Method of heterological polypeptide purification
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to biotechnology. Method includes addition to fermentation broth or homogenate from E. coli of efficient quantity of ethacridinlactate solution for sedimentation of contamination from host-cells in conditions, when greater part of polypeptide remains dissolved, and isolation of heterological polypeptide from broth or homogenate.
EFFECT: simplification of target polypeptide purification and obtaining it with high degree purity.
23 cl, 15 dwg, 3 tbl
The basis for the creation of inventions
1. The scope to which the invention relates.
This invention relates to a method of purification of polypeptides of interest from the broth after microbial fermentation or from the homogenate. More specifically, in the broth or homogenate injected precipitating agent, for example, to effect the removal of protein, DNA or cellular debris.
2. Description of the prior art,
The development of recombinant technology now allows you to get in appropriately transformed cells, high levels of proteins. In the result there is an increased need for fast, reliable and efficient methods of production created by recombinant method proteins. Typically, proteins are produced by culturing such cells as mammalian cell lines, insect, fungi and bacteria designed for the production of protein of interest by inserting a recombinant plasmid carrying the gene of this protein. As used cell lines are living organisms, they need to provide food in the form of an integrated environment for growth, containing sugars, amino acids and growth factors, as a rule, obtained from preparations of animal serum. The separation of the desired protein from a mixture of components for power cells and from by-products of the cells is about purity, sufficient for use as a drug for humans, is a significant problem.
Procedure purification of proteins from cell debris source depends on the site of expression of the protein. Some proteins can secretariats directly from cells into the surrounding nutrient medium; others are synthesized intracellularly. For proteins produced in mammalian cells, the purification scheme is much simpler than for polypeptides produced in other types of host cells. Mammalian cells are exported polypeptides so that they can be collected from the culture medium, where they exist in a relatively pure form. However, if the polypeptide is produced in cells, non-mammalian cells, such as cells of microorganisms such as fungi orE. coliprotein will be obtained inside the cell or in periplasmatic space (Kipriyanov and Little,Molecular Biotechnology,12: 173-201 (1999); Skerra and Pluckthun,Science,240: 1038-1040 (1988)). Therefore, it is necessary to release the protein from the cells in the extracellular environment through extraction, such as lysis of the cells. This destruction makes the entire contents of the cells in the homogenate and additionally forms a subcellular fragments that are difficult to remove due to their small size. They usually are removed by differential centrifugation or what exploits filtering.
Lysis of cells, usually accompanied by the use of mechanical methods of destruction, such as homogenization or milling head. Although protein of interest, as a rule, allocated effectively, these methods have several disadvantages (Engler,Protein Purification Process Engineering, Harrison eds., 37-55 (1994)). The temperature rise that occurs frequently in the course of treatment, can lead to protein interaction. In addition, the resulting suspension contains a wide range of contaminating proteins, nucleic acids and polysaccharides. Nucleic acids and polysaccharides increase the viscosity of the solution, potentially complicating subsequent processing by centrifugation, filtering, cross-flow or chromatography. Complex associations of these pollutants with the protein of interest can complicate the treatment process and result in unacceptably low yields.
Essentially, more selective methods of release of intracellular proteins facilitate further processing. For violations of the cell membrane permeability and/or extraction of cellular proteins described several ways. These methods include the use of solvents, detergents, chaotropic funds, antibiotics, enzymes and chelating means to increase cell permeability and/or facilitate the extraction. is written, what supplements during growth of the culture to the environment fermentation of certain compounds, such as glycine, also contribute to the release of certain intracellular enzymes. Finally, it is shown that methods such as the processing method of freezing-thawing or osmotic shock, unleash subclasses of intracellular proteins.
However, these methods are not necessarily applicable to all intracellular proteins of microorganisms, and they all have limited use for large-scale processing and/or other disadvantages. For example, although solvent, such as toluene or chloroform, contribute to the release of intracellular proteins, it is known that these substances are toxic and/or carcinogenic (Windholtzet al.,The Merck Index10th Edition: 300 and 1364 (1983)). Ionic detergents such as SDS, often irreversibly denature secreted proteins. Although non-ionic detergents, as a rule, not denatured selected proteins is often associated with micelles of detergent that may require additional processing to get protein without detergent. Chaotrope tools, such as urea and guanidine hydrochloride may be denaturing at concentrations required for complete release, and their effectiveness may depend on the growth phase of the culture. The use of lysozyme, which provides relative to the positive soft tool for the separation of proteins, limited due to its relatively high cost and because of the need for further purification protein of interest from the enzyme. In addition, chelating tools, often used to increase the effectiveness of other ways to breach the membrane permeability/allocation, such as extraction using lysozyme or toluene, suffer from the lack of non-specific separation of proteins of the host.
Other methods for isolating proteins, there are also disadvantages. For example, osmotic shock, in which cells are suspended in a medium with high osmotic pressure, remove, and then placed in a buffer with low osmotic pressure required additional stages of processing in relation to other alternatives for extraction (Moiret al.,Separation Processes in Biotechnology, Asenjo eds: 67-94 (1990)) or you need to support large volumes of liquid at low temperatures. This makes the method unattractive for large-scale processing.
Processing by freezing-thawing also releases intracellular proteins, however, relatively low outputs often lead to repeated cycles and additional necessary conditions for processing. In addition, the freezing cell mass is an additional non-trivial necessary condition for processing in comparison with other the alternatives for extraction.
Finally, during the fermentation of added reagents, such as glycine, to facilitate the release of proteins in the extracellular environment (Aristidouet al.,Biotechnology Letters15: 331-336 (1993))HOTA reported partial release of some intracellular proteins, this approach requires direct coupling strategies fermentation and separation and subsequent separation protein of interest from a potentially complex extracellular broth.
After the release of the polypeptide of interest from the host cell, it must be purified from other cellular components. Unfortunately, most methods of extraction, such as lysis of the cells, not only leave the protein is protected from possible degradation by cellular proteases, but also make the separation of protein from other elements of the mixture is more complex. For example, the presence of negatively charged molecules such as DNA, RNA, phospholipids and lipopolysaccharides (LPS), often requires the use of anion-exchange chromatography (Sassenfeld,TIBTECH,8: 88-93 (1990); Spears,Biotechnologyvol. 3--Bioprocessing, Rehm eds:40-51 (1993)) and/or deposition of polycation, such as preteenslut (Kelleyet al.,Bioseparation,1: 333-349 (1991); Scopes,Protein Purification Principles and Practice, 2nd edition, Cantor eds., pp. 21-71 (1987)), streptomycinresistant (Wanget al., eds,Fermentation and Enzyme Technology: 253-256 (1979)), polyethylenimine (PEI) (Kelleyet al., above; Sassenfeld, you the e ;Cumminget al.,Bioseparation,6: 17-23 (1996); Jendrisak,The use of polyethyleneimine in protein purification. Protein purification: micro to macro, ed. Alan R, Liss, Inc., 75-97 (1987); Saltet al.,Enzyme and Microbial Technology,17: 107-113 (1995)), and/or aqueous two-phase extraction in immiscible polymer systems, such as polyethylene glycol (PEG)/phosphate or PEG/dextran (Kelleyet al.above, Strandberget al.,Process Biochemistry26: 225-234 (1991)).
Alternative protein of interest can be separated from non-protein polyanionic pollutants by adding a neutral salt such as ammonium sulfate or potassium chloride (Wheelwright,Protein Purification: Design and Scale up of Downstream Processing: 87-98 (1991); Englardet al.,Methods in EnzymologyVolume 182, Deutscher eds.: 285-300 (1990)), and/or polymer, such as PEG or dextran sulfate (Wanget al.,above; Wheelwright, above). When the protein of interest is charged positively, he seeks contact with negatively charged molecules located close by, making the protein purification virtually impossible.
As a rule, for the Department of disrupting the work of polianionov from the protein of interest, the researchers used the above-described initial stage of fractionation. Unfortunately, each of these initial separation methods has serious drawbacks, especially when they are used in the production of pharmaceutical agents. For example, large amounts of non-protein poly is niennah pollutants in bacterial lysates, led to a decrease in the binding ability of the resin to anion-exchange chromatography. In addition, the regeneration protocols are often ineffective due to the strong binding of polianionov with the resin (Spears, above). In conclusion, the conditions of low ionic strength, enabling protein binding, ineffective in the destruction of the interactions of the polyanion-protein and lead to the lack of separation (Scopes,Protein Purification Principles and Practice, 3rd edition, Cantor eds., p. 171 (1994)). Drugs preteenlolita dirty ProcessName and viral contaminants. In addition, when using this reagent may occur unwanted precipitation (Scopes,Protein Purification Principles and Practice, 2nd edition, Cantor eds., 21-71 (1987)).
Upon receipt of pharmaceutical proteins, as a rule, streptomycinresistant not apply due to the General fear to use antibiotics as reagents to obtain (Scawenet al.,Handbook of Enzyme Biotechnology2nd edition, Wiseman eds.: 15-53 (1985)). Drugs PEI is often contaminated with varying amounts of monomer etilenimina, who is suspected of having carcinogenic properties (Scawenet al.,above). PEI also leads to irreversible binding with many chromatographic resins, thereby limiting its effektivnosti and the number of potentially available to apply what I am after cleansing with the use of PEI chromatographic resins. In General, the behavior of the systems water two-phase extraction difficult to predict and often to determine the conditions under which the protein of interest is spent in an appropriate aqueous phase, an empirical approach (Kelleyet al.above).
The ways in which specifically precipitated protein of interest, often lead to the capture sediment non-protein contaminants, making the separation ineffective (Scopes, above; Wheelwright, above).
Examples of patents describing the receipt and clearance of protein include:
In U.S. Patent No. 5665866 described a method of producing antibodies in soluble and correctly laid and collected form. It includes a stage simultaneously raising the operating temperature from 34 to 60°C in the process that is selected to facilitate subsequent extraction of soluble, properly stacked and assembled antibodies, substantially purified from other related antibody substances.
In U.S. patent No. 5760189 describes how to release such thioredoxin hybrid protein with negatively charged non-protein substance from theE. coliin solution by adding to a solution of chelator and deposition of negatively charged non-protein substances from solution by adding to the solution a solution of divalent cation/alcohol with the formation of the first soluble fraction content the total protein, and the first insoluble fraction containing undesirable contaminants. Optional temperature before adding the chelator may be significantly colder than after addition of the chelator. Divalent cation includes, for example, magnesium, manganese and calcium, separately or in combination.
In U.S. patent No. 5714583 described methods purification of factor IX in solution, comprising the stage of application containing factor IX solution on anion-exchange resin, washing the anion-exchange resin solution with an electrical conductivity less than that needed for elution from the resin of factor IX, elution anion exchange resin first allanton with the formation of the first eluate, applying the eluate to a heparin or similar to heparin resin (for example, negatively charged matrix), elution of heparin or similar to heparin resin second allanton with the formation of the second eluate, the application of the second eluate and hydroxyapatite resin, and then elution hydroxyapatite resins third allentow with the formation of the third eluate containing the purified factor IX.
In U.S. patent No. 6322997 described a method of obtaining a polypeptide comprising an impact on containing the polypeptide composition of the reagent, binding or modifying the polypeptide, where the reagent is immobilized on a solid phase; and then the transmittance of the composition h is cut filter, bearing a charge opposite to that of the reagent in the composition, to remove from the composition of leached reagent.
In U.S. patent No. 6214984 described chromatography based on hydrophobic interactions at low pH for the purification of antibodies.
Specifically, this patent relates to a method of purification of antibodies from pollution, including the application contains antibodies and pollutant mixtures by column chromatography based on hydrophobic interactions and the elution of antibodies from the column buffer with a pH of approximately 2.5 to 4.5. Typically, the pH of the mixture applied to the column, approximately the same as eluting buffer.
U.S. patent No. 6121428 relates to a method for producing a polypeptide, comprising an impact on containing the polypeptide composition to bind or modifying the polypeptide reagent, where the reagent is immobilized on a solid phase; and then passing the composition through a filter carrying a charge opposite to that of the reagent in the composition to remove from the composition of leached reagent.
U.S. patent No. 5641870 relates to a method of purification of antibodies, where containing antibodies and contaminant mixture is subjected LPHIC, not necessarily at low salt concentrations. Antibody elute from the column in not communicating with her faction. At the stage of extraction, precipitation frozen cells resuspending is at room temperature in 20 mm MES buffer, pH of 6.0, containing 5 mm EDTA and 20 mm 4,4'-DTP, pre-dissolved in ethanol (3 l of buffer/kg cell sediment). Suspended cells destroy the double passage through the homogenizer Mantin Gaulin at a speed of from 5500 to 6500 pounds/inch2. The homogenate was adjusted to 0.25% (vol./about.) polyethylenimine (PEI) and dilute with an equal volume of purified water with a temperature of 2-8°C. Diluted homogenate was then centrifuged. Fragments of antibodies are found in the supernatant.
Historically, the immunoglobulin G (IgG) was purified from human serum and plasma (Putnam, ed.,The Plasma Proteinsvol. 1 (Academic Press, 1975)). The cleaning process often involves one or several stages of deposition. The most commonly used scheme to obtain the IgG represents the fractionation Cohen (Cohnet al.,J. Amer. Chem. Soc.,72: 465 (1950)). However, informed about other methods of deposition (Niederauer and Glatz,Advances in Biochemical Engineering Biotechnologyv. 47 (Springer-Verlag Berlin Heidelberg, 1992); Steinberg and Hershberger,Biochim. et Biophys. Acta,342: 195-206 (1974)). The first work on the purification of IgG from plasma using reference to ha cationic dye 6,9-diamino-2-ethoxyacrylate (USAN name, and here called ethacridine and also known under the names ETHODIN™ or RIVANOL™) printed Horsjsi and Smetana,Acta Med. Scand.,155: 65 (1956). In the subsequent decade was released a number of publications show the General ability 6,9-diamino-2-ethoxyacrylate to purify IgG and other proteins (Miller, Nature,184: 450 (1959); Steinbuch and Niewiarowski,Nature,186: 87 (1960); Neurath and Brunner,Experientia,25: 668 (1969)) from biological substances, such as plasma and the environment growth. Published application ethacridine to generate antibodies and other proteins from other sources. Cm. Tchernovet al.,J. Biotechnol.,69: 69-73 (1999); SU 944580 published on July 28, 1982; Franek and Dolnikova,Biotech-Forum-Eur,7: 468-470 (1990); EP 250288 published December 23, 1987; DE3604947 published August 20, 1987; Rothwellet al.,Anal. Biochem.,149: 197-201 (1985); Lutsik and Antonyuk,Biokhimiya,47: 1710-1715 (1982); and Aizenmanet al.,Mikrobiol-Zh.,44: 69-72 (1982).
In the first stage receiving the polypeptides of the microorganisms most frequently conducts the removal of solids, such as cells or cellular debris. It is important to understand the need for separation of the desired product present in the conditioned medium components with which it specifically interacts. When the protein of interest is charged positively, this leads to the binding of any present negatively charged molecules, thus making very difficult the protein purification traditional ways. Additional removal during this stage of contaminating soluble protein from the rough microbial extracts, for example homogenateE. colicould simplify further phase chromatography. T is some additional removal could be particularly useful for industrial production, reducing the size of the chromatographic columns and time of receipt.
The invention relates to cleaning, as specified in the claims.
Specifically, in one aspect the invention relates to a method of purification of the desired heterologous polypeptide from the broth or homogenate after microbial fermentation, in which the acquisition and dissolution of this polypeptide comprises adding to the broth or homogenate effective amount of a solution of 6,9-diamino-2-ethoxyacrylate (ethacridine) for the deposition of contaminating cells are the owners of substances in the conditions, when a large portion of the polypeptide remains soluble, and the allocation of the desired polypeptide from the broth or homogenate.
In another aspect the invention relates to a fermentation broth or homogenate microbial cells containing ethacridine and a polypeptide that is heterologous to the cells.
Adding ethacridine as precipitating means unexpectedly leads to a significant removal of debris of the owner, including proteins of the host. In this process, the majority of proteins of the host together with cellular debris goes into the sediment, and the polypeptide remains in purified supernatant. Increased when using ethacridine the purity of the purified extract leads to kumansenu volume of medium or resin for chromatography, necessary for the columns, thereby reducing the size needed for further treatment. It also leads to the exclusion of a particular stage(s) chromatography, which improves processing time and cost. In addition, this method leads to a stable starting materials and it can be used at neutral pH.
BRIEF DESCRIPTION of DRAWINGS
Figure 1 is a schematic illustration of the construction of plasmids pS1130 (one promoter) and pxCD18-7T3 (dual promoter)encoding associated latinboy clasp F(ab')2to CD18.
The figure 2 shows the inserted nucleic acid sequence (designated as Anti-CD18-TTC; SEQ ID No. 1) design pxCD18-7T3 dual promoter.
In figures 3A and 3B depicts the amino acid sequence (collectively designated as Anti-CD18-teak)encoded by two translational units in structure pxCD18-7T3 (SEQ ID No. 2 and 3), designated as STII + Anti-CD18 light chain (Fig. 3A) and STII + Anti-CD18 heavy chain (Fig. 3B), respectively. N-terminal secretory signal sequence STII underlined.
Figure 4 is a diagram of plasmid paTF130 (promotersphoA/phoA) and pxTF-7T3FL (promotersphoA/tacII)encoding IgG1 to tissue factor.
The figure 5 shows the inserted nucleic acid sequence (designated as Anti-TF-7T3FL.; SEQ ID No. 4)design pxTF-7T3FL with promoter phoA/tacII.
In figures 6A and 6B depicts the amino acid sequence (collectively designated as Anti-TF-7T3FL.)encoded by two translational units in structure pxTF-7T3FL (SEQ ID No. 5 and 6), denoted as STII + Anti-TF light chain (Fig. 6A) and STII + Anti-TF heavy chain (Fig. 6B), respectively. N-terminal secretory signal sequence STII underlined.
In figure 7 a schematic structure of ethacridine.
In figures 8A-8C shows the analysis of three supernatants after deposition accredidation in the gel by way of non SDS-PAGE with Coomassie blue staining blue. The deposition was carried out at different pH values as indicated for each lane. Paths, denoted as X, are cleared supernatant corresponding homogenateE. colii.e. F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF (figures 8A, 8B and 8C, respectively). The homogenates were diluted 4 times of 0.8% solution of ethacridine, i.e. to a final concentration of ethacridine in each experiment to 0.6%. In all samples before applying to the gel compensated volume. Therefore, if you have reached 100% of the certificates, the intensity of the bands should be comparable to extract (X). Arrows indicate the band of the product.
