Antibody purification method

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. A method is proposed for isolating antibodies from other compound(s) in a liquid sample where a mobile phase containing the said sample is brought into contact with a multimodal separation matrix in order to adsorb undesirable compounds, while the antibody remains in the liquid in free state, where the multimodal separation matrix contains first type groups, which can interact with negatively charged sites of target compounds and second type groups which are capable of at least one interaction which is different from charge-charge interaction with the said target compounds. Also proposed is a chromatographic column filled with the multimodal separation matrix described above and a filter which has multimodal groups adsorbed on its surface.

EFFECT: design of an efficient method of isolating antibodies from other compound(s) in a liquid sample.

26 cl, 5 ex, 9 tbl, 20 dwg

 

The technical field

The present invention relates to a method of purification of antibodies. This method can be, for example, applied to the raw source material or as a stage following the affinity chromatography, to remove any remaining impurities and substances coming from resin for affinity chromatography. The present invention also includes a kit for purification of antibodies.

Prior art

The immune system consists of a large number of independent cell types that collectively protect the body from bacterial, fungal, viral infections, infections caused by parasites, and from the growth of tumor cells. The "guards" of the immune system are macrophages, which are constantly wander the bloodstream of their host. On the ingested substances as a result of infection or immunization macrophages respond absorption "occupiers"marked alien molecules known as antigens. This event is mediated by helper T-cells, triggers a complex chain of responses that result in the stimulation of b-cells. These b-cells, in turn, produce proteins called antibodies that are associated with the alien "invader". As a result of binding of antibody and antigen alien "invader" is marked for destruction by phagocytosis or activation systemcomponent. There are several 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 their structure. From a structural point of view widely studied IgG-antibodies possibly due to the fact that they play a major role in natural immune response. Polyclonal antibodies receive in accordance with the standard methods of immunization of the animal with the relevant antigen. In response to this, the animal will produce antibodies, which is polyclonal. However, for many tasks it is desirable to have a single clone specific antibodies, known as monoclonal antibodies. Monoclonal antibodies (mAb) produced hybrid or fused cell, formed by the merger of normal b-cells, producing only a single antibody with abnormal tumor cell myeloma. The resulting hybrid, known as hybridoma, these days used in the standard methods for producing antibodies. Biological activity, which have the antibodies currently used in several different applications in the field of diagnostics, health and the treatment of people and animals. In fact, in recent years, monoclonal antibodies and recombinant constructs antibodies were the most the vast class of proteins, passing current research in clinical trials and receive FDA approval (Food and Drug Administration; Department for quality control of food, drugs and cosmetics (USA)) as therapeutic and diagnostic tools. In addition to the development of expression systems and production strategies for obtaining highly purified antibodies simple and effective from the point of view of cost method it is necessary to develop effective protocols for purification of antibodies.

Traditional methods of isolation of immunoglobulins based on selective reversible precipitation of the protein fraction containing the immunoglobulins, in which other groups of proteins remain in solution. Typical agents for the deposition are ethanol, polyethylene glycol, lyotropic salt such as ammonium sulfate and potassium phosphate, and Caprylic acid. Usually the use of such methods of deposition results in products of very low purity, at the same time requires much time and is labor intensive. In addition, calling the precipitation agent to the raw material makes it difficult to use supernatant for other purposes and creates the problem of disposal of residue, which is especially acute when it comes to large-scale purification of immunoglobulins.

Al is ernational way of selecting immunoglobulins is chromatography, which includes a family of closely related methods of separation. A characteristic feature that distinguishes chromatography from most other physical and chemical methods of separation, is the presence of two mutually immiscible phases are brought into contact, and one phase is stationary and the other movable. A mixture of samples entered in the sliding phase, undergoes a series of interactions with the stationary and mobile phases as it is transferred through the system by the mobile phase. Interactions are based on differences in physical and chemical properties of the components of the sample. These differences affect the speed of movement of the individual components under the influence of the mobile phase, moving through a column containing a stationary phase. Separated components appear at the output depending on the increasing strength of interaction with the stationary phase. The least retained component is eluted first, most strongly retained material eluted last. Separation is achieved when one component is held sufficiently in order to prevent the overlap area with the adjacent dissolved substances as components of the sample suiryudan with speakers. Constantly made efforts to create the optimum stationary phase for each specific objectives of the division. This stationary phase in the General case contains a substrate or core matrix to which is attached a ligand containing functional groups, i.e. the groups involved in the binding. The link for each type of chromatography, usually given on the basis of the used principle of interaction, such as, for example, ion exchange chromatography, chromatography hydrophobic interaction and affinity chromatography.

In the protocols of isolation of immunoglobulins often used ion-exchange chromatography. When anion exchange chromatography negatively charged side chains of amino acids immunoglobulin will interact with the positively charged ligands chromatographic matrix. When cation exchange chromatography, conversely, positively charged side chains of amino acids immunoglobulin will interact with negatively charged ligands chromatographic matrix.

The hydrophobic interaction chromatography (HIC) is another method described and used in protocols allocation of immunoglobulins. If the objective is to obtain highly purified immunoglobulin product, it is generally recommended to combine HIC with one or more additional stages of treatment. When HIC, in order immunoglobulin effectively contacted HIC matrix, neo is should add lyotropic salts to the mobile phase. Associated immunoglobulin next release from the matrix by reducing concentrations of lyotropic salts. Thus, the disadvantage of this procedure is the need to add lyotropic salt to the raw material, as this can create problems and increase the cost of large-scale application. For example, the addition of lyotropic salts such raw materials as serum, plasma and egg yolk, will in many cases be difficult for large-scale applications, because salt can prevent any economically possible using separated from the immunoglobulin source material. An additional problem with large-scale applications will be utilization of a few thousand gallons of waste.

Affinity chromatography is based on specific interactions between biomolecules target and biospecific ligand on the principle of recognition "lock-key". Thus, the target and the ligand will be affine pair such as antigen/antibody, enzyme/receptor, and so forth. Well-known affinity ligands based proteins, such as protein a and protein G affinity chromatography with the use of which is widespread way as both the selection and purification of antibodies. It is well known that the chromatography with the use of the protein And provides outstanding specificity, particularly in relation to monoclonal antibodies, and as a consequence, obtaining a high degree of purity. Based on the use of protein And methods used in conjunction with the stages of ion exchange, hydrophobic interaction purification on hydroxyapatite and/or gelfiltration, became the subject of choice for many biopharmaceutical companies as methods purification of antibodies, see, for example, WO 8400773 and US 5151350. However, due to the presence of peptide bonds in a protein matrix with protein And are to some extent sensitive to alkalis. Moreover, when the matrix protein And used for the purification of antibodies from cell culture medium, cell protease can cause leakage of the protein or its fragments.

An attempt to reduce leakage of the ligand from the matrix for affinity chromatography are presented in WO 03/041859 (Boehringer Ingelheim Pharma KG), which offers pre-processing, for example, matrix protein And at least one surface-active substance to reduce leakage of the ligand. Affine matrix can be processed, for example, surface-active agent in the amount of 5-15 free volumes. The duration of contact is critical for the effectiveness of the process. For example, at room temperature to reduce leakage is required, the duration of exposure of at least 16 hours

Alternate wny approach to the problem of leakage of the ligand from the matrix for affinity chromatography is proposed in US 4983722 (Miles Inc.), where protein And selectively separated from the liquid containing the antibody and protein a, by keeping them with the anion exchange material. Both components are adsorbed on the anion exchange material and then antibodies and protein And elute each other in conditions of increasing ionic strength. Illustrative anionoobmennika is diethylaminoethyl (DEAE) Trisacryl M DEAE Sepharose™.

WO 2004/076485 (Lonza Biologics Pic.) relates to the purification of antibodies using protein a and ion exchange chromatography. Stage ion exchange comprises applying antibodies, purified on protein a, ion-exchange material under conditions that allow binding of the protein and the acquisition of antibodies in the ongoing thread. Aminoalkenes is aminoalkenes based Quaternary amine, most preferably Sepharose™ Q (Amersham Biosciences, now GE Healthcare).

US 5429746 (SmithKline Beecham Corp.) refers to the manner in which antibodies initially adsorb on associated with protein And chromatographic substrate and elute; then adsorb on the cation-exchange chromatographic substrate and selectively elute with her; and finally adsorb on the HIC substrate and elute. Printed on HIC-column mixture after affine and/or cation exchange chromatography may contain aggregates of immunoglobulins, incorrectly curved types of molecules, cell proteins households the ins and residual material from the stage affinity chromatography.

