Chromatographic ligand

FIELD: chemistry.

SUBSTANCE: claimed invention relates to chromatographic ligand representing N-benzyl-N-methylethanolamine, as well as to method of obtaining separation matrix, containing said ligand and chromatographic column for antibody purification.

EFFECT: elaboration of efficient method of obtaining chromatographic ligand.

10 cl, 4 tbl, 4 ex, 10 dwg

 

The scope of the invention

The present invention relates to a new chromatographic ligands, which are applicable for the purification of biomolecules, such as proteins. These ligands are applicable, for example, for the purification of antibodies and preferably immobilized on a porous base, such as particles or membrane. Therefore, this invention also includes a chromatographic matrix, containing the new ligands, its preparation and purification of antibodies.

Prior art

The immune system consists of many interdependent cell types that collectively protect the body against bacterial, parasitic, fungal, viral infections and from the growth of tumor cells. The sentinels of the immune system are macrophages, which are constantly moving in the bloodstream of the host. When infection or immunization macrophages react, capturing pathogens, marked alien molecules known as antigens. This event is mediated by helper T-cells, defines a complex chain of responses, which leads to stimulation of b-cells. These b-cells, in turn, produce proteins called antibodies, which bind with the foreign agent. This binding between antibody and antigen marks the alien pathogen destruction by phagocytosis or is ctivitie of the complement system. There are several different classes of antibodies, also known as immunoglobulins, such as IgA, IgD, IgE, IgG and IgM. They differ not only its physiological role, but also its structures. From a structural point of view, have been widely investigated IgG-antibodies, possibly due to the major role they play in a Mature immune response. Polyclonal antibodies are produced according to standard techniques by immunizing an animal with a suitable antigen. In response, the animal produces antibodies are polyclonal. However, for many reasons it is desirable to have a single clone specific antibodies, known as monoclonal antibodies. Monoclonal antibodies (mAbs) are produced hybrid or fused cell, representing the product of the merger between normal B-cell, which produces only one antibody, and abnormal myeloma tumor cell. The resulting hybrid, known as hybridoma, currently used in the standard methods of obtaining antibodies.

Biological activity, which have the antibodies currently used in many different applications in the diagnosis of human and veterinary diagnostics, medical care and therapeutic sector. In fact, in the last few years, monoclonal anti-Christ. ate and recombinant constructs antibodies have become the largest class of proteins, investigated currently in clinical trials and receive FDA approval (Management under the control over products and medicines of the USA) as therapeutic and diagnostic tools. In addition to the expression and production strategies required for effective cleaning protocols to obtain high-purity antibody simple and cost-effective way.

Traditional methods of isolation of immunoglobulins based on the selective reversible precipitation of the protein fraction containing immunoglobulins, while other groups of proteins remain in solution. Typical precipitating agents are ethanol, polyethylene glycol, lyotropic salt such as ammonium sulfate and potassium phosphate, and Caprylic acid. Usually, such deposition methods give very contaminated products and at the same time, are time-consuming and laborious. In addition, adding a precipitating agent to the crude substance complicates the use supernatant for other purposes and creates the problem of waste disposal, which becomes especially important when it comes to large-scale purification of immunoglobulins.

An alternative method of separation of immunoglobulins is chromatography, which includes close family separation techniques. The feature that distinguishes chromatography from most other physical and the mental and chemical methods of separation, is that the contact lead two mutually immiscible phases, one phase is stationary and the other movable. The mixture model, introduced in the sliding phase, undergoes a series of interactions with the stationary and mobile phases, while it flows through the system using a mobile phase. Interaction is based on differences in physical or chemical properties of the components in the sample. These differences determine 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 in ascending order of interaction with the stationary phase. Least delayed component is eluted first, most strongly held substance eluted last. Separation is achieved when one component is retained to a degree sufficient to prevent overlap with the area adjacent the solute when the sample components suiryudan with speakers. Constantly attempt to create the optimal stationary phase for each specific purpose of the separation. This stationary phase usually consists of a base or core matrix to which is attached a ligand containing a functional, i.e. linking group. Each type of chromatography is usually specified based on the principle of interaction, it uses, for example, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography.

Affinity chromatography is based on specific interactions between the target biomolecule and biospecific ligand in accordance with the principle of recognition of the key-lock. Thus, the target and the ligand are affine pair such as antigen/antibody, enzyme/receptor, etc. Protein affinity ligands are well known, for example, affinity chromatography with protein a and protein G, both of which are common methods of isolation and purification of antibodies. It is well known that chromatography with protein And provides exceptional selectivity, particularly in relation to monoclonal antibodies and, therefore, allows to obtain a high degree of purity. When used in combination with ion exchange, hydrophobic interactions, stages on the hydroxyapatite and/or gel filtration, methods based on protein And became the preferred method of purification of antibodies for many biopharmaceutical companies, see, for example, WO 8400773 and US 5151350. However, due to the peptide bonds of proteins matrix with protein And have somewhat sensitive to alkalis. In addition, when the matrix with protein And used for purification of antibodies from cell is ulturally environment, dentures originating from these cells can cause cleavage of the protein or its peptide fragments.

Ion exchange chromatography is often used in the protocols of isolation of immunoglobulins. When ion-exchange chromatography negatively charged amino acid side chains of the immunoglobulin will interact with the positively charged ligands chromatographic matrix. When cation exchange chromatography, on the other hand, positively charged amino acid side chains of the immunoglobulin interact with negatively charged ligands chromatographic matrix.

The hydrophobic interaction chromatography (HIC) represents another described method and is used in the protocols of isolation of immunoglobulins. If the goal is immunoglobulin product of high purity, it is generally recommended to combine HIC with one or more additional stages. When HIC, in order immunoglobulin effectively contacted hic key matrix, requires the addition of lyotropic salts to the mobile phase. Linked hemoglobin is then released from the matrix at lower concentrations of lyotropic salts. Thus, a disadvantage of this method is the need to add lyotropic salt to the raw substance, as this can cause problems and, consequently, increase the component cost for large-scale consumers. For example, raw materials such as serum, plasma and egg yolk, the addition of lyotropic salts to the raw substances in many cases is unacceptable in large-scale applications, as salt can prevent any economically feasible to use depleted by immunoglobulin raw substances. An additional problem in large-scale applications is the elimination of several thousand gallons of waste.

In the US 5945520 (Burton et al.) described chromatographic resin mixed mode, which have a hydrophobic nature at pH binding and hydrophilic and/or electrostatic in nature with a pH of desorption. This resin is designed to be contacted with the target compound from the aqueous solution, both at low and at high ionic strength. This is achieved by using selected an ionisable ligands containing spacer elements shoulder and at least one of an ionisable functional group, where the density inisheer ligands on the solid matrix is based on more than the lower of either approximately 150 µmol/ml resin, or 1 mmol/g dry weight of resin. Furthermore, the hydrophobic nature of the resin containing an ionisable these ligands are sufficient to bind at least 50% of the target compounds in the aquatic environment at high and low ionic strength at first the om pH. Illustrative examples of an ionisable functional groups are 4-(aminomethyl)pyridine, 3-(aminomethyl)pyridine, 2-(aminomethyl)pyridine, 1-(3-aminopropyl)imidazole, 2-(aminomethyl)benzimidazole, 4-(3-aminopropyl)morpholine.