In figures 9A-9C shows an analysis of supernatants after deposition of ethacridine the DG gel method non SDS-PAGE with Coomassie blue staining blue. The deposition was carried out at various concentrations of ethacridine, as indicated on each track. Paths, denoted as X, are cleared supernatant corresponding homogenate of E. coli, i.e. F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF (figures 9A, 9B and 9C, respectively). pH F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF was 8.5, and 7.5 and 6.0, respectively. Specific conductance in samples was 3.2±0,2 MSM/see all samples before applying to the gel compensated volume. Therefore, if you have reached 100% of the certificates, the intensity of the bands should be comparable to extract (X). Arrows indicate the band of the product.
In figures 10A and 10B shows an analysis of two supernatants after dilution with water or accredidation respectively in the gel by way of non SDS-PAGE with Coomassie blue staining blue. The deposition was carried out at various levels of electrical conductivity. For this study used E. coli homogenates containing F(ab')2to CD18. The homogenate was diluted 4 times or water (figa) or 0.8% solution of ethacridine, i.e. to a final concentration of ethacridine in each experiment 0,6% (figure 10), and the pH was brought to 8.3. To change the electric conductivity of the samples was added NaCl at various concentrations in the range of 0-400 the M (as shown in the figures). Arrows indicate the band of the product.
The figure 11 shows a graph of the solubility of ethacridine with increasing concentrations of sodium chloride. Samples before determining the concentration of soluble ethacridine three hours incubated at room temperature. Empty symbols indicated a 1.2% solution of ethacridine, and the solid symbols - 0,6% solution. The solid line represents a 0.6% solution of ethacridine at pH 6.0, the dotted line represents a 1.2% solution of ethacridine at pH 6, the dotted line represents a 0.6% solution of ethacridine at pH 9, and the dashed line with dots represents a 1.2% solution of ethacridine at pH 9.
In figures 12A-12C shows the analysis of three supernatants after deposition accredidation in the gel by way of non SDS-PAGE with Coomassie blue staining blue. The deposition was carried out at elevated temperatures. Paths, denoted as X, are cleared supernatant corresponding homogenate of E. coll, i.e. F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF (figures 12A, 12B and 12C, respectively). The homogenate was diluted 4-fold to a final concentration of ethacridine 0.6%, while the pH for F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF brought to 8.5, and 7.5 and 6.0, respectively. The temperature and time of incubation are indicated on the hurah. Arrows indicate the band of the product.
Figure 13 is a diagram of the turbidity as a function of time for three different supernatants. Supernatant of the homogenate with F(ab')2to CD18, treated with 0.6% accredidation marked as shaded circles (4° (C) or an empty circle (21°C), and samples treated with 0.2% PEI, represented as shaded squares (4° (C) and empty squares (21°C). The supernatant obtained from purified homogenate with F(ab')2to CD18, which before the concentration was diluted with water, represented as shaded triangles (4° (C) and empty triangles (21°C). In all cases, the homogenate with F(ab')2to CD18 were diluted 4 times, and the pH was 7.2.
DETAILED DESCRIPTION AND PREFERRED IMPLEMENTATION
The expression "the broth or homogenate after microbial fermentation" refers to the broth, paste or extract, preferably resuspending obtained from microogranisms, which includes yeast, fungi and prokaryotes, such as bacteria, cultivated and which consume nutrients, regardless of the used vessel for culturing, for example, shake flask or fermentor. Preferably the broth or homogenate obtained from yeast or prokaryotes. Most preferably the broth Il is the homogenate is obtained from bacteria. Here the preferred homogenate. In some cases, if the solution has a very high electrical conductivity, it may be preferable to collect cells and their resuspendable, but in other cases it is preferable to apply the homogenate, such as the homogenate directly from the fermenter. Components of the broth or homogenate include cell debris, protein, host cell DNA, RNA, etc. So adding here lactate leads to selective deposition of proteins of the host cell and so on, providing the best degree of treatment than without the use of lactate.
The expression "conditions, when a large portion of the polypeptide remains soluble" refers to adding ethacridine in the broth or homogenate in quantities and at a temperature and electric conductivity, which prevents the precipitation of the broth or homogenate of mostly protein of interest. Preferably such condition prevents from precipitation approximately more than 60% of the polypeptide, more preferably approximately more than 70%, even more preferably approximately more than 75%, even more preferably approximately more than 80%, even more preferably approximately more than 85%, even more preferably approximately more than 90%, and most the e preferably approximately more than 95%. This degree of solubility is measured by an appropriate analysis, such as RP-HPLC, affinity chromatography, ELISA, RIA and the combination of SDS-PAGE and high-performance affinity chromatography (NRAS). The choice of analysis depends on such factors as the type of host cell and the resulting peptide.
"Bacteria" for the purposes of this description include eubacteria and archaebacteria. Preferred of them are eubacteria, including gram-positive and gram-negative bacteria. More preferred are gram-negative bacteria. One of the preferred types of bacteria areEnterobacteriaceae. Examples of bacteria belonging toEnterobacteriaceaeincludeEscherichia,Enterobacter,Erwinia,Klebsiella,Proteus,Salmonella,SerratiaandShigella. Other types of suitable bacteria includeAzotobacter,Pseudomonas,Rhizobia,VitreoscillaandParacoccus. Here the preferredE. coli. Suitable hostsE. coliincludeE. coliW3110 (ATCC 27325),E. coli294 (ATCC 31446),E. coliB andE. coliX1776 (ATCC 31537). These examples are illustrative and not limiting, and it is preferable W3110. You can also use mutant cells of any of the above bacteria. Naturally, you need to choose the appropriate bacteria, whereas re is literwest replicon in these bacterial cells. For example, as the owner accordingly, you can use the formsE. coli,SerratiaorSalmonellawhen to maintain replicon apply well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Regarding examples of suitable bacterial host cells, see further below.
As used here, the expression "cell", "cell line", "strain" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the original cell and cultures derived from it, regardless of the number of transplants. It is also understood that all progeny may not be absolutely identical in DNA content, due to deliberate or accidental mutations. Here include mutant progeny that have the same function or biological activity, as shown for the original and transformed cells. When talking about the special designations, it will be clear from the context.
As used here, "polypeptide"generally refers to peptides and proteins from any cell source, consisting of more than about ten amino acids. "Heterologous" polypeptide are polypeptides alien to the used host cell, such as a human protein, the product of the dummy E. coli. Although heterologous polypeptide may be prokaryotic or eukaryotic, preferably it is eukaryotic, more preferably mammals and most preferably humans. Preferably it is produced by recombinant means or by recombinant polypeptide.
The polypeptide is produced and dissolved in the fermentation broth or homogenate, meaning that he obtained in the broth or homogenate is or is already in a soluble fraction obtained in the process of production, or is in the insoluble fraction, or the form or phase of that process and bring into contact with the solvent medium, such as chaotropes tool (for example, urea or guanidine) or detergent (such as sodium dodecyl sulphate (SDS)), with regenerating means (such as dithiothreitol or beta mercaptoethanol)to dissolve the polypeptide. "Soluble", "dissolved", "dissolution", "diluted" or "dilution" in the sense used here means that the polypeptide after centrifugation is in the supernatant and not in the solid fraction. You can determine the sedimentation rate or degree of solubility, for example, by using appropriate analyses, as noted above.
Examples of mammalian polypeptides include molecules like, n is an example, renin, a growth hormone, including human growth hormone; bovine growth hormone; a factor that stimulates growth hormone; parathyroid hormone; thyrostimulin hormone; lipoproteins; 1-antitripsin; A-chain, insulin B-chain of insulin; proinsulin; thrombopoietin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and the factor a background of Villebranda; factors, anti coagulation, such as protein C; trially natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue plasminogen activator (t-PA) and its variants, such as RETEVASE™ and TNKASE™; bombezin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and-beta; antibodies to the domain(s) ErbB2, such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697), communicating with a region in the extracellular domain of ErbB2 (e.g., one or more residues in the region of ErbB2 from about residue 22 to about 584 inclusive), enkephalinase; serum albumin, such as human serum albumin; inhibitory Mullerova ducts substance; A-chain relaxin; B-chain relaxin; prolactin; mouse associated with gonadotropin peptide; a microbial protein, such as beta-lactamase; D. the Casa; inhibin; activin; growth factor vascular endothelial (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; neurotropic factor, such as neurotrophic factor brain (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF, cardiotrophin (factor hipertrofia heart), such as cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF)such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and-II (IGF-I and IGF-II); des(1-3)-IGF-I (IGF-I and brain), proteins that bind insulin-like growth factor; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factors; immunotoxins; morphogenic protein bone (BMP); an interferon such as interferon-alpha, -beta and-gamma; serum albumin such as human serum albumin (HSA) or bovine serum albumin (BSA); colony stimulating factors (CSFs)such as M-CSF, GM-CSF and G-CSF; interleukins (IL), for example, from IL-1 to IL-10, antibody to HER-2; Apo2 ligand; superoxide dismutase; receptors of T-cells; surface membrane proteins; complementability stimulator hemolysis; viral antigen such as, for example, part of the membrane of HIV; the salvage proteins; receptors "homing"; Addressin; regulatory proteins; antibodies; and fragments of any of the above polypeptides.
Preferred here polypeptides include human serum albumin (HSA), 2C4, tissue factor antibody to tissue factor antibody to CD20, antibody to HER-2, heregulin, antibody to IgE antibody to CD11a, the antibody to CD18, VEGF and its receptors and antibodies thereto, such as rhuFab V2 and AVASTIN™, growth hormone and its variants, such as hGH, receptors of growth hormone, a protein that stimulates growth hormone (GHRP), LIV-1 (EP 1263780), TRAIL, the tumor necrosis factor (TNF) and antibodies thereto, the TNF receptor and the bound antibodies, TNF receptor-IgG, associates with TNF receptor factors (TRAF) and their inhibitors, factor VIII, domain B of factor VIII, interferon, such as interferon-gamma, transforming growth factors (TGF)such as TGF-beta antibody to TGF, such as antibody to TGF-beta, activin, inhibin, antibody to activin, antibody to inhibin, activators tissue plasminogen and their variants, such as t-PA, RETEPLASE™ and TNKase, antibodies to Fas, the ligand Apo-2; inhibitor of ligand Apo-2; receptor for Apo-2, Apo-3, apoptosis factors, Ced-4, DcR3, the death receptor and antibody agonists (DR4, DR5), lymphotoxin (LT), prolactin, prolactin receptor, proteins SOB, WISP (induced wnt secreted proteins), neurotoxin-3 (NT-3), nerve growth factor (NGF) and antibody to NGF, Ankasa, the antigen of the hepatitis b virus antigen of herpes simplex virus, leptin, insulin-like growth factors (IGFs), such as IGF-1 and IGF-2 binding proteins and their receptors, such as IGFBP-1 to IGFBP-6, insulin, fibroblast growth factors (FGF)such as FGF-17, a protein Toll, TIE ligands, CD40 and antibody to CD40, immunoadhesin, subtilisin, a growth factor for hepatocytes (HGF), thrombopoietin (TPO), the interleukins, such as IL-2, IL-12, IL-17, IL-22, IL-8, IL-9, and antibodies thereto, and which are specific for a prostate tumor antigen (PSCA).
Examples of antibodies that bind HER2 include 4D5, 7C2, 7F3 and 2C4, and their humanized variants, which include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 as described in table 3 of U.S. patent 5821337; and humanized mutant 2C4№№ 560, 561, 562, 568, 569, 570, 571, 574 or 56869, as described in WO01/00245. 7C2 and 7F3 and humanized variants described in WO98/17797.
Examples of antibodies that bind the CD20 antigen include "C2B8", now called "Rituximab" ("RITUXAN®") (U.S. patent No. 5736137); labeled with yttrium- murine antibody 2B8, designated "Y2B8" (U.S. patent No. 5736137); murine IgG2a "B1", not necessarily labeled131I obtaining antibodies131I-B1 (BEXXAR™) (U.S. patent No. 5595721); murine monoclonal antibody "1F5" (Presset al.,Blood,69(2): 584-591 (1987)); antibody "hybrid 2H7" (U.S. patent No. 5677180) and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available from the International Leukocyte Typing Workshop (Valentineet al.In:Leukocyte TypingIII (McMichael, Ed., p. 440, Oxfor University Press (1987)).
Preferred polypeptides are 2C4, antibody to tissue factor antibody to CD20, antibody to HER-2, heregulin, antibody to IgE antibody to CD11a, the antibody to CD18, an antibody to VEGF, such as rhuFab V2, hGH, GHRP, LIV-1, TRAIL, antibodies to TNF and TNF receptor and related antibodies, inhibitors TRAF, TNF receptor-IgG, factor VIII, domain B of factor VIII, interferon-gamma, TGF-beta and antibody to TGF-beta, activin, inhibin, antibody to the activin, antibody to inhibin, t-PA, TNK, antibodies to Fas, the ligand Apo-2; inhibitor of ligand Apo-2; receptor for Apo-2, Apo-3, DcR3, the death receptor and antibody agonists (DR4, DR5), lymphotoxin (LT), prolactin, prolactin receptor, WISP, antibody to NGF, NGF, NT-3, antibody to IL-8, antibody to IL-9. IL-17, IL-22, Tnkase, GHRP, an antigen of the hepatitis b virus antigen herpes simplex virus, leptin, IGF-1, IGFBP1-6, insulin, FGF-17, a protein Toll, TIE ligands, CD40, immunoadhesin, subtilisin, HGF and TPO.
More preferred polypeptides are 2C4, antibody to tissue factor antibody to CD20, antibody to HER-2, antibody to IgE antibody to CD11a, the antibody to CD18, an antibody to VEGF, such as rhuFab V2, hGH, LIV-1, TRAIL, antibodies to TNF and TNF receptor and related antibodies, TNF receptor-IgG, factor VIII, domain B of factor VIII, interferon-gamma, TGF-beta and antibody to TGF-beta, activin, inhibin, antibody to activin, antibody to inhibin, t-PA, TNK, ligand Apo-2; inhibitor of ligand Apo-2; receptor for Apo-2, Apo-3, DcR3, the death receptor and antibody agonists (R4, DR5), WISP, inhibitors TRAF, antibody to NGF, NGF, NT-3, antibody to IL-8, antibody to IL-9. IL-17, IL-22 antibody to TGF, Tnkase, GHRP, an antigen of the hepatitis b virus antigen herpes simplex virus, leptin, IGF-1, IGFBP1-6, insulin, FGF-17, a protein Toll, TIE ligands, antibody to CD40, HGF and TPO.
Particularly preferred polypeptides are recombinant polypeptides, more preferably antibodies, including monoclonal antibodies and humanized antibodies. Such antibodies can be a full-length antibodies or antibody fragments. More preferably, these antibodies are human or humanitarianism antibodies. They include, for example, particularly preferred polypeptides 2C4, Fab'2 and a full-sized antibodies against tissue factor antibodies against CD20 antibodies against HER-2, antibodies against IgE, antibodies against CD11a, Fab'2 and a full-sized antibodies against CD18, full-size antibodies and rhuFab V2 against VEGF, LIV-1, DR4, DR5 and TRAIL.
Even more preferably, the antibody was an antibody against IgE, the antibody against CD18, anti-VEGF antibody, antibody against tissue factor, 2C4, antibody against Her-2, an antibody against CD20, antibody against CD40 or antibody against CD11a. Antibody fragments covered by the definition of the polypeptide, preferably contain a light chain, and more preferably light chain Kappa. The same is preferred fragments include, for example, Fab, Fab', F(ab')2or protein associated latinboy zipper (LZ) F(ab')2and most preferably represent F(ab')2.The most preferred antibodies are F(ab')2against CD18 F(ab')2against tissue factor, a full-sized antibody against tissue factor and the anti-VEGF antibody.
Here, the term "antibody" is used in the broadest sense, and specifically it refers to intact monoclonal antibodies, polyclonal antibodies, antibodies with multiple specificity (for example, antibodies with dual specificity) formed from at least two intact antibodies and fragments of antibodies, provided that they exhibit the desired biological activity.
Here, the term "monoclonal antibody" refers to an antibody obtained from a combination of substantially homogeneous antibodies, i.e constituting the aggregate of individual antibodies are identical except for possible natural mutations that may be present in small quantities. Monoclonal antibodies are highly specific, aimed at one antigenic site. In addition, in contrast to the preparations of polyclonal antibodies, which include different antibodies against different determinants (epitopes), each monoclonal ant the body is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can synthesize uncontaminated by other antibodies. Additive "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and it should not be seen as an antibody, which requires obtaining any particular way. For example, monoclonal antibodies for use according to the invention can be obtained by the hybrid method, first described by Koehleret al.,Nature,256: 495 (1975), or they can be obtained by means of recombinant DNA (see, for example, U.S. patent No. 4816567). "Monoclonal antibodies" also can be isolated from phage libraries of antibodies using methods described, for example, Clacksonet al.,Nature,352: 624-628 (1991) and Markset al., J. Mol. Biol.,222: 581-597 (1991).
The monoclonal antibodies herein include, in particular, "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a specific class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, and the e fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. patent No. 4816567 and Morrisonet al.,Proc. Natl. Acad. Sci. USA,81: 6851-6855 (1984)). Interested chimeric antibodies herein include "primaryservername" antibodies comprising antigennegative sequence of the variable domain obtained from a Primate, non-human (e.g., Old world monkey, APE etc) and sequences of human constant region.
"Antibody fragments" include a portion of an intact antibody, preferably including his antigennegative or variable region. Examples of fragments of antibodies include Fab, Fab', F(ab')2and Fv fragments; di-antibodies; linear antibodies; single-stranded molecules of antibodies and antibodies with multiple specificity generated from the fragment(s) of antibodies.
"Intact" antibody is an antibody containing antigennegative variable region and the constant domain of the light chain (CL) and the constant domains of the heavy chain, CH1, CH2 and CH3. The constant domains may be a natural sequence constant domains (e.g., human constant domains with the natural sequence or variant of the amino acid sequence. Preferably intact the e antibody has one or more effector functions.
"The effector functions of antibodies are types of biological activities attributable to the Fc region of an antibody (Fc region with the natural sequence or a Fc region with a change in amino acid sequence). Examples of effector functions of antibodies include the binding of C1q-dependent complement cytotoxicity, receptor binding to Fc-dependent antibody-mediated cell cytotoxicity (ADCC), phagocytosis, decreasing regulation of cell surface receptors (e.g. B cell receptor; BCR), etc
Depending on the amino acid sequence of the constant domain of their heavy chains of intact antibodies can be classified according to different "classes". There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM heavy chains, marked α, δ, ε, λ and μ sootvetstvenno. Classes λ and α additionally divided into subclasses on the basis of relatively minor differences in sequence and function CHfor example, people expressed the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.
