In clinical studies conducted with a wide range of indications and with a large dose range (from 6 million IU / m2 per week for hairy cell leukemia; up to 100 million IU / m2 per week for melanoma), the most common adverse events were fever, fatigue, headache, myalgia. Fever and fatigue subsided 72 hours after stopping the drug administration. Although fever can be one of the symptoms of flu-like syndrome often seen with interferon treatment, testing should be done to rule out other possible causes of persistent fever.
The safety profile below was obtained from 4 clinical studies in patients with chronic hepatitis C who received Intron A alone or in combination with ribavirin for 1 year. All patients received 3 million IU of Intron A 3 times a week.
Table 2 shows adverse events observed with a frequency of greater than or equal to 10% in patients who were previously untreated and received Intron A (or Intron A in combination with ribavirin) for 1 year. In general, the reported adverse events were mild to moderate.
Table 2.
| Adverse events | Intron A (n = 806) | Intron A + ribavirin (n = 1010) |
| Local reactions | ||
| Inflammatory reactions at the injection site | 9–16% | 6–17% |
| Other reactions at the injection site | 5–8% | 3–36% |
| General reactions | ||
| Headache | 51–64% | 48–64% |
| Fatigue | 42–79% | 43–68% |
| Chills | 15–39% | 19–41% |
| Fever | 29–39% | 29–41% |
| Flu-like syndrome | 19–37% | 18–29% |
| Asthenia | 9–30% | 9–30% |
| Weight loss | 6–11% | 9–19% |
| Gastrointestinal reactions | ||
| Nausea | 18–31% | 25–44% |
| Anorexia | 14–19% | 19–26% |
| Diarrhea | 12–22% | 13–18% |
| Stomach ache | 9–17% | 9–14% |
| Vomit | 3–10% | 6–10% |
| Musculoskeletal system reactions | ||
| Myalgia | 41–61% | 30–62% |
| Arthralgia | 25–31% | 21–29% |
| Bone and muscle pain | 15–20% | 11–20% |
| CNS reactions | ||
| Depression | 16–36% | 25–34% |
| Irritability | 13–27% | 18–34% |
| Insomnia | 21–28% | 33–41% |
| Anxiety | 8–12% | 8–16% |
| Impaired ability to concentrate | 8–14% | 9–21% |
| Emotional lability | 8–14% | 5–11% |
| Skin reactions | ||
| Alopecia | 22–31% | 26–32% |
| Itching | 6–9% | 18–37% |
| Dry skin | 5–8% | 5–7% |
| Rash | 10–21% | 15–24% |
| Respiratory reactions | ||
| Pharyngitis | 3–7% | 7–13% |
| Cough | 3–7% | 8–11% |
| Dyspnoea | 2–9% | 10–22% |
| Other | ||
| Dizziness | 8–18% | 10–22% |
| Viral infection | 0–7% | 3–10% |
Adverse events observed in patients with viral hepatitis C correspond to those observed when using Intron A for other indications with some dose-dependent increase in the frequency of development.
When using Intron A for other indications (in clinical and non-clinical trials), rarely (| 1/10000,< 1/1000) или очень редко (.
On the part of the body as a whole. Very rarely - swelling of the face.
Asthenic conditions (asthenia, malaise and fatigue), dehydration, palpitations, psoriasis, fungal infection and bacterial infection (including sepsis) have been reported.
From the immune system. Very rarely, sarcoidosis or its exacerbation.
With the use of alpha interferons, the development of various autoimmune and immune-mediated disorders has been reported, including idiopathic or thrombotic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, vasculitis and Vogt-Koyanagi-Harada syndrome.
Cases of acute hypersensitivity reactions have been reported, including urticaria, angioedema, and anaphylaxis.
From the side of the cardiovascular system: rarely - arrhythmia (usually occurred in patients with a history of previous diseases of the cardiovascular system or with previous cardiotoxic therapy), transient reversible cardiomyopathy (noted in patients without a history of the cardiovascular system); very rarely - arterial hypotension, myocardial ischemia and myocardial infarction.
From the central nervous system and peripheral nervous system. Rarely - suicidal tendencies; very rarely - aggressive behavior, including directed at other people, suicidal attempts, suicide, psychosis (including hallucinations), impaired consciousness, neuropathy, polyneuropathy, encephalopathy, cerebrovascular ischemia, cerebrovascular hemorrhage, peripheral neuropathy, seizures.
On the part of the organ of hearing. Very rare - hearing impairment.
From the endocrine system. Very rarely - diabetes mellitus, worsening of the course of existing diabetes mellitus.
From the gastrointestinal tract. Very rarely - pancreatitis, increased appetite, bleeding gums, colitis.
From the liver and biliary tract. Very rarely - hepatotoxicity (including death).
Changes in the teeth and periodontium. In patients receiving combined therapy with Nitron A and ribavirin, there were pathological changes in the teeth and periodontium. Dry mouth with long-term combination therapy with ribavirin and Intron A can contribute to damage to the teeth and oral mucosa. Patients should brush their teeth 2 times a day and have regular dental check-ups. In addition, vomiting may occur in some patients.
From the side of metabolism. Rarely - hyperglycemia, hypertriglyceridemia.
From the musculoskeletal system. Rarely - rhabdomyolysis (sometimes severe), leg cramps, back pain, myositis.
On the part of the skin. Very rarely - erythema multiforme, Stevens-Johnson syndrome, toxic epidermal necrolysis, necrosis at the injection site.
From the respiratory system. Rarely pneumonia; very rarely - pulmonary infiltrates, pneumonitis.
From the urinary system. Very rarely - nephrotic syndrome, impaired renal function, renal failure.
From the hematopoietic system. Very rarely, when using Intron A as monotherapy or in combination with ribavirin, aplastic anemia and complete aplasia of the red bone marrow were noted.
On the part of the organ of vision. Rarely - hemorrhage in the retina, focal changes in the fundus, thrombosis of the retinal arteries and veins, decreased visual acuity, decreased visual fields, optic neuritis, edema of the optic nerve head.
