Method of separating one or more antibodies from one or more other compounds in a liquid sample, kits for purifying antibodies from one or more other components in a liquid, and for capturing antibodies from one or more other components in a liquid , chromatography column, and, disposable filter for antibody purification

Abstract

METHOD OF SEPARATING ONE OR MORE ANTIBODIES FROM ONE OR MORE COMPOUNDS IN A NET SAMPLE, KITS FOR THE PURIFICATION OF ANTIBODIES FROM ONE OR MORE OTHER COMPONENTS IN A NET, AND FOR CAPTURING ANTIBODIES FROM ONE OR MORE COMPONENTS IN A NET , CHROMATOGRAPHY COLUMN, AND, DISPOSABLE FILTER FOR ANTIBODY PURIFICATION. The present invention relates to a method of separating antibodies from other compound (s) in a liquid sample, a mobile phase comprising said sample being contacted with a multimodal separation matrix to adsorb unwanted compounds, while antibodies remain free in the liquid, with the multimodal separation matrix comprising first groups, which are able to interact with negatively charged sites of the target compounds, and second groups, that are capable of at least one different charge-charge interaction with said target compounds. The invention also relates to a chromatography column filled with the multimodal separation matrix described above and a filter showing said multimodal groups adsorbed on its surface.

Description

10518207-7 METHOD OF SEPARATING ONE OR MORE ANTIBODIES FROM A OR OTHER COMPOUNDS IN A LIQUID SAMPLE, KITS For the purification of antibodies from one or more other components in a liquid, and for the capture of 5 ANTIBODIES OF ONE OR MORE OTHER COMPONENTS IN A LIQUID, CHROMATOGRAPHIC COLUMN, AND DISPOSABLE FILTER FOR ANTIBODY PURIFICATION Technical field The present invention relates to a method of purifying 10 antibodies. The method can be used, for example, in raw feed, or as a subsequent step to affinity chromatography to remove remaining contaminants and substances that have leaked from the affinity resin. The present invention also comprises a kit for the purification of antibodies. Previous The immune system consists of many types of interdependent cells that collectively protect the body from bacterial, parasitic, fungal, and viral infections, and from the growth of tumor cells. The guardians of the immune system are the macrophages, which continuously roam the bloodstream of their host. When exposed to infection or immunization, macrophages respond to invaders by engulfing those marked with foreign molecules known as antigens. This event, mediated by helper T cells, imposes a complicated chain of responses that result in the stimulation of B cells. These B cells, in turn, produce proteins called antibodies, which bind to the foreign invader. The antibody-antigen binding event marks the foreign invader for destruction via phagocytosis or activation of the complement system. There are a number of different classes of antibodies, also known as immunoglobulins, such as IgA, IgD, IgE, IgG and IgM. They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies have been extensively studied, perhaps due to the dominant role they play in a mature immune response. Polyclonal antibodies are produced according to conventional methods by immunizing an animal with the appropriate antigen. In response, the animal will produce antibodies that are polyclonal. However, for many purposes, it is desirable to have a single clone of a given antibody, known as monoclonal antibodies. Monoclonal antibodies (MAbs) are produced using hybrid or fused cells consisting of a fusion between a normal B cell, which produces only a single antibody, and an abnormal myeloma tumor cell. The resulting hybrid, known as a hybridoma, is currently used in conventional methods for antibody production. The biological activity exhibited by immunoglobulins is currently being explored in a range of diverse applications in the human and veterinary diagnostic, health treatment, and therapeutic sectors. Indeed, in the last few years, monoclonal antibodies and recombinant antibody constructs have become the largest class of proteins currently investigated in clinical trials and receiving FDA approval as therapeutics and diagnostics. Complementing expression systems and production strategies, efficient purification protocols are needed to obtain highly pure antibodies in a simple and economical way. Traditional methods for isolating immunoglobulins rely on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other protein groups in solution. Typical precipitating agents are ethanol, polyethylene glycol, lyotropic salts such as ammonium sulfate and potassium phosphate, and acid. Caprylic acid. Typically, these precipitation methods yield highly impure products while being time-consuming and laborious to produce. Additionally, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem, which is particularly relevant when discussing large-scale purification of immunoglobulins. An alternative method for isolating immunoglobulins is chromatography, which comprises a family of closely related separation methods. The characteristic that distinguishes chromatography from most other physical and chemical separation methods is that it involves two mutually miscible phases, one stationary and the other miscible. The sample mixture, introduced into the mobile phase, undergoes a series of interactions with the stationary and mobile phases as it travels through the system. These interactions exploit differences in the physical or chemical properties of the components in the sample. These differences determine the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in order of increasing interaction with the stationary phase. The most retarded component elutes first, the most strongly retained material elutes last.Separation is achieved when a component is sufficiently retarded to prevent overlapping with the zone of an adjacent solute as sample components elute from the column. Continuous efforts are made to design the optimal stationary phase for each specific separation purpose. Commonly, a stationary phase of this type consists of a support or base matrix to which a ligand comprising linking groups, i.e., functional groups, is attached. Reference is commonly made to each type of chromatography based on the interaction principle it uses, such as ion-exchange chromatography, hydrophobic interaction chromatography, and so on. Affinity chromatography. Ion-exchange chromatography is frequently used in protocols for the isolation of immunoglobulins. In ion-exchange chromatography, negatively charged amino acid side chains of the immunoglobulin will interact with positively charged ligands of a chromatography matrix. Conversely, in cation-exchange chromatography, positively charged amino acid side chains of the immunoglobulin will interact with negatively charged ligands of a chromatography matrix. Hydrophobic interaction chromatography (HIC) is another method described and used in protocols for the isolation of immunoglobulins. If the object is a highly pure immunoglobulin product, it is commonly recommended to combine HIC with one or more additional steps. In HIC, aiming to efficiently bind the immunoglobulin with the HIC matrix, the addition of lyotropic salts to the mobile phase is necessary. The bound immunoglobulin is subsequently released from the matrix by reducing the concentration of lyotropic salt. Thus, the disadvantage of this procedure is the need to add lyotropic salt to the raw material, as this can cause problems and consequently increase costs for the user on a large scale.For example, in the case of raw materials such as serum, plasma, and egg yolk, the addition of lyotropic salts to the raw materials could, in some cases, be prohibitive in large-scale applications, as the salt could prevent any economically feasible use of the immunoglobulin-depleted raw material. An additional problem in large-scale applications could be the disposal of several thousand liters of waste. Affinity chromatography is based on specific interactions between a target biomolecule and a biospecific ligand in a lock-and-key recognition principle. Thus, the target and ligand will constitute an affinity pair, such as antigen / antibody, enzyme / receptor. etc. Protein-based affinity ligands are well known, such as Protein A and Protein G affinity chromatography, which are both widespread methods for antibody isolation and purification. It is generally known that Protein A chromatography provides remarkable specificity, particularly with respect to monoclonal antibodies, and consequently, high purities can be obtained. Used in combination with ion exchange, hydrophobic interaction, hydroxyapatite steps and / or gel filtration, Protein A-based methods have become the method of choice for antibody purification for many biopharmaceutical companies, see e.g. WO 8400773 and US 5,151,350. However, due to the peptide bonds of the proteins, Protein A matrices exhibit a certain degree of alkaline sensitivity.Additionally, when using Protein A matrices to purify antibodies from cell culture media, proteases originating from the cells can cause leakage of Protein A, or peptide fragments thereof. An attempt to reduce ligand leakage from affinity chromatography matrices was presented in WO 03 / 041859 (Boehringer Ingelheim Pharma KG), suggesting pre-treating, for example, Protein A matrices with at least one surfactant to reduce ligand leakage. The affinity matrix can be treated, for example, with 5-15 volumes of surfactant bed. Contact time is crucial for the effectiveness of the process. For example, at room temperature, a contact time of at least 16 h is required for leakage reduction. An alternative approach to the problem of ligand leakage from affinity chromatography matrices is provided in US 4,983,722 (Miles Inc.), whereby Protein A is selectively isolated from a liquid containing antibody and Protein A by exposing the liquid to an anion exchange material. Both components are adsorbed onto the anion exchange material, and the antibodies and Protein A are then eluted. 