"Dependent antibody-mediated cell cytotoxicity" and "ADCC" refer to mediated cell reaction in which nonspecific toxic the e cells, expressing Fc receptors (FcR) (e.g., natural killer cells (NK), neutrophils, and macrophages) recognize bound antibody on the target cell, and further cause lysis of the target cells. Primary cells mediating ADCC, NK cells, Express only FcRIII, whereas monocytes Express FcRI, FcRII and FcRIII. Data about the expression on hematopoietic cells FcR are summarized in table 3 on page 464 in Ravetch and Kinet,Annu. Rev. Immunol.,9: 457-492 (1991). To assess ADCC activity of specific molecules can analyze ADCCin vitrofor example, such as described in U.S. patent No. 5500362 or 5821337. Suitable for such analysis effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additionally, ADCC activity of specific molecules can be estimatedin vivofor example, in animal models, for example, as described in Clyneset al.Proc. Natl. Acad. Sci. USA95: 652-656 (1998).
"Human effector cells are leukocytes expressing one or more FcR and perform effector functions. Preferably, the cells Express at least FcRIII and perform effector function is ADCC. Examples of human leukocytes mediating ADCC include mononuclear cells of peripheral blood (PBMC), natural killer cells (NK), monocyte is, cytotoxic T cells and neutrophils, are preferred where PBMC and NK-cells. Effector cells can be isolated from their natural sources, e.g. from blood or PBMC, as described here.
"Natural antibodies", as a rule, are heterotetrameric glycoproteins weight of approximately 150000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each easy part is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains in different isotypes of immunoglobulins varies. Each heavy and light chain also has regularly spaced megamachine disulfide bridges. Each heavy chain at one end has a variable domain (VH) with the following next constant domains. Each light chain at one end has a variable domain (VL), and at the other end of the constant domain. Constant domain of the light chain is aligned with the first constant domain of the heavy chain and the variable domain of the light chain is aligned with the variable domain of the heavy chain. I believe that between the variable domains of light and heavy chains of communication form a specific amino acid residues.
The term "variable" refers to the fact that certain parts of the variable domain is in the antibodies differ significantly in sequence and are used for binding and recognition of each particular antibody for its particular antigen. However, the variability is distributed throughout the variable domains of antibodies unevenly. It is concentrated in three segments called hypervariable region located in the variable domains of light and heavy chains.
The most highly conserved region of variable domains are called the frame regions (FR). Each variable domain of the natural heavy and light chain contains four FR, predominantly host configuration beta layer, corresponding hypervariable regions, which form loops connecting, and in some cases forming part of the structure of the beta-layer. Hypervariable region in each chain are held together in close proximity by the FRs and, with the hypervariable regions of the other circuit involved in the formation antigennegative site of antibodies (see Kabatet al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains in binding the antibody to the antigen is not directly involved, but perform a variety of effector functions, such as loss of antibody in the precipitate-mediated cell cytotoxicity (ADCC).
Here, the term "hypervariable region" refers to amino acid residues of the antibody responsible for binding antigen. Hypervariable region, as the rights of the lo, contains amino acid residues from a "complementarity determining region"or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the variable domain light chain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of the heavy chain; Kabatet al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the variable domain light chain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable domain of the heavy chain; Chothia and Lesk,J. Mol. Biol.,196: 901-917 (1987)). The remains of the "framework region" or "FR" represents the remains of the variable domain of the different residues from a hypervariable region, as defined here.
Cleavage of antibodies with papain leads to the formation of two identical antigen binding fragments, called " fragments "Fab", each with one antigennegative plot and residual fragment "Fc", whose name reflects its ability to easily crystallize. Treatment with pepsin results in the formation of fragment F(ab')2with two antihistamine areas and still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment that contains a complete antipersonnel and antigennegative plot. This region consists of a dimer of one heavy chain and variable home is and one light chain, United non-covalent bonds. In this configuration three hypervariable region of each variable domain interact to define antigennegative area on the surface of the dimer VH-VL. Together the six hypervariable regions provide the binding specificity of antigen-antibody. However, even a single variable domain (or half of an Fv, containing only three hypervariable region that is specific for an antigen) has the ability to recognize and bind antigen, although with less than the full binding site affinity.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fragments, Fab' differ from Fab fragments by the addition of a few residues at the carboxyl end of the CH1 domain of the heavy chain comprising one or more cysteine residues of the hinge region of the antibody. Fab'-SH here is a designation for Fab'in which the cysteine residue(s) of the constant domains bear at least one free Tilney group. Antibody fragments F(ab')2the source was given as pairs of Fab fragments' with the hinge cysteine residues between them. Also known for other types of chemical compounds fragments of antibodies.
The "light chains" of antibodies of any species of mammal on the basis of and inability sequences of their constant domains can be attributed to one of two clearly distinct types, called Kappa (6) and lambda (8).
Fragments of antibodies, "single-chain Fv" or "scFv" contain domains of the antibody VHand VLwhere these domains are in the same polypeptide chain. Preferably, the Fv polypeptide between domains VHand VLfurther comprises a polypeptide linker, enabling scFv to form the desired binding of the antigen structure. For a review of scFv, see Pluckthunin The Pharmacology of Monoclonal Antibodiesvol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315. In WO93/16185, U.S. patent No. 5571894 and U.S. patent No. 5587458 described fragments of scFv antibodies to ErbB2.
The term "di-antibody" refers to small fragments of antibodies with two antihistamine areas where the fragments contain the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) in the same polypeptide chain (VH- VL). Through the use of a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific plot. Di-antibodies are more fully described in, for example, in EP 404097; WO 93/11161 and Hollingeret al.,Proc. Natl. Acad. Sci. USA,90: 6444-6448 (1993).
"Humanized" forms of antibodies, non-human (e.g. rodent)are ant the body, contain minimal sequence derived from an antibody which does not belong to man. Typically, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a hypervariable region of the recipient are replaced by residues from having the desired specificity, affinity and capacity hypervariable region (donor antibody) species that are not human, such as mouse, rat, rabbit or non-human primates, non-human. In some cases, the relevant residues not belonging to the person, replace the remnants of the framework region (FR) of a human immunoglobulin. Furthermore, humanized antibodies may contain residues that are not present in the recipient or the donor antibody. Data modification is performed to further improve the characteristics of the antibodies. As a rule, humanitariannet antibody contains essentially the entire at least one, and typically two, variable domain, in which all or substantially all of the hypervariable loops correspond to loops immunoglobulin derived not from man, and all or substantially all of the FR represent the FR sequence of the human immunoglobulin. Humanitariannet antibody optionally also contains at least part of a constant about the Asti immunoglobulin (Fc), typically, Fc human immunoglobulin. For additional details, see Joneset al.,Nature,321: 522-525 (1986); Riechmannet al.,Nature,332: 323-329 (1988) and Presta, Curr. Op. Struct. Biol.,2: 593-596 (1992).
"Isolated" antibody is an antibody that has been identified and separated and/or from a component of its natural environment. Contaminant components of its natural environment are substances that prevent diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones and other protein and non-protein soluble substances. In the preferred implementation of the antibody must be cleaned (1) to more than 95% by weight of antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of the sequencing machine with rotating cups or (3) to homogeneity with SDS-PAGE in reducing or non conditions using color Kumasi blue or, preferably, silver. The selected antibody includes antibodyin situwithin recombinant cells, since at least one component of the natural environment antibody is not present. However, as a rule, is dedicated antibody receive through at least one stage of cleaning.
"Lacinova clasp is a peptide (often about 20 to 40 amino acid residues in length) with multiple repeating amino acids, in which every seventh amino acid is Lazenby balance. Such sequences latinovich fasteners form an amphipatic alpha-helix with latinoware residues located in the hydrophobic side for the formation of the dimer. Examples latinovich buckles here include lacinova clasp Fos-Jun (O'sheaet al.,Science,245: 646 (1989)), which can be used for the formation of heterodimers (for example, antibodies with dual specificity); lacinova zipper GCN4 of yeast (Landschulzet al.,Science,240: 1759-1764 (1988)), which can be used for the formation of homodimers (e.g., monospecific antibodies), and lacinova fasteners located in other DNA binding proteins, such as C/EBP and c-myc, and any of their options.
The expression "control sequences" refers to DNA sequences necessary for the expression of functionally related coding sequences in a particular organism, the host. Control sequences that are suitable for bacteria, include a promoter, optionally a sequence operator and the binding site of the ribosome.
Nucleic acid is functionale related", when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA predpolagavshegosja or secretory leader sequence functionally linked to DNA polypeptide if it is expressed as probelec involved in secretion of the polypeptide; a promoter functionally linked to the coding sequence if it affects the transcription of the sequence; or binding of the ribosome a site linked to the coding sequence if it is to contribute to the broadcast. As a rule, "functionally linked" means that the associated DNA sequence is continuous and in the case of a secretory leader sequence is continuous at the stage of reading. Binding is carried out, for example, by ligating by suitable restriction sites. If such sites do not exist, using synthetic oligonucleotide adaptors or linkers in accordance with the usual practice.
The term "receipt" of a polypeptide, usually means getting polypeptide, without cells in which it is produced.
"Pollution from host cells" refers to the contamination of host proteins and other biomolecular pollutants, such as DNA and cellular debris, in a fermentation broth or homogenate.
The method of the embodiment of the invention
In one aspect the invention relates to a method of purification of the desired heterologous polypeptide from the broth or homogenate after microbial fermentation, in which it is produced and dissolved. The polypeptide can be obtained in the soluble fraction or may be insoluble (for example, produced in the insoluble fraction, phase or form) and therefore result in contact or treated so as to dissolve the polypeptide. If the polypeptide is produced in an insoluble state, it before adding ethacridine dissolved by exposure to or brought into contact with the solvent vehicle (as described above), for example, adding this tool to the fraction containing insoluble polypeptide. Preferably, the polypeptide was already in the soluble fraction. This method involves adding to the broth or homogenate effective amount of a solution of ethacridine for the deposition of contaminants from host cells contained in the broth or homogenate. This addition is performed in conditions in which most proteins remain soluble. In the next stage of the broth or homogenate containing cellular debris, proteins, host cells, DNA, RNA and the like, produce the desired polypeptide.
Since most proteins Ho who Aina, besieged by accredidation, carry a negative charge, whereas the polypeptide of interest borne on the surface of the positive charge, preferably the polypeptide of interest had a pI greater than the average pI of the proteins of the host contained in the contamination of the host cell so that it was possible to separate the supernatant from the precipitated proteins of the host. This average pI can be determined by two-dimensional gel protein owner, where, for example, for a range of bands pI from about 7.5 to 5.0, the average is 6.25. An alternative to this definition can be applied only isoelectrofocusing (representing the first dimension of two-dimensional gel), as well as chromatofocusing and calculations on amino acid composition. The most preferred polypeptides are polypeptides with a pI of at least about 7, and preferably approximately 7-10.
Preferred polypeptides for use stated above.
The applied concentration of ethacridine depends on the number of negative charges in the solution on the surface of most cell impurities present in the solution. Therefore, the concentration of ethacridine depends on at least the number of such impurities from host cells as the concentration of DNA and protein owner in Rast is the op. The higher the concentration of protein owner and DNA in the homogenate, the more ethacridine necessary. Therefore, the more negatively charged components are available for the formation of a complex with accredidation and deposition thus, the greater the number of ethacridine need to maximize sediment. The preferred concentration of ethacridine, as a rule, is more than about 0.1% wt./volume. More preferred is a concentration of ethacridine approximately 0.1-5%, even more preferably about 0.4-5%, and most preferably 0.6 to 5% wt./volume.
As a rule, the smaller the conductivity of the solution, when conducting the deposition, the more effective is the purification of the polypeptide from the cell debris and DNA. Conductivity can be controlled, for example, the amount of salt in the homogenate or broth, or by dilution of the homogenate or broth with water or other suitable solvent. Preferably, the electrical conductivity of the broth or homogenate after adding ethacridine was less than approximately 16 millisiemens (MSM), more preferably about 1-15 MSM, even more preferably about 1-10 MSM, and most preferably - PR is approximately 1-5 MSM.
The electrical conductivity of the solution during the deposition depends at least partly on the type present in it salt. Halides (for example chlorides or bromides) are not preferred anions for salt, but if they are present, they are preferred in a concentration of less than approximately 100 mm in the solution before adding ethacridine and below about 50 mm after adding ethacridine. Some illustrative salts for use herein include buffer salts, TRIS, MES, MOPS, acetate and citrate. The concentration of salts present may not be greater than the amount, which may upset ethacridine. The exact number depends on the type of salt and the stoichiometric relationship between salt and accredidation, and the limit is the lower limit of the stoichiometric ratio, i.e. the lower limit means a greater amount of salt relative to ethacridine.
the pH of the broth or homogenate after adding ethacridine depends on the pI of the polypeptide, the number of negative surface charges on the polypeptide, the amount of contaminants from the host cells in solution and the concentration of ethacridine. the pH is preferably not greater than the pI of the polypeptide. Typically, the pH range is approximately 4-10; however, for the effective precipitation polluted by the th of host cells the pH of the broth or homogenate after adding ethacridine preferably does not exceed approximately 9, as ethacridine becomes less charged at pH above this, with a preferred range of about 4-9. More preferably the pH of the broth or homogenate after adding ethacridine approximately 5-9 and even more preferably about 6-9. The more negative surface charge on the polypeptide, the lower pH in this range, with the preferred pH range for such polypeptides of approximately 6-7.
The broth or homogenate after adding ethacridine optional for some period of time incubated at elevated temperatures. To raise the temperature of and for what period of time depends on many factors, including the type of interest protein type, if any, modifications of the protein of interest that occur when exposed to high temperatures during this period in the process, and the like, for Example, for cleaning F(ab')2to tissue factor preferred elevated temperature, whereas for full-length antibody, preferably not apply heat or to ensure that the temperature did not rise above about 25°C. Taking into account these factors, as a rule, the temperature of the broth or homogenate after adding ethacridine approximately from room temperature the temperature to about 70° C, more preferably from about room temperature to about 65°C with a retention time of approximately 1-60 minutes. If the temperature must be raised, the preferred range is from about 50 to 65°C with a retention time of approximately 1-60 minutes.
In another aspect the invention relates to compositions of matter, representing a fermentation broth or homogenate from microbial cells containing ethacridine and a heterologous polypeptide. Preferably the polypeptide is dissolved in such a broth or homogenate. Cells, the polypeptide concentration and conditions for broth or homogenate described above. The degree of dissolution of the polypeptide can determine the appropriate form of analysis, such as the above analyses. The cultivation parameters apply and receive polypeptide carried out in the usual way, such as the procedure described below.
A. Selection of nucleic acid and its modification
Although these polypeptides, such as an antibody, can be obtained from any source (e.g., peptide cleavage of intact antibodies), it is preferable to obtain them by recombinant means. Encoding the desired polypeptide, nucleic acid, respectively, is an RNA, cDNA or genomic DNA from any source, in terms of the AI that it encodes the desired polypeptide(s). Methods for selection of suitable nucleic acids for expression of heterologous polypetides (including their variants) in microbial hosts are well known. The selection of suitable nucleic acid to obtain a non-antibody polypeptides in microbial cell culture are well known in this field.
If you get a monoclonal antibody encoding a monoclonal antibody DNA is easy to isolate and identify the sequence using conventional procedures (e.g., using oligonucleotide probes capable of specific binding to genes encoding the heavy and light chains of murine antibodies). For this DNA is the preferred source are hybridoma cells. After DNA extraction can be placed in expressing vectors, which are then transformed into microbial cells-owners, here for the organization of the synthesis of monoclonal antibodies in the recombinant cell host. Review articles on recombinant expression of the coding DNA antibodies in bacteria include Skerraet al.,Curr. Opinion in Immunol.,5: 256-262 (1993) and Pluckthun,Immunol. Revs.,130: 151-188 (1992).
In this area describes how humanitarian non-human antibodies. Preferably humanitariannet antibody has one or more amino acid residues introduced into it employee group from, not a person. These non-human amino acid residues are commonly referred to as "imported" residues, which, as a rule, taken from the variable domain for "import". Humanitarian essentially can be performed according to the method of Winter and colleagues (Joneset al.,Nature,321: 522-525 (1986); Riechmannet al.,Nature,332: 323-327 (1988); Verhoeyenet al.,Science,239: 1534-1536 (1988)), by replacing sequences of the hypervariable region for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibody is a hybrid antibodies (U.S. patent No. 4816567), where the corresponding sequences of species that are not human, being replaced with less than an intact human variable domain. In practice, humanized antibodies, as a rule, is a human antibodies in which some hypervariable residues region and possibly some FR residues replaced by residues from analogous sites of antibodies rodents.
The choice of human variable domains of heavy and light chains for use in the creation of humanized antibodies is very important to reduce antigenicity. In the so-called method of "optimal approximation of the sequence of the variable domain of the antibody of the rodent is miniroot in relation to the full library of known sequences of human variable domains. Then the human sequence that is closest to the sequence of the rodent, is chosen as a framework region (FR) for gumanitarnogo antibody (Simset al.,J. Immunol.,151: 2296 (1993); Chothiaet al.,J. Mol. Biol.,196: 901 (1987)). In another method used concrete frame region, derived from a consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same frame can be used for several different humanized antibodies (Carteret al.,Proc. Natl. Acad. Sci. USA,89: 4285 (1992); Prestaet al.,J. Immunol.,151: 2623 (1993)).
In addition, it is important that antibodies humanitarianly with retention of high affinity towards the antigen and other favorable biological properties. To achieve this objective, the preferred method humanized antibodies produced by the process of analysis of the source sequences and various abstract humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulins, generally available and known to experts in this field. Available computer programs that illustrate and demonstrate probable three-dimensional conformational structures of selected candidate immunoglobulin serial is Inesta. The study of these images allows to analyze the possible role of the residues in the functioning of the immunoglobulin sequences of the candidate, i.e. to analyze the remains, affecting the ability of selected candidate immunoglobulin to bind antigen. This way, FR residues of the recipient and sequences to import, you can select and combine to achieve the desired characteristics of the antibodies, such as increased affinity for the antigen(s)target(s). In General terms, the remains of the hypervariable region are directly and most substantially involved in the effect on binding to the antigen.