Clinically significant changes in laboratory parameters.(more often observed when prescribing the drug in doses of more than 10 million IU / day) - a decrease in the number of granulocytes and leukocytes, a decrease in the level of hemoglobin and the number of platelets, an increase in the activity of alkaline phosphatase, LDH, the level of creatinine and serum urea nitrogen. An increase in the activity of ALT and ACT in blood plasma is noted as pathological when used for all indications, except for hepatitis, as well as in some patients with chronic hepatitis B in the absence of HBV DNA.
If during the use of Intron A for any indication, undesirable effects develop, the dose should be reduced or the treatment should be temporarily interrupted until the undesirable effects are eliminated. If persistent or repeated intolerance develops with the use of an adequate dosing regimen or the disease progresses, Intron A therapy should be canceled.
Altevir, Alfarona, Viferon, Intron-A, Realdiron, Eberon alfa R.
Composition and form of release
Interferon alpha-2b. Lyophilized powder for injection (in 1 vial - 3 million ME, 5 million IU, 10 million ME, 30 million ME). Recombinant interferon alpha-2b.
Solution for injection (pen-syringe - 10 million ME, 18 million ME, 25 million ME; in 1 vial - 10 million ME, 18 million ME, 25 million ME; 1 dose - 3 million ME, 5 million ME, 10 million ME). Human recombinant interferon alpha-2b. Rectal suppositories (150,000 ME, 500,000 ME).
pharmachologic effect
The drug is a highly purified recombinant interferon alpha-2b for parenteral administration. Obtained from the Escherichia coli clone by hybridization of a bacterial plasmid with a human leukocyte gene encoding interferon synthesis. It is a water-soluble protein with a molecular weight of 19,300 daltons.
The biological activity of interferons is manifested through their binding to specific membrane receptors of cells. Interferon alpha-2b has an antiproliferative effect on tumor cells, as well as antiviral and immunomodulatory effects.
Pharmacokinetics
With subcutaneous and intramuscular administration, bioavailability is 100%. - with subcutaneous administration 2-3 hours, with i / m - 6-7 hours, with i / v - 2 hours. The concentration of interferon in plasma was not determined after 16.24 and 4 hours, respectively. Alpha interferons are capable of disrupting oxidative metabolic processes, reducing the activity of microsomal liver enzymes of the cytochrome P450 system. It is excreted in the urine.
Indications
Multiple myeloma (generalized forms), hairy cell leukemia, chronic myeloid leukemia, malignant melanoma, bladder cancer, superficial genital warts, laryngopapillomatosis, Kaposi's sarcoma, AIDS, chronic hepatitis C, chronic hepatitis B.
Application
The application and treatment regimen depend on the type of disease. During pregnancy, interferon alpha-2b is used only in cases where the expected effect of treatment for the mother outweighs the potential risk to the fetus.
The components of the drug penetrate into the timing belt. Therefore, during lactation, based on the importance of using interferon alpha-2b for the mother, either breastfeeding or drug treatment is stopped. The experience of using the drug in children is not great: the appointment of the drug to children should be carefully justified.
Side effect
On the central nervous system, psyche: often - a feeling of fatigue, headache; possible impairment of consciousness, dizziness, ataxia, anxiety, depression, hyperexcitability, drowsiness, paresthesia; rarely - insomnia; individual cases of development of paralysis of the oculomotor nerves, visual impairments are described.
On CVS: possible hypertension or hypotension; rarely - tachycardia; individual cases of development of orthostatic hypotension, shortness of breath are described.
On PS: often - anorexia, nausea, increased levels of ACT and ALT (when using a dose of more than 100 million IU / day), alkaline phosphatase; vomiting is possible; rarely - constipation, stomatitis; individual cases of the development of dyspepsia, increased salivation, ulcerative stomatitis, flatulence are described.
On the SC: often - thrombocytopenia, granulocytopenia; in some cases - coagulation disorders (increased prothrombin and partial thromboplastin time), epistaxis; individual cases of the development of purpura are described.
On the skin: alopecia, transient rash, itching; rarely - urticaria, furunculosis, herpetic eruptions, blister lichen; individual cases of the development of erythema are described.
Local reactions: individual cases of the development of inflammation at the injection site are described.
Others: often - fever, myalgia; arthralgias are possible; rarely - cramps of the calf muscles, paroxysmal fever, dehydration, cough, increased creatinine content; individual cases of the development of sneezing, disturbances in the outflow of secretions from the nose, and hyperglycemia are described.
The invention relates to genetic engineering, biotechnology, medicine, pharmacology. New recombinant multicopy plasmid DNA pSX50 encoding the synthesis of human leukocyte alpha-2b interferon, the expression of which is under the control of lactose and tryptophan promoters and transcription terminator. As a result of the transformation of the cells of the recipient E. coli BL21 strain with the recombinant plasmid DNA pSX50, E. coli SX50 strain was obtained - the producer of recombinant human leukocyte alpha-2b interferon with a productivity of up to 0.9-1.0 g of alpha-2b interferon from 1 liter of culture medium. The method of obtaining recombinant alpha-2b interferon is based on the use of the created recombinant strain of E. coli SX50 and providing for its deep cultivation on a nutrient medium with a reduced content of tryptophan with continuous addition of nutrient substrates during biosynthesis, mechanical destruction of microorganism cells at high pressure, dissolution of the aggregated protein in concentrated solution of guanidine hydrochloride followed by renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents and its purification using three-stage chromatographic purification of interferon on resins like Chelating Sepharose Fast Flow immobilized with Cu +2 ions, ion exchange chromatography on ion exchange resins such as CM Flow and Seph gel filtration chromatography on resins of the Superdex 75 type. The method makes it possible to obtain an interferon substance of more than 99% purity according to electrophoresis data under reducing and non-reducing conditions when staining gels with silver and more than 98% according to RF HPLC and pyrogen-free (LAL-test) in quantities of at least 400-800 mg from 1 liter of culture medium. 3 n. and 3 s.p f-ly, 6 ill.
