The method of separating a mixture of human gnkazy (options), purified detalizirovannaya human tnkase purified nedetekovana human ankasa, the pharmaceutical composition to reduce the viscosity of pus (options), a method of treating a patient suffering from a collection of pus (options), the method of treatment of a patient suffering from cystic fibrosis, bronchitis, pneumonia
The invention relates to biotechnology, concerns the identification and characterization of two components of recombinant drug Gnkazy. These components are purified detalizirovannoi and purified necessarily.banno human Tnkase. Methods of separation of these Dinkas performed using polydentate cation exchange resin or by using a resin with immobilized therein by heparin or by using resin with immobilized therein digitalisieren analogue of DNA. Use necessarily.banno Gnkazy as part of a pharmaceutical composition effective for use in patients suffering from pulmonary disorders nature. 13 N. p. F.-ly, 10 ill., 5 table.
This application is related essentially of the stated material with the material contained in International Publication WO 90/07572. The contents of the specified previous proposal specifically provides for this reason, in the present invention by reference.
The technical field to which the present invention
The present invention relates to the results obtained in the study of deoxyribonuclease (Gnkazy)-phosphodiesterase capable of hydrolysis polyethoxyethanol kalorik can be made useful medicinal compositions as well as to methods of using Dinkas and created on the basis of their compositions.
Background of invention
Tnkase is a phosphodiesterase that can hydrolyze polyethoxyethanol acid. Tnkase have been isolated from various sources with varying degrees of purity. The first complete amino acid sequence for Gnkazy of mammals was identified in 1973, Cm., Liao, et al., J. Biol. Chem.:1489 (1973).Tnkase characterized by a set of already known useful properties and has been used in this regard for therapeutic purposes. The main therapeutic use of Gnkazy was associated with the ability to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, which contributed to cleanse the respiratory tract. Cm. Lourenco, et al., Arch. Intern. Med.:2299 (1982); Shak, et al., Proc. Nat. Acad. Sci.:9188 (1990); Hubbard, et al., New Engl. J. Med.:812 (1992).
DNA encoding Tnkase man, was isolated and sequenced, this was achieved by the expression of the DNA in recombinant cells, which opened the possibility of obtaining human Gnkazy in commercially significant quantities. See, Shak, et al., piticescu respect, especially when used in pure form, when she was released from proteases and other proteins normally associated in the natural state. See, Hubbard, et al., New Engl. J. Med.:812 (1992).
In the above cited patent describes methods and techniques for obtaining human Gnkazy in pharmaceutically acceptable form. The existing prior art includes various specific methods of cleaning Gnkazy. See, Khouw, et al., U. S. Patent No.4065355 (published December 27, 1977); Markey, FEBS Letters:155 (1984); Nefsky, et al., Eur. J. Biochem.:215 (1989).
Although it was not known at the time of submitting the above application for the grant of a patent, as has now become clear, Tenkasu-containing product obtained from the culture of recombinant host cells, typically includes a mixture of detalizirovannoi and necessarily.banno forms Gnkazy. The existence of detalizirovannym forms Gnkazy remained unknown, although it was known that some proteins is desametasone aspartic and glutamic residues. Cm. Eipper et al., Ann. Rev. Physiol.:333 (1988); Kossiakoff, Science:191 (1988); Bradbury et al., Trends in Biochem, Sci.The invention
The present invention relates to a method of separation detalizirovannoi and necessarily.banno form h is using resin or other supporting medium, capable of binding with cationic polymer such as heparin or similar non-hydrolyzed deoxyribonucleic acid (DNA), or the chromatography was carried out using the so-called fuzzy cation exchange resin. The present invention relates also to the use of such chromatographic methods other than human Gnkazy, in particular for the bullish Gnkazy.
The present invention relates also to detalizirovannoi human DNase as a purified product, essentially not containing necessarily.banno human Tnkase.
The present invention relates also to necessarily.banno human DNase as a purified product, essentially not containing detalizirovannoi human Tnkase. Using the present invention it was shown that purified nedetekovana human Tnkase has more full enzymatic activity in comparison with detalizirovannoi Dnazol.
The present invention relates also to pharmaceutical compositions containing as active ingredient either purified detalizirovannoi human Tnkase or purified necessarily.banno human Tnkase, optionally in combination with pharmaceutically who piticescu effective amount of purified detalizirovannoi Gnkazy or purified necessarily.banno Gnkazy for treatment, for example, such patients with marked accumulation of viscous DNA-containing material. The preferred method of introduction of such treated Dinkas is inhalation of the drug directly into the lungs.
The present invention relates in particular to a method of treatment of patients with lung diseases such as chronic bronchitis, cystic fibrosis, or emphysema, introducing a therapeutically effective amount of purified necessarily.banno Gnkazy, preferably in the respiratory tract.
The present invention relates also to pharmaceutical compositions comprising necessarily.banno human Tnkase placed in a plastic vessel, optionally in combination with a pharmaceutically acceptable carrier.
Brief description of figures
The figure 1 presents the amino acid (SEQ ID No.1) and the DNA nucleotide sequence (SEQ ID No. 2) corresponding to human DNase I. Native signal sequence is underlined, the potential initiator codons are circled, and the Mature sequence enclosed in parentheses.
