7.4. IgG was eluted at pH 3.0 using 0.1M citrate buffer, while IgG fractions were immediately neutralized by Tris pH 8.0. Buffer exchange was performed by centrifugation using ultracel (millipore). Such samples were finally in a final buffer at PBS pH 7.4.

Cation exchange Chromatography (CIEX)

CEX-HPLC chromatography was performed using an ion exchange column of the TSKgel SP-STAT series (7 μm particle size, 4.6mM I.D.. times.10 cm L, Tosoh 21964). Such columns are packed with non-porous resin particles that are additionally separated for velocity and high resolution analysis of biomolecules. The particles in the TSKgel STAT column contain an open access network of multiple layers of ion exchange groups for loading capacity, and the particle size makes these columns suitable for HPLC and FPLC systems.

TSKgel SP-STAT (7 μm particle size, 4.6mM I.D. times.10 cm L, Tosoh 21964) was equilibrated with buffer A (sodium phosphate buffer, 25mM, pH 6.0), after which the antibody was drained from the column by increasing the salt concentration and running a gradient of buffer B (25mM sodium phosphate, 1mM NaCl, pH 6.0). The flow rate was set at 0.5 mL/min. The injected sample mass for all test samples as well as the control group (in PBS) was 10. mu.g, while the injection volume was 10-100. mu.L. The chromatogram was analyzed with respect to the observed peak pattern, retention time and peak area of the main peak based on the 220nm results.

Results

CIEX plots of bispecific antibodies PB4516p08 and PB6892p04 were compared (see fig. 2). What is observed is: the production of PB6892 contained a significant amount of impurities. Also, the residence time of the bispecific antibody fraction of PB6892 was significantly lower than that of the bispecific antibody fraction of PB 4516. The residence times of the two bispecific antibodies are preferably closer together for co-production by the same cell and subsequent co-purification using CIEX. For this reason, the variable region of the HER2 arm of PB6892 was replaced with a different variable region. The heavy chain with variable region MF2032 was selected and used to generate EGFR x HER2 bispecific antibody PB 11244. CIEX plots of PB4516p10 and PB11244p01 are shown in fig. 3. The residence time of such bispecific antibody fractions was 16.310 and 16.950, respectively. These residence times were sufficiently identical to allow co-purification using CIEX under the conditions indicated. Further, the figure shows: the residence times of the impurities are sufficiently different to allow effective separation in both the analytical and preparative columns. The bispecific antibody preparation described above was generated in HEK293 cells.

For co-production, CHO-K1 cells were used. CHO cells were transfected to contain the three heavy chain constructs with the corresponding variable regions of MF3755(EGFR), M2032(HER2) and MF3178(HER3) along with the sequence identification numbers: 26 to transfect CHO-K1 cells. Vector positive cells were selected and pooled. 10 independent pools (identified as A-J) consisting of transfected CHO-K1 cells were generated.

Table 1 shows the number of bispecific antibodies PB4516(EGFR × HER3) and PB11244(EGFR × HER2) produced by the respective pools. Again, the ratio of such amounts is further shown by the total amount of IgG produced. Pools F and J were selected for sub-colonization.

Table 2 shows the number of bispecific antibodies PB4516(EGFR × HER3) and PB11244(EGFR × HER2) produced by the corresponding clones.

Antibodies generated by the clone FST2cp12 were used to analyze the CIEX plot (see figure 4). Clearly visible are: the two bispecific antibodies were co-eluted efficiently in the same CIEX eluate fraction.

The colonizer FST2cp09 was further sub-colonized to ensure that the cell line was unigenic, and a further CIEX plot identified such antibodies as being produced. Fig. 5 shows the CIEX plot. Clearly visible are: the two bispecific antibodies were co-eluted efficiently in the same CIEX eluate fraction. The relative contribution of the two bispecific antibodies in the co-elution was analyzed by ELISA and/or by hydrophobic interaction column. Bispecific antibody specific ELISA indicated the presence of the EGFR/HER2 bispecific antibody PB11244 at 743. mu.g/mL and the EGFR/HER3 bispecific antibody PB4516 at 1134. mu.g/mL.

