Method for promotion of active conformation of glycosylated recombinant protein, method for preparing active glycosylated protein and method for preparing composition of this protein for administration to user and/or patient or for using by user and/or patient
FIELD: medicine, biotechnology, pharmaceutical industry.
SUBSTANCE: method involves increasing part of the most active conformation of a glycosylated recombinant protein secreted by mammalian cell by its contact with a reagent for coupled oxidation-reduction. The proposed promotion method of the most active conformation of protein is used in a method for preparing a glycosylated recombinant protein in its the most active conformation. Configuration isomer of protein prepared by the indicated method for preparing a glycosylated recombinant protein in it's the most active conformation used in a method for preparing the protein composition for its administration to user and/or a patient or for consumption by user and/or patient. Using of the proposed invention provides enhancing activity of glycosylated protein prepared by a method of recombinant DNAs using a mammalian cell.
EFFECT: improved preparing method, valuable properties of protein.
27 cl, 9 dwg, 2 tbl, 3 ex
This application claims the benefits of provisional application U.S. 60/271033, filed February 23, 2001, the description of which is incorporated in its entirety by reference.
The technical FIELD TO WHICH THIS INVENTION
This invention relates to the field of medicine and biotechnology, in particular to the field of processing and producing proteins with the desired biologically active conformation, as well as the preparation of the formulations and pharmaceutical compositions.
The LEVEL of TECHNOLOGY
High levels of expression of many proteins of eukaryotic origin were achieved in prokaryotic expression hosts. Such eukaryotic proteins often incorrectly stacked and accumulated in the form of insoluble Taurus inclusion in the prokaryotic host. For the biologically active protein proteins enclosed in inclusion bodies, must be deployed and re-laid under severe conditions, including chaotrope agents and restoration of thiols.
Expression of proteins of eukaryotic origin in eukaryotic hosts avoids these problems. Provided that expressing vector was constructed correctly (for example, with secretory signal peptides and so on), the line of eukaryotic cells tend to correctly processional and secrete extracellular eUK is roticheskie proteins in the form of soluble products.
However, while expression systems and vectors have been improved to maximize levels of expression in eukaryotic cells, not the entire recombinant protein expressed and the secretory of these owners is desirable, the most active conformation. This invention is intended to overcome such problems of expression and maximize outputs of biologically active protein.
This invention is based in part on the discovery that not all of the recombinant protein, which is expressed in eukaryotic cells masters laid the native tertiary conformation. In addition, it was found that the areas or domains of recombinant proteins can be properly laid, whereas other areas or domains may have an undesirable conformation. Thus, in one aspect, the invention provides a method for contacting the recombinant protein, which contains a mixture of at least two isomers of the recombinant protein conjugate reagent for oxidation-reduction in a period of time sufficient to increase the relative proportion of the desired conformational isomer, and determine the relative proportion of the desired conformational isomer in the mixture. In another aspect, the invention describes kontaktirovanije of recombinant protein which was produced by mammalian cells with a conjugate reagent for oxidation-reduction at pH from about 7 to about 11 and the allocation fractions of the recombinant protein with the desired conformation. Preferred recombinant proteins are glycosylated recombinant proteins, such as, for example, proteins produced by eukaryotic cells. This invention relates also to a method of preparation in a sterile dosage form and the compositions obtained by the methods of this invention.
BRIEF DESCRIPTION of FIGURES
Figure 1. Hydrophobic chromatography (HIC) TNFR:Fc. This drug TNFR:Fc eluted during the HIC in the form of three separate peaks collected in fraction 2 and fraction No. 3 as specified.
Figure 2. Analysis of circular dichroism fractions # 2 and # 3. Measurement of circular dichroism in the near-UV region of the spectrum, expressed as the average ellipticity of the balance are shown in figure 2. Figure 2A represents the spectral data; the line for a fraction No. 3 is closest to that indicated by the arrow a negative shift at approximately 270 nm, attributed to the contribution of disulfide bonds, and the line for fractions of No. 2 is darker solid line. Figure 2B presents data in the form of a smoothed curve for fraction No. 2 (small dashed line) is the fraction No. 3 (large dashed line).
Figure 3. Determination of molecular weight in the on-line mode by using gel-filtration chromatography (SEC), with sequential detection in the ultraviolet (UV), light scattering (LS) and refractive index (RI) (On-line SEC/UV7LS/RI). Figure AT is a fraction No. 3, and figure 3B - fraction No. 2. Vertical dotted lines indicate the sections that were evaluated for the determination of molecular weight in the neighborhood surrounding the main peak.
Figure 4. Differential scanning calorimetric analysis of fractions # 2 and # 3. Figure 4A represents the unadjusted data, and figure 4B is adjusted in relation to the background data. Thermal melting transitions marked by the vertical dashed lines. Arrows indicate the shift of enthalpy. The horizontal dotted lines in figure 4B is used as the reference background level.
Figure 5. Correlation of fraction No. 2 and binding activity. Six different drugs TNFR:Fc (denoted A-F) from six different cell lines was tested for correlation between the increase in the proportion of fraction No. 2 in percent (dark diamonds) and the increase in units of the binding of TNF-alpha in percent (white diamonds).
Figure 6. The effect of the concentration of cysteine on the conversion of fraction No. 3 in fraction No. 2. The protein samples were treated with various concentrations of cysteine (0.25 to 5.0 mm) and changes in FR is work No. 3 was estimated using HIC. Four different parties TNFR:Fc were treated for 18 hours with the indicated on the x-axis the concentration of cysteine. The percentage of fraction No. 3 in every game, turned in fraction No. 2, shown on the y-axis.
Figure 7. The effect of the concentration of cysteine in the proportion of fraction No. 3. The protein samples from four different batches were treated with various concentrations of cysteine (0-50 mm) and the resulting level of fraction # 3 was estimated using HIC.
Figure 8. The effect of temperature on disulfide exchange. Protein fractions were processed at room temperature or at 4°in the presence or in the absence of copper during different periods of time. Figure 8A presents the changes in fraction No. 3 according to the results of HIC after 6 hours, and figure 8B presents the changes in fraction No. 3 according to the results of HIC after 18 hours.
INFORMATION CONFIRMING the POSSIBILITY of carrying out the INVENTION
This invention provides methods of increasing output active recombinant proteins. In particular, this invention includes the promotion of desirable conformation of the protein in the recombinant protein. The important point is that the invention provides a soft ways of changing the structure of the protein without the necessity of applying hard chaotropic treatments (such as, for example, a strong denaturing agents, such as LTO, guanidinium the reed or urea). Using the methods of the present invention the recombinant protein leads to a higher percentage, or a larger relative fraction of the recombinant protein in the product with the desired conformation. Desirable conformation for recombinant protein is the three-dimensional structure of the protein that is most similar to the structure and/or duplicates the functionality of the natural domain of this protein. These soft methods are particularly useful when a recombinant protein is suitable for use in vivo as a drug or biological product.
Generally, when a recombinant protein contains the domain of the receptor protein, it is desirable conformation will have a higher binding affinity of (and, hence, a lower dissociation constant) in relation to cognate ligand of this receptor. For example, the desired conformation of TNF-binding molecules will have a higher affinity binding and a lower dissociation constant in relation to TNF (e.g., TNF-alpha).
In addition, the desired conformation of the recombinant protein preferably is more thermostable than undesirable conformation (when measured in terms of the same solution). thermal stability can be measured by any method, such as, for example, the transition at the melting point (Tm). Desire is part conformation of the recombinant protein may or may not have a great location disulfide bonds, although preferably this conformation contains native disulfide bonds. The desired conformation of the recombinant protein may have other characteristics of the tertiary structure. For example, it is desirable conformation may be a monomer, dimer, trimer, tetramer, or some other form of protein higher order. For the purposes of this invention "conformation" of a protein is its three-dimensional structure. Two different structures of the polypeptide with the same primary amino acid sequence are "conformers" each other when they have different conformations, corresponding to the minima of the energy, and they differ from each other only in how their atoms are oriented in space. The conformers can be vzaimoprevrascheny (with regard to the freedom of rotation around the links except break ties). Two different structures of the polypeptide with the same primary amino acid sequence are "configuration isomers", when they have different conformations, corresponding to the minima of the energy, they differ from each other in how their atoms are oriented in space, and they are neusamariabrasil without breaking covalent bonds. In the practice of this invention configurational isomers can be vzaimoprevrascheny, for example, through R is sriva and, if necessary, re-formation of disulfide bonds.
Thus, the invention provides for contacting the glycosylated recombinant protein, which contains a mixture of at least two configurational isomers of this recombinant protein, conjugate reagent for oxidation-reduction over a period of time sufficient to increase the relative proportion of the desired configurational isomer, and determining the relative proportion of the desired configurational isomer in the mixture. In another aspect, the invention provides for the contacting of the recombinant protein, which was produced by mammalian cells with a conjugate reagent for oxidation-reduction at pH from about 7 to about 11 and the allocation of a fraction of the preparation of this recombinant protein with the desired conformation. Preferred recombinant proteins are glycosylated recombinant proteins, such as, for example, proteins produced by eukaryotic cells.