US 6498236 (Upfront Chromatography) is aimed at solving specific problems caused by the slight difference in molecular weights of affinity ligands on the basis of protein compared to immunoglobulins target. Thus, the described method of selection or purification of immunoglobulins from a solution, for example supernatant of cultured hybridoma cells, plasma or serum of animals, which has been proposed as an alternative to the use of protein a, protein G, synthetic peptides and other relatively high molecular weight ligands. Solid-phase matrix used in the described method, defined by the formula M-SP1-X-A-SP2-ACID, where M denotes the basis matrix, SP1 designates a spacer, X denotes O, S or NH, And denotes a mono - or bicyclic, possibly substituted aromatic or heteroaromatic group, SP2 indicates a possible Speiser and ACID refers to an acid group. It is argued that critical whether the matrix is effective to bind the antibodies are specific substituents.

In the US 5945520 (Burton and others) described chromatographic resin mixed actions that demonstrate hydrophobic at pH values corresponding to the binding, and hydrophilic and/or electrostatic in nature with pH values corresponding to desorption. Resin specifically designed to bind soedineniya from aqueous solution at low and at high ionic strength. Thus, at the stage of adsorption is used HIC, while desorption is based on the repulsion of charges.

In the US 6702943 (Johansson and others) described a method of removing substances target of fluid through its adsorption on the matrix bearing a variety of ligands containing anion-exchange groups and having a hydrophobic structure. More specifically, the ligands contain an aromatic ring in the vicinity of positively charged anion-exchange groups. It is argued that the inclusion of other groups capable of participating in electron-electron interactions may increase the degree of interaction between the substance and the adsorbent. It is alleged that the desired substances are cells, parts of cells and substances containing peptide structure. The storage capacity of the matrix is determined by the proteins of the comparison, such as bovine serum albumin and IgG. Described ligands are referred to as "high salt ligands because of their ability to adsorb a substance to a target at high salt concentrations, for example, 0.25 M NaCl.

In addition, in WO 01/38228 (Belew and others) described another way to remove negatively charged substance from a liquid by its binding to the matrix containing the anion-exchange ligands are of mixed nature. Each ligand contains put the nutrient-charged nitrogen and thioester bond at a distance of 1-7 atoms from the specified charged nitrogen. Similarly to the above, such desired substance, such as cells, parts of cells and substances containing peptide structure, adsorbed at concentrations of salts in the field of 0.25 M NaCl.

It has been suggested that the ceramic hydroxyapatite is a useful stage for purification of immunoglobulins. More specifically, it was reported (Chromatography, tech note 2849; S.G. Franklin, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA), IgG1 can be distinguished from the complex IgG1 protein And nefrackzionirovannam environment on ceramic hydroxyapatite CHT (Bio-Rad). More specifically, hydroxyapatite (CA10(PO4)6(OH)2) is a form of calcium phosphate, which, as shown, has unique properties division. However, it is also known that the matrix on the basis of hydroxyapatite have several disadvantages. For example, due to leakage of CA they are unstable at acidic pH values and sensitive to chelating agents such as EDTA (ethylenediaminetetraacetic acid). Moreover, it was shown that it is difficult to develop and scale a reliable and reproducible method of treatment using matrices based on hydroxyapatite, for example, because of the difficulty in packaging hydroxyapatite and maintain a working speaker to the large size. Finally, there is the risk of changes in media properties, obukov is the R impurity metal ions and the exchange of calcium ions, which are of serious concern to regulatory authorities.

Johansson and others (Journal of Chromatography A, 1016 (2003) 21-33: "Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions") describe the screening prototype multimodal ligands to capture negatively charged proteins of mobile phases a high conductivity. It was found that non-aromatic multi-modal anion-exchange ligands on the basis of weak ion-exchange ligands (primary and secondary amines) were optimal for capture of proteins by adsorption under conditions of high concentrations of salts.

Brief description of the invention

According to one aspect of the present invention, a method for separation of antibodies from other components of the fluid, which requires less time and fewer ongoing stages than methods of the prior art. This can be achieved by a method in which a liquid containing antibodies lead in contact with the multi-modal separation matrix and substantially pure antibodies extracted in the unconjugated form. For example, if the liquid is applied to the chromatographic column containing the specified matrix, the antibodies are easily extracted from the passing stream.

According to another aspect of the present invention, a method for separating the Titel from other components of the fluid with new specific features in comparison with the methods of the prior art.

According to further aspect of the present invention, a method for separation of antibodies from other components of the fluid, whereby an improved degree of separation from impurities present in the raw source material, such as proteins of the host cell.

Additional aspects and advantages of the invention will be apparent from the following further detailed description.

A brief description of graphic materials

On Figa)-(d) shows an illustrative selection of multi-modal anion-exchange ligands, useful in the method according to the present invention: N-benzyl-N-methyl-ethanolamine, N,N-dimethylbenzylamine, 2-aminobenzimidazole and timecamera.

Figure 2 shows the chromatogram, corresponding to the separation of monoclonal antibodies for multi-modal separation matrix containing N-benzyl-N-methyl-ethanolamine, immobilized on Sepharose™ 6 FF; N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF; and, for comparison, on a strong aminoalkanoic Q Sepharose™ FF, as described in Example 1, below.

On Figa) and (b) shows the chromatogram of monoclonal antibodies deposited on the separating matrix containing timicuan and 2-aminobenzimidazole immobilized on Sepharose™ 6 FF with different densities, as described in Example 3, below.

On Figa)-(g) shows the results of chromatography of a mixture of mAb1-rProtein A (recomb nanny protein a), conducted on the prototypes.

On Figa)-(h) shows the results of the analytical exclusion (gel) chromatography (SEC) of the sample containing mAb1 and 1% recombinant protein a, and collected fractions flowing and collected fractions of the eluate from the chromatographic separations shown in Figure 4.

Figure 6 shows the separation of molecules of monoclonal antibodies using multi-modal matrix Q is Phenyl Sepharose™ Fast Flow, as described in Example 5.

Definition

The terms "antibody" and "immunoglobulin" are used in this description are interchangeable.

The term "separation matrix" is used herein to denote a material consisting of a substrate, which are connected one or more ligands containing functional group.

The term "multimodal" separation matrix refers to a matrix that is able to ensure the availability of at least two different, but acting together sites that interact with the connection that should be connected. For example, one of these sites can give the type of interactions charge-charge, causing attraction between the ligand and the substance of interest. Another site can give electron-donor-acceptor interactions and/or hydrophobic and/or hydrophilic interactions. Electron-acceptor the interactions include interactions such as the formation of hydrogen bonds, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, and so on. "Multimodal" separation matrix is also known as the separation matrix "mixed type".

The term "surface" means in this specification, all external surfaces and includes, in the case of the porous substrate, the outer surface and pore surface.

The phrase "donor-acceptor interaction" means that an electronegative atom with a free electron pair acts as a donor and is associated with electrondeficient atom, which acts as acceptor for the electron pair of the donor (see, for example Karger et al., An Introduction into Separation Science, John Wiley & Sons (1973), page 42).

The term "anion-exchange group" means in this description of a group that is positively charged or can be charged positively.

The term "eluent" is used in its usual meaning in the art, that is, to denote the buffer with a suitable pH and/or ionic strength to release one or more compounds with the separation matrix.

The term "stage capture" in the context of liquid chromatography refers to the initial stage of the procedure division. In the most General sense stage capture includes clarification, concentration, stabilizati and substantial purification from soluble impurities. After the capture stage can be followed by intermediate treatment, which additionally reduces the number of remaining impurities, such as proteins of the host cell, DNA, viruses, endotoxins, nutrients, components of the cell culture medium, such as antifoams and antibiotics, and the associated product impurities such as aggregates, incorrectly curved types of molecules and assemblies.

The term "stage of treatment" in the context of liquid chromatography refers to the final stage of purification, which removes trace amounts of impurities and remains active safe product. Impurities are removed at the stage of purification, often represent the conformational isomers of a target molecule or suspicious seeping products.

The term "Fc-binding protein" means a protein that is able to contact the crystallizing part (Fc) antibodies include, for example, protein a and protein G, or any fragment or protein, which saves the specified property to bind.