Also WO 01/38228 (Belew et al.) refers to the manner in anion-exchange adsorption in which to retrieve the negatively charged substances from liquids by means of their binding uses the thioester-exchangers. Each ligand contains positively charged nitrogen and thioester bond at a distance of 1-7 atoms from the specified charged nitrogen. Target substances such cells, parts of cells and substances containing peptide structure, adsorbed when the salt concentration of about 0.25 M NaCl.

Finally, in the US 6702943 (Johansson et al.) discovered a way to extract a target substance from a fluid by adsorption on the matrix bearing a variety of ligands containing anion-exchange groups and a hydrophobic structure. More specifically, the ligands contain an aromatic ring near the positively charged anion-exchange groups. The target substances are identified as cells, parts of cells and substances containing peptide structure. Described ligands labeled as "high salt" ligands, due to their ability to adsorb a target substance at high concentrations of salt, such as 0.25 M NaCl.

However, for optimizing the process, related to the cleanup of specific target molecules, require unique working conditions, and better separation matrix will in each case be subject to change. For example, in the biotechnology industry to develop specific processes for the purification of peptides and proteins; nucleic acids, virus, etc. in Addition, when cleaning antibodies to select the separation matrix will be decisive type antibodies. Thus, there is still a need in the field of alternative separation matrix to provide a wide choice for treatment of many new products that are constantly being developed.

A brief description of the invention

In one aspect of the invention proposes a new ligand, which is applicable to the separation of antibodies from other components of the liquid.

In a particular aspect of the invention proposed such a ligand, which is able to adsorb impurity proteins, but not the target antibody.

Additional aspects and advantages of this invention will become clear from the detailed description that follows.

A brief description of graphic materials

Figure 1 shows illustrative chromatographic ligand according to the invention, namely N-benzyl-N-methylethanolamine connected to the base via its amine.

Figure 2 shows the chromatogram of the separation of monoclonal antibodies is and the separation matrix, containing N-benzyl-N-methylethanolamine ligands immobilized on Sepharose™ 6 FF and, for comparison, a strong aminoalkanoic Q Sepharose™ FF, as described below.

On Figa) and b) shows the results of chromatography conducted on prototypes of ligands with a mixture of mAb1-rProtein A.

On Figa)-C) shows the results of the analytical exclusion chromatography (SEC) samples with mAb 1, 1%rPrA and the joint flow and suetnyh fractions from chromatographic separations with 3.

On Figa presents the results of the analytical exclusion chromatography (SEC) for timecamera.

On Figb presents the results of the analytical exclusion chromatography (SEC) for 2-aminobenzimidazole.

Figure 6 presents the results of the analytical exclusion chromatography (SEC) for N-benzyl-N-methylethanolamine.

Definition

The terms "antibody" and "immunoglobulin" in this application are used interchangeably.

The term "separation matrix" is used herein to denote a material consisting of a base to which is attached one or more than one ligand containing a functional group. Sometimes the separation matrix in this field uses the term "resin".

The term "multimodal" separation matrix refers to a matrix capable of providing at least two different, but together deistvujushsaja, which interact with the connection that you want to link to. For example, one of these sites can provide favorable type charge-charge interaction between the ligand and the target substance. Another site may provide electronic acceptare-donor interaction, and/or hydrophobic and/or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen bond, TT-TT, cation-TT, charge transfer, dipole-dipole, induced dipole, etc. "Multimodal" separation matrix is also known as the separation matrix "mixed type".

The term "surface" means here, all external surfaces and includes in the case of porous bases of the outer surface and pore surface.

The term "eluent" is used in its conventional in the art value, i.e. the buffer with a suitable pH and/or ionic strength to release one or more components of the separation matrix.

The term "stage capture" in the context of liquid chromatography refers to the initial stage of the separation process. Often stage capture includes purification, concentration, stabilization and significant purification from soluble impurities. After the capture stage can be followed by intermediate treatment, which fill the nutrient reduces the amount of remaining impurities, such as proteins and host cells, DNA, viruses, endotoxins, nutrients, components of the cell culture medium, such as defoamers and antibiotics, and additives, a related product, such as aggregates varieties with improper folding and aggregation.

The term "disposable" means in this description, in the context of chromatographic columns and other separation matrix, a matrix that is designed for single use or for a limited number of applications. Disposable products are primarily used to remove contaminants that are harmful even in small quantities, and in this case it is convenient to adsorb the specified pollution on the matrix and then to throw this matrix. Another situation where it is desired disposable products, is sterile processing, when the matrix is sterile or at least aseptic.

The term "fine cleaning stage" refers, in the context of liquid chromatography to the final stage of purification, which removes trace impurities with getting active, safe product. Impurities are removed at the stage of fine purification, often represent the conformational isomers of the target molecule or the products of the proposed diversion.

The term "Fc-binding protein" means a protein capable svyazyvatsyas kristallisoituu part (Fc) antibodies and includes, for example, protein a and protein G, or any fragment or protein, which retains the specified property of the binding.

Detailed description of the invention

In the first aspect of the present invention is a chromatographic ligand containing aromatic ethanolamine. The ligand in accordance with the invention is particularly useful in the purification of antibodies, as is discussed in greater detail below.

In the first embodiment of the present ligand defined by the following formula:

R1-R2-N(R3)-R4-R5where

R1represents a substituted or unsubstituted aromatic ring system such as phenyl group;

R2represents a hydrocarbon chain containing 0-4 carbon atoms;

R3represents a hydrocarbon chain containing 1-3 carbon atoms;

R4represents a hydrocarbon chain containing 1-5 carbon atoms; and

R5HE is a or N.

As follows from the above, the group R1connected with the amine through a carbon chain R2that may not contain carbon atoms, that is, to create a link between R1and an amine; or contain 1-4 carbon atoms, for example 2-3 carbon atoms, which may be substituted. The carbon chain R4connecting with Amin R5can erati 1-5 carbon atoms, for example of 2-4 carbon atoms, possibly substituted. R3amine may contain 1-3 carbon atoms, for example 2 carbon atoms, possibly substituted.

Aromatic ring system R1may contain one or more than one substituted or unsubstituted phenyl group, provided that the substitution(I) does not impair the binding properties of the ligand to any significant degree. Thus, R1may contain one or more than one aromatic ring, for example fenelonov, biphenylene or naftalanovoy structure and other aromatic ring system. The aromatic ring may be a heterocyclic containing one or more than one nitrogen atom, oxygen or sulfur, for example pyridine, pyrimidine, pyrrole, imidazole, pyrrole, imidazole, thiophene or Piran. Illustrative of the substituted groups R1selected from the group consisting of hydroxyphenyl (2-, 3 - and 4-); 2-benzimidazolyl; methylthiophenyl (2-, 3 - and 4-); 3-indolyl; 2-hydroxy-5-nitrophenyl; AMINOPHENYL (2-, 3 - and 4-); 4-(2-amino-ethyl)phenyl; 3,4-dihydroxyphenyl; 4-nitrophenyl; 3-triptoreline; 4-imidazolyl; 4-aminopyridine; 6-aminopyrimidine; 2-tanila; 2,4,5-diaminophenyl; 4-aminotriazole and 4-sulfonatophenyl.

In the preferred embodiment R1represents unsubstituted phenyl. In an alternative embodied the attachment R 1represents phenyl substituted by one or more groups.

