Methods of purifying single-domain antigen-binding molecules

FIELD: chemistry.

SUBSTANCE: invention relates to the field of obtaining and separation of single-domain molecules (SDAB). Described is a method of the separation or purification of the SDAB molecule, which represents a trivalent molecule of a ATN-103 nanobody, targeting TNFα and HAS, from a mixture, containing the said SDAB molecule and one or more polluting substances. The mixture is brought in contact with a cation-exchange carrier under conditions, which make it possible for the SDAB molecule to bind with the carrier or be absorbed on the carrier. One or more polluting substances are removed and SDAB is selectively eluted from the carrier. The conductivity of a conditioning medium (CM), used for the carrier loading, constitutes from approximately 12 to 9 mS/cm and pH under conditions of loading is corrected to a value from 4.0 to 4.3. The buffer for elution corresponds to approximately 50 mM of sodium chloride or less and has pH from approximately 5.5 to 7.2. Disclosed is a method or a process of obtaining recombinant SDAB of ATN-103. A host-cell is supported in the conditions at which recombinant ATN-103 SDAB is expressed. The mixture of molecule SDAB and one or more polluting substances is obtained. ATN-103 SDAB is purified or separated with the application of cation-exchange chromatography, as said above.

EFFECT: application of the invention provides new methods of the separation or purification of the nanobody, which can be applied in obtaining the ATN-103 nanobody.

19 cl, 4 dwg, 6 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

In this application claimed priority under U. S. serial No. 61/109481, filed October 29, 2008, the full content of which is included in this application by reference in its entirety.

Sequence listing

The present application contains a sequence listing which has been submitted via EFS-Web and hereby incorporated in this application by reference in its entirety. The said ASCII copy, created on October 28, 2009, marked w223738w.txt and it has a size 12853 bytes.

PRIOR art

Recombinant proteins such as antibodies, usually contain various impurities that must be removed before the protein product will be pharmaceutically acceptable. Some of these impurities may include proteins of the host cell (HCPs), DNA molecules of different and/or misfolded forms of the protein product and high molecular weight aggregates (HMWA). The formation of aggregates is problematic in obtaining antibodies, as this may adversely affect the safety of the product, causing activation of complement or anaphylaxis after injection. Formation of aggregates can also prevent the production processes, leading to reduced product yield, peak broadening and loss of activity. These impurities can have Shi�of Oki range of paintings holding at the various types of chromatography. The removal of such a wide range of impurities is often difficult, usually requiring multiple stages with various types of chromatography.

Traditional methods of protein purification are based on the differences in size, charge and solubility between the protein and contaminants. Protocols based on these parameters include, but are not limited to, affinity chromatography, ion exchange chromatography, size exclusion chromatography and hydrophobic interaction chromatography. These chromatographic methods, however, sometimes exhibit technical difficulties in the separation of aggregated or multimeric species of antibodies. Methods such as ion-exchange chromatography and hydrophobic interaction chromatography, for example, can induce formation of aggregates due to the increased concentration of the protein or the necessary changes in buffer concentration and/or pH during elution. In addition, in some cases, antibodies demonstrate the differences in isoelectric points that are too small to allow their separation by ion exchange chromatography (Tarditi, J. Immunol. Methods 599:13-20 (1992)). Size exclusion chromatography is usually time consuming and leads to a significant dilution of the product that is a hindrance to large-scale, effective proizvodstvennaya. Can also occur the leaching of ligands of columns for affinity chromatography, which leads to undesirable contamination-eluted product (Steindl, J. Immunol. Methods 235:61-69 (2000)).

Although during the purification of recombinant proteins can be used several different methods of chromatography, there is still a need to develop cleaning methods that reduce the number of stages used chromatography and which do not destroy or substantially reduce the biological activity of the recombinant protein.

A BRIEF SUMMARY of the INVENTION

The present invention is based in part on the discovery that single-domain antigen-binding (SDAB, from the English. single domain antigen binding molecules) molecules interact, e.g., bind to protein A or its functional variant, which allows you to use affinity chromatography on protein A for the purification of SDAB molecules. In another embodiment of the SDAB molecule can be cleaned using other chromatographic methods such as ion-exchange (e.g., cationic) chromatography. SDAB molecule can include one or more single antigen-binding domains that interact, for example, be contacted with one or more target proteins (e.g. tumor necrosis factor and/or human serum albumin). In some embodiments SAB molecule is a single-chain polypeptide consisting of one or more molecules nanotesla essentially deprived of a complementary domain of the antibody and/or constant region of an immunoglobulin. Thus, the present invention relates to processes and methods of purification or separation of the SDAB molecules that include one or more single binding domains (e.g., one or more molecules nanotesla), using chromatographic methods such as affinity chromatography based on protein A and ion-exchange (e.g., cationic) chromatography, alone or in combination.

[Note: Napothera™ and Nanotesla™ are registered trademarks of Ablynx N. V.]

Accordingly, in one aspect, the invention relates to a method or process of separating or purifying SDAB molecule (e.g., one or more molecules nanotesla) from a mixture containing the SDAB molecule and one or more contaminants (also referred to in this application as "the drug SDAB molecule"). Method or process includes: bringing the mixture into contact with a native protein A or ion (e.g., cation) exchange (SEH) a carrier under conditions that allow the SDAB molecule to contact or be absorbed by the carrier; removing one or more contaminants, for example, by washing related media in conditions when the SDAB molecule remains associated with the media, for example, flushing bound carrier by at least one buffer for washing the protein A or SEH); and selective elution of the SDAB molecule from the media, for example, by elution of adsorbed SDAB molecule at least one buffer for washing the protein A or SEH.

In one of the embodiments of the method of separation or purification of the SDAB molecule comprises bringing a mixture of the SDAB molecule and one or more contaminants into contact with a cation exchange media.

In another embodiment of the method of separation or purification of the SDAB molecule comprises bringing a mixture of the SDAB molecule and one or more contaminants in contact with the resin-based protein A.

Method or process can be used alone or in combination with at least one other method of cleaning, including, but not limited to, one or more of: hydroxyapatite, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration, viral inactivation (e.g. low pH) and/or filtration to remove viruses. For example, the method or process can be used in combination with one or more of hydroxyapatite chromatography, ultrafiltration, viral inactivation (e.g. low pH) and/or filtering for UD�tion of viruses. In those embodiments that use a carrier on the basis of protein A, method or process may further comprise ion (e.g., cation or anyone-exchange chromatography.

In embodiments of the method or process further includes bringing the mixture into contact with a hydroxyapatite resin and selective elution of the SDAB molecule from the hydroxyapatite resin. In other embodiments where you use the media on the basis of protein A, method or process further includes bringing the mixture into contact with a cation-exchange (SEH) column and selective elution of the SDAB molecule from the column.

Implementing the above methods and processes may include one or more of the following features:

In one of the embodiments of the SDAB molecule is isolated or purified by the method or process according to the invention, is a recombinant protein obtained in the form of a product of cell culture, for example, a host cell (e.g., mammalian, e.g., cells of the Chinese hamster ovary (Cho) cells) in a mixture that includes SDAB molecule and contaminants of cell culture. Cell culture can be small or large scale culture.

In other embodiments of the pollutant in the mixture, is isolated or purified by the method or process according to the invention, include one or more of the high-molecular balkaniadriano, proteins of the host cell, DNA and/or protein A (e.g. washed protein A). In embodiments of the SDAB molecule is purified to at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher purity.

In another embodiment the carrier protein And used in the method or process according to the invention, includes a carrier, for example, a resin with immobilized protein a (e.g., recombinant or dedicated protein A or its functional variant. In one embodiment, the immobilized protein A is a full-staphylococcal protein A (SpA), consisting of five domains of about 50-60 amino acid residues, known as domains E, D, A, b and C in order from N-Terminus. For example, protein A includes the amino acid sequence of SpA (SEQ ID NO:11) shown in Fig.4A, or an amino acid sequence essentially identical (for example, amino acid sequence at least 85%, 90%, 95% or more identical to the amino acid sequence SEQ ID NO:11 shown in Fig.4A). In other embodiments, the immobilized protein A is a functional variant SpA, which includes at least one domain selected from E, D, A, b and/or S, or modified form. For example, a functional variant SpA can include at least a domain of SpA domain or variant, having one or more zamestnaneckou asparagine, also referred to in this application domain Z. In one of the embodiments of the functional SpA variant comprises the amino acid sequence of SEQ ID NO:12 shown in Fig.4B, or an amino acid sequence essentially identical (for example, amino acid sequence at least 85%, 90%, 95% or more identical to the amino acid sequence SEQ ID NO:12 shown in Fig.4B). Can be used other permutations of the functional variants of the protein And that includes the domain or In a domain option and In one or more of: domain A and/or C; domains E, A and/or S; or domains E, D, A and/or C. Any combination of E, D, A, b and/or C or their functional variants can be used as long as the combination is able to contact the SDAB molecule. Typical resin with the carrier on the basis of protein A, which can be used include column MabSELECT™ columns MabSELECT™ SuRe, MabSELECT™ Xtra (GE Healthcare Products) and ProSep™ Va Ultra Plus (Millipore Corporation, Billerica MA).

In one of the embodiments, which uses a carrier protein And the method or process according to the invention, mixtures of SDAB molecules and contaminants are brought into contact with the carrier protein And, for example, is applied to the carrier protein, under conditions that allow the SDAB molecule to contact or be absorbed on the carrier on the basis of protein A. In some voploscheni�x using protein A-boot buffer which includes air-conditioned environment. Protein-A column can be trimmed using protein A-balancing solution that includes about 10 to about 250 mm NaCl and from about 10 to about 100 mm Tris at pH in the range from about 6 to 8; from about 50 to about 200 mm NaCl and from about 20 to about 75 mm Tris at pH in the range from about 6.5 to 7.5; from about 100 to about 175 mm NaCl and from about 40 to about 60 mm Tris at pH in the range from about 7 to 7.5; from about 125 to about 160 mm NaCl and from about 45 to about 55 mm Tris at pH in the range from about 7 to 7.5; from about 50 to about 150 mm NaCl and about 50 mm Tris at pH in the range from about 7.5; or about 150 mm NaCl and about 50 mm Tris at pH in the range of about 6.5, about 7.0, and 7.5 or 8.0.

In yet another embodiment, which uses a carrier protein And the method or process according to the invention, removing one or more contaminants from a mixture, for example, by washing with binding media in conditions when the SDAB molecule remains associated with the media (for example, washing of the bound carrier by at least one buffer for washing protein A). In some embodiments, the buffer for washing the protein And includes from about 10 to about 250 mm NaCl and from about 10 to about 100 mm Tris at pH in the range from about 6 to 8; from about 50 d� about 200 mm NaCl and from about 20 to about 75 mm Tris at pH in the range from about 6.5 to 7.5; from about 100 to about 175 mm NaCl and from about 40 to about 60 mm Tris at pH in the range from about 7 to 7.5; from about 125 to about 160 mm NaCl and from about 45 to about 55 mm Tris at pH in the range from about 7 to 7.5; from about 50 to about 150 mm NaCl and about 50 mm Tris at pH in the range from about 7.5; or about 150 mm NaCl and about 50 mm Tris at pH in the range of about 6.5, about 7.0, and 7.5, or 8.0. In some embodiments, the buffer for washing includes 50 mm NaCl and 50 mm Tris at pH 7.5. In some embodiments, the buffer for washing includes 10 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm or 500 mm NaCl. In some embodiments, the buffer for washing includes 10 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm or 500 mm CaCl2. In some embodiments, the buffer for washing includes 10 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm or 500 mm Tris. In some embodiments, the buffer for washing includes 10 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm or 500 mm citrate. In some embodiments, the buffer for washing includes 10 mm, 25 mm, 50 mm, 75 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm or 500 mm HEPES. In some embodiments, the buffer for washing has a pH 6,0; 6,5; 7,0; 7,5; 8,0; 8,5 or 9.0.

In yet another embodiment, which uses a carrier protein And the method or process according to the invention, SDB molecule selectively eluted from the media for example, by elution of absorbed SDAB molecule at least one protein And elution buffer. In some embodiments of the elution buffer comprises from about 5 to about 50 mm NaCl and from about 5 mm to about 100 mm glycine at pH 4.0 or less. In some embodiments of the elution buffer comprises about 10 mm, about 25 mm, about 50 mm, about 75 mm, about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, about 400 mm, about 450 mm, or about 500 mm NaCl; about 10 mm, about 25 mm, about 50 mm, about 75 mm, about 100 mm, about 150 mm, about 200 mm, or about 250 mm glycine. In some embodiments of the elution buffer has a pH 2,0; 2,5; 3,0; 3,5 or 4,0. In some embodiments the protein A-elution buffer comprises about 10 mm NaCl and about 50 mm glycine at a pH of approximately 3,0.

In one of the embodiments chromatography on ceramic hydroxyapatite used in combination with chromatography on protein And in the method or process according to the invention. Chromatography on ceramic hydroxyapatite can be used before, or more often, after chromatography on protein A. In such embodiments, the method comprises bringing a mixture of the SDAB molecule (e.g., a mixture after separation or purification by chromatography on protein A) into contact with a hydroxyapatite resin and selective�e elution SDAB molecule from the resin. Alternatively, the mixture can be pushed equilibrating buffer and then to ensure its flow through a hydroxyapatite resin. Any of these methods can also be used in combination with the cleaning mixture. In one of the embodiments of the elution and loading buffers include from about 1 to about 20 mm sodium phosphate and from about 0.2 to about 2.5 M sodium chloride, where the elution and loading buffers have a pH of from about 6.4 to about 7.6. In another embodiment of the equilibrating buffer and the buffer for washing includes from about 1 to about 20 mm sodium phosphate, from about 0.01 to about 2.0 M sodium chloride, from about 0 to about 200 mm of arginine and from about 0 to about 200 mm HEPES, where the equilibrating buffer and the buffer for washing have a pH of from about 6.2 to 8.0. In embodiments of the obtained purified SDAB molecule contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of high molecular weight aggregates.

In other embodiments the ion exchange chromatography is used in combination with one or both of: chromatography on protein A and/or hydroxyapatite chromatography, as described in this application. A typical method or process, wherein the chromatography-based protein And perform in front of ion-exchange chromatography, include: bringing a mixture containing the SDAB molecule and one or more contaminants in contact�CT with a native protein A, allow the SDAB molecule to be adsorbed on the media, washing media and adsorbed SDAB molecule at least one buffer for washing the protein A, the elution of adsorbed SDAB molecule at least one protein And elution buffer, thereby obtaining the drug SDAB molecules. Method or process further includes bringing the drug SDAB molecules in contact with the ion-exchange media, ensuring the flow of SDAB molecules through the media, washing media, at least one buffer for washing the ion exchanger, thereby obtaining the flow-through fraction from the ion exchanger. In some embodiments of the method or process further includes bringing the flow-through fraction from the ion exchanger into contact with hydroxyapatite resin, providing adsorption flow-through fraction to the resin, washing the resin at least one buffer for washing the hydroxyapatite and elution of purified SDAB molecule from the resin with at least one hydroxyapatite-elution buffer.