Detailed description of the invention

In the first aspect of the present invention relates to a method of separating antibodies from one or more than one other connection liquid sample, in which a mobile phase containing a specified liquid sample is brought into contact with multimodal the ow separation matrix to adsorption of one or more compounds of targets, while antibodies remain in a free state in the mobile phase, where the multi-modal separation matrix contains the group of the first type, capable of interacting with negatively charged sites of the compound(s)target(s), and groups of the second type, capable of at least one interaction that is different from the interaction of the charge-charge with the specified(s) connection(s)target(s). The present invention also covers a method, where in addition to the groups of the first and second type type group of the third or next type.

In the preferred embodiment of the method according to the present invention carried out using the principles of liquid chromatography, i.e. by passing the mobile phase through the chromatographic column containing a multi-modal separation matrix. The substrate may be in the form of a porous or nonporous particles, such as essentially spherical particles, in the form of a monolith, filter, membrane, surface, capillary, or any other commonly used form. In an alternative embodiment of the method according to the present invention is performed with the use of the principles of chromatography in a larger volume of the layer, i.e. by adding the mobile phase to the increased volume of layer separation matrix in the form of particles, such as on the substance of the spheres is ical particles, containing a filler with a high density. In another alternative embodiment of the method according to the present invention is carried out using a batch process, wherein the separation matrix is added to the vessel containing the liquid sample.

Thus, in the method of purification of antibodies in accordance with this invention one or more undesirable compounds adsorb on the separating matrix, while the desired antibodies remain in the mobile phase without adsorption. In the context of the method according to the present invention can understand that the term connection-"target" refers to the compounds adsorbed on the separating matrix. It is obvious that the nature and characteristics of adsorbed compounds will depend on the origin of the liquid sample. Examples of compounds of the targets are cells and cell debris; proteins and peptides; nucleic acids such as DNA and RNA; endotoxins and viruses.

According to one embodiment of the present invention proposed a multi-modal separation matrix in the chromatographic column and the mobile phase passes through the said column under the action of gravity and/or by using a pump, while antibodies are removed from the flow through the column. The advantage of the method according to the present invention is that it does not require nikakog the elution of the antibody from the column. The absence of a separate stage of elution is an advantage of the process, because fewer stages will lead to the Protocol faster cleaning and, consequently, to reduce production costs. Moreover, antibodies are sensitive to certain conditions, which can damage their curved picture or to cause their degradation as a result of exposure to the peptide bond in them. Even if the conditions of elution for-exchangers in General do not include any extreme chemicals, any modification of salt and pH can affect sensitive antibody, the effect varies from species to species depending on PI, charge distribution, and so forth. Therefore, another advantage of the method according to the present invention is that it is possible to avoid adding eluent and application of the conditions of elution of the antibodies.

As mentioned above, in the method according to the present invention, the connection-target, from which it is desirable to separate the antibodies adsorbed on multi-modal separation matrix. To achieve the most suitable conditions for adsorption of compounds to target liquid sample together with a suitable buffer or other liquid receiving mobile phase. The method according to the present invention typically the military carry out in the conditions, traditional anion exchange chromatography, which usually include adsorption at relatively low concentrations of salt. Thus, in one embodiment of the method according to the present invention, the conductivity of the mobile phase is in the range from 0 to 25, for example from 10 to 15 MS/cm.) In one embodiment the pH of the mobile phase is about 5-6. The specialist in this field of technology can easily find the conditions for obtaining antibodies in a passing stream, for example by adjusting the pH or conductivity, which will depend, for example, the charge and charge distribution of the purified antibodies. If you need to, it can be entered in one or more stages of washing, to any or between any of these stages of the passage of flow. If after this it is desirable release of adsorbed compounds, for example, for re-use matrix, it can be done elution at higher salt concentrations, for example, by increasing the salt concentration gradient. For elution of the adsorbed compounds can also, or alternatively, to shift the pH value, for example, by lowering the pH gradient.

As mentioned above, the multi-modal separation matrix contains the group of the first type, which are capable of interacting with negatively charged site of the mi connections-targets and groups of the second type, which is capable of at least one interaction that is different from the interaction of the charge-charge with the specified connection target. In this context, it is clear that on the same connection-target focus group separation matrix with different types of interaction, i.e. each connection target in the ideal case is adsorbed by two or more types of interaction. Multimodal ligands containing positively charged or capable of being positively charged anion-exchange group, known in this technical field, see, for example, US 6702943 (Johansson et al), WO 01/38228 (Belew et al) and WO 02/053252 (Belew et al).

In one embodiment of the groups of the first type, that is, anion-exchange group multi-modal separation matrix, constitute a strong anion-exchangers. In this context, the term "strong" - exchangers understand groups that remain charged to a wide range of pH. In an advantageous embodiment strong anion-exchange groups are Quaternary amines, also known as group Q. In an alternative embodiment of the groups of the first type of multi-modal separation matrix are weak anion-exchangers. In this context, the term "weak" - exchangers is understood as meaning groups that are charged p and certain pH values, but they may lose power when the change of the pH value. In a specific embodiment of the group of the first type include a mixture of anion-exchange groups and additional functional groups, such as anion-exchange and groups forming hydrogen bonds. Thus, in this embodiment group of the first type may be a TRIS (TRIS; Tris(hydroxymethyl)aminomethan).

In one embodiment of the groups of the second type of multi-modal separation matrix include aromatic groups and/or groups forming hydrogen bonds. In one embodiment of the above aromatic groups include ring systems containing aromatic or heteroaromatic structure. In an advantageous embodiment of the groups of the second type include phenyl groups. Alternatively, the groups of the second type may include a mixture of aromatic and non-aromatic hydrophobic groups such as alkyl groups. Thus, in a specific embodiment, the group of the second type include alkyl groups. The separation matrix used in accordance with the invention may contain two or more than two functional groups of the same type, for example two or more different types of hydrophobic groups; or two or more different types of multimodal-exchangers.

Specialists in this field it is clear that the functional group full separation is s, used in the method according to the present invention, can be presented on the same ligand, in this case, each is a multi-modal ligand or different ligands, in this case, multimodal is the General nature of the separation matrix.

Thus, in one embodiment, the separating matrix contains a group of the first and second type associated with the same ligands. Any of the groups of the first and of the second type discussed above may be used in this embodiment, for example groups of the Quaternary amine and phenyl groups. In one embodiment, the ligands are bound to the substrate through its groups of the first type, for example, through amino groups, which leads to the formation of Quaternary amines. In one embodiment group of the first and second type are separated from each other hydrocarbon chain containing 1-6, for example 1-3, preferably 1-2 carbon atoms. In a particular embodiment, the ligands are selected from the group consisting of N-benzyl-N-methyl-ethanolamine, N,N-dimethylbenzylamine; 2-aminobenzimidazole; timecamera and Q is phenyl (Q is Phenyl).

In an alternative embodiment, the separating matrix contains a group of the first and second type associated with different ligands. Any of the groups of the first and of the second type discussed above may be used in this embodiment, for example groups of the Quaternary amine and fineliner. In this embodiment, in the case of the separation matrix in the form of particles, these different ligands can be immobilized on different or on the same particles essentially in the same or different quantities. Alternative or in addition, the separation matrix in the form of particles may contain different types of groups of the first type or of different types of groups of the second type, immobilized on different particles.

Multimodal chromatographic matrix used in the method according to the present invention is easily manufactured by the expert in this area. Briefly, the matrix is composed of ligands associated with the substrate (in this area, also known as the basis matrix) directly or indirectly through a common spacer, providing a suitable distance between the surface of the substrate and interacting groups. To obtain a high adsorption capacity of the substrate in the preferred case, is porous, and then the ligands associated with the external surfaces, and surfaces of pores. Methods of immobilization of ligands on porous or non-porous surfaces are well known in the art; 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 ligands on the surface of the substrate is in the range close to that usually is used for traditional ion-exchange matrix. The ligands can be directly connected to the substrate through a linker element, formed as a result of the applied chemical reaction, or through a longer item, known as the extender (extender), tentacle or flexible hand; see, for example US 6428707 included thus in this description by reference. Briefly, the extender may be in the form of a polymer, for example Homo - or copolymer. Hydrophilic polymer extenders can be of synthetic origin, i.e. it should be with a synthetic frame, or of biological origin, that is, to be a biopolymer with existing in nature frame. Typical synthetic polymers selected from the group consisting of polyvinyl alcohols, polyacrylic and polymethacrylamide; and polyvinyl ethers. Typical biopolymers selected from the group consisting of polysaccharides, such as starch; cellulose; dextran; and agarose.