Figure 2 shows the correlation between enzymatic activity and the level of desametasone samples of human Gnkazy. The specific activity of the medium concentration, close to the standard curve) to the concentration of Gnkazy, measured on the basis immunosorbent assay for the binding of the enzyme (ELISA). The percentage of desametasone was determined by mapping using trypsin. Samples of human Gnkazy corresponding marks the "Day of collection of cells" ("Day of Harvest"), was isolated, followed by purification from the culture of recombinant cells of the Chinese hamster ovary (Cho) capable of expression of DNA that encodes a human Tnkase I. These samples were selected on 3, 5, 7, 9, 11, 13 and 20 days from the beginning of crop growth. Samples designated as "High pH" ("Higt pH) represent samples of the purified Gnkazy 13 days, which were incubated in vitro for two days at pH 8 and 37°. Samples designated as "Stability" ("Stability"), represent samples of the purified Gnkazy 13 days, which was stored in vitro at a temperature of 5, 25or 37C for different periods of time.
The figure 3 presents an example tripticase mapping Gnkazy done to determine the level of desametasone. In the example shown 65% detalizirovannaya DNA. The abbreviation "mAU" indicates meladinine absorption at 214 nm.
On f is(Asn-74) in native human DNase. Desametasone turns Asn-74 or aspartic acid (Asp) or in the rest itsprevious acid (iso-Asp). Each of these three forms Dinkas gives the splitting of trypsin couple of peptides that indicates the identity of these forms Dinkas.
Figure 5 shows a chromatogram of a sample of human Gnkazy, fractionated on a column with fleecy cation-exchanger (TLC). Shows the sample represents 67% detalizirovannoi Tnkase.
The figure 6 shows maps cleavage by trypsin fractions of the two peaks, taken in the process of separation on TLC, shown in figure 5. The absence of peptide T6-7 on tripticase map splitting of the peak 2 indicates the lack detalizirovannoi Gnkazy.
The figure 7 shows the chromatogram of a few samples of human Gnkazy, fractionated by TLC column. Samples designated as "MI-28 STD", are drugs human Gnkazy obtained from cell culture Chinese hamster ovary (Cho), transformed with DNA encoding native human Tnkase I. Samples designated as "Asn-Mutant Gnkazy" ("DNase has ASP Mutant"), represent Tnkase had in position 74 amino acid chain residue aspartic acid (not mod the trated Gnkazy, shown in figure 4. Asp Mutant Gnkazy was obtained from a culture of cells transformed by DNA encoding the mutant form of the human Gnkazy. DNA encoding the Asp mutant Gnkazy, was obtained using the site-directed mutagenesis of the DNA encoding the native human DNA. Comparison of the chromatograms shows that one of the forms of human Gnkazy is a MI-28 STD, which eluted with TLC column in the same position as Asp mutant Gnkazy.
The figure 8 presents the performance of several samples of human Gnkazy, fractionated on a TSK-heparin column (Toso Haas, Montgomeryville, Pennsylvania). The sample designated as "12K #8", is a human drug Gnkazy obtained from cell culture Chinese hamster ovary (Cho) transformed by DNA encoding the native human Tnkase I. the Sample designated as "Dezaminirovanie Standard" ("Deamidated Standard"), is a purified detalizirovannoi human Tnkase. The sample designated as "Nezamedlitelbnyj Standard" (Non-deamidated Standard"), refers to purified necessarily.banno human DNase. Purified detalizirovannoi human Tnkase and purified necessarily.banno human Tnkase get with fraktsionirovannyj on a column with immobilized analog of DNA. The sample designated as "MI-28", is a human Tnkase obtained from cell culture Chinese hamster ovary (Cho) transformed by DNA encoding the native human Tnkase. The sample designated as "Dezaminirovanie Standard" ("Deamidated Standard"), is a purified detalizirovannoi human Tnkase. The sample designated as "Nezamedlitelbnyj Standard" (Non-deamidated Standard"), refers to purified necessarily.banno human DNase. Peeled desametasone human Gnkazy and peeled nezamechennye human Gnkazy get by TLC chromatography. The sample designated as "Asp mutant Gnkazy" ("DNase has ASP Mutant"), represents Tnkase, bearing residue aspartic acid (and not the rest of the aspartic acid at position 74 amino acid sequence.
The term "human Tnkase" in the present description refers to a polypeptide having the same amino acid sequence as shown above in figure 1 amino acid sequence of human Mature Gnkazy I, the same applies to the amino acid sequence of its variants including allelic variants), oblagayuschiesya Tnkase" refers to a wide range of materials, the disclosure or receipt of which is shown in the above patents.
The term "human Tnkase" necessarily includes native Mature human Tnkase with the rest of the asparagine (Asn) in 74 the position of the amino acid sequence of the polypeptide. As shown in the present invention, the asparagine residue is sensitive to dezaminirovanie, which can result in a mixture detalizirovannoi and necessarily.banno forms of human Gnkazy. Instead residue (Asn) at position 74 amino acid sequence, detalizirovannaya Tnkase contains the residue aspartic acid (Asp) or ISO-aspartic acid (iso-Asp) (Cm. figure 4).
Used in this invention, the term "detalizirovannaya human Tnkase" means human Tnkase, detalizirovannoi the residue asparagine, with the specified desametasone takes place in 74 the position of the amino acid sequence of native Mature human Gnkazy. It was shown that detalizirovannaya Tnkase can arise when human Gnkazy using recombinant methods and can be detected in preparations of human Gnkazy derived from recombinant host cells the human Gnkazy.
Despite the fact that the asparagine residue at position 7 the amino acid sequence of native Mature human Gnkazy can also be detalizirovan (in addition to the asparagine residue 74 of the position of the amino acid chain), it is shown that this dual desametasone results in enzymatic inactive Gnkazy.