In other embodiments of the ion (e.g., cation) exchange chromatography (SEH) is used alone or in combination with another resin, for example, one or both of: chromatography on protein A and/or chromatography on ceramic hydroxyapatite. Method or process comprises bringing a mixture containing the SDAB molecules� and one or more contaminants, in contact with the ion-exchange media, ensuring the flow of SDAB molecules through the media, washing media, at least one buffer for washing ion (e.g., cation) of the exchanger. In one of the embodiments of the cation-exchange media is selected from: Capto™ S (GE Heathcare), Fractogel® S03-(M) (EMD Chemicals), Toyopearl® Gigacap S-650M (Tosoh Bioscience) or Poros® HS 50 (Applied Biosystems). In one of the embodiments SEH resin demonstrates the capacity of at least about 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l or 60 g/L. In another embodiment the conductivity of the medium for conditioning (CM) used for application to the column is from about 15 to 5 MS/cm, from 14 to 6 MS/cm, from 13 to 8 MS/cm, 12 to 9 MS/cm, or from 11 to 10 MS/cm, or about 7 MS/cm, 8 MS/cm, 9 MS/cm, 10 MS/cm to 11 MS/cm, 12 MS/cm or 13 MS/cm In another embodiment the pH of the load conditions, adjust to a value less than about 6, 5,5, 5, 4,5, 4,4, 4,3, 4,2, 4,1, 4, 3,9, 3,8 or 3.7. In embodiments of buffer for elution corresponds to about 100 mm sodium chloride or less, about 90 mm sodium chloride or less, about 80 mm sodium chloride or less, about 70 mm sodium chloride or less, about 60 mm sodium chloride or less, about 50 mm sodium chloride or less, about 40 mm sodium chloride or less, or about 30 mm sodium chloride or less, 20 mm sodium chloride or less, about 10 mm sodium chloride or less, about 5 mm sodium chloride �or less, approximately 1 mm of sodium chloride or less, and has a pH from about 4 to 8, from about 5 to 7.5, from about 5.5 to 7.2, from about 6 to 7.1 or from about 6.5 to 7, or about 5, 5,5, 6, 6,5 or 7. In other embodiments SEH column can also be eluted using a top-down sleep-balancing buffer.

In some embodiments the cation exchange chromatography is the only chromatographic method used to clean the SDAB. In another embodiment the cation exchange chromatography is used in combination with other chromatographic methods (e.g., hydroxyapatite chromatography). A typical method or process in which perform cation exchange chromatography, include: bringing a mixture containing the SDAB molecule and one or more contaminants in contact with the cation-exchange medium under conditions that reduce the conductivity of the loading buffer or air-conditioned environments (e.g., from about 15 to 5 MS/cm, from 14 to 6 MS/cm, from 13 to 8 MS/cm, 12 to 9 MS/cm, or from 11 to 10 MS/cm, or about 7 MS/cm, 8 MS/cm, 9 MS/cm, 10 MS/cm to 11 MS/cm, 12 MS/cm or 13 MS/cm), providing adsorption SDAB molecules on the media, washing media and adsorbed SDAB molecule at least one buffer for washing Latinoamerica, elution of adsorbed SDAB molecules by men�her least one buffer for elution, through this drug SDAB molecules. Method or process may additionally include the reduction of drug SDAB molecules in contact with another media or resin, for example, the method or the process may further include bringing the flow-through fraction from the ion exchanger into contact with hydroxyapatite resin, providing adsorption flow-through fraction to the resin, washing the resin at least one buffer for washing the hydroxyapatite and elution of purified SDAB molecule from the resin with at least one hydroxyapatite-elution buffer.

In other embodiments of the method or process further includes concentrating-eluted SDAB molecule, e.g. through the implementation stage UF/diafiltration, to a specified target volume. Stage of concentration can also be used to replace the buffer-eluted SDAB molecule. For example, a concentrated, - eluted SDAB molecule can be filtered, for example through diafiltration, in the presence histidinemia buffer or Tris buffer. In embodiments that use histidinemia buffer, the buffer is present in a concentration of at least from about 5 to 30 mm, from about 7.5 to 28 mm, from about 10 to 20 mm, from about 12 to 15 mm, or about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 �M, about 20 mm, about 25 mm, about 28 mm at a pH of about 7, about 6, about 5, about 4, about 3, or in the range from about 4 to about 6.5, from about 5 to 6, some 5.9, about 5,8, about 5.7, about 5.6 or about 5.5. In embodiments, a small volume of concentrated buffer of the drug-eluted add to concentrated SDAB molecule (e.g., at least 2, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20% about./about. concentrated washing buffer preparation. In embodiments of the concentrated buffer of drug is from about 10 to 50 mm histidine (e.g., about 20 mm, about 30 mm histidine, from about 10 to 60% sugar (e.g. sucrose, sorbitol or trehalose), for example about 50% sucrose, and a surfactant (e.g., Polysorbate 80) in the range from about 0.001 to about 0.1%, for example about 0.06 per cent). Typical drugs for SDAB molecules described in USSN 12/608553, filed October 29, 2008 in the name of Wyeth, the content of which is incorporated by reference into this application.

In embodiments of the SDAB molecule is concentrated to at least about 20 g/l, 30 g/l, 40 g/l, 80 g/l, 90 g/l g/l 100 g/l 150 g/l 200 g/l, 210 g/l, 220 g/l, 230 g/l, 240 g/l 250 g/l 260 g/l 270 g/l 280 g/l 290 g/l, 300 g/l 310 g/l 320 g/l 330 g/l 340 g/l, 350 g/l or higher.

In some embodiments of the method or process includes: assessment (e.g., detection, quantification and/or monitoring) p� least one parameter of purity, activity, toxicity, pharmacokinetics and/or pharmacodynamics of the SDAB molecule; (possibly) comparing at least one parameter with a reference value to thereby evaluate or choose SDAB molecule. The comparison may include determining whether at least one parameter preselected relationship with a reference value, such as determining whether it is located within the reference values (either including or aluca endpoints of the range); he is reference value or exceeds it. In some embodiments, if at least one parameter meets a preselected relationship, for example, is within a reference value, the SDAB molecule are selected.In other embodiments, the assays, methods, or an indication that the preselected relationship between the at least one parameter and a reference value exists, write or record, for example, on computer readable media. Such methods, tests or indications of the existence of pre-selected relationship can be listed on the package insert, the guide (for example, the United States Pharmacopoeia, or any other material, such as labeling, which can be distributed, e.g., for commercial use or for submission to regulatory about�Gan U.S. or foreign regulatory authority.

In one of the embodiments of the method or process further includes comparing a predetermined value with a reference value for analyzing thus the manufacturing process.

In one of the embodiments of the method further includes maintaining the manufacturing process based at least in part, on the analysis. In one of the embodiments of the method further includes changing the manufacturing process on the basis of the analysis.

In another embodiment the method comprises the assessment process, for example, the manufacturing process for the SDAB molecules, for example molecules of the TNF nanotesla obtained by a selected process, which includes determination of the process based on the method or analysis described in this application. In one of the embodiments of the method further includes maintaining or changing the manufacturing process based at least in part, on the method or analysis. Thus, in another embodiment of the evaluating party does not practice a method or analysis described in this application, but only take into account the results obtained by using method or analysis described in this application.

In another embodiment the method includes comparing two or more drugs in the way of monitoring or controlling the differences between the parties or to compare the drug with the reference standard.

Still water embodiment of the method may further include issuing a decision, for example, to classify, select, accept or reject, release or suspension, processing into drug product, shipping, moving to another place, cooking in the form of a preparation, labeling, packaging, release, turnover, sales or offers for sale of the preparation, based, at least in part, on the conclusion.

In another aspect, the invention relates to a method of compliance with mandatory requirements, for example, post-registration requirements of a regulatory body, for example, the FDA (the U.S. quality supervision food and drug). The method includes providing estimates of the parameter of the SDAB molecule, as described in this application. Post-registration requirement may include measuring one or more of the above parameters. The method also includes, perhaps, to determine whether the observed permission setting is pre-selected criteria, or whether the parameter to a pre-selected range; perhaps a documentary fixing the value or result of the analysis or communication with the body, for example, by passing the value or result in a regulatory authority.

In another aspect, the invention relates to one or more of: providing the report to the Agency receiving the report, evaluation of�sample of the SDAB molecule, for example, molecules of the TNF nanotesla, in conformity with a reference standard, such as FDA regulations, search the instructions from the other party that the drug SDAB molecules satisfies a certain prescribed requirement, or provide information about the drug SDAB molecule to the other party. Typical receiving bodies or other parties include the government, such as the U.S. Federal government, for example, a government Agency such as the FDA. The method includes one or more (or all) of the following stages: the manufacture and/or testing of the SDAB molecules in the first country, for example USA; sending at least an aliquot of the sample outside of the first country, for example, sending her out of the United States in the second country; preparation or receipt of the report, which includes data on the structure of the drug SDAB molecule, e.g., data relating to the structure and/or circuits described in this application, for example, data obtained using one or more of the methods described in this application; and submitting of this report the entity receiving the report.

SDAB molecule, e.g. a molecule of nanotesla (e.g., TNF-binding molecule of nanotesla) isolated or purified by the method or process according to the invention, may include one or more single binding domains (e.g., one or more of NanoTec). For example, mole�ula nanotesla may contain a polypeptide, for example, single-chain polypeptide containing at least one immunoglobulin variable domain (comprising one, two or three complementarity determining region (CDR)), or may consist of such polypeptide. Examples of SDAB molecules include molecules that are naturally devoid of light chains (e.g., VHH, nanotesla or antibodies derived from camelids). Such SDAB molecules can occur or can be obtained from animals of the camelid such as camel, llama, DROMEDARY, Alpaca and guanaco. In other embodiments of the SDAB molecule can include single-domain molecules, including but not limited to, other natural single-domain molecules, such as single-domain polypeptides sharks (IgNAR); and single-domain frames (for example, fibronectine frames). Single-domain molecules can occur from sharks.

In one of the embodiments of the SDAB molecule is isolated or purified by the method or process according to the invention, is a single-chain polypeptide consisting of one or more single-domain molecules. In embodiments of the molecule nanotesla is a monovalent or polyvalent (e.g., divalent, trivalent or tetravalent). In other embodiments the molecule nanotesla is monospecifičeskoj or polyspecific (e.g. bespecifically, thespecifics or terspecific�tion). SDAB molecule can contain one or more single-domain molecules which are recombinant, CDR-grafted, humanized, verludzutune, daimonizomai and/or obtained in vitro (e.g., selected by phage display). For example, the SDAB molecule can be a single-stranded fused polypeptide containing one or more single-domain molecules that bind to one or more target antigens. Typically, the target antigen is an antigen of the mammal, for example, the human protein. In some embodiments of the SDAB molecule binds to serum protein, e.g., human serum proteins, selected from one or more serum albumin (human serum albumin (HSA)), fibrin, fibrinogen or transferrin.

In one typical embodiment of the SDAB molecule is isolated or purified by the method or process according to the invention is a trivalent, bespecifically molecule consisting of the merger of single-chain polypeptides of two single-domain molecules (for example, two variable regions of camelids) that bind to a target antigen, such as tumor necrosis factor a (TNFα), and one single-domain molecules (e.g., variable regions camelids), which is associated with serum b�lcom, for example, HSA. Single-domain molecules SDAB molecules can be arranged in the following order from N - to C-Terminus: TNFα-binding single-domain molecule - HSA-binding single - domain molecule TNFα-binding single-domain molecule. It should be borne in mind that any order or combination of single-domain molecules against one or more targets can be obtained, as described in this application.

In one of the embodiments of the SDAB molecule is isolated or purified by the method or process according to the invention, referred to in this application as "ATN-103, contains the amino acid sequence of SEQ ID NO:1 shown in Fig.2, or an amino acid sequence essentially identical (for example, amino acid sequence at least 85%, 90%, 95% or more identical to the amino acid sequence SEQ ID NO:1 shown in Fig.2), or consists of it. Examples of additional trivalent molecules, especificacao nanotesla that can be obtained, as described in this application include TNF24, TNF25, TNF26, TNF27, TNF28, TNF60 and TNF62 disclosed in Table 29 of WO 2006/122786.

In some embodiments, at least one of single-domain molecules SDAB molecule is isolated or purified by the method or process according to the invention, which binds to TNFα, includes one, two or three CDRs having the amino acid sequence: DYWMY (CDR1), EINNGLITKYPDSVKG (CDR2) and/or SPSGFN (CDR3), or having a CDR that differs by less than 3, 2 or 1 amino acid substitutions (e.g., conservative substitutions) from one of the CDR. In other embodiments of single-domain molecule contains a variable region having the amino acid sequence of amino acids from about 1 to 115 of SEQ ID NO:1 shown in Fig.2, or an amino acid sequence essentially identical (for example, amino acid sequence at least 85%, 90%, 95% or more identical to the amino acid sequence SEQ ID NO:1 shown in Fig.2). In embodiments of TNFα-binding single-domain molecule has one or more of the biological activities of the molecule TNFα-binding single-domain antibody of SEQ ID NO:1 shown in Fig.2. For example, the TNFα-binding single-domain molecule binds with the same or similar epitope as the epitope recognized by TNFα-binding single-domain molecule of SEQ ID NO:1 shown in Fig.2 (e.g., binds to TNFα in its trimeric form; associated with the site of TNFα, which interacts with TNF receptor; binds to the epitope in the TNFα trimer containing Gin at position 88 and Lys at position 90 on the first TNF monomer (monomer A and Glu at position 146 in the second TNF monomer (monomer B), or epitope described in WO 06/122786). In another embodiment of the TNFα-binding single-domain molecule has �aktivnosti (for example, the binding affinity, dissociation constant, binding specificity, TNF-inhibitory activity) similar to any of the TNFα-binding single-domain molecules disclosed in WO 06/122786.

In other embodiments, the molecule TNFα-binding nanotesla contains one or more of nanutel disclosed in WO 2006/122786. For example, a molecule TNFα-binding nanotesla can be a monovalent, divalent, trivalent molecule TNFα-binding nanotesla disclosed in WO 2006/122786. Typical TNFα-binding nanotesla include, but are not limited to, TNF1, TNF2, TNF3, their humanized forms (for example, TNF29, TNF30, TNF31, TNF32, TNF33). Additional examples of monovalent TNFα-binding of nanutel disclosed in Table 8 of WO 2006/122786. Typical divalent molecules TNFα-binding nanotesla include, but are not limited to, TNF55 and TNF56 that contain two nanotesla TNF30 connected via a peptide linker with the formation of a single fused polypeptide (disclosed in WO 2006/122786). Additional examples of bivalent TNFα molecules connecting nantel disclosed in Table 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, at least one of single-domain molecules SDAB molecule is isolated or purified by the method or process according to the invention, which binds to HSA, comprises one, two or three CDRs having amino acid is consistent�here: SFGMS (CDR1), SISGSGSDTLYADSVKG (CDR2) and/or GGSLSR (CDR3), or having a CDR that differs by less than 3, 2 or 1 amino acid substitutions (e.g., conservative substitutions) from one of the CDR. In other embodiments of single-domain molecule contains a variable region having the amino acid sequence of amino acids from about 125 to 239 of SEQ ID NO:1 shown in Fig.2, or an amino acid sequence essentially identical (for example, amino acid sequence at least 85%, 90%, 95% or more identical to the amino acid sequence SEQ ID NO:1 shown in Fig.2). In embodiments of the HSA-binding single-domain molecule has one or more of the biological activities of the HSA-binding single-domain molecule of SEQ ID NO:1 shown in Fig.2. For example, the HSA-binding single-domain molecule binds with the same or similar epitope as the epitope recognized by HSA-binding single-domain molecule of SEQ ID NO:1 shown in Fig.2. In another embodiment of the HSA-binding single-domain molecule has activity (e.g., binding affinity, dissociation constant, binding specificity) similar to any of the HSA-binding single-domain molecules disclosed in WO 06/122786.