This invention can be used to handle almost any protein for promotion of desirable conformation. The term "protein" usually meant a polypeptide of at least 10 amino acids, more preferably at least about 25 amino acids, more preferably at least priblizitelen the 75 amino acids and most preferably at least about 100 amino acids. The methods of this invention find particular application in the processing of proteins that have at least about 3 cysteine residue, more preferably at least about 8 cysteine residues, even more preferably at least about 15 cysteine residues, even more preferably at least about 30, and even more preferably at least approximately 50-150 cysteine residues.
Typically, the methods of the present invention is applicable to improve methods of production of recombinant proteins. Recombinant proteins are proteins produced by genetic engineering. The term "genetic engineering" refers to any method of recombinante DNA or RNA used to generate a host cell that expresses the gene at high levels, at low levels and/or produces a mutant form of this gene. In other words, this cell was transliterowany, transformed or transducible recombinant polynucleotide molecule and thereby modified so as to cause the cell to change the expression of the desired protein. Method and vectors for genetically engineered cells and/or cell lines for the expression of protein of interest are well known to specialists in this field the tee; for example, numerous methods are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988 and quarterly updates) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, 1989). The methods of genetic engineering include, but are not limited to, expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Patent No. 5272071 issued by the Chapel) and TRANS-activation of genes engineered transcription factors (see, for example, Segal et al., 1999, Proc. Natl. Acad. Sci. USA 96(6): 2758-63).
The invention finds special application in improving the production of proteins that are glycosylated. Specifically proteins that are secreted systems on the basis of the fungal cell (e.g., yeast, filamentous fungi) and systems based on mammalian cell, will be glycosylated. Preferably, these proteins are secreted production by mammalian cells adapted to growth in cell culture. Examples of such cells commonly used in the industry, the cells are Cho, VERO, BHK, HeLa, V1 (including Cos), MDCK, 293, ZTZ, lines of myeloma cells (in particular, mouse), PC 12, and WI38. Particularly preferred cells host cells are Chinese hamster ovary (Cho), which are widely used to produce more complex recombinant proteins, for example, is Fokino, clotting factors and antibodies (Brasel et al., 1996, Blood 88:2004-2012; Kaufinan et al., 1988, J Biol Chem 263:6352-6362; McKinnon et al., 1991, J Mol Endocrinol 6:231-239; Wood et al., 1990, J Immunol 145:3011-3016). Insufficient dihydrotetrazolo (DHFR) mutant cell line (Uriaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44 are the preferred cell lines-owners SNO as effective DHFR-breeding and amplificatory the expression system allows high level expression of recombinant protein in these cells (Kaufinan R.J., 1990 Meth En2ymol 185:527-566). In addition, these cells are easy to handle as attached or suspension cultures, and they are relatively good genetic stability. Cells SNO and expressed them in recombinant proteins were thoroughly characterized and approved for use in clinical production of regulatory agencies.
It was found that the present invention is a gentle and effective way to improve the method of obtaining proteins that can adopt multiple conformations and/or contain more than one domain. "Domain" is a continuous region of the polypeptide chain, which adopts a defined tertiary structure and/or has a certain activity, which can be localized in this region of the polypeptide chain. For example, one domain of the protein may have an affinity svyazyvanie is in one ligand and one domain of the protein may have a binding affinity of another ligand. In terms of thermal stability domain can be called a unit of the cooperative unfolding of the protein. Such proteins that contain more than one domain that can occur in nature in the form of one protein or can be genetically engineered in the form of a fused protein. In addition, the domains of the polypeptide can have sub domains.
In one aspect, the methods of this invention can be used in preparations of recombinant proteins in which at least one domain of the protein is stable conformation and at least one domain of the protein is unstable conformation. The terms "stable" and "unstable" are used in comparative terms. Domain protein with a stable conformation will have, for example, a higher melting temperature (Tm)than the unstable domain of this protein when measured in the same solution. The domain is stable in comparison with another domain, when the difference in Tm is at least about 2°C, more preferably about 4°S, even more preferably about 7°S, even more preferably about 10°S, even more preferably about 15°S, even more preferably about 20°S, even more preferably about 25°and most preferably priblisitelno° Since when measured in the same solution.
This invention is also generally applicable to proteins that have a Fc-domain and another domain (e.g., antibodies and Fc-fused proteins). For example, in one non-restrictive embodiments, illustrated below, are related to TNFR:Fc, Tm for the Fc part of the molecule is of 83.4 69.1 and°whereas Tm for TNFR-part of this molecule is in the range from 52,5°With (more desirable conformation) to Tm 49,7°With (less desirable conformation).
Particularly preferred proteins are drugs based on proteins, also known as biologics. Preferably, these proteins are expressed in the form of extracellular products. Proteins that can be obtained using the methods of this invention include, but are not limited to, flt3-ligand (described in WO 94/28391, which is incorporated herein in its entirety by reference), CD40-ligand (described in US6087329, which is incorporated herein in its entirety by reference), erythropoietin, thrombopoietin, calcitonin, Fas ligand, ligand for receptor activator of NF-Kappa b (RANKL), related to the tumor necrosis factor (TNF) induce apoptosis ligand (TRAIL described in WO 97/01633, included here in its entirety by reference), derived from the stroma of the thymus lymphopoietin, granulocyte colony-stimulating factor, granulocyte-mA is rafaelly colony-stimulating factor (GM-CSF, described in Australian patent No. 588819, which is incorporated herein in full by reference), a growth factor, mast cell growth factor, stem cells, epidermal growth factor, RANTES (cytokine subfamily, produced by T-cells), growth hormone, insulin, insulinotropic, insulin-like growth factors, parathyroid hormone, interferons, nerve growth factors, glucagon, interleukins 1 through 18, colony stimulating factors, lymphotoxin-R, tumor necrosis factor (TNF), inhibitory factor leukemia, oncostatin-M, and various ligands for molecules the cell surface ELK and Hek (such as the ligands for eph-related kinases (proteincontaining) or LERK.S). Descriptions of proteins that can be purified in accordance with the methods of the present invention may be found, for example, Human Cytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, MA, 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993); and The Cytokines Handbook (A.W. Thompson, ed.. Academic Press, San Diego, CA, 1991).
Drugs receptors, in particular the soluble forms of these receptors, for any of the aforementioned proteins can also be improved using the methods of the present invention, including both forms of TNFR (referred to as P55 and P75)receptors type I and type II interleukin-1 (as described in EP 0460846, US 4968607 and US 5767064, which are incorporated herein in their entirety by reference) receptor, interleukin-2, receptor interleukin-4 (as described in EP 0367566 and US 5856296, which are incorporated herein in their entirety by reference), receptor, interleukin-15, receptor, interleukin-17, receptor, interleukin-18 receptor, granulocyte-macrophage colony-stimulating factor receptor, granulocyte colony-stimulating factor, receptors for oncostatin-M and inhibitory factor leukemia, receptor activator of NF-Kappa b (RANK, described in US 6271349, incorporated herein in its entirety by reference), receptors for TRAIL (including receptors 1, 2, 3 and 4 TRAIL), and receptors that contain death domains, such as Fas-receptor or apoptose-inducing receptor (AIR).
Other proteins synthesis method which can be improved using the methods of this invention include cluster of differentiation antigens (referred to as CD proteins), for example, described in Leukocyte Typing VI (Proceedings of VIth International Workshop and Conference; Kixhimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD molecules disclosed in the subsequent workshops. Examples of such molecules include CD27, CD30, CD39, CD40 and ligands thereto (CD27 ligand, CD0-ligand and CD40-ligand). Some of them are members of the family of TNF receptors, which also includes W and H; these ligands are often members of the TNF family (for example, the ligand VW and ligand H); accordingly, members of the families of TNF and TNFR m which may be obtained from the use of this invention.
Proteins that are enzymatically active, can also be obtained in accordance with this invention. Examples include members of the family of metalloproteinases-disintegrin (destructive integrins), various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I, the antagonist of IL-2, alpha-1-antitripsin, TNF-α-converting enzyme and numerous other enzymes. Ligands for enzymatically active proteins can also be expressed using the present invention.
Compositions and methods of the present invention is also applicable to other types of recombinant proteins, including molecules of immunoglobulins or parts thereof, and chimeric antibodies (for example, antibodies having a constant region of a person associated with antigennegative site mouse) or their fragments. There are many ways in which you can manipulate with DNA encoding the immunoglobulin molecule, to obtain DNA that can encode recombinant proteins, such as single-chain antibodies, antibodies with high affinity or other polypeptides on the basis of antibodies (see, for example, Larrick et al., 1989, Biotechnology 7:934-938; Reichmann et al., 1988, Nature 332:323-327; Roberts et al., 1987, Nature 328:731-734; Verhoeyen et al., 1988, Science 239:1534-1536; Director et al., 1989, Nature339:394-397). Preparations are fully human antibodies (such as preparations produced using transgenic animals, and, if necessary, then modified in vitro), as well as humanized antibodies can also be used in this invention. The term humanitariannet antibody also includes single-chain antibodies, See, for example, Cabilly et al., U.S. Pat. No. 4816567; Cabilly et al., European Patent No. 0125023 Bl; Boss et al., U.S. Pat. No. 4816397; Boss et al., European Patent No. 0120694 Bl; Neuberger, M.S. et al., WO 86/1533; Neuberger, M.S. et al., European Patent No. 0194276 B1; Winter, U.S. Patent No. 5225539; Winter, European Patent No. 0239400 Bl; Queen et al., European Patent No. 0451216 Bl and Padlan, E.A. et al., EP 0519596 Al. The method of this invention can also be used while obtaining conjugates containing the antibody and cytotoxic or luminescent substance. Such substances include: derivatives of maytansine (such as DM1); enterotoxins (such as staphylococcal enterotoxin); isotopes of iodine such as iodine-125); isotopes of technetium (such as TC-t); cyanine fluorochromes (such as Cu); and ribosome inactivating proteins (such as Bugarin, gelonin or saporin-86).