Used herein, the term "mixture" as applied to preparations of human Gnkazy means the presence detalizirovannoi and necessarily.banno forms Dinkas. It was shown, for example, that in preparations of human Gnkazy, obtained by expression in recombinant cells, dezaminirovanie exposed from 50 to 80%, and possibly higher percentage Gnkazy.
The term "purified detalizirovannaya human Tnkase" used in the present invention, means detalizirovannoi human Tnkase, which essentially does not contain impurities necessarily.banno human Gnkazy. In other words, nedetekovana human Tnkase is approximately less than 10%, preferably less than about 5%, and in the most preferred embodiment, less than about 1% of the total mass of Gnkazy in the drug treated detalizirovannoi human Gnkazy.
The term "ochisanu human Tnkase, which essentially does not contain impurities detalizirovannoi human Gnkazy. In other words, detalizirovannaya human Tnkase is approximately less than 25%, preferably less than about 5%, and in the most preferred embodiment, less than about 1% of the total mass of Gnkazy in the drug treated necessarily.banno human Gnkazy.
Used in the present description, the term "filler" means a pharmaceutically acceptable material, used in conjunction with Dnazol for the preparation of suitable drugs to improve his actions with the introduction of Gnkazy patients. Suitable for purposes such fillers are well known and described, for example, in the Physicians Desk Reference, the Merck Index and Remington's Phrmaceutical Sciences.
A preferred pharmaceutical composition for human Gnkazy is buffered or neseborainey aqueous solution, preferably in an isotonic salt solution, such as 150 mm sodium chloride containing 1.0 mm calcium chloride at pH 7. Such solutions are particularly suitable for use in commercially significant production of sprayers, including jet nebulizers and ultrasonic nebulizers, especially applicable in particular when spraying leporacanthicus compositions as well as introducing them to obtain effective results, can be found in the above-cited patent applications.
Under used here, the term "therapeutically effective amount" refers to the dosage of 1 μg to about 100 mg human Gnkazy per 1 kg of body weight of the patient, and enter in the composition of the described pharmaceutical compositions. A therapeutically effective amount of human Gnkazy depends, for example, from therapeutic objectives, the route of administration and the condition of the patient. In this regard, the treating physician must conduct the titration the right dosage and to vary the route of administration of the drug to obtain the optimal therapeutic effect. In connection with the above-described differences in enzymatic activity detalizirovannoi and necessarily.banno forms Dinkas it may happen that the amount of purified necessarily.banno Gnkazy required to achieve a therapeutic effect will be less than the amount of purified detalizirovannoi human Gnkazy or a mixture of these two forms needed to achieve the same effect under the same conditions.
Peeled Gnkazy, especially necessarily.banno its shape, is used to change the viscosity of mucous from what euonymi, characterized by the presence of abnormally viscous purulent secretions, as well as to treat conditions such as acute or chronic bronchopulmonary diseases, including infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, tuberculosis, and fungal infections. For this treatment applies burial in the bronchi solution well pulverized dry preparation or purified detalizirovannoi human Gnkazy or purified necessarily.banno human Gnkazy using traditional methods, such as aerosol spray.
Century Preferred options
As a result of successful cloning and achieve expression of human Gnkazy in recombinant cell hosts have been shown through rigorous analysis that obtained when such expression in recombinant cells Dncanny the product is a typical case of the mixture to that time not yet certain components. In particular, the study of human Gnkazy isolated from cell culture Chinese hamster ovary (Cho) and further purified using the method of isoelectric focusing (IEF), revealed a complex set of different kinds of Gnkazy. Defined in this way, various types and desametasone.
To determine the presence and level detalizirovannym Dinkas in such preparations used two methods of analysis. One of them included the splitting of the original drugs Gnkazy with trypsin and further analysis of the resulting peptides by HPLC with treatment phases. In this method, the number detalizirovannoi Gnkazy in the original drug was determined by the number six characteristic peptides obtained by cleavage with trypsin.
Another method included the chromatography was carried out of the original drug Gnkazy on ion-exchange column with fleecy cation exchange resin (TLC). It was shown that the use of TLC column gives the ability to resolve peaks detalizirovannoi human Gnkazy and necessarily.banno human Gnkazy, so that each of the existing forms Gnkazy can be effectively separated from the other and obtained in pure form. In this method, the number detalizirovannoi and necessarily.banno Dinkas in the original preparation was determined by measuring the areas of the chromatographic peaks corresponding to individual forms Dinkas.
Despite the fact that both of the above method is equally effective in the detection and quantitative determination detalizirovannoi Gnkazy, TLC method are preferred due to the fact that he was a t the IU, method chromatography on TLC provides separation detalizirovannoi and necessarily.banno forms of DNA, even in cases when traditionally used cation exchange resin, and other resins used in chromatographic technique, did not give the desired separation.
The basic principles of chromatography on TLC were described, in particular, Miller, J. Chromatography:133 (1990); Janzen et al., J. Chromatography:77 (1990); and Hearn et al., J. Chromatography:117 (1991). Without wishing to limit the invention offer the only mechanism of action or theoretical frameworks, the authors suggest, by analogy with the known crystal structure bullish Gnkazy that residue Asn-74 in human DNase sensitive dezaminirovanie located in enzyme site, convenient to associate with the groove in the DNA helix. The corresponding groove in the enzyme contains a basic amino acid residues (for binding to DNA and, apparently, it is sensitive to ligands fleecy cation-exchange resin, but not to much shorter ligands traditional cation exchange resins. Apparently, the mechanism of action is based on the fact that the League is zi can be put, that ion-exchange chromatography on a fuzzy resin is useful for cleaning other nucleases, such as ribonuclease (Mcasa) or restriction endonuclease, as well as proteins that bind to DNA.