In addition, one or more of R1, R2, R3and R4can be replaced by any suitable Deputy to until the binding properties of the ligand does not deteriorate to any significant degree. For example, if you want more hydrophobic ligand, it may contain one or more than one hydrophobic group, such as group IT. Alternatively, the substitution may increase the hydrophobicity of the ligand, and in this case, the ligand can contain one or more hydrophobic group, such as alkyl and/or fluorine. Finally, the substitution can be used to introduce one or more additional functional elements, such as the charged elements to enhance the multimodal nature of the ligand. In addition, the carbon chain R2and R3can be linear or branched up until branching does not affect the binding properties of the ligand to any significant degree.

In a particular embodiment of the present ligand R2represents-CH2-. In another embodiment R3represents-CH3. In another embodiment R4represents-CH2-CH2-CH2- or-CH2-CH2-. In another embodiment R1represents unsubstituted phenyl.

The ligand according to the invention can be easily synthesized by a person skilled in the art by standard methods of organic chemistry.

Another aspect of the present invention is a method for the separation matrix comprising the immobilization of a variety of ligands, as described above, on the basis of. To obtain the matrix, suitable for a single use, especially in the field of medicine or diagnostics, the separation matrix made according to the invention, at a later stage also sterilized. Thus, in one embodiment of this method involves the preparation of a matrix, as described above; placing the matrix obtained in this way, the column and sterilization thus prepared matrix. The person skilled in the art can easily carry out sterilization in suitable conditions, for example by heat processing; radiation or any other traditionally used method.

As follows from the above equation, it neimmunizirovannah state, the ligand according to the invention contains a Quaternary amine, which forms a suitable tool for associating it with basis, thus creating tie the config ligand, which contains Quaternary amine and phenyl group. Therefore, it is believed that the immobilization of the ligand according to the invention is a multi-modal anion-exchange ligand, as in addition to the positively charged Quaternary amine group, it also contains an aromatic ring structure, which is hydrophobic. Methods of immobilization of ligands on porous or non-porous surface is 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 substrate surface is in the range close to the interval, typically used for traditional ion-exchange matrix.

In the preferred embodiment, the binding of ligand to the basis provided by the introduction of a linker between the base and the linker. Linking can be carried out after any of the traditional method of covalent binding, for example when using epichlorohydrin; epibromohydrin; allyl-glitsinovogo ether; bis-epoxides, such as potentiallly ether; halogen-substituted aliphatic substances, such as dichloropropanol; and diphenylsulfone. These methods are well known in the field and easy to carry out specialist.

In a specific embodiment, the ligand according to the invention is associated with a through D. the other linker molecule, also known as the extender. The extenders are well known in this field and are widely used for spatial distances between ligand and substrate. The extenders are sometimes referred to as probe or flexible shoulder, a more detailed description of possible chemical structures can be found, for example, in US 6428707, which is included in this description by reference. Briefly, the extender may be in the form of a polymer, such as Homo - or copolymer. Hydrophobic polymer extenders can be of synthetic origin, i.e. to have a synthetic skeleton, or of biological origin, that is, the biopolymer having a skeleton found in nature. Typical synthetic polymers are polyvinyl alcohols, polyacrylic and polymethacrylamide, polyvinyl ethers, etc. Typical biopolymers are polysaccharides, such as starch, cellulose, dextran, agarose.

The base may be made of organic or inorganic substances and can be porous or non-porous. In one embodiment the base is prepared from a natural polymer, such as cross-linked carbohydrate material, for example agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, 'gellan, alginate, pectin, starch, etc. the Basics of the native polymer is easy to make and can be custom made standard with the persons, such as reverse gelation in suspension (S Hjertén: Biochem Biophys Acta 79(2), 93-398 (1964). In a particularly preferred embodiment the base is a type of relatively rigid but porous agarose, which is prepared in a way that reinforces its fluid properties, see for example US 6602990 (Berg) or SE 0402322-2 (Berg et al.). In an alternate embodiment the base 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 ethers, vinylamide etc. Such synthetic polymers are easy to prepare and can be made by standard methods, see, for example Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L lndustria 70(9), 70-75 (1988)). Native or synthetic polymer base also available commercially, for example from GE Healthcare, Uppsala, Sweden, for example, in the form of porous particles. In yet another alternative embodiment the base is made from an inorganic polymer, such as silica gel. Inorganic porous or non-porous bases are well known in this area and can be easily prepared by standard methods.

Suitable particle sizes of the separation matrix of the present invention can be in the range of diameters of 5-500 μm, for example, 10-100 μm, for example 20-80 μm. In the case with the society of 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 person skilled in the art can easily select a suitable particle size and porosity depending on how you intend to use. For example, for large-scale process, for economic reasons it may be preferable to more porous but rigid base, which will allow the processing of large volumes, especially at the capture stage. In chromatography, the choice will be influenced by such method as the size and shape of the column. In the way of expanded layer matrix usually contains fillers of high density, preferably made from stainless steel. For other ways on the nature of the matrix may be affected by other criteria.

Thus, the second aspect of the present invention is a separation matrix, which contains the ligands described above, associated with the basis. As is obvious to a person skilled in the art, each basis will usually contain a variety of ligands. In a particular embodiment, the base contains a ligand, as described above, in combination with a second type of ligand, where the ligand according to the invention is present in amounts of at least about 30%, preferably at least about 50%, more preferably at least arr is siteline 70% and most preferably at least about 90% of the total number of ligand. Such a combined ligand separation matrix can be constructed for a specific case, when additional interactions improves its separation properties. The second type of ligand can contain one or more than one charged group, such as a cation-exchanger used for elution of compounds through charge repulsion; hydrophobic group; groups capable of forming hydrogen bonds; affine group or similar.

In the first embodiment of the matrix according to the invention is in the form of particles, such as essentially spherical, elongated particles, or particles of irregular shape. In a particular embodiment of the separation matrix is dried, for example, is in the form of dry particles that are using immersed in the liquid to maintain their original shape. In the illustrative embodiment of this dry separation matrix consists of dried agarose particles. However, the matrix according to the invention alternatively can take any other form, traditionally used in the separation, such as monoliths, filters or membranes; the capillaries, chips, surface, etc.

Therefore, in the second embodiment, the matrix contains a membrane structure, such as a single membrane, the package membranes or filter.

The third aspect of the invention is when is the change of the separation matrix, described above. In the first embodiment of the present invention uses the separation matrix as described above, in the purification of proteins. In the preferred embodiment of the present application, the protein is an antibody; an antibody fragment; or a fused protein containing the antibody. In another embodiment of the present invention using the separation matrix as described above, in the separation of any other connection, for example selected from the group consisting of polypeptides; nucleic acids such as DNA, RNA or oligonucleotides, plasmids; virus; prion; cells, such as prokaryotic or eukaryotic cells; lipids; carbohydrates; organic molecules such as small organic molecules; drug target; diagnostic marker molecules. The application is discussed in greater detail below. In yet another embodiment of the present invention using the separation matrix as described above, as the basis in cell culture, that is, for immobilization of cells that grow on surfaces. As is obvious to a person skilled in the art, in this application the term division is used for purification; separation and extraction of compounds, and also includes the identification of target compounds, for example for diagnostic purposes.