6 I ;• : : : 7: : : : : sequentially under conditions of increasing ionic intensity. An illustrative anion exchanger is diethylaminoethyl (DEAF) Trisacryl M or DEAF Sepharose™. WO 2004 / 076485 (Lonza Biologies Pic.) refers to the purification of antibody by Protein A ion-exchange chromatography. The ion-exchange step comprises loading the purified Protein A antibody onto an ion-exchange material under conditions that permit Protein A binding and collecting the antibody in the percolated material. The anion exchanger is a quaternary amine-based anion exchanger, most preferably Sepharose™ Q (Amersham Biosciences, now GE Healthcare). US Patent 5,429,746 (SmithKline Beecham Corp.) refers to a process in which antibody is first adsorbed onto a Protein A chromatographic support and eluted; then adsorbed onto a cation exchange support and selectively eluted from it; and finally adsorbed onto an HIC support and eluted. The mixture applied to the EHC column, after affinity chromatography and / or cation exchange chromatography, may contain immunoglobulin aggregates, misfolded species, host cell proteins, and residual material from the affinity chromatography step. US Patent 6,498,236 (Upfront Chromatography) refers to specific problems caused by small differences in molecular weight between protein-based affinity ligands and target immunoglobulins. Therefore, a method for isolating or purifying immunoglobulins from a solution, such as a hybridoma cell culture supernatant, serum, or animal plasma, is disclosed. This method is suggested as an alternative to the use of Protein A, Protein G, synthetic peptides, and other ligands with relatively high molecular weights. The solid-phase matrices used in the disclosed method are defined by the formula M-SP1-XA-SP2-acid, where M indicates the matrix backbone, SP1 indicates a... Spacer, X indicates O, S, or NH, A indicates an aromatic or heteroaromatic, mono- or bicyclic moiety, optionally substituted, SP2 indicates an optional spacer, and acid indicates an acid group. The specific substituents are indicated as being decisive in whether the matrix will bind immunoglobulins efficiently. US 5,945,520 (Burton et al.) discloses mixed-mode chromatographic resins that exhibit a hydrophobic character at the binding pH and a hydrophilic and / or electrostatic character at the desorption pH. The resin is specifically designed to bind the target compound from an aqueous solution at both low and high ionic concentrations. Thus, the adsorption step employs HIC, while desorption is based on charge repulsion. US Patent 6,702,943 (Johansson et al.) discloses a method for removing a target substance from a liquid by adsorbing it onto a matrix bearing a plurality of ligands comprising anion-exchange groups and a hydrophobic structure. More specifically, the ligands contain an aromatic ring in the vicinity of the positively charged anion-exchange groups. It is claimed that the inclusion of other groups capable of electron-acceptor-electron-donor interactions can enhance the intensity of the interaction between the substance and the adsorbent. It is claimed that the desired substances are cells, cell parts, and substances comprising peptide structures. Matrix traversal capacity is defined by reference proteins such as bovine serum albumin and IgG. The disclosed ligands are indicated as "high-salt ligands" due to their ability to adsorb target substances at high salt concentrations, such as 0.25 M NaCl. Additionally, WO 01 / 38228 (Belew et al.) discloses another method for removing a negatively charged substance from a liquid by binding it to a matrix comprising mixed-mode anion exchange ligands. Each ligand comprises a nitrogen • • • • ••• • ••• • • positively charged and a thioether bond at a distance of 1 to 7 atoms from said charged nitrogen. Similarly to what is indicated above, the desired substances, such as cells, cell parts and substances comprising peptide structures, are adsorbed at saline concentrations in the region of 0.25 M NaCl. Ceramic hydroxyapatite has been suggested as useful for polishing immunoglobulin. More specifically, it has been reported (Chromatography, technical note 2849; SG Franklin, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA) that IgG1 can be resolved from an IgG1-Protein A complex in unfractionated medium on CHT ceramic hydroxyapatite (Bio-Rad). More specifically, hydroxyapatite (CaI(PO4)6(OH)2) is a form of calcium phosphate, which, as has been shown, possesses unique separation properties. However, hydroxyapatite-based matrices are also known to involve certain disadvantages. For example, due to Ca leakage, they are unstable at acidic pH values, and are sensitive to chelating agents such as EDTA. Additionally, it has been shown that it is difficult to develop, and scale up, a robust and reproducible purification method using hydroxyapatite-based matrices, e.g.Because it was difficult to pack hydroxyapatite and maintain performance in large columns. Finally, there is a risk of changes in the resin's properties caused by contamination with metal ions and calcium ion exchange, and this alteration is a serious problem for regulatory authorities. Johansson et al (Journal of Chromatography A, 1016 (2003) 21-33: "Preparation and characterization of prototypes for multimodal separation media aimed for capture of negatively charged biomolecules at high salt conditions") describe the selection This study investigated the development of multimodal ligand prototypes for capturing negatively charged proteins from highly conductive mobile phases. It was found that non-aromatic multimodal anion exchange ligands based on weak ion exchange ligands (primary and secondary amines) were optimal for capturing proteins via adsorption under high salinity conditions. Brief description of the invention One aspect of the present invention consists in preparing a method for separating antibodies from other components of a liquid, which requires less time and fewer process steps than prior art methods. This can be achieved by means of a method in which the liquid comprising antibodies is contacted with a multimodal separation matrix, and substantially pure antibodies are recovered in non-binding mode. For example, if the liquid is applied to a chromatography column comprising said matrix, the antibodies are readily recovered from the percolated material. Another aspect of the invention consists of preparing a method for separating antibodies from other components of a liquid, obtaining unprecedented specificity compared to state-of-the-art methods. An additional aspect of the invention consists in preparing a method for separating antibodies from other components of a liquid, whereby the elimination of contaminants present in raw feed, such as host cell proteins, is improved. Additional aspects and advantages of the invention will appear in the detailed description that follows. Brief description of the drawings Figure 1 a) - d) shows an illustrative selection of multimodal anion exchange ligands useful in the method of the present invention: N-benzyl-N-methyl ethanolamine, N,N-dimethylbenzylamine, 2-aminobenzimidazole and thiomycamine. Figure 2 shows a chromatogram of monoclonal antibody separation in multimodal separation matrices comprising N-benzyl-N-methylethanolamine immobilized on Sepharose™ 6 FF; N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF; and, for reference, the strong anion exchanger Q Sepharose™ FF, as described in Example 1 below. Figure 3 a) and b) shows chromatograms of monoclonal antibodies loaded onto separation matrices comprising thiomycamine and 2-aminobenzimidazole immobilized on Sepharose™ 6 FF at different densities, as described in Example 3 below. Figure 4 a)-g) shows the results of chromatography performed on prototypes with a mixture of mAbl-rProtein A. Figure 5 a)-h) shows the results of analytical size exclusion chromatography (SEC) on the sample with MAbl, 1% rPrA and combined percolated material and elute fractions from the chromatographic operations in Figure 4. Fig. 6 shows the separation of monoclonal antibody molecules using the Q Phenila Sepharose™ Fast Flow multimodal array, as described in Example 5. Definitions The terms "antibody" and "immunoglobulin" are used interchangeably in this description. The term "separation matrix" is used here to indicate a material consisting of a support onto which one or more binders comprising functional groups have been attached. The term "multimodal" separation matrix refers to a matrix capable of providing at least two different, yet cooperative, sites that interact with the compound to be bound. For example, one of these sites may provide an attractive type of charge-charge interaction between the ligand and the substance of interest. The other site may provide electron donor-acceptor interactions and / or hydrophobic and / or hydrophilic interactions. Donor-acceptor electron interactions include interactions such as hydrogen bonding, tctt, cation-7r, charge transfer, dipole-dipole, induced dipole, etc. "Multimodal" separation matrices are also known as "mixed-mode" separation matrices. The term "surface" here means all external surfaces, and includes, in the case of a porous support, both external surfaces and pore surfaces. The expression "donor-acceptor electron interactions" means that an electronegative atom with a lone pair of electrons acts as a donor