In other embodiments of the HSA-binding SDAB molecule contains one or more nantel disclosed in WO 2006/122786. For example, HSA-binding�sort of SDAB molecule can be a monovalent, divalent, trivalent molecule HSA-binding nanotesla disclosed in WO 2006/122786. In other embodiments of the HSA-binding SDAB molecule can be a monospecifičeskoj or polyspecific molecule with at least one of specificdate binding, which binds to HSA. Typical TNFα-binding nanotesla include, but are not limited to, ALB1, their humanized forms (for example, ALB6, ALB7, ALB8, ALB9, ALB10) disclosed in WO 06/122786.

In other embodiments, two or more single-domain molecules SDAB molecules are fused, with a binding band or without it, in the form of genetic or polypeptide fusion. Linking group can be any linking group known to the person skilled in the art. For example, the linking group can be a biocompatible polymer with length from 1 to 100 atoms. In one of the embodiments of the linking group comprises or consists of polyglycine, Polverino, polylysine, polyglutamate, politicaleconomic or poliorgannoj residues, or combinations thereof. For example, polyglycine or polizeirevier linkers can include at least five, seven, eight, nine, ten, twelve, fifteen, twenty, thirty, thirty-five and forty residues of glycine and serine. Typical linkers that may be used include Gly-Ser repeats, �of primer, (Gly)4-Ser repeats in one, two, three, four, five, six, seven or more repeats (SEQ ID NO:8). In embodiments, the linker has the following sequence: (Gly)4-Ser-(Gly)3-Ser or ((Gly)4-Ser)n, where n is 4, 5 or 6 (SEQ ID NO:10).

SDAB molecule isolated or purified by the method or process according to the invention, can be further modified by attaching, for example, covalent or non-covalent, the second grouping. For example, the molecule nanotesla can be covalently attached to a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or its derivative (such as methoxypoly(ethylene glycol) or mPEG). Examples paglierani molecules nantel disclosed as TNF55-PEG40, TNF55-PEG60, TNF56-PEG40 and TNF56-PEG60 in WO 06/122786.

In one embodiment of the method or process further includes one or more of ion (e.g., cation or anyone-exchange chromatography, hydroxyapatite chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration and/or filtration to remove viruses.

In one of the embodiments of the method or process further includes the preparation of the recombinant SDAB molecule in the form of pharmaceutical compositions. The composition may include the SDAB Molek�in one or in combination with a second agent, for example, the second therapeutically or pharmacologically active agent that is useful in the treatment of TNFα-associated disorders, e.g., inflammatory or autoimmune disorders, including, but not limited to, rheumatoid arthritis (RA) (e.g., rheumatoid arthritis moderate to severe), arthritic conditions (e.g., psoriatic arthritis, juvenile idiopathic arthritis (LA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease and/or multiple sclerosis. For example, the second agent may be an antibody against TNF or TNF-binding fragment, where the second TNF antibody binds to a different epitope than TNF-binding SDAB molecule preparation. Other non-limiting examples of agents that can be prepared in the form of a preparation with TNF-binding SDAB molecule include, but are not limited to, an inhibitor of a cytokine inhibitor, growth factor, immunosuppressant, anti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent and cytotoxic agent. In one of the embodiments the additional agent is a standard treatment of arthritis, including but not limited to this, non-steroidal anti-inflammatory drugs (NSAIDs); Corti�asteroidi, including prednisolone, prednisone, cortisone and triamcinolone; and Antirheumatic drugs, disease-modifying (DMARDs) such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, Leflunomide (Arava®), inhibitors of tumor necrosis factor, including etanercept (Enbrel®), infliximab (Remicade®) (with or without methotrexate) and adalimumab (Humira®), an antibody against CD20 (e.g., Rituxan®), a soluble receptor of interleukin-1 such as anakinra (Kineret®), gold, minocycline (Minocin®), penicillamine, and cytotoxic agents, including azathioprine, cyclophosphamide and cyclosporine. In such combination therapies may advantageously be used lower dosages are administered therapeutic agents, thus avoiding possible toxicities or complications associated with different monoterpene.

In another aspect, the invention relates to SDAB molecule, obtained by the method or process described in this application. Compositions, e.g., pharmaceutical compositions, and medicaments containing the SDAB molecule, obtained by the method or process described in this application, are also within the scope of this invention. For example, drugs can include SDAB molecule described in this application, in a pharmaceutically acceptable carrier.

In one of the embodiments of the SDAB molecule, obtained by the method or p�the process, described in this application, are suitable for administration to a subject, for example, the subject-person (e.g. a patient having a TNFα-related disorder). For example, the SDAB molecule or drug can be administered to a subject via injection (e.g., subcutaneous, intravascular, intramuscular or intraperitoneal) or by inhalation.

In another aspect, the invention relates to methods of treating or preventing in a subject (e.g., subject-person) disorders associated with SDAB molecule, described in this application (e.g., a TNFa-related disorder, e.g., inflammatory or autoimmune disorders, including, but not limited to, rheumatoid arthritis(KA) (e.g., rheumatoid arthritis moderate to severe), arthritic conditions (e.g., psoriatic arthritis, juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease and/or multiple sclerosis). The method comprises administering to a subject, e.g. patient, person, pharmaceutical compositions containing the TNF-binding SDAB, obtained by the method or process described in this application, separately or in combination with any of combination therapy, described in this application, in quantities�, such that one or more symptoms of TNFα-associated disorders are reduced.

In another aspect, the invention relates to a kit or article, including a device, a syringe or vial containing the SDAB, obtained by the method or process described in this application.

All publications, patent applications, patents, and other references mentioned in this application are incorporated by reference in its entirety.

Detailed information about one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects and advantages of the invention will be apparent from the description and graphic materials, and from the claims.

BRIEF DESCRIPTION of GRAPHIC MATERIALS

Fig.1 is a diagram of the predicted structure of ATN-103.

Fig.2 shows the amino acid sequence of the polypeptide chain ATN-103 (SEQ ID NO:1).

Fig.3 presents a block diagram of the purification process for ATN-103.

Fig.4A presents the amino acid sequence of the full-size staphylococcal protein A (SpA) (SEQ ID NO:11). Fig.4B presents the amino acid sequence of the modified domain In SpA (SEQ ID NO:12). the α-Helical region in bold.

DETAILED description of the INVENTION

The present invention is based, at least an hour�icno, on the discovery that the SDAB molecule that includes one or more single binding domains (e.g., one or more molecules nanotesla), interacts, e.g., binds, the protein a or its functional variant, which allows the use of affinity chromatography methods based on protein A for the purification of SDAB molecules. Thus, the present invention relates to processes and methods of cleaning or releasing fused antigen-binding polypeptides that include one or more single binding domains (e.g., one or more molecules nanotesla) deprived of a complementary domain of the antibody and the Fc-region of an immunoglobulin, using affinity chromatography on protein A.

In order to more easily understand the present invention, first define some terms. Additional definitions are set out in the detailed description.

As used in this application, the singular refers to one or more than one (e.g., at least one) of the grammatical object.

The term "or" is used in this application to refer to the term "and/or" and is used interchangeably with it, unless the context clearly indicates otherwise.

The terms "proteins" and "polypeptides" are used interchangeably in this application.

"About" and "approximately" shall generally denote acceptable �level errors for the measured quantities according to the nature and accuracy of measurements. Typical levels of error are within 20 percent (%), typically within 10% and more typically within 5% of the specified value or range of values.

The term "drug SDAB molecule" refers to any composition containing the SDAB molecule and/or one or more unwanted contaminants. The drug may be partially isolated or purified, e.g., by passing through a chromatographic column, as described in this application, for example, a carrier on the basis of protein A or cation exchange media.

The term "chromatography" refers to the separation of chemically different molecules in a mixture from each other by bringing said mixture into contact with the adsorbent, where one class of molecules reversibly binds to the adsorbent or adsorbed on the adsorbent. Molecules that are the least strongly adsorbed or retained by the adsorbent are released from the adsorbent under conditions, when the molecules are more strongly adsorbed or held, does not release them.

The term "flow-through mode" refers to the method of release of the drug SDAB molecule, wherein at least one SDAB molecule contained in the product, as suggested, flows through a chromatographic resin or media, while at least one potential contaminant or impurity in contact with chromatographies�Oh resin or carrier. The flow-through mode can be used, for example, hydroxyapatite chromatography and ion exchange chromatography.

"Pairing mode" relates to a method for preparation of the SDAB molecule, wherein at least one antibody molecule contained in the product, binds to the chromatographic resin or media, while at least one potential contaminant or impurity flow through them. Pairing mode can be used, for example, hydroxyapatite chromatography and ion exchange chromatography.

"Contaminant" refers to any foreign or objectionable molecule, in particular biological macromolecules such as DNA, RNA or protein different from the protein that is clear, which is present in the protein sample, which cleanse.Contaminants include, for example, other proteins of the host cell from the cells used for expression of the recombinant protein, which is purified, the proteins that are part of an absorbent used on stage affinity chromatography, which can leach into a sample during the preliminary stages of affinity chromatography such as protein A, as well as misfolded variants of the target protein.

"Proteins of the host cell include proteins encoded by the natural genome of the host cell, in which� injected DNA encoding a protein that must be cleared. Proteins of the host cell can be a pollutant protein treated, the levels of which can be reduced by cleaning. Proteins of the host cell can be analyzed, among others, by any suitable method, including gel electrophoresis and staining and/or ELISA (ELISA) analysis. Proteins of the host cell include, for example, proteins (CHOP) Chinese hamster ovary (Cho) cells, produced in the form of the product of expression of recombinant proteins.

The term "high molecular weight aggregates" or "HMWA" refers to the Association of at least two antibody molecules. The Association may occur by any method, including, but not limited to, covalent, non-covalent, disulfide or non-repairable stitching. At least two molecules can reach the same or different antigens.

Used in this application, the term "protein A" and related expressions such as "the media on the basis of protein A", intended to include a protein (e.g., recombinant or dedicated protein A or its functional variant. In one of the embodiments of the protein And is a full-staphylococcal protein A (SpA), consisting of five domains of about 50 to 60 amino acid residues, known as domains E, D, A, b and C, RA�required in order from N-Terminus (Sjodhal EurJ Biochem 78: 471-490 (1977); Uhlen et al. J. b/o/. Chem. 259: 1695-1702 (1984)). These domains contain about 58 residues, and each has about 65% -90% homologous amino acid sequence. Studies of binding between protein a and antibodies showed that although all five domains of SpA (E, D, A, b and C) bind to IgG through its Fc-region, the domains D and E show significant binding Fab (Ljungberg et al. Mol Immunol. 30(14):1279-1285 (1993); Roben et al. J. Immunol 154:6437-6445 (1995); Starovasnik et al. Protein Sci 8:1423-1431 (1999). Showed that the Z-domain, functional equivalent and variant domain with minimized energy (Nilsson et al. Protein Eng 1:107-113 (1987)), has negligible binding to the variable region domain of the antibody (Cedergren et al. Protein Eng 6(4):441-448 (1993); Ljungberg et al. (1993) above; Starovasnik et al. (1999) supra). Protein And may include the amino acid sequence of SpA (SEQ ID NO:11) shown in Fig.4A, or an amino acid sequence essentially identical to it. In other embodiments the protein And represents a functional variant SpA, which includes at least one domain selected from E, D, A, b and/or S, or modified form. For example, a functional variant SpA can include at least a domain of SpA domain or variant, having one or more substituted aspartic residues, also referred to in this application domain Z. In one of the embodiments of the functional variant SpA incl�et amino acid sequence of SEQ ID NO:12), shown in Fig.4B, or an amino acid sequence essentially identical to it. You can use other permutations of the functional variants of the protein And containing domain, or a domain option, and one or more of: the domains A and/or C; domains E, and and/or or domains E, D, A and/or C. Any combination of E, D, A, b and/or C or its functional variant may be used as long as the combination is able to contact the SDAB molecule.

"Ceramic hydroxyapatite" or "sleep" refers to insoluble gidrauxilirovannogo the calcium phosphate, for example, having the formula [Cao(PO4)6(OH)2or Sa10(PO4)6(OH)2], which is sintered at high temperatures in a spherical, macroporous ceramic form. The term "sleep" includes, but is not limited to this, ceramic hydroxyapatite type I and type II. Unless otherwise noted, "sleep" refers to a particle of any size; including, but not limited to, 20, 40 and 80 µm.

"Clear" polypeptide means a reduced number of alien and undesirable elements, especially of biological macromolecules, such as proteins or DNA that may be present in the protein sample. The presence of foreign proteins can be assessed by any suitable method, including gel electrophoresis and staining and/or ELISA analysis. The presence of DNA can PR�to analyze any suitable method, including gel electrophoresis and staining and/or analysis using the polymerase chain reaction. In embodiments of the polypeptide, for example, the SDAB molecule, purified to at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher purity.

A polypeptide is "isolated" (or "remote") from a mixture containing protein and other contaminants, when the mixture is subjected to such a process that the concentration of the target polypeptide obtained above in the product than in the original product.

The methods and compositions of the present invention encompass polypeptides having a specific sequence, or a sequence essentially identical or similar, for example, sequences at least 85%, 90%, 95% or more identical to a specific sequence. In the context of amino acid sequence, the term "substantially identical" is used in this application with respect to the first amino acid sequence which contains a sufficient or minimum number of amino acid residues that are 1) identical or (2) represent conservative substitutions of aligned amino acid residues in a second amino acid sequence, such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. Example�, amino acid sequences that contain a common structural domain having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a reference sequence.

As the polypeptides of the present invention also includes fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination of them. The terms "fragment", "variant", "derivative" and "analog" when talking about proteins of the present invention include any polypeptides which retain at least some of the functional properties of the corresponding native antibody or polypeptide. Fragments of the polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to the specific antibody fragments discussed elsewhere in this application. Variants of the polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants may occur naturally or artificially. Artificial versions can be obtained using known methods of mutagenesis. Variants of polypeptides may contain conservative or non-conservative amino acid substitutions Affairs�functions or add. Derivatives of the fragments of the present invention are polypeptides that have been modified so as to exhibit additional features not found in the native polypeptide. Examples include fusion proteins. Variants of the polypeptides can also be referred to in this application as "polypeptide analogs". Used in this application as a "derivative" of a polypeptide refers to the polypeptide having one or more residues chemically modified by reaction of a functional side group. As "derivatives" include polypeptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, Proline may be substituted 4-hydroxyproline; lysine can be substituted 5-hydroxylysine; histidine may be substituted 3-methylhistidine; serine can be replaced by homoserine; and lysine can be replaced by ornithine.