Alternative separation detalizirovannoi and necessarily.banno forms Dinkas can be achieved by chromatography on a resin or other carrier matrix, covalently associated with cationic polymers, such as heparin or synthetic neytralizuya similar DNA.
Column chromatography on immobilized heparin commercially available (for example, from Toso Haas Co., Montgomeryville, Pennsylvania).
Neytralizuya analogues of DNA have been described, in particular, Spitzer et al., Nuc. Acid. Res.:11691 (1988). Immobilized neytralizuya similar DNA get typically the synthesis of similar DNA with amino group at the 3'-end of one or both of the complementary strands. The amino group is then available for binding with epoxyoctadecane column as described in materials published Rainin Biochemical Elsie products (Rainin Biochemical LC Products, Woburn, Massachusetts).
After successful separation detalizirovannoi and necessarily.banno forms Dinkas according to the method of this izobreteny is no reduced compared to necessarily.banno human Dnazol enzymatic activity. Kurnick, Arch. Biochem.:41 (1950). It was shown that detalizirovannaya human Tnkase has half enzymatic activity in comparison with necessarily.banno human Dnazol. Thus, combining purified desametasone Gnkazy and peeled nezamechennye Gnkazy of the present invention in different proportions, you can create a pharmaceutical composition based on human Gnkazy having the desired specific activity in a wide range of specific activity of the individual components, with the possibility of choosing the best options for the treatment of certain diseases.
The following examples are given for illustration only and does not purport to limit in any way the present invention.
1. Mapping using trypsin
The procedure used to tripticase mapping human Gnkazy, can be summarized as follows:
Stage 1. The DNA sample weighing 1 mg is brought to a concentration of 4 mg/ml by concentration on the device Amicon Centricon-10 (Amicon Method-10) or by dilution media. Final volume: 250 µl.
Stage 2. To the sample add 250 ál of pre-treated s 3. Change the buffer in which the sample, splitting buffer (100 mm Tris, pH 8) using a column Pharmacia NAP-5. Final volume: 1 ml
Stage 4. To the sample add 10 ál of trypsin (1 mg/ml trypsin, 1 mm Hcl) and incubated for 2 hours at 37C.
Stage 5. To the sample add a second aliquot trypsinogen solution of 10 μl, and incubated for 2 hours at 37C.
Stage 6. Stop the digestion by adding 6 μl of trichloroacetic acid (THU). Until the chromatography was carried out keeping the samples at temperatures below 5C.
Stage 7. Divide mixture of peptides using HPLC method under the following conditions:
Column: Nucleosil (Nucleosil) C18, 5 μm, 100 A, 2,0150 mm (Alltech, Co., Deerfield, Illinois - Alltech, Co. Deerfield, Illinois).
The column temperature: 40C.
Eluent A: 0,12% THU in water
Eluent A: 0.10% THU in acetonitrile.
The gradient profile (see tab. :
The flow rate: 0.25 ml/min Volume of the applied sample: 250 µl.
The time of equilibration of the column after separation at 100% A: 20 minutes
Temperature automatic otbornye samples: 5. the aptidon comparing the time of their retention with the standard.
Stage 9. Integrate peaks in the chromatogram obtained at 280 nm. Check the quality of the chromatogram on the position of the baseline and the division next eluruumiks peaks. Special attention should be paid to early eluruumis peptides T7 and (D)T7, which may very well leave during elution.
Stage 10. Relate zone six peaks above peptides containing tyrosine. Each of the peptides T7, (D)T7, T7-8 and (D)T7-8 contains a single tyrosine residue, while the T6-7-8, T6-7 contains three tyrosine residue. Calculate the proportion of detalizirovannym species based on normalized, i.e. correlated with the content of tyrosine areas of the peaks (D)T7, (D)T7-8, T6-7-8, T6-7, relative to the total amount of the normalized areas of the peaks of all six peptides.
For the careful execution of the method tripticase mapping to determine detalizirovannoi Gnkazy in accordance with the process described above requires 1 mg Gnkazy in volume of 250 μl. In this regard, the initial preparation for this procedure includes either the concentration or dilution of the sample to obtain the desired result. Tnkase highly resistant to the action of proteases, including trypsin, in the presence of calcium. For this reason, the next stage of the procedure for the red acid (EGTA). Overdose in the processing of EGTA can lead to denaturation and aggregation of Gnkazy, so that this stage must be done very carefully. For EGTA-treated sample in a volume of 0.5 ml replace next Wednesday at splitting the buffer (1 ml), add trypsin and incubate the sample at 37 ° C for 2 hours Then add a second aliquot of trypsin and incubate the sample for 2 h Splitting is stopped by acidification of the mixture, and the sample or left for further analysis or loaded for research directly on the column for VSGH.
250 ál (250 micrograms) of a Mixture of peptides obtained after cleavage with trypsin, separated on the HPLC column with the treatment phases of compliance with the above conditions. Typical map tripticase splitting human Gnkazy presented on figure 3. HPLC was carried out on the model Hewlett-Packard (Hewlett Packard) 1090M HPLC. Eluruumis with the column material was immediately analyzed for the presence of absorption at 214 and 280 nm, using the device of the diode detector. Because the peptide map obtained on the basis of the early portions aliremove material, is not possible to quantify the level detalizirovannoi Gnkazy, below opisyvalitseli in studies of large, for the analysis volume. Each analysis in accordance with such procedure requires 70 min gradient for separation and 20 min for equilibration of the column for the total cycle HPLC of 90 minutes theoretical foundations and practical approaches for integrating peak to determine detalizirovannoi Gnkazy in the sample are presented below.