Drawings for RF patent 2242516
The invention relates to genetically engineered drugs obtained by biotechnological means, namely to methods of industrial production of recombinant human leukocyte interferon alpha-2b for medical purposes (hereinafter interferon), as well as to recombinant strains producing Escherichia coli (E. coli) and plasmid DNA, coding for the synthesis of interferon.
Interferons are protein molecules with a molecular weight of 15,000 to 21,000 daltons, produced and secreted by cells in response to a viral infection or other pathogens. There are three main groups of interferons: alpha, beta and gamma. By themselves, these groups are not homogeneous and may contain several different molecular species of interferon. Thus, more than 14 genetic varieties of interferon alpha have been identified, which are of interest and are widely used in medicine as antiviral, antiproliferative and immunomodulatory agents.
Known methods for producing human leukocyte interferon from human donor blood leukocytes induced by viruses and other inducers (SU1713591, RU 2066188, RU 2080873).
The main disadvantage of these methods for producing interferons is the likelihood of contamination of the final product with human viruses, such as hepatitis B and C virus, immunodeficiency virus, etc.
At present, the method of obtaining interferon by microbiological synthesis is recognized as more promising, which makes it possible to obtain the target product with a significantly higher yield from a relatively inexpensive feedstock. The approaches used in this case make it possible to create variants of the structural gene that are optimal for bacterial expression, as well as the regulatory elements that control its expression.
Various constructs of Pichia pastoris, Pseudomonas putida and Escherichia coli strains are used as starting microorganisms.
The disadvantage of using P. pastoris as an interferon producer (JN Garcia, JA Aguiar et. Al. // High level expression of human IFN-2b in Pichia pastoris. // Biotecnologia Aplicada, 12 (3), 152-155, 1995), is extremely difficult conditions for fermentation of this type of yeast, the need to strictly maintain the concentration of the inducer, in particular methanol, in the process of biosynthesis. The disadvantage of using Ps. putida (SU1364343, SU1640996, SU1591484, RU1616143, RU2142508) is the complexity of the fermentation process at a low level of expression (10 mg of interferon per 1 liter of culture medium). More productive is the use of strains of Escherichia coli (Semin. Oncol., 1997, Iun; 24 (3 Suppl. 9): S9-41-S9-51).
A large number of plasmids and E. coli strains based on them expressing interferon are known: E. coli strains ATCC 31633 and 31644 with plasmids Z-pBR322 (Psti) HclF-11-206 or Z-pBR 322 (Pstl) / HclN SN 35 -AHL6 (SU 1764515), E. coli strain pINF-AP2 (SU 1312961), E. coli strain pINF-F-Pa (AU 1312962), E. coli SG 20050 strain with plasmid p280 / 21FN (Kravchenko V.V. et al. Bioorganic chemistry, 1987, v. 13, No. 9, p. 1186-1193), strain E. coli SG 20050 with plasmid pINF14 (SU 1703691), strain E. coli SG 20050 with plasmid pINF16 (RU 2054041) and others. The disadvantage of technologies based on the use of these strains is their instability, as well as an insufficient level of expression of interferon.
Along with the characteristics of the strains used, the effectiveness of the process largely depends on the technology used for the isolation and purification of interferon.
A known method for producing interferon, including the cultivation of Ps cells. putida, destruction of biomass, treatment with polyethyleneimine, fractionation with ammonium sulfate, hydrophobic chromatography on phenylsilochrome C-80, pH fractionation of the lysate, its concentration and diafiltration, ion-exchange chromatography on cellulose DE-52, elution in a pH gradient, ion-exchange eluent on cellulose -52, concentration by passing through a filter cassette and gel filtration on Sephadex G-100 (SU 1640996). The disadvantage of this method, in addition to complex multi-stage fermentation, is the multi-stage process in obtaining the final product.
There is also known a method of producing interferon, including the cultivation of the E. coli strain SG 20050 / pIF16, in LB-broth in flasks in a thermostated shaker, centrifugation of the biomass, washing it with a buffer solution and sonication to destroy cells. The resulting lysate is centrifuged, washed with 3M urea solution in buffer, dissolved in a solution of guanidine chloride in buffer, sonicated, centrifuged, oxidative sulfitolysis, dialysis against 8 M urea, renaturation and final two-stage chromatography on CM-52 cellulose and Sephadec RU 2054041). The disadvantages of this method is its relatively low productivity of the main stages of the isolation and purification process. In particular, this applies to ultrasonic treatment of the product, dialysis and oxidative sulfitolysis, which leads to instability of the interferon yield, as well as to the impossibility of using this method for the industrial production of interferon.
As the closest analogue (prototype), a method for producing human leukocyte interferon can be indicated, which consists in cultivating a recombinant strain of E. coli, freezing the resulting biomass at a temperature not exceeding -70 ° C, defrosting, destroying cells of a microorganism with lysozyme, removing DNA and RNA by introducing into lysate of DNase and purification of the isolated insoluble form of interferon by washing with a buffer solution with detergents, dissolving the interferon precipitate in guanidine hydrochloride solution, renaturation and one-stage purification by ion exchange chromatography. As a producer used strain E. coli SS5, obtained using the recombinant plasmid pSS5 containing three promoters: P lac, P t7 and P trp, and the alpha-interferon gene with introduced nucleotide substitutions.
The expression of interferon by the E. coli strain SS5 containing this plasmid is controlled by three promoters: P lac, P t7 and P trp. The expression level of interferon is about 800 mg per 1 liter of cell suspension (RU 2165455).
The disadvantage of this method is the low manufacturability of the use of enzymatic destruction of cells, DNA and RNA of the microorganism and one-stage chromatographic purification of interferon. This causes the instability of the interferon release process, leads to a decrease in its quality and limits the possibility of using the above scheme for the industrial production of interferon. The disadvantages of this plasmid and the strain based on it are the use in the plasmid of a strong unregulated promoter of the T7 phage in the E. coli BL21 (DE3) strain, in which the T7 RNA polymerase gene is located under the lac operon promoter and which always "flows". Consequently, the synthesis of interferon continuously occurs in the cell, which leads to dissociation of the plasmid and a decrease in the viability of the cells of the strain, and as a result, a decrease in the yield of interferon.