The substrate may be made of organic or inorganic material. In one embodiment the substrate is made from a natural polymer, such as cross-linked hydrocarbon material, such as agarose, agar, cellulose, dextran, chitosan, konjac (konjac), carrageenan, 'gellan, alginate, and so forth. Natural polymeric substrate is easily manufactured and may carry out cross-stitching in accordance with undertime ways, such as facing suspension gilotinirovaniya (s Hjertén: Biochim. Biophys. Acta 79(2), 393-398 (1964)). In a particularly preferred embodiment the substrate is a type of relatively rigid but porous agarose, which is made by way of increasing its rheological properties, see for example US 6602990 (Berg) or SE 0402322-2 (Berg and others). In an alternative embodiment the substrate is made from a synthetic polymer or copolymer, such as cross-linked synthetic polymers, for example styrene or derivatives of styrene, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinylamides and so on. Such synthetic polymers are easily made and may carry out cross-stitching in accordance with standard methods, see, for example Styrene based polymer supports developed by suspension polymerization" (R. Arshady: Chimica e L'industria 70(9), 70-75 (1988)). Natural or synthetic polymeric substrate is also available from commercial sources, such as GE Healthcare (Uppsala, Sweden), for example, in the form of porous particles. In yet another alternative embodiment the substrate is made from an inorganic polymer, such as silicon dioxide. Inorganic porous and non-porous substrates is well known in this field and they are easily made in accordance with standard methods.

The appropriate size of the particles of the separation matrix of the present invention can be in the range for the diameter of 5-500 μm, such as 10-100 μm, for example 20-80 μm. In the case of essentially spherical particles, the average particle size may be in the range of 5-1000 μm, for example 10-500. In a particular embodiment, the average particle size is in the range of 10-200 μm. The specialist in this field of technology can easily choose the appropriate size and porosity of the particles depending on the method that will be used. For example, for large-scale process for economic reasons it may be preferable to more porous but rigid substrate, which enables you to handle large volumes, especially for the capture stage. In chromatography the choice will be affected by such process parameters as the size and shape of the column. The method is increased by the volume of the layer matrix usually contains fillers of high density, preferably made from stainless steel. For other processes on the nature of the matrix may be affected by other criteria.

Antibodies, separated in accordance with the present invention, can be from any of the commonly used source, such as cells cultured on the surface, or from cell cultures grown in fermentation vats or vessels periodic or continuous manner. Thus, in one embodiment the fluid is a supernatant prepared by the fermentation of cells. Examples of Adso berhemah compounds are proteins, DNA, viruses, endotoxins, nutrients, components of the cell culture medium, such as antifoams and antibiotics, and the associated product impurities, such as improperly curved types of molecules and aggregates. The stage of bringing into contact of the mobile phase and multi-modal separation matrix, i.e. the stage of adsorption, can be preceded by a stage mechanical filtration, centrifugation and/or chromatography. For example, if a liquid sample is a fermentation broth, before multimodal chromatography is preferred mechanical removal of cell debris, the whole Klykov and other relatively large components.

In one embodiment of the method according to the present invention, the purification Protocol involves the step of capturing. In a particular embodiment the liquid sample is a crude starting material, which is filtered before bringing into contact with multimodal chromatographic matrix. Therefore, this embodiment will still include the stage of capture, despite the fact that the liquid sample was pre-cleaned by mechanical means. As is well known, the cells of the host, which produce antibodies will also contain several other proteins, usually known as the proteins of the host cell (NDS). Such NDS in luchot such enzymes, as proteases and other proteins produced by the host cells. In the present invention it has been unexpectedly discovered that proteins of the host cell could be adsorbed on multi-modal separation matrix, while antibodies remained free in the mobile phase. Thus, in one embodiment, essentially all of the proteins of the host cell liquid sample was adsorbiroval on multi-modal separation matrix.

In alternative embodiments of the method according to the present invention is applied as a second, third or even fourth stage in the purification Protocol, for example as an intermediate purification step or stage of purification. Thus, in one embodiment of the mobile phase applied to multi-modal separation matrix contains antitelomerase the eluate from the separation matrix. In one embodiment the liquid sample is the eluate from the previous affinity chromatographic matrix. In the preferred embodiment of the separation matrix, which receive the eluate contains one or more Fc-binding protein ligands, such as ligand-based protein A. In this context, the term "ligand-based protein And includes natural and recombinant protein a or their functional fragments. In this context, the term "functional fragment means a fragment that retains the original the global properties of protein binding. Such affinity matrices are commercially available, such as MabSelect™ from GE Healthcare. Therefore, in this embodiment of the adsorbed compounds can be one or more than one selected from the group consisting of: released protein A; complexes formed between proteins and antibodies, such as protein complexes And MAb, which may contain some amount of antibodies to the protein molecule And, for example 2-4 molecules of antibody in complex with one molecule of protein A; and units of released protein a or antibodies. As will be obvious to the expert in this field of technology, depending on the specific conditions used in the previous stage, such as affinity chromatography, the eluate may require conditioning by suitable additions or regulation. So, for preparation of mobile phase the eluate combined with a suitable buffer or liquid. It should be noted that, if there must be subjected to cleaning the eluate from the protein A-containing column, even though it may be preferred for practical purposes, the method according to the present invention is not necessarily carried out immediately after affinity chromatography or even the same technical equipment.

In a particular embodiment of the method according to the present invention is a multistage process, in the stage with capture on affinity chromatographic matrix, such as protein A-containing chromatographic matrix, and the stage of purification of multi-modal separation matrix, which is described above. Liquid sample applied to the affinity chromatography matrix may be a cell culture fluid or a fermentation broth, which may have been pretreated, for example by filtration and/or by changing the conditions by summing up the pH and/or conductivity, for preparation of the mobile phase. In this process, as a result of the capture stage will be removed one or more proteins of the host cell and residual host cell, such as cell debris and proteins, DNA, endotoxins, and the like. At a later stage of treatment will be mainly adsorbed compounds in the form of balances with the capture stage, such as aggregates of the protein a-antibody.

The method according to the present invention is useful for extracting any monoclonal or polyclonal antibodies, such as antibodies originating from mammalian hosts, such as mice, rodents, primates and humans, or antibodies derived from hybridomas. In one embodiment recoverable antibodies are human or humanized antibodies. In the preferred embodiment the antibodies are Monomeric antibodies. Antibodies may be ubago class that is selected from the group consisting of IgA, IgD, IgE, IgG and IgM. In one embodiment of the subject cleaning antibodies are antibodies capable of binding to protein A, or Fc-containing antibody fragments or fused proteins. In a particular embodiment recoverable antibodies are immunoglobulin G (IgG, such as IgG1. In one embodiment of the method according to the present invention is used for purification of antibodies having a pI in the range of 6-9, for example in the range of 7-8. In a particular embodiment, the pI of the purified antibody of approximately 9. In the context of the present invention should be understood that the term "antibody" also includes antibody fragments and any protein that contains antibody or antibody fragment. Thus, the present invention also includes cleaning of the fragments of any of the above antibodies, as well as fused proteins containing such antibodies. In one embodiment, the antibodies are monoclonal antibodies.

As follows from the above, in the method according to the present invention unwanted compounds adsorb on the multi-modal separation matrix and obtain essentially pure fraction readsorbing antibodies. In this context, the term "essentially pure" is understood as meaning that essentially all non-antibody compounds removed. The most is her preferred to at least about 80%, for example at least about 95%, i.e. in the range of 95-100%, for example 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 amounts of impurities were removed on multi-modal separation matrix. However, as is clear to the expert in this field of technology, it is possible purity will depend on the concentration of antibodies in a liquid sample applied to the separation matrix, and also from other common conditions. Thus, in one embodiment the antibody is separated in accordance with the method according to the present invention are antibodies categories for therapeutic applications. Thus, antibodies are purified in accordance with the invention, are useful in scientific research and also for the preparation of drugs based on antibodies, such as mAb drugs. Alternative use of purified antibodies is diagnostic application. In addition, purified antibodies are also useful in food products such as nutritional supplements for people. For example, bovine antibodies, purified in accordance with the present invention are useful in food products.