Desametasone human Gnkazy takes place, at least for aspartic residue 74 of the position of the amino acid chain (Asn-74) of native Mature human DNA. As you can see from the list of expected peptides with trypsin cleavage of human Gnkazy, are shown in table 1, Asn-74 is located on the C-terminal section of the site of cleavage by trypsin next to the arginine residue in 73 the position of the amino acid chain (Arg-73).
Instead of Asn (denoting only the letter "N") residue at position 74 native necessarily.banno human Gnkazy, desametasone human Gnkazy, may take place or Asp or iso-Asp residue, as shown in figure 4. Iso-Asp is an isomer, beta-amino acid form of aspartic acid. The peptide bond between AGD-73 and iso-Asp resistant to cleavage by trypsin, so desamuduru is Amy T6-7, because it is a peptide, United on the basis of peptides T6 and T7. In the conditions used for tripticase cleavage, the peptide bond Arg-73-Asn-74 in necessarily.banno human DNase and the peptide bond Arg-73-Asn-74 in Asp form detalizirovannoi human Gnkazy split by trypsin. Hence the presence of necessarily.banno Gnkazy indicates the presence tripticase map T7 peptide, are shown in table 1, whereas the presence of Asp-74 forms of human detalizirovannoi Gnkazy indicates the presence tripticase map dezaminirovanie T7 peptide, called (D)T7. These three are described peptide are indicated on figure 3. Unfortunately, trypsin only partially cleaves the peptide bond on the C-terminal site T7, between residues 77 and 78, so that each of the described peptides T7, (D)T7 and T6-7 have T7-conjugate, T7-8 and T6-7-8 respectively. These six reporter peptides should be taken into account for the quantitative determination detalizirovannoi human Gnkazy using the method tripticase mapping.
In principle, (D)T7, (D)T7-8, T6-7, T-6-7-8 peptides indicate detalizirovannoi human Gnkazy, and T7 and T7-8 peptides indicate necessarily.banno human Gnkazy the th for this drug Gnkazy. To calculate the number fraction detalizirovannoi Gnkazy you must have knowledge of molar ratios detalizirovannoi and necessarily.banno kinds Dinkas. However, in the procedure tripticase mapping there are two additional problems that must be overcome: one - chromatographic problem and the second problem detection. Chromatographic problem is that the T2 peptide eluted with T6-7 and this complicates the integration of the clear areas of the peaks that are characteristic dezaminirovanie material of the peptide. This problem may be overcome by integrating chromatograms obtained at 280 nm, since all six relevant peptides have at least one of the tyrosine residue (Tight), and therefore is strongly absorb at 280 nm, while T2 contains Tight or tryptophan (Thr) residue, and therefore its absorption at this wavelength is negligible. The problem of detection is that the T6-7 and T6-7-8 peptides each contain three (Tight) residue, whereas each of the remaining four peptides contain only one residue Tight. Thus, T6-containing peptides have a greater ratio of polar absorption than peptides, Sogo species in the sample. This problem can be solved due to the normalization, i.e., conversion of areas of peaks of the six peptides based on the number of Tight residues in the peptide. Normalization thus the values of the areas of the peaks implies that all the tyrosine residues in each peptide are equal to the chemical environment, which, as you might expect, quite well suited for relatively small peptides, in particular those which are considered here. When carrying out the normalization of the adjusted area of the peaks detalizirovannym and medialiteracy peptides can be compared to assess the content detalizirovannoi Gnkazy in the sample.
2. Ion-exchange chromatography on a fuzzy cation
Fleecy trail resin (TLC) for ion-exchange chromatography unlike traditional resin-cation has polyionic ligands, bind to the silicon surface. Used in the example column with ligands of Lichrospher® 1000 SO3(LiChrospher®, EM Separations, Gibbstown, New Jersey) contains, as presented in advertising, from 25 to 50 sulfopropyl groups along the plastic rods that are joined by silicon surface.
TLC chromatogram of a sample of recombinant human Gnkazy provole followed by purification from cell culture Chinese hamster ovary (Cho), the transformed DNA that encodes a human Tnkase, Shak, et al., Proc. Nat. Acad. Sci.:9188-9192 (1990); Shak, et al., International Patent Application Publication No.WO 90/07572 (published 12 July 1990).
Both the obtained peak is collected and subjected to several tests to identify forms Dinkas that differ only in the balance of 74 positions of amino acid sequence. The figure 6 shows maps tripticase splitting of the two peaks, highlighted by TLC column to confirm that they are respectively detalizirovannoi and necessarily.banno forms of human Gnkazy. Map tripticase splitting also shows that both forms detalizirovannoi Gnkazy (with Asp and iso-Asp at position 74 amino acid sequence) appear already in the first peak when the separation on TLC. Table 2 shows that measured for these two peaks specific activity, confirming the link between dezaminirovanie and specific activity, deduced on the basis shown in figure 2 correlation, which serves, in addition, for additional identification of TLC fractions. Activity faction Gnkazy was determined using the method of the study with methyl green (MG).
The mutant form of challenge mutagenesis DNA the coding of native Mature human DNA. This mutant form eluted together with the first peak obtained by the above procedure chromatography was carried out (see figure 7).