The objective of this invention is to construct a recombinant industrial E. coli producer strain using a new recombinant plasmid DNA with a high level of interferon biosynthesis, and to develop an effective industrial technology for producing an interferon substance for medical purposes, corresponding in quality to "European Pharmacopoeia" for interferon alpha-2b substance.
This problem was solved by creating recombinant plasmid DNA pSX50 and Escherichia coli SX50 strain, deposited in the All-Russian collection of industrial strains of the Federal State Unitary Enterprise GosNII Genetics, number VKPM B-8550,
as well as a method for producing recombinant alpha-2b interferon based on the use of the recombinant E. coli SX50 strain and providing for its deep cultivation on a nutrient medium with a reduced tryptophan content with continuous addition of nutrient substrates during biosynthesis, mechanical destruction of microorganism cells at high pressure, dissolution of aggregated protein in a concentrated solution of guanidine hydrochloride, followed by renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents and three-stage chromatographic purification of interferon on resins of the Chelating Sepharose Fast Flow type, immobilized with Cu +2 ions, ion exchange chromatography on ion exchange resins of the CM Flow type and CM Sepharose gel filtration chromatography on Superdex 75 resins.
According to the invention, there is provided a new recombinant multicopy plasmid DNA pSX50 encoding the synthesis of human leukocyte alpha-2b interferon, the expression of which is under the control of the lactose and tryptophan promoters and the transcription terminator. Plasmid pSX50 has 3218 base pairs (bp) and is characterized by the following fragments:
The sequence from 1 nucleotide to 176 nucleotide (nt) includes a DNA fragment of 176 bp containing the tryptophan promoter (P trp);
Sequence from 177 n. to 194 N. includes a synthetic DNA fragment of 18 bp containing the Shine Delgarno sequence responsible for the initiation of translation;
Sequenced from AD 195 to 695 N. includes a DNA fragment of 501 bp containing the sequence of the interferon gene with the following nucleotide substitutions: at position 37, a change from A to C, at position 39, a change from G to T, at position 40, a change from A to C, at position 42, a change from G to T , in position 67, replacement of A with C, in position 69, replacement of G with T, in position 70, replacement of A with C, in position 72, replacement of A with T, in position 96, replacement of G with A, in position 100, replacement of A with C, in at position 102 change A to T, at position 114 change A to C, at position 120 change C to G, at position 126 change G to A, at position 129 change G to A, at position 330 change C to G, at position 339 replacement of G by A, at position 342 replacement of G by A, at position 487 replacement of A by C, at position 489 replacement of A by T, at position 495 replacement of G by A;
Sequence from 696 n. to 713 N. includes a synthetic DNA fragment of 18 bp containing a synthetic polylinker;
Sequence from 714 n. until 1138 n. includes a DNA fragment of the plasmid pKK223-3 with 4129 nt. to 4553 N. size 425 bp, containing the sequence of the strict transcription terminator rrnBT 1 T 2;
Sequence from 1139 AD until 1229 AD includes a DNA fragment of plasmid pUC19 with 2487 nt. until 2577 n. 91 bp in size, containing the promoter of the β-lactomase gene (ampicillin resistance gene - Amp R);
Sequence from 1230 AD until 2045 n. includes a DNA fragment of plasmid pUC4K with 720 nt. until 1535 n. size 816 bp, containing the structural region of the kan gene;
Sequence from 2046 AD until 3218 n. includes a DNA fragment of plasmid pUC19 from 1625 to 453 nt. size 1173 bp, containing the sequence responsible for the replication of the plasmid (ori) and the lac promoter (P lac).
Figures 1-5 show the construction schemes and physical map of plasmid pSX50.
Figure 6 shows the complete nucleotide sequence for plasmid pSX50.
The Escherichia coli SX50 strain was obtained by transforming Escherichia coli BL21 cells with the pSX50 plasmid using traditional genetic engineering technology. E. Coli SX50 strain is characterized by the following features.
Cultural and morphological characteristics
The cells are small, straight, thickened rod-shaped, gram-negative, non-sportive. Cells grow well on simple nutrient media. When growing on Difco agar, round, smooth, convex, cloudy, shiny, gray colonies with smooth edges are formed. When growing in liquid media (in a minimal medium with glucose or in LB broth), they form an intense, even haze.
Physico-biological signs
Aerobe. The temperature range for growth is 4-42 ° C with an optimum pH of 6.5-7.5.
As a nitrogen source, both mineral salts in ammonium and nitrate forms and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc. are used.
Amino acids, glycerin, carbohydrates are used as a carbon source. Antibiotic resistance. The cells show resistance to kanamycin (up to 100 μg / ml).
Escherichia coli 8X50 strain is an interferon producer.
Method, conditions and composition of the medium for storing the strain
In L-arape with the addition of kanamycin to a concentration of 20 μg / ml under oil, in L-broth containing 15% glycerol and the corresponding antibiotics in ampoules at a temperature of minus 70 ° C, in a lyophilized state in ampoules at a temperature of plus 4 ° C.
The Escherichia coli SX50 strain is identified by Bergi's Guide (1974) as a strain of the species Escherichia coli.
A method for the industrial production of alpha-2b interferon
A feature of the proposed method is the development of a technology that allows you to isolate interferon from the insoluble form that accumulates during fermentation, which can significantly simplify the technological scheme of the isolation process and increase the yield of the target product.
The method consists in the cultivation of the Escherichia coli SX50 strain in a nutrient medium, with the constant addition of nutrient substrates, preferably glucose and yeast extract, in the process of biosynthesis, preferably with a reduced tryptophan content, mechanical destruction of microorganism cells at a high pressure of 700-900 bar, dissolution of interferon in buffer guanidine hydrochloride solution, renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents, followed by three-stage chromatographic purification of interferon on Chelating Sepharose Fast Flow resins immobilized with Cu +2 ions, ion-exchange chromatography on ion-exchange resins of the CM type gel Sepharose Flow resins of the Superdex 75 type.