The fourth aspect of the present invention is the fast way to split, when the target connection, such as an antibody, is separated from one or more other compounds in a liquid sample by bringing the mobile phase containing the specified liquid sample in contact with the separation matrix as described above. In the preferred embodiment of the present method is carried out using the principles of liquid chromatography, i.e. by passing the mobile phase through the chromatographic column containing a separation matrix according to the invention. In yet another alternative embodiment of the present method is carried out using periodic chromatographic process, wherein the separation matrix is added to the vessel containing the liquid sample. In a particular embodiment of the separation matrix is added to a periodic process, contains dry particles, such as dry particles of agarose. In yet another embodiment the method is carried out using the principles of chromatography in expanded layer, that is, by adding the mobile phase to the expanded layer, for example fluidized layer, the separation matrix, which is in the form of essentially spherical particles containing a high density filler.

In the first embodiment of the present method is undesirable compounds adsorb on the separation matrix, while the desired connection, such as antibodies, does not deviate from the mobile phase neadsorbirovanne. As is obvious to a person skilled in the art, the nature and character of the adsorbed compounds will depend on the origin of the liquid sample. Examples of compounds adsorbed in this embodiment, where the desired antibodies are not adsorbed, are cells and cell debris; proteins and peptides; nucleic acids such as DNA and RNA; endotoxins, viruses, remnants of cultural media, etc. In a specific embodiment, the present separation matrix is presented in the chromatographic column and the mobile phase passes through the column under the action of gravity and/or pumping, antibodies get into the flowing fluid column. Thus, the advantage of this embodiment is that it does not require any elution of the product - antibodies from the column. The elimination of a separate stage of elution is attractive from the point of view of method, as fewer stages will lead to a more rapid purification Protocol and, therefore, reduce the cost of this method. In addition, antibodies sensitive to certain conditions, which will, for example, have a negative influence on the pattern of folding; or to split them, attacking their peptide bonds. Thus, even though the conditions for elution of-exchangers in General do not include any extreme chemical agents, changing the salt and the pH can affect sensitive antibody moreover, this effect may vary from species to species, depending on P1, charge distributions, etc. Therefore, another advantage of this embodiment is that it allows you to avoid adding eluent and applications eluting conditions to the target compounds. To obtain the most suitable conditions for adsorption of compounds in a liquid sample together with a suitable buffer or other liquid with obtaining the mobile phase. The present embodiment mainly carried out under conditions suitable for anion exchange chromatography, which usually includes adsorption at relatively low concentrations of salt. Thus, in one embodiment of this method, the conductivity of the mobile phase is in the range of 0-25, for example 10-15 MS/see In one embodiment the pH of the mobile phase is about 5-6. If it is desirable then to release the adsorbed compounds, for example for re-use matrix, elution can be done at higher salt concentrations, for example, by increasing the ingredient salt. For elution of adsorbed compounds is pH also or alternatively can be shifted, for example to represent a decreasing pH gradient.

In the second or alternative embodiment of this method the target compounds adsorb on mA is ritsu, as in conventional liquid chromatography. The matrix then can be reused after selective elution of the product. Elution easy to implement, passing a suitable buffer through the column. If you want to or between such(and) transmission(s) you can use one or more stages of washing. In one embodiment the working conditions of this embodiment, such as when traditional ion exchange, i.e. adsorption using a mobile phase having a low conductivity, and elution using buffer with a high conductivity, as discussed above. The person skilled in the art can easily adjust these conditions by testing different conditions and analysis adsorbed(s) connection(s) and flow. In a particular embodiment the desired compounds are antibodies.

Selecting between the first and second embodiment described above, the person skilled in the art can easily adapt the conditions for adsorption of specific compounds, mainly by regulating the pH and/or conductivity. For example, the separation of antibodies of different classes of antibodies have different charges and pattern of charge distribution that, together with the purpose of separation, will decide whether it is more preferred to adsorb antibodies or allow them to pass through the column without adsorption.

Antibodies, separated by the accordance with one embodiment of the present invention, can happen from a well known source, such as cells cultured on the surface, or from periodic or continuous cell culture in a fermentation tanks or vessels. Thus, in one embodiment the fluid is a supernatant obtained by fermentation of the cells. Examples of compounds from which you want to purify antibodies are proteins, DNA, viruses, endotoxins, nutrients, components of the environment for culturing cells, such as defoamers and antibiotics, and related product impurities, such as improperly folded species and aggregates. Stage of contact between the mobile phase and the separation matrix of the present invention, 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, preferably mechanically to remove cellular debris, unbroken cells and other relatively large components to the stage of use of this matrix.

In one embodiment of this method represents a stage of capture in the purification Protocol. In a particular embodiment the liquid sample is a crude mixture, which is filtered before contact with the chromatographic matrix according to the invention. Therefore, given the second invention will be still present stage of capture, even if the liquid sample was mechanically cleaned. As is well known, the cells of the host, which produce antibodies, also contain several other proteins, traditionally known as the proteins of the host cell (NDS). Such NDS include enzymes, such as proteases, and other proteins produced by cells of the host. Thus, in one embodiment, essentially all of the proteins of the host cell of the liquid sample is removed in this way, for example by adsorption on the separation matrix.

In alternative embodiments, this method uses as a second, third or even fourth chromatographic stage in the purification Protocol, such as the purification of intermediate compounds or fine cleaning stage. Thus, in one embodiment the mobile phase used with this separation matrix, contains the eluate from the separation matrix containing the antibody. 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 than one Fc-binding protein ligand, such as ligand-protein A. the Term ligand-proteins And in this context includes native and recombinant protein or its functional fragments. In this context the term "functional fragment means a fragment, still retaining the original properties of the binding proteins. Such affinity matrices are commercially available, such as MabSelect™ from GE Healthcare. Therefore, in this embodiment extracted, preferably adsorbed compound may be one or more selected from the group which consists of released protein A; complexes formed by protein a and antibodies, such as complexes of protein a-mAb, and these complexes may contain a number of antibody binding sites on the protein molecule And, for example 2-4 antibodies in complex with one molecule of protein A; and units of released protein a or antibodies. As is obvious to a person skilled in the art, depending on the specific conditions used in the previous phase, such as affinity chromatography, the eluate may require conditioning by appropriate additions or zavedenii. Thus, the eluate combined with a suitable buffer or fluid with obtaining the mobile phase.

This method is useful for separating any monoclonal or polyclonal antibodies, such as antibodies originating from the hosts-mammals, such as mice, rodents, primates and humans, or antibodies derived from hybridomas. In one embodiment of the separated antibodies are human or humanitarianism antibodies. Antibodies can be of any Klah is sa, that is, can be selected from the group consisting of IgA, IgD, IgE and IgM. In one embodiment the antibodies are antibodies which are able to bind with protein A, or Fc-containing antibody fragments or fused proteins. In a specific embodiment the antibody is an immunoglobulin G (IgG), such as lgG1. In one embodiment the method is used for the 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 is approximately 9. In this context, it 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 the separation of fragments of any of the above antibodies, as well as fused proteins containing such antibodies. In one embodiment, the antibodies are monoclonal antibodies. In a specific embodiment, the antibodies are humanized antibodies.

As follows from the above, in the method according to the present invention to obtain essentially pure fraction readsorbing antibodies. In this context, the term "essentially pure" means that essentially all connections that are not antibodies, are removed. Most preferably this separation matrix is removed at least at listello 80%, for example at least 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 content of impurities. However, as is obvious to a person skilled in the art, the purity will depend on the concentration of antibodies in a liquid sample applied to the separation matrix, as well as other common conditions. Thus, in one embodiment the antibodies that are shared in this way are antibodies therapeutic quality. Thus, antibodies are purified according to the invention, are useful in research, as well as for the manufacture of pharmaceutical drugs based on antibodies, such as medicines on the basis of mAb. 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 according to the present invention, useful in food products.