Below is the method of filling the column fleecy cation exchange resin Lichrospher® (LiChrospher®) 1000 SO3. Another close to it as fleecy cation-exchange resin, suitable for separation detalizirovannoi and necessarily.banno forms Gnkazy is fleecy cation exchange resin Fractogel (Fractogel®) (EM Separations, Gibbstown, New Jersey). Resin Lichrospher and Fractogel are registered trademarks of EM industries, Inc., Hawthorn N. Th. EM (EM Industries, Inc., Hawthorne, N. Y., or E. Merck, Darmstadt, Germany (E. Merck, Darmstadt, West Germany). Currently, the authors believe that the "strong" form fleecy cation exchange resins having SO3functional groups (regardless of Lichrospher or Fractogel), can give the best separation results.
3. The method of filling the columns for HPLC resin Lichrospher® 1000 SO3
A. Materials and equipment:
1. Glass cartridge with the best resolution parameters and dimensions 1.0 cm5,0 see
2. The buffer to fill: 100 mm sodium acetate, 1 mm CaCl2pH ech part #9501 or equivalent option).
4. Empty column stainless steel size 4.6 mm50 mm Frit size limit of 0.5 ám.
5. Pump for HPLC, is able to maintain a back pressure equal to 2000 lb/in2(Model wouterse 510 (Watters) or equivalent option).
b. The procedure of filling columns
1. Preparation of resin to the content of:
a) Unpack the glass column for high performance separation size 1.0 cm5.0 cm (net = 3,93 ml resin). To resuspendable resin in 20 ml of pure glass, pour into a vessel the buffer to fill (packing buffer). The resulting suspension is shaken to homogeneity and divided into two aliquots: 210 ml of each aliquot was added 10 ml of a packing buffer to obtain suspensions of the approximate composition of 1.95 ml of resin in 20 ml packing buffer.
b) the Resin is shaken in order to achieve a homogeneous suspension. Then allow to pass the deposition process up until the particles on the bottom of the vessel do not form a solid layer (2-4 hours). Carefully decant the supernatant containing small particles.
c) Add to the resin 20 ml packing buffer and repeat stage b). This procedure is repeated at least four times for sure50 mm with a packing capacity. Again shake the resin in the packing buffer.
b) Pour the mixed resin in the packaging capacity and quickly close it. Pump packing buffer in this mode, so that the pressure does not exceed 2000 pounds/inch2. Set the flow rate such that the filling pressure remained at the level of 2000 pounds/inch2and the flow continued for 15 min after the pressure is stabilized. Next, disconnect the column and close the upper end. Column can be used immediately or can be stored in 0.02% sodium azide.
For most samples, including Tnkase prepared in 150 mm NaCl, does not require any sample preparation prior to application to the column. Column balance acetate buffer, pH 4.5, containing calcium ions, making the sample and then spend the elution of the column using a salt gradient. In the case of small scale processes separation detalizirovannoi and necessarily.banno forms Gnkazy can be used the following procedure. The ratio of the areas of the peaks, which appeared on the chromatogram corresponds to the ratio detalizirovannoi and necessarily.banno forms Gnkazy in the sample.
Stage 1. Download a sample containing up to 150 will gorhaut summing the pH to 4.5 and centrifugation to remove these proteins, which is insoluble in used according to this method the buffer.
Stage 2. Share two forms of Gnkazy by HPLC under the following conditions:
Column: TLC of Lichrospher® 1000 SO3loaded in a stainless steel column. The size of the column 4.6 mm50 mm 4,60 mm150 mm, already filled and ready to use.
The temperature of the column: corresponds to the ambient temperature.
Eluent A: 10 mm sodium acetate, 1 mm CaCl2, pH 4.5.
Eluent B: 1 M NaCl in buffer A.
The gradient profile (see tab. B):
The flow rate: 0.8 ml/min (50 mm column), 0.5 ml/min (150 mm column).
The volume of the applied sample: up to 250 µl.
The time of equilibration of the column after working at 100% A: 20 minutes
The temperature inside the automatic sampler: 5.
Detection: absorbance at 280 nm.
Stage 3. Integrate the chromatogram. Calculate the proportion of dezaminirovanie species on the basis of peak area elyuirovaniya before detalizirovannoi Gnkazy relative to the total area of the peaks of both forms.
Chromatography on a fuzzy cation-exchanger is also a way of sharing in large scale detalizirovannoi and necessarily.banno forms of human Gnkazy. Wide is described for small parties analytical separation of the two forms of Tnkase. In this regard, large-scale separation is done with the use of fuzzy cation exchanger of Fractures on the basis of the following procedure, the pH elution.
Stage 1. Fill 31,6 column (1.6 cm EXT. dia.15,7 cm height) Fractogel EMD SO3650-M fleecy cation exchange resin (EM Separations, Gibbstown, New Jersey).
Stage 2. Tnkase after diafiltration load balancing buffer (30 mm sodium acetate (NaAc). 1 mm of calcium Chloride (CaCl2), 50 mm sodium chloride (NaCl), pH 5). Concentrated using ultrafiltration to a volume of 355 ml and concentration of 2.5 mg/ml
Stage 3. Wash the column with 2.5 column volumes (CV) 2% solution of sodium hydroxide (NaOH).
Stage 4. Wash the column with 2.5 CV pre-equilibrated buffer (300 mm NaAc, 1M NaCl, pH 5).
Stage 5. Wash the column with 2.5 CV-equilibrating buffer.
Stage 6. Load column 1-1,3 g diafiltration/ultrafiltration Gnkazy (from stage 2).
Begin collecting fractions resulting from the column effluent after the application of Gnkazy.
Stage 7. Wash the column with 5 CV-equilibrating buffer.
Stage 8. Wash the column with 5 CV wash buffer, pH of 5.3 (25 mm succinate, 1 mm CaCl2pH of 5.3).