Proposed various forms gumanitarnogo antibodies or affinity Mature antibodies. For example, humanitariannet antibody or affinity Mature the antibody can be an antibody fragment such as Fab, which is optional anywhereman with one or more guide means for receiving immunoconjugate. Alternative humanitariannet antibody or affinity Mature the antibody can be an intact antibody, such as an intact IgG1 antibody.
Fragments, Fab'-SH can be directly retrieved fromE. coliand chemically bind with the formation of fragments F(ab')2(Carteret al.,Bio/Technology,10: 163-167 (1992)). Another approach fragments F(ab' 2you can select directly from a culture of the recombinant host cells. Practitioners demonstrate other ways to obtain fragments of antibodies. In other implementations, the selected antibody is a single-chain Fv fragment (scFv) (WO 93/16185; U.S. patent No. 5571894 and 5587458). The antibody fragment may also be a "linear antibody", e.g., as described in U.S. patent No. 5641870. Such fragments, linear antibodies can be a monospecific or be antibodies with dual specificity.
Antibodies with dual specificity are antibodies, specific binding at least two different epitopes. Illustrative antibodies with dual specificity can communicate with two different epitopes of the protein Dkk-1. Antibodies with dual specificity can be obtained as full-length antibodies or antibody fragments (for example, F(ab')2antibodies with dual specificity).
According to another approach, the variable domains of the antibodies with the desired specificnosti binding (areas of interaction of the antibody-antigen) merge sequences of the constant domains of immunoglobulins. The fusion preferably is carried out with a constant domain of the heavy chain of immunoglobulin containing at least part of the hinge region regions CH2 and CH3. Suppose the equipment to be included with the first constant region of the heavy chain (CH1), containing the site necessary for binding to the light chain present in at least one of the mergers. DNA encoding the Association of the heavy chains of immunoglobulins and, if desirable, of immunoglobulin light chain, are inserted into separate expressing vectors and cotransfected in a suitable bacterial organism-owner. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in the implementation, when used in the construction of unequal relationships of three polypeptide chains provide optimal outputs. However, when the expression of at least two polypeptide chains in equal ratios results in high outputs or when the relationship does not matter, one expressing the vector it is possible to insert the coding sequences for two or all three polypeptide chains.
In the preferred implementation of this approach, antibodies with dual specificity consist of a hybrid heavy chain immunoglobulin with the first specific binding site on the same branch and hybrid pair of heavy chain-light chain (providing a second specific binding site) on the other branch. Found that this asymmetric structure facilitates the separation of the desired compounds with dual specificity from unwanted combination of the immunoglobulin chains, because the presence of the light chain of immunoglobulin in only half of the molecule with dual specificity facilitates the path selection. This approach is described in WO 94/04690. For more details about how to obtain antibodies with dual specificity, see, for example, Sureshet al.,Methods in Enzymology,121: 210 (1986).
According to another approach described in U.S. patent No. 5731168, it is possible to construct the surface interaction between a pair of antibody molecules to maximize the percentage of heterodimers allocated from the culture of recombinant cells. The preferred surface interaction contains at least part of the domain CH3 constant domain of the antibody. In this way, the side chain of one or more amino acids of the surface of interaction between the molecules of the first antibody replace larger side chains (e.g. tyrosine or tryptophan). At the interface of the second antibody molecule by replacing large side chains of amino acids with smaller side chains (e.g., alanine or threonine) create compensatory "cavities" of identical or similar with a large side chain(s) size. This provides a mechanism to increase the output of heterodimers compared to other unwanted end-products such as homodimers.
Antibodies with dual specificity in luchot cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in heteroconjugate may be associated with Avidya, and the other with Biotin. Such antibodies, for example, proposed to direct the immune system cells to unwanted cells (U.S. patent No. 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be obtained using any suitable cross-linking methods. Suitable cross-linking means well known in this area and along with a number of methods for cross-linking are described, for example, in U.S. patent No. 4676980.
Also the literature describes methods for producing antibodies with dual specificity of the fragments of antibodies. For example, antibodies with dual specificity can be obtained with the use of chemical binding. Brennanet al.,Science,229: 81 (1985) describe the way in which the intact antibody proteoliticeski split with obtaining fragments F(ab')2. These slices are in the presence of complexing agents dithioles of sodium arsenite to stabilize neighboring dithioles and prevent the formation of intermolecular disulfide bonds. Then the resulting fragments Fab' is converted into derivatives of dinitrobenzoate (TNB). One of the derivatives of Fab'-TNB then turn back in the Fab'-thiol by restoring mercaptoethylamine and mixed with ecvim the regular number of other derived Fab'-TNB with the formation of antibodies with dual specificity. The obtained antibodies with dual specificity can be used as tools for selective immobilization of enzymes.
In addition, fragments of Fab'-SH can be directly retrieved fromE. coliand chemically bind with the formation of antibodies with dual specificity (Shalabyet al.,J. Exp. Med.,175: 217-225 (1992)).
Also describes various methods for obtaining and selection of antibodies with dual specificity directly from a culture of recombinant cells. For example, antibodies with dual specificity obtained with the use of latinovich fasteners (Kostelnyet al.,J. Immunol.,148: 1547-1553 (1992)). Peptides with latinoware clasps of proteins Fos and Jun bind with parts of the Fab' of two different antibodies by gene fusion. Homodimeric antibodies restore in the hinge region to form monomers and then re-oxidized with the formation of heterodimeric antibodies. This method can also be used to obtain homodimeric antibodies. Technology "di-antibodies described Hollingeret al.,Proc. Natl. Acad. Sci. USA,90: 6444-6448 (1993), provides an alternative mechanism for obtaining fragments of antibodies with dual specificity. The fragments contain the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VL) by a linker which is too short to allow Sarianidi domains on the same chain. Thus, the domains of the VHand VLone fragment are forced to pair with complementary domains of the VLand VHanother fragment, thereby forming two antigenspecific plot. Also described another strategy to obtain fragments of antibodies with dual specificity through the use of dimers odnotsepochechnykh Fv (sFv) (Gruberet al.,J. Immunol.,152: 5368 (1994)).
Consider antibodies with more than two valencies. For example, you can get thespecification antibodies (Tuttet al.,J. Immunol.,147: 60 (1991)).
The nucleic acid molecules encoding variants of polypeptides, get different well-known in this field means. These methods include as non-limiting examples of selection from a natural source (in the case of naturally occurring amino acid sequence variants) or receiving mediated by oligonucleotides (or site-specific) mutagenesis, PCR mutagenesis or cassette mutagenesis previously received options or betweenthey version of the polypeptide.
It may be desirable to modify the antibody according to the invention in respect to effector function, e.g. so as to enhance binding of the Fc receptor. This can be achieved by introducing one or more amino acid substitutions in the Fc region of antibodies. The alternative is actively or additionally, the Fc region can be entered cysteine residue(s), thereby providing the education miaocheng disulfide bond in this field.
To extend the half-life of the antibody in the antibody (especially an antibody fragment), you can enter the epitope binding receptor recycling, such as described in U.S. patent 5739277. As used here, the term "epitope-binding receptor recycling" refers to the epitope of the Fc region of the IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4)that is responsible for increasing the half-life of the IgG molecule in the serum ofin vivo.
Here consider other modifications of the antibody. For example, the antibody can be linked to one of the many non-protein polymers, such as polyethylene glycol, polypropyleneglycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylenglycol.
B. inserting a nucleic acid into a replicable vector
Heterologous nucleic acid (e.g., cDNA or genomic DNA) is suitably inserted into a replicable vector for expression in the organism under the control of a suitable promoter. For this purpose suitable set of vectors, and the choice of an appropriate vector mainly depends on the size inserted into the vector nucleic acid and a specific host cell transformed by the vector. Each vector contains various components, depending on the Conques is to maintain host cell, with which it is compatible. Depending on the specific type of host vector components generally include, as non-limiting examples of one or more of the following: a signal sequence, the starting point of replication, one or more marker genes, the promoter and the termination sequence of transcription.
In General terms, in connection with microbial hosts used plasmid vectors containing the sequence of the replicon and control sequences derived from a species compatible with the host-cell. Typically, the vector is site replication, as well as marking sequences capable of providing phenotypic selection in transformed cells. For example,E. coliusually transformed using pBR322 plasmids derived fromE. coli(see, for example, Bolivaret al.,Gene,2: 95 (1977)). pBR322 contains genes for resistance to ampicillin and tetracycline, and thus provides easy means for identifying transformed cells. Also plasmid pBR322 or other microbial plasmid or phage, as a rule, contain or be modified to contain, promoters which can be used in the host for the expression of the selective marker gene.
(i) Component signal sequence
DNA encoding the interest is in store here polypeptide, you can Express not only directly, but also in the form of a hybrid with another polypeptide, preferably a signal sequence or other polypeptide with a specific cleavage site at the N-end of the Mature polypeptide. In General, the signal sequence may be a component of the vector, or it may constitute a part of the polypeptide of the DNA inserted into the vector. Select heterologous signal sequence must be a recognizable and processed (i.e. split the signal peptidase) the host-cell sequence.
For prokaryotic host cells that do not recognize and do not processorbased natural signal sequence or signal sequence of the eukaryotic polypeptide, a signal sequence can substitute a prokaryotic signal sequence selected from the group consisting of a leader sequence lamB, ompF, alkaline phosphatase, penitsillinazy, lpp, or thermostable enterotoxin II. For secretion in yeast signal sequence may represent, for example, a leader sequence of yeast invertase, a leader sequence factor-alpha (including the leader sequence of factorsSaccharomycesandKluyveromyceswhere is posledni described in U.S. patent No. 5010182), or leader sequence of acid phosphatase, a leader sequence glucoamylaseC. albicans(EP 362179, published 4 April 1990), or the signal described in WO 90/13646, published November 15, 1990.
(ii) Component start replication
Expressing the vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected cells of the host. Such sequences are well known to many microorganisms. Start replication from the plasmid pBR322 is suitable for most gram-negative bacteria such asE. coli.
(iii) Component gene selection
Expressing the vectors typically contain gene selection, also referred to as selective marker. This gene encodes a protein necessary for the survival or growth of transformed host cells growing in culture on selective medium. Cells are the masters, not transformed by a vector containing selective gene do not survive in the environment for cultivation. This selective marker is different from genetic markers, and it is used and defined in this invention. Typical genes breeding encode proteins that (a) provide resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement of auxotrophs the th deficiency, other than the deficit caused by the presence of a genetic marker(s), or (c) supply critical nutrients not available from complex environments, such as the gene encoding racemase D-alanine atBacilli.
In one example schema selection for growth inhibition of host cell used drug. In this case, those cells that are successfully transformed interested nucleic acid to produce the polypeptide, giving stability to the drug and, thus, survive in the mode selection. Examples of such dominant selection use the drugs: neomycin (Southernet al.,J. Molec. Appl. Genet.,1: 327 (1982)), mycophenolate acid (Mulliganet al.,Science,209: 1422 (1980)) or hygromycin (Sugdenet al.,Mol. Cell. Biol.,5: 410-413 (1985)). In the three examples above, the use of bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycofenolate acid), or hygromycin respectively.
(iv) promoter Component
Expressing the vector to obtain the polypeptide of interest contains a suitable promoter, which learns a host organism and which is functionally linked to a nucleic acid that encodes a protein of interest. Suitable for use prokarioticheskih the owners promoters include the promoter of beta-lactamase and lactose (Chang et al.,Nature,275: 615 (1978); Goeddelet al.,Nature,281: 544 (1979)), the promoter system arabinose (Guzmanet al.,J. Bacteriol.,174: 7716-7728 (1992)), the promoter system, the alkaline phosphatase, the tryptophan (trp) (Goeddel,Nucleic Acids Res.,8: 4057 (1980) and EP 36776) and hybrid promoters such as the promoter oftac(deBoeret al.,Proc. Natl. Acad. Sci. USA,80: 21-25 (1983)). However, suitable and others well-known promoters. Their nucleotide sequences are published, thereby enabling professionals to ligitamate them to DNA encoding the desired polypeptide (Siebenlistet al.,Cell,20: 269 (1980))using linkers or adapters to provide any required restriction sites.
Also promoters for use in bacterial systems generally contain a Shine-dalgarno sequence (S.D.), functionally linked to DNA that encodes a desired polypeptide. The promoter can be removed from DNA bacterial source through cleavage by the restriction enzyme and inserted into a vector containing the desired DNA.
Promoters suitable for use in yeast is well known in this field. Examples of suitable promoter sequences for use in yeast hosts include the promoters for 3-phosphoglycerate (Hitzemanet al.,J. Biol. Chem.,255: 2073 (1980)) or other glycolytic enzymes (Hesset al. J. Adv. Enzyme Reg.,7: 149 (1968); Holland,Biochemistry,17: 4900 (1978)), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-phosphatization, 3-phosphoglyceromutase, piruwatkinaza, triazolopyrimidine, phosphoglucomutase and glucokinase.
Other yeast promoters, which represents an inducible promoters with the additional advantage of transcription controlled by growth conditions, are the promoter region of the alcohol dehydrogenase 2, sociogram C, acid phosphatase, destructive enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use in gene expression in yeast is additionally described in EP 73657.
(v) Design and analysis of vectors
Construction of suitable vectors containing one or more of the components listed above, involves the application of standard methods of ligation. Selected plasmids or DNA fragments are cleaved, tailored, and again are ligated in the form desired for obtaining the necessary plasmids.
For analysis to confirm correct sequences in the constructed plasmid mixture after which Yerevani used for transformation of strain 294 E. coliK12 (ATCC 31446) or other strains and cells with successful transformation are selected by resistance to ampicillin or tetracycline, when appropriate. Plasmids for transformants receive, analyze the splitting of the restriction endonucleases and/or determine posledovatelnosti way Sangeret al.,Proc. Natl. Acad. Sci. USA,74: 5463-5467 (1977) or Messinget al.,Nucleic Acids Res.,9: 309 (1981) or by the method of Maxamet al.,Methods in Enzymology,65: 499 (1980).
C. Selection and transformation of host cells
Appropriate cell hosts for cloning or expression of the DNA in the vectors according to the invention are any microbial cells, including prokaryotic cells or cells of fungi, including yeast. Suitable for this purpose, the prokaryotes include bacteria, as defined above, preferably eubacteria, such as gram-negative or gram-positive organisms. Examples include theEnterobacteriaceaesuch asEscherichiafor example,E. coli,Enterobacter,Erwinia,Klebsiella,Proteus,Salmonellafor example,Salmonella typhimurium,Serratiafor example,Serratia marcescansandShigellaandBacillisuch asB. subtilisandB. licheniformis(for example,B. licheniformis41P described in DD 266710, published 12 April 1989),Pseudomonassuch asP. aeruginosaandStreptomyces. One of the preferred as the host for reflection is Denmark E. colirepresents aE. coli294 (ATCC 31, 446), although it is also suitable for other strains, such asE. coliB,E. coliX1776 (ATCC 31537), andE. coliW3110 (ATCC 27325). These examples are illustrative and not restrictive. Also as initial owners can apply mutant cells of any of the above strains, which are then optionally subjected to mutations, to contain at least the minimum required here genotype.
The strain ofE. coliW3110 is the preferred parent hostE. colibecause it is a common strain host for fermentation products of recombinant DNA. Examples of source hostsE. colifor use as a parent owners along with their genotypes included in the table below:
|27A7||W3110)fhuA ptr3 phoA)E15 )(argF-lac)169|
|27C6||W3110)fhuA ptr3 phoA)E15 )(argF-lac)169 )ompT|
|27C7||W3110)fhuA ptr3 phoA)E15 )(argF-lac)169 )ompT degP41::kanR|
|33D3||W3110)fhuA ptr3 lacIq lacL8 )ompT degP41:: kanR|
|36F8||W3110)fhuA phoA)E15 )(argF-lac)169 ptr3 degP41> :: kanRilvG2096|
|41H1||W3110phoS* (T104) (argF-lac)169 degP41:: kanRptr3 ilvG2096(Valr)|
|43D3||W3110)fhuA ptr3 phoA)E15 )(argF-lac)169 )ompT degP41kanRilvG2096|
|43H1||W3110phoA(argF-lac) 169 degP41ilvG2096(Valr) ptr3 prc::kanRsprW148R|
|43E7||W3110)fhuA )(argF-lac)169 )ompT ptr3 phoA)E15 degP41ilvG2096|
|44D6||W3110)fhuA ptr3 )(argF-lac)169 degP41:: kanR)ompT ilvG2096|
|45F8||W3110)fhuA ptr3 )(argF-lac)169 degP41 )ompT phoS* (T10Y) ilvG2096|
|45F9||W3110)fhuA ptr3 )(argF-lac)169 degP41 )ompT ilvG2096 phoS* (T10Y) )cyo::kanR|
|49A5||W3110phoA (argF-lac)169 deoC2 degP41 ilvG2096 (Valr)|
|58B3||W3110phoA(argF-lac)169 deoC degP41 ilvG2096(Valr)|
|58H2||W3110phoA(argF-lac)169 degP41ilvG2096(Valr) ptr3 sprW148R|
|58H7||W3110() ptr3 lac Iq|
|59A7||W3110phoA(argF-lac)169 deoC degP41 ilvG2096(Valr) sprW148R|
|60H4||W3110phoA(argF-lac)169 deoC2 degP41 ilvG2096(Valr) prc-suppressor|
|61D6||W3110ptr3 lac Iq lacL8 (nmpc-fepE) degP41|
|62A7||W3110ptr3 lac Iq lacL8 (nmpc-fepE) degP41 ilvG2096|
Also suitable intermediate form upon receipt of the strain 36F8, i.e. 27B4 (U.S. patent No. 530472) and 35E7 (isolate spontaneous temperature resistant colonies, growing better than 27B4). Additional suitable strain is a strain ofE. coliwith mutant periplasmatic the protease(s), described in U.S. patent No. 4946783, issued August 7, 1990.
The above strains can be obtained by chromosomal integration of the parent strain or by other means, including those indicated in the examples below.
Full-length antibodies can be obtained inE. colithe guidelines WO 02/061090, published August 8, 2002.