and bonds to an electron-deficient atom that acts as an acceptor for the donor's electron pair. (See, for example, Karger et al., An Introduction into Separation Science, John Wiley & Sons (1973), page 42.) The term "anion exchange group" here means a group that is positively charged or chargeable. The term "eluent" is used in its conventional meaning in this field, i.e., a buffer of appropriate pH and / or ionic strength to release one or more compounds from a separation matrix. In the context of liquid chromatography, the term "capture step" refers to the initial step in a separation procedure. Most commonly, a capture step includes clarification, concentration, stabilization, and significant purification of soluble impurities. Following the capture step, an intermediate purification may occur, which further reduces remaining impurities such as host cell proteins, DNA, viruses, endotoxins, nutrients, components of a cell culture medium such as antifoaming agents and antibiotics, and product-related impurities such as aggregates, malformed species, etc. Folded and aggregated. The term "polishing step" refers, in the context of liquid chromatography, to a final purification step where trace impurities are removed, leaving an active and safe product. Impurities removed during the polishing step are often conformers of the target molecule or products suspected of leakage. The term "Fc-binding protein" means a protein capable of binding to the crystallizable (Fc) portion of an antibody and includes, for example, Protein A and Protein G, or any fragment or fusion protein thereof that has retained said binding property. Detailed description of the invention In a first aspect, the present invention relates to a method or antibodies for separating one or more other compounds from a liquid sample, wherein a mobile phase comprising said liquid sample is contacted with a multimodal separation matrix to adsorb one or more target compounds, while the antibodies remain free in the mobile phase, the multimodal separation matrix comprising first groups capable of interacting with negatively charged sites of the target compound(s) and second groups capable of at least one interaction other than charge-charge interaction with said target compound(s). The present invention also comprises a method in which third groups or additional groups are added in addition to the first and second groups. In an advantageous embodiment, the present method is carried out using the principles of liquid chromatography, i.e., passing a mobile phase over a chromatography column comprising the multimodal separation matrix. The support may be in the form of porous or non-porous pairs, such as essentially spherical particles, a monolith, filter, membrane, surface, capillaries, or any other commonly used shape. In an alternative embodiment, the present method is This method is carried out using the principles of expanded bed chromatography, i.e., by adding the mobile phase to an expanded bed of separation matrix in the form of particles, such as essentially spherical particles, comprising a high-density charge. In another alternative embodiment, the present method is carried out using a batch process, wherein the separation matrix is ​​added to a vessel comprising the liquid sample. Thus, in the method for antibody purification according to the invention, one or more undesirable compounds are adsorbed onto the separation matrix while the desired antibodies remain in the mobile phase without being adsorbed. In the context of the present method, the term "target compounds" refers to the compounds adsorbed onto the separation matrix. Obviously, the nature and identity of the adsorbed compounds will depend on the origin of the liquid sample. Examples of target compounds are cells and cell fragments; proteins and peptides; nucleic acids, such as DNA and RNA; endotoxins; and viruses. In one embodiment of the present invention, the multimodal separation matrix is ​​provided in a chromatography column, and the mobile phase is passed through said column by gravity and / or pumping, with the antibodies being recovered in the percolated material from the column. An advantage of the present method is that it does not require any elution of the antibodies from the column. Avoiding a specific elution step is advantageous from a process standpoint because fewer steps will result in a faster purification protocol and, consequently, reduce process costs. Additionally, antibodies are sensitive to certain conditions that can impair their folding pattern or degrade them by attacking their peptide bonds. Although elution conditions for anion exchangers generally do not involve any extreme chemicals, each change in salinity and pH This can affect the sensitive antibody, with the effect varying from species to species depending on pH, charge distribution, etc. Consequently, another advantage of the present method is that it avoids adding an eluent and applying elution conditions to the antibodies. As mentioned above, in the method according to the invention, the target compounds, from which the antibodies are to be separated, are adsorbed onto the multimodal separation matrix. To obtain the most advantageous conditions for adsorption of target compounds, the liquid sample is combined with an appropriate buffer or other liquid to provide a mobile phase. The present method is advantageously carried out under conventional conditions for ion-exchange chromatography, which commonly involves adsorption at a relatively low salt concentration. Thus, in one embodiment of the present method, the conductivity of the mobile phase is in the range of 0-25, such as 10-15 MS / cm. In one embodiment, the pH of the mobile phase is about 5-6. A person skilled in this field can easily adapt conditions to obtain percolated material from the antibodies, e.g., by adjusting the pH or conductivity, which will depend, for example, on the charge and charge distribution of the antibodies to be purified.If necessary, one or more washing steps can be applied before or between any of the aforementioned passes. If it is desired to subsequently release the adsorbed compounds, e.g., for matrix reuse, elution can be carried out at a higher salt concentration, e.g., by using an increasing salt gradient. The pH value can be shifted, equally or alternatively, e.g., as a decreasing pH gradient, to elute adsorbed compounds. As mentioned above, the multimodal separation matrix comprises first groups, which are capable of interacting with negatively charged sites of the target compounds, and second groups, which are capable of at least one interaction other than charge-charge interaction with referred to as target compounds. In this context, it is understood that the different interaction modes of the separation matrix groups are directed towards the same target compound, i.e., each target compound is ideally adsorbed by two or more interaction modes. Multimodal ligands comprising positively charged or chargeable anion exchange groups are known in this field, see, e.g., US 6,702,943 (Johansson et al), WO 01 / 38228 (Belew et al), and WO 02 / 053252 (Belew et al). In one embodiment, the first groups, i.e., the anion-exchange groups of the multimodal separation matrix, are strong anion exchangers. In this context, the term "strong" anion exchangers is understood as groups that remain charged over a wide pH range. In an advantageous embodiment, the strong anion-exchange groups are quaternary amines, also known as Q groups. In an alternative embodiment, the first groups of the multimodal separation matrix are weak ion exchangers. In this context, the term "weak" anion exchangers is understood as meaning groups that are charged at certain pH values ​​but can lose charge via pH exchange. In a specific embodiment, the first groups comprise a mixture of anion-exchange groups and additional functionalities such as anion exchangers and hydrogen bonding groups. Thus, in this embodiment, the first groups may be TRIS (tris(hydroxymethyl)aminomethane). In one embodiment, the second groups of the multimodal separation matrix comprise aromatic groups and / or hydrogen bonding groups. In one embodiment, said aromatic groups comprise ring systems comprising aromatic or heteroaromatic structures. In an advantageous embodiment, the second groups comprise phenyl groups. Alternatively, the second group may comprise a mixture of hydrophobic aromatic and non-aromatic groups. Aromatic compounds, such as alkyl groups. Thus, in a specific embodiment, the first groups comprise alkyl groups. The separation matrix used according to the invention may comprise two or more functional groups of the same type, such as two or more different types of hydrophobic groups; or two or more different types of multimodal anion exchangers. As anyone versed in this field will understand, the functional groups of the separation matrix used in the present method may be present in the same ligand, in which case each ligand is multimodal, or in different ligands, in which case the overall nature of the separation matrix is ​​multimodal. Thus, in one embodiment, the separation matrix comprises first and second groups coupled to the same ligands. Any of the first and second groups discussed above can be used in this embodiment, such as quaternary amine groups and phenyl groups. In one embodiment, the ligands were coupled to the support via their first groups, such as via amines resulting in quaternary amines. In another embodiment, the first and second groups are spaced apart by a hydrocarbon chain comprising 1-6, such as 1-3, preferably 1-2 carbon atoms. In a specific embodiment, the ligands are selected from the group consisting of N-benzyl-N-methylethanolamine; N,N-dimethylbenzylamine; 