Examples of antibodies or conjugates of the antibody/cytotoxin or antibody/phosphor provided by the present invention include antibodies and conjugates, which recognize any of the above-mentioned protein or a combination of the above-mentioned proteins and/or following antigen is: CD2, CD3, CD4, CD8, CDlla, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (V), CD86 (V), CD147, IL-ld. IL-lβ, IL-4, IL-5, IL-8, IL-10 receptor, IL-2, IL-4 receptor, the receptor for IL-6, IL-13 receptor, receptor subunit, IL-18, PDGF-p, VEGF, TGF, TGF-p2, TGF-pl, EGF receptor, VEGF receptor, 5-complement, IgE, tumor antigen CA, a tumor antigen MUC1, antigen REM, LCG (which is the product of the gene, which is expressed in lung cancer), HER-2 is associated with tumor glycoprotein TAG-72 antigen SK-1, associated with tumor epitopes that are present in elevated amounts in the sera of patients with colon cancer and/or pancreatic cancer associated with cancer epitopes or proteins expressed on cancer cells of the breast, colon, flat cells, cancer cells of the prostate, pancreas, lung and/or kidney and/or on melanoma cells, glioma, or neuroblastoma, the necrotic core of the tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, the adhesion molecule VAP-1, an adhesion molecule of epithelial cells (Ersam), molecule (factor) intercellular adhesion 3 (ICAM-3), adhesin lacondeguy, the platelet glycoprotein gp IIb/IIIa, a heavy chain myosin heart, parathyroid hormone, rNAPc2 (which is an inhibitor of tissue factor Vila), MHC I, cancer-embryonic antigen (CEA), alpha fetoprotein (AP), the tumor necrosis factor (TNF), CTLA-4 (which is associated with cytotoxic T-lymphocyte antigen), Fc receptor-γ-1, HLA-DR 10 beta, HLA antigen-DR, L-selectin, IFN-y, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis b virus (HBV), Streptococcus mutans and Staphylococcus aureus.
Using the methods of this invention can be obtained preparations of various fused proteins. Examples of such fused proteins include proteins, expressed in the form merged with part of the molecule of the immunoglobulin proteins, proteins, expressed in the form of a merged component type "zipper" (zipper) protein, and the new multifunctional proteins, such as fused proteins of the cytokine and growth factor (i.e. GM-CSF and IL-3, MGF and IL-3). In WO 93/08207 and WO 96/40918 described for various soluble oligomeric forms of the molecule, called CD40L, which includes fused with the immunoglobulin protein and merged with the "zipper" protein, respectively; methods discussed here are applicable to other proteins. Any of the above molecules can be expressed in the form of a fused protein, including, but not limited to, the extracellular domain of a cellular receptor molecule, an enzyme, a hormone, a cytokine, a part of the molecules of the immunoglobulin domain "zipper" and the epitope.
The recombinant protein may be a supernatant of cleoc the second culture, extract cells, but preferably partially purified fraction of them. Under "partially purified" mean there were some procedure or some fractionation procedure, but there are additional types of polypeptides (at least 10%), other than the desired protein or desirable conformation of the protein. One advantage of the methods of the present invention is that the recombinant protein can be relatively high concentration. Preferred concentration ranges are 0.1-20 mg/ml, more preferably from 0.5 to 15 mg/ml and even more preferably from 1 to 10 mg/ml
The recombinant protein may be initially obtained by culturing recombinant host cells in culture conditions suitable for the expression of this polypeptide. The polypeptide may also be expressed as a product of transgenic animals, for example in the form of a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the polypeptide. Then, the resulting expressed polypeptide may be purified or partially purified from such culture or its component (e.g., from culture medium or extracts of cells or liquid body is ISM) using known methods. The fractionation procedure may include, but are not limited to, one or more stages of filtration, centrifugation, sedimentation, phase separation, affinity purification, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography (HIC; using such resins as simple phenyl ether, butyl ether, or propyl ether), HPLC, or any combination of the above.
For example, purification of the polypeptide can include an affinity column containing agents which will contact with the polypeptide; one or more column stages with the use of such affinity resins as concanavalin a-agarose, heparin-toyopearl®or Cibacron blue, sea-Sepharose®; one or several stages, including elution; and/or immunoaffinity chromatography.
The polypeptide can be expressed in a form that facilitates cleaning. For example, it can be expressed in the form of a fused polypeptide such as a fusion polypeptide with malesurvivor a polypeptide, a glutathione-8-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of the fused polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The polypeptide may be labeled epitope and then cleaned with specificity the ski antibodies aimed at such epitopes this epitope (FLAG®) is commercially available from Kodak (New Haven, Conn.). You can also use affinity column containing polypeptidesee polypeptide, for example a monoclonal antibody to this recombinant protein for affinity purification of expressed polypeptides. Other types of affinity purification can be protein a - or protein G-column, affinity agents which bind to proteins containing the Fc domains. The polypeptides can be removed from the affinity column using conventional methods, for example, in high salt buffer for elution, and then cialisbuy against nizkosoleva buffer for use or by changing pH or other components depending on the affinity matrix, or can be competitive removed using the natural substrate of the affine component. In one embodiment of the present invention, illustrated below, the recombinant protein was partially purified by protein a-affinity column.
Some or all of the previous stages of purification in various combinations can be used to produce a suitable preparation of recombinant protein for use in the methods of the present invention and/or for additional purification of the recombinant polypeptide after contacting preparationincome protein conjugate reagent for oxidation-reduction. The polypeptide which is essentially free of other mammalian polypeptides, is defined as an "isolated polypeptide".
The polypeptide may also be obtained well-known conventional chemical synthesis. The methods of constructing polypeptides by synthetic methods known to experts in this field. Synthetically engineered polypeptide sequence can be glycosylated in vitro.
The desired final degree of purification depends on the intended application of this polypeptide. For example, a relatively high degree of purity is desired, when the polypeptide should be administered in vivo. In such case, the polypeptide is cleaned so that no protein bands corresponding to other polypeptides that are not detected in the analysis of electrophoresis in LTO-polyacrylamide gel (LTOs-SDS page). Specialist in the appropriate field will be clear that multiple bands corresponding to that polypeptide, may be visualized during electrophoresis LTO-page, due to differential glycosylation, differential post-translational processing and the like, More preferably the polypeptide of the present invention purified to substantial homogeneity, as a single polypeptide band upon analysis by electrophoresis in DS is-PAG. This polypeptide band can be visualized by staining with silver, Kumasi blue and/or (if the polypeptide is radioactively labeled) autoradiographies.
Under "contacts" mean exposure, and/or the exposure step in the solution. The protein or polypeptide may also be subjected to contact, when it is bound to a solid carrier (e.g., affinity column or chromatographic matrix). Preferably this solution is buffered. To maximize the yield of protein with the desired conformation of the pH of the solution is chosen to protect the stability of this protein and the optimal disulfide exchange. In the practice of this invention the pH of the solution preferably is not strongly acidic. Thus, the preferred pH ranges are greater than pH 5, preferably from about pH 6 to about pH 11, more preferably from about pH 7 to about pH 10 and even more preferably from about pH 7.6 to about a pH of 9.6. In one non-restrictive embodiment of the present invention using TNFR:Fc, which is illustrated below, it was found that the optimum pH should be around pH to 8.6. However, the optimal pH for a specific embodiment of the present invention can easily be determined experimentally by specialists in this field.
agent for paired oxidation-reduction is a source of reducing agents. Preferred reducing agents are free thiols (SH). The conjugate reagent for oxidation-reduction preferably includes a compound of the group consisting of reduced and oxidized glutathione, dithiothreitol (DTT), 2-mercaptoethanol, getinitialstate, cysteine and cystine. For ease of use and economy can be used restored glutathione and/or re-cysteine.
The conjugate reagent for oxidation-reduction is present in sufficient concentration to increase the relative proportion of the desired conformation. The optimal concentration of the conjugate reagent for oxidation-reduction depends on the concentration of protein and the number of disulfide bonds in a given protein. For example, it was found using protein (TNFR:Fc) 29 disulfide bonds at a concentration of 2 mg/ml (approximately 14 μm protein or 400 μm disulfide)that the conjugate reagent for oxidation-reduction with 2 mm restored thiol worked well, increasing the relative proportion of the desired conformation. This corresponds to a ratio of approximately 35 free thiols on 1 disulfide bonds. However, it was also found that the relationship between 20 and 400 free thiols to disulfide also worked well. Of course, the number of thiol used in the particular to the centration, may slightly vary depending on restoring the ability of the thiol and can be easily determined by the expert in this field.