In a particular embodiment of this method the separation matrix of the present invention are presented in the form of a disposable chromatographic column or a disposable filter. The advantage of using disposable products in the method of purification of therapeutic connect the developments such as antibodies, is that it avoids cross-contamination between two different processes. Thus, in one embodiment, this separation matrix proposed in the form of sterile chromatographic column or filter. In one embodiment the method is carried out as a periodic process in which a disposable separation matrix is added to the vessel containing the liquid, from which to extract antibodies. The target compounds allow for appropriate time to adsorb to the matrix, after which the liquid phase containing the antibodies are removed from the vessel. Used matrix can then be eliminated, without releasing the adsorbed compounds, which again can be useful from a security standpoint, since compounds such as endotoxin and/or some proteins, host cells, do not require further processing. In an alternative embodiment of the matrix according to the invention is proposed in the form of a single product in the chromatographic column, which is used in the way in which adsorbed antibodies. In the preferred embodiment of the column and the matrix is sterilized, which allows the user to clear the product-antibody in aseptic and even sterile conditions.

In the second embodiment of the present invention relates to a kit for the purification of antibodies on the one or more other components in the fluid, the set contains the individual cells of the chromatographic column Packed with a separation matrix as described above; one or more than one buffer and written instructions. The separation matrix may be such as described above. These instructions mainly describe the method, as described above.

Detailed description of graphic materials.

Figure 1 shows a sample of the ligand, N-benzyl-N-methylethanolamine immobilized via a nitrogen atom on a carrier in the form of granules. Attached ligand to the left, shown schematically drawn linker; and on the right with illustrative hydrophilic linker. In the experimental part, the prototype ligand linked 6%agarose matrix Sepharose™ 6 FF (GE Healthcare, Upsala, Sweden).

2 shows the chromatogram of a sample containing 50 mg of mAb 1, applied to the separation matrix containing ligands: N-benzyl-N-methylethanolamine immobilized on Sepharose™ 6 FF (901035A); N,N-dimethylbenzylamine immobilized on Sepharose™ 6 FF; and Q Sepharose™ FF 25 mm bis-Tris, 100 mm NaCl (~ 12 MS/cm), pH 6.5. Elution was carried out by 25 mm bis-Tris, 0.5 M NaCl, pH 6.5.

On Figa) and b) shows the results of chromatography conducted on prototypes with mAb1-r-Protein A. buffer consisted of 25 mm bis-Tris, 50 mm NaCl, pH to 6.0. Conductivity was approximately 7 MS/cm For elwira the project used the B-buffer 0.5 M Na-acetate, pH 4.0. The flow rate was 0.5 ml/min (150 cm/h). The sample was a 10 mg mAb1, 0.10 mg rPrA at a concentration of 4 mg/ml mAb1 and 1% of r-Protein (wt./wt.) 3A) cf. Q Sepharose™ FF and b) N-benzyl-N-methylethanolamine, 146 μmol/ml (901035A).

On Figa)-C) presents the results of the analytical exclusion chromatography (SEC) of the sample with mAb 1, 1% PrA and the joint flow and eluate fractions from the chromatographic runs of figure 3. The blue curve represents the flow-through fraction (FT), and the red - eluate. More specifically, Figa) shows a sample of 4 mg/ml mAb1, 0.04 mg/ml rPrA, giving a 1% (wt./wt.); on 4B) shows FT and eluate with Figa) Q Sepharose™ FF and 4B) shows the FT and eluate with Figb) N-benzyl-N-methylethanolamine, 146 μmol/ml (A).

EXPERIMENTAL PART

These examples are presented solely for illustrative purposes and should not be interpreted as limiting in any way the scope of the invention as defined in the attached claims.

Example 1. Obtaining reparation matrix according to the invention

Getting VMEA Sepharose Fast Flow (N-benzyl-N-methylethanolamine derived sepharose for fast flow)

One of the embodiments of the method of obtaining the separation matrix according to the invention are shown below, starting with crosslinked agarose gel (Sepharose™ 6 Fast Flow, GE Healthcare, Upsala, Sweden).

A. Introduction of allyl groups on the matrix is.

Sepharose 6 Fast Flow activated allylglycidyl ether as follows: 100 ml of Sepharose 6 Fast Flow was dried by suction, was mixed with 0.3 g of 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 allylglycidyl ether, the suspension was left at 50°C under vigorous stirring for a further 16 hours. After filtering 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.

Titration showed the degree of substitution 0.22 mmol allyl/ml of gel.

B. Activation of allyl-Sepharose 6 Fast Flow through synthesized.

Bromine was added to a stirred suspension of 50 ml of activated allyl Sepharose 6 Fast Flow (0.22 mmol allyl groups/ml of dried gel), 1 g of sodium acetate and 15 ml of distilled water until a permanent yellow color. Then added sodium formate, and the suspension was completely bleached. The reaction mixture was filtered and the gel was washed with 500 ml of distilled water. The activated gel was then directly transferred into the reaction vessel and then interacted with N-benzyl-N-methylethanolamine.

Century, the Introduction of groups of UMEA (N-benzyl-N-methylethanolamine) on the activated matrix.

Amine groups were introduced to full the CC directly via the nitrogen atom of amine groups. In a typical procedure, the binding matrix was carried out by means of synthesized allyl group and nucleophilic substitution under alkaline conditions. 25 ml of gel, activated bromine (0.22 mmol allyl groups/ml of dried gel), transferred into a reaction vessel containing a solution

N-benzyl-N-methylethanolamine (16.0 ml). Added 5 ml of water and the pH of the reaction solution was brought up to 12.0 with sodium hydroxide solution. The reaction mixture was left for 16 hours under stirring at 50°C. After filtering the reaction mixture, the gel is then washed 3×10 ml of distilled water, 3×10 ml of water with 0.5 HCl and finally 3×10 ml of distilled water. Gel VMEA Sepharose 6 Fast Flow was received with a degree of substitution of 0.15 mmol amines/ml of gel.

Example 2: Purification of antibodies in the thread

Example 2A) Disposition

In nesvezhih conditions samples containing approximately 50 mg mAb1, were applied to the prototype 901035 And (N-benzyl-N-methylethanolamine) at approximately 5 and 12 MS/see the Flow-through fraction (FT) was collected at 5, 10 and 15 column volumes (CV). The fraction of the peak elution were combined. FT-fractions were analyzed for the content of the NDS and protein A.

To confirm that the chromatographic characteristics were not unique to one specific mAb, chromatographic runs were repeated using a sample containing mAb2 at pH 6.0 and about 12 MS/cm'hare is the statistics of the prototype was first evaluated by analytical SEC (exclusion chromatography). Selected fractions were analyzed for contents of the NDS and protein A. After extraction of the fractions with the SEC selected fractions were sent for analysis NDS and protein A.

To check the clearance of r-Protein And on a test specimen in mAb1 was injected with 1% (wt./wt.) recombinant protein A (r-PrA). The prototype were injected with with a sample volume corresponding to 10 mg mAb1, 1% r-Protein And, at a pH of 6.0 and a conductivity of about 7 MS/see Supply and eluate fractions were pooled separately and analyzed by SEC.