Stage 9. Wash column with 10 CV of wash buffer, the pH of 5.4 (25 mm succinate, 1 m is Diya 11. To obtain a pool, consisting mainly of detalizirovannoi Gnkazy, combine the fractions collected at the stages 6-8. To obtain a pool consisting of necessarily.banno Gnkazy, combine the fractions collected during stage 10. Fractions collected at stage 9, contain a mixture of two forms Gnkazy and may be again subjected to separation.
The procedure described above represents only one example of the use of fuzzy ion-exchange resin is a cation exchanger to separate the two forms of recombinant human Gnkazy under conditions suitable for the large-scale allocation of purified detalizirovannoi and purified necessarily.banno Dinkas.
4. Chromatography using heparin and immobilized similar DNA
The figure 8 shows the data by chromatography on a TSK-heparin columns (Toso Haas, Montgomeryville, Pennsylvania) samples containing either a mixture of detalizirovannoi and necessarily.banno forms or purified detalizirovannoi human Tnkase or purified necessarily.banno human Tnkase. Chromatography on a TSK-heparin column was carried out under the same conditions as were described for analytical options on TLC column. Aligned in Fig.8 chromatogram demonstrate that Colo is noted above, another way to separate detalizirovannoi and necessarily.banno forms Dinkas is the use of a column containing immobilized analog of DNA that is resistant to hydrolysis by Dnazol. One example of this approach using a column with immobilized analog of DNA involves the synthesis phosphorothioates of the oligonucleotide: 5’-GCGCGCGCGCGCGCGCGCGCGC-NH3-3'. This semicomplementary sequence can be converted into double-stranded form and then entered into Renin-Hydrophor-EP Column (Rainin Co., Woburn, Massachussetts). The figure 9 presents data on chromatographicaliy on this column samples containing either a mixture of detalizirovannoi and necessarily.banno forms of human Gnkazy purified detalizirovannoi human Tnkase purified necessarily.banno human Tnkase or purified mutant human Tnkase with the aspartic acid residue (and not the rest of the aspartic acid at position 74 amino acid sequence. Column worked with the purpose of the analysis in buffer containing 1 mm calcium chloride, 5 mm MES at pH 6, was suirable gradient with salt concentration up to 1 M sodium chloride over 20 min at a flow rate of 1 ml/min. As shown in figure 9, under these conditions detalizirovannaya and nnoi Gnkazy, they differ by the position of the 74 amino acid sequences that have either aspartic acid or ISO-aspartic acid, are also resolved by this column. Thus, an additional advantage of this chromatographic method is that it allows separation of the two isomers arising from dezaminirovanie human Gnkazy.
5. Enzymatic activity detalizirovannoi human Gnkazy and necessarily.banno human Gnkazy
Used several methods to identify and study the effects of desametasone on the enzymatic activity of human Gnkazy.
Purified detalizirovannoi human Tnkase and purified necessarily.banno human Tnkase for use in the following tests were obtained using chromatography on TLC in the above-described method.
In one of the methods for determination of enzyme activity Gnkazy used synthesized double-stranded DNA-25 base pairs in length, which were marked at one end by dinitrophenol (DNP), and at the other end - Biotin. Hydrolysis of the substrate by Dnazol was determined by the binding of the reaction products on the microtiter wells plateau, covered with antibody to TNF, and the t with the stability of the samples was correlated (r2=0,613: n=5) with the level of detalizirovannostyu Gnkazy (ranging from 27% to 93%). Extrapolation of the linear equation gives an estimate of the specific activity detalizirovannoi human Gnkazy approximately 77% lower than the corresponding figure for necessarily.banno human Gnkazy.
Another method for determining the enzymatic activity Gnkazy involves the hydrolysis of a chromogenic substrate p-nitrophenylphosphate (PNPP) by the method of: M. Liao et al. (Liao, et al., Biochem. J.:781-787 (1988)). Kinetics of hydrolysis PPF human Dnazol presents sigmoid curve described by the equation nonlinear regression hill. Using this method it was shown that Vmaxfully detalizirovannoi human Gnkazy 77% lower than necessarily.banno human Gnkazy. The substrate concentration for activity equal to half the maximum (S0,5) was not significantly different for dezaminirovanie and necessarilybeing samples Gnkazy.
The following method of determining the enzymatic activity is a test that is described by Conitzer (Kunitz, J. Gen. Physiol.:349 (1950), heavily modified so that the enzymatic reaction is carried out at pH below than that of necessarily.banno human Gnkazy.
6. Storage of human Gnkazy in vitro
Purified human Tnkase from recombinant Cho cells is dissolved to a concentration of 4 mg/ml in nezaboravnom aqueous solution of 150 mm NaCl and 1 mm l2. Then the samples obtained solution Gnkazy placed in a glass and plastic ampoules. They use two types of plastic vials: one of them is prepared on the basis of a plastic resin DuPont 20 (Dupont 20) (produced by E. I. du Font de Nemours & Co., Inc., Wilmington, Delaware USA), and the other is made on the basis of plastic resin Escort (Escorene) (production Exxon Corp.). Both types of plastic materials are low-density polyethylene, however, can also be used in containers, prepared on the basis of other plastics, such as polypropylene, polystyrene or other polyolefins. Ampoules containing solution Gnkazy are stored at temperatures of -70, 2-8 or 25C. Initially, the solutions contain up to 60-65% Gnkazy in detalizirovannoi form.
The solutions in the vials were studied repeatedly after the beginning of storage to determine the level of detalizirovannostyu Gnkazy.
The results are shown in table 3.
The village is trated Gnkazy in plastic, and in glass vials. In each case, approximately 64% (2%) Gnkazy in ampoules were presented detalizirovannoi Dnazol.