In addition to prokaryotes suitable hosts for expressing encoding polypeptides vectors are eukaryotic microorganisms such as filamentous fungi or yeast. Commonly used microorganism belonging to the lower eukaryotes, isSaccharomyces cerevisiae. Other kinds includeSchizosaccharomyces pombe(Beach and Nurse,Nature,290: 140 (1981); EP 139383, published 2 may 1985); hostsKluyveromyces(U.S. patent No. 4943529; Fleeret al.,Bio/Technology,9: 968-975 (1991)), such asK. lactis(MW98-8C, CBS683, CBS4574; Louvencourtet al.,J. Bacteriol., 737 (1983)),K. fragilis(ATCC 12424),K. bulgaricus(ATCC 16045),K. wickeramii(ATCC 24178),K. waltii(ATCC 56500),K. drosophilarum(ATCC 36906; Van den Berget al.,Bio/Technology,8: 135 (1990)),K. thermotoleransandK. marxianus; yarrowia(EP 402226);Pichia pastoris(EP 183070; Sreekrishnaet al.,J. Basic Environ.,28: 265-278 (1988));Candida; Trichoderma reesia(EP 244234);Nerospora crassa (Caseet al.,Proc. Natl. Acad. Sci. USA,76: 5259-5263 (1979));Schwanniomycessuch asSchwanniomyces occidentalis(EP 394538 published 31 October 1990) and filamentous fungi, such asNeurospora,Penicillium,Tolypocladium(WO 91/00357 published 10 January 1991) and the hostsAspergillussuch asA. nidulans(Ballanceet al.,Biochem. Biophys. Res. Commun.,112: 284-289 (1983); Tilburnet al.,Gene,26: 205-221 (1983); Yeltonet al.,Proc. Natl. Acad. Sci. USA,81: 1470-1474 (1984)) andA. niger(Kelly and Hynes,EMBO J.,4: 475-479 (1985)). Here suitable methylotrophic yeast, and they include as non-limiting examples of yeast capable of growth on methanol selected from the genera, includingHansenula,Candida,Kloeckera,Pichia,Saccharomyces,TorulopsisandRhodotorula. A list of specific species that are examples of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).
Nucleic acid encoding a polypeptide of interest, making the cells of the host. Preferably this is done by transforming cells of the hosts described above expressing vectors and cultivating in conventional nutrient medium, modified as necessary to include different promoters.
Depending on the host cell transformation carried out using standard methods suitable for such cells. For prokaryotes the x cells and other cells, containing significant barriers in the form of cell walls, as a rule, use the treatment with calcium using calcium chloride as described in part 1,82 in Sambrooket al.,Molecular Cloning:A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). In another method of transformation used glycol/DMSO, as described by Chung and Miller,Nucleic Acids Res.,16: 3580 (1988). Transformation in yeast, as a rule, carried out according to the method of Van Solingenet al.,J. Bact.,130:946 (1977) and Hsiaoet al.,Proc. Natl. Acad. Sci. (USA),76:3829 (1979). However, for the introduction of DNA into cells can also be used in other ways, such as by microinjection, electroporation, fusion of bacterial protoplasts with intact cells or polycation, such as polybrene, poliarnaia.
D. Culturing host cells
Employed to produce polypeptides according to the invention prokaryotic cells are grown in well-known in this field and are suitable for culturing the selected cells of host environments, including environments, generally described in Sambrooket al., above. Environment for bacteria include as non-limiting examples of environment AP5, nutrient broth, broth, Luria-Bertani (LB), minimum environment Neidhardt and minimal or complete medium C.R.A.P. with the necessary nutritional supplements. In the preferred implementation of the environment shall also contain a means for selection, choose based on design expressing vector for selective growth of prokaryotic cells containing expressing vector. For example, for the growth of cells expressing the gene of resistance to ampicillin, environment add ampicillin. Besides sources of carbon, nitrogen and inorganic phosphate can include any necessary additives in appropriate concentrations, administered separately or as a mixture with other additives or environment, such as a complex nitrogen source. Optional media for cultivation may contain one or more reducing means selected from the group consisting of glutathione, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.
Examples of suitable media are given in U.S. patent No. 5304472 and 5342763. Wednesday C.R.A.P. with limiting phosphate consists of 3.57 g (NH4)2(SO4), 0.71 g of Na citrate-2H2O, 1.07 g KCl, are 5.36 g yeast extract (certified), are 5.36 g HycaseSF™-Sheffield increase pH by KOH to 7.3, brought the necessary amount to 872 ml of H2O SQ and paavolainen; cooled to 55°C and added 110 ml 1 M MOPS pH 7.3, 11 ml 50% glucose, 7 ml of 1 M MgSO4). You can then add of carbenicillin at a concentration of 50 μg/ml for the induction culture.
Prokaryotic cells are the owners of cultivated under suitable is emperature. For example, for the growth ofE. colithe preferred temperature range is from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably about 30°C.
When using the alkaline phosphatase promoter, used to obtain the interest of the polypeptide of the present invention cellsE. colicultivated in a suitable medium, which can be partially or fully to induce alkaline phosphatase promoter, for example, as generally described in Sambrooket al.above. Cultivation should never have happened in the absence of inorganic phosphate or pressure levels of phosphate. First, the environment contains inorganic phosphate in a quantity greater than the level of induction of protein synthesis and sufficient for the growth of bacteria. As cells grow and disposed of phosphate, they reduce the level of phosphate in the medium, thereby causing the induction of synthesis of the polypeptide.
If the promoter is an inducible promoter, for the purpose of induction cells are usually cultivated to achieve a certain optical density, for example, to A550approximately 200 using high cell density, where the start induction (for example, adding a range of complete the ora, by reducing the component of the environment, and etc) for the induction of expression of the gene encoding the desired polypeptide.
In appropriate concentrations, you can add any necessary additives, known to specialists in this field, introduced separately or as a mixture with other additives or environment, such as a complex nitrogen source. the pH of the medium may be any pH from about 5 to 9, mainly depending on the host organism. ForE. colipH preferably represents from about 6.8 to about 7.4 for, and more preferably approximately 7,0.
One of the selective environments that can be used for the cultivation of yeast, is a synthetic complete agar with dextrose absence of uracil (SCD-Ura), obtained as described in Kaiseret al.,Methods in Yeast Genetics(Cold Spring Harbor Press, Cold Spring Harbor, NY, 1994), p. 208-210.
E. Detecting expression
Gene expression in the sample can be measured directly, for example through traditional blotting on the Southern, "Northern"blot, quantitative counting of transcription of mRNA (Thomas,Proc. Natl. Acad. Sci. USA,77: 5201-5205 (1980)), dot blotting (DNA analysis), or hybridizationin situ, using appropriately labeled probe, based on the sequences of the polypeptide. You can apply again the ranks of the label, the most commonly used radioisotopes, specifically32P. But you can also use other methods, such as using a modified Biotin nucleotides for introduction into polynucleotide. Then, Biotin serves as a binding site avidin or antibodies, which can be marked by a wide variety of labels, such as radioisotopes, fluorescent substances, enzymes and other Alternative you can apply tests or gels to detect the protein.
F. Purification of polypeptides
When using recombinant methods polypeptides according to the invention receive inside the cell or in periplasmatic space. If the polypeptide is formed inside the cell, as a first stage for receiving the cell broth or homogenate, for example, by centrifugation or ultracentrifugation to remove debris from the host cells or lysed cells (for example, the resulting homogenization)
Then according to the invention defined above cell contamination is removed from the homogenate or broth by precipitation using ethacridine in the conditions specified above, and the resulting mixture is treated to obtain the desired polypeptide in a soluble form.
Department of the polypeptide of interest from the broth or homogenate can wire shall be by any suitable means, including the methods well known in the field, such as centrifugation or filtration. Preferably the separation is carried out with the use of centrifugation or filtration tangential flow, for example, applying a filter from about 300 kilodaltons to 1 micron.
After the polypeptide separated from the broth or homogenate, it can be cleaned using any known methods, including chromatography or filtration, such as ultrafiltration/diafiltration or filtering in the tangential flow. Specific examples of suitable filtering methods are the following, either alone or in combination with specific method(s)used, depending on the type of polypeptide: affinity chromatography with immobilized metal (IMAC); the separation of the two aqueous phases (ATPS); fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; HPLC with reversed phase chromatography based on hydrophobic interaction (HIC); chromatography on silica; chromatography on ion-exchange resin such as S-SEPHAROSE™ and DEAE; chromatofocusing; SDS-PAGE; precipitation ammonium sulfate; ultrafiltrate/diafiltrate filtering in a tangential flow and gel filtration using, for example, SEPHADEX™ G-75.
For example, as part of the overall process of obtaining protein, polypeptid what you can bring into contact with the immobilized reagent, linking or modifying the polypeptide. Thus, the polypeptide can be subjected to affinity chromatography, where the immobilized reagent that specifically bind to the polypeptide, such as antibody capture antibody, and impurities pass through the column affinity chromatography. Further, the polypeptide can be eluted from the column by changing conditions, so that the polypeptide is no longer bound to the immobilized reagent. The immobilized reagent may also be an enzyme, such as protease, which modifies the polypeptide (multi-storieset al.,Anal. Biochem.,193: 178-185 (1991) and Voyksneret al.,Anal. Biochem.,188: 72-81 (1990)).
Another type of cleaning process is filtration. Filtering of liquids impurities with small particle size is carried out with the use of various porous filter media through which the contaminated composition passes so that the impurities remain on the filter. Retention of impurities may occur as a result of mechanical filtration or electrokinetic capture and adsorption of particles. Mechanical filtration particle is held as a result of the delay, when it tries to pass through the pore smaller than she. In the case of electrokinetic mechanisms of capture particle hits the surface of the porous filter and zaderzhivaet the surface forces of attraction with a small radius. To achieve the electrokinetic capture to change the surface charge characteristics of the filter can be applied system charge (see, for example, WO 90/11814). For example, when the impurity removal are anions, to change the charge characteristics of the filter, you can apply the modifier with the charge of the cation, so that the impurity is retained by the filter.
Monoclonal antibodies can accordingly be separated from the precipitate by conventional procedures purification of antibodies, such as gel filtration or electrophoresis, dialysis, HIC, affinity chromatography, for example SEPHAROSE™ protein A chromatography with protein-G affinity to the antigen or the affinity for IgG, homogenization, cleaning the filter by centrifugation, sedimentation, for example by treatment with ammonium sulfate, polyethylene glycol or Caprylic acid, ion exchange chromatography, for example, with the use of such resins as hydroxyapatite, for example, resins containing calcium phosphate, such as ceramic hydroxyapatite and BIOGEL HT™and anion exchange resins, including resins with positively charged residues (at neutral pH), such as diethylaminoethyl (DEAE), polyethylenimine (PEI) and Quaternary aminoethane (QAE), for example resin, Q-SEPHAROSE FAST FLOW™ (Pharmacia)resin DEAE-SEPHAROSE FAST FLOW™, resin DEAE-TOYOPEARL™, resin QAE-TOYOPEARL™, POROS resin-Qࡊ the FRACTOGEL resin-DMAE™, resin FRACTOGEL EMD-TMAE™, MATREX CELLUFINE DEAE™ and other Methods of isolation and purification of antibodies are additionally described inAntibodies: A Laboratory Manual"; Harlow and Lane, eds. (Cold Spring Harbor Laboratories, New York: 1988).
In one particular implementation stage of obtaining includes the conversion of dissolved polypeptide into contact with a solid phase on which an immobilized reagent that binds or modifying the polypeptide. In one implementation of the solid phase is placed in a column, and the immobilized reagent captures the polypeptide. In another implementation of the reagent chemically and/or physically modifies the polypeptide and mobilizes on the solid phase, which, for example, placed in a column, and the composition passes through the column. For example, the polypeptide may contain domain-predecessor, which immobilized reagent removes as part of the process of obtaining, for example, the polypeptide is a predecessor is an antibody domain dimerization latinboy clasps, which removes immobilized pepsin in the production process.
In this implementation, the composition comprising the polypeptide and leached reagent (and optionally one or more additional impurities), passes through the filter carrying a charge opposite to that of the reagent at a pH of the composition to remove leached reagent of the composition. Filtrage be positively charged to remove impurities, charged when the pH of the composition is negative, such as an acid protease, protein A, protein G, or other reagents that may be leaching from the affinity columns. Alternative filter can be charged negatively to remove impurities, charged at the pH of the composition is positive, such as the main protease. Preferably the characteristics of the charge of interest of the polypeptide in the composition passing through the filter, such that the polypeptide filter does not significantly retained and passes through it. The filter can be placed "in line" with the flow treated at the previous stage (i.e. runoff, directly passing through the filter). This can be achieved by connection of the filter directly to the output port of the column, before the runoff is collected in a collection tank. The filter can be regenerated by applying suitable for the type of filter methods.
For purification of fragments of antibodies will also apply HIC. See, for example, Inouyeet al.,Protein Engineeringpp. 6, 8 and 1018-1019 (1993); Inouyeet al.,Animal Cell Technology: Basic & Applied Aspects,5: 609-616 (1993); Inouyeet al.,Journal of Biochemical and Biophysical Methods,26: 27-39 (1993); Morimotoet al.,Journal of Biochemical and Biophysical Methods,24: 107-117 (1992) and Reaet al.,Journal of Cell. Biochem.,Suppl. 0, Abstract No. X1-206 (17 Part A), p. 50 (1993). Column HIC, as a rule, contain the fundamental matrix (for example, agarose and cross with what ukami or synthetic copolymer material), which swyazawatisa hydrophobic ligands (e.g., alkyl or aryl group). Most speakers for HIC available commercially. Examples include as non-limiting examples of a column of Phenyl SEPHAROSE 6 FAST FLOW™ with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); a column of Phenyl SEPHAROSE™ High Performance (Pharmacia LKB Biotechnology, AB, Sweden); column Octyl SEPHAROSE™ High Performance (Pharmacia LKB Biotechnology, AB, Sweden); FRACTOGEL column™ EMD Propyl or FRACTOGEL™ EMD Phenyl (E. Merck, Germany); MACRO-PREP™ Methyl or MACRO-PREP™ t-Butyl Supports (Bio-Rad, California); column WP HI-Propyl (C 3)™ (J. T. Baker, New Jersey) and, or phenyl or butyl TOYOPEARL column™ (TosoHaas, PA).
Examples of sets of matrices for hydrophobic chromatography is well known in this field and include alkyl chain length of C18associated with the supporting matrix, such as SEPHAROSE™, agarose or silica, for example, butyl-, phenyl-, or octyl-SEPHAROSE™or such polymers as cellulose or polystyrene. In U.S. patent No. 6214984 described the use of chromatography based on hydrophobic interactions at low pH values (LPHIC) for the purification of antibodies and fragments of antibodies. This method is particularly suitable for the purification of fragments of antibodies, particularly correctly installed and connected by disulfide bonds fragments of antibodies (e.g., Fab fragments) from contaminating fragments of antibodies that n is properly stowed and/or have formed incorrect disulfide bonds. To LPHIC obtained from cells the antibody composition is preferably subjected to at least one stage of treatment, with examples including chromatography on hydroxyapatite, gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any of the Fc domain of immunoglobulin present in the antibody. Protein A can be used for purification of antibodies, based on certain human heavy chains (Lindmarket al.,J. Immunol. Meth.,62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human isotype (Gusset al.,EMBO J.,5: 1567-1575 (1986)). The matrix to which is attached an affine ligand, often represents the agarose, but available and other matrices. Mechanically stable matrices such as glass with controlled pore size or poly(Stradivari)benzene, provide faster flow rates and shorter processing time than that which can be achieved with the use of agarose. When the antibody contains a CH3 domain, suitable for cleaning resin BAKERBOND ABX™ (J. T. Baker, Phillipsburg, N.J.). Depending on the antibody to obtain also available other methods of protein purification such as fractionation on an ion-exchange column, ethanol precipitation, HPLC with reversed phase chromatography on silica, chromatography is the raffia on the heparin-SEPHAROSE™ , chromatography on anyone - or cation-exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.
G. Use of polypeptides
Thus obtained polypeptide can be formulated in pharmaceutically acceptable carrier and apply for various diagnostic, therapeutic and other purposes, known for such molecules. For example, these antibodies can be used for immunological assays such as enzyme immunological tests.
Also provided therapeutic use of purified using the method described here polypeptides. For example, to enhance growth if necessary, you can apply a growth factor or hormone, and the antibody can be used for oriented cytotoxicity (e.g., for the destruction of tumor cells), as adjuvants in vaccines for delivery of thrombolytic agents to blood clots, to deliver immunotoxins to the tumor cells, to convert the activated enzymes proletarienne funds in the area of the target (e.g. a tumor), for the treatment of infectious diseases or for directions immune complexes to cell surface receptors.
Therapeutic preparations of polypeptide receive for storage by mixing the polyp is Chida with desired levels of purity with optional physiologically acceptable carriers, fillers or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980)) in the form of a lyophilized pellet or water solutions.
Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; polypeptides of low molecular weight (approximately less than 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such glycine, glutamine, asparagine, arginine or lysine; monosaccharide, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
The polypeptide can also be enclosed in microcapsules, for example, obtained by means koatservatsii or by interfacial polymerization (for example, hydroxymethylcellulose or microcapsules with gelatin or capsules with poly-(methylmetacrylate) teams respectively), in colloidal drug delivery systems means (n is an example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsion. Such methods are described in Remington'sPharmaceutical Sciencesabove.
The polypeptide for use for the introduction ofin vivomust be sterile. This is easily achieved by filtration through sterile filtration membranes, prior to or after lyophilization and receiving. Typically, the antibody should be stored in lyophilized form or in solution.
Therapeutic polypeptide compositions generally are placed into a container with a sterile access hole, for example a bag of IV solution or bottle with stopper, sharp needle for subcutaneous injection.
The route of introduction of the polypeptide corresponds to known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial route or oral administration of the hearth damage or through systems with delayed release, as noted below. The polypeptide is administered continuously by infusion or by bolus injection.
Suitable examples of drugs with a slow release include a semi-permeable membrane of solid hydrophobic polymers containing the polypeptide, where the matrices are in the form of shaped p is the FL, for example, films or microcapsules. Examples of matrices for sustained release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate)as described by Langeret al.,J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer,Chem. Tech.,12: 98-105 (1982) or poly(vinyl alcohol)), polylactide (U.S. patent No. 3773919, EP 58481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidmanet al.,Biopolymers,22: 547-556 (1983)), neraspadayuschikhsya the ethylene vinyl acetate (Langeret al.,above), disintegrating copolymers of lactic acid-glycolic acid such as LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid-glycolic acid and leuprolide), and poly-D-(-)-3-hydroxipropionic acid (EP 133988).