2-aminobenzimidazole; thiomycamine; and Q-phenyl. In an alternative embodiment, the separation matrix comprises first and second groups coupled to different ligands. Any of the first and second groups discussed above can be used in this embodiment, such as quaternary amine groups and phenyl groups. In this embodiment, in the case of a particulate separation matrix, said different ligands can be immobilized to different particles or identical particles in substantially equal or different amounts. Alternatively, or additionally, a particulate separation matrix It can comprise different types of first groups; or different types of second groups; immobilized on different particles. The multimodal chromatography matrix used in the present method is easily prepared by a person skilled in this field. In summary, the matrix consists of ligands coupled to a support, known in this field as a base matrix, directly or indirectly via a conventional spacer to provide an appropriate distance between the support surface and the interacting groups. To obtain high absorption capacities, the support is preferably porous, and ligands are then coupled to the outer surfaces and also to the pore surfaces. Methods for immobilizing ligands to porous or non-porous surfaces are well known in this field; see, e.g., Immobilized Affinity Ligand Techniques, Hermanson et al., Greg T. Hermanson, A. Krishna Mallia, and Paul K. Smith, Academic Press, INC, 1992. In one embodiment, the density of the ligands on the support surface is in a range close to that commonly used for conventional ion-exchange matrices.Binders can be coupled directly to the support simply via the binding element resulting from the chemistry used; or via a longer element known as an extender, tentacle, or flexible arm, see, e.g., US 6,428,707, incorporated herein by reference. In short, the extender can be in the form of a polymer, such as a homopolymer or a copolymer. Hydrophilic polymeric extenders can be of synthetic origin, i.e., with a synthetic backbone, or of biological origin, i.e., a biopolymer with a naturally occurring backbone. Typical synthetic polymers are selected from the group consisting of polyvinyl alcohols; polyacryl- and polymethacrylamides; and polyvinyl ethers. Typical biopolymers are selected from the group consisting of polysaccharides, such as starch; cellulose; dextran; and agarose. The support can be made from an organic material or Inorganic. In one embodiment, the support is prepared from a native polymer, such as a cross-linked carbohydrate material, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc. Native polymer supports are readily prepared and optionally crosslinked according to conventional methods such as inverted suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)). In a particularly advantageous embodiment, the support is a relatively rigid, yet porous, type of agarose, which is prepared by a method that enhances its flow properties, see, e.g., US 6,602,990 (Berg) or SE 0402322-2 (Berg et al). In an alternative embodiment, the support is prepared from a copolymer or synthetic polymer, such as crosslinked synthetic polymers, p.The aforementioned synthetic polymers are readily prepared and optionally crosslinked according to conventional methods, see e.g. "Styrene-based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Native or synthetic polymer supports are also obtainable from commercial sources, such as GE Healthcare, Uppsala, Sweden, for example in the form of porous particles. In a further alternative embodiment, the support is prepared from an inorganic polymer, such as silica. Porous and non-porous inorganic supports are well known in this field and readily prepared according to conventional methods. Advantageous particle sizes of the present separation matrix may have a diameter in the range of 5-500 µm, such as 10-100 µm, e.g., 20-80 µm. In the case of essentially spherical particles, the average particle size may be in the range of 5-1000 µm, such as 10-500 µm. In a specific embodiment, the average particle size is in the range of 10-200 µm. A person skilled in this field may determine The size and porosity of the particles are easily chosen to be advantageous depending on the process to be used. For example, for a large-scale process, for economic reasons, a more porous, yet rigid, support may be preferred to allow the processing of large volumes, particularly for the capture step. In chromatography, process parameters such as column size and shape will affect the choice. In an expanded bed process, the matrix commonly contains high-density feedstocks, preferably stainless steel feedstocks. For other processes, other criteria may affect the nature of the matrix. The antibodies separated according to the present invention can originate from any commonly used source, such as surface-cultured cells or continuous or batch tissue culture in fermentation vessels or tanks. Thus, in one embodiment, the liquid is a supernatant obtained from cell fermentation. Examples of compounds that are adsorbed are proteins, DNA, viruses, endotoxins, nutrients, components of a cell culture medium such as anti-foaming agents and antibiotics, and product-related impurities such as misfolded species and aggregates. The contact step between the mobile phase and the multimodal separation matrix, i.e., the adsorption step, can be preceded by a mechanical filtration, centrifugation, and / or chromatography step. For example, if the liquid sample is a fermentation broth, it is advantageous to mechanically remove cell fragments, whole cells, and other relatively large components before multimodal chromatography. In one embodiment, the present method constitutes the capture step of a purification protocol. In a specific embodiment, the liquid sample is a raw feed that is filtered before contact with the multimodal chromatography matrix. Consequently, this embodiment could also constitute a capture step, although the liquid sample has been purified by mechanical means. As is generally known, the Host cells that produce antibodies will also comprise a number of other proteins commonly known as host cell proteins (HCPs). Said HCPs include enzymes, such as proteases, and other proteins produced by host cells. According to the present invention, it has been unexpectedly found that host cell proteins could be adsorbed onto the multimodal separation matrix while antibodies remain free in the mobile phase. Thus, in one embodiment, substantially all host cell proteins from the liquid sample are adsorbed onto the multimodal separation matrix. In alternative embodiments, the present method is used as a second, third, or even fourth step in a cleaning protocol, as an intermediate purification or polishing step. Thus, in one embodiment, the mobile phase applied in the multimodal separation matrix comprises an antibody-containing eluate from a separation matrix. In another embodiment, the liquid sample is an eluate from a preceding affinity chromatography matrix. In an advantageous embodiment, the separation matrix from which the eluate is obtained comprises one or more Fc-binding protein ligands, such as Protein A ligands. The term Protein A ligands includes in this context native or recombinant Protein A, or its functional fragments. In this context, the term "functional" fragment means a fragment that has retained the protein-binding properties. Such affinity matrices are commercially obtainable, such as MabSelect™ from GE Healthcare.Consequently, in this embodiment, the adsorbed compounds may be one or more selected from the group consisting of released Protein A; complexes formed between Protein A and antibodies, such as Protein A-MAb complexes, wherein said complexes may comprise a quantity of antibodies per Protein A molecule, such as 2-4 antibodies complexed with a Protein A molecule. Protein A molecule; and aggregates of Protein A or released antibodies. As a person versed in this field will understand, depending on the specific conditions used in the preceding step, such as affinity chromatography, the eluate may require conditioning by means of additions or appropriate adjustment. Thus, the eluate is combined with an appropriate buffer or liquid to provide a mobile phase. Note that, although it may be preferred for practical reasons, if an eluate from a Protein A column has to be purified, the present method is not necessarily carried out directly after affinity chromatography, or even in the same facilities. In one specific embodiment, the present method is a multi-step process comprising a capture step in an affinity chromatography array, such as Protein A chromatography, and a polishing step in a multimodal separation array, as described above. The liquid sample applied to the affinity chromatography array can be a cell culture liquid or a fermentation broth, which has optionally been pretreated, such as filtration and / or conditioning by adjusting the pH and / or conductivity to provide a mobile phase. In this process, the capture step will remove one or more host cell proteins and host cell radicals, such as cell and protein fragments, DNA, endotoxins, and analogs. In the subsequent polishing step, primarily, radical-shaped compounds formed in the capture step, such as Protein A-antibody aggregates, will be adsorbed. The present method is useful for recovering any monoclonal or polyclonal antibody, such as antibodies originating from mammalian hosts, e.g., mice, rodents, primates, and humans, or antibodies originating from hybridomas. In one embodiment, the recovered antibodies are human or humanized antibodies. Advantageously, the antibodies are monomeric antibodies. The antibodies can be of any class, i.e., selected