The term "functional variant" refers to polypeptides which have the amino acid sequence essentially identical to the natural sequence, or encoded is essentially identical to the nucleotide sequence and is able to have one or more activities of natural sequence.

Calculations of homology or identity between after�euteleostomi (the terms are used interchangeably in this application) are as follows.

To determine the percent identity of two amino acid sequences, sequences are aligned for optimal comparison (for example, can be introduced gaps into one or both of the first and second amino acid sequence or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% the length of the reference sequence. Amino acid residues in the corresponding positions of the amino acids are then compared. When the position in the first sequence is occupied by the same amino acid residue, and the corresponding position in the second sequence, then the molecules are identical at that position (as used in this application, the "identity" of amino acids or nucleic acids equivalent to "homology" of amino acids or nucleic acids).

The percent identity between two sequences depends on the number of identical provisions common to these sequences, subject to�of icesta gaps and the length of each gap, to be introduced for optimal alignment of these two sequences.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch ((1970) J. Mol. In/about/. 48:444-453), which was incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum matrix 62, or the matrix RAM, and a lot of the gaps 16, 14, 12, 10, 8, 6 or 4 and a lot of length 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna matrix.CMP and a lot of the gaps 40, 50, 60, 70, or 80 and a lot of length 1, 2, 3, 4, 5 or 6. Particularly preferred set of parameters (and which should be used if not specified otherwise) is a scoring matrix Blossum 62 with a penalty for a gap 12, the penalty for lengthening the gaps 4 and the penalty for a gap with a shift of the reading frame 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), the cat�which was incorporated into the ALIGN program (version 2.0), using table mass balances RAM, the penalty for the length of the gap 12 and the penalty for the breach 4.

Nucleic acid sequences and protein sequences described in this application can be used as a "query sequence" to perform a search on public databases to, for example, identify other family members or related sequences. Such searches can be performed using programs NBLAST and XBLAST (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The search for a nucleotide BLAST can be performed using the NBLAST program, scale = 100, word length = 12, to obtain nucleotide sequences homologous to nucleic acid molecules (SEQ ID NO:1) according to the invention. The search for proteins in the BLAST can be performed using the XBLAST program, scale = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules (SEQ ID NO:1) according to the invention. To obtain alignments with gaps for comparison purposes, you can use Gapped BLAST, as described in Altschul et al., (1997) Nucl Acids Res. 25:3389-3402. When using the programs BLAST and Gapped BLAST you can use the values of the default parameters of the respective programs (e.g., XBLAST and NBLAST).

"Conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with the amino acid�now the remnant, having a similar side chain. Family of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Various aspects of the invention are described in more detail below.

Single-domain antigen-binding (SDAB) molecules

In some embodiments of the SDAB molecule, purified by the methods according to the invention, are single-stranded fused polypeptides consisting of one or more molecules nanotesla. For example, the SDAB molecule can be a single-stranded fused polypeptide consisting of one or more molecules nanotesla that is associated with one or more target antigens, connected via a linker, e.g., a peptide linker.

Used in this application "fusion polypeptide" relative�tsya to protein, containing two or more functionally related, such as United, fragment, such as protein fragments. Typically, the fragments are covalently linked. Fragments can be directly connected or connected via a spacer or linker (e.g., a linking group, as described in this application). Fused polypeptide can be obtained using standard recombinant DNA methods. For example, DNA fragments encoding different polypeptide sequence, be ligated together within the reading frame in accordance with conventional methods, for example, with blunt or step (stagger-ended) ends for ligation, restriction digestion enzymes to ensure appropriate ends, filling in the sticky ends as appropriate, alkaline phosphatase treatment to avoid undesirable compounds and enzymatic ligation. In another embodiment of the fused gene can be synthesized by conventional methods, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using zakurivaya primers, which give complementary overhangs between two consecutive gene fragments that can subsequently be subjected to annealing and re-amplification to create a follower�spine chimeric gene (see, for example, Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, 1992). Moreover, many expression vectors that encode the merged fragment, are commercially available. In some embodiments of the fused polypeptides exist as oligomers, such as dimers or trimers single contiguous polypeptide or two or more noncontiguous polypeptides. In another embodiment, additional amino acid sequences can be added to the N-or C-Terminus fused protein to ensure the expression of steric flexibility, detection and/or separation or purification.

Single-domain antigen-binding (SDAB) molecules include molecules, complementarity determining region which are part of single-domain polypeptide. Examples include, but are not limited to, the variable domains of the heavy chain of the binding molecules, naturally devoid of light chains, single domain, originating from conventional 4-chain antibodies, engineered domain and single-domain frames, different from that derived from antibodies. SDAB molecule can be any of the prior art or any future single-domain molecules. SDAB molecules can be from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit and cattle. The term also includes natural single-domain mo�ecoli antibodies of the species different from the camelidae family and sharks.

In one aspect of the invention, the SDAB molecule can be derived from variable regions of immunoglobulin found in fish, such as, for example, which derives from immunoglobulin isotype known as new antigen receptor (NAR). Novel Antigen Receptor), detected in the serum of sharks. Methods of obtaining single-domain molecules occurring in their variable region of NAR ("IgNARs") described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

In accordance with another aspect of the invention, the SDAB molecule is a natural single-domain antigen-binding molecule, known as heavy chain devoid of light chains. Such single-domain molecules disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example. For clarity, this variable domain derived from molecules of the heavy chain of naturally devoid of light chain, known in this application as VHH or nanotesla, in order to distinguish it from conventional VH of chetyrehyacheechny of immunoglobulins. Such a VHH molecule can be derived from species of the camelidae family, for example, camel, llama, DROMEDARY, Alpaca and guanaco. Other species besides camelidae family, can produce molecules of the heavy chain of naturally devoid of light chain; such VHHs are in the scope of the invention.

In some embodiments of the SDAB molecule includes at least one variable house� immunoglobulin (including one, two and/or three complementarity determining region (CDR)), in the absence of a complementary variable chain of an antibody (for example, variable regions of the heavy chain (VH) in the absence of variable regions corresponding light chain (VL) and/or constant region of immunoglobulin, for example, the Fc-region (or constant region or its part, is able to bind to protein A).

In some embodiments of the SDAB molecule comprises antibody molecules having the variable domains of the heavy and light chains of antibodies (e.g., full length antibodies or their antigen-binding fragments, with fragments of the heavy and light chains of the antibody (e.g., Fab, F(ab')2-a fragment, scFv, having variable regions of the light and heavy chain in a single polypeptide chain, or Fv fragment consisting of the VL and VH domains of a single arm of an antibody).

SDAB molecule can be a recombinant CDR-grafted, humanized, verludzutune, daimonizomai and/or obtained in vitro (e.g., selected by phage display), as described in more detail below.

The term "antigen-binding" is intended to include part of a polypeptide, for example, single-domain molecules described in this application, which contains determinants that form a contact area, which is associated with the target antigen or epitope. What Casa�tsya proteins (or protein mimetics), then the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid mimetics), which form a contact area, which is associated with the target antigen. Typically, antigen-binding portion of the polypeptide, for example, a molecule of single-domain antibody includes at least one or two CDR or more typically at least three, four, five, or six CDR.

VH - and VL-region can be subdivided into the field of hypervariability called "areas that define complementarity" (CDR), interspersed with regions that are more conservative and are called "framework regions" (FR). The length of the frame region and CDRs has been precisely defined in various ways (see Kabat, E. A., et al. (1991) Sequences of Proteins of tmmunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and AM the definition used software for modeling antibody Oxford Molecular's AbM. Cm. typically, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In; Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). As a rule, unless specifically stated, the following definitions are used:

AbM definition variable domain CDR1 of the heavy chain and the Kabat definition for other CDR. In addition, embodiments of the invention, described in relation Kaba or AbM CDR, you can also perform using hypervariable loops Chothia. Each VH and VL, as a rule, consists of three CDR and four FR located from amino end to carboxy-Terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "immunoglobulin variable domain" is often understood in the art as identical or essentially identical to a VL - or VH-domain of human or animal origin. It should be understood that the immunoglobulin variable domain can evolve in some species, such as sharks and Lam, therefore, to differ in amino acid sequence from the VL or VH of a human or mammal. However, these domains are mainly involved in the binding to the antigen. The term "immunoglobulin variable domain" typically includes at least one or two CDR or more typically at least three CDR.

"The constant domain of an immunoglobulin or a constant region" is intended to include immunoglobulin domain that is identical or substantially similar CL-, CH1-, CH2-, CH3 - or CH4-domain of human or animal origin. See, for example, Charles A Hasemann and J. Donald Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, Second Edition, 209, 210-218 (1989). The term "Fc region" refers to the Fc-plot of the constant domain of immunoglobulin, which includes domains CH2 and CH3 of an immunoglobulin or domains they�of noglobulin, essentially similar to them.

In some embodiments of the SDAB molecule is a monovalent or polyspecific molecule (e.g., divalent, trivalent or tetravalent molecule). In other embodiments of the SDAB molecule is a monospecifičeskoj, bespecifically, thespecifics or retrospections molecule. Whether the molecule "monospecifičeskoj" or "polyspecific", for example, "bespecifically" refers to the number of different epitopes that react binding polypeptide. Polyspecific molecule can be specific against different epitopes of the target polypeptide described in this application or may be specific for a target polypeptide, as well as against heterologous epitope, such as a heterologous polypeptide or solid material substrate.

Used in this application, the term "valency" refers to the number of potential binding domains, e.g., antigen-binding domains present in the SDAB molecule. Each binding domain specifically binds to one epitope. When SDAB molecule contains more than one binding domain, each binding domain may specifically contact the same epitope, for an antibody with two binding domains, called "bivalent �nespetsificheskie", or to different epitopes, for SDAB molecule with two binding domains, called "bivalent bespecifically". SDAB molecule can also be bespecifically and bivalent for each specificity (called "bespecifically tetravalent molecule"). Bespecifically bivalent molecules and methods for their preparation are described, for example, in us Pat. U.S. No. 5731168; 5807706; 5821333; and us Pat.publ. U.S. No. 2003/020734 and 2002/0155537, the descriptions of which are incorporated by reference into this application. Bespecifically tetravalent molecules and methods for their preparation are described, for example, in WO 02/096948 and WO 00/44788, the descriptions of which are incorporated by reference into this application. See, generally PCT publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

In some embodiments of the SDAB molecule is a single-stranded fused polypeptide containing one or more single-domain molecules (for example, nantel) deprived of a complementary variable domain or constant region of immunoglobulin, for example, the Fc-region, which is associated with one or more target antigens. Typical target antigen recognized by the antigen-binding polypeptide comprises a tumor necrosis factor a (TNFα). In some embodiments of the single-domain antigen-binding molecule of the con�ivalsa with whey protein for example, human serum proteins, selected from one or more of serum albumin (human serum albumin (HSA)) or transferin.

TNFα

The tumor necrosis factor alpha, as is well known in this area, associated with inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis and multiple sclerosis. As TNFα and receptors (CD120a and CD120b) have been studied in great detail. TNFα in its bioactive form is a trimer. Several strategies antagonise the action of TNFα using antibodies against TNFα have been developed and are currently commercially available, such as Remicade® and Humira®. Known molecules of antibodies against TNFα. Numerous examples of TNFα-binding single-domain antigen-binding molecules (e.g., nantel) are disclosed in WO 2004/041862, WO 2004/041865, WO 2006/122786, the contents of which are incorporated by reference into this application in its entirety. Additional examples of single-domain antigen-binding molecules are disclosed in U.S. 2006/286066, USA 2008/0260757, WO 06/003388, USA 05/0271663, USA 06/0106203, the contents of which are incorporated by reference into this application in its entirety. In other embodiments, mono-, bi-, tri - and other polyspecific single-domain antibodies against TNFα and serum protein, e.g., HSA, disclosed in these piss�tuples.

In specific embodiments, the molecule TNFα-binding nanotesla contains one or more nantel disclosed in WO 2006/122786. For example, a molecule TNFα-binding nanotesla may be monovalent, divalent, trivalent TNFα molecule-binding nanotesla disclosed in WO 2006/122786. Typical TNFα-binding nanotesla include, but are not limited to, TNF1, TNF2, TNF3, their humanized forms {e.g., TNF29, TNF30, TNF31, TNF32, TNF33). Additional examples of monovalent TNFα-binding of nanutel disclosed in table 8 of WO 2006/122786. Typical divalent molecules TNFα-binding nanotesla include, but are not limited to, TNF55 and TNF56 that contain two nanotesla TNF30 coupled via a peptide linker to form a single fused polypeptide (disclosed in WO 2006/122786). Additional examples of bivalent TNFα molecules binding nanotesla disclosed in table 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, the molecule of HSA-binding nanotesla contains one or more nantel disclosed in WO 2006/122786. For example, a molecule of HSA-binding nanotesla may be monovalent, divalent, trivalent molecule of HSA-binding nanotesla disclosed in WO 2006/122786. In other embodiments, the molecule of HSA-binding nanotesla can be monospecifičeskoj or polyspecific molecule having at least one of the tie�equation describing specificdate, which binds to HSA. Typical TNFα-binding nanotesla include, but are not limited to, ALB1, their humanized forms (for example, ALB6, ALB7, ALB8, ALB9, ALB10) disclosed in WO 06/122786.

In other embodiments, two or more single-domain molecules nanotesla merged, with a binding band or without it, in the form of genetic or polypeptide fusion. Linking group can be any linking group known to specialists in this field. For example, the linking group may be a biocompatible polymer with length from 1 to 100 atoms. In one of the embodiments of the linking group comprises or consists of the remains of polyglycine, polyserena, polylysine, polyglutamate, polysilazane or polyalanine or combinations thereof. For example, polyglycine or polizeirevier linkers can include at least five, seven, eight, nine, ten, twelve, fifteen, twenty, thirty, thirty-five and forty residues of glycine and serine. Typical linkers that may be used include Gly-Ser repeats, for example, (Gly)4-Ser repeats in one, two, three, four, five, six, seven or more repeats (SEQ ID NO:8). In embodiments, the linker has the following sequence: (Gly)4-Ser-(Gly)3-Ser (SEQ ID NO:9) or ((Gly)4-Ser)n, where n is 4, 5 or 6 (SEQ ID NO:10).

In one typical embodiment the antigen-binding polypeptide, SOS�Mr sage from single-chain polypeptide fusion of two single-domain antibody molecules (e.g., two camel variable regions) that bind to a target antigen, such as tumor necrosis factor (TNFα), and one single-domain antibody molecules (e.g., camel variable region) that binds to serum protein, e.g., HSA, named in this application "ATN-103, as shown, binds to protein A or its functional variant. ATN-103 is a trivalent humanized bespecifically TNFα-inhibitory protein. The antigen for this protein is a tumor necrosis factor-alpha (TNFα). Fig.1 shows a schematic representation of the predicted structure of ATN-103. This fused protein derived from camelids and has a high degree of sequence homology and structural homology with the VH domains of human immunoglobulins. Its single polypeptide chain consists of two binding domains for TNFα and one for human serum albumin (HSA), with two consisting of nine amino acids (G-S linkers connecting these domains. Detailed description of ATN-103 is presented in WO 06/122786.