Thus, typically, the concentration of free thiols of conjugate reagent for oxidation-reduction can be from about 0.05 to about 50 mm, more preferably approximately 0.1 to approximately 25 mm and even more preferably approximately 0.2 to approximately 20 mm.
In addition, the conjugate reagent for oxidation-reduction may contain oxidized thiols at a higher, equal or lower concentrations in comparison with the restored thiol component. For example, the conjugate reagent for oxidation-reduction can be a combination of reduced glutathione and oxidized glutathione. Through real business examples, it was found that the ratio of reduced glutathione to oxidized glutathione from about 1:1 to approximately 100:1 (reduced thiols:oxidized thiols) can work equally well. In another alternative embodiment, the conjugate reagent for oxidation-reduction may be a cysteine or a combination of cysteine and cystine. Thus, with the inclusion of oxidized thiols in the original conjugate reagent for oxidation-reduction attributed the e thiols to oxidized thiols may be in a preferred embodiment, from about 1:10 to about 1000:1, more preferably about 1:1 to about 500:1, more preferably about 5:1-100:1, even more preferably about 10:1.
The contacting of the recombinant protein conjugate reagent for oxidation-reduction is performed in a period of time sufficient to increase the relative proportion of the desired conformation. Any relative increase in this proportion is desired, but preferably at least 10% protein with undesirable conformation turns into a protein with the desired conformation. More preferably at least 20, 30, 40, 50, 60, 70 and even 80% of this protein make out of junk in the desired conformation. Typical outputs that have been achieved by the methods of the present invention were in the range from 40 to 80%. If the phase of the probe carried out on partially purified or highly purified preparation of recombinant protein, the stage of contacting can be performed within a short period of time as about 1 hours, about 4 hours, and during such a long period of time, as approximately 6 hours to approximately 4 days. It was found that the phase of the probe approximately 4 hours, about 16 hours, or about 18 hours works well. Stage contact may also have the place during another stage, for example, on a solid phase or during filtration or during any other stage of treatment.
The methods of this invention can be performed in a wide temperature range. For example, the methods of the present invention has been successfully carried out at temperatures from about 4 to about 37°With, however, the best results were obtained at lower temperatures. A typical temperature for contacting partially or completely purified recombinant protein is the temperature of approximately 4 to about 25°C (ambient temperature), but communication can also be performed at lower temperatures and at higher temperatures.
The recombinant protein can be contacted with the conjugate reagent for oxidation-reduction in various amounts depending on feasibility. For example, the methods of the present invention carried out successfully in the analytical laboratory scale (1-50 ml), preparative scale (50 ml-10 l) and industrial scale (10 or more liters). Thus, the methods of this invention can be carried out in small scale and large scale reproducible.
In preferred aspects of stage contacting is performed in the absence of significant quantities of chaotropic Agay is tov, such as, for example, LTOs, urea and guanidine-HCl. A significant number chaotropic agents are required to observe tangible expander. Usually, there is less than 1 M chaotropic agent, more preferably less than 0.5 M, more preferably less than 0.1 M chaotropic agent. The solution is essentially free from chaotrope (for example, LTO, urea and guanidine-HCl), when chaotrope not added to this solution, and may contain only trace amounts (e.g. less than 10 mm) (for example, from the vessel or as cellular by-product).
Disulfide exchange can be stopped by any of the methods known to experts in this field. For example, the conjugate reagent for oxidation-reduction may be removed or its concentration can be reduced through the stage of purification, and/or it can be chemically inactivated, for example, by acidification of the solution. Usually, when the reaction is stopped by acidification, the pH of the solution containing the conjugate reagent for oxidation-reduction, will be reported below pH 7. Preferably the pH is adjusted to a value of less than pH 6. Typically, the pH is reduced to between about pH 2 and about rn.
Determination of the conformation of the protein and the relative degree of conformation of the protein in the mixture can be is performed using any of a variety of analytical and/or qualitative methods. If there is a difference in activity between the two conformations of the protein, determining the relative proportion of the conformation of the mixture can be performed using the analysis activity (e.g., binding to a ligand, enzyme activity, biological activity etc). For example, in one non-restrictive embodiments described below, at least two different conformations TNFR:Fc may be separated using solid-phase analysis of binding TNF. This analysis is basically the same as described for IL-1R (Slack, et al., 1993, J. Biol. Chem. 268:2513-2524), can distinguish between the relative shares of different conformations of the protein through changes received constants of Association, dissociation or inhibition of binding of the ligand-receptor. Alternative the results of the binding can be expressed as units of activity per mg of protein.
If these two conformations are separated in various ways during chromatography, electrophoresis, filtration or other purification techniques, the relative share of conformations in the mixture can be determined using such methods of cleaning. For example, in non-restrictive embodiments described below, at least two different conformations TNFR:Fc could be separated using a hydrophobic chromatography. Further, as for the calculation of the composition of the secondary structure Bel is s used circular dichroism in the far UV region of the spectrum (Perczel et al., 1991, Protein Engrg. 4:669-679), this method can determine whether alternative conformations of the protein. Another method used to determine the conformation is fluorescence spectroscopy, which can be used to identify additional differences in tertiary structure that may be associated with the fluorescence of tryptophan and tyrosine. Other methods that can be used to determine the differences in conformation and, consequently, the relative share of conformation, are SEC on-line for measuring the aggregation state, differential scanning calorimetry to measure the melting transitions (TM) and enthalpies of the components and chaotrope unwrapping.
The term "selection" mean a physical separation of at least one component of the mixture from the other components of the mixture. The separation of the components or a specific conformation of a protein can be achieved using any method of cleaning that has a tendency to share similar components. Thus, it is possible to carry out one or several steps of chromatography, including, but not limited to, HIC, hydroxyapatite chromatography, ion exchange chromatography, affinity chromatography and SEK. Other cleaning methods are filtering (for example, filtering with tangential flow), elektroforeticheskie ways (for example, electrophoresis, electrolyte, isoelectric focusing) and phase separation (for example, phase separation using PEG-dextran), to name some of them. In addition, the fraction of the recombinant protein, which contains protein in an undesirable conformation, can be processed again by the methods of the present invention to further optimize the outputs of the protein with the desired conformation.
For example, after processing, can be prepared protein samples for hydrophobic chromatography (HIC) as follows. Equal volume of 850 mm sodium citrate, 50 mm sodium phosphate, pH 6.5, added to the treated sample and give be balanced at room temperature. After filtration (for example, using a 0.22 μm filter) are HIC chromatography on Toyopearl resin®Butyl 650 M (Tosoh Biosep LLC, Montgomeryville, PA), at a speed of current of 150 cm/h and the load weight 2.1 mg per ml of resin. Column pre-balance 3 column volumes 425 mm sodium citrate, 50 mm phosphate, pH 6.5, put the sample and then the column was washed with 3 column volumes of 425 mm sodium citrate, 50 mm phosphate, pH 6.5. Elution can be performed with a gradient of 425 mm sodium citrate, 50 mm phosphate, pH 6.5 - 0 mm sodium citrate, 50 mm phosphate, pH 6.5) in a total volume equal to 5 column volumes. During the elution can be collected fractions. Column can be tmit 3 column volumes of water followed by washing with 3 column volumes of 0.1 M NaOH. Using the methods of the present invention can be thus obtained preparations TNFR:Fc, which contain more than 85, 90, and even more than 95% of TNFR:Fc present in this preparation, the most active conformation (fraction No. 2). Thus, compositions, including pharmaceutical compositions, TNFR:Fc containing such proportion of fraction No. 2, also provided by this invention.
The invention also optionally includes the formulation of these proteins. The term "formulation" mean that these proteins may be subjected to change buffer, sterilized, packaged in a gross form and/or packaged for the final consumer. For the purposes of this invention, the term "sterile gross form" means that this form is not containing or essentially does not contain microbial contamination (to a degree that is acceptable for food and/or medicinal purposes) and has a specific composition and concentration. The term "sterile dosage form" means a form that is suitable for introduction to the consumer and/or patient or consumption by the consumer and/or patient. Such compositions may contain an effective amount of the protein in combination with other components such as a physiologically acceptable diluent, carrier and/or excipient. Those who min "physiologically acceptable" means a non-toxic material, which does not prevent the effectiveness of the biological activity of the active ingredient (the active ingredients). Formulations suitable for administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostatic agents and dissolved substances that make the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspendresume agents or thickening agents. In addition, sterile gross forms and sterile dosage forms may contain a small concentration (approximately 1 to approximately 10 mm) of the conjugate reagent for oxidation-reduction (e.g., glutathione, cysteine, and so on). These polypeptides can be prepared in a formulation according to known methods to prepare pharmaceutically applicable compositions. They can be combined in a mixture, either as the only active substance or with other known active substances suitable for specific medical indications, with pharmaceutically acceptable diluents (e.g., saline, buffered Tris-HCl, acetate or phosphate solutions), preservatives (e.g. thimerosal, gasoline, alcohol, parabens), emulsifiers, soljubilizatorami, adjus nami and/or carriers. Suitable formulations of pharmaceutical compositions include formulations described in Remington's Pharmaceutical Sciences, 16thed. 1980, Mack Publishing Company, Easton, PA. In addition, such compositions can be in complex with polyethylene glycol (PEG), metal ions, and/or can be incorporated into polymeric compounds such as polixena acid, polyglycolic acid, hydrogels, dextran etc. or can be incorporated into liposomes, microemulsions, micelles, single-layer or multilayer vesicles, the shade of red blood cells or spheroblast. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, etc.