But suddenly there was a marked difference during storage of the drug for 83 or 174 days at 25With the number detalizirovannoi Gnkazy in plastic and in glass vials. In plastic vials attended significantly fewer detalizirovannoi Gnkazy. In particular, after 83 days of storage at 25With 78% Gnkazy glass ampoules were presented detalizirovannoi Dnazol, whereas in plastic ampoules - only about 70% of Gnkazy was detalizirovannaya Ankasa. After 174 days storage at 25With 81% Gnkazy glass ampoules were presented detalizirovannoi Dnazol, whereas only about 71% Gnkazy in plastic vials were presented detalizirovannoi Dnazol.
Without limiting the invention to a specific mechanism or a certain theoretical basis, nevertheless we hypothesized that differences in the magnitude detalizirovannoi Gnkazy in plastic and glass vials may be the result of different pH solutions in different vials. Initially the pH RAS), the pH of the solution Gnkazy glass ampoules continued over time slightly increase (up to about pH 6.9 after 83 days of storage at 25With and about to 7.0 after 174 days storage at 25With), perhaps as a consequence of dissolution of silicates or ions from the surface of the glass. At higher pH the rate of desametasone human Gnkazy increases. Because it was not previously appreciated the fact that with the increase of pH is desametasone Gnkazy, one of the variants of the present invention is to develop a method of preparation and/or storage of human Gnkazy in solutions having an acidic pH, typically at pH 4.5-6.8 and in the most preferred cases, at pH 5.0 to 6.8.
Thus, there has been a significant improvement of the stability properties of the solution of human Gnkazy when placing this solution in plastic, not glass ampoules, because if such a change is much smaller desametasone Gnkazy over time. This discovery is significant for the choice of packaging for drug human Gnkazy used for therapeutic purposes, especially when it is desirable that the drug human Gnkazy was SPO is to be glass ampoules with desteklenen lining layer will be equally useful for this purpose. However, it is important to avoid when storing Gnkazy contact with the glass, especially when stored over a period of more than 15-30 days.
It is widely known that Tnkase I is a protein used to reduce the viscosity of pus by hydrolytic cleavage of DNA in the specified pus. Under the trade name Pulmozyme® Tnkase I approved for such use in cystic fibrosis.
The following is a description of specific details relating to the application of the present invention. Having detailed specific methods for identification, characterization, separation and use of treated detalizirovannoi and necessarily.banno human Dinkas, as well as knowledge of specific model systems to maintain them in an active state, each with an average level of knowledge in this area can develop their own alternative ways to use the fruits of the present invention on the basis of available information. Despite the fact that in the following text may appear unnecessary details, examples should not be interpreted in terms of any restrictions in the area.
1. The method of separating a mixture of human Gnkazy, detalizirovannoi amino acid residue corresponding to Asn74 native human Gnkazy, and human Gnkazy, necessarily.banno at the specified amino acid residue, providing for the separation of mixtures detalizirovannoi and necessarily.banno human Gnkazy using polydentate cation exchange resin with getting treated detalizirovannoi and necessarily.banno forms of human Gnkazy.
2. The method of separating a mixture of human Gnkazy, detalizirovannoi amino acid residue corresponding to Asn74 native human Gnkazy, and human Gnkazy, necessarily.banno at the specified amino acid residue, providing for the separation of mixtures detalizirovannoi and necessarily.banno human Gnkazy using resin with immobilized therein heparin with getting treated detalizirovannoi and necessarily.banno forms of human Gnkazy.
3. The method of separating a mixture of human Gnkazy, detalizirovannoi amino acid residue corresponding to Asn74 native human Gnkazy, and human Gnkazy, necessarily.banno at the specified amino acid residue, with pre-immobilized on it digitalisieren similar DNA with getting treated detalizirovannoi and necessarily.banno forms of human Gnkazy.
4. Purified detalizirovannaya human Tnkase having the amino acid sequence of native human Gnkazy, in which the Asn residue corresponding to Asn74 native human Gnkazy, detalizirovan.
5. Pharmaceutical composition to reduce the viscosity of pus containing detalizirovannoi human Tnkase described in paragraph 4, as an active ingredient and a pharmaceutically acceptable filler.
6. A method of treating a patient suffering from a collection of pus, introducing the indicated patient a pharmaceutical composition, described in paragraph 5, in an amount therapeutically effective to reduce the viscosity of pus.
7. Purified nedetekovana human Tnkase having the amino acid sequence of native human Gnkazy, in which the Asn residue corresponding to Asn74 native human Gnkazy, nudesonideros.
8. A method of treating a patient suffering from a collection of pus, introducing a specified patient treated necessarily.banno human Gnkazy described in paragraph 7, in an amount therapeutically effective to reduce the viscosity of pus.
9. A method of treating a patient suffering from the breeding necessarily.banno human Gnkazy, described in paragraph 7.
10. The method of treatment of a patient suffering from bronchitis, introducing the indicated patient a therapeutically effective amount of purified necessarily.banno human Gnkazy described in paragraph 7.
11. A method of treating a patient suffering from pneumonia, introducing the indicated patient a therapeutically effective amount of purified necessarily.banno human Gnkazy described in paragraph 7.
12. Pharmaceutical composition to reduce the viscosity of pus containing as the active ingredient necessarily.banno human Tnkase described in paragraph 7, and a pharmaceutically acceptable filler.
13. A method of treating a patient suffering from a collection of pus, introducing the indicated patient a pharmaceutical composition, described in paragraph 12, in an amount therapeutically effective to reduce the viscosity of pus.