Complete amino acid sequence of the polypeptide chain ATN-103, predicted on the basis of DNA sequence expressing the corresponding vector shown in Fig.2 (SEQ ID NO:1) (residues are numbered starting with NH2-end as the Rest index1). The last amino acid residue encoded by the DNA sequence, is a S363and forms the COOH-end of the protein. Predicted molecular weight disulfide-linked ATN-103 (without any post-translational modifications) is 38434,7 Yes. ATN-103 does not contain N-linked consensus sequence for glycosylation. The molecular mass observed for the predominant isoforms by quadrupole time-of-flight mass spectrometry with ionization by nanoelectrospray corresponds 38433,9 Yes, that confirms the absence of post-translational modifications.

Fig.2 region, complementarity determining (CDR) are underlined (SEQ ID NO:2-7). Predicted intramolecular disulfide bonds are illustrated by compounds involved in cysteine residues. Domains that binds to TNF, bold, and a domain that binds to HSA in bold italics. Amino acid linkers connecting these binding domains in italics. For a given polypeptide chain also shows the signal peptide (-19MGW VHS...-1).

Getting SDAB molecules

SDAB molecules can consist of one or more single-domain molecules (for example, NanoTec), which are recombinant, CDR-grafted, humanized, verludzutune, daimonizomai and/or semi�enny in vitro (for example, selected by phage display). Methods of producing antibodies and SDAB molecules and recombinant modification known in this field and are described in detail below.

To generate antibodies are available in numerous ways known to specialists in this field. For example, monoclonal antibodies can be obtained by producing hybridomas in accordance with known methods. The hybridomas thus created is then subjected to screening using standard methods such as enzyme-linked immunoassay (ELISA) and analysis method overhasty plasma resonance (BIACORE™), to identify one or more hybridomas that produce nanotesla that specifically binds with a specified antigen. Any form of a given antigen can be used as an immunogen, for example, recombinant antigen, natural forms, any of its variants or fragments, as well as its antigenic peptide.

One typical way to create antibodies and SDAB molecules includes screening of protein expressmusic libraries, e.g., phage or ribosomal display libraries. Phage display is described, for example, in Ladner et al., U.S. patent No. 5223409; Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690 and WO 90/02809.

In addition to the use of display libraries. you can use set�th antigen for immunization of an animal, non-human, e.g., a rodent such as a mouse, hamster or rat. In one of the embodiments of the animal, not human, includes at least a portion of a human immunoglobulin gene. For example, you can construct a mouse line defect in production of murine antibodies, with large fragments of the human Ig loci. With the use of hybrid technology can be obtained and the selected antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, USA 2003-0070185, WO 96/34096, published October 31, 1996, and PCT application no PCT/US96/05928, filed April 29, 1996.

In another embodiment of the SDAB molecule derived from animal, not human, and then modified, for example humanitariannet, neimmunizirovannah, chimeric, can be obtained using recombinant DNA methods known in this field. Have been described various approaches to create chimeric antibodies and SDAB molecules. See, for example, Morrison et al., Proc. Natl. Acad. Sci. U. S. A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. patent No. 4816567; Boss et al., U.S. patent No. 4816397; Tanaguchi et al., European patent publication EP 171496; European patent publication 0173494, UK patent GB 2177096 V. Humanized antibodies and SDAB molecules can also be obtained, for example, I�p, using transgenic mice that Express human genes of heavy and light chains, but not capable of expressing endogenous murine genes of heavy and light chains of immunoglobulins. Winter describes a typical method of CDR grafting, which can be used for obtaining humanized antibodies and SDAB molecule described in this application (U.S. patent No. 5225539). All CDRs of a particular human antibody may be replaced with at least part of nonhuman CDR or only some of the CDRs may be replaced with nonhuman CDR. You must only replace the number of CDRs required for binding of the humanized antibody and SDAB molecules with the predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Typical methods for obtaining humanized antibodies or fragments thereof are presented in Morrison (1985) Science 229:1202-1207; Oi et al. (1986) V/o Techniques 4:214; and US 5585089; US 5693761; US 5693762; US 5859205 and US 6407213. These methods include the selection, manipulation and expressiona nucleic acid sequences that encode all or part of the Fv variable domains of immunoglobulin at IU�e one heavy or light chain. Such nucleic acids can be obtained from the hybridomas producing nanotesla against a predetermined target, as described above, as well as from other sources. Recombinant DNA that encodes humanisierung SDAB molecule, e.g. a molecule, nanotesla, can then be cloned into a suitable expression vector.

In some embodiments humanitariannet SDAB molecule, e.g. a molecule of nanotesla optimized by the introduction of conservative substitutions, the substitutions from the consensus sequence of substitutions from germ line and/or back mutations. Such altered immunoglobulin molecules can be created using any of several methods known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. U. S. A., 80: 7308-7312, 1983; Kozbor et al., 1 gtipod Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982), and can be created according to the ideas of PCT publication WO 92/06193 or EP 0239400).

Methods of humanizing SDAB molecules, for example molecules of nantel, are disclosed in WO 06/122786.

SDAB molecule, e.g. a molecule of nanotesla, can also be modified by specific deletion of human T cell epitopes or "daemonization" using the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the variable domains of the heavy and light chains, such as nanotesla, can be analyzed for peptides that bind to MHC cluster�sa II (major histocompatibility complex); these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). To identify potential T-cell epitopes can be used a computer simulation approach, called "peptide broaching ("peptide threading"), and, in addition, a database of human binding peptides MHC class II can be investigated in relation to motifs present in the sequence VHand VLas described in WO 98/52976 and WO 00/34317. These motifs are associated with any of 18 major allotypes DR MHC class II and, thus, represent potential T-cell epitopes. Detected potential T-cell epitopes can be eliminated by replacing a small number of amino acid residues in the variable domains, or, preferably, by single amino acid substitutions. Generally make conservative substitutions. Often, but not exclusively, you can use the amino acid specific to the position in the sequences of human antibodies embryonic type. The human sequence of the embryonic type, for example, are disclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol.16 (5): 237-242; Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Directory V BASE is a comprehensive guide sequences of variable regions of immuno�of abulanov (compiled by Tomlinson, I. A. et al. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for frame regions and CDR. The consensus of the human frame region can also be used, for example as described in US 6300064.

SDAB molecules, e.g., molecules nantel, are produced by living cells-the hosts that have been genetically engineered to produce the protein. Methods of genetic engineering of cells for the production of proteins are well known in this field. See, for example, Ausabel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include the introduction of nucleic acids that encode and make possible the expression of a protein in living cells-hosts. These the host cell can be a bacterial cell, fungal cell, or preferably animal cells growing in culture. Bacterial cell hosts include, but are not limited to, cells of Escherichia coli. Examples of suitable E. coli strains include: NV, DH5a, GM2929, JM109, KW251, NM538, NM539 and any strain of E. coli that is unable to cleave foreign DNA. Fungal cells-owners, which can be used include, but are not limited to, cells of Saccharomyces cerevisiae, Pichia pastoris and Aspergillus. Some examples of animal cell lines that can be used�used, are Cho, VERO, BHK, HeLa, Cos, MDCK, 293, ztd and WI38. New animal cell lines can be established using methods well known to specialists in this field (e.g. by transformation, viral infection or breeding). Maybe protein can secretariats cells-the hosts on Wednesday.

Modified SDAB molecule

SDAB molecule, e.g. a molecule of nanotesla, purified using the methods of the invention may have amino acid sequence which differs by at least one amino acid position in one frame from areas of the amino acid sequence of the natural domain, e.g., VH domain.

It should be understood that the amino acid sequence of some of the SDAB molecules of the invention such as humanized SDAB molecules can differ by at least one amino acid position in at least one of the frame regions from the amino acid sequences of the natural domain, such as natural domains VHI-I.

The invention also includes methods of cleaning derived SDAB molecules. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g., enzymatic) modification, SDAB molecules and/or one or more amino acids�s residue, which form SDAB molecule described in this application.

Examples of such modifications, as well as examples of amino acid residues within the sequence of the SDAB molecule, which may be modified in such a manner (i.e. either on the protein skeleton, but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the specialist.

For example, such modifications can include the introduction (e.g. by covalent linking or in another suitable manner) of one or more functional groups, residues or groups in the SDAB molecule or SDAB molecule and, in particular, one or more functional groups, residues or groups which impart one or more desired properties or functional activities SDAB molecules. An example of such functional groups will be clear to the specialist.

For example, such modification may involve the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or absorption of SDAB molecules that reduce the immunogenicity and/or toxicity of the SDAB molecules that eliminate or reduce unwanted its�STV SDAB molecule and/or which provide other favorable properties and/or reduce the undesired properties of the SDAB molecule; or any combination of two or more of the above elements. Examples of such functional groups and methods for their introduction will be obvious to the expert and can, as a rule, include all functional groups and techniques mentioned in the General prior art cited in this application above, as well as functional groups and methods known in themselves for the modification of pharmaceutical proteins, and in particular, for the modification of antibodies or fragments of antibodies (including ScFv's and 148-single-domain antibody), to which reference is made, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups can be linked, for example, directly (e.g., covalently) with nanotesla according to the invention, or optionally via a suitable linker or spacer, which also will be clear to the specialist.

One of the widely used ways of increasing the half-life and/or reduce immunogenicity of pharmaceutical proteins comprises attaching a suitable pharmacologically acceptable polymer, such as gender and (ethylene glycol) (PEG) or its derivatives (such as methoxypoly(ethylene glycol) or mPEG). Typically, you can use any appropriate form of paglierani such as paglierani used in this region for the antibodies and fragments of antibodies (including, but not limited to, (�bottom)domain antibodies and ScFv's); reference is made, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for paglierani proteins are also commercially available, e.g. from Nektar Therapeutics, USA.

Preferably, use the site-directed paglierani, in particular via a cysteine residue (see, e.g., Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to the cysteine residue that naturally occurs in the SDAB molecule, the SDAB molecule can be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence containing one or more cysteine residues for attachment of PEG may be fused to the N - and/or-the end of nanotesla according to the invention, where all the used design methods proteins are known to those skilled in themselves.

Preferably, for SDAB molecules using PEG with a molecular weight of more than 5000, such as more than 10,000 and fewer than 200,000, such as less than 100,000; for example in the range 20000-80000.

As to paglierani, it should be noted that, generally, the invention also covers any SDAB molecule that was paglinawan in one or more amino acid positions, preferably in such a way that paglierani or (1) took�Ivan doesn half-life in vivo; (2) reduces immunogenicity; (3) provides one or more further beneficial properties known to paglierani as such; (4) essentially no effect on the affinity of the SDAB molecule (e.g., does not reduce the specified affinity by more than 90%, preferably more than 50% and more than 10%, as determined by a suitable analysis, such as described in the examples below); and/or (4) does not affect any of the other desirable properties of SDAB molecules. Suitable PEG-groups and methods for their adherence or non-specific, will be clear to the specialist.

Fitting kits and reagents for such paglierani can be obtained, e.g., from Nektar (CA, USA).

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part cotranslational and/or post-translational modification, depending on the host cell used to ekspressirovali SDAB molecule.

Chromatographic methods

The method of purification of a protein often requires numerous stages, where each stage leads to a further decrease in output. Chromatography-based protein And are one of many commonly used methods. The protein purification by chromatography-based protein And can be carried out on a column containing immobilized ligand Bel�to A (usually column filled with modified media methacrylate copolymer or agarose beads, which are attached to the adsorbent, consisting of protein A or its functional derivative). Typically, the column is equilibrated with buffer with high salt concentration, and a sample containing a mixture of protein (target protein plus contaminating proteins) in compatible sedentarism high salt solution applied to the column. As the passage of the mixture through the column, the target protein binds to the adsorbent inside the column, whereas the unbound contaminants are derived. The bound protein is then eluted in a column with low salt concentration. Typically, the target protein can be removed by elution of the column the concentration of salt used in a gradual or graded decreasing gradient for selective release of various proteins at a certain salt concentration suitable for their release, and the collection of light fractions until then, until it receives a fraction containing a greater amount of purified protein. Collecting fractions of the eluate after certain periods of time, it is possible to allocate fractions containing specific proteins. In the method, when a target protein binds to the column (ensuring the flow of pollutants), the adsorbent having a greater affinity for protein A, to�to the rule, used to bind a wider range of proteins that collect in specific fractions, contributing to the release of the protein.

The method of the invention can be used in combination with other protein purification methods such as precipitation with salts, affinity chromatography, hydroxyapatite chromatography, liquid chromatography with reversed phase, ion-exchange chromatography, or any other commonly used method for protein purification. However, it is anticipated that the method of the present invention eliminates or substantially reduces the need for other stages of treatment.

Any or all chromatography steps according to the present invention can be realized by mechanical means. For example, chromatography can be performed on the column. The column can be run under pressure or without pressure and top-down or bottom-up. The direction of flow of liquid in the column can be reversed during the chromatographic process. Chromatography can also be carried out using a batch process in which a solid medium is separated from the liquid used for loading, washing and elution of the sample by any appropriate sposobami, including gravity, centrifugation or filtration. Chromatography can also be performed by bringing the sample into contact with the filter, which� absorbs or retains some molecules in the sample are stronger than others. In the following description of various embodiments of the present invention is described in the context of chromatography, performed on the column. However, it is clear that the use column is just one of several chromatographic methods that can be used and the illustration of the present invention using the column does not limit the application of the present invention separated by column chromatography, as experts in this field can easily use the information about other methods, such as methods using a batch process or filter.

Suitable carriers may be any currently available or later developed materials having the characteristics required for practical implementation of the claimed method, and can be osnovana on any synthetic, organic or natural polymers. For example, the commonly used substances-carriers include organic substances such as cellulose, polystyrene, agarose, sepharose, the polyacrylamide polymethacrylate, dextran and starch, and inorganic substances such as charcoal, silica (glass granules or sand) and ceramic materials. Suitable solid carriers are disclosed, for example, Zaborsky in "Immobilized Enzymes" CRC Press, 1973, table IV on page 28-6.

Before equilibration and chromatography of the medium for chromatography (carrier and an adsorbent attached to the carrier) can be pre-equilibrated in the selected solution, such as solution of salt and/or buffer solution. Pre-trim performs the function of substitution of the solution used for regeneration and/or storage of the chromatographic medium. The person skilled in the art it is clear that the composition of the solution prior to equilibration depends on the composition of the storage solution and the solution used for subsequent chromatography. Thus, appropriate solutions for pre-equilibration may include the same buffer or salt used to implement chromatography, possibly with a higher concentration than used for chromatography. Buffers and salts that can be used for chromatography, are discussed below. For example, when the solution used for the chromatography, contains sodium phosphate at a given concentration, pre-equilibration can occur in a solution containing sodium phosphate at higher concentrations. To illustrate this, if the solution used for the chromatography, contains sodium phosphate from about 0.5 mm to about 50 mm, pre-trim the bottom side o�eshiwani can occur in solution, containing sodium phosphate at a concentration from about 0.2 M to about 0.5 M, more preferably at a concentration of sodium phosphate from about 0.3 M to about 0.4 M, inclusive.