Example 2

Generation of a pool of stable cell lines co-expressing two bispecific antibodies

Cell lines representing the double paired (two by two) bispecific antibodies listed in figure 7 were generated as follows. CHO cells were transfected with 3 heavy chain constructs and 1 common light chain construct. The 3 heavy chains were identified by the heavy chain variable regions (MFXXXX) indicated in the box. The light chain includes a light chain having the serial identification number: 26 of IgV κ 1 × 39/jk 1. The two bispecific antibodies have one heavy chain in common and one heavy chain each different. For example, the first pair indicated in fig. 7 shares a common heavy chain (comprising the same HER3 binding arm comprising a heavy chain variable region (MF3178)) and a non-identical 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 (MF 3958). The shared heavy chain has the KK CH3 region of a compatible DE/KK heterodimerization domain. The shared heavy chain arm may also have the DE CH3 region. For example, at fig. 3-5, two bispecific antibodies were co-purified, PB11244 and PB 4516. As shown at figure 3c, PB11244 and PB4516 share the same EGFR binding arm with a heavy chain variable region (MF3755), and PB11244 has a HER2 binding arm with a heavy chain variable region (MF2032), while PB4516 has a HER3 binding arm with a heavy chain variable region (MF 3178). The shared heavy chain arm in the bispecific antibody of this pair has the DE CH3 region, while such non-identical HER2 and HER3 binding arms have the KK CH3 region.

Common light chain construct (cclc): shared heavy chain construct: non-identical heavy chain construct 1: the moir ratio of the non-identical heavy chain construct 2 was 2.5:2:1: 1. A pool of stably transfected cells was obtained. ELISA analysis of antigens was performed on supernatant collected from such wells. All 3 antigen binding species were determined in this pool. The CIEX retention time of the bispecific antibody located in each pair of fig. 7 was determined under similar CIEX conditions and is indicated in the 8 th straight line. The deviation from the mean residence time was calculated using the formula 100x (a-B)/(a + B)), where a is the residence time of the bispecific antibody with the longest residence time. For example, the deviation for the first pair is 100 × ((16.46-16.24)/(16.46+16.24)) -0.67 or 0.7%.

The antibody in the collected supernatant is first extracted by protein a followed by acid extraction and rapid neutralization, while the other proteins in the supernatant are separated. The buffer of the collected antibody was then exchanged for PBS. Such samples were then loaded onto a CIEX column and washed and eluted by administering an increased salt gradient. The absorption of the eluate was measured at 220nm, while the retention time was calculated from the onset of the salt gradient and observation of the peak of the bispecific antibody. Such bispecific antibodies were collected and the corresponding bispecific antibodies in the collected eluate were validated by ELISA (verified). The retention time of the corresponding bispecific antibody is indicated in the last straight line. Clearly visible are: many pairs have residence times that effectively co-elute in one CIEX column. It is also clear that: the CIEX chromatography provided a good separation of the co-eluted bispecific antibody and the corresponding homodimer (if any). FIG. 6 is a bar listing the residence times of various antibodies with homodimers of heavy chains (including the heavy chain variable region) present in the coeluting bispecific antibody. Clearly visible are: the residence time of such homodimers is sufficiently different from the corresponding bispecific antibody. For example, in the first box of fig. 7, such homodimers PG3178, PG3958, and PG4003 may be present in one product. The retention times for such corresponding homodimers were about 22, 12 and 13 (fig. 6, rows 1-3), while the retention times for such bispecific antibodies including the heavy chain variable region were about 19 and 19.5 (fig. 7, rows 1 and 2).

TABLE 1

Table 1: quantification of EGFRxHER2 and EGFRxHER3 bispecific antibody production in cell pools. The supernatant of the culture from 10 wells (A-J) was evaluated. ELISA assays were based on EGFR-Fc coating, binding of the generated antibodies, and detection using either labeled HER2-Fc or labeled HER 3-Fc. The A-J pool was analyzed using the two ELISA assays. Bispecific antibodies PB4516 and PB11244 are IgG1 heavy chain antibodies with compatible DE/KK heterodimerization domains. Such heavy chains are combined with the common light chain. Such bispecific antibodies share the MF3755 heavy chain variable region on one heavy chain, and each have a non-identical heavy chain variable region on the other IgG1 heavy chain (MF3178 for PB4516, and MF2032 for PB 11244).