Preparation of such liposomal formulations is within the level of skill in this area, as described, for example, in U.S. patent numbers 4235871, 4501728, 4837028 and 4737323. Such compositions will influence the physical state, solubility, stability, rate of release in vivo and speed of clearance in vivo and, therefore, can be selected in accordance with the intended use, so that the characteristics of the carrier will depend on the selected route of administration. Forms with delayed release, suitable for use include, but are not limited to, polypeptides that are encapsulated in slow the dissolving biocompatible polymer (such as microparticles of alginate described in U.S. patent number 6036978), mixed with such a polymer (including applied topically hydrogels) or encased in a biocompatible semi-permeable implant.
After the description of this invention the following examples are given to illustrate but not to limit the invention.
Biophysical evaluation of fractions # 2 and # 3 TNFR:Fc
TNFR:Fc eluted with columns for hydrophobic chromatography (HIC) in the form of three separate peaks indicated the fraction No. 1, fraction 2 and fraction No. 3 (see figure 1). Fraction No. 2 is the desired fraction. Fraction No. 3 was of particular interest because it may contain from 20 to 60% of the sample, and it was shown that it exhibits a low TNF-binding activity and biological activity A3 75 compared to fraction No. 2. Thus, in the interests of understanding the differences between the two factions and to determine what factors contribute to the loss of activity in fraction No. 3 in connection with the structure and conformation, conducted biophysical studies. In this example, the inventors analyzed the fraction 2 and fraction No. 3 using circular dichroism, fluorescence, SEC/UV/LS/RI in the on-line mode and differential scanning calorimetry (DSC).
Materials and methods:
Materials: the raw material was TNFR:Fc in TMS buffer (10 mm Tris, 4% mannitol, 1% sucrose). Elwer the bathrooms at HIC fractions of this material was obtained in the form of fractions # 2 and # 3 for the following experimental studies.
Circular dichroism: the Study was carried out in the near (250-340 nm) and far (190-250 nm) UV spectral regions. Research in the near UV region was performed to determine differences in the tertiary structure, whereas research in the far UV region were used to characterize differences in the secondary structure.
Measurement of circular dichroism in the near-UV region were carried out in TMS solutions with the following concentrations. The source material was diluted to 6.25 mg/ml, whereas fractions # 2 and # 3 were evaluated at their existing concentrations of 9.4 and 5.4 mg/ml, respectively. Used the cell for circular dichroism with optical path length of 0.1 cm and the scan was performed from 340 to 250 nm.
Measurement of circular dichroism in the far-UV region were carried out in buffer for protein, replaced with 10 mm sodium phosphate (pH 7.0), and then evaluated using a cell with optical path length of 0.1 cm and the scan was performed from 250 to 190 nm. The secondary structure was evaluated using analysis of restrictions on the convexity (convex constraint analysis, CCA) (Perczel et al., 1991, Protein Engrg. 4:669-679).
Fluorescence spectroscopy: the Specimens were examined after dilution to about 50 micrograms per ml using two different excitation wavelengths. The fluorescence of tyrosine and tryptophan was investigated using excitation at 270 nm, while fluore is Cencio exclusively tryptophan was estimated, using excitation at 295 nm (Lakowicz, J. R. in Principles of fluorescence spectroscopy". Plenum Press, 1983. New York, N.Y., 342-343). Scanning fluorescence wavelengths were performed from 300 to 440 nm for excitation at 270 nm and from 310 to 440 nm for excitation at 295 nm. Four consecutive scans were averaged signal for each spectrum. Provides normalized data to assess differences in fluorescence originating from different samples.
SEC/UV/LS/RI in the on-line mode: Molecular weight eluruumiks components using gel-filtration chromatography was determined using sequential detection in the UV (280 nm UV), scattering (90° (C) and refractive index (RI). This method has been well documented (see Arakawa et al., 1992, Anal. Biochem. 203:53-57 and Wen et al., 1996, Anal. Biochem. 240:155-166) and has the advantage that the measurement deglycosylated molecular masses of proteins and peptides, which are glycosylated. Data SEC and UV were collected using HPLC system Integral (PerSeptive Biosystems, Inc.) with column BioSil-400-5 (BioRad) at a speed of current of 1 ml/min Buffer for elution consisted of 100 mm phosphate (pH 6.8) and 100 mm Nad. Multi-angle light scattering detector DAWN DSP and Refractometer Optilab DSP were both purchased from Wyatt Technology, Inc. Calibration standards for determining the instrument constants included dimer BSA monomer BSA and ovalbumin (figure ).
Differential scanning calorimetry (DSC): the Physical characteristics of the expander was measured using a device MicroCal MC-2 DSC mode the upward scan. Samples were prepared by replacing the buffer at the same TMS buffer with pH 7.4. The sample contained approximately 4 mg/ml of protein, and they are evaluated against buffer (no protein) as a control. The scan rate was 67°/h when covering the temperature range from 20 to 90°C. Then the obtained scan data were converted into normalized concentration data for a better comparison of the behavior of the enthalpies of unfolding transitions taking into account differences in concentration (data are given in kcal/mol). The results:
Circular dichroism. Measurement of circular dichroism in the near-UV region, expressed as the average ellipticity of the balance are shown in figure 2. Changes in the broad area near 270 nm were evident between fractions # 2 and # 3, as shown greater share of negative ellipticity in the spectrum of fraction No. 3 (indicated by the arrow in figure 2A). It was noted that the spectral behavior of the source material closely coincides with the spectral behavior of fraction # 2, but really shows a weak negative shift in the same area near 270 nm. This result seems consistent, as the fraction No. 3 pillar is t a small part of the source material, and, therefore, its contribution to the overall ellipticity in this area was greatly reduced, but in the same direction of the shift. The reproducibility of the spectrum of fraction # 3 was confirmed that the observed shift for this sample was true. With this in mind and knowing that the disulfides lead to a characteristic broad negative ellipticity in this region of the spectrum circular dichroism (see Kahn, R.S., 1978, Methods Enzymol. 61:339-378 and Kosen et al., 1981, Biochemistry 20:5744-5754), the spectrum of the circular dichroism in the near-UV region was represented by a smoothed curve to estimate that the mean of the observed changes in this region from the positions of the tertiary structure. The results of smoothing the data presented in figure B2, and they showed a small shift in the red region (3 nm) and increased negative shift, consistent with the contribution arising from changes in the tertiary structure involving disulfides, when comparing fraction # 3 fraction No. 2.
Circular dichroism in the far UV region of the spectrum used to determine the composition of the secondary structure of proteins (Perczel et al., 1991, Protein Engrg. 4:669-679). Correlation with secondary structure was performed using SSA. Calculated spectra, consisting of the sum of the elements of secondary structure, was compared with the experimentally observed spectra, and they found good agreement. Secondary structure of both coat the s were comparable within the experimental accuracy (within 10%). Thus, this experiment did not find any differences in secondary structure for each of these two factions.
Fluorescence spectroscopy. Knowing that there were significant differences observed in the spectrum of the circular dichroism in the near-UV region, used fluorescence spectroscopy to detect more differences in the tertiary structure associated with the fluorescence of tryptophan and tyrosine. Using two excitation wavelengths were able to determine that the spectra for all the considered three cases (SM, fraction 2 and fraction No. 3) were superimposed on each other with the maximum fluorescence near 338 nm. Since the three-dimensional structure of a protein is responsible for the emission maxima of native proteins, these results suggest that the average structure, which includes natural fluorophores, tryptophan and tyrosine, was undisturbed.
SEC/UV/LS/RI-mode on-line. Studies of light scattering performed in the on-line mode with the SEC, given the molecular weight of the main peak of elution, which is consistent with the molecular weight replicationmanager polypeptide TNFR:Fc (for example, 102 KD). Although there were clear differences in the compositions eluruumid molecular forms that are measured in this way, when comparing the elution profile of fraction No. 3 elution profile of fraction No. 2 (figures is OVER) measurement showed the main peak containing the main component had a molecular weight 102,5±1,6 kDa (held volume=8,4 ml) and 101,9±2,1 kDa (held volume = 8,3 ml), respectively. Precision was expressed as the standard deviation for 23 slices through the peak elution, limited by the vertical dashed lines in the figure 3. It was also noted that a significant signal of the downward shoulder for fraction # 3 was possible to determine that the molecular weight of this polypeptide was 78,1±3,7 KD (this estimate took into account 8 slices surrounding the peak, marked on cent to 8.85 ml). As shown by the precision associated with the determination of the molecular weight of this component, this peak showed a higher heterogeneity, and consequently it was assumed most polidispersity than the main peak. Fraction No. 3 also contained a significant amount of high molecular weight forms of the molecules, which is consistent with the volume of elution predominantly dimeric forms of TNFR:Fc (near to 7.5). Thus, it was determined that the fraction No. 3 consists of several forms, including aggregates and fragmented parts of this molecule.