In a particular embodiment of the FPIC of the BA of the present invention proposed a multi-modal separation matrix in the form of a single(CSOs) chromatographic(CSOs) column or filter. The advantage of using disposable products in the method of purification of therapeutic compounds, such as antibodies, is that by throwing the separation matrix after use eliminates the risk of mutual contamination between two different processes. For many of these ways there is a requirement to maintain aseptic conditions. Thus, in one embodiment of the method according to the present invention the multi-modal separation matrix was sterilized, and this sterile multi-modal separation matrix proposed in the form of sterile(CSOs) filled(CSOs) chromatographic(CSOs) column or filter. In one embodiment of the method according to the present invention is carried out as a batch process, where one-time multimodal separation matrix is added to the vessel containing the liquid, which should be extracted antibodies. In the preferred embodiment of the disposable separation matrix further comprises dried particles, such as dried particles of agarose, which are easily swell upon contact with aqueous fluid. Choose the length of time that is appropriate for adsorption of compounds targets in the matrix, after which the liquid phase containing the antibodies are removed from the vessel. Used matrix can then be removed without releasing adsorb the skilled compounds, that again can be an advantage from a security perspective, because in the future it will not be necessary to work with such compounds as endotoxins; prions and/or certain proteins of the host cell.

In a second aspect the present invention relates to a kit for the purification of antibodies from one or more than one other component in the liquid containing different cells of the first chromatographic column filled with the first separation matrix; the second chromatographic column Packed with multi-modal separation matrix, which contains groups of the first type, capable of interacting with negatively charged sites of the connection target, and groups of the second type, capable of at least one interaction that is different from the interaction of the charge-charge with the specified connection target; one or more buffers; and written instructions. In the preferred embodiment, the instructions are designed to teach the purification of antibodies from a stream passing through the multi-modal separation matrix. The ligands; the substrate and other components of the multi-modal separation matrix can be the same as described above. The instructions preferably describe the method, as defined above. In one embodiment of the set of first separating matrix is the Wallpaper matrix for affinity chromatography and preferably contains protein ligands, such as ligand-based protein a or G. In another embodiment the first and/or second chromatographic column are sterile and/or disposable columns.

Finally, the present invention also relates to a disposable chromatographic column for the purification of antibodies, which contains multi-modal separation matrix containing groups of the first type, capable of interacting with negatively charged sites of the target, and groups of the second type, capable of at least one interaction that is different from the interaction of the charge-charge. Ligands, substrate and other components of the multi-modal separation matrix can be the same as described above. In one embodiment of the separation matrix capable of adsorbing proteins other than antibodies of the mobile phase, the conductivity of which is in the range from 0 to 50, such as from 0 to 25, for example from 0 to 15 MSM/see Alternative embodiment of this aspect is a disposable filter for the purification of antibodies containing groups of the first type, capable of interacting with negatively charged sites of the target, and groups of the second type, capable of at least one interaction that is different from the interactions charge-charge, and these groups are connected with the surface of the filter. In a particular embodiment of the filter according to the present invention is pasaban to adsorb proteins, other than antibodies of the mobile phase, the conductivity of which is in the range from 0 to 50, such as from 0 to 25, for example from 0 to 15 MSM/see

Detailed description of graphic materials

Figure 1 shows a) a multimodal ligand prototype 2-aminobenzimidazole; (b) multimodal ligand prototype timicuan; (C) multimodal ligand prototype N-benzyl-N-methyl-ethanolamine, immobilized on a substrate in the form of granules, and (d) multimodal ligand prototype N,N-dimethylbenzylamine. In the experimental part, the ligands of the prototype linked 6%agarose matrix Sepharose™ 6 FF.

Figure 2 shows the chromatogram of a sample containing 50 mg mAb1 deposited on the multi-modal separation matrix containing ligands N-benzyl-N-methyl-ethanolamine, immobilized on Sepharose™ 6 FF (A); N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF (901035), and Q Sepharose™ FF 25 mm Bis-Tris, 100 mm NaCl (about 12 MS/cm), pH 6.5. Elution was carried out using 25 mm Bis-Tris; 0.5 M NaCl, pH 6.5.

On Figa) and (b) shows the chromatogram of a sample containing 20 mg mAb2 applied to the prototypes and matrix comparison, as described below in Example 3. The buffer consisted of 25 mm Bis-Tris, 100 mm NaCl (about 12 MS/cm), pH 6.0, for balancing and application. Eluting buffer consisted of 0.5 M Na-acetate, pH 4.0. Figa) timicuan (1282004, green), 65 µmol/m is, timicuan (1282002, blue), 128 μmol/ml and Q Sepharose™ FF (black); (b) 2-aminobenzimidazole (1282045, blue), 65 μmol/ml, 2-aminobenzimidazole (1282030, green), 146 μmol/ml and Q Sepharose™ FF (black).

Figure 4 (a)-(g) shows the results of chromatography, performed on the prototypes, to a mixture of mAb1-recombinant protein a (rPrA). A-buffer consisted of 25 mm Bis-Tris, 50 mm NaCl, pH to 6.0. Conductivity was approximately 7 MSM/see-buffer, 0.5 M Na-acetate, pH of 4.0, was used for elution. The flow rate was 0.5 ml/min (150 cm/h). The sample contained 10 mg mAb1, 0.10 mg rPrA at a concentration of 4 mg/ml mAb1 and 1% recombinant protein A (wt./mass.). Figa) timicuan, 65 μmol/ml (1282004); (b) timicuan, 128 μmol/ml (1282002); (C) matrix comparison Q Sepharose™ FF; d) 2-aminobenzimidazole, 65 μmol/ml (1282045); (e) 2-aminobenzimidazole, 146 μmol/ml (1282032); (f) N-benzyl-N-methylethanolamine, 146 μmol/ml (A), and (g) N,N-dimethylbenzylamine, 175 μmol/ml (901035).

Figure 5 (a)-(h) shows the results of the analytical exclusion (gel) chromatography (SEC) of the sample containing MAb1 and 1% recombinant protein a, and collected fractions flowing and collected fractions of the eluate from the chromatographic separations shown in Figure 4. The blue curve corresponds to the fractions passing flow (FT), and the red eluate. More specifically, Figa) shows a sample of 4 mg/ml mAb1, 0.04 mg/ml rPrA that gives 1% (wt./wt is,); on 5b) shows FT and eluate with Figa) timicuan, 65 μmol/ml (1282004); 5C) shows FT and eluate with Fig.4b) timicuan, 128 μmol/ml (1282002); 5d) shows FT and eluate with Figs) Q Sepharose™ FF; 5e) shows FT and eluate with Fig.4d) 2-aminobenzimidazole, 65 μmol/ml (1282045); 5f) shows FT and eluate with Fige) 2-aminobenzimidazole, 146 μmol/ml (1282032); 5g) shows FT and eluate with Fig.4f) N-benzyl-N-methylethanolamine, 146 μmol/ml (A), and 5h) shows FT and eluate with Pigd) N,N-dimethylbenzylamine, 175 μmol/ml (901035).

Figure 6 shows the results of the following Example 5. More specifically, shows the chromatogram of a sample containing 50 mg of MAb deposited on Q Phenyl Sepharose™ 6 Fast Flow. Elution was carried out using 25 mm Tris, 0.5 M NaCl, pH 8.0. From Fig.6 shows that the molecules are monoclonal antibodies adsorbed to Q Phenyl Sepharose™ Fast Flow, as in a gradient elution on the chromatogram observed only a very small peak.

EXPERIMENTAL PART

These examples are offered for illustrative purposes only and in no way should be interpreted as limiting the scope of the present invention, which is defined in the attached claims. All the links provided below and elsewhere in another place of the present description, are included in this description by reference.

The sequence of actions

In conditions that do not result in binding of the Oia, sample containing about 50 mg mAb1, inflicted on prototypes 901035 And (N-benzyl-N-methylethanolamine) and 901035 In (N,N-dimethylbenzylamine) at approximately 5 and 12 MSM/see Collected fractions passing flow (FT) at 5, 10 and 15 column volumes (CV). Fractions corresponding to the peak elution were combined. FT-fractions were analyzed for the content of the NDS and protein A.