Before applying sample to column, the column can be equilibrated in buffer or salt, to be used for protein chromatography. As discussed below, chromatography (and protein deposition, treated) can occur in a variety of buffers or salts, including salts of sodium, potassium, ammonium, magnesium, calcium, chloride, fluoride, acetate, phosphate and/or citrate and/or Tris buffer. Citrate buffers and salts are preferred for specialists in this area due to the simplicity of their utilization. Such buffers or salts may have a pH of at least about 5.5. In some embodiments, the equilibration can occur in a solution containing Tris or sodium phosphate buffer. Perhaps the sodium phosphate buffer is at a concentration from about 0.5 mm to about 50 mm, more preferably in a concentration of from about 15 mm to about 35 mm. Preferably, the equilibration occurs at a pH of at least about 5.5. Balancing can occur at a pH from approximately 6.0 to approximately 8.6, preferably at pH from about 6.5 to 7.5. Most preferably, the solution contains sodium phosphate buffer at a concentration of about� approximately 25 mm and at a pH of approximately 6.8.

Suitable buffers include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers and/or citrate buffers. Acceptable salts may include, but are not limited to, sodium chloride, ammonium chloride, potassium chloride, sodium acetate, ammonium acetate, sodium sulphate, ammonium sulphate, ammonium thiocyanate, sodium citrate, sodium phosphate, and potassium, magnesium and calcium salt and a combination of these salts. In another embodiment the salts include sodium citrate and sodium chloride. Acceptable levels of salt concentrations used for chromatographic systems are typically in the range of from 0 to about 2 M of sodium citrate, from 0 to about 4 M of sodium chloride, from 0 to about 3 M ammonium sulfate, from 0 to about 1 M of sodium sulfate and from 0 to about 2 M of sodium phosphate. The salt concentration ranges may include from 0 to about 1 M of sodium citrate, from 0 to about 2 M sodium chloride, from 0 to about 1.5 M ammonium sulfate, from 0 to about 1 M of sodium sulfate and from 0 to about 1.5 M sodium phosphate. Can also be used other buffers and salts. After application, the adsorbent may be flushed with large amounts of the same solution in order to cause the flow of a target protein (unrelated to the adsorbent) through the adsorbent. Then the protein is collected in the eluate fraction. Conditions for binding of pollutants,while the target protein will not be contacted can be easily optimized by specialists in this field. Salt concentrations discussed in this application are exemplary, and other salts and salt concentrations can be used by changing the flow rates, temperatures and elution time, as is well known in this field.

The conditions under which use columns vary depending on specific columns, as is well known in this field. For most of the proteins in the pH range may be from about 6.0 to about 8.6 or, alterantive, from about 6.5 to about 7.5. However, some proteins are known that are resistant to extreme pH values, and a wider range may be possible. Typical conditions include a pH range from 5 to 7 and the range of concentration of sodium citrate, from 0 to about 0.8 M (e.g., 0.5 M sodium citrate, pH 6.0).

The person skilled in the art will be guided by the knowledge in this field to determine which buffer or salt suitable for a specific protein, of treated. In addition, the specialist can easily determine the optimal concentration of the selected buffer or salt for use, for example, by creating a specific buffer or saline conditions under which pollutants are associated with column, while for protein arrives in a fraction of eluate. FR�functions of runoff from the column can be collected and analyzed to determine the concentration of the buffer or salt, in which the target protein and contaminants eluted. Suitable assays include, for example, the measurement of electrical conductivity using conductivity meter (to determine the salt concentration in the sample), as well as gel electrophoresis or ELISA analysis (to determine the identity of proteins in the sample). Perhaps the column can be flushed with large amounts of the same solution, which is applied to the protein sample, and the solution for cleaning you can also collect and combine with the flowing liquid.

After collecting the eluate and possibly washing liquid, which consisted of treated protein, the proteins that remained bound to the column, can be released by removing the chromatographic medium using a solution containing a buffer or salt used for chromatography, but at a lower ionic strength for the release of contaminating proteins. Then the column can be regenerated using a solution that will affect the release of most or all proteins from a chromatographic medium, and the reduction or elimination of any microbial contamination that may be present in the chromatographic medium. In one of the embodiments of such a solution may include sodium hydroxide. Can also be used with other reagents. Then the column can be rinsed and stored in solution, to�which can impede the growth of microbes. Such solution may contain sodium hydroxide, but other reagents can also be appropriate.

The protein concentration in the sample at any stage of purification can be determined by any suitable method. Such methods are well known in this field and include: 1) colorimetric methods of analysis by Lowry, analysis by Bradford analysis by Smith and the analysis of colloidal gold; and 2) the ways using properties of proteins absorb UV; and (3) visual assessment based on a comparison of stained protein bands on the gels with standard proteins in known quantities on the same gel. See, for example, Stoschek (1990), Quantitation of Protein, in Guide to Protein Purification, Methods in Enzymol. 182: 50-68.

Target protein and contaminating proteins that may be present in the sample, can be controlled by any suitable methods. Preferably, the method should be sensitive enough to detect contaminants in the range of from about 2 parts per million (mn-1) (calculated as nanogrammy per milligram of purified protein), 500 million-1. For example, solid-phase enzyme-linked immunosorbent assay (ELISA) method, well known in this field can be used to detect contamination of the protein with a second protein. See, for example, Enzyme-Linked Immunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols, Methods Mol. Biol. 32: 461-466, which is included in this proposal through�Ohm reference in its entirety. In one aspect, the contamination of protein other proteins can be reduced by the methods described in this application, preferably at least about twice, more preferably at least about three times, more preferably at least about five times, more preferably at least about ten times, more preferably at least about twenty times, more preferably at least about thirty times, more preferably at least about forty times, more preferably at least about fifty times, more preferably at least about sixty times, more preferably at least about seventy times, more preferably at least about 80 times, more preferably at least about 90 times and most preferably at least about 100 times.

In another aspect, the contamination of protein other proteins after the methods described in this application, is not more than about 10,000 mn-1preferably is not more than about 2,500 million-1more preferably is not more than about 400 m-1more preferably is not more than about 360 million-1more preferably is not more than primerno mn -1more preferably is not more than about 280 million-1more preferably is no greater than about 240 million-1more preferably is not more than about 200 mn-1more preferably is not more than about 160 million-1more preferably is no greater than approximately 140 million-1more preferably is not more than approximately 120 million-1more preferably is not more than about 100 million-1more preferably is not more than about 80 million-1more preferably is not more than about 60 million-1more preferably is not more than about 40 mn-1more preferably is not more than about 30 mn-1more preferably is not more than about 20 mn-1more preferably is not more than about 10 million-1and most preferably is not more than about 5 million-1. Such contamination can vary from undetectable levels to approximately 10 million-1or from about 10 million-1up to about 10,000 mn-1. If purified protein for pharmacological application, the person skilled in the art understand that the preferred level of the second protein may depend on e�endeley dose of protein, enter the patient so that the patient has not received more than a certain number of polluting of protein per week. Thus, if the required weekly dose of protein decreases, the level of contamination of the second protein may increase.

The amount of DNA that may be present in the sample of the purified protein can be determined by any suitable method. For example, you can use the analysis using the polymerase chain reaction. Perhaps this method can detect DNA contamination level is 10 picograms per milligram of protein and more. The DNA levels can be reduced by using HIC (chromatography hydrophobic interaction), maybe about two times, preferably five times, more preferably about ten times, more preferably approximately fifteen times, most preferably about 20 times. Perhaps the DNA levels after hydroxyapatite chromatography is less than about 20 picogram per milligram of protein, preferably less than 15 picogram per milligram of protein, more preferably less than 10 picograms per milligram of protein, most preferably less than 5 picograms per milligram of protein.

Chromatography-based protein And

In one of the embodiments of the collected medium containing a preparation of antibodies can be purified by chromatogr�fee-based protein A. Staphylococcal protein A (SpA) is a protein of 42 kDa, consisting of nearly five homologous domains, named E, D, A, b and C and arranged in order from N-Terminus (Sjodhal EurJ Biochem 78: 471-490 (1977); Uhlen et al. J. Biol. Chem. 259: 1695-1702 (1984)). These domains contain about 58 residues, and each has about 65% -90% identity amino acid sequence. Studies on the binding between protein a and antibodies showed that although all five domains of SpA (E, D, A, b and C) bind to IgG via its Fc-region, the domains D and E show significant binding Fab (Ljungberg et al. Mol. Immunol. 30(14): 1279-1285 (1993); Roben et al. J. Immunol. 154:6437-6445 (1995); Starovasnik et al. Protein Sci 8:1423-1431 (1999). It was shown that the Z-domain, functional analog and minimized energy option In the domain (Nilsson et al. Protein Eng 1:107-113 (1987)), had negligible binding to the variable region domain of the antibody (Cedergren et al. Protein Eng 6(4):441-448 (1993); Ljungberg et al. (1993) above; Starovasnik et al. (1999) above).

Until recently, commercially available protein A stationary phases used the SpA (Staphylococcus aureus isolated from or recombinante expressed) as immobilized ligand. The application of these columns was not possible to use the alkaline conditions for regeneration of the column and rehabilitation, as is usually done with other types of chromatography with the use of non-protein ligands (Ghose et al. Biotecnology and Bioengineering Vol.92 (6) (2005)). Developed a new resin (MabSELECT™ SuRe), more resistant to harsh alkaline conditions (Ghose et al. (2005) above). Using methods of protein engineering, has replaced a number of aspartic acid residues in the Z-domain of protein A and created a new ligand in the form of a tetramer of four identical modified Z-domain (Ghose et al. (2005) above).

Accordingly, the cleaning methods can be implemented using commercially available columns with protein a in accordance with the manufacturer's instructions. As described in the appended examples, can be used column MabSELECT™ column MabSELECT™ SuRe (GE Healthcare Products). MabSELECT™ is a commercially available resin containing recombinant SpA as its immobilized ligand. It captures molecules of antibodies of the many mediums through chromatography in the dense layer. Recombinant protein ligand As MabSELECT™ designed in favor of the orientation of the ligand protein A, which has enhanced ability to bind IgG. The specificity of the ligand protein And the binding region of IgG is similar to the specificity of the native protein A. the Column MabSELECT™ SuRe had similar highly crosslinked agarose matrix used for MabSELECT™, the ligand used was a tetramer of four identical modified Z-domains (GE Healthcare Products). Other commercially available sources of protein A-column, which can be EF� - objective use, include, but are not limited to, PROSEP-ATM (Millipore, U.K.), which consists of protein A covalently bound to porous glass with controlled pore size. Other protein used And the compositions include a Protein A Sepharose FAST FLOW™ (Amersham Biosciences, Piscataway, NJ) and TOYOPEARL™ 650M Protein A (TosoHaas Co., Philadelphia, PA).

Hydroxyapatite resin

Various hydroxyapatite chromatography resins are commercially available and any available form of the material can be used in the practice of this invention. Detailed description of the conditions suitable for hydroxyapatite chromatography, is presented in WO 05/044856, the contents of which are incorporated by reference into this application in its entirety.

In one of the embodiments of the invention, the hydroxyapatite is a crystalline form. Hydroxyapatite for use in the present invention may constitute hydroxyapatite, which are subjected to sintering with the formation of particles and sintered at high temperatures in a stable porous ceramic mass. The particle size of hydroxyapatite can vary greatly, but a typical particle size ranges from 1 µm to 1000 µm in diameter and can be from 10 μm to 100 μm. In one of the embodiments of the invention, the particle size is 20 microns. In another embodiment of the invention, the particle size is 40 μm. In yet another embodiment of the Fig�of moving the particle size is 80 microns.

A number of chromatographic media can be used to prepare columns of sleep, the most widely used are the hydroxyapatite type I and type II. Type I has a high protein binding capacity and a higher capacity for acidic proteins. Type II, however, has a lower protein binding capacity, but has a better resolution of nucleic acids and some proteins. Material type II also has a very low affinity to albumin, and is particularly suitable for cleaning many types and classes of immunoglobulins. The person skilled in the art can determine the choice of a particular type of hydroxyapatite.

The present invention can be used with hydroxyapatite resin that is loose, Packed in a column or in a continuous periodic chromatography. In one of the embodiments of the invention, a ceramic hydroxyapatite resin Packed in the column. The specialist can determine the range of column sizes. In one of the embodiments of the invention for small-scale purification can be used column with a diameter of at least 0.5 cm, with a depth of approximately 20 cm.

In an additional embodiment of the invention can be used column with a diameter of about 1 cm to about 60 cm In yet another embodiment of the invention can be used column with a diameter from 60 cm to 85 cm � some embodiments of the invention may be used in the slurry of ceramic hydroxyapatite resin in 200 mm Na 2HPO4solution at pH 9.0 for the packing of the column at a constant flow rate of approximately 4 cm/min, or under the influence of gravity.

Buffer composition and the processing conditions for hydroxyapatite resins

Before the conversion of a hydroxyapatite resin in contact with the preparation of the antibody, it is necessary to adjust parameters such as pH, ionic strength and temperature, and in some cases, the addition of substances of various kinds. Thus, it is possible stage to perform trim hydroxyapatite matrix by washing with a solution (e.g., a buffer for adjusting pH, ionic strength, etc. or for the introduction of a detergent), which imparts the necessary characteristics of drug purification of antibodies.

In the case of the combined binding/hydroxyapatite chromatography in flow-through mode, hydroxyapatite matrix trim and wash solution, which gives the necessary characteristics of drug purification of antibodies. In one of the embodiments of the invention the matrix can be balanced with the use of a solution containing from 0.01 to 2.0 M NaCl from slightly alkaline to slightly acidic pH. For example, balancing the buffer can contain from 1 to 20 mm sodium phosphate, in another embodiment it may contain from 1 to 10 mm sodium phosphate, in another embodiment it may contain from 2 d� 5 mm sodium phosphate, in another embodiment it may contain 2 mm sodium phosphate, and in another embodiment it may contain 5 mm sodium phosphate. Equilibrating buffer may contain from 0.01 to 2.0 M NaCl. In one of the embodiments of from 0.025 to 0.5 M NaCl, in another embodiment 0.05 M NaCl and in another embodiment 0.1 M NaCl. the pH of the loading buffer may range from 6.2 to 8.0. In one of the embodiments the pH may be from 6.6 to 7.7, and in another embodiment the pH may be a 7.3. Equilibrating buffer may contain from 0 to 200 mm of arginine, in another embodiment it may contain 120 mm arginine, and in another embodiment it may contain 100 mm arginine. Equilibrating buffer may contain from 0 to 200 mm HEPES, in another embodiment it may contain 20 m HEPES, and in another embodiment it may contain 100 mm HEPES.

In the preparation of the SDAB molecule is also possible to replace the buffer with a suitable buffer or boot buffer in preparation for hydroxyapatite chromatography in flow-through mode. In one of the embodiments of the invention in the preparation of the antibody is possible to replace the buffer in the loading buffer containing from 0.2 to 2.5 M NaCl at pH from slightly acidic to slightly alkaline. For example, the loading buffer may contain from 1 to 20 mm sodium phosphate, in another embodiment it may contain from 2 to 8 mm sodium phosphate, in another embodiment it may contain from 3 to 7 mm sodium phosphate, and in another embodiment about� may contain 5 mm sodium phosphate. Boot buffer may contain from 0.2 to 2.5 M NaCl, in one embodiment from 0.2 to 1.5 M NaCl, in another embodiment from 0.3 to 1.0 M NaCl, and in another embodiment 350 mm NaCl. the pH of the loading buffer may range from 6.4 to 7.6. In one of the embodiments the pH can range from 6.5 to 7.0, and in another embodiment the pH may be at 6.8.