The optimal conditions for the individual stages of obtaining interferon are as follows:
Fermentation is carried out with continuous addition of substrates throughout the entire process, which leads to a high level of expression of interferon;
The destruction of cells is carried out in a Gaulin-type disintegrator at a pressure of 900 bar;
Removal of soluble cellular components (DNA, RNA, proteins, lipopolysaccharides, etc.) is performed by washing the insoluble form of interferon with buffer solutions containing detergents (Triton XI00, urea, etc.);
The resulting precipitate containing interferon is dissolved in a buffer solution of 6 M guanidine hydrochloride;
The renaturation of interferon is carried out in a physiological buffer solution containing chaotropic agents;
Three-stage chromatographic purification of interferon is carried out on Chelating Sepharose Fast Flow, immobilized by Cu +2 ions, on a CM Sepharose Fast Flow cation exchange resin, and gel filtration chromatography on a Superdex 75 type resin;
After each chromatographic purification, sterilizing filtration is carried out through pyrogen-free filters with a pore size of 0.22 μm.
The yield of interferon as a result of the application of the described method is approximately 400-800 mg of interferon from 1 liter of culture medium. The quality of the product obtained meets the standards and requirements of the "European Pharmacopoeia" for the substance alpha-2b interferon.
The essential differences between the proposed method and the prototype are:
Use of a strain construct with a higher productivity, which makes it possible to obtain a larger amount of interferon from 1 liter of culture medium during biosynthesis;
The use of effective mechanical destruction of cell biomass, which allows you to obtain a more pure extract of the insoluble form of interferon in a shorter time, with less losses;
The use of physiological buffer solutions during renaturation in the presence of chaotropic agents increases the yield of the correctly renatured form of interferon;
Three-stage chromatographic purification of interferon makes it possible to obtain interferon substance of more than 99% purity according to electrophoresis in reducing and non-reducing conditions when staining gels with silver and more than 98% according to RF HPLC and practically free of pyrogens (LAL test).
The essence and advantages of the claimed group of inventions is illustrated by the following examples.
Example 1. Construction of the recombinant plasmid pSX50
The method for constructing plasmid pSX50 includes the following steps:
Construction of vector plasmid pSX10;
1.construction of plasmid pSX3 (2641 bp)
2.construction of vector plasmid pSX10 (2553 bp)
Construction of the recombinant plasmid pSX41 (3218 bp);
Construction of the recombinant plasmid pSX43 (3218 bp);
Construction of the recombinant plasmid pSX45 (3218 bp);
Construction of the recombinant plasmid pSX50 (3218 bp).
Construction of vector plasmid pSX10
The vector plasmid pSX10 is a vector pUC19, in which the coding sequence of the beta-lactomase gene, which confers resistance to ampicillin, is replaced by the coding sequence of the kan gene and contains a transcriptional terminator from the plasmid pKK223-3.
The construction of the vector plasmid pSS10 is carried out in two stages:
Obtaining plasmid pSX3 (2641 bp), which is a plasmid pUC19, in which the coding region of the amp gene is replaced by the coding region of the kan gene;
Obtaining the wind plasmid pSX10 (2553 bp), which is a plasmid pSX3, in which a DNA fragment encoding the transcription terminator rBT 1 T 2 is inserted behind the BamHI site.
To obtain plasmid pSX3, five rounds of DNA amplification by PCR (polymerase chain reaction) are performed. During the first round, using the DNA of the plasmid pUC19 as a template, amplification of a DNA fragment of 1828 bp is carried out. (fragment PU1-PU2) using primers:
This and subsequent PCR reactions are carried out under the following conditions: 20 mM Tis-HCl, pH 8.8, 10 mM (NH 4) 2 SO 4, 10 mM KCl, 2 tM MgCl 2, 0.1% Triton X100, 0.1 mg / ml BSA, 0.2 mM of each dNTP, 1.25 units. Pfu DNA polymerase, 100 ng DNA. The amplification process consists of the following stages: heating at 95 ° C for 5 min, 35 PCR cycles (30 sec at 95 ° C, 30 sec at 56 ° C, 2 min at 72 ° C) and incubation for 10 min at 72 ° C. After amplification (and after subsequent amplifications), the DNA fragment is purified by electrophoresis in 1% agarose gel. During the second and third rounds, using the pUC4K plasmid DNA as a template, a 555 bp DNA fragment is amplified. (fragment KM1-KM2) using primers:
and amplification of a 258 bp DNA fragment. (KMZ-KM4) with primers
In the fifth round of PCR, the fragments (PU1-PU2) and (KM1-KM4) are combined under the following conditions: heating at 95 ° C for 5 min, 5 PCR cycles (30 sec 95 ° C, 30 sec 56 ° C, 10 min 72 ° C) and incubation for 10 min at 72 ° C. The DNA obtained after the last PCR is directly transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, clones are screened out, plasmid DNA is isolated and restriction analysis is performed. As a result, the plasmid pSX3 with a size of 2641 bp is obtained.
To obtain the vector plasmid pSX10, three rounds of DNA amplification by PCR are performed. During the first round, using the DNA of the plasmid pSX3 as a template, amplification of a DNA fragment of 2025 bp is carried out. (fragment 10.1-10.2) using primers:
During the second round, using the DNA of the plasmid pKK223-3 as a template, amplification of a DNA fragment of 528 bp is carried out. (fragment KK1-KK2) using primers:
In the third round of PCR, the fragments (10.1-10.2) and (KK1-KK2) are combined under the following conditions: heating at 95 ° C for 5 min, 5 PCR cycles (30 sec 95 ° C, 30 sec 56 ° C, 10 min 72 ° C) and incubation for 10 min at 72 ° C. The DNA obtained after the last PCR is directly transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, clones are screened out, plasmid DNA is isolated and restriction analysis is performed. As a result, plasmid pSX10 with a size of 2553 bp is obtained.