Created prototypes with high and low densities of ligands for multimodal ligands 2-aminobenzimidazole and timecamera. The sample containing 20 mg mAb1, were applied to the column at pH 6.5 and about 5 and 12 MSM/see the performance of the original prototypes were evaluated using analytical SEC. Selected fractions were analyzed for the content of the NDS and protein A. After screening fractions using SEC selected fractions were sent for analysis NDS and protein A.

To ensure that the chromatographic performance characteristics are not unique to one specific mAb, chromatographic separation was repeated using a sample containing mAb2, at pH 6.0 and about 12 MSM/see the performance of the original prototypes were evaluated using analytical SEC. Selected fractions were analyzed for the content of the NDS and protein A. After screening fractions using SEC selected fractions were sent for analysis of the NDS and the protein is more easily determine which of the prototype gives the best separation of the recombinant protein And, mAb1 was supplemented with 1% (wt./mass.) recombinant protein A (rPrA). Each prototype was injected sample volume corresponding to 10 mg MAb1, 1% recombinant protein a at pH 6.0 and a conductivity of about 7 MSM/see Faction flowing and the eluate was collected separately and analyzed using SEC.

Materials/analyzed samples

Columns and gels were obtained from GE Healthcare (Uppsala, Sweden)

HiPrep™ 26/10 for desaltingNo. kat-5087-01CV=53,09 ml
(Desalting)
Tricorn™ 5/50No. kat-1163-09CV=1 ml
HR 5/5™No. kat-0338-01CV=1 ml
Superdex™ 200 10/300 GLNo. kat-5175-01CV=23,56 ml

Devices

Chromatographic system:ÄKTAExplorer™ 10
SpectrophotometerSpectra MAX plus

Chemicals

All the used helices is their reagents were of analytical purity. Use water that has been filtered using MilliQ.

Chromatographic environment

Matrix comparison was a Q Sepharose™ Fast Flow (FF) (GE Healthcare, Uppsala, Sweden). Prototypes of multi-modal separation matrix contained ligands are described below in Table 1.

Table 1
Multi-modal anion-exchange ligands
The link to the prototypeThe ligandThe capacity of Cl-(µmol/ml)
AN-benzyl-N-methyl-ethanolamine146
WN,N-dimethylbenzylamine175
1282002timicuan128
1282004timicuan65
12820322-aminobenzimidazole (ABI)146
12820452-aminobenzimidazole (ABI)65

Preparation of prototype N-benzyl-N-m is telethonin-Sepharose™ Fast Flow

A. Introduction to matrix allyl group

Sepharose™ 6 Fast Flow (GE Healthcare, Uppsala, Sweden) activated allylglycidyl simple ether as follows: 100 ml of Sepharose™ 6 Fast Flow was dried by suction, was mixed with 0.3 g (NaBH4, 12 g of Na2SO4and 35 ml of 50%aqueous NaOH solution. The mixture was stirred for 1 hour at 50°C. After adding 100 ml allylglycidylether simple ether suspension was left at 50°C. with intensive stirring for an additional 16 hours. After filtration of the mixture, the gel is then washed 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.

After titration received the degree of substitution equal to 0.22 mmol allyl per 1 ml of gel.

C. Activation of allyl-Sepharose™ 6 Fast Flow through the synthesized

To a stirred suspension of 50 ml allelectronic Sepharose™ 6 Fast Flow (0.22 mmol allyl groups per 1 ml of dried gel), 1 g of sodium acetate and 15 ml of distilled water was added bromine to obtain a stable yellow color. Then add the sodium formiate until complete discoloration of the suspension. The reaction mixture was filtered and the gel was washed with 500 ml of distilled water. Then the activated gel was transferred directly into the reaction vessel and resulted in further interaction with N-benzyl-N-methylethanolamine.

Sweenie groups VMEA (N-benzyl-N-methylethanolamine) in the activated matrix

Amino groups were introduced into the matrix directly via the nitrogen atom of these amino groups. According to a typical method, the combination with a matrix was carried out by means of synthesized allyl group and nucleophilic substitution under alkaline conditions. 25 ml of activated bromine gel (0.22 mmol allyl groups per 1 ml of dried gel) was transferred into a reaction vessel containing a solution of N-benzyl-N-methylethanolamine (16.0 ml). Added 5 ml of water and with a solution of sodium hydroxide was established pH of the reaction solution of 12.0. The reaction mixture was left for 16 hours with stirring at 50°C. After filtration of the reaction mixture, the gel is then washed 3×10 ml of distilled water, 3×10 ml aqueous 0.5 M HCl and, finally, 3×10 ml of distilled water. Got the gel BMEA-Sepharose™ Fast Flow with a degree of substitution of 0.15 mmol (amines/ml gel).

Prototype 2-aminobenzimidazole and timecamera with high and low density of the ligands were obtained in accordance with standard techniques (see US 6702943 (Johansson et al), WO 01/38228 (Belew et al) and WO 02/053252 (Belew et al)).

Samples

Used two different humanized IgG antibody, subclass 1, denoted by MAb1 and MAb2, with the extinction coefficient of 1.46 and 1.50, respectively. Both antibodies expressed in cultures of Cho (Chinese hamster ovary), and then, before carrying out experiments on the right is bretania, was purified by using conventional affinity chromatography on protein A.

Shift buffer was performed on the column for desalting HiPrep™ Desalting (GE Healthcare, Uppsala, Sweden), equilibrated need a buffer, through the introduction of appropriate amounts (5-15 ml) using a Superloop™ (GE Healthcare, Uppsala, Sweden). The flow rate was 5 ml/min was Collected fraction volume of 5 ml. Fractions containing eluruumis peak were combined and two repeats was determined by absorption at 280 nm in order to calculate the concentration in accordance with equation 1:

A280=ε·C·I (Equation 1),

where a280is the absorption at 280 nm;

ε (ml·mg-1·cm-1) is the extinction coefficient for a particular protein;

(Mg/ml) is the concentration of protein;

I (cm) is the length of the path.

Exclusion (helpanimals) chromatography (SEC) was performed on a column of Superdex™ 200 10/300 (GE Healthcare, Uppsala, Sweden) at a flow rate of 0.5 ml/min. as a buffer used PBS (phosphate buffered saline)solution: 10 mm phosphate, 0,M NaCl, 2.7 mm KCl, pH 7.4, prepared from tablets (Sigma P-4417).

Way

Trim2/0,1 CV;2 CV initial use; 0,1 CV between divisions
Introduction sample50 µl
Isocratic elution1,5 CV

Chromatography mAb prototypes

A-buffer consisted of 25 mm Bis-Tris, pH 6.0 or 6.5. Depending on the desired conductivity of about 5 or 12 MS/cm, buffer consisted of 35 or 100 mm NaCl. For prototypes A and 901035 In an eluting buffer (In buffer) consisted of 25 mm Bis-Tris, 0.5 M NaCl, pH 6.5. For prototypes with timecamera and ABI as ligands eluting buffer (In buffer) consisted of 0.5 M Na-acetate, pH 4.0. The flow rate was 0.5 ml/min (150 cm/h).

Method:Trim5CVA-buffer
Introduction sample5-25 mlthe sample containing 20 or 50 mg mAb
Flushing5CVA-buffer
Grade is the things 10 CV0-100%B-buffer
elution
Elution10 CV100%In-buffer
Regeneration5CVA-buffer

Chromatography of the mixture of MAb recombinant protein And prototypes

A-buffer consisted of 25 mm Bis-Tris, pH of 6.0. By adding 50 mm NaCl conductivity was approximately 7 MSM/see-buffer consisted of 0.5 M Na-acetate, pH of 4.0. The flow rate was 0.5 ml/min (150 cm/h). The concentration of the sample was as follows: 4 mg/ml MAb1 and 0.04 mg/ml for rPrA, which corresponds to 1% (wt./mass.).

Method:Trim5CVA-buffer
Introduction sample2.5 ml10 mg MAb, 1% rPrA
Flushing5CV
Gradient elution10 CV0-100%B-buffer
Elution10 CV100%In-buffer
Regeneration5CVA-buffer

Cleaning in place (CIP; cleaning in place)

After each chromatographic separation prototypes and matrix comparison Q Sepharose™ FF were subjected to the following procedure CIP:

30%isopropanol5 CV (column volumes)
H2O5CV
of 1.0 M NaOH4 CV (including 15 minute break)
H2O5CV
A-buffer5CV
H2O5CV
20%EtOH5CV

Analysis of protein And

Selected fractions were mixed with sample diluent SPA in proportions 800 ál of sample diluent SPA+200 MK the sample. After stirring fraction was heated in a heating block at 99°C for 10 minutes, then the mixture was stirred. Next, the samples were analyzed for recombinant protein A.