from the group consisting of IgA, IgD, IgE, IgG, and IgM. In one embodiment, the antibodies to be purified are antibodies capable of binding to Protein A, or antibody fragments containing Fc or fusion proteins. In a specific embodiment, the recovered antibodies are immunoglobulin G (IgG), such as IgG1. In one embodiment, the present method is used to purify antibodies exhibiting a p1 in the range of 6-9, as in the range of 7-8. In a specific embodiment, the p1 of the purified antibodies is about 9. In the present context, it should be considered that the term "antibodies" also includes antibody fragments and any fusion protein comprising an antibody or an antibody fragment.Thus, the present invention also comprises the purification of fragments of any of the antibodies indicated above and also fusion proteins comprising said antibodies. In one embodiment, the antibodies are monoclonal antibodies. As can be seen from the above, in the present method, undesirable compounds are adsorbed onto the multimodal separation matrix, and a substantially pure fraction of non-adsorbed antibodies is recovered. In this context, the term "substantially pure" is understood to mean that substantially all non-antibody compounds have been removed. Most advantageously, at least about 80%, as well as at least about 95%, i.e., in the range of 95-100%, as well as at least about 98%, i.e., in the range of 98-100%, and preferably at least about 99%, i.e., in the range of 99-100%, of the total amount of contaminants are removed in the multimodal separation matrix. However, as a person versed in this field will realize, the possible purities will depend on the antibody concentration in the liquid sample applied to the separation matrix and also on other conditions employed. Thus, in one embodiment, the Antibodies separated according to the present method are therapeutic class antibodies. Thus, the purified antibodies according to the invention are useful in research and also for the preparation of antibody-based pharmaceuticals, such as MAb drugs. An alternative use of the purified antibodies is for diagnostic use. Additionally, the purified antibodies are also useful in food products, as food additives for humans. For example, bovine antibodies purified according to the present invention are useful in food products. In a specific embodiment of the present method, the multimodal separation matrix is ​​provided as a chromatography column or disposable filter. An advantage of using disposable products in a method for purifying therapeutic compounds, such as antibodies, is that discarding the separation matrix after use eliminates the risk of cross-contamination between two processes. In many methods of this type, it is necessary to maintain aseptic conditions. Thus, in one embodiment of the present method, the multimodal separation matrix has been sterilized, and the sterile multimodal separation matrix is ​​provided as a sterile packed chromatography column or filter. In another embodiment, the present method is carried out as a batch process, wherein a disposable multimodal separation matrix is ​​added to a vessel comprising the liquid from which the antibodies will be recovered.In an advantageous embodiment, the disposable separation matrix consists of dry particles, such as dry agarose particles, which swell readily upon contact with the aqueous liquid. An appropriate time is allowed for target compounds to be adsorbed onto the matrix, after which the liquid phase comprising the antibodies is removed from the vessel. The used matrix can then be discarded without releasing the adsorbed compounds, which, again, can be advantageous from a safety standpoint because compounds such as endotoxins, prions, and / or certain cell proteins may be released. Host organisms no longer need to be handled. In a second aspect, the present invention relates to a kit for the purification of antibodies from one or more other components in a liquid, said kit comprising, in separate compartments, a first chromatography column packed with a first separation matrix; a second chromatography column packed with a multimodal separation matrix, comprising first groups capable of interacting with negatively charged sites of the target compounds, and second groups capable of at least one interaction other than a charge-charge interaction with said target compounds; one or more buffers; and written instructions. In an advantageous embodiment, the instructions teach the purification of antibodies from the percolated material of a multimodal separation matrix. The ligands, support, and other details of the multimodal separation matrix may be as described above. The instructions advantageously describe a method as defined above.In one embodiment of the kit, the first separation matrix is ​​an affinity chromatography matrix and preferably comprises protein ligands, such as protein A or G ligands. In another embodiment, the first and / or second chromatography columns are sterile and / or disposable columns. Finally, the present invention also relates to a disposable chromatography column for antibody purification, said column comprising a multimodal separation matrix comprising first groups capable of interacting with negatively charged target sites and second groups capable of at least one interaction other than charge-charge interaction. The ligands, support, and other details of the multimodal separation matrix may be as described above. In one embodiment, the separation matrix is ​​capable of adsorbing proteins other than antibodies from a mobile phase with conductivity in the range of 0-50, such as 0-25, e.g., 0-15 mS / cm. One embodiment An alternative to this aspect is a disposable filter for antibody purification, wherein said filter comprises first groups capable of interacting with negatively charged target sites and second groups capable of at least one interaction other than charge-charge interaction, said groups being coupled to the filter surface. In a specific embodiment, the present filter is capable of adsorbing proteins other than antibodies from a mobile phase, with conductivity in the range of 0-50, such as 0-25, e.g., 0-15 mS / cm. Detailed description of the drawings In Figure 1, a) shows the prototype multimodal ligand 2-aminobenzimidazole; b) shows the prototype multimodal ligand thiomycamine; c) shows the prototype multimodal ligand N-benzyl-N-methylethanolamine immobilized on a bead-shaped support; d) shows the prototype multimodal ligand N,N-dimethylbenzylamine. In the experimental part, the prototype ligands were coupled with a 6% Sepharose™ 6 FF agarose matrix. Figure 2 shows a sample chromatogram containing 50 mg of Mabl applied to multimodal separation matrices comprising N-benzyl-N-methylethanolamine ligands immobilized on Sepharose™ 6 FF (901035A); N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF (901035B); and Q Sepharose™ FF in 25 mM Bis-Tris, 100 mM NaCl (~12 mS / cm), pH 6.5. Elution was performed with 25 mM Bis-Tris, 0.5 M NaCl, pH 6.5. Figures 3a) and 3b) show sample chromatograms containing 20 mg of MAb 2, loaded onto prototypes and reference, as described in Example 3 below. Buffers were 25 mM Bis-Tris, 100 mM NaCl (~12 mS / cm), pH 6.0 for equilibrium and loading. Elution buffer was 0.5 M sodium acetate, pH 4.0. 3a) thiomycamine (1282004, green), 65 µmol / ml, thiomycamine (1282002, blue), 128 µmol / ml and Q Sepharose™ FF (black), b) 2-aminobenzimidazole (1282045, blue), 65 pmol / ml, 2-aminobenzimidazole (1282030, green), 146 pmol / ml and Q Sepharose™ FF (black). Figure 4 a)-g) shows the chromatography results performed on prototypes with mAb 1-rProtein A. Buffer A was 25 mM Bis-Tris, 50 mM NaCl, pH 6.0. The conductivity was approximately 7 mS / cm. Buffer B, 0.5 M sodium acetate, pH 4.0, was used for elution. Flow rate was 0.5 ml / min (150 cm / h). Sample was 10 mg of mAb, 0.10 mg of rProtein A at a concentration of 4 mg / ml of mAb and 1% rProtein A (w / w). 4a) thiomycamine, 65 pmol / ml (1282004); b) thiomycamine, 128 pmol / ml (1282002); c) ref. Q Sepharose™ FF; d) 2-aminobenzimidazole, 65 pmol / ml (1282045); e) 2-aminobenzimidazole, 146 pmol / ml (1282032); f) N-benzyl-N-methylethanolamine, 146 pmol / ml (901035A); eg) N,N-dimethylbenzylamine, 175 pmol / ml (901035B). Figure 5a)-h) shows the results of analytical size exclusion chromatography (SEC) on a sample with MAbl, 1% rPrA, and combined percolated material and eluate fractions from the chromatographic cycles in Figure 4. The blue curve represents percolated material (FP) fractions and the red curve is eluate. More specifically, Figure 5a) shows a sample of 4 mg / ml mAbl, 0.04 mg / ml rPrA giving 1% (w / w); 5b) shows FP and eluate from Fig. 4a) thiomycamine, 65 pmol / ml (1282004); 5c) shows FP and eluate from Fig. 4b) thiomycamine, 128 gmol / ml (1282002); 5d) shows FP and eluate from Fig. 4c) Q Sepharose™ FF; 5e) shows FT and eluate from fig. 4d) 2-aminobenzimidazole, 65 pmol / ml (1282045); 5f) shows FT and eluate from fig. 4e) 2-aminobenzimidazole, 146 gmol / ml (1282032); 5g) shows FT and eluate from fig. 4f) N-benzyl-N-methylethanolamine, 146 gmol / ml (901035A); and 5h) shows FT and eluate of fig. 4g) N,N-dimethylbenzylamine, 175 pmol / ml (901035B). Fig. 6 shows the results of Example 5 below. More Specifically, the chromatogram resulting from the sample containing 50 mg of Mab applied to Q Phenyl Sepharose™ 6 Fast Flow is shown. Elution was performed with 25 mM Tris, 0.5 M NaCl, pH 8.0. Figure 6 appears to show that the monoclonal antibody molecules are not adsorbed onto Q Phenyl Sepharose™ Fast Flow, because only a small peak was observed in the chromatogram during gradient elution. Experimental part The examples provided herein are for