TABLE 2

Table 2: the selected pool was used for single cell colonization. 18 communities were selected from 3 pools. Two separate F pools (FST1 and FST2) and one J pool (JST1) were used for single cell colonization. Indicating "cp" in turn identifies the individual communities of a pool by a number. The selected colonies were allowed to grow and used for antibody collection. Individual colonies from the same pool produced different amounts and ratios of the corresponding bispecific antibody.

Claims (23)

1. A method for generating at least two antibodies, comprising:

-providing cells carrying nucleic acids encoding such antibodies;

-culturing such cells;

-collecting such antibodies from the culture; and

-separating the generated antibodies from the half-antibodies by ion exchange chromatography (IEX);

the method is characterized in that: such antibodies exhibit an IEX retention time that deviates 10% or less from the mean value of the retention times of the individual antibodies under the IEX conditions used.

2. The method according to claim 1, wherein collecting such antibodies from the culture comprises purifying antibodies from other proteins by antibody affinity purification, preferably by protein a extraction.

3. The method of claim 2, further comprising introducing the affinity-purified antibody to size-exclusion chromatography (gel filtration chromatography and/or anion exchange chromatography).

4. The method according to claims 1-3, wherein after the IEX, the collected antibodies are quantitatively analyzed for relative performance levels by Hydrophobic Interaction Chromatography (HIC).

5. The method of claim 4, wherein the specificity of the collected antibodies is verified by ELISA.

6. The method of claims 1-5, wherein the residence time of the respective half-antibodies falls outside the range spanned by the residence times of such antibodies.

7. The method of claim 6, wherein such cells produce 3 heavy chains.

8. The method of claim 7, wherein such heavy chains comprise a domain for efficient heterodimerization of the heavy chains.

9. The method of claims 1-8, wherein at least two of such antibodies are bispecific antibodies.

10. The method of claims 1-9, wherein at least two of such antibodies share one identical heavy chain.

11. The method of claims 1-10, wherein such antibodies have an isoelectric point (pI) which differs from the average pI value of the at least two antibodies by 0.4 units or less.

12. The method of claims 1-11, wherein such antibodies are selected for having heavy and light chain combinations that have retention times under the IEX conditions used that are significantly different from the retention times of intact antibodies.

13. The method of claim 12, wherein the pI value of the heavy chain in combination with the light chain differs from the average pI value of the at least two antibodies by 0.4 units or less.

14. The method according to claims 1-13, wherein such heavy chains comprise a CH3 domain that facilitates heterodimerization of the heavy chains.

15. The method of claims 1-14, wherein the heavy chain of such antibody is an IgG heavy chain.

16. The method of claims 7-15, wherein one heavy chain comprises amino acid substitutions L351K and T366K (EU numbering) located in the CH3 region, and the other heavy chain comprises amino acid substitutions L351D and L368E located in the CH3 region.

17. A method for generating at least an antibody, comprising:

-providing cells carrying nucleic acids encoding such antibodies;

-culturing such cells;

-collecting such antibodies from the culture; and

-separating the generated antibodies from the half-antibodies by ion exchange chromatography (IEX);

the method is characterized in that: such antibodies exhibit an IEX retention time that deviates 10% or less from the mean value of the retention times of the individual antibodies under the IEX conditions used, and wherein after the IEX the collected antibodies are quantitatively analyzed for relative performance levels by Hydrophobic Interaction Chromatography (HIC), and the specificity of the collected antibodies is verified by ELISA.

18. A composition comprising 2-10 recombinant antibodies obtainable by a method according to claims 1-17.

19. A composition comprising 2-10 recombinant antibodies, characterized in that: under such IEX conditions, the IEX retention time of at least two of such antibodies deviates by 10% or less from the mean of the retention times of the individual antibodies.

20. A composition comprising 2-10 recombinant antibodies, characterized in that: the pI values of at least two of such antibodies differ from the average pI value of the at least two antibodies by 0.4 units or less.

21. The composition according to claims 18-20, characterized in that: such IEX retention times and/or such pI values are substantially the same for all such antibodies.

22. The composition of claims 18-21, wherein at least two of such antibodies are bispecific antibodies.

23. The composition of claim 22, wherein at least two of such antibodies share one identical heavy chain.

Images