Differential scanning calorimetry. The DSC measurements conducted on two factions, gave significant differences in the deployment of TNFR-part molecules TNFR:Fc (figure 4). As more clearly follows from the adjusted background data (figure 4B), the offset is of 2,8° With in the direction of lower temperatures in the melting transition (Tm) when comparing Tm 52,5° (fraction No. 2) Tm 49,7° (fraction No. 3). This transition is slightly wider for a fraction No. 3 with a width of 8°at half maximum of the transition in comparison with the fraction No. 2, having a width of 6.5°S. Of experiments on thermal unfolding of the monomer TNFR:Fc was found that this low-temperature transition due to the TNFR domain of this molecule. Thermal transitions of 83.4 69.1 and°were included in the Fc-part of the molecule. These last two transitions expand well comparable and are comparable in respect to Tm and enthalpies of the components.
Among the tested methods, the differences were observed in the measurements of circular dichroism in the near-UV region of the spectrum and DSC. The differential scanning calorimetry confirmed the loosening of the structure, which can be attributed to the receptor part of this molecule, with a small modification in the Fc region. The determination results of circular dichroism in the near-UV region of the spectrum suggest that the disulfides are involved in the changes of the tertiary structure associated with the fraction No. 3. These changes can occur as a consequence shipped disulfides, acquiring greater openness to solvent and is responsible for the increase in hydrophobicity, as should the duty to regulate from a small increase in the withholding amount, observed in the elution fraction No. 3 in HIC. Interestingly, there was no discernible difference detected by fluorescent data that would indicate such a change in the conformational structure. If we analyze the primary structure of TNFR:Fc in connection with the distribution of tyrosines (Y) and tryptophane (W), it becomes apparent that the area extending from the C-terminal side of residue 104 domain TNFR to balance 296 N-terminal of the Fc part (containing 40% of a linear sequence of TNFR:Fc), deprived of these natural fluorophores. Thus, one possible explanation consistent with these data, it may be that the tertiary structure, remote from the hinge region of Fc, is relatively unchanged, whereas the structure of the region from about residue S to S may change conformation. This region of the molecule contains 10 possible cysteines that may be affected are likely to be small effects of the structural changes that affect the local structure of tyrosines and tryptophane. It is noted that it is currently unknown how fit this molecule, and it seems likely that cysteine, which form disulfide bonds, which are more remote from any specific residue of tryptophan or tyrosine, would be the logical suspects izmeneniya tertiary structure, which lead to the observed results of determination of circular dichroism in the near-UV region of the spectrum, but have little effect on vizualnoy structure, including tyrosines and tryptophane. This idea does not exclude the possibility that there is some unusual change in the structure in one or both shoulders TNFR, which does not cause significant changes in the total effect of fluorescence caused by tyrosine and tryptophan. The fact that fluorescence data (which are insensitive to disulfides) do not detect changes and near-UV region of the spectrum (which is sensitive to disulfides, tyrosines and tryptophane) shows a small negative shift, consistent with disulfide structural modification, indeed suggests that the disulfide responsible for the differences between the fractions of No. 2 and No. 3.
Summarize other results concerning the fraction No. 3, it was found that all indicators related to the molecular weight and secondary structure are indistinguishable from the performance of fraction No. 2.
Experiments disulfide exchange into fractions No. 3 TNFR:Fc with glutathione
This experiment was designed to assess the ability of various treatments to translate fraction No. 3 TNFR:Fc in conformation fraction No. 2 in the process, suitable for large stanoi commercial operation.
Materials and methods:
Materials. The source material was TNFR:Fc in the form of the eluate from the column with protein And pure eluate fraction No. 3 after HIC and a mixture of 50:50 eluates fraction 2 and fraction 3 after HIC. Buffers were 0.1 M citrate or 0.1 M Tris/glycine at pH of 7.6, pH 8.6 and pH of 9.6. The concentration of protein TNFR:Fc was from 0.2 to 4.5 mg/ml of Conjugated redox system of glutathione and glutathione (GSH/GSSG ratio 10:1) was added at 0.1-5 mm GSH. The incubation temperature was varied at 4, 22 ili°C.
The ways. Disulfide exchange was stopped by acidification of the sample to pH 6 1 M acetic acid. Processed recombinant protein was characterized by analytical HIC, SEC (retention time, concentration units) and solid phase analysis of the binding of TNF to determine the percentage and yield of fraction No. 2.
Results and discussion:
Processing efficiency as a function of pH and concentration of GSH. A significant% of the protein in fraction No. 3 (at least 10%) turned in fraction No. 2, when the processing is performed as 0.1 mm GSH/pH of 7.6, and 0.1 mm GSH/pH to 8.6. However, the efficiency was significantly improved (from 45 to 70%)when the processing was performed with 0.1 mm GSH/pH 9,6; 1 mm GSH/pH of 7.6; 1 mm GSH/pH 8.6 and 1 mm GSH/pH of 9.6. Thus, although the processing efficiency is sensitive to pH and the concentration of free thiols (SH), it can effectively be performed in a wide diaphaneity variables.
Temperature effects. Fraction No. 3 were treated at three different temperatures: 4, 22 and 31°C. the Concentration of GSH was maintained at 1 mm and a pH of 8.6. After 16 hours when all treatment options were found significant transformation of the fraction No. 3 in fraction # 2, but this transformation seemed to be slightly more effective at the two lower temperatures.
Clonal effects. Six different clones, cell lines, all of which produce TNFR:Fc was tested in a standardized Protocol based on the above results. Specifically, the eluate from the column with protein And containing 0.4 to 0.7 mg/ml of TNFR:Fc (approximately pH 4), brought to a pH of 8.6 with 1 M Tris/glycine (final concentration 0.1 M Tris/glycine). These solutions were brought to 1 mm EDTA and 2.5 mm GSH/0.25 mm GSSG, and incubated at room temperature for about 16 hours. Disulfide exchange was stopped by acidification, as described above.
Each of the six different clones showed improvement in the generation and output of fraction No. 2. The decrease in fraction No. 3 after HIC as a result of processing in different clones was 64, 72, 77, 78, 78, and 83%. The increase in fraction No. 2 after HIC in the same clones was 37, 64, 78, 70, 44, and 54%, respectively. The increase in fraction No. 2 after HIC, expressed in percent, is well correlated with the increase of the binding units in percentage, as shown n the figure 5. Thus, these methods proved to be generally applicable to all tested clones.
Analyses of the binding. Three different drug TNFR:Fc were analyzed in solid-phase analysis of binding TNF. Samples 11-6 and 12 were eluates from the column with protein A. the Sample 8085-47 also suirable with a column with protein a and then were subjected to additional purification stages with the use of HIC; this sample contained only the fraction No. 3. The samples were tested in the analysis of binding before and after disulfide exchange, as described above. The results presented below in table 1, show an increase in the binding activity of the ligand after processing all samples glutathione.
TNF-binding activity of TNFR:Fc before and after disulfide exchange
|Sample||To exchange||After exchange||Change|
|(activity per mg protein) (%)|
Experiments disulfide exchange rate is well on TNFR:Fc, treated with L-cysteine
This experiment was designed to assess the cysteine/cystine as reagents for conjugate oxidation-reduction TNFR:Fc. This procedure allows us to estimate the change in fraction No. 3 after HIC in conformation fraction No. 2 in the process suitable for large-scale industrial operation. This procedure can be carried out on purified fractions No. 3, mixture fractions # 2 and # 3 and/or other separation methods such as chromatography on protein A, with similar results.
Materials and methods:
The source material was TNFR:Fc in the form of pure eluate fraction No. 3 after HIC or lirovannomu with a column with protein AND TNFR:Fc containing fraction No. 2, and fraction No. 3. Buffers were 0.1 M citrate or 0.2 M Tris at pH 8.5. The concentration of protein TNFR:Fc was from 2.5-3 mg/ml
Used the coupled redox-system of L-cysteine (in the range from 0 to 50 mm). This procedure was also evaluated +/- L-cystine (0,025-0,5 mm) and +/- 1 mm EDTA. The incubation temperature was evaluated at 4, 15 and 22°for 6, 18 and 48 hours. Disulfide exchange was stopped by acidification of the sample to pH 7 NaH2PO4or 0.85 M citrate. Processed recombinant protein was characterized by analytical HIC and SEC (retention time, concentration units) to determine the percentage and yield of fraction 2 and fraction No. 3, analysis containerbase of analisa free sulfhydryl groups. Results and discussion:
Processing efficiency as a function of the concentration of L-cysteine (0-5 mm). A significant percentage of protein TNFR:Fc in fraction No. 3 after HIC (average 10%) turned in fraction No. 2, when the processing was performed with 0.25 mm L-cysteine in the absence of L-cysteine or EDTA in four replications of samples (figure 6). However, the efficiency was greatly improved (from 45 to 70%)when treatment was performed 1 mm L-cysteine or 5 mm cysteine (figure 6). Effect of cystine in these reaction conditions were changed depending on the presence of EDTA (see below). For a particular batch of cell culture was treated samples from four different batches of cell cultures) processing method was reproducible.