illustrative purposes only and should not be construed in any way that limits the scope of the invention as defined by the appended claims. All references made below and elsewhere in this description are incorporated herein by reference. Disposition Under non-binding conditions, samples containing approximately 50 mg of mAbl were loaded onto prototypes 901035 A (N-benzyl-N-methylethanolamine) and 901035 B (N,N-dimethylbenzylamine) at approximately 5 and 12 mS / cm. Percolated material (TP) fractions were collected at 5, 10, and 15 column volumes (CV). Elution peak fractions were combined. TP fractions were analyzed for HCP and Protein A content. Prototypes with high and low ligand densities were prepared for the multimodal ligands 2-aminobenzimidazole and thiomycamine. At pH 6.5, sample(s) containing 20 mg of mAbl were loaded onto columns at approximately 5 and 12 mS / cm. The performance of the prototypes was first evaluated with analytical SEC. Selected fractions were analyzed for HCP and Protein A content. After selection with SEC, the fractions selected were sent for HCP and Protein A analysis. To confirm that the chromatographic performance was not unique to a particular mAb, the chromatographic cycles were repeated using a sample containing mAb2 at pH 6.0 and approximately 12 mS / cm. The performance of the prototypes was first evaluated with analytical SEC. Selected fractions were analyzed for HCP and Protein A content. After selection, the fractions selected with SEC were sent for HCP and Protein A analysis. To more easily distinguish which of the prototypes provided the best elimination of rProtein A, MAbl showed a peak with 1% (w / w) recombinant Protein A (rPrA). Each prototype was injected with a sample volume corresponding to 10 mg of MAbl, 1% rProtein A at pH 6.0 and a conductivity of approximately 7 mS / cm. Fractions of percolated material and eluate were combined separately and analyzed with SEC. Materials / Units investigated Columns and gels were obtained from GE Healthcare, Uppsala. Sweden HiPrep™ 26 / 10 cat. no. 17-5087-01 Desalination Tricorn™ 5 / 50 cat. no. 18-1163-09 HR 5 / 5™ cat. no. 18-0338-01 Superdex™ 200 10 / 300 cat. no. 17-5175-01 GL, Instruments Chromatography systems: Spectrophotometer CV = 53.09 ml CV = 1 ml CV = 1 ml CV = 23.56 ml AKTAExplorer™ 10 Spectra MAX plus Chemical products All chemicals were of analytical grade. Water It was filtered using MilliQ. Chromatography medium The reference matrix was Q Sepharose™ Fast Flow (FF) (GE Healthcare, Uppsala, Sweden). The multimodal separation matrix prototypes carried ligands as described in Table 1 below: Table 1: Multimodal anion exchange ligands Prototype ref. ligand capacity Cf (pmol / ml) 901035A N-benzyl-N-methyl ethanolamine 146 901035B NN-dimethylbenzylamine 175 1282002 thiomycamine 128 1282004 thiomycamine 65 1282032 2-aminobenzimidazole (ABI) 146 1282045 2-aminobenzimidazole (ABI) 65 Preparation of N-benzyl-N-methyl ethanolamine prototype Sepharose™ Fast Flow A. Introduction of an allyl group into the matrix Sepharose™ 6 Fast Flow (GE Healthcare, Uppsala, Sweden) was activated with allyl glycidyl ether as follows: 100 ml of Sepharose™ 6 Fast Flow were suction-dried, mixed with 0.3 g of NaBH4, 12 g of Na2SO4 and 35 ml of 50% aqueous NaOH solution. The mixture was stirred for 1 hour at 50°C. After adding 100 ml of allyl glycidyl ether, the suspension was left at 50°C under vigorous stirring for a further 16 hours. After filtering the mixture, the gel was washed successively with 500 ml of distilled water, 500 ml of ethanol, 200 ml of distilled water, 200 ml of 0.2 M acetic acid and 500 ml of distilled water. Titration yielded a degree of substitution of 0.22 mmol of allyl / ml of gel. B. Activation of Sepharose™ 6 Fast Flow allyl via bromination Bromine was added to a stirred suspension of 50 ml of allyl-activated Sepharose™ 6 Fast Flow (0.22 mmol allyl groups / ml of drained gel), 1 g of sodium acetate, and 15 ml of distilled water until a persistent yellow color was obtained. Sodium formate was then added until the suspension was completely decolorized. The reaction mixture was filtered, and the gel was washed with 500 ml of distilled water. The activated gel was then transferred directly to a reaction vessel and further reacted. as N-benzyl-N-methylethanolamine. C. Introduction of BMEA (N-benzyl-N-methylethanolamine) groups into the activated matrix. The amine groups were introduced into the matrix directly via the nitrogen atom of the amine groups. In a typical procedure, matrix coupling was performed via bromination of the allyl group and nucleophilic substitution under basic conditions. 25 ml of bromine-activated gel (0.22 mmol of allyl groups / ml of drained gel) were transferred to a reaction flask containing a solution of N-benzyl-N-methylethanolamine (16.0 ml). 5 ml of water were added and the pH of the reaction solution was adjusted to 12.0 with a solution of Sodium hydroxide. The reaction was left for 16 hours under stirring at 50°C. After filtering the reaction mixture, the gel was successively washed with 3 x 10 ml of distilled water, 3 x 10 ml of 0.5 ml aqueous HCl, and finally, 3 x 10 ml of distilled water. Sepharose™ Fast Flow BMEA gel was obtained with a degree of substitution of 0.15 mmol of amines / ml of gel. Prototypes of 2-aminobenzimidazole and thiomycamine with high and low ligand densities were prepared according to conventional procedures (see US 6,702,943 (Johansson et al), WO 01 / 38228 (Belew et al), and WO 02 / 053252 (Belew et al)). Samples Two different humanized IgG antibodies, subclass 1, designated as MAbl and MAb 2, with extinction coefficients of 1.46 and 1.50 respectively, were used. Both antibodies were expressed in CHO cultures and subsequently purified using conventional Protein A affinity chromatography prior to the present experiments. Buffer exchange was performed in a HiPrep™ desalination column (GE Healthcare, Uppsala, Sweden), equilibrated with the buffer of interest, by injecting an appropriate volume (5-15 ml) with a Superloop™ (GE Healthcare, Uppsala, Sweden). The flow rate was 5 ml / min and 5 ml fractions were collected. Fractions containing the eluted peak were combined and the absorbance at 280 nm was determined in duplicate, to calculate the concentration according to equation 1: A28o = eC 1 (equation 1) where A28o is the absorbance at 280 nm. £ (mUmg'^cnf') is the extinction coefficient for a particular protein. C (mg / ml) is the protein concentration. 1 (cm) is the length of the path. Size exclusion chromatography (SEC) was performed on a Superdex™ 200 10 / 300 column (GE Healthcare, Uppsala, Sweden) at a flow rate of 0.5 ml / min. The buffer was PBS (phosphate-buffered saline); 10 mM phosphate, 0.137 M NaCl, 2.7 mM KCl, pH 7.4 prepared from tablets (Sigma, P-4417). Method Equilibrium 2 / 0.1 CV; Sample injection 50 pl Isocratic elution 1.5 CV 2 CV first use; 0.1 HP between cycles Chromatography in prototypes with mAb Buffer A was 25 mM Bis-Tris, pH 6.0 or 6.5. Depending on the For the desired conductivity, approximately 5 or 12 mS / cm, 35 or 100 was included. mM of NaCl. For prototypes 901035 A and 901035 B, elution buffer (Buffer B) was 25 mM Bis-Tris, 0.5 M NaCl, pH 6.5. For prototypes with Thiomycamine and ABI were used as ligands, and the elution buffer (Buffer B) was 0.5 M. Sodium acetate, pH 4.0. The flow rate was 0.5 ml / min (150 cm / h). Method: Equilibrium 5 CV Buffer A Sample injection 5-25 ml sample content 20 or 50 mg MAb washing 5 CV Buffer A gradient elution 10 CV 0-100% of B buffer elution 10 CV 100% of B buffer regeneration 5 CV Buffer A Chromatography in prototypes with MAb-rProtein A Buffer A was 25 mM Bis-Tris, pH 6.0. Conductivity was approximately 7 mS / cm by adding 50 mM NaCl. Buffer B was 0.5 M sodium acetate, pH 4.0. Flow rate was 0.5 ml / min (150 cm / h). Sample concentration was 4 mg / ml MAbl - 0.04 mg / ml of rPrA giving 1% (weight / weight). Method: Equilibrium 5CV Buffer A Sample injection 2.5 ml 10 mg MAb, 1 wash 5CV % rPrA Buffer A Gradient elution 10 CV 0-100% Buffer B Elution 10 CV 100% Buffer B Regeneration 5CV Buffer A CIP (clean-in-place) After each chromatographic cycle, the prototypes and the matrix of 5 CV (column volumes) 5HP 4 CV (including 15-minute break) 5HP 5HP 5 CV 5HP Reference Q Sepharose™ FF samples underwent the following CIP procedure; 30% isopropanol H2O 1.0 M of NaOH H2O Plug A H2O 20% EtOH Protein A Analysis Selected fractions were mixed with diluent of The SPA sample was prepared in proportions of 800 µl of SPA sample diluent + 200 µl of sample. After mixing, the fractions were heated in a heating block at 99°C for 10 minutes, then mixed again. The samples were then analyzed for recombinant Protein A. Host cell protein analysis (HCP) The samples (min. 600 pl) were analyzed for the content of HCP. The lower detection limit is 10 ng / ml. Example 1: Sample containing purified MAbl in prototype ligands N-benzyl-N-methylethanolamine (901035A) and N,N-dimethylbenzylamine (901035B) In Example 1, a sample containing 50 mg of MAbl was applied to N-benzyl-N-methyl ethanolamine immobilized on Sepharose. rM6 FF (901035A), N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF (901035B), and the Q Sepharose™ FF reference matrix in 25 mM BisTris, 100 mM NaCl (~12 mS / cm), pH 6.5. Elution was performed with 25 mM Bis-Tris, 0.5 M NaCl, pH 6.5. The chromatograms of Example I are shown in Figure 2, which shows the two prototypes N-benzyl-N-methylethanolamine Sepharose™ 6 FF (90103 5A) and N,N-dimethylbenzylamine Sepharose™ 6 FF (901035B) compared with Q Sepharose. rM FF. Fractions of percolated material (FT) selected for analysis are indicated with arrows. The results for HCP and Protein A elimination shown in Tables 