Although such polymers as vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. For example, when encapsulated antibodies remain in the body for a long period, they can denaturing or aggregate as a result of exposure to moisture at 37°C, leading to loss of biological activity and possible changes immunogenicity. Depending on the involved mechanisms to stabilize antibodies can develop rationalisation. For example, if the aggregation mechanism is the formation of intermolecular bridge S--S through the exchange of citicoline, stabilization can be achieved by modifying sulfhydryl residues, lyophilisation from the acidic solution, the control of moisture content using appropriate additives, and developing specific matrix compositions.
Polypeptide compositions with delayed release also include enclosed in liposomes polypeptides. Containing antibodies liposomes get essentially known methods: DE 3218121; Epsteinet al.,Proc. Natl. Acad. Sci. USA,82: 3688-3692 (1985); Hwanget al.,Proc. Natl. Acad. Sci. USA,77: 4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; patent application Japan 83-118008; U.S. patent No. 4485045 and 4544545 and EP 102324. Typically, liposomes are small single-layer liposomes (about 200-800 angstroms), where the concentration of the lipid is a bómore than about 30 mol.% cholesterol, the selected proportion, adjustable for the most effective therapy polypeptide.
An effective amount of the polypeptide for therapeutic use depends on therapeutic objectives, the route of administration and the condition of the patient. Thus, there is a need for the physician to titrate the dosage and modify the route of administration, required to receive the of the most beneficial therapeutic effect. The usual daily dosage can vary from about 1 μg/kg to 10 mg/kg or more, depending on the factors mentioned above. Typically, the Clinician enters the polypeptide to achieve a dose that provides the desired effect. The progress of this treatment is easy to control by traditional methods of analysis.
The invention will be more clear from the following examples. However, they should not be construed as limiting the scope of the invention. All literature and patent citations included here as a reference.
Materials and methods
A. Plasmids, transformation, fermentation
1. Getting rhuFab'2 (xCD18)
a. Construction of plasmids
The control plasmid pS1130 designed for dahlstrand expression F(ab')2to CD18 F(ab')2and based on the vector described Carteret al.,Bio/Technology,10: 163-167 (1992). In this construct, the transcription of genes of two light chains and one fragment of the heavy chain with C-terminal latinboy buckle is placed under the control of one promoterphoA. Transcription is terminated by a terminator of transcription λt0below coding lacinova clasp heavy chain sequence (Scholtissek and Grosse,Nucleic Acids Res.,15(7): 3185 (1987)). The signal sequence of thermostable enterotoxin (STII) preaches is there coding sequence of each chain and directs secretion of the polypeptide in periplasm (Lee et al,Infect. Immun.,42: 264-268 (1983); Pickenet al.,Infect. Immun.,42: 269-275 (1983)). To ensure the dimerization of the two branches of Fab' to the C-end fragment of a heavy chain attached lacinova clasp.
A plasmid with two promoters containing two separate translation units, pxCD18-7T3, splits the light chain transcription from the transcription of the heavy chain over time. As pS1130, light chain remains under control of the promoter ofphoA. However, in pxCD18-7T3 for the coding sequence of the light chain is followed by a transcription terminatorλt0. Below this terminator to control transcription of a fragment of the heavy chain/C-terminal latinboy clasps added promotertacII(DeBoeret al.,Proc. Natl. Acad. Sci. USA,80: 21-25 (1983)). For this coding sequence, a second transcription terminatorλt0. To direct the secretion of both circuits have applied the silent options codons STII signal sequences (Simmons and Yansura,Nature Biotechnology,14: 629-634 (1996)).
A schematic comparison of the plasmids with one promoter from the plasmid with two promoters is shown in figure 1. The sequence of the expression cassette pxCD18-7T3 presented in figure 2 (SEQ ID No. 1)and amino acid sequence (SEQ ID No. 2 and 3) two translational units shown in figures 3A (light chain) and 3B (heavy chain) according to the respectively.
Strain-host used for fermentation derived fromE. coliW3110, meant 59A7. Full genotype 59A7 represents W3110fhuA phoAE15 (argF-lac)169 deoC degP41 ilvG2096(Valr) prc sprW148R. Cells of host-59A7 transformed with plasmid pxCD18-7T3 and cells with successful transformation were selected and grown in culture. Together with pxCD18-7T3 together transformed the additional plasmid pMS421. This additional plasmid, pMS421, represents based on pSC101 plasmid providing lacIq to gain control of the promoter oftacIIalso carrying resistance to spectinomycin and streptomycin.
For every 10 liters of fermentation solution in 1-liter shake flask containing 500 ml of LB medium, supplemented with 0.5 ml of tetracycline solution (5 mg/ml) and 2.5 ml of 1M solution of sodium phosphate, dissolve one vial containing 1.5 ml of culture in 10-15% DMSO. The original culture was grown for approximately 16 hours at 30°C and then used for inoculation of a 10-liter fermenter.
First, the fermenter was launched approximately 6.5 liters of medium containing approximately 4.4 g of glucose, 100 ml of 1M magnesium sulfate, 10 ml of trace element solution (100 ml hydrochloric acid, 27 g of uranyl ferric chloride, 8 g of heptahydrate zinc sulfate, 7 g of uranyl cobalt chloride, 7 g of dihydrate of sodium molybdate, 8 g of the pentahydrate Sul the veil of copper, 2 g of boric acid, 5 grams of monohydrate, manganese sulfate in a final volume of 1 liter), 20 ml of tetracycline (5 mg/ml in ethanol), 10 ml FERMAX Adjuvant 27™ (or any equivantage of antifoam), 1 packet salt HCD (37,5 g of ammonium sulfate, 19.5 g of dibasic potassium phosphate, 9,75 g of monobasic sodium phosphate, 7.5 g of sodium citrate, 11.3 g of monobasic potassium phosphate and 200 g of NZ Amine A (hydrolyzed protein). Fermentation was carried out at 30°C with 10 SL/min air flow and pH was kept at 7.0±0,2 (although in some cases was random deviations from this range). The back pressure of the fermentor and the stirring speed was varied to manipulate the velocity of the oxygen in the fermenter and, consequently, control over the level of cell respiration.
After inoculation of the fermenter containing cells from a shake flask culture was grown in a fermenter with a high density of cells with the use of computer-based algorithm for introduction into the fermenter solution of concentrated glucose. If necessary, the pH control in the fermenter was introduced ammonium hydroxide (58% solution) and sulfuric acid (24% solution). Also in some cases used additional antifoam additives for foam control. When the culture reached a cell density of OD550 of approximately 40 in the fermenter we use the and an additional 100 ml of 1M manganese sulfate. Additionally in the fermenter when the culture reached approximately 20 OD550 was started at a speed of 2.5 ml/min to add a concentrated salt (consisting of approximately 10 g of ammonium sulfate, 26 g of dibasic potassium phosphate, 13 g of monobasic dihydrate phosphate, 2 g of sodium citrate and 15 g of nonoonono potassium phosphate in 1 l of water) and continued to be added to the fermentation solution approximately 1250 ml. of Fermentation, usually continued for 72-80 hours.
During fermentation, as soon as control was achieved for fermentation point dissolved oxygen, based on the signal test dissolved oxygen, for controlling the concentration of dissolved oxygen in the control point was added a concentrated solution of glucose. Therefore, in this control scheme, the manipulation of the functioning parameters of the fermenter, such as the mixing rate or back pressure affecting the ability to move oxygen during fermentation, respectively, changed the capture rate of the oxygen or the metabolism of the cells.
To control the composition of the emitted when fermentation gas used mass spectrometer, and he allowed the calculation of the speed of the capture of oxygen and excretion of carbon dioxide when fermentation.
When the culture reached a cell density AP is sustained fashion 220 OD550, the stirring was reduced from an initial velocity of 1000 rpm to approximately 725 Rev/min for approximately 12 hours. For the induction of the synthesis of the heavy chain of approximately 12 hours after the culture cell density 220 OD550) was added and fifty ml of 200 mm isopropyl-β-D-thiogalactopyranoside (IPTG).
2. Obtain F(ab')2to tissue factor
a. Construction of plasmids
A plasmid with two promoters, pxTF7T3, created like a plasmid with two promoters pxCD18-7T3, and used to provide temporary separation expression of light chains and heavy chains of the antibody to tissue factor. In pxTF7T3 to create a new plasmid with two promoters, pJVG3IL also inserted sequencelacIfrom a plasmid pMS421.
Strain-host used for data fermentati, was a derivative ofE. coliW3110 marked 60H4. Full genotype 60H4 represents: W3110fhuAmanA phoAE15 (argF-lac)169 deoC2 degP41 ilvG2096(Valr) prc prc suppressor. Cell owners 60H4 transformed through pJVG3IL, and the cells pass through the transformation were selected and grown in culture.
Fermentation was carried out in conditions similar to the conditions for F(ab')2to CD18, as described above, with the principal exception that the duration of the process varied from approximately 72 to 114 h the owls, and a heavy chain using OPTG induced approximately the period of time from 4 to 12 hours after the culture OD550 220.
3. Getting a full-sized antibodies to TF
a. Construction of plasmids
The expression cassette plasmid pxTF-7T3FL contains, from 5' to 3': (1) the promoterphoA(Kikuchiet al.,Nucleic Acids Res.,9(21): 5671-5678 (1981)); (2) the Shine-dalgarno sequence fortrp(Yanofskyet al.,Nucleic Acids Res.,9: 6647-6668 (1981)); (3) silent version of the codon in the signal sequence STII (relative power TIR approximately 7) (Simmons and Yansura,Nature Biotechnology,14: 629-634 (1996)); (4) the coding sequence of the light chain of the antibody to tissue factor; (5) the terminator λt0(Scholtissek and Grosse,Nucleic Acids Res.,15: 3185 (1987)); (6) the promotertacII((DeBoeret. al.,Proc. Natl. Acad. Sci. USA,80: 21-25 (1983)); (7) the second Shine-dalgarno sequence fortrp; (8) the second silent variant of codon signal posledovatelnosti STII (relative strength TIR approximately 3); (9) the coding sequence of a full-sized heavy chain antibodies to tissue factor and (10) the second terminator λt0. This expression cassette was cloned in frame plasmidsE. colipBR322 (Sutcliffe,Cold Spring Harbor Symp. Quant. Biol.,43: 77-90 (1978)).
Thus, vector design pxTF-7T3FL provides temporary is OTDELENIE expression of each chain through the application of two different, not two identical promoters. In this plasmid light chain is under the control of the promoter ofphoA. However, to control the transcription of the heavy chain promoter usedtacII. As is well known in this area, promotersphoAandtacIIinduced in significantly different conditions. Schematic comparison of plasmids with a single promoter paTF130 and pxTF-7T3FL shown in figure 4. The sequence of the nucleic acid expression cassette pxTF-7T3FL (SEQ ID No. 4) is shown in figure 5, and the encoded she polypeptide sequence (SEQ ID No. 5 and 6) are presented in figures 6A (light chain) and 6B (heavy chain), respectively. Cells of the hosts below in conjunction transformed pxTF-7T3FL and pJJ247. pJJ247 encodes a promotertacIIthat controls the expression of DsbA and DsbC, with DsbA first in the series, and its design is described in WO 02/061090.
For the limited expression as host cells used a strain ofE. coli61D6 with genotype W3110fhuA (tonA) ptr3 lacIq lacL8 ompT(nmpc-fepE) degP41. After transformation, the selected transformants were inoculable in 5 ml of medium, Luria-Bertani supplemented with carbenicillin (50 μg/ml) and kanamycin (50 μg/ml) and were grown in culture plates at 30°C during the night. 10-liter fermentation was carried out using the environments, as described in WO 02/061090, and the basic conditions of fermentation are also described in WO 02/061090 the exception is the group of in the fermentation process was performed the following modifications: approximately 40 hours to obtain a concentration of about 30 mm was added 300 ml of 1M NaPO4, pH 7.0. Approximately 44 hours to obtain a concentration of approximately 2 mm was added 100 ml of a 200 mm solution of IPTG. The collection of cells after fermentation was performed at 80 hours after inoculation.
B. identification of proteins
Conducted one-dimensional gel electrophoresis SDS-PAGE linear gradient of acrylamide 4-12% Novex. Specifically, the implemented system was a NOVEX® NUPAGE™ System consisting of NUPAGE™ Bis-TRIS Pre-Cast Gels (for proteins with low and medium molecular weight).
Precipitating agent ethacridine was 98% purity and were purchased at Sigma (St. Louis, MO, USA). The molecular mass of ethacridine is 361,4 Yes. All other chemical reagents were of analytical purity.
Containing antibodies and F(ab')2substanceE. colihomogenized with the use of microfluidizer acquired in Watts Fluidair Inc. (model B12-04DJC, Kittery, MN, USA). The cells three times was passed through microfluidizer at a pressure of 4 bar. To eliminate thermal destruction of the protein substance was passed through a bath of ice water on each pass through microfluidizer. The total protein concentration in the homogenates of F(ab')2 was 30 mg/ml of the Homogenate full-size antibodies to TF obtained from resuspending pasty mass, had a total protein concentration of 18 mg/ml Pasty mass of antibodies to TF resuspendable in 25 mm buffer TRIS-HCl, pH 7.5.
Precipitating agent ethacridine was dissolved in water to the desired final concentration (wt./vol.).
1. Research pH:
Experiments on the deposition was performed at a constant concentration of ethacridine in the amount of 0.6% (wt./vol.). Received a 0.8% solution of ethacridine and mixed with the homogenate of E. coll in the ratio 3:1, for example 3 ml ethacridine and 1 ml of homogenate of E. coll. the pH was made using HCl or NaOH depending on the desired pH.
2. Research concentration ethacridine:
The homogenate was diluted 4 times the original solutions ethacridine (1:3, as in the study of pH), and the pH is maintained at a set value for each protein target. For antibodies to CD18 pH was 8.5, and for antibodies to TF pH was 7.5. Final concentration of ethacridine in precipitating systems amounted to 0.15, of 0.30, 0.45, and of 0.60, 0.75 and 0.9 percent (wt./vol.). As a comparison conducted a series of experiments with a concentration of ethacridine to 0%.
3. Study of electrical conductivity/dilution:
The homogenate antibodies against CD18 was added in various concentrations of NaCl for evaluating the effects of electrical conductivity on the precipitation of protein. Studied NaCl concentration was 0, 50, 100, 150, 200 and 400 mm. As a comparison we also carried out a number of studies without ethacridine to determine whether deposition of a protein due to the high concentration of salt. the pH in this study, peaks salt was 8.5, and the homogenate antibodies against CD18 were diluted 4 times. The concentration of ethacridine in the samples was 0.6% and 0% for control experiments.
To change the electrical conductivity of the sample homogenates were diluted in increasing degrees, pH is kept constant at pH of 8.5 and 7.5 for antibodies against CD18 and antibodies against TF, respectively. The final concentration of ethacridine in all experiments was 0.6% (wt./vol.). The homogenates were diluted in 2, 3, 4, 5, 6 and 7 times.
4. The study of temperature:
Some experiments were performed at elevated temperatures. The homogenate of E. coli was diluted 4 times, and the final concentration of ethacridine was 0.6%. pH for antibodies against CD18 and antibodies against TF was 8.5 or 7.5, respectively. Samples were incubated in a thermostat water bath at the desired temperature i.e. 50, 60 and 70°C. the Samples were incubated for 20-120 minutes at elevated temperatures. One long-term incubation for 16 hours was carried out at 50°C.
After mixing the precipitating environments is TBA and homogenate of E. coli and bringing the pH of the samples incubated in the conditions of mixing within 30-60 minutes. Experiments on the deposition was carried out in glass vials at 4 ml All experiments were performed in duplicate and reported average values.
E. Analysis of proteinsE. coli
Ethacridine interacts with most frequently used tests measure protein, for example, Bradford, BCA and measuring the absorption at 280 nm. Thus, the total protein concentrations were measured using the General analysis of proteinsE. coliby ELISA. Samples were diluted in containing gelatin buffer fish (0.15 M NaCl, 0.1 M NaPO4, of 0.1% fish gelatin, 0.05% of TWEEN 20™, of 0.05% PROCLIN™ 300) to reduce nonspecific binding with antibodies to proteinsE. coli. Covering the antibody was a goat antibody to a ECP. Forming a conjugate antibody was an antibody for the antibody to the whole ECP attached to horseradish peroxidase. Then controlled absorption using a tablet reader from Molecular Devices model SPECTRA MAX PLUS™ (Sunnyvale, CA, USA).
F. Analysis of protein G
To measure the obtained concentrations of F(ab')2and the antibodies were applied to the analysis of affinity chromatography with protein g Column and protein G were purchased PerSeptive Biosystems (Framingham, MA, USA). The column was balanced phosphate-saline buffer (PBS) and were suirable PBS with pH, increased to 2.2 with p the physical alteration of HCl. To minimize the impact of ethacridine samples before analysis were treated to exclusive rotating columns (BIO-SPIN® 6 Tris columns (Bio-Rad Laboratories, Hercules, CA, USA). Rotating column was used, as recommended by the manufacturer. In the chromatography method for minimising any impact of residual ethacridine in the sample has entered the stage of washing Tetramethylammonium (TMAC) (Fahrneret al.,Biotechnology and Genetic Engineering Reviews,18: 302-327 (2001)). The analysis was carried out using HPLC (liquid chromatograph HP 1090™) from Hewlett Packard (Mountain View, CA, USA). The samples were diluted with PBS. For each protein using purified protein (from Genentech, Inc.) build standard curves.
G. Analysis of DNA
The concentration of DNA in supernatant after deposition was measured with a set of Pico Green Kit from Molecular Probes (Eugene, OR, USA). He is a fluorescent analysis, where the fluorescent reagent binds to double-stranded DNA. Reagent Pico green leaves at 502 nm and emission at 523 nm was recorded. The analysis was carried out using the tablet reader fluorescence SPECTRA MAX GENINI XS™from Molecular Devices (Sunnyvale, CA, USA). Ethacridine interacts with the analysis of Pico green and, thus, the precipitating agent are removed from the solution prior to analysis. Ethacridine removed from the sample using columns BIO-SPIN®6 TRIS, described in the section on affine chromatog is the her protein G.
Obtained after deposition accredidation supernatant analyzed by SDS-PAGE. To render cleaning and generate antibodies to CD18 and antibodies to TF used non gel 4-12% NUPAGE™ from Novex (San Diego, CA, USA). Used gels factory, and a buffer for holding was MOPS (pre-made concentrate was purchased in Novex). Gels were stained with a filtered solution of COOMASSIE BRILLIANT BLUE R250™. Supernatant compensated by volume relative to the corresponding purified extract ofE. coli. Thus, if supernatant after deposition accredidation received 100% output, the intensity of the bands of protein in the extract ofE. coliand the samples should be identical. Thus, the gels can be applied to accurately determine the degree of purification obtained after deposition.