Construction of the recombinant plasmid pSX41
The recombinant plasmid pSX41 is a Hind III - BamHI DNA fragment of the vector plasmid pSX3 (2529 bp), Hind III - EcoRI a 168 bp DNA fragment encoding the E. coli tryptophan operon promoter (P trp), EcoRI-XbaI synthetic DNA fragment of 20 bp encoding SD sequence (Shine-Delgarno) and XbaI-BamHI DNA fragment of 501 bp encoding the human interferon alpha 2b gene.
To obtain Hind III - BamHI, a DNA fragment of the vector plasmid pSX3 (2529 bp), the DNA of the plasmid pSX3 is treated with restriction enzymes HindIII and BamHI, followed by electrophoretic purification in 1% agarose gel. Hind III EcoRI DNA fragment of 168 bp, encoding the tryptophan operon promoter (P trp), was obtained by PCR using total E. coli DNA as a template and primers TRP1 and PRP2, followed by processing of the amplified fragment with restriction enzymes Hindlll and EcoRI:
To obtain EcoRI-Xbal, a 20 bp synthetic DNA fragment encoding the SD sequence (Shine-Delgarno), the following complementary oligonucleotides are synthesized:
A 501 bp XbaI-BamIII DNA fragment encoding the human interferon alpha 2b gene is obtained by PCR using total human DNA as a template and primers IFN1 and IFN2, followed by treatment of the amplified fragment with restriction endonucleases Xbal and BamIII:
Then the electrophoretically purified fragments are combined, ligated with the T4 phage ligase enzyme, the DNA is transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml of kanamycin. After incubation for 12 hours at 37 ° C, the clones are sifted out, plasmid DNA is isolated, restriction analysis is carried out, and the primary structure of the DNA is determined. As a result, a 3218 bp plasmid pSX41 is obtained. Then, stepwise mutagenesis of the interferon gene is carried out to increase the level of expression of the target product. Mutagenesis of the interferon gene consists in the replacement of triplets, which are rarely found in E. coli, coding for the corresponding amino acids, with triplets, which are often found in E. coli, coding for the same amino acids. DNA mutagenesis of the interferon gene is performed by PCR.
Construction of the recombinant plasmid pSX43
To obtain the recombinant plasmid pSX43, one round of DNA amplification by PCR is carried out using the DNA of the plasmid pSX41 as a template and primers IFN3 and IFN4:
PCR is carried out under the following conditions: heating at 95 ° C for 5 min, 20 PCR cycles (30 sec at 95 ° C, 30 sec at 56 ° C, 10 min at 72 ° C) and incubation for 20 min at 72 ° C. The DNA obtained after PCR is directly transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, the clones are sifted out, plasmid DNA is isolated, restriction analysis is carried out, and the primary structure of the DNA is determined. As a result, the plasmid pSX43 with a size of 3218 bp is obtained.
Construction of the recombinant plasmid pSX45
To obtain the recombinant plasmid pSX45, one round of DNA amplification by PCR is carried out using the DNA of the plasmid pSX43 as a template and primers IFN5 and IFN6:
PCR is carried out under the following conditions: heating at 95 ° C for 5 min, 20 PCR cycles (30 sec at 95 ° C, 30 sec at 56 ° C, 10 min at 72 ° C) and incubation for 20 min at 72 ° C. The DNA obtained after PCR is directly transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, the clones are sifted out, plasmid DNA is isolated, restriction analysis is carried out, and the primary structure of the DNA is determined. As a result, a 3218 bp plasmid pSX45 is obtained.
Construction of the recombinant plasmid pSX50.
To obtain the recombinant plasmid pSX50, one round of DNA amplification by PCR is carried out using the DNA of the plasmid pSX45 as a template and primers IFN7 and IFN8:
PCR is carried out under the following conditions: heating at 95 ° C for 5 min, 20 PCR cycles (30 sec at 95 ° C, 30 sec at 56 ° C, 10 min at 72 ° C) and incubation for 20 min at 72 ° C. The DNA obtained after PCR is directly transformed into cells of the E. coli DH5 strain and plated on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, the clones are sifted out, plasmid DNA is isolated, restriction analysis is carried out, and the primary structure of the DNA is determined. As a result, a 3218 bp plasmid pSX50 is obtained.
Example 2. Obtaining a strain of E. coli SX50 - producer of interferon
The strain producing interferon E. coli SX50 is obtained by transforming the cells of the E. coli BL21 strain with the recombinant plasmid pSX50. The interferon-producing strain is grown in a 30 L fermenter to an optical density of 25.0-30.0 o.u. in M9 medium containing 1% acid casein hydrolyzate (Difco), 1% glucose, 40 μg / ml kanamycin, at a temperature of 38-39 ° C. During the fermentation, the nutrient substrate is continuously added using a gravitometric controller.
Example 3. Method for isolating interferon from E. coli SX50 strain
Interferon was obtained in 4 stages:
Stage 1. Cultivation of E. coli SX50 strain.
Stage 2. Isolation and purification of the insoluble form of interferon.
Stage 3. Dissolution and renaturation of interferon.
Stage 4. Chromatographic purification of interferon.
Stage 1. Cultivation of E. coli SX50 strain
The grown inoculum of the E. coli SX50 strain in a volume of 3 l of rich LB medium for 12 h at 26 ° C is aseptically introduced into a fermenter containing 27 l of sterile medium containing M9, 1% acid hydrolyzate of casein, 1% glucose, 1 mM MgCl 2, 0.1 mM CaCl 2, 40 mg / ml kanamycin. Cultivation in a fermenter is carried out at a temperature of 38-39 ° C, maintaining a pH of 7 ± 0.15 by automatic titration with 40% sodium hydroxide solution. The concentration of dissolved oxygen in the range (50 ± 10)% of saturation is maintained by changing the speed of the stirrer from 100 to 800 rpm and air supply from 1 to 15 l / min. The concentration of substrates, in particular glucose and yeast extract, is measured during fermentation and their concentration is maintained by varying the feed rate of concentrated solutions through peristaltic pumps using a gravimetric controller.