Analysis of the proteins of the host cell (NDS)

Samples (minimum of 600 μl) were analyzed on the content of the NDS. The lower detection limit is 10 ng/ml.

Example 1

MAb1-containing sample, purified on prototypes with ligands N-benzyl-N-methylethanolamine (901035A) and N,N-dimethylbenzylamine (901035 In)

In Example 1, a sample containing 50 mg b1, were applied to N-benzyl-N-methylethanolamine immobilized on Sepharose™ 6 FF (901035A), N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF (901035B), and a matrix comparing the Q Sepharose™ FF 25 mm Bis-Tris, 100 mm NaCl (about 12 MS/cm), pH 6.5. Elution was performed using 25 mm Bis-Tris, 0.5 M NaCl, pH 6.5.

Chromatogram of Sample 1 is shown in figure 2, which shows two prototype N-benzyl-N-methylethanolamine-Sepharose™ 6 FF (901035A) and N,N-dimethylbenzylamine-Sepharose™ 6 FF (901035B) compared to Q Sepharose™ FF. The fraction passing flow (FT), selected for analysis, indicated by arrows. Results purification from the NDS and the protein As shown below in Tables 2 and 3 show that the prototypes exceed Q Sepharose™ FF in this regard.

Table 2
The results of the analysis NDS
ColumnpHStartFT1FT2FT3
(ng/ml)(ng/ml)(ng/ml)(ng/ml)
Q Sepharose™ FF (comparison)6,5890160200180
N-benzyl-N-methylethanolamine,6,5890102035
146 μmol/ml (901035A)
N,N-dimethylbenzylamine,6,5890273945
175 µmol/ml (901035B)

Table 3
The results of the analysis of protein And
ColumnpHStartFT1FT2FT3
(ng/ml)(ng/ml)(ng/ml)(ng/ml)
Q Sepharose™ FF (comparison)6,50,400,690,460,31
N-benzyl-N-methylethanolamine,6,50,40000
146 μmol/ml (901035A)
N,N-dimethylbenzylamine, 175 μmol/ml (901035 In),5 0,400,110,100,08

Example 2

MAb1-containing sample, purified on prototypes with ligands by timecamera and 2-aminobenzimidazole

In this Example, the sample containing 20 mg MAb1, inflicted on the prototypes and the separating matrix comparison. Buffers for balancing and application consisted of 25 mm Bis-Tris, 35 mm NaCl (about 5 MS/cm), pH 6.5. Eluting buffer consisted of 0.5 M Na-acetate, pH 4.0. a) Timicuan, 65 μmol/ml (1282004), (b) timicuan, 128 μmol/ml (1282002), (C) Q Sepharose™ FF, d) 2-aminobenzimidazole (ABI), 65 μmol/ml (1282045) and (e) 2-aminobenzimidazole (ABI), 146 μmol/ml (1282032). The results of the NDS and the protein As shown below in Tables 4 and 5.

Table 4
The results of the analysis NDS
ColumnPHStartFT1FT2
(ng/ml)(ng/ml)(ng/ml)
Timicuan, 65 μmol/ml (1282004) 351≤10≤10
Q Sepharose™ FF6,53511111
2-amino-benzimidazole (ABI), 65 μmol/ml6,5351≤10≤10
(1282045)

Table 5
The results of the analysis of protein And
ColumnpHStartFT1FT2
(ng/ml)(ng/ml)(ng/ml)
Timicuan, 65 μmol/ml (1282004)6,50,390,000,00
Q Sepharose™ FF 6,50,390,090,21
2-aminobenzimidazole (ABI), 65 μmol/ml6,50,390,000,00
(1282045)

Example 3

MAb2-containing sample, purified on prototypes with ligands by timecamera and 2-aminobenzimidazole

The sample containing 20 mg MAb2, inflicted on prototypes and matrix comparison. The buffer consisted of 25 mm Bis-Tris, 100 mm NaCl (about 12 MS/cm), pH 6.0. Elution was performed using 0.5 M Na-acetate, pH 4.0. The resulting chromatogram is shown in Figure 3.

3A) Timicuan (1282004, green), 65 μmol/ml, Timicuan (1282002, blue), 128 μmol/ml and Q Sepharose™ FF (black); (b) 2-aminobenzimidazole (1282045, blue), 65 μmol/ml, 2-aminobenzimidazole (1282030, green), 146 μmol/ml, and Q Sepharose™ FF (black). For analysis of the NDS and protein in selected fractions used analytical SEC, as shown below in Tables 6 and 7.

Table 6
The results of the analysis NDS
ColumnPHStart (ng/ml)FT1 (ng/ml)FT2 (ng/ml)
Timicuan, 65 μmol/ml (1282004)6,0170≤10≤10
Q Sepharose™ FF6,01706655

Table 7
The results of the analysis of protein And
ColumnpHStart (ng/ml)FT1 (ng/ml)FT2 (ng/ml)
Timicuan, 65 μmol/ml (1282004)6,05,420,000,24
Q Sepharose™ FF6,05,423,90is 4.93

Example 4

Purification of MAb1 from a sample containing MAb1 and recombinant protein a (rPrA), on the prototypes with ligands N-benzyl-methylethanolamine. N,N-dimethylbenzylamine, timecamera and 2-aminobenzimidazole

In this Example, conducted a chromatography sample containing mAb1-recombinant protein And, on the prototypes. A-buffer consisted of 25 mm Bis-Tris, 50 mm NaCl, pH to 6.0. Conductivity was approximately 7 MSM/see-buffer consisted of 0.5 M Na-acetate, pH of 4.0. The flow rate was 0.5 ml/min (150 cm/h). The sample contained 10 mg mAb1, 0.10 mg rPrA at a concentration of 4 mg/ml mAb1 and 1% recombinant protein A (wt./mass.). The results are shown in figure 4.

At the end of the conducted analytical SEC of sample containing mAb1 and 1% recombinant protein a, and collected fractions flowing and collected fractions of the eluate from the chromatographic separations corresponding to Figure 4. The results are shown in Figure 5. On Figa shaded peak is a complex of MAb1-protein A. the Blue curve corresponds to the fractions passing flow (FT), and the red eluate.

Example 5: Purification of antibodies on Q Phenyl Sepharose 6 Fast Flow

The sequence of actions

In conditions that do not lead to binding, sample, containing approximately 50 mg of mAb, were applied to the prototype Q Phenyl Sepharose™ 6 Fast Flow. The fraction passing flow (FT) was collected at 5, 10 and 15 column volumes (CV). Analyzed the fractions from the peak elution.

Q Phenyl Sepharose™ 6 Fast Flow was prepared by incorporating a Q-group (-N(CH3)3to Phenl Sepharose™ 6 Fast Flow (45 μmol of phenyl groups per ml of gel) in accordance with the standard method (see below). Ion-exchange capacity Q Phenyl Sepharose™ 6 Fast Flow was $ 108 μmol per ml of gel. At pH 7.0 or 8.0 sample containing 50 mg mAb (purified on MabSelect), was applied on the column and operating characteristics Q Phenyl Sepharose™ 6 Fast Flow was evaluated by analyzing selected fraction flowing to the protein of the host cell (NDS) and protein A.

Materials/analyzed samples

Speakers and Phenyl Sepharose™ 6 Fast Flow was obtained from GE Healthcare (Uppsala, Sweden)

HR 5/5™No. kat-0338-01CV=1 ml

Devices

Chromatographic system: ÄKTAExplorer™ 10

Spectrophotometer: Spectra MAX plus

Chemicals

All used chemicals were of analytical purity. Used water filtered with the use of MilliQ.

Obtaining Q Phenyl Sepharose™ 6 Fast Flow

In one of the ways to obtain the separation matrix according to the invention, which is shown in the example below, the quality of the source material used cross-linked agarose gel (Phenyl Sepharose™ 6 Fast Flow (high sub (high substitution)), GE Healthcare, Uppsala, Sweden).