I. the solubility of ethacridine:
Examined two solution ethacridine, i.e. of 0.6%and 1.2%. Each solution was divided into two aliquots and the pH is brought to 6.0 and 9.0, respectively. To obtain a minor buffer capacity in the system ethacridine was dissolved in 10 mm buffer Tris-HCl. For each solution ethacridine was affected by increasing amounts of NaCl, i.e 0, 50, 100, 150, 200, 300 and 600 mm. Samples were incubated for 3 hours, then centrifuged for 20 minutes pri g on microcentrifuge (SORVALL MC12V™ , DuPont, Wilmington, DE, USA). Supernatant evaluated on accredidated by measuring the absorption at 270 nm. Used the spectrophotometer was a HP8453 UV-VIS™ from Hewlett Packard (Wilmington, DE), now Aligent Technologies (Palo Alto, CA) and is known as a spectrophotometer AGILENT 8453 UV-VIS™. From a solution with a known concentration of ethacridine received standard curve.
To measure the stability of supernatant as a function of time and temperature were measured viscosity. Applied measuring viscosity purchased in HACH model 2100N, Ames, Iowa, USA). The samples were measured at room temperature and without dilution of the sample.
The homogenate antibodies to CD18 processed or 0.6% accredidation, and 0.2% PEI or only water. In all three samples of the homogenate antibodies to CD18 were diluted 4 times, and the pH was 7.2+0,2. After centrifugation for one hour at 4000 g was obtained supernatant and divided into two aliquots. One part of each sample were incubated at room temperature (21°C)and the other at 4°C.
Results and discussion
The effect of pH
Molecules ethacridine positively charged at most pH interval (Miller, above; Neurath and Brunner, above; Franek,Methods in Enzymology, ed. Langone, J.J., Van Vunakis, H.,121: 631-638 (1986)). However, since the change in pH affects the charge of the polypeptide, and there is a correlation between the pI polyp is Chida and pH, when it is deposited under the influence of ethacridine (Neurath and Brunner, above), investigated the effect of pH on the degree of purification method according to the present invention.
The homogenates containing F(ab')2to CD18 F(ab')2to TF and a full-sized antibodies to TF, respectively, were subjected to 0.6% solution of ethacridine brought to cover the pH range of 4-10. Figures 8A-8C show a purified phase after treatment with accredidation and centrifugation of each of these proteins, respectively.
In table 1 antibody to TF studied, and as a full-size antibody, and as F(ab')2. The homogenatesE. coliprocessed to 0.8% (wt./about.) solution ethacridine in the ratio of 1:3, i.e. to a final concentration of ethacridine in the sample in the amount of 0.6%. pH brought HCl and NaOH, respectively, to obtain the desired pH. Calculated outputs and efficiencies for each of the treated cell homogenates. The concentration of DNA in vosstanovlennykh supernatant also presented in the table.
The effect of pH on treatment and release of antibodies to CD18 and antibodies to TF's handling accredidation
|F(ab')2to CD18||F(ab')2to TF||the full sized plans for Ab to TF|
|pH||The purification coefficient*||Output(%)||The purification coefficient*||Output (%)||The purification coefficient*||Output (%)||Conc. DNA (µg/ml)|
|9||of 5.4||84||the 4.7||22||5,3||24||,001|
|* A value of 1 represents the same degree of treatment as received in the system, n is processed by accredidation|
You can see that for all three proteins bell curve obtained with respect to the purification and concentration of DNA in the range of pH from 4 to 10. In the middle of the pH values, i.e. pH 5-9, received approximately 5 times more cleaning. The output F(ab')2to CD18 and a full-sized antibodies to TF and F(ab')2decreased with increasing pH, while the full-sized antibody to TF and F(ab')2have a stronger dependence on pH than the dependence of F(ab')2to CD18. The output F(ab')2to CD18 reduced from 100 to 85%, and F(ab')2to TF from 93% to 18% in the pH range of 4-10. Without limiting any one theory, this may partly be due to the smaller pI antibodies to TF compared with antibody to CD18, for example, a pI of 7.5 and 8.9, respectively; however, they represent theoretically calculated values.
Full-TF proteins have even more strict dependence than F(ab')2the protein. The purity of full-size antibodies to TF greatest at pH 7.0. At pH above 8 see significant loss of output (Fig. 8C). For full-length antibodies to TF preferred pH of approximately 7,0, i.e. 7.1 times more cleaning and yield of 86%. Without limitation to any theory, one possible explanation for the large losses of antibodies to TF compared with antibodies to CD18 is the fact that antibodies to TF are more negative surface ZAR is Dov, than antibodies to CD18 during incubation above its pI. Without limitation to any theory, similarly large full-sized antibodies to TF can carry more negative surface charge than the corresponding F(ab')2and, therefore, have a significantly greater yield loss observed with increasing pH.
However, from the polypeptide-target it is necessary to separate the components other than the cellular debris and proteins of the host. One such component is DNA. A big disadvantage of the presence of high concentrations of DNA with a polypeptide-target is the increase in solution viscosity. This will have a negative impact on further below processing. In addition, if the first exciting speakers used anion-exchange column, negatively charged DNA will be contacted with the resin and, thus, will reduce the capacity of the column for protein.
Therefore, after the deposition of accredidation in supernatant determined the concentration of DNA. The results show that the concentration of DNA in supernatant after deposition accredidation significantly decreased in comparison with the initial concentration of DNA obtained homogenateE. coli. However, the DNA concentration in the supernatant increased with increasing pH of the supernatant, i.e. 0.1 and 0.2 µg/ml at pH 5.0 and 4.0, respectively. Without limiting the Oia any theory, this can happen due to the fact that the phosphates in DNA become less negatively charged at low pH. At very high pH, for example pH of 10.0, the DNA concentration was significantly increased (0.3 ág/ml), and the purity of the protein purification was also decreased. Without limitation to any theory, it is partly due to the fact that the pH is above the pI of the antibody and F(ab')2; however, this may partly be due to the fact that ethacridine less charged at this pH.
Effect of concentration of ethacridine
The homogenatesE. colimixed with solutions of ethacridine in the ratio (1:3). The concentration of ethacridine in the samples increased with the increase of 0.15% from 0 to 0.9% (wt./vol.). This study was carried out at pH 8.5, and 7.5 and 6.0 for antibodies to CD18 F(ab')2to TF and a full-sized antibodies, respectively.
Table 2 presents the effect of the concentration of ethacridine at clearance and outlet. In table 2, antibody to TF was studied as a full-sized antibodies as F(ab')2. The homogenatesE. coliworked in the ratio 1:3 different concentrations of ethacridine; shows the final concentration of ethacridine in the sample. the pH of the antibody to CD18 F(ab')2to TF and a full-sized antibodies to TF was 8.5, and 7.5 and 6.0, respectively. Also in this table, p is avodat the DNA concentration in the obtained supernatant.
The effect on the purity and the yield of antibodies to CD18 and antibodies to TF when processing the increasing concentration of ethacridine
|F(ab')2to CD18||F(ab')2to TF||the full sized plans for Ab to TF|
|ethacridine (% wt./about.)||The purification coefficient*||Output|
|The purification coefficient*||Output (%)||The purification coefficient*||Output (%)||Conc. DNA (µg/ml)|
|* A value of 1 represents the same degree of treatment as received in the system, not processed by accredidation|
It is shown that the purity of the antibodies strictly korrelirovana with the concentration of ethacridine (figures 9A-9C and table 2). At concentrations of ethacridine higher than approximately 0.6% of the effect of increasing concentrations of ethacridine to increase the cleaning was not dramatic. However, at lower concentrations, i.e. when ethacridine is in deficit, even a small addition of precipitating means leads to significant additional purification of F(ab')2.
The addition of various concentrations of ethacridine no effect on the yield of F(ab')2to CD18. Stage retrieving both F(ab')2approximately 90% for all experiments. However, it seems easier to achieve quantitative obtain F(ab')2to CD18 than F(ab')2to TF. Without limitation to any theory, this may be because on the surface F(ab')2to TF what about the comparison with the F(ab') 2to CD18 there are more negative charges in the studied pH. Full-size antibodies to TF reached its maximum cleaning at lower concentrations ethacridine than the corresponding protein, 0.3% and 0.6% respectively. Without limitation to any theory, it is likely to occur due to the lower total protein concentration in the homogenate with a full-size antibodies to TF, ie 18 and 30 mg/ml for full-length antibodies to TF and F(ab')2respectively. Full-size antibodies to TF also obtained from resuspending a pasty mass, and F(ab')2obtained directly from the broth from the fermenter. Therefore, the soluble components of cultural media present in the brothE. colinot present in the material resuspending full-size antibodies to TF, which may partially explain the observed differences without limitation to any one theory.
The concentration of DNA in supernatant strictly correlated with the data obtained for protein purification. Adding 0.6% or above ethacridine DNA supernatant was not revealed. Also, there is a clear trend of decreasing concentrations of DNA, i.e., from 78 to 0 μg/ml, supernatant, when the concentration of ethacridine increases from 0 to 0.6%.
The effect of electrical conductivity
As ethacridine OS the claims proteins partially due to the characteristics of the charge of the molecule (Neurath and Brunner, above), the electrical conductivity of the sample can affect the purity of the antibodies and F(ab')2after deposition. Therefore, if the sample has a high concentration of salts, i.e., high electrical conductivity, salt can protect proteins from ethacridine and, thus, reduce the cleaning effect.
The homogenate antibodies against CD18 was subjected to two series of experiments to distinguish between the effect of electrical conductivity on the effect of protein concentration in the sample. In both series of experiments was added NaCl (0-400 mm). In one series of experiments the content of ethacridine was 0.6%, and the other used water. Water-containing system was used as a control, so that if NaCl caused any precipitation, it could be distinguished from deposition associated with accredidation. In systems without ethacridine, i.e. water systems in the range of NaCl concentrations from 0 to 400 mm of precipitation of the proteins were not observed (figa). In experiments it was shown that in the presence of ethacridine there is a clear increase in cleaning anti-D18-antibodies in the reduction of the electrical conductivity (pigv).
Without limitation to any theory one of the arguments in favor of improving purification of antibodies against CD18 at a lower electric conductivity may be lower shielding capacity dawn the military ethacridine at low salt concentrations. Similar shielding effects can be observed when protein purification with increasing salt concentrations used PEI (Jendrisak, above). However, even more important factor is the low solubility of ethacridine at high salt concentrations (Miller, above; Neurath and Brunner, above; Franek, above). When 100 mm NaCl was observed deposition of ethacridine in the system for extraction, and as the salt concentration increased, deposited more ethacridine. Therefore, the system remains soluble and available to precipitate proteins and other biological molecules fewer ethacridine.
When we examined the solubility of ethacridine as a function of NaCl concentration, the observed dependence on pH (11). At pH 6 significant differences in solubility between 0.6 and 1.2 percent solutions of ethacridine not observed. Both solution remains soluble at 50 mm NaCl, but almost completely deposited at 100 mm. For solutions with pH 9, where ethacridine was less charged, between the two concentrations ethacridine observed a significant difference in solubility. 0,6% solution of ethacridine was deposited at lower salt concentrations than the more concentrated solution of ethacridine (11). It is shown that the chloride precipitated ethacridine especially effective (Franek, above).
In practice the automatic electrical conductivity in experiments on the deposition must be determined by the dilution factor. Therefore, conducted a series of experiments where we studied the effect of dilution ratio homogenate of E. coll. The total concentration of ethacridine kept constant, i.e. of 0.6% (wt./vol.), but the electrical conductivity of the sample was reduced, increasing dilution. The results are shown in table 3. In table 3 antibodies against TF was studied both in the form of full-size antibodies and F(ab')2. The concentration of ethacridine in each experiment was 0.6% (wt./vol.). pH antibodies against CD18 F(ab')2against TF (F(ab')2) and a full-sized antibodies against TF was 8.5, and 7.5 and 6.0, respectively. Outputs and efficiencies were calculated for each treated cell homogenates. The table below shows the concentration of DNA in the resulting supernatant.
|The effect of different degrees of dilution homogenates of E. coll purity and the yield of antibodies against CD18 and antibodies against TF|
|F(ab')2to CD18||F(ab')2to TF||Polnorazmernyi Ab to TF|
|Dilution (fold)||Electic. conductivity (MSM)||The purification coefficient *||Output (%)||The purification coefficient*||Output (%)||The purification coefficient*||Output (%)||Conc. DNA (µg/ml)|
|* A value of 1 represents the same degree of treatment as received in the system, not processed by accredidation|
The results showed that the purification of F(ab')2 was increased with decreasing electric held the tee by increasing cultivation. However, when the electrical conductivity of 3.5 MSM or less the effect of electrical conductivity was negligible. For homogenate of E. coli used in this example, conducted a 4-fold dilution to obtain an electrical conductivity of less than 3.5 MSM. For full-length antibodies against TF can be a little lower dilution than in homogenates with F(ab')2. Without limitation by any theory, this may happen due to the fact that the protein concentration in the homogenate with a full-size antibody is lower. In addition, as the substance of a full-sized antibodies obtained from resuspending pasty mass, some components of environments, which may affect deposition, removed prior to deposition accredidation.
In these experiments, the protein concentration was reduced, increasing thinning (reducing the electrical conductivity). Thus, even if added 0.6% ethacridine for which studies concentration ethacridine revealed that it is present in excessive concentrations, less diluted samples to maximize the removal of protein and DNA of the host is not achieved. This is due to the increased concentration of the homogenate, i.e. total protein and DNA in these samples compared to the sample presented in the study of concentration is the radio of ethacridine. Therefore, the total concentration of protein, DNA and other components will affect the concentration of ethacridine or, alternatively, the need for cultivation. The output of the anti-CD18 F(ab')2and full-size antibodies and F(ab')2against TF increases with decreasing electrical conductivity.
It is revealed that the electrical conductivity also affects the removal of DNA. Two breeding, i.e. when 5,0 MSM in the supernatant were given a high concentration of the DNA component of 20.7 µg/ml. However, if you did a three-fold dilution, i.e. 4,0 MSM, the concentration was decreased to 0.5 μg/ml, and at a higher dilution of DNA was not detected. As mentioned earlier, in this case, the total number of ethacridine in relation to protein and DNA increases with dilution, i.e. the reduction of the electrical conductivity. Thus, observed in this example, the effect is reflected in the reduction of the electrical conductivity and the increase in the number of ethacridine.
The effect of temperature
Temperature is a factor. for which it is known that it is important when conducting experiments on the deposition. Thus, studying a slight increase in temperature in combination with accredidation. Also investigated the effect of time of incubation at increased temperature.
Incubation with ovalicin the first temperature, i.e. 50-70°had a positive effect on the purification of the two proteins F(ab')2(figures 12A and 12 B). The higher the temperature, the more efficient cleaning. Incubation at 70°With significantly increased the protein purity of F(ab')2. Incubation of the sample at 70°C for longer periods of time, i.e. within 40 minutes compared with 20 minutes did not increase the purity of F(ab')2(figures 12A and 12B), but was observed in approximately 10% yield loss. However, when the sample is incubated at a temperature of more than 70° (C, F(ab')2did not receive what was happening due to the temperature of deposition of the F(ab')2as well as other proteins of E. coll.
To study the effect of time of incubation in more detail we conducted an experiment where samples were incubated for 16 hours and 30 minutes at 50°C. the Results showed that significant improvements cleaning of the sample, inkubiruemykh for 16 hours, compared with a 30-minute incubation at this temperature does not occur. This indicates that the temperature of the deposition is a rapid phenomenon and suggests that the rapid heating to the appropriate temperature may be more appropriate than a long time of incubation.
Full-size antibodies against TF also studied at elevated temperatures. The 15-minute incubation at 50°on azula insignificant positive effect on the purity of the antibody, and compared to the incubation of the sample at room temperature was not observed losses in the output material (figs). However, if the temperature was increased to 60°With almost all antibodies against TF precipitated. These data indicate that full-size anti-TF antibodies have less stability at increased temperatures than protein F(ab')2against TF.
As the temperature increases can lead to modifications of the polypeptide target, you must first assess the stability of the specific interest of the polypeptide at an elevated temperature.
A steady flow of incoming substances
It is important to obtain the most uniform flow of incoming substances possible. However, another important characteristic of the flow of incoming substances is its stability over time. Therefore, compared the stability of supernatants after treatment with accredidation or PEI or supernatant after centrifugation. Each of supernatants incubated at two temperatures, i.e. at room temperature (21° (C) and 4°C. Stability was controlled by changing the viscosity of the corresponding sample.
The figure 13 shows the viscosity over time for three different supernatants, i.e. supernatant with accredidation, PEI and nearabout the aqueous purified supernatant, respectively. You can clearly see that the polyelectrolytes, i.e. ethacridine and PEI, significantly reduce the viscosity of the supernatant. Immediately after cleaning the raw purified supernatant had a viscosity constituting approximately 700 NTU, whereas processed PEI sample had a viscosity, which is only half of the specified viscosity, i.e. 327 NTU, a supernatant with accredidation had a viscosity, component 1 NTU. When investigated the viscosity of the treated PEI supernatant, large differences over time or depending on the temperature of incubation was observed. To the supernatant, which was centrifuged, observed the tendency to decrease the viscosity of the sample at room temperature during the first 48 hours. However, for the sample at room temperature for 72 hours to measure proved impossible due to the high viscosity of the sample. Treated accredidation supernatant had a very low viscosity, which by incubation at 4°was not significantly increased. When the sample is incubated at 21°S, the viscosity is significantly increased, namely, from 1 to 100 NTU for 72 hours. However, 100 NTU is still less than the viscosity obtained for the other two supernatants directly after cleaning. Therefore, we can conclude that the supernatant obtained field processing ethacridine what kratom at pH 7, is a steady stream of incoming substances.
Ethacridine can be successfully used as a precipitating means for initially obtaining heterologous polypeptide from the culture broth or homogenate. When the besiegers tools used ethacridine, perfect polypeptide-target preferably has a higher pI value than the arithmetic mean of the proteins of the host. Therefore, the majority of proteins can be negatively charged and deposited by accredidation, at the same time as the protein target is positively charged and, thus, remains in the supernatant.