The accumulation of insoluble interferon is monitored by phase contrast microscopy, 15% polyacrylamide gel electrophoresis (SDS-PAAG) and reverse phase high performance chromatography (RF HPLC). Fermentation is stopped after reaching the maximum optical density (~ 25-30 pu) and stopping the synthesis of interferon. At the end of the fermentation, the culture liquid is separated by centrifugation in a flow-through rotor at a rotation speed of 5000-10000 rpm. The biomass is packed in plastic bags and frozen at minus 70 ° C.
Stage 2. Isolation and purification of the insoluble form of interferon
300-400 g of frozen biomass of E. coli SX50 strain are suspended in 3000 ml of buffer 1 (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.1% Triton X100). The suspension is passed through a Gaulin-type flow-through homogenizer, maintained at 900 bar and centrifuged in a flow-through rotor at 15,000 rpm. The resulting precipitate is washed under similar conditions sequentially with buffers 2 (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 3 M urea) and buffer 3 (20 mM Tris-HCl pH 8.0, 1 mM EDTA) and finally the interferon precipitate is suspended in 200 ml of buffer 3. In this case, the time for isolation and purification of the insoluble form of interferon is no more than 5 hours.
Stage 3. Dissolution and renaturation of interferon
To the suspension of the insoluble form of interferon obtained at the previous stage, dry guanidine hydrochloride is added to a concentration of 6 M, dithiothreitol is added to a concentration of 50 mM, Tris-HCl pH 8.0 to a concentration of 50 mM, NaCl to a concentration of 150 mM, and Triton X100 to a concentration of 0.1%, incubated at room temperature for 2 hours. The undissolved material is separated by sterilizing filtration through membranes with pore diameters of 0.22 microns.
Renaturation of interferon is carried out by slowly diluting the resulting solution 100-200 times with buffer 4 (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1 mM EDTA). Then the renaturation mixture is incubated with constant stirring for 12-15 hours at a temperature of 4-8 ° C. Then magnesium sulfate is added to a concentration of 1 mM and the aggregated material is removed by sterilizing filtration through a membrane filter with a pore diameter of 0.22 microns.
Stage 4. Chromatographic purification of interferon
Chromatographic purification of interferon is carried out in three stages.
1. The resulting renatured interferon is purified in the first stage by affinity chromatography on a Chelating Sepharose Fast Flow resin (Amersham Biosciences) immobilized with Cu +2 ions. For this, an interferon solution is applied to a column with Cu +2 Chelating Sepharose Fast Flow, and interferon is eluted with a 0.1 M citric acid buffer, pH 2.2.
2. At the second stage of chromatographic purification, an interferon solution is applied onto a CM Sepharose Fast Flow cation-exchange resin (Amersham Biosciences) and interferon is eluted with a gradient of solutions (0.0-0.5 M NaCl) in a buffer of 50 mM Na (CH 3 COO), pH 5.5.
3. Purification of the monomeric form of interferon from residues of polymeric forms of interferon is carried out at the third stage of purification of interferon by gel filtration on a Superdex 75 resin (Amersham Biosciences). Chromatography is performed in 50 mM Na (CH 3 COO) buffer, pH 5.0, containing 0.15 M NaCl.
The described method of isolation and purification of interferon allows you to obtain 4-8 g of highly purified interferon in one isolation cycle for 7-10 days from the biomass obtained from 10 liters of culture medium. The quality of the obtained interferon fully complies with the requirements of the "European Pharmacopoeia" for the substance of interferon alpha-2b, namely:
The concentration of interferon is not less than 2 × 10 8 IU / ml;
Specific activity of interferon is not less than 2.0 × 10 8 IU / mg;
Electrophoretic purity of the preparation is not less than 99% under reducing and non-reducing conditions when staining gels with silver;
The isoelectric point of the isolated interferon is in the region of pH 5.8-6.3;
The peptide map of the isolated interferon does not fundamentally differ from the peptide map for the European standard for interferon alpha 2b CRS;
As follows from the above examples, the claimed group of inventions makes it possible to obtain interferon alpha-2b in a high yield with a relatively simple and reliable technology.
CLAIM
1. Recombinant plasmid DNA pSX50, encoding the synthesis of recombinant human alpha-2b interferon, characterized in that it has a size of 3218 base pairs (bp) and consists of the following fragments: sequence from 1 to 176 nucleotide (bp) includes a fragment DNA with a size of 176 bp, containing the tryptophan promoter (P trp), sequence from 177 to 194 nt. includes a synthetic DNA fragment of 18 bp containing the Shine Delgarno sequence responsible for the initiation of translation, the sequence from 195 to 695 nt. includes a 501 bp DNA fragment containing the interferon alpha-2b gene with nucleotide substitutions: 37 (A> C), 39 (G> T), 40 (A> C), 42 (G> T), 67 ( A> C), 69 (G> T), 70 (A> C), 72 (A> T), 96 (G> A), 100 (A> C), 102 (A> T), 114 (A > C), 120 (C> G), 126 (G> A), 129 (G> A), 330 (C> G), 339 (G> A), 342 (G> A), 487 (A> C), 489 (A> T), 495 (G> A), sequence from 696 to 713 n. includes a synthetic DNA fragment of 18 bp containing a synthetic polylinker, sequence from 714 to 1138 bp. includes a DNA fragment of plasmid pKK223-3 from 4129 to 4553 nt. size 425 bp, containing the sequence of the strict transcription terminator rrnBT 1 T 2, sequence from 1139 to 1229 nt. includes a DNA fragment of plasmid pUC19 from 2487 to 2577 nt. size 91 bp, containing the promoter of the gene β-lactomase (gene resistance to ampicillin -Amp R), sequence from 1230 to 2045 nt. includes a DNA fragment of plasmid pUC4K with 720 nt. until 1535 n. 816 bp in size, containing the structural region of the kan gene, sequence from 2046 bp. until 3218 n. includes a DNA fragment of plasmid pUC19 from 1625 to 453 nt. size 1173 bp, containing the sequence responsible for the replication of the plasmid (ori) and the lac promoter (P lac).