Materials/researchable units

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

HiPrep™ 26/10 Desaltingcat. No. 17-5087-01CV=53,09 ml
Tricorn™ 5/50cat. No. 18-1163-09CV=1 ml
HR 5/5™cat. No. 18-0338-01CV=1 ml
Superdex™ 200 10/300 GLcat. No. 17-5175-01CV=23,56 ml

Tools

Chromatographic system:ÄKTAExpforer™ 10
Spectrophotometer Spectra MAX plus

Chemicals

All chemical reagents were of analytical quality. The water was filtered through a MilliQ.

Chromatographic environment

Q Sepharose™ Fast Flow (FF) (GE Healthcare, Uppsala, Sweden). The ligands of the separation matrices are experienced by the samples as described in Table 1 below.

Table 1
The ligand
Test sampleThe ligandCl-capacity (µmol/ml)
AN-benzyl-N-methylethanolamine146

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 the culture of Cho (ovaries of Chinese hamsters) and then purified using conventional affinity chromatography protein And before these experiments.

Changed buffer on HiPrep™ Desalting column (GE Healthcare, Uppsala, Sweden), equilibrated interested buffer, by injection of a suitable amount (5-15 ml) using a Superloop™ (GE Healthcare, Uppsala, Sweden). The flow rate was 5 ml/min, collecting fractions of 5 ml Fractions, sod is readie suirvey peak, United and Parallels were determined by absorption at 280 nm in order to calculate the concentration according to equation 1:

where A280the absorption at 280 nm;

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

C (mg/ml) protein concentration;

l (cm) is the length of the run.

Exclusion 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 Buffer consisted of PBS (phosphate buffer saline); 10 mm phosphate, 0,137 M NaCl, 2.7 mm KCl, pH of 7.4, prepared from tablets (Sigma P-4417).

Way

Balance 2/0,1 CV 2 CV applying for the first time of 0.1 CV between runs Injection of sample 50 ál

Isocratic elution with 1.5 CV

Chromatography on prototypes with mAb

A-buffer consisted of 25 mm bis-Tris, pH of 6.0 or 6.5. Depending on the desired conductivity of about 5 or 12 MS/cm, consisted of 35 or 100 mm NaCl. Eluting buffer (G-buffer) consisted of 25 mm bis-Tris, 0.5 M NaCl, pH 6.5. The flow rate was 0.5 ml/min (150 cm/h).

The way the Balance5 CVA-buffer
introduction sample5-25 mlthe sample content is l 20 or 50 mg mAb
flushing5 CVA-buffer
gradient
elution10 CV0-100% B-buffer
elution10 CV100% B-buffer
regeneration5 CVA-buffer

Chromatography on prototypes with mAb-r-Protein And

A-buffer consisted of 25 mm bis-Tris, pH of 6.0. Conductivity was approximately 7 MS/cm by adding 50 mm NaCl, B-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 4 mg/ml mAb 1 - 0.04 mg/ml rPrA with getting 1% (wt./wt.).

The way the Balance5 CVA-buffer
introduction sample2.5 ml10 mg mAb, 1% rPrA
flushing5 CVA-buffer
gradient elution10 CV0-100% B-buffer
elution10 CV100% B-buffer
regeneration5 CVA-buffer

CIP (cleaning in place)

After each chromatographic run test sample and the reference matrix Q Sepharose™ FF were subjected to the following CIP procedure

30% isopropanol5 CV (column volumes)
H2O5 CV
of 1.0 M NaOH4 CV (including 15 min break)
H2O5 CV
A-buffer5 CV
H2About5 CV
20% EtOH5 CV

Analysis of protein And

Selected fractions were mixed with SPA diluent for the sample in the ratio of 800 μl SPA of sample diluent to 200 ál of sample. After mixing fraction was heated on a heating block at 99°C for 10 minutes and then again mixed. Then the samples were analyzed against recombinant protein A.

Analysis of the proteins of the host cell (NDS)

Samples (a minimum of 600 µl) was analyzed in relation to the content of the NDS. The lower detection limit is 10 ng/ml.

Example 2B) mAb1-containing sample, purified samples - ligands, N-benzyl-N-methylethanolamine (A).

Sample containing 50 mg mAb1, were applied to N-benzyl-N-methylethanolamine immobilized on Sepharose™ 6 FF (901035A), prepared as described in Example 1 above, and on the comparative matrix Q Sepharose™ FF 25 mm bis-Tris, 100 mm NaCl (~12 MS/cm), pH 6.5. Elution was performed by 25 mm bis-Tris, 0.5 M NaCl, pH 6.5.

Chromatogram of sample 2 is shown in Figure 2, which shows the prototype N-benzyl-N-methylethanolamine on Sepharose™ 6 FF (901035A) compared to Q Sepharose™ FF. Flow-through (FT) fractions selected for analysis, indicated by the arrows. The results of the NDS and the clearance of protein And presented in tables 2 and 3 below indicate that the prototype is superior to Q Sepharose™ FF in this regard.

Table 2
The results of the analysis NDS
ColumnpHStart (ng/ml)FT1 (ng/ml) FT2 (ng/ml)FT3 (ng/ml)
Q Sepharose™ FF (standard)6,5890160200180
N-benzyl-N-methylethanolamine, 146 μmol/ml (901035A)6,5890102035

Table 3
The results of the PrA analysis
ColumnpHStart (ng/ml)FT1 (ng/ml)FT2(ng/ml)FT3 (ng/ml)
Q Sepharose™ FF (standard)6,50,400,690,460,31
N-benzyl-N-methylethanolamine, 146 μmol/ml (901035A)6,50,40000

Example 3

Purification of mAb1 in about the Oka from the sample, containing mAb1 and recombinant protein A (rPrA), the prototype ligand N-benzyl-N-methylethanolamine

This example was carried out by chromatography on prototypes of the sample containing mAb1-r-Protein. A-buffer consisted of 25 mm bis-Tris, 50 mm NaCl, pH to 6.0. Conductivity was approximately 7 MS/see B-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 was a 10 mg mAb1, 0.10 mg rPrA at a concentration of 4 mg/ml mAb1 and 1% of r-Protein A (wt./wt.) The results are presented in Table 3.

Finally, was carried out by analytical SEC on the sample with mAb1, 1% rPrA and the joint flow and boatname fractions from the chromatographic runs figure 4. The results are presented in figure 4. On Figa shaded peak is a complex of mAb1-protein A. the Blue curve represents the flow-through (FT) fractions, and the red represents the eluate.

Example 4: the Mode of adsorption

4A) Disposition

To test the selectivity of UMEA SEpharose Fast Flow (BMEA; N-benzyl-N-methylethanolamine) in the adsorption mode, check the retention time of human IgG and eight different proteins. The results were compared with a commercially available anionoobmennika Q Sepharose Fast Flow. The basis of this method of testing was that proteins were injected with at HR5/5 column (containing VMEA ligands immobilized on Sepharose the Fast Flow), balanced And-buffer (containing piperazine as a buffer component). For elution of the proteins used salt gradient (see method below).

Materials/researchable units

Speakers and Q Sepharose Fast Flow was obtained from GE Healthcare, Uppsala, Sweden.

HR 5/5™, catalog number 18-0338-01 the column Volume (CV=1 ml)

Tool

Chromatographic system: ÄKTAExplorer™ 10

Chemical reagents and samples

Proteins, ovalbumin, β-lactoglobulin, bovine serum albumin, α-lactalbumin, myoglobin, lactoferrin, ribonuclease a and cytochrome C were purchased from Sigma, and human IgG (Gammanorm) was purchased from Octapharma. Proteins were dissolved in A-buffer at a concentration of 1-10 mg/ml Q Sepharose Fast Flow was purchased from GE Healthcare, Uppsala, Sweden. All chemical reagents were of analytical purity and the water was filtered through a MilliQ.