The preferred concentration of ethacridine for deposition strongly depends on the concentration of the protein and the DNA of the host in the broth or homogenate. The higher the concentration of protein and DNA in the broth or homogenate, the more ethacridine necessary. Therefore, the more negatively charged components available for ethacridine for the formation of the complex and, thus, deposition, the higher the preferred number of ethacridine for deposition. The smaller the electrical conductivity of the solution when conducting the deposition, the more effective is the purification of the polypeptide. Stage deposition provides a significant purification of the polypeptide and Eisenia DNA at the same time, if you remove cellular debris.
For effective precipitation of the proteins and DNA of the host pH typically ranged from approximately 4 to 10, and preferably not more than about pH 9, as the molecule becomes less charged at higher pH. Ethacridine when cleaning polypeptide, more preferably at a pH in the range of about 5-9. To improve cleaning can be a short incubation at elevated temperature. However, it is necessary to determine the stability of the specific interest of the polypeptide at high temperatures in order to avoid precipitation of the polypeptide target. It is also necessary to investigate the quality of the polypeptide target, to verify that no modifications of the polypeptide target has not occurred.
1. The method of purification of the desired heterologous polypeptide, providing added to the fermentation broth or homogenate of E. coli, in which it was obtained and dissolved, effective quantities of ethacridine for the deposition of contaminants from the host cells under conditions when a large portion of the polypeptide remains soluble, and secretion of the desired polypeptide from the broth or homogenate, and an effective amount of ethacridine is approximately 0.1-5 wt.%/volume, electrical conductivity of the solution components that are contributing to the t approximately 1-15 MSM, the temperature of the broth or homogenate after adding ethacridine approximately from room temperature to 70°C, pH after adding ethacridine is not higher than 9, the polypeptide has a pI value that exceeds the average pI of the proteins of the host contained in the contamination of the host cells, and the polypeptide is an antibody.
2. The method according to claim 1, where the polypeptide isolated from the homogenate.
3. The method according to claim 1, where the polypeptide has a pI value in the interval from 7 to 10.
4. The method according to claim 1, where the polypeptide is a recombinant polypeptide.
5. The method according to claim 1, where the polypeptide is humanitariannet antibody.
6. The method according to claim 1, where the polypeptide is a full-size antibody.
7. The method according to claim 1, where the polypeptide is an antibody fragment.
8. The method according to claim 7, where the polypeptide is a fragment of an antibody containing light chain.
9. The method of claim 8, where the polypeptide is a fragment of an antibody containing light chain Kappa.
10. The method according to claim 7, where the polypeptide is a Fab, Fab', F(ab')2or hybrid F(ab')2with latinboy clasp.
11. The method according to claim 7, where the polypeptide is a F(ab')2.
12. The method according to claim 1, where the polypeptide is an antibody against IgE, the antibody against CD 18 antibody against VEGF antibody against tissue factor, S, antibody against Her-2, an antibody against CD20, antibody against CD40 or antibody against CD11 or antibody fragment.
13. The method according to claim 1, where the polypeptide is a F(ab')2against CD 18 F(ab')2against tissue factor, a full-sized antibody against tissue factor, or an antibody against VEGF.
14. The method according to claim 1, where the concentration of ethacridine is approximately 0.4 to 5 wt.%/volume.
15. The method according to claim 1, where the concentration of ethacridine is approximately 0.6 to 5 wt.%/volume.
16. The method according to claim 1, where the pH of the broth or homogenate after adding ethacridine approximately 5-9.
17. The method according to claim 1, where the pH of the broth or homogenate after adding ethacridine approximately 6-9.
18. The method according to claim 1, where the temperature of the broth or homogenate after adding ethacridine is approximately in the range from room temperature to 65°C, held for about 1-60 minutes
19. The method according to claim 1, where the temperature of the broth or homogenate after adding ethacridine is approximately in the range from 50°to 65°C, held for about 1-60 minutes
20. The method according to claim 1, where the selection is carried out by centrifugation or filtering.
21. The method according to claim 1, where after isolation of the polypeptide from the broth or homogenate gadopentate purify by chromatography or filtration.
22. The method according to claim 1, where the polypeptide get in the soluble fraction before adding ethacridine.
23. The method according to claim 1, where the polypeptide is insoluble, and it is dissolved by contact with a solvent to add ethacridine.
FIELD: chemistry, biotechnology.
SUBSTANCE: invention relates to field of biotechnology and preparation chemistry and can be used in biopharmacology and medicine. Cells of yeast P.pastoris are successively transformed by two different genetic structures, containing gene of human serum albumin (HAS) precursor. Obtained strain-producent is cultivated in nutrient medium. Recombinant HAS is isolated from cultural medium by clarification of said medium, as well as carrying out stages of successive centrifuging at 2000 and 10000 g, ultrafiltration, dialysis and cation-exchanging chromatography on column Source S. Target product represents eluate, including recombinant human serum albumin, 50 mM phosphate buffer, containing 400 mM of sodium chloride, with pH 9. Application of said iclaimed invention allows to extend arsenal of means, directed at production of recombinant HAS, and to obtain recombinant HAS in form of product, which in addition to recombinant HAS contains 50 mM phosphate buffer, containing 400 mM of sodium chloride and has pH 9.
EFFECT: extension of arsenal of means directed at obtaining recombinant HAS.
2 cl, 7 dwg
SUBSTANCE: invention concerns biotechnology, specifically production of new polypeptides regulating carbohydrate metabolism, and can be used in medicine. New polypeptides reacting as both GLP-1 receptor agonists and glucagon receptor antagonists. Polypeptides and coding nucleic acids are used as components of pharmaceutical compositions for treatment of diabetes type 2 and metabolism disorders.
EFFECT: production of compounds providing effective glucose homeostasis for patients, suffering from carbohydrate metabolism disorders.
18 cl, 1 dwg, 4 tbl, 20 ex
SUBSTANCE: invention relates to genetic engineering, namely, to obtaining inhibitors of TGF-β1 and can be used in medicine. Obtained peptides are capable of binding with transforming growth factor TGF-β1 and are potential inhibitors of biological activity of TGF-β1, binding with this cytokine directly. Peptides are obtained by recombinant method using transformed host-cell, by cultivating host-cell in conditions which ensure production of the said peptide, and its separation. The invention allows for efficiently treatment of diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
EFFECT: possibility to efficiently treat diseases or pathological disorders connected with hyperexpression or disregulated expression of TGF-β1.
12 cl, 6 dwg, 4 tbl, 4 ex
FIELD: technological processes; pharmacology.
SUBSTANCE: antagonist of human interleukine-1 receptor is prepared with the help of recombinant strain E. coli that contains plasmid, which provides production of this protein. For this purpose the strain is cultivated. Then from bacterial cells the target product is separated with application of three-stage chromatography and concentration on hydrophobic sorbent. At that the first and the third stage of mentioned chromatographic purification is performed on cation-exchanging resin, and in the second stage of chromatography the anion-exchanging resin is used.
EFFECT: application of invention allows to prepare antagonist of human receptor of high purity.
7 cl, 1 tbl, 4 ex
FIELD: technological processes; medicine.
SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.
EFFECT: highly active and stable preparation of deltaferon is produced.
2 cl, 6 dwg, 1 tbl, 3 ex
FIELD: technological processes.
SUBSTANCE: method suggests protein of adipocyte plasma membrane, method of its preparation and complex based on this protein. Protein has molecular mass of 115 kilodaltons and has the ability to start-up tyr-phosphorylation of insulin-receptor proteins substrate in adipocyte. Method of protein preparation provides for adipocytes preparation out of rat, mouse or human tissues and plasma membranes extraction out of them. Then plenty of domains are isolated with high content of cholesterol hcDIG, which are treated with solution trypsin/NaCl. Centrifugation is done and protein fraction SDS-polyacrylamide gel is segregated with electrophoresis. Prepared protein fraction in amount of 115 kilodaltons is eluated from this gel. Complex constitutes activated protein and is formed during its combination with one of compounds from group: YCN-PIG, YMN-PIG, YCN or lcGcel.
EFFECT: protein in its activated form allows regulating glucose utilization bypassing insulin signal chain.
7 cl, 20 dwg, 1 tbl
SUBSTANCE: invention concerns glycoforms of VII factor and compositions of VII factor, characterized by modified configurations on basis of asparaginic oligosaccharide chains. In addition invention includes detection method applied for polypeptide glycoforms of VII factor, receiving method and disease treatment method as well.
EFFECT: identification of biologically active forms of recombinant VII factor.
53 cl, 5ex
FIELD: biotechnology, molecular biology, proteins.
SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.
EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.
24 cl, 21 tbl, 32 ex
SUBSTANCE: invention relates to biologically active peptides possessing antibacterial effect that can be used in biotechnology and medicine. Peptide latarcin shows the following amino acids sequence: H2N-Gly1-Phe2-Phe3-Gly4-Lys5-Met6-Lys7-Glu8-Tyr9-Phe10-Lys11-Lys12-X13-Gly14-Ala15-Ser16-Phe17-Lys18-Arg19-Arg20-Phe21-Ala22-Asn23-Leu24-Lys25-Lys26-Arg27-Leu28-NH2 wherein X represents phenylalanine Phe13 or tryptophan Trp13 residue, and possesses the broad spectrum of antibacterial activity and expressed toxic effect with respect to human promyelocyte leukemia. Using the invention allows expanding assortment of natural peptide antibiotics not containing cysteine residues and possessing high antibacterial activity.
EFFECT: valuable medicinal properties of peptide.
2 tbl, 5 dwg, 8 ex
FIELD: biotechnology, microbiology, peptides.
SUBSTANCE: invention relates to a method for preparing dipeptide from L-amino acid ester and L-amino acid using culture of microorganisms possessing ability for producing dipeptide from L-amino acid ester and L-amino acid. Method involves using cells of microorganisms of genus Pseudomonas or Corynebacterium isolated from culture or disrupted cells of these microorganisms, or lyophilized cells of such microorganisms. By other variant method involves using a protein possessing proline iminopeptidase activity instead microorganism culture. Invention provides preparing dipeptides with high effectiveness degree using inexpensive parent substance by industrially economy and simple way.
EFFECT: improved preparing method.
13 cl, 1 dwg, 4 tbl, 14 ex
FIELD: medicine, immunology, chemistry of peptides.
SUBSTANCE: invention discloses peptides GNA33 representing mimetics of epitopes of microorganisms Neisseria meningitides of the serogroup B showing definite amino acid sequences given in the description and able to induce production of antibodies eliciting complement-mediated bactericidal activity and/or opsonic activity against indicated microorganisms in a mammalian subject. Indicated peptides are used as components of a composition used in a method for production of the immune response reaction against indicated microorganisms in a mammalian subject. Also invention concerns a polynucleotide encoding indicated peptide and recombinant expression vector comprising indicated polynucleotide. Also invention discloses a method for preparing peptide GNA33 by culturing the cell-host comprising recombinant expression vector and assay for the presence of antibodies raised against meningococci B in biological sample using GNA33 peptide. Using the invention provides effectiveness of vaccine raised against MenB and safety for its using for a patient.
EFFECT: valuable biological and medicinal properties of peptides.
11 cl, 6 tbl, 8 dwg, 6 ex
FIELD: medicine, oncology, genetic engineering, pharmacy.
SUBSTANCE: invention relates to a peptide or polypeptide representing a Fv-molecule, its genetic engineering construction, fragment of Fv-molecule or its genetic engineering construction able to bind leucosis cells and cells expressing glycocalicine. The selective and/or specific binding with target-cell is determined firstly by the first hypervariable site being Fv-molecule is a single-chain Fv-molecule or disulfide Fv-molecule (scFv or dsFv) and can be labeled by one or more labels. Fv molecule comprises variable sites of heavy and light chain wherein variable site of heavy chain comprises CDR3, CDR2 and CDR1 sites with amino acids sequences given in the invention description. Invention provides using peptides or polypeptides for design of an antitumor pharmaceutical compositions based on the specific direction for enhanced binding on essentially exposed and/or superexpressed binding site in target-cell or inside of its. Also, binding with a target-cell is carried out for benefit of other cells on which or inside of these cells this binding site is not essentially available and/or expressed.
EFFECT: valuable biological and medicinal properties of antibodies.
11 cl, 25 dwg, 11 tbl, 10 ex
FIELD: pharmaceutical chemistry and biotechnology.
SUBSTANCE: invention relates to highly purified lipopeptide antibiotic and pharmaceutical compositions comprising this compound. Invention also discloses daptomycin purification method including a series of consecutive stages of anion-exchange chromatography, hydrophobic chromatography, and anion-exchange chromatography as well as a method for purification of daptomycin using anion-exchange chromatography enhanced by modified buffer. Invention discloses improved method for production of daptomycin via fermentation of Streptomyces roseosporus followed by purification stage. Invention further discloses using high-efficiency liquid chromatography methods for checking purity of daptomycin. Disclosed are likewise lipopeptide micelles and methods for making these micelles as well as use of lipopeptide micelles in purification of lipopeptide antibiotics, such as daptomycin, and therapeutical application thereof. Invention allows preparation of lipopeptide antibiotics with 95 to 98% purity.
EFFECT: increased antibacterial activity of lipopeptide antibiotics against gram-positive microflora.
68 cl, 19 dwg, 3 tbl, 18 ex
SUBSTANCE: invention relates to water soluble polypeptides (SEQ ID NO.12) and (SEQ ID NO.7), derived from full-length tryptophanyl-tRNA-synthetase and having angiostatic activity in relation to eye neovasculatisation. Also disclosed are polynucleotides, encoding truncated polypeptide forms (SEQ ID NO.12) and (SEQ ID NO.7), and E.coli cell, expressing abovementioned polypeptides. Said polypeptides are useful in injecting angiostatic composition and kit for inhibiting of eye neovasculatisation.
EFFECT: polypeptides having non-immunogenic angiogenic properties.
22 cl, 5 dwg, 2 tbl, 5 ex
FIELD: biotechnology, vaccines.
SUBSTANCE: invention relates to producing vaccines and describes anti-anthrax vaccine that comprises mutant protein toxin from Bacillus anthracis chosen from mutant PA or mutant LF, or mutant EF or their combinations. Mutations of toxins provide the retained immunogenicity in decreasing the level of their toxicity. For the development of vaccine with reduced reactivity invention proposes recombinant DNA-construction for expression of said protein-toxins. DNA-construction comprises the expressing vector and DNA fragment comprising, in turn, genes encoding the corresponding protein-toxin (PA, LF or EF). Invention describes a method for preparing mutant proteins by using the transformed prokaryotic host. Prepared mutant protein-toxin possessing immunogenic properties and absence of toxicity is used for preparing anti-anthrax vaccine comprising one or more mutant protein-toxins and in combination with protein-toxin PA, LF or EF of wild type. Using the invention provides to develop the safety and effective vaccine against anthrax.
EFFECT: improved preparing method of vaccine.
13 cl, 2 dwg
FIELD: biotechnology, medicine.
SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.
EFFECT: valuable medicinal properties of allergens.
33 cl, 62 dwg, 10 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: invention proposes a nutrient medium containing components chosen in the following ratio, g/1 l: casaminic acids, 10-12; yeast extract, 3-5; potassium hydrogen phosphate, 7-8; magnesium sulfate, 0.1-0.2; sodium citrate dihydrate, 0.5-0.6; ammonium sulfate, 2-2.5, and glucose, 1-2. Using a liquid nutrient medium provides the intensive growth of staphylococci since first hours of cultivation and allows attainment of maximal yield of antibacterial compound after 8 h growth of culture. Invention is designated for culturing staphylococci in aim for preparing antibacterial peptide compounds.
EFFECT: improved and valuable properties of nutrient medium.
1 dwg, 2 tbl, 3 ex
FIELD: biotechnology, medicine, in particular treatment, prevention and diagnosis of diseases, associated with Neisseria meningitides.
SUBSTANCE: Claimed protein includes one or more N. meningitides protein fragments with known amino acid sequences, wherein said fragment contains one or more antigen determinants and has not more than 1977 amino acid from SEQ ID NO:1 and/or not more than 1531 amino acid from SEQ ID NO:2 which are described in specification with the proviso, that general protein sequence is not characterized by amino acid sequences represented in NO:1 and NO:2. Each claimed protein is encoded by nuclear acid (NA) with nucleotide sequence that defines protein amino acid sequence according to gene code. Peptides and nuclear acid of present invention are useful in drug production for treatment and prophylaxis of infections induced neisseria, as well as in production of diagnostic reagent for detection of neisseria or specific antibodies. Aldo disclosed is peptide- or NA-based pharmaceutical composition in effective amount with acceptable carrier. Said composition is useful for treatment of N. meningitides mediated infection. For prophylaxis composition is applied in vaccine form. Invention makes it possible to overcome diversity of neisseria antigen properties.
EFFECT: improved method for neisseria infection prophylaxis and treatment.
24 cl, 2 tbl
FIELD: immunotherapeutic agents.
SUBSTANCE: antigenic preparations are obtained from keratinophilic fungi Trichophiton or Microsporum species or yeast species Candida by alkali hydrolysis techniques. Thus obtained preparations can be, in particular used, as vaccines and for treating allergy and modulating immune response.
EFFECT: expanded immunotherapeutic possibilities.
17 cl, 5 dwg, 12 tbl, 20 ex
FIELD: biotechnology, medicine, proteins.
SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.
EFFECT: valuable medicinal properties of polypeptide.
19 cl, 33 dwg, 1 ex
FIELD: chemistry, pharmaceutics.
SUBSTANCE: invention relates to immunology and biotechnology. Described is polypeptide of protein P17 of HIV virus with amino acid sequence, consisting of peptide, corresponding to neutralising epitope of protein p17 of HIV, and peptide, corresponding to positions 23-32 of protein p17 of HIV. Peptide is bound to peptide of neutralising in carboxy-terminal position of epitope and makes it soluble. Described are vaccine compositions based on polypeptide, versions of polypeptide application such as, for preparation of medication against HIV, and as specific reagent in test on detecting neutralising anti-p-17-antibodies. Described are monoclonal and polyclonal anti-p 17-antibodies, able to recognise neutralising polypeptide epitope in specific way and neutralise its biological activity. Described is application of said antibodies to polypeptide for detecting p17 in biological material and for preparation of medication inhibiting p17 protein activity in HIV-infected people. Application of invention allows to induce neutralising anti- p17-antibodies and inhibit p17 activity which can be applied in medicine for preparation of vaccine of injection material against HIV.
EFFECT: obtaining composition which can be applied in medicine for preparation of vaccine or injection material against HIV.
17 cl, 9 ex