Bringing drug SDAB molecules in contact with hydroxyapatite resin or binding mode, flow mode, or their combinations, can be realized in a column of a dense layer, column pseudouridines/porous layer containing a solid-phase matrix, and/or in simple batch mode, where tverdofaznoe matrix mixed with a solution within a specified time.

After bringing hydroxyapatite resin in contact with the preparation of the antibody may perform the washing procedure. However, in some cases, where very high purity of the immunoglobulin is not fatal or no additional duct antibody, the washing procedure can be omitted, saving at the stage of the method and solution for cleaning. Used buffers for washing will depend on the nature of the hydroxyapatite resin, mode used by hydroxyapatite chromatography and therefore may be determined by the person skilled in the art. In flow-through mode and combination binding/flow-through re�ima-eluted purified antibody, obtained after washing the column, can be combined with other purified fractions of antibodies.

In the mode of binding of the SDAB molecule can be eluted from the column after the washing procedure. For elution of the antibody from the column in the present invention, use of a phosphate buffer with a high ionic strength containing from about 0.2 to 2.5 M NaCl at pH from slightly acidic to slightly alkaline. For example, the elution buffer may contain from 1 to 20 mm sodium phosphate, in another embodiment it may contain from 2 to 8 mm sodium phosphate, in another embodiment it may contain from 2 to 6 mm sodium phosphate, in another embodiment it may contain 3 mm sodium phosphate, and in another embodiment it may contain 5 mm sodium phosphate. Elution buffer may contain from 0.2 to 2.5 M NaCl, in one of the embodiments from 0.2 to 1.5 M NaCl, in another embodiment from 0.3 to 1.1 M NaCl, in another embodiment of the 1.0 M NaCl and in another embodiment 0.35 M NaCl. the pH of the elution buffer may range from 6.4 to 7.6. In one of the embodiments the pH can range from 6.5 to 7.3, in another embodiment the pH may be 7.2, and in another embodiment the pH may be at 6.8. Elution buffer can be changed for elution of antibody from the column in a continuous or stepwise gradient.

In both modes, the binding mode and flow mode, as well as their combinations, solid-phase matrix can, if necessary�ti, clear, i.e., subjected to desorption and regeneration after elution or flow antibodies. This procedure is typically performed regularly to minimize the accumulation of impurities on the surface of the solid phase and/or to sterilize the matrix to avoid contamination of the product with microorganisms.

Components of the buffer can be adjusted in accordance with specialist knowledge in this field.

Additional possible stage

Although it was found that hydroxyapatite chromatography can be used by itself for the separation of Monomeric IgG from aggregates, as mentioned above, the purification method according to the invention can be used in combination with other methods of protein purification. In one of the embodiments of the invention, one or more stages prior to hydroxyapatite stage, it may be desirable to reduce the load of contaminants or impurities. In another embodiment of the invention, one or more purification steps following the hydroxyapatite phase, it may be desirable to remove additional contaminants or impurities.

Describes how sleep-cleaning can be combined with other stages of purification, including, but not limited to, chromatography based on protein A, affinity chromatography, hydrophobic interaction chromatography, affinity of chromatogr�FIU with immobilized metal size exclusion chromatography, diafiltration, ultrafiltration, filtration for virus removal and/or ion exchange chromatography.

In one embodiment, before the stage of sleep-clean, gathered Wednesday may perhaps be cleared first by a phase chromatography on protein A. for Example, can be used effectively PROSEP-ATM (Millipore, U.K.), which consists of protein A covalently linked to glass with controlled pores. Other useful protein drugs And include Protein A Sepharose FAST FLOW™ (Amersham Biosciences, Piscataway, NJ), TOYOPEARL™ 650M Protein A (TosoHaas Co., Philadelphia, PA) and column MABSELECT™ (Amersham Biosciences, Piscataway, NJ).

As a possible stage before sleep-cleaning may be used ion-exchange chromatography. In this regard, various anionic or cationic substituents may be attached to matrices in order to form anionic or cationic carriers for chromatography. Anion-exchange substituents include diethylaminoethyl (DEAE), Triethylenetetramine (TMAE), Quaternary aminoaniline (QAE) and Quaternary amine (Q) groups. Cationic exchange substituents include carboxymethyl (CM) sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U. K. the ion exchangers are Also known on the basis of Sephadex and cross stitched �ionoobmennika. For example, DEAE-, QAE-, CM -, and SP Sephadex and DEAE-, Q-, CM - and S-Sepharose, and Sepharose are available from Amersham Biosciences, Piscataway, NJ. In addition, both DEAE and CM-derivationally ethylene glycol-methacrylate copolymer such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or M are available from Toso Haas Co., Philadelphia, PA.

In one of the embodiments of the invention ion-exchange chromatography can be used in binding mode or flow mode.

In some embodiments phase chromatography-based protein And is the first stage anion-exchange chromatography is performed second, and the sleep stage is the third.

The removal of additional impurities

In addition to the removal HMWA shown that sleep chromatography useful in the removal of other impurities from preparations of antibodies. Other impurities that can be removed via sleep chromatographic methods of the invention include, but are not limited to, DNA, protein host cell, random viruses and protein And contaminants from the previous purification steps.

In one of the embodiments of the invention it is possible to remove the protein And the drug antibody. In some embodiments of this invention the amount of protein A present in the final preparation can be substantially reduced, for example, from 300 million-1to less than 1 million-1.

Introduction and method of treatment

Preparations containing the SDAB molecule, PTS�perturbed ways disclosed in this application, you can enter the subject (e.g., subject-person), alone or in combination with a second agent, e.g., a second therapeutically or pharmacologically active agent for treating or preventing (e.g., reducing or alleviating one or more symptoms associated with a) TNFα-associated disorders, e.g., inflammatory or autoimmune disorders. The term "treatment" refers to therapy in the amount, manner and/or mode effective to improve a condition, symptom, or parameter associated with the disorder, or to prevent the development of disorders in either a statistically significant degree or to a degree detectable by a person skilled in the art. An effective amount, method or mode may vary depending on the subject and can be adapted to the subject.

Non-limiting examples of immune disorders that can be treated include, but are not limited to, autoimmune disorders such as arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis associated with lupus arthritis or ankylosing spondylitis), scleroderma, systemic lupus erythematosus, sindorim Sjogren's syndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis, dermatitis (including�traveler atopic dermatitis and eczematous dermatitis), asthenic bulbar paralysis, inflammatory bowel disease (IBD), Crohn's disease, colitis, diabetes mellitus (type I); inflammatory conditions, e.g., skin (e.g., psoriasis); acute inflammatory conditions (e.g., endotoxicosis, sepsis and septicaemia, toxic shock syndrome and infectious disease); transplant rejection and Allergy. In one of the embodiments of the TNFα-related disorder is an arthritic disorder, e.g., a disorder chosen from one or more of rheumatoid arthritis, juvenile rheumatoid arthritis (RA) (e.g., rheumatoid arthritis moderate to severe), osteoarthritis, psoriatic arthritis or ankylosing spondylitis, juvenile idiopathic arthritis (LA); or psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease and/or multiple sclerosis.

In some embodiments, the drugs comprise a second therapeutic agent. For example, TNF-Manotel the second agent may be an antibody against TNF or TNF-binding fragment, where the second TNF epitope antibody has a specificity different from TNF-binding SDAB molecules of the drug. Other non-limiting examples of agents that can be prepared together with TNF-binding SDAB include, e.g.�, inhibitor of a cytokine inhibitor, growth factor, immunosuppressant, anti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, a cytotoxic agent and cytotoxic agent. In one of the embodiments the additional agent is a standard treatment of arthritis, including, but not limited to, nonsteroidal anti-inflammatory agents (NSAIDs); corticosteroids, including prednisolone, prednisone, cortisone and triamcinolone; and disease-modifying Antirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, Leflunomide (Arava®), inhibitors of tumor necrosis factor, including etanercept (Enbrel®), infliximab (Remicade®) (with or without methotrexate) and adalimumab (Humira®), anti-CD20 antibody (e.g., Rituxan®), a soluble receptor of interleukin-1 such as anakinra (Kineret), gold, minocycline (Minocin®), penicillamine, and cytotoxic agents, including azathioprine, cyclophosphamide and cyclosporine. In such combination therapies may advantageously be used a lower dose of administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

Drugs can be in the form of a liquid solution (e.g., solutions for injection and infusion). Such drugs can enter�ü parenteral route (for example, by subcutaneous, intraperitoneal, or intramuscular injection) or through inhalation. The phrases "parenteral administration" and "introduced parenterally" as used in this description, means modes of administration, than enteral and local administration, usually by injection, and include subcutaneous or intramuscular, and intravenous, intraarticular, vnutriglaznogo, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In one of the embodiments of the drugs described in this application, is administered subcutaneously.

Pharmaceutical preparations are sterile and stable under conditions of manufacture and storage. Pharmaceutical composition can also be tested to ensure that it meets regulatory and industry standards of administration.

The pharmaceutical preparation can be prepared in the form of a solution, microemulsion, dispersion, liposome, or other organized structure suitable for high concentration of protein. Sterile solutions for injection can be prepared by incorporating the agent described in this application, in the required amount in an appropriate solvent with one or a combination of ingredients, ne�chislennyh above, if required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the agent described in this application, into a sterile vehicle, which contains an alkaline dispersion medium and the required other ingredients from those listed above. The proper fluidity of a solution can be maintained, for example, by applying a coating such as lecithin, by maintenance of required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be attributed to the inclusion in the composition of an agent that slows the absorption of, for example, monostearate salts and gelatin.

EXAMPLES

The following examples are provided for illustrative purposes only.

Example 1: description of the sequence encoding the ATN-103

ATN-103 is a trivalent molecule nanotesla towards TNFα and HSA. Nanotesla was isolated from ragovoy libraries obtained from llamas by breeding on TNFα or HSA, as described in WO 06/122786. Nanotesla tested in relation to the specific activity and chose TNF1 as nanotesla inhibitor of human TNFα and ALB1 human anti-HSA nanotesla to increase the half-life period. TNF1 and ALB1 humanitarianly by transferring on CDR closest to the human frame (DP51/DP53). PR� humanization TNF1 kept 2 camel residue (R and R103), this option is outlined TNF30. When humanization ALB1 kept 7 camel residues (N16, N73, t, R, T, 194 and S103), and this version marked the ALB8. Each of the two TNF30 of nanutel linked with 9-amino acid glycine-serine linker (Gly4SerGly3Ser (SEQ ID NO:9)) with Central ALB8 nanotesla with getting trivalent molecules, designated in this application as "ATN-103." Amino acid sequence of ATN-103, shown in Fig.3, has the following configuration-THF30-(glycine-serine linker)-ALB8-(glycine-serine linker)-TNF30. Fig.2, complementarity determining region (CDR) are underlined (SEQ ID NO:2-7). Predicted intramolecular disulfide bonds are illustrated by compounds involved in cysteine residues. Domains that binds to TNF, bold, and a domain that binds to HSA in bold italics. Amino acid linkers connecting these binding domains in italics. The signal peptide (-19MGW VHS...-1also shown to the polypeptide chain.

Example 2: Method of purification of ATN-103

Method of purification of ATN-103 consists of two chromatographic stages and three stages of membrane filtration (see Fig.3). All of the stages performed at room temperature unless otherwise indicated.

The principles, objectives, and description of each purification step is shown below.

Affine chromatography on OS�ove MabSelect protein A and inactive low pH virus

The main goals of phase chromatography based on protein MabSelect™ include capturing product from osvetleni cell-free conditioned media and the Department of ATN-103 from impurities produced in the process (e.g., protein and DNA of host cells, components of the environment and adventitious agents).

MabSelect Protein A is an affinity resin comprising highly crosslinked agarose matrix that is covalently derivatization via thioether bond with recombinant protein A, produced as a result of Escherichia coli (E. coli) fermentation.

Column (MabSelect Protein And balanced, in a Tris-buffered sodium chloride solution and loaded osvetleni cell-free conditioned medium (CM). All buffers were driven at a speed of 300 cm/h. Balancing the buffer contains 150 mm NaCl and 50 mm Tris at pH 7.5. ATN-103 is associated with MabSelect™ Protein A resin and impurities flow through the column. The loaded resin is washed with Tris-buffered saline (150 mm NaCl and 50 mm Tris at pH 7.5) to further reduce the level of impurities, and then the Trio-a buffer with low concentration. Tris-buffer with low concentration for washing contains 10 mm NaCl and 10 mm Tris at pH 7.5. Related product alumroot column with glycine buffer with low pH. Glycine elution buffer with a low pH containing 10 mm NaCl and 50 mm glycine at pH 3.0. The resin is regenerated and desinfiziert races�thief hydroxide and then stored in solution, containing 16% ethanol. Several cycles stage MabSelect Protein A can be obtained from a single collection.

The combined product is maintained at a pH of not more than 3.8 for 1.5±0.5 h at 18°C - 24°C. the low pH Inactivation after MabSelect™ Protein A column was designed to inactivate enveloped viruses. - Eluted product is then neutralized with concentrated HEPES buffer (2 mm HEPES at pH 9,0) and filtered using a 0.2 μm deep and filtering.

Chromatography on ceramic hydroxyapatite Macro-Preo

The main objectives of the stage on a ceramic hydroxyapatite Macro-Prep™ (sleep) is to remove high molecular weight aggregates (HMWA), washed protein A and originating from host cells impurities such as DNA and proteins of host cells (NDS).

Ceramic hydroxyapatite Macro-Prep is newpathname matrix consisting of hexagonal crystal lattice. Molecules of calcium, phosphate and hydroxide form the matrix with stoichiometry (CA5(PO4)3(IT))2. sleep resin is a multimode matrix capable of providing a cationic exchange, anionic exchange and coordination of interaction of metals. The elution associated protein, usually achieved by increasing the concentration of salt or phosphate.

sleep first column trim buffer containing a high concentration of chloride on�Riya, then, a buffer containing a low concentration of sodium chloride. Load sleep column represents a neutralized MabSelect™ Protein A pool. After loading the column was washed with equilibrating buffer with low salt and ATN-103 is extracted using a buffer containing a high salt concentration. After elution ATN-103, HMW and other impurities are removed from the column much higher concentrations of salt and phosphate. The column is regenerated and then stored in a solution of sodium hydroxide.

Virus retentive filter Planova 20N

Stage the virus retentive filter (VRF) Planova 20N provides a significant level to clean the virus to ensure the safety of the product by removing particles that may represent random potential viral contamination.

Disposable device Planova 20N VRF balance sleep elution buffer and load assembled with ceramic hydroxyapatite Macro-Prep product. The product is collected in the ongoing flow. After the processing load of wash buffer used to extract more of the product remaining in the system.

Ultrafiltration/diafiltration and drug

Stadiou UF/diafiltration (clipping MW 10 kDa) used for the concentration and replacement of the buffer in the VRF product at the buffer of the drug.