The introduction of the group Q in the Phenyl Sepharose™ 6 Fast Flow (high sub)

Group of Q (-N(CH3)3) were introduced in the Phenyl Sepharose™ 6 Fast Flow (high sub) by reacting with chloride glycidyl-trimethylammonium (G-MAC) as follows: 15 g visus is authorized by suction 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 NaBH4and 40 ml G-MAC. The mixture was stirred for 16 hours at 30°C. After filtration of the mixture, the gel is then washed with 100 ml of distilled water. 100 ml ethanol and 100 ml of distilled water.

After titration received the degree of substitution equal to 0.11 mmol amines/ml of gel.

Samples

Used monoclonal antibodies expressed in cultures of Cho, and then, before conducting the experiments of the present invention, purified using conventional affinity chromatography on protein A.

The concentration of mAb

The mAb sample was diluted ten times with buffer. The sample solution in two iterations were measured at A. To calculate the concentration in accordance with the law of Lambert-Baer used average:

C=A/(I×ε),

where C represents the concentration of IgG;

And is the absorption at 280 nm;

I is the path length;

ε is the molar extinction coefficient for mAb, mg-1ml=1,46.

Chromatography on Q Phenyl Sepharose™ 6 Fast Flow

Department of mAb proteins from the host cell and the protein And tested in conditions that do not lead to binding. Applied to the column, the sample was a mAb, purified on MabSelect. The flow rate was 0.5 ml/min (150 cm/h). The absorption at 280 nm were detected throughout the section the definitions. Tested two different buffer (see below). Before each division were replaced buffer a-buffer. Depending on sample volume used column HiPrep desalting and HiTrap desalting.

Buffers: buffer: 25 mm Tris/HCl, pH 8.0

In-buffer: 25 mm Tris/HCl, 0.5 M NaCl, pH 8.0

A-buffer: 25 mm phosphate buffer, pH 7.0

In-buffer: 25 mm phosphate buffer, 0.5 M NaCl, pH 7.0.

Method: as source material used eluate with MabSelect with a defined pH value.

Trim5CVA-buffer
Introduction sample16 CV(50 mg mAb)
Flushing5CVA-buffer
Gradient5CV100%In-buffer
Cleanup gradient5CVA-buffer

During introduction of the sample, washing and elution were collected fractions of 1 ml

After each separation was performed CIP (will isdu in place) using 1 M NaOH. The processing time was approximately 25 minutes.

Analysis of protein And

Selected fractions were mixed with sample diluent SPA in proportions 800 ál of sample diluent SPA+200 μl of the sample. After stirring fraction was heated in a heating block at 99°C for 10 minutes, then the mixture was stirred. Next, the samples were analyzed for recombinant protein A.

Analysis of the proteins of the host cell (NDS)

Samples (minimum of 600 μl) were analyzed on the content of the NDS. The lower detection limit is 10 ng/ml.

Results

In conditions that do not lead to binding of approximately 50 mg mAb was applied on the column HR 5/5, filled Q Phenyl Sepharose™ Fast Flow, at two different pH values (pH 7.0 and 8.0). Collected fractions flowing at 5, 10 and 15 column volumes (CV) according to Figure 1. In Tables 8 and 9 presents the results of the analysis of protein and NDS in the fractions passing flow. No residual protein And were not able to fix in these fractions. In addition, there have been no proteins of the host cell in FT1 and FT2, when used the sample with pH 8.0. A small number of proteins of the host cell was observed when using a sample with a pH of 7.0, but the content of the NDS was decreased by about 50 times compared with the contents of the NDS in the sample. 6 also shows that the molecules of monoclonal antibodies adsorbed to Q Phenl Sepharose™ Fast Flow, because in a gradient elution on the chromatogram observed only a very small peak (6).

Table 8
The results of the analysis of protein And
ColumnpHStart (ng/ml)FT1 (ng/ml)FT3 (ng/ml)The eluate (ng/ml)
Q Phenyl Sepharose™ FF8,06,980,000,0048,25
Q Phenyl Sepharose™ FF7,05,030,000,0036,15

Sample volume was 16 ml and fractions FT1-FT3 was 1 ml of the Collected volumes of elution was 2 ml.

Table 9
The results of the analysis of the proteins of the host cell
ColumnPHStart (ng/ml) FT1 (ng/ml)FT2(ng/ml)FT3 (ng/ml)The eluate (ng/ml)
Q Phenyl Sepharose™ FF8,01100<10<10124900
Q Phenyl Sepharose™ FF7,012001623265100

Sample volume was 16 ml and fractions FT1-FT3 was 1 ml of the Collected volumes of elution was 2 ml.

1. A way of separating one or more than one antibody from one or more than one of the other compounds in a liquid sample, in which a mobile phase containing a specified liquid sample is brought into contact with the multi-modal separation matrix to adsorption of one or more compounds of the targets, while the antibodies remain in the free state in the mobile phase, where the multi-modal separation matrix contains the group of the first type, capable of interacting with negatively charged sites of the compound(s)target(s), and groups of the second type, capable of at least one interaction, on the Sabbath.personal interactions charge-charge, with the specified(s) connection(s)target(s).

2. The method according to claim 1, where the multi-modal separation matrix proposed in the chromatographic column, the mobile phase passes through the said column under the action of gravity and/or by using a pump, and antibodies are removed from the flow through the column.

3. The method according to claim 1, where the liquid sample contains supernatant obtained by fermentation of a cell.

4. The method according to claim 1, where the contact with the multi-modal separation matrix is preceded by a stage mechanical filtration and/or chromatography.

5. The method according to claim 1, where the liquid sample contains the raw source material.

6. The method according to claim 5, where the compound(I)-target(s) is(are) a protein of the host cell and essentially all of these proteins adsorb on the multi-modal separation matrix.

7. The method according to claim 4, where the liquid sample contains the eluate from the separation matrix.

8. The method according to claim 7, where the separation matrix, which receive the eluate contains protein ligands, preferably the ligands on the basis of protein a or G.

9. The method according to claim 1, where the conductivity of the mobile phase is in the range from 0 to 25, for example from 0 to 15 MSM/see

10. The method according to claim 1, where the group of the first type are Quaternary amines.

11. The method according to claim 1, where the group of the second type represent groups, prasouda hydrogen bonds.

12. The method according to claim 1, where the group of the second type represent a hydrophobic group, such as groups containing aromatic(s) or heteroaromatic(s) ring(s) structure(s).

13. The method according to claim 1, where the separation matrix contains the group of the first and second type associated with the same ligands.

14. The method according to claim 1, where the first group and the group of the second type are separated from each other hydrocarbon chain of 1-3 carbon atoms.

15. The method according to claim 1, where the ligands immobilized on the substrate through its groups of the first type.

16. The method according to claim 1, where the separation matrix contains the group of the first and second type associated with different ligands.

17. The method according to claim 1, where the separation matrix in the form of particles and contains a mixture of particles of the first type, in which the immobilized ligands containing groups of the first type; and particles of the second type, in which the immobilized ligands containing groups of the second type.

18. The method according to claim 1, where the separation matrix is a filter on which an immobilized mixture of ligands of the first type, containing groups of the first type; and ligands of the second type containing groups of the second type.

19. The method according to claim 1, where the separation matrix contains the group of the third type, capable of interaction of the third type with the connection-target.

20. The method according to claim 1, where the antibodies are monoclonal antibodies.

21. The method according to claim 20, where the antibodies are humanized antibodies.

22. The method according to claim 1, where the multi-modal separation matrix proposed in a disposable chromatographic column.

23. The method according to item 22, wherein the disposable column sterilized before bringing into contact with the mobile phase.

24. Kit for purification of antibodies from one or more than one other component in the liquid containing different cells of the first chromatographic column filled with the first separation matrix; the second chromatographic column Packed with multi-modal separation matrix, which contains groups of the first type, capable of interacting with negatively charged sites of the connection target, and groups of the second type, capable of at least one interaction that is different from the interaction of the charge-charge with the specified connection target; one or more buffers; and written instructions for the purification of antibodies from a stream passing through the multi-modal separation matrix.

25. Set point 24, where in the first chromatographic separation column matrix contains protein ligands, preferably the ligands on the basis of protein a or G.

26. Set point 24, where the first and/or second chromatographic columns are disposable columns.



 

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