2 and 3 below reveal that the prototypes are superior to Q Sepharose™ FF with respect to that. Table 2: HCP analysis results Column pH start (ng / ml) FT 1 (ng / ml) FT2(ng / ml) FT3 (ng / ml) Q Sepharose 1MFF (ref.) 6.5 890 160 200 180 N-benzyl-N-methylethanolamine, 146 pmol / ml (901035A) 6.5 890 10 20 35 N,N-dimethylbenzylamine 175 pmol / ml (901035B) 6.5 890 27 39 45 Table 3: Results of PrA analysis Column pH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) FT3 (ng / ml) Q Sepharose 1M FF (ref.) 6.5 0.40 0.69 0.46 0.31 N-benzyl-N-methylethanolamine, 146 pmol / ml (90103 5A) 6.5 0.40 0 0 0 N,N-dimethylbenzylamine 175 pmol / ml(901035B) 6.5 0.40 0.11 0.10 0.08 Example 2: Sample containing purified MAbl in prototype ligands thiomicamine and 2-aminobenzimidazole In this example, a sample containing 20 mg of MAbl was loaded into prototype and reference separation matrices. Buffers were 25 mM Bis-Tris, 35 mM NaCl (-5 mS / cm), pH 6.5 for equilibrium and loading. Elution buffer was 0.5 M sodium acetate, pH 4.0, a) Thiomycamine, 65 pmol / ml (1282004), b) Thiomycamine 128 pmol / ml (1282002), c) Q Sepharose™ FF, d) 2-aminobenzimidazole (ABI), 65 pmol / ml (1282045) e) 2-aminobenzimidazole (ABI), 146 pmol / ml (1282032). The results for the HCP and Protein A analyses are shown below in Tables 4 and 5. Table 4: HCP analysis results Column pH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) Thiomicamine, 65 pmol / ml (1282004) 6.5 351 <10 <10 Q Sepharose IM FF 6.5 351 11 11 2-amino-benzimidazole (ABI), 65 pmol / ml (1282045) 6.5 351 <10 <10 Table 5: Results of PrA analysis Column pH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) Thiomicamine, 65 pmol / ml( 1282004) 6.5 0.39 0.00 0.00 Q Sepharose* M FF 6.5 0.39 0.09 0.21 2-aminobenzimidazole (ABI), 65 pmol / ml( 1282045) 6.5 0.39 0.00 0.00 Example 3: Sample containing purified MAb2 in prototype ligands thiomicamine and 2-aminobenzimidazole A sample containing 20 mg of MAb2 was applied to prototypes and a reference. The buffer was 25 mM Bis-Tris, 100 mM NaCl (-12 mS / cm), pH 6.0. Elution was performed with 0.5 M sodium acetate, pH 4.0. The resulting chromatograms are shown in Figure 3. a) Thiomycamine (1282004, green), 65 pmol / ml, Thiomycamine (1282002, blue), 128 pmol / ml and Q Sepharose™ FF (black), b) 2-aminobenzimidazole (1282045, blue), 65 pmol / ml, 2-aminobenzimidazole (1282030, green), 146 pmol / ml and Q Sepharose™ FF (black). Analytical SEC was used to select fractions for HCP and Protein A analyses as shown in Tables 6 and 7 below. Table 6: HCP analysis results Column PH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) Thiomicamine, 65 pmol / ml 1282004 6.0 170 <10 <10 Q Sepharose™ FF 6.0 170 66 55 Table 7: Results of PrA analyses Column PH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) Thiomicamine, 65 pmol / ml (1282004) 6.0 5.42 0.00 0.24 Q Sepharose™ FF 6.0 5.42 3.90 4.93 Example 4: Purification of MAbl from a sample comprising MAbl and recombinant protein A (rPrA) in prototype ligands N-benzyl-N-methylethanolamine, N,N-dimethylbenzylamine, thiomicinamine and 2-aminobenzimidazole In this example, prototype chromatography was performed with a sample containing mAbl-rProtein A. Buffer A was 25 mM Bis-Tris, 50 mM NaCl, pH 6.0, conductivity was approximately 7 mS / cm. Buffer B was 0.5 M sodium acetate, pH 4.0. The flow rate was 0.5 ml / min (150 cm / h). The sample consisted of 10 mg of mAbl, 0.10 mg of rProtein A at a concentration of 4 mg / ml of mAbl and 1% rProtein A (w / w). The results are shown in Figure 4. Finally, analytical SEC was performed on a sample with mAbl, 1% rPrA, and combined percolated material and eluate fractions from the chromatographic cycles in Figure 4. The results are shown in Figure 5. In Figure 5a, the shaded peak is the mAbl-Protein A complex. The blue curve represents the percolated material (FP) fractions, and the red curve represents the eluate. Example 5: Antibody purification in Q arrangement Phenylalanine Sepharose 6 Fast Flow Under non-binding, sample conditions] containing Approximately 50 mg of mAb were loaded into a prototype Q Phenila Sepharose™ 6 Fast Flow. Fractions of percolated material (FP) were collected at 5, 10, and 15 column volumes (CV, column volumes). Elution peak fractions were analyzed. Q Phenyl Sepharose™ 6 Fast Flow was prepared by linking Q (-N(CH3)3) groups to Phenyl Sepharose™ 6 Fast Flow (45 pmol phenyl groups / ml gel) according to standard procedure (see below). The ion exchange capacity of Q Phenyl Sepharose™ 6 Fast Flow was 108 pmol / ml gel. At pH 7.0 or 8.0, samples containing 50 mg of mAb (purified with MabSelect) were loaded onto the column and the performance of Q Phenyl Sepharose™ 6 Fast Flow was evaluated by analyzing selected fractions of percolated material with respect to host cell protein (HCP) and Protein A content. Materials / Units investigated Columns and Phenyl Sepharose™ 6 Fast Flow were obtained from GE Healthcare, Uppsala, Sweden. HR 5 / 5™ cat no. 18-0338-01 CV = 1 ml Instruments Chromatography systems: AKTAExplorer™ 10 Spectra MAX plus spectrophotometer Chemical products All chemicals were of analytical grade. Water was filtered with MilliQ. Preparation of Q Phenila Sepharose™ 6 Fast Flow One method for preparing a separation matrix according to the invention is exemplified below, starting from a cross-linked agarose gel (Phenyl Sepharose™ 6 Fast Flow (high sub), GE Healthcare, Uppsala, Sweden). Introduction of group Q in Phenyl Sepharose™ 6 Fast Flow (high sub): Q (-N(CH3)3 groups were introduced into Phenyl Sepharose™ 6 Fast Flow (high sub) via reaction with glycidyl trimethylammonium chloride (G-MAC) as follows: 15 g of suction-dried Phenyl Sepharose™ 6 Fast Flow (high sub) was mixed with 5 ml of water, 5 ml of 50% aqueous NaOH solution, 0.02 g of NaBH4, and 40 ml of G-MAC. The mixture was stirred for 16 hours at 30°C. After filtration of the mixture, the gel was washed successively with 100 ml distilled water, 100 ml ethanol, and 100 ml distilled water. Titration yielded a degree of substitution of 0.11 nunol amines / ml of gel. Samples The monoclonal antibody used was expressed in CHO cultures and subsequently purified using conventional Protein A affinity chromatography prior to the present experiments. Determination of mAb concentration The mAb sample was diluted ten times with buffer. Two replicates of the sample solution were measured at A280. The average value was used to calculate the concentration according to Lambert-Beer's law: C = A / ( 1 xs) C = IgG concentration A = absorbance at 280 nm 1 = length of the route 8 = molar extinction coefficient for mAb, mg' 1 ml = 1.46 Phenyl Sepharose™ 6 Fast Flow Q Chromatography The separation of mAb from host cell proteins and protein A was tested under non-ligand conditions. The sample applied to the columns was mAb purified with MabSelect. The flow rate was 0.5 ml / min (150 cm³ / h). Absorbance at 280 nm was detected during all cycles. Two different buffers were tested (see below). Buffer switching to Buffer A was performed before each cycle. HiPrep and HiTrap desalination columns were used depending on the sample volume. Plugs: Buffer A: 25 mM Tris / HCl pH 8.0 Buffer B: 25 mM Tris / HCl, 0.5 M NaCl, pH 8.0 Buffer A: 25 mM phosphate buffer pH 7.0 Buffer B: 25 mM phosphate buffer, 0.5 M NaCl, pH 7.0 Method: MabSelect eluate with adjusted pH was used as starting material. Equilibrium 5 CV Buffer A 16 CV sample injection (50 mg of mAb) 5 HP Washing Machine, Plug A gradient 5 CV 100% Buffer B Clean after 5 CV Plug A gradient 1 ml fractions were collected during sample injection, washing, and elution. CIP (clean-in-place) with 1 M NaOH was performed after each cycle. The dwell time was approximately 25 minutes. Protein A Analysis Selected fractions were mixed with SPA sample diluent in proportions of 800 µl SPA sample diluent + 200 µl sample. After mixing, the fractions were heated in a heating block at 99°C for 10 minutes, and then mixed again. The samples were then analyzed for recombinant Protein A. Host cell protein analysis (HCP) The samples (min. 600 l) were analyzed for HCP content. The lower detection limit is 10 ng / ml. Results Under non-binding conditions, approximately 50 mg mAb were loaded onto an HR 5 / 5 column packed with Q Phenyl Sepharose™ Fast Flow at two different pHs (pH 7.0 and 8.0). Fractions of percolated material were collected at 5, 10, and 15 column volumes (CV, column volumes) as shown in Figure 1. Tables 8 and 9 present the results of Protein A and HCP analysis of the percolated material fractions. No traces of Protein A were detected in the fractions. Additionally, no host cell proteins were detected in FT1 and FT2 when using a sample with pH 8.0. Small amounts of host cell proteins were observed when using a sample with pH 7.0, however, the reduction in HCP was approximately 50 times compared to the HCP content in the sample. Fig. 6 also shows that monoclonal antibody molecules are not adsorbed onto Q Phenyl Sepharose™ Fast Flow because a very small peak was observed in the chromatogram during gradient elution (Fig. 6). Table 8. Protein A analysis results Column pH start (ng / ml) FT1 (ng / ml) FT3 (ng / ml) eluate (ng / ml) Q Phenyl Sepharose 1M FF 8.0 6.98 0.00 0.00 48.25 Q Phenyl Sepharose 1M FF 7.0 5.03 0.00 0.00 36.15 The sample volume was 16 ml and FT1-FT3 were fractions. 1 ml. The combined elution volumes were 2 ml. Table 9. Results of host cell protein analysis. Column pH start (ng / ml) FT1 (ng / ml) FT2 (ng / ml) FT3 (ng / ml) eluate (ng / ml) Q Phenyl Sepharose 1M FF 8.0 1100 <10 <10 12 4900 Q Phenyl Sepharose IM FF 7.0 1200 16 23 26 5100 The sample volume was 16 ml and FT1-FT3 were fractions of 1 ml. The combined elution volumes were 2 ml.