Chromatography

The column was balanced And the buffer at a flow rate of 0.6 ml/min, then put 100 ál of the sample solution. Simultaneously analyzed, only one protein. Proteins were suirable using a linear gradient from buffer a to buffer B gradient volume 21 volume of the column (see method below). Buffer And consisted of 25 mm piperazine, pH of 10.0, and buffer B consisted of 25 mm piperazine, 1.0 M NaCl, pH of 10.0. During all the runs were detected absorption at 280 nm.

Method:

Balance:5 CV buffer A.
Injection of the sample:100 μl (approximately 0.2 mg of protein)
Gradient:21 CV 100% B-buffer
Balance after gradient:5 CV buffer A.

Results

To register, communicates whether WMEA-ligand to immunoglobulin, human IgG was applied to a 1 ml column (HR 5/5), filled the new environment. In addition, it also inflicted proteins: ovalbumin, β-lactoglobulin, bovine serum albumin, α-lactalbumin, myoglobin, lactoferrin, ribonuclease a and cytochrome C. the Results were compared with the retention time of proteins observed for Q Sepharose Fast Flow. Q Sepharose Fast Flow is a strong anionoobmennika and is used as the reference anion-exchanger, as it has the same matrix-based (substance basis, granule size, size of pores, pore volume, filling etc) and has essentially the same degree of substitution (measured as ion-exchange capacity). As can be seen from Table 1, UMEA Sepharose Fast Flow was keeping all the studied proteins more strongly compared to Q Sepharose Fast Flow. In addition, IgG was a squirrel that had the longest retention time on VMEA Sepharose Fast Flow (table 1) This indicates a much stronger link with WMEA environment, than with Q Sepharose Fast Flow. Compared to Q Sepharose Fast Flow, the retention time of IgG exceeded 27,3 min when used VMEA Sepharose Fast Flow (table 1). These results clearly indicate that VMEA Sepharose Fast Flow can be used to capture and elution of IgG in a selective manner.

4,3
Table 1
Retention time (trdifferent proteins on Q Sepharose Fast Flow, FMEA Sepharose Fast Flow.
ProteinMolecular weightpItron Q Sepharose Fast Flow (min)tron VMEA Sepharose Fast Flow (min)Δtr(trBMEA-trQ)
Cytochrome C124009,615,316,81,5
Ribonuclease And137009,415,822,66,8
Lactoferrin750007,915,119,4
Myoglobin176007,216,120,54,4
Human IgG16000016,543,827,3
α-Lactalbumin144005,224,6of 40.315,7
Bovine serum albumin690005,1to 25.332,67,3
β-Lactoglobulin350005,125,137,1to 12.0
β-Lactoglobulin435005,130,0x37,17,1
Ovalbumin43500the 4.7 21,830,89,0
on = not analyzedxObserved two peaks

a) Obtaining reparation membrane

Epoxy-activation

1920 g of distilled water and 198 ml NaOH 50% within a few seconds mixed in a rotating flask 1000 ml with suspended magnetic stir bar. Then continuously for about 25 minutes using a pump was added 300 ml of epichlorohydrin. In a rotating flask around the rod stirrer vertically placed dry layer DIN A4 membrane Sartobind Epoxy (Sartorius-Stedim, Gottingen Germany, art. No 94EPOX06-001), wrapped in polypropylene mesh, and the reaction was continued for 120 minutes under stirring at 30°C. the Membrane roll 6 times washed with water and rinse time of 2 minutes.

Combination

1810 g of distilled water, and 3.3 ml NaOH 50% and 180 ml of N-benzyl-N-methylethanolamine mixed in a rotating flask with a volume of 1000 ml Then activated membrane roll (wet) vertically immersed in the reaction mixture around the rod stirrer and the reaction mixture was left overnight under stirring at 50°C. the Membrane roll once washed with ethanol and 6 times with water.

Results

The flow of water (defined in 25 mm perforated circle in the filter funnel under vacuum 0.7 bar (70 kPa)1.67 DM3 /min/cm2. Dynamic binding capacity in relation to nitrate ions (defined through the stop from 5 membrane disks (16 mm) in column (GE Healthcare XK 16/5) was 59 micromoles per ml volume of the membrane and dynamic capacity (QB, 10% at a pH of 7.4) against bovine serum albumin (BSA) was 3.0 mg per ml volume of the membrane.

b) Removing the protein host cell using membrane

Supernatant of cells Chinese hamster ovary (Cho)expressing the monoclonal antibody was purified on a column of protein a and then filtered through a syringe filter Sarstedt Filtropur S 0.2 microns. The antibody was 8 mg/ml and the residual concentration of protein in the host cell was 400 ng/ml After adjusting the pH to 6.5 and adding NaCl to obtain a conductivity of 7 MS/cm solution was pumped through five discs of cellulose membranes with the ligand N-benzyl-N-methylethanolamine, obtained as described above, and Packed in a column, GE Healthcare XK 16/5. After passing through the membrane, the concentration of protein in the host cell decreased to 160 ng/ml.

b) Sterilization

50 g of the washed spherical agarose pellets with 104 micromoles per ml of N-benzyl-N-methylethanolamine attached, as described in example 1, was placed into the vial with a volume of 500 ml. Vial autoclaved in an autoclave Getinge GE EC-1 control system PACS 2000 for 10 cycles, each of the ikl corresponds to 17 minutes at 121, 1million°C at 2.5 to 2.7 bar (250-270 kPa). Capacity in relation to chloride-ion content ligand) gel before autoclaving was 104 micromoles per ml of gel and after 10 cycles, it was 96 micromoles per ml of gel. Therefore, the loss of ligand in each cycle was negligible and the gel was considered suitable for sterilization.

Comparison with ligands described in WO 01/38227

Prototypes

Three ligand was attached to crosslinked agarose beads Sepharose 6 Fast Flow method disclosed in example 1. These ligands and the resulting capacity in respect of the chloride ions (ligands) in the table below. Timicuan and 2-aminobenzimidazole disclosed in WO 01/38227.

Ligand-matrixCl-capacity (µmol/ml)
N-benzyl-N-methylethanolamine146
Timicuan128
2-aminobenzimidazole146

Methods

Humanitariannet antibody against lgG1 with isoelectric point ≈ 9 expressed in cells SNO, was partially purified on a resin with protein And MabSelect (GE Healthcare) and strengthened recombinant protein A (RepliGen). The buffer was replaced with 25 mm Bis-Tris, pH 6,0, 7 MS/cm in column (GE Healthcare HiPrep Desalting.

Column GE Healthcare HR /5 Packed prototype environments and balanced 25 mm Bis-Tris, pH of 6.0, 7 MS/cm (buffer A). Put the sample and washed with 5 column volumes (CV) of buffer A. the Column was suirable gradient of 0-100% buffer B and buffer B consisted of 0.5 M Na-acetate, pH of 4.0. Samples of the flow-through fractions of the column (FT) and pooled eluates were analyzed by analytical exclusion chromatography (SEC) on a column of Superdex 200 10/300 (GE Healthcare).

Results

The SEC chromatogram presented on Figa, 5B and 6, show a good selectivity of N-benzyl-N-methylethanolamine environment. The greatest number of antibodies selected in flow-through fractions, while the greatest number of contaminants detected in the eluate. When using tomikanosal and aminobenzimidazole environments antibody is bound and eluted together with impurities.