Processing efficiency as a function of the higher concentration of L-cysteine (5-50 mm). Higher concentrations of L-cysteine (5, 15 and 50 mm L-cysteine), used for treatment of TNFR:Fc, led to a decrease in fraction No. 3 after HIC from the source material in every case, but 5 mm L-cysteine was the most effective in stimulating the accumulation of fraction No. 2 (figure 7). It is believed that higher concentrations of L-cysteine or significantly restored the sulfhydryl group in the molecule, or required too much time to re-oxidation.
Processing efficiency as a function of the additional application of L-cysteine. In attempt to increase the program efficiency disulfide exchange TNFR:Fc was treated with 5 mm L-cysteine, and incubated at 4° C for 18 hours. Then add an additional amount of L-cysteine (0-5 mm) and samples were incubated at 4°within two days. When these conditions were not noted to have a significant influence on the ratio of the fraction No. 3 after HIC fraction No. 2 after HIC with additional introduction of L-cysteine.
The effect of EDTA, cysteine and L-cysteine. Effect of cystine (0-0,4 mm) in combination with L-cysteine (5 mm) on TNFR:Fc was assessed in the presence or absence of 1 mm EDTA. Optimal results in the presence of 1 mm EDTA occurred at concentrations of cystine in the range of 0.1 to 0.2 mm.
The introduction of copper, temperature and time. TNFR:Fc were treated with 5 mm L-cysteine at 4°C for 6 or 18 hours. The completion of the treatment TNFR:Fc was assessed by adding copper with subsequent HIC. After 6 hours incubation disulfide exchange is more complete in 4°C than at 22°and treatment is clearly more complete after 18 hours at 4°With (figure 8A and 8B).
Comparison of the effectiveness of L-cysteine in analytical scale with efficiency in preparative scale. On the basis of the processing conditions, optimized on a small scale, TNFR:Fc (2.5 mg/ml in 0.2 M Tris, pH 8.5) in quantities of either 3 ml or 20 l was treated with 5 mm L-cysteine (in the absence of cysteine or EDTA), incubated at 4°C for 18 hours, diluted with an equal volume of 850 mm sodium citrate, 50 mm is osveta sodium, pH 6.5 to stop processing and chromatographically on HIC. Control samples TNFR:Fc preparative and analytical scale was 63% and 68% fraction No. 3, respectively. After processing using the above-mentioned conditions, the fraction # 3 was decreased to 28% as in preparative and analytical scale. Thus, the processing efficiency was 56 and 59% for preparative and analytical samples, respectively (table 2). This experiment shows that this method is applicable to large-scale processing.
Thus, although the redox processing efficiency is affected by the concentration of free thiols, temperature and time, it can be effectively optimized and performed in a wide range of variables. The Protocol processing can also be performed in small and large scale with good reproducibility.
This invention is not limited in the amount described herein specific embodiments, which are intended only to illustrate certain aspects of the present invention, and functionally equivalent methods and components are within the scope of this invention. Indeed, various modifications of the present invention, in addition to the shown and described herein will be obvious to experts in the Anna area from the preceding description and accompanying drawings. It is assumed that such modifications are within the scope of the attached claims.
1. Method of promotion the most active conformation glycosylated recombinant protein that was secreted from mammalian cells in the form of a preparation containing a mixture of at least two configurational isomers, where specified, the most active conformation is a three-dimensional structure of a protein that is most similar to the structure and/or duplicates the functionality of the natural domain of this protein, including
the contacting of the specified drug conjugate reagent for oxidation-reduction in a period of time sufficient to increase the relative proportion of the desired configurational isomer, and then determining the relative proportion of the desired configurational isomer in the mixture.
2. The method according to claim 1, where the glycosylated recombinant protein contains at least two domains.
3. The method according to claim 2, where at least one domain is glycosylated recombinant protein is stable conformation and at least one domain is glycosylated recombinant protein is unstable conformation with respect to each other.
4. The method according to claim 1, where the glycosylated recombinant protein contains an extracellular domain of the receptor.
5. The method according to the .4, where desired configurational isomer glycosylated recombinant protein has a higher affinity binding of a cognate ligand of this receptor compared to the other isomers mixture.
6. The method according to claim 1, where the glycosylated recombinant protein is a soluble form of the TNF-receptor.
7. The method according to claim 6, where the desired configurational isomer glycosylated recombinant protein has a higher binding affinity of TNF compared with other isomers mixture.
8. The method according to claim 7, where TNF is TNF-alpha.
9. The method according to claim 1, where the glycosylated recombinant protein is fused with Fc protein.
10. The method according to claim 9, where the drug is glycosylated recombinant protein purified on a column of protein a or protein G.
11. The method according to claim 1, where the glycosylated recombinant protein selected from the group consisting of soluble IL-4 receptor, soluble receptor of IL-1 type II soluble Flt3 ligand, soluble CD40 ligand, CD 39, CD30, CD27, TEK/Ork, IL-15, soluble receptor of IL-15, Ox 40, GM - CSF, RANKL, RANK, TRAIL, soluble TRAIL receptor, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I receptor, IL-2, an antagonist of IL-2, alpha-1-antitrypsin, calcitonin, growth hormone, insulin, insulinopenia, insulinopenia growth factor, parathyroid mountains is she, interferon, superoxide dismutase, glucagon, erythropoietin, antibodies, glucocerebrosidase, Fc-fused protein, globin, nerve growth factor, interleukin, and colony-stimulating factor.
12. The method according to claim 1, where the pH is from 7 to 10, preferably from about 7.6 to 9.6, or a pH of 8.6.
13. The method according to claim 1, where the conjugate reagent for oxidation-reduction selected from the group consisting of glutathione, cysteine, DTT (dithiothreitol), 2-mercaptoethanol and getinitialstate.
14. The method according to item 13, where the conjugate reagent for oxidation - reduction contains restored glutathione.
15. The method according to 14, where the restored glutathione is in a concentration of from 1 to 10 mm.
16. The method according to item 13, where the conjugate reagent for oxidation - reduction contains restored cysteine.
17. The method according to item 13, where the ratio of reducing thiols in the conjugate reagent for oxidation-reduction to disulfide linkages in the protein ranges from 320:1 to 64000:1 (reducing thiol: disulfide bonds).
18. The method according to claim 1, where the protein concentration is from 0.5 to 10 mg/ml
19. The method according to claim 1, where the stage contacting is conducted within 4-16 hours
20. The method according to claim 1, where the stage contacting is carried out at 25 or 4°C.
21. The method according to claim 1, where the phase of the probe is stopped by acidification.
22. The method according to claim 1 where the stage of determining includes one or several steps of chromatography.
23. The method according to claim 1, where the stage of definition involves the reaction of binding of glycosylated recombinant protein.
24. The method according to claim 1, in which stage contacting is carried out in the absence of sodium dodecyl sulfate, urea or guanidine hydrochloride.
25. The method of obtaining glycosylated recombinant protein in the most active conformation where the specified most active conformation is a three-dimensional structure of a protein that is most similar to the structure and/or duplicates the functionality of the natural domain of this protein, including the promotion of the most active conformation glycosylated recombinant protein that was secreted from the cells of a mammal, the method according to claim 1, and the allocation fraction of the drug glycosylated recombinant protein with the desired configurational isomer from the mixture obtained.
26. The method according A.25, in which stage contacting is carried out in the absence of sodium dodecyl sulfate, urea or guanidine hydrochloride.
27. A method of obtaining a composition glycosylated recombinant protein in the most active conformation for the introduction of the consumer and/or patient or consumption by the consumer and/or patient, where this most active conformation is a three-dimensional structure of a protein that is most similar to the structure of the St and/or duplicates the functionality of the natural domain of this protein, including the production of glycosylated recombinant protein in the most active conformation way A.25, the combination with a pharmaceutical acceptable diluent, carrier and/or excipient and obtaining sterile dosage forms.
FIELD: biotechnology, immunology, biochemistry, medicine.
SUBSTANCE: invention proposes peptide concatemer inducing production of antibodies against apolipoprotein B-100 that inhibit lipase effect and inhibit binding LDL with LDL receptors. This concatemer consists of amino acid sequence of peptide repeating four times. Amino acid sequence is given in the invention description. Also, invention describes a concatemer-base vaccine used in treatment and prophylaxis of obesity and a method for preparing concatemer in E. coli cells using a vector. Invention discloses a polynucleotide encoding concatemer and expressing vector comprising the indicated polynucleotide. Using the invention provides inhibition of obesity.
EFFECT: valuable medicinal properties of concatemer and vaccine.
7 cl, 16 dwg, 1 tbl, 6 ex
FIELD: immunology, biotechnology.