After equilibration of the membrane module loaded�full-time the solution is first concentrated to a predetermined target volume, and then subjected to diafiltration using 14 mm histidinol buffer with a pH of 5.8. After further concentration to about 110 g/l pool is removed from the system by washing histidinol buffer to achieve a final concentration of target protein of approximately 90 g/l. Small volume (11,1% vol./about.) concentrated stock solution (10 mm histidine, 50% sucrose and 0.1% Polysorbate 80) was added to the pool of product. Received final drug substance (DS) is a 80 g/l ATN-103 in 10 mm histidine, pH 6.0, 5% sucrose, 0.01% Polysorbate.

Final filter

Obtained in the form of a preparation of drug substance is passed through a disposable 0.2 µm filter to remove any potential accidental microbial contamination and particles.

Example 3: Comparison of capture protein And

The following matrix protein based And evaluated on their ability to capture ATN-103: MabSelect™ (GE Healthcare), MabSelect Xtra™ (GE Healthcare), ProSep® Va Ultra Plus (Millipore) and MabSelect SuRe™ (GE Healthcare). In MabSelect™ protein is used As the ligand containing the Z-domain, and the base resin is more hydrophobic due to the cross-linkers. In MabSelect Xtra™ uses the same ligand, and MabSelect, with increasing density of 30% or more small balls and bolism pore size. ProSep® Va Ultra Plus has a glass base and native protein And ligand. It is designed for higher capacity at higher flow rates. MabSelect SuRe™ binds Fc-containing Molek�crystals (ATN-103 does not have Fc-region) and her new ligand is more stable to alkali.

When used peak pooled protein A, which was purified using MabSelect™, as the boot substance (pH=7.0, diluted to 1 g/l (the expected concentration of conditioned media)), ProSep® Va Ultra Plus showed the highest binding capacity (16 g/l g), and the binding capacity of MabSelect™ showed a 20% increase compared to the previously demonstrated binding capacity.

Investigated the effect of flow velocity on the ability to dynamic linking (DBC, from the English. Dynamic Binding Capacity). For MabSelect™ binding capacity was 7.4 g/l g at 600 cm/h compared with 8.0 g/l g at 60 cm/h. a Similar trend was observed when testing MabSelect Xtra™ and ProSep® Va Ultra Plus. Thus, the influence of flow velocity on DBC in the tested conditions was minimal.

Also investigated the effect of the modifier on the binding capacity of protein A. the Results showed that the addition of 0.5 M Na2SO4can increase the DBC by strengthening hydrophobic interactions. For example, the binding capacity for MabSelect™ (along with CM) increased to 12.5 g/l g at a flow rate of 150 cm/h. Additional linked material was subsequently suirable solution without Na2SO4. High precipitation was determined in CM with Na2SO4. Similar results were obtained when testing MabSelect Xtra™ (DBC=16 g/l g) and ProSep® Va Ultra Plus (DBC=17.5 g/LH).

To�to follow the effect of PEG on protein binding capacity And, added 6% PEG (4000 Da) in CM. Additional related material was then suirable in the buffer without PEG. The results showed a slight increase in binding capacity and lack of precipitation.

Example 4: Evaluation of MabSelect SuRe™ ATN-103

During the development stage MabSelect, MabSelect Sure™ was used in binding experiments to obtain a better understanding of the mechanism of binding protein And with ATN-103. Suddenly ATN-103 was associated with MabSelect Sure™ and demanded a solution with pH 4.5 to remove an associated product. MabSelect Sure is a protein A-resin, which is engineered to bind only molecules containing the Fc-region, such as antibodies. ATN-103 does not contain the Fc-region. Later in the development determined that MabSelect Sure™ can bind ATN-103 up to 8 g/l resin at 10% breakthrough of the initial concentration.

Example 5: Evaluation of cation exchange (SEH) stage of ATN-103

Stage-based capture of Latinoamerica (SEH) was evaluated with the aim of increasing the capacity of a fascinating column in the purification method ATN-103. By reducing the conductivity of the conditioned media (CM) from 12 to 9 MS/cm and pH titration to a value of not more than 4.3 watched a capacity of 40 g/l g ATN-103 communicates very poorly with SEH environment due to the low number of charge/mol at these pH levels. Because of this weak binding of ATN-103 can be eluted with SEH resin in solutions with low conductivity. Conditions �of layoune was usually not more than 50 mm NaCl at pH 6.5-7.0. SEH column can be subjected to elution using flow down sleep equilibrating buffer.

Screening capacity SEH

Four Latinoamerica Capto™ S (GE Heathcare), Fractogel® S03-(M) (EMD Chemicals), Toyopearl® Gigacap S-650M (Tosoh Bioscience) and Poros® 50 MS (Applied Biosystems) were tested with respect to their binding capacities for ATN-103. 0.75 ml CM TS2 with 10 μl of resin (target resin 75 g/l) was used for screening. The column was washed with buffer containing 50 mm sodium acetate at the same pH, which in conditions of load. Proteins were suirable buffer containing 1M NaCl (pH 5.5). Linked mass was measured by spectrophotometry at A. Then the column was subjected to desorption by using buffer containing 1M NaCl and urea. Spectrophotometry, measured at A showed that after desorption was not observed significant binding mass. Up to about 25 g/l g binding capacity was observed for all the studied resins. Capto™ S and Toyopearl® Gigacap S-650M showed relatively weak binding, a Fractogel® SO3-(M) and Poros® HS 50 showed a more durable binding. This study shows that the elution pH in very low conductivity.

Binding capacity SEH

CM was titrated to pH 4.0 and diluted with 3:1 Rodi water (3 parts of CM was added to 1 part Rodi) to 0.75 full dilution (ratio 8 MS/cm). Binding capacity of Capto™ S, Toyopearl® Gigacap S-650M, Poros® HS 50 and Fractogel® S03-(M) per�oncale evaluated against the target binding capacity of more than 40 g/l, using a 2 ml column (0.5 cm × 10 cm). Binding capacity of Capto™ S, Toyopearl® Gigacap S-650M and Fractogel® SO3-(M) were additionally determined in different conditions in the pH range from 4.0 to 4.3. For example, the binding capacity for Capto™ S was measured at pH of 4.3, 9 MS/cm; pH 4,0, 11 MS/cm, pH of 4.0, 9 MS/cm, and pH of 4.1, 8 MS/cm, respectively. Binding capacity for Toyopearl® Gigacap S-650M was measured at pH of 4.3, 9 MS/cm and pH of 4.1, 8 MS/cm Binding capacity for Fractogel® SO3-(M) was measured at pH of 4.3, 12 MS/cm; pH of 4.3, 9 MS/cm; pH 4,0, 12 MS/cm, pH of 4.1, 9 MS/cm and pH of 4.0, 9 MS/cm, respectively. The results showed that Fractogel® SO3-(M) can be used without dilution CM water. Without dilution Fractogel® SO3-(M) showed good binding capacity at a pH of from 4.0 to 4.3 (DBC range: from 25 to more than 40 g/l g). With 23% dilution Fractogel® SO3-(M) showed excellent capacity at pH from 4.0 to 4.3 (DBC range: 40 to more than 50 g/l d).

Approach SEH elution

Gradient elution was performed as described in the experiments for SEH binding capacity. Citrate buffer was used for pH elution. Tested Capto™ S, Toyopearl® Gigacap S-650M, Fractogel® S03-(M) and Poros® HS 50. The elution from the size exclusion chromatography (SEC) showed a high purity of ATN-103 and the low level of HMW and LMW samples. The degree of purity is at least comparable to protein A- - eluted substances. HTS (high-performance, high-throughput screening) screening can be done using the information obtained�th of gradient elution.

Example 6: Ultrafiltration/diafiltration to a high concentration and the drug

Example 6.1: Ultrafiltration/diafiltration to a high concentration and the drug

Stage UF/diafiltration (clipping MW 10 kDa) was used for the concentration and replacement of the buffer in the VRF product at the buffer of the drug.

After equilibration of the membrane module boot the solution is first concentrated to a predetermined target volume, and then subjected to diafiltration with 28 mm histidinol buffer, pH of 5.8. After further concentration to about 200 g/l pool is removed from the system by washing histidinol buffer to achieve a final concentration of target protein of approximately 150 g/l. Small volume (17,6% vol./about.) concentrated stock solution (20 mm histidine, 50% sucrose and 0,0667% Polysorbate 80) was added to the pool of product. Received final DS represents 80 g/l ATN-103 in 10 mm histidine, pH 6.0, 5% sucrose, and 0.01% PS80. Received end-DS is a 125 g/l ATN-103 in 20 mm histidine, pH 6.0, and 7.5% sucrose, 0.01% Polysorbate.

Example 6.2: Ultrafiltration/diafiltration to high concentrations

Stage UF/diafiltration (clipping MW 10 kDa) was used for the concentration and replacement of the buffer in the VRF product at the buffer of the drug.

After equilibration of the membrane moduledirectory the solution is first concentrated to a preset target amount of 40 g/l and then subjected to diafiltration with 30 mm histidinol buffer, pH of 5.8, containing 8.5% of sucrose. After further concentration to about 300 g/l pool was extracted from the system buffer for diafiltration to achieve a final concentration of target protein of approximately 175 g/L.

Example 6.3: Ultrafiltration/diafiltration to a high concentration and the drug

Stage UF/diafiltration (clipping MW 10 kDa) was used for the concentration and replacement of the buffer in the VRF product at the buffer of the drug.

After equilibration of the membrane module boot the solution is first concentrated to a preset target amount of 40 g/l and then subjected to diafiltration with 30 mm histidinol buffer, pH of 5.8. After further concentration to about 320 g/l pool was extracted from the system buffer for diafiltration to achieve a final concentration of target protein of about 210 g/L.

If the target DS was at least 200 g/l, a combination of Example 6.2 and Example 6.3 can be implemented as follows: stage UF/diafiltration (clipping MW 10 kDa) can be used for concentrating and replace the buffer in the VRF product at the buffer of the drug. After equilibration of the membrane module boot the first solution can be concentrated to a preset target amount of 40 g/l and then expose diafiltration with histidine-Zahorodny� buffer. After further concentration to about 320 g/l pool can be removed from the system buffer for diafiltration to achieve a final concentration of target protein not less than approximately 200 g/l, where sucrose is already present. Then the peak of the drug would be of 1.01%./vol., thus, UF (citratecitrate) pool will not be significantly diluted and end DS will be at least 200 g/L.

Equivalents

All references cited herein, incorporated herein by reference in its entirety and for all purposes to the same extent as if each individual publication or patent or patent application were specifically and separately identified for inclusion by reference in its entirety for all purposes.

The present invention should not be limited to the specific embodiments described in this application. Indeed, various modifications of the invention in addition to described in this application will be clear to experts in the art from the foregoing description and accompanying graphic materials. It is assumed that such modifications fall under the scope of the attached claims.

1. A method of separating or purifying single-domain antigen-binding (SDAB) molecules having SEQ ID NO:1, from a mixture containing the SDAB molecule and one or more load�znajomych substances including:
bringing the mixture into contact with a cation-exchange medium under conditions that allow the SDAB molecule to contact the media or be absorbed on the media;
removing one or more contaminants; and
selective elution of the SDAB molecule from the media,
where the conductivity of the medium for conditioning (CM) used for loading media varies from approximately 12 to 9 MS/cm and pH in the load conditions adjusted to a value from 4.0 to 4.3,
where a buffer for elution corresponds to about 50 mm sodium chloride or less and has a pH from about 5.5 to 7.2.

2. A method according to claim 1, wherein the pH in the correct load conditions to a value equal to approximately 4,3; 4,2; 4.1 or 4.

3. A method according to claim 1, wherein the stage of removing one or more contaminants includes flushing bound media in conditions when the SDAB molecule remains associated with the media.

4. A method according to claim 1, wherein the cation exchange medium is a cation exchange column.

5. A method according to claim 1, including:
bringing a mixture containing the SDAB molecule and one or more contaminants into contact with a cation-exchange (SEH) resin, where SEH resin demonstrates the capacity of at least about 40 g/l;
ensuring the flow of SDAB molecules through the media, washing media, at least one buffer for washing Latinoamerica; and elution of the SDAB molecules of the buffer for elution.

6. A method according to claim 1, wherein the contaminants in the mixture contain one or more of the high molecular protein aggregates, protein, host cell or DNA.

7. A method according to claim 1, wherein the SDAB molecule is purified to at least 90% purity or higher.

8. A method according to claim 1, further comprising one or more of: hydroxyapatite chromatography, cation exchange chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration, viral inactivation or filtration to remove viruses.

9. A method according to claim 8, including bringing the mixture into contact with a hydroxyapatite resin and selective elution of the SDAB molecule from the hydroxyapatite resin.

10. A method according to claim 9, comprising pre-processing the mixture of equilibrating buffer and ensuring the flow of pre-treated mixture through a hydroxyapatite resin.

11. A method according to claim 9, where the stage of bringing the mixture into contact with a hydroxyapatite resin and elution stage is carried out in buffer solutions containing from about 1 to 20 mm sodium phosphate and from about 0.2 to 2.5 Μ sodium chloride, at pH from about 6.4 to about 7.6.

12. A method according to claim 10, wherein equilibrating buffer contains from about 1 to 20 mm sodium phosphate from about 0.01 to 2.0 Μ sodium chloride, from about 0 to 200 mm of arginine, from about 0 to 200 mm HEPES, at a pH of from about 6.2 to 8.0.

13. A method according to claim 1, wherein the purified SDAB molecule contains less than 10% high molecular weight aggregates.

14. Method or process of obtaining recombinant single-domain antigen-binding (SDAB) molecules having SEQ ID NO:1, wherein:
take a host cell of a mammal, containing a nucleic acid which encodes the recombinant SDAB molecule;
support a host cell under conditions in which the expressed recombinant SDAB molecule;
a mixture of recombinant SDAB molecule and one or more contaminants;
purified or isolated recombinant SDAB molecule from the specified mix using cation exchange chromatography,
where the specified stage of purification or selection includes bringing the mixture into contact with the medium under conditions that allow the SDAB molecule to contact or be absorbed by the carrier; and
where the conductivity of the medium for conditioning (CM) used for loading media varies from approximately 12 to 9 MS/cm and pH in the load conditions adjusted to a value from 4.0 to 4.3;
removing one or more contaminants; and
selective elution of the SDAB molecule from the media, whereby the drug-eluted SDAB molecule,
where a buffer for elution corresponds to PR�mately 50 mm sodium chloride or less and has a pH from about 5.5 to 7.2.

15. A method according to claim 14, in which the drug-eluted SDAB molecule optionally subjected to one or more of: hydroxyapatite chromatography, affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, metal affinity chromatography, diafiltration, ultrafiltration or filtration to remove viruses.

16. A method according to claim 14, further comprising a preparation of the recombinant SDAB molecule in the form of pharmaceutical compositions.

17. A method according to any one of claims. 1-16, further comprising a concentration-eluted SDAB molecule to a pre-selected target volume.

18. A method according to claim 17, where the step of concentrating is carried out by performing stage UF/diafiltration in the presence histidinemia buffer or Tris buffer.

19. A method according to any one of claims. 1-16 or 18, where the SDAB molecule is concentrated to at least about 80 g/l - 350 g/L.



 

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22 cl, 5 dwg, 11 tbl

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