1. Chromatographic ligand, representing N-benzyl-N-methylethanolamine.

2. A method of obtaining a separation matrix comprising the immobilization of the ligand according to claim 1 onto the canvas.

3. The method according to claim 2, where the ligand immobilized via amine group.

4. The method according to claim 2 or 3, where the base is porous.

5. The separation matrix containing the ligand according to claim 1, attached to the base.

6. The matrix according to claim 5, where the basis is a particle, such as essentially spherical particles.

7. The separation matrix according to claim 5, where the basis is a membrane structure.

8. The method of making chromatographic column, comprising the manufacture of a matrix as defined in claim 5 or 6; the space thus obtained matrix in a column; and sterilization of the column containing the matrix.

9. Disposable chromatographic column for the purification of antibodies containing the separation matrix according to any one of pp.5-7.

10. Column according to claim 9, which was subjected to sterilization.



 

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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

Cancer treatment // 2389507

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, particularly to new drugs and preparations containing effective anticancer agent with anti-Hsp90 antibody.

EFFECT: invention improves clinical effectiveness in treating cancer and leukemia.

48 cl, 25 tbl

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.

EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.

9 cl, 14 dwg, 2 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: proposed method of purification of human recombinant interferon beta-1b (rIFN beta-1b) provides for optimisation of processes of purification of inclusion bodies, their dissolution and refolding of target protein Purification of human rIFN beta-1b is conducted with zwittehent 3-14 as a main detergent for purification and dissolving of inclusion body of Ecoli with the following refolding of rIFN beta-1b. After that prechromatographic processing of a sample is conducted and after that chromatographic processing of Ceramic S and of Source S30 and oxidation using a mixture of cysteamine/cystamine are performed. Then gel-filtration on Superdex 75 Prep grade, chromatographic processing on Source Q30, gel-filtration on Sephacril S200HR are made and finally the effectiveness of purification of the target protein by high performance liquid chromatography is monitored.

EFFECT: raise of the target protein yields and obtaining human rIFN beta-1b which meets the requirements of normative documents with high levels of specific antiviral activity.

5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: proposed method of purification of human recombinant interferon beta-1b (rIFN beta-1b) provides for optimisation of processes of purification of inclusion bodies, their dissolution and refolding of target protein Purification of human rIFN beta-1b is conducted with zwittehent 3-14 as a main detergent for purification and dissolving of inclusion body of Ecoli with the following refolding of rIFN beta-1b. After that prechromatographic processing of a sample is conducted and after that chromatographic processing of Ceramic S and of Source S30 and oxidation using a mixture of cysteamine/cystamine are performed. Then gel-filtration on Superdex 75 Prep grade, chromatographic processing on Source Q30, gel-filtration on Sephacril S200HR are made and finally the effectiveness of purification of the target protein by high performance liquid chromatography is monitored.

EFFECT: raise of the target protein yields and obtaining human rIFN beta-1b which meets the requirements of normative documents with high levels of specific antiviral activity.

5 tbl, 3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. The method involves successive steps for ultrafiltration of a first antibody preparation to obtain a second antibody preparation, diafiltration of the second antibody preparation to obtain an intermediate preparation and second ultrafiltration of the intermediate preparation to obtain a third antibody preparation. All steps are carried out at temperature ranging from approximately 30°C to approximately 50°C.

EFFECT: design of an efficient method of concentrating antibody preparations.

52 cl, 25 dwg, 25 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there is prepared identification peptide with amino acid sequence: Gly-Pro-Ala-Pro-Gln-Pro-Asp-Glu-Asp-Leu-Lys-Arg-Gln. The prepared peptide is applied to identify, purify and recover the recombinant proteins containing it. For making the recombinant proteins with a polypeptide label, an expression vector pDED is applied. Said vector contains a nucleotide sequence coding amino acid sequence of the identification protein.

EFFECT: invention allows extending range of the methods to identify the recombinant proteins.

14 cl, 6 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and can be used for removing proteins and amino nitrogen from aqueous solutions. The method involves adsorption of proteins on a hydroaluminosilicate natural sorbent, which contains such minerals as clay, zeolite, feldspars, mica, calcite, and filtration. Adsorption takes place at pH ranging from 1 to 3 for 1 to 10 minutes.

EFFECT: invention allows for fast and quality deproteinisation of a solution.

3 tbl, 2 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns biotechnology. Method involves: reduction of plasmatic Cohn IV1 fraction; anion exchange chromatography of solution containing alpha-1-antitrypsin on anion exchange gel, preferably DEAE Sepharose® fast flow; concentration of obtained eluate by ultrafiltration; additional chromatography on hydrophobic carrier and processing of fraction containing alpha-1-antitrypsin by material containing immobilised heparin form; further inactivation of viruses covered with lipidic capsule by adding detergents and optional solvent; salting-out of detergents by further salt concentration increase to ≥ 0.5 M; separation of detergents and virus particles by nanofiltration with pore grade of 15-20 nm.

EFFECT: enhanced purity and safety of obtained product.

17 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and application of proline-specific endoproteases for peptide and protein hydrolysis. Invention involves application of proline-specific endoprotease with pH optimum under 5.5 for production of diet additive or medicine for in vivo treatment of celiac disease or diseases related to presence of proline-rich peptides in food, or as diet additive or medicine for celiac disease prevention.

EFFECT: enhanced hydrolysis of forage proteins with high proline content.

13 cl, 12 ex,10 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining somatotrophic hormone (STH) with decreased content of aggregate of its isoforms involves separation of STH isoforms by means of anion exchange chromatography by using anion exchange resin for decreasing content of the above aggregate up to not more than 10% (wt), on the basis of total mass of the above isoforms and the above aggregate. At that, there performed is loading of the above STH including dephenylalanine and/or trisulphide impurity and/or its aggregate on anion exchange resin chosen from the group including diethylaminoethyl cellulose and Q - sepharose. Loading is performed at the value of loading conductivity of anion exchange resin less or equal to 10 m cm/cm, at pH of anion exchange resin loading of 5 to 10 and at loading of anion exchange STH resin, which includes the above impurity or the above aggregate comprising not more than 10 g of protein/l of the volume filled with anion exchange resin.

EFFECT: invention allows obtaining somatotrophic hormone with decreased content of aggregate of its isoforms, its antagonist with decreased content of aggregate of its isoforms and total content of trisulphide impurity and dephenylalanine impurity.

8 cl, 5 dwg, 8 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: proposed method of purification of human recombinant interferon beta-1b (rIFN beta-1b) provides for optimisation of processes of purification of inclusion bodies, their dissolution and refolding of target protein Purification of human rIFN beta-1b is conducted with zwittehent 3-14 as a main detergent for purification and dissolving of inclusion body of Ecoli with the following refolding of rIFN beta-1b. After that prechromatographic processing of a sample is conducted and after that chromatographic processing of Ceramic S and of Source S30 and oxidation using a mixture of cysteamine/cystamine are performed. Then gel-filtration on Superdex 75 Prep grade, chromatographic processing on Source Q30, gel-filtration on Sephacril S200HR are made and finally the effectiveness of purification of the target protein by high performance liquid chromatography is monitored.

EFFECT: raise of the target protein yields and obtaining human rIFN beta-1b which meets the requirements of normative documents with high levels of specific antiviral activity.

5 tbl, 3 ex

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