Claims

CLAIMS 1. A method for separating one or more antibodies from one or more other compounds in a liquid sample, characterized in that a mobile phase comprising said liquid sample is contacted with a multimodal separation matrix to adsorb one or more target compounds while the antibodies remain free in the mobile phase, wherein the multimodal separation matrix comprises first groups capable of interacting with negatively charged sites of the target compound(s) and second groups capable of at least one interaction other than charge-charge interaction with said target compound(s).

2. Method according to claim 1, characterized in that the multimodal separation matrix is ​​provided in a chromatography column, the mobile phase is passed through said column by gravity and / or pumping, and the antibodies are recovered in the percolated material from the column.

3. Method according to claim 1 or 2, characterized in that the liquid sample comprises a supernatant obtained from cell fermentation.

4. A method according to any of the preceding claims, characterized in that contact with the multimodal separation matrix is ​​preceded by a mechanical filtration and / or chromatography step.

5. A method according to any of the preceding claims, characterized in that the liquid sample comprises a raw feed.

6. Method according to claim 5, characterized in that the target compound(s) are host cell proteins and substantially all of said proteins are adsorbed onto the multimodal separation matrix.

7. Method according to claim 4, characterized in that the liquid sample comprises an eluate from a separation matrix.

8. Method according to claim 7, characterized in that the separation matrix from which the eluate is obtained comprises protein binders, preferably protein A or G binders.

9. Method according to claim 8, characterized in that protein A and / or protein G ligands are adsorbed onto the multimodal separation matrix.

10. A method according to any of the preceding claims, characterized in that the conductivity of the mobile phase is in the range of 0-25, such as 0-15 mS / cm.

11. A method according to any of the preceding claims, characterized in that the first groups are quaternary amines.

12. A method according to any of the preceding claims, characterized in that the second groups are hydrogen bonding groups.

13. Method according to claim 12, characterized in that the second groups are hydrophobic groups, such as groups comprising aromatic or heteroaromatic ring structure(s).

14. A method according to any one of the preceding claims, characterized in that the separation matrix comprises first and second groups coupled to the same ligands.

15. Method according to claim 14, characterized in that the first and second groups are separated from each other by a hydrocarbon chain of 1-3 carbon atoms.

16. A method according to any of the preceding claims, characterized in that the binders were immobilized in support via their initial groups.

17. A method according to any one of the preceding claims, characterized in that the separation matrix comprises first and second groups coupled to different ligands.

18. Method according to claim 17, characterized in that the separation matrix is ​​particulate and comprises a mixture of first particles, in which ligands comprising the first groups have been immobilized; and second particles, in which ligands comprising the second groups have been immobilized.

19. Method according to claim 17, characterized in that the separation matrix is ​​a filter in which a mixture of first ligands comprising the first groups; and second ligands comprising the second groups have been immobilized.

20. A method according to any of the preceding claims, characterized in that the separation matrix comprises third groups capable of a third interaction with a target compound.

21. A method according to any of the preceding claims, characterized in that the antibodies are monoclonal antibodies.

22. Method according to claim 21, characterized in that the antibodies are humanized antibodies.

23. A method according to any of the preceding claims, characterized in that the separated antibodies are therapeutic-class monoclonal antibodies.

24. A method according to any of the preceding claims, characterized in that the multimodal separation matrix is ​​provided in a disposable chromatography column.

25. A method according to any of the preceding claims, characterized in that the disposable column was sterilized before contact with the mobile phase.

26. Kit for the purification of antibodies from one or more other components in a liquid, characterized in that said kit comprises, in separate compartments, a first chromatography column packed with a first separation matrix; a second chromatography column packed with a multimodal separation matrix, comprising first groups capable of interacting with negatively charged sites of the target compounds, and second groups capable of at least one interaction other than charge-charge interaction with said target compounds; one or more buffers; and written instructions that teach the purification of antibodies from the percolated material of a multimodal separation matrix.

27. Kit according to claim 26, characterized in that in the first chromatography column, the separation matrix comprises protein ligands, preferably protein A or G ligands.

28. Kit according to claim 26 or 27, characterized in that the separation matrix comprises first and second groups coupled to the same ligands.

29. Kit according to claim 26 or 27, characterized in that the separation matrix comprises first and second groups coupled to different ligands.

30. Kit according to any one of claims 26 to 29, characterized in that the first and / or second chromatography columns are disposable columns.

31. Kit for capturing antibodies from one or more other components in a liquid, characterized in that said kit comprises, in separate compartments, a chromatography column packed with a multimodal separation matrix, comprising first groups capable of interacting with negatively charged sites. of the target compounds, and second groups capable of at least one interaction other than charge-charge interaction with said target compounds; one or more buffers; and written instructions.

32. Kit according to any one of claims 26 to 31, characterized in that the first groups are quaternary amines.

33. Kit according to any one of claims 26 to 32, characterized in that the second groups are hydrophobic and / or hydrogen bonding groups.

34. Kit according to any one of claims 26 to 33, characterized in that the separation matrix comprises first and second groups coupled to the same ligands.

35. Kit according to any one of claims 26 to 34, characterized in that the separation matrix comprises first and second groups coupled to different ligands.

36. Chromatography column, characterized in that it comprises a multimodal separation matrix comprising first groups capable of interacting with negatively charged sites of the target compound(s) and second groups capable of at least one interaction other than charge-charge interaction with said target compound(s).

37. Disposable chromatography column for antibody purification, characterized in that said column comprises a multimodal separation matrix comprising first groups capable of interacting with negatively charged target sites and second groups capable of at least one interaction other than charge-charge interaction, said groups being coupled to the surface of a porous support.

38. Chromatography column according to claim 36 or 37, characterized in that the separation matrix is ​​capable of adsorbing proteins other than antibodies from a mobile phase, wherein the Conductivity ranges from 0 to 25, as well as from 0 to 15 mS / cm.

39. Disposable filter for antibody purification, characterized in that said filter comprises first groups capable of interacting with negatively charged target sites and second groups capable of at least one interaction other than charge-charge interaction, said groups being coupled to the surface of the filter.

40. Filter according to claim 39, characterized in that it is capable of adsorbing proteins other than antibodies from a mobile phase, with conductivity ranging from 0 to 25 mS / cm, as well as from 0 to 15 mS / cm.