SUBSTANCE: invention relates to variants of nucleic acid construct (NK-construct) encoding of MUC1 antigen based on seven full repeated VNTR-units. Variants include NK-constructs selected from group containing MUC1 based on seven full repeated VNTR-units, MUC1 based on seven full repeated VNTR-units without signal sequence, MUC1 based on seven full repeated VNTR-units without signal sequence, transmembrane and cytoplasm domains, full MUC1 based on seven full repeated VNTR-units without transmembrane and cytoplasm domains, as well as mutants of abovementioned variants, wherein at least one VNTR is mutated to reduce of glycosylation potential. Disclosed are NK-constructs additionally containing epitopes selected from group: FLSFHISNL, NLTISDVSV or NSSLEDPSTDYYQELQRDISE. Also described are variants of expressing plasmide carrying NK-construct represented as DNA, protein having anti-tumor activity, encoded with NK-construct and pharmaceutical composition with anti-tumor activity based on said protein, NK-construct or plasmide. Application of NK-construct and protein for producing of drug for treatment or prevention of MUC-1 expressing tumors; method for therapy by using NK-construct, protein, or plasmide also are disclosed.
EFFECT: NK-constructs with increased anti-tumor activity.
20 cl, 25 dwg, 5 ex
FIELD: medicine, biotechnology, pharmacy.
SUBSTANCE: invention relates to exchangers of ligand/receptor specificity delivering antibodies to receptors on pathogen. In particular, invention describes variants related to manufacturing and using exchangers of ligand/receptor specificity. Exchangers comprise at least one specificity domain containing ligand for receptor wherein ligand is not antibody or its part, and at least one antigenic domain combined with abovementioned specificity domain wherein antigenic domain comprises epitope of pathogen or toxin. Advantage of the invention involves enhanced specificity in delivery of drug.
EFFECT: improved and valuable properties of exchangers.
30 cl, 5 tbl, 6 ex
FIELD: medicine; biology.
SUBSTANCE: method involves introducing vector containing DNA-sequence into area under protection encoding T-cadgerin and being capable of providing its expression in target tissue or cells compatible with target tissue cells transected with said vector in advance and selected with respect to T-cadgerin expression level. The method has been tested on model system by implanting Matrigel to mice. Reliable implant mass-, implant hemoglobin contents- and capillary and medium-sized blood vessel number reduction has been observed in experimental animals, receiving cells L929 (clone TC3) expressing T-cadgerin, in two weeks after the injection.
EFFECT: enhanced effectiveness in treating pathological states with new blood vessel formation being suppressed.
7 cl, 8 dwg
FIELD: genetic engineering, biotechnology, biochemistry, medicine, pharmacy.
SUBSTANCE: invention relates to DNA encoding glycoprotein VI (GP VI) used as a base for synthesis of recombinant GP VI by method of recombinant DNAs followed by its using for the development of pharmaceutical compositions possessing with capacity for inhibiting or blocking interaction of platelets with collagen. Using the invention provides preparing GP VI in the recombinant form and using this agent for therapeutic aims.
EFFECT: valuable medicinal properties of glycoprotein VI.
6 cl, 4 dwg, 6 ex
SUBSTANCE: invention relates to soluble CTLA4, which represents mutant variant of wild type CTLA4 and conserves binding ability to CD80 and/or CD86. Molecules of soluble CTLA4 have the first amino acid sequence containing extracellular CTLA4 region, which includes some mutant amino acid residues in S25-R33 region and M97-G107 region. According the present invention mutant molecules also may include second amino acid sequence, enhancing solubility of mutant molecule. Nucleic acid (NA) molecules encoding said CTLA4 and including NA-vectors also are described. Invention also relates to method for production of mutant CTLA4 and uses thereof in controlling of interaction between T-cell and CD80 and/or CD86-positive cell; suppression of graft-versus-host reaction; and treatment of immune system diseases. Soluble mutant CTLA4 according to present invention binds to CD80 and/or CD86 antigen with higher avidity than wild type CTLA4 or non-mutant CTLA41g.
EFFECT: new preparation for treatment of immune system diseases.
65 cl, 19 dwg, 2 tbl, 2 ex
FIELD: biotechnology, medicine.
SUBSTANCE: cells are contacted with protein containing fibronectin EDb-domen in presence of one or more chemical substances. As check experiment reaction between the same cells and abovementioned protein in absence of said substances. Expression of certain protein or presence of nucleic acid encoding the same makes it possible to select compounds bonding to fibronectin EDb-domen.
EFFECT: new biotechnological method.
1 tbl, 3 ex
FIELD: biotechnology, genetic engineering, immunology.
SUBSTANCE: invention proposes: isolated nucleic acid encoding feline ligand CD86; diagnostic oligonucleotide; cloning vector; vaccine for modulation of the immune response in cat; method for induction, enhancement and suppression of immune response in cats. Proposed group of inventions allows designing effective vaccines used in prophylaxis of immunodeficiency in felines and infectious peritonitis in domestic cats. Invention can be used in veterinary science.
EFFECT: valuable properties of nucleic acid.
27 cl, 13 dwg, 5 tbl, 8 ex
FIELD: biotechnology, preparative biochemistry.
SUBSTANCE: invention proposes a method for preparing the recombinant human tumor necrosis factor-aplha (TNF-alpha). Method involves culturing the strain-producer E. coli SG20050/pTNF311Δ, disruption of cells by ultrasonic oscillation, extraction of the end protein and 3-step chromatography purification procedure on DEAE-cellulose column in the linear gradient concentrations of NaCl at pH 8.0, on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 8.0 and on hydroxyapatite in the linear gradient concentrations of potassium phosphate at pH 6.7. The recombinant TNF-alpha prepared by the proposed method shows the reduced content of impurity proteins, nucleic acids and lipopolysaccharides especially that provides its direct medicinal using. Invention can be used in medico-biological industry.
EFFECT: improved preparing method, enhanced quality of polypeptide.
2 tbl, 5 dwg, 2 ex
man, is able to induce the formation of neutralizing antibodies to human tnfdna, its coding, vector (options), a method of obtaining a vaccine tnf(options), method of testing for the presence of tnfthe way to test body fluids of a person, the method of diagnosis, method of treatment and prophylaxis medication for the treatment of" target="_blank">
FIELD: organic chemistry, peptides.
SUBSTANCE: invention proposes a method for labeling phosphorylated peptides, method for adsorption of phosphorylated peptides and compounds possessing high coordination capacity with respect to phosphorylated peptides that are used in such methods, and compounds used as parent substances. Chelating compound is represented by the formula (I): wherein X represents linker group; Y represents fluorescent group or biotin as labeling group. Compound of the formula (I) provides easy detection and identification of phosphorylated peptide in a sample prepared from a living body.
EFFECT: improved methods of preparing and analysis.
13 cl, 9 dwg, 8 ex
FIELD: pharmaceutical chemistry, chemistry of peptides, hormones.
SUBSTANCE: invention relates to a method for preparing analogs of adrenocorticotropic hormone (ACTH) (4-10) possessing neurotropic activity. Method for preparing analogs of adrenocorticotropic hormone (ACTH), a sequence (4-10), of the general formula (I): A-Glu-His-Phe-Pro-Gly-Pro-OH (I) wherein A means hydrogen atom (H), Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr is carried out by liquid-phase method by step-by-step splicing peptide chain beginning from C-terminal protected tetrapeptide of the formula: H-Phe-Pro-Gly-Pro-OH (II) wherein X means a protective group and using corresponding fully protected amino acids in activated form followed by removal of protective groups at each step and purification of the end product by liquid chromatography. Method provides simplifying the process and to enhance the yield of the end product.
EFFECT: improved preparing method.
5 cl, 1 tbl, 5 ex
FIELD: biochemistry, medicine, allergology.
SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.
EFFECT: improved and valuable properties of allergen, improved preparing method.
27 cl, 6 dwg, 1 tbl, 6 ex
FIELD: biochemistry, medicine, allergology.
SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.
EFFECT: improved and valuable properties of allergen, improved preparing method.
27 cl, 6 dwg, 1 tbl, 6 ex
SUBSTANCE: invention relates to protein deposition by overcritical fluid medium as well as stabilization and protection from melting thereof. Method for codeposition of protein or polypeptide with stabilizing agent thereof by gas antisolvent process includes feeding overcritical fluid medium into vessel for particle formation individually or in mixture with modification agent and solution containing said protein or polypeptide and said modification agent dissolved in solvent differ from said modification agent. In such a manner than abovementioned solvent is extracted from solution with said overcritical fluid medium and substance and stabilizing agent (such a sugar) are simultaneously deposited. Device for such method and obtained product also are disclosed.
EFFECT: increased protein stability.
18 cl, 2 tbl, 7 dwg
FIELD: biotechnology and genetic engineering.
SUBSTANCE: in order to renature membrane protein, homogenous solution of the protein and phospholipid or phospholipid mixture in organic solvent blend or solvent/water mixture is prepared, after which solvents are removed and remaining mixture is hydrated. Method can also be applied in pharmaceutical industry.
EFFECT: increased yield of renatured membrane protein.
3 dwg, 3 ex