2. The bacterial strain Eschcerichia coli SX50 transformed with the recombinant plasmid according to claim 1 - the producer of recombinant human leukocyte interferon alpha-2b.
3. A method of obtaining human interferon alpha-2b, including the cultivation of the Escherichia coli SX5 strain according to claim 2 in a nutrient medium with the constant addition of nutrient substrates during biosynthesis, mechanical destruction of microorganism cells at a pressure of 700-900 bar, dissolution of interferon in a buffer solution of guanidine hydrochloride , renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents, three-stage chromatographic purification of interferon on Chelating Sepharose Fast Flow resins immobilized with Cu +2 ions, ion exchange chromatography on CM Sepharose Fast Flow ion exchange resins and gel filtration chromatography on Superdex 75 resins ...
4. The method according to claim 3, in which the cultivation is carried out on a nutrient medium with a reduced content of tryptophan with the continuous addition of nutrient substrates, preferably glucose and yeast extract.
5. The method according to claim 3, in which, before dissolving the interferon, it is purified by removing soluble cellular components, including DNA, RNA, proteins, lipopolysaccharides, washing with buffer solutions containing detergents such as Triton XI 00, ureas.
6. The method according to claim 3, wherein after each chromatographic purification, sterilizing filtration is carried out through filters with a pore size of 0.22 µm.
- Clinical pharmacology
Pharmacological action - antiviral, antitumor and immunomodulatory.
It is a highly purified recombinant protein with a molecular weight of 19,300 daltons. Obtained from an E. coli clone by hybridization of a bacterial plasmid with a human leukocyte gene encoding interferon synthesis. Unlike interferon, alpha-2a has arginine at position 23. It has an antiviral effect, which is due to the interaction with specific membrane receptors and the induction of RNA synthesis and ultimately proteins. The latter, in turn, interfere with the normal reproduction of the virus or its release. It has immunomodulatory activity, which is associated with the activation of phagocytosis, stimulation of the formation of antibodies and lymphokines. It has an antiproliferative effect on tumor cells.
- Pharmacokinetics
When administered intramuscularly, 70% enters the systemic circulation. It is biotransformed mainly in the kidneys and to a small extent in the liver. Interferon alpha-2b is excreted by the kidneys.
- Pharmacokinetics
- Indications for use
- Chronic hepatitis B.
- Chronic hepatitis C.
- Mushroom mycosis.
- Primary T-cell lymphosarcoma.
- Hairy cell leukemia.
- Multiple myeloma (generalized forms).
- Chronic myeloid leukemia.
- Malignant melanoma.
- Bladder cancer (superficially located).
- Basal cell carcinoma.
- Pointed condylomatosis.
- Kaposi's sarcoma (including AIDS).
- Non-Hodgkin's lymphomas (as part of combination therapy).
- Method of administration and dosage
Individual, depending on the indications and treatment regimen.
- With hairy cell leukemia
Adults intramuscularly or subcutaneously administer 2 million IU / m 2 3 times a week.
- With Kaposi's sarcoma
30 million IU / m 2 3 times a week.
- With hairy cell leukemia
- Contraindications
- Severe cardiovascular disease.
- Severe violations of liver and / or kidney function.
- Epilepsy and / or serious functional disorders of the central nervous system.
- Chronic hepatitis with the threat of liver cirrhosis.
- Liver disease in the phase of decompensation.
- Chronic hepatitis on the background or after previous therapy with immunosuppressants (except for the condition after the cancellation of short-term therapy with corticosteroids).
- Autoimmune hepatitis.
- History of autoimmune diseases.
- Immunosuppressed transplant recipients.
- Previous diseases of the thyroid gland.
- Hypersensitivity to interferon alpha-2b.
- Application during pregnancy and lactation
Application during pregnancy is possible in cases where the expected benefit to the mother outweighs the potential risk to the fetus. Women of childbearing age should use reliable methods of contraception while using interferon alfa-2b.
If necessary, use during lactation should decide on the termination of breastfeeding.
- Interaction
Interferons can enhance the neurotoxic, myelotoxic or cardiotoxic effects of drugs previously prescribed or concurrently with them.
- Special conditions
Should not be used in patients with a history of mental illness. Use with caution in patients with a history of pulmonary disease (including chronic obstructive pulmonary disease), diabetes mellitus with a tendency to ketoacidosis, increased blood coagulation (including a history of thrombophlebitis and pulmonary embolism), conditions of severe myelodepression.
Before starting and systematically during the period of treatment, control of liver function, peripheral blood patterns, blood biochemical parameters, creatinine should be carried out. During the treatment period, adequate hydration of the body should be carried out. In patients with chronic hepatitis C, thyroid-stimulating hormone levels should be monitored during treatment.
In chronic hepatitis B, accompanied by a decrease in the synthetic function of the liver (which is manifested in a decrease in albumin levels or an increase in prothrombin time), the expected benefit and possible risk of therapy should be assessed. The use of concomitant psoriasis is justified in cases where the expected benefit of therapy outweighs the potential risk. With concomitant diabetes mellitus or arterial hypertension, an examination of the fundus is necessary before and during treatment. If there is a history of chronic heart failure, myocardial infarction and / or previous or existing arrhythmias, treatment with interferon alfa-2b should be carried out under the strict supervision of a physician.
- Influence on the ability to drive vehicles and use mechanisms
Influence on the ability to drive vehicles and control mechanisms At the beginning of therapy, one should refrain from potentially hazardous activities requiring increased attention, rapid psychomotor reactions, up to the period of stabilization of the effect of interferon alfa-2b.
Interferon alfa-2b in the form of powder for injection and solution for injection is included in the VED List.
- Influence on the ability to drive vehicles and use mechanisms
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