Children's health. All about medicines What is interferon alpha 2b human
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 within 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 others. possible reasons persistent fever.
The safety profile below was obtained from 4 clinical research in patients with chronic hepatitis C who received Intron A as monotherapy 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 more 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 \u003d 806) | Intron A + ribavirin (n \u003d 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% |
| Vomiting | 3–10% | 6–10% |
| Musculoskeletal 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 (.
From the body as a whole.Very rarely - facial edema.
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 worsening of it.
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 allergic edema and anaphylaxis.
From the side of cardio-vascular 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 burdened 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.
From the organ of hearing.Very rare - hearing impairment.
From the endocrine system.Rarely - diabetes, 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 combination therapy with Nitron A and ribavirin, pathological changes in the teeth and periodontium were noted. Dry mouth with long-term combination therapy with ribavirin and Intron A can damage 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.
From 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, renal dysfunction, renal failure.
From the hematopoietic system.Very rarely, when using Intron A alone 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 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 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.
- 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 interaction with specific membrane receptors and 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. Has an antiproliferative effect on tumor cells.
- Pharmacokinetics
With i / m administration, 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 liver and / or kidney dysfunction.
- Epilepsy and / or serious functional disorders of the central nervous system.
- Chronic hepatitis with the threat of liver cirrhosis.
- Liver disease in the decompensation phase.
- Chronic hepatitis with 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 mental disorders in the anamnesis. 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 clotting (including a history of thrombophlebitis and pulmonary embolism), states of severe myelodepression.
Before starting and systematically during the treatment period, control of liver function, peripheral blood patterns, blood biochemical parameters, creatinine should be carried out. During the period of treatment, 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 liver synthetic function (which manifests itself in a decrease in albumin levels or an increase in prothrombin time), the expected benefit should be assessed and possible risk therapy. Use with 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 required 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
Impact on the ability to drive vehicles and control mechanisms At the beginning of therapy, you should refrain from potentially dangerous species activities requiring increased attention, rapid psychomotor reactions, up to the period of stabilization of the action of interferon alpha-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
Release form, composition and packaging
Injection transparent, colorless.
Excipients:
0.5 ml - ampoules (5) - contoured cell packs (1) - cardboard packs.
0.5 ml - ampoules (5) - contoured cell packs (2) - cardboard packs.
0.5 ml - bottles (1) - cardboard packs.
0.5 ml - bottles (5) - contoured cell packs (1) - cardboard packs.
0.5 ml - glass syringes (1) - contoured cell packs (1) - cardboard packs.
0.5 ml - glass syringes (1) - contoured cell packs (3) - cardboard packs.
0.5 ml - glass syringes (3) - contoured cell packs (1) - cardboard packs.
0.5 ml - glass syringes (3) - contoured cell packs (3) - cardboard packs.
Injection transparent, colorless.
Excipients: sodium acetate, sodium chloride, tetraacetic acid ethylenediamine, disodium salt, tween-80, dextran 40, water d / i.
1 ml - ampoules (5) - contoured cell packs (1) - cardboard packs.
1 ml - ampoules (5) - contoured cell packs (2) - cardboard packs.
1 ml - bottles (1) - cardboard packs.
1 ml - bottles (5) - contoured cell packs (1) - cardboard packs.
1 ml - glass syringes (1) - contoured cell packaging (1) - cardboard packs.
1 ml - glass syringes (1) - contoured cell packs (3) - cardboard packs.
1 ml - glass syringes (3) - contoured cell packs (1) - cardboard packs.
1 ml - glass syringes (3) - contoured cell packs (3) - cardboard packs.
Clinical and pharmacological group
Interferon. Antineoplastic, antiviral and immunomodulatory drugpharmachologic effect
Interferon. Altevir ® has antiviral, immunomodulatory, antiproliferative and antitumor effects.
Interferon alpha-2b, interacting with specific receptors on the cell surface, initiates a complex chain of changes within the cell, including the induction of the synthesis of a number of specific cytokines and enzymes, disrupts the synthesis of viral RNA and virus proteins in the cell. The result of these changes is nonspecific antiviral and antiproliferative activity associated with the prevention of viral replication in the cell, inhibition of cell proliferation and the immunomodulatory effect of interferon. Interferon alpha-2b stimulates the process of antigen presentation to immunocompetent cells, has the ability to stimulate the phagocytic activity of macrophages, as well as the cytotoxic activity of T cells and "natural killer cells" involved in antiviral immunity.
Prevents the proliferation of cells, especially tumor cells. It has a depressing effect on the synthesis of some oncogenes, leading to inhibition of tumor growth.
Pharmacokinetics
Suction
With s / c or i / m administration of interferon alfa-2b, its bioavailability ranges from 80% to 100%. After the introduction of interferon alfa-2b, T max in the blood plasma is 4-12 hours, T 1/2 - 2-6 hours. 16-24 hours after administration, the recombinant interferon is not detected in the blood serum.
Metabolism
Metabolism is carried out in the liver.
Alpha interferons are capable of disrupting oxidative metabolic processes, reducing the activity of microsomal liver enzymes of the cytochrome P450 system.
Withdrawal
It is excreted mainly by the kidneys by glomerular filtration.
Indications for the use of the drug
As part of complex therapy in adults:
- with chronic viral hepatitis B without signs of liver cirrhosis;
- with chronic viral hepatitis C in the absence of symptoms liver failure (monotherapy or combination therapy with ribavirin);
- with papillomatosis of the larynx;
- with genital warts;
- with hairy cell leukemia, chronic myeloid leukemia, non-Hodgkin's lymphoma, melanoma, multiple myeloma, Kaposi's sarcoma against the background of AIDS, progressive kidney cancer.
Dosage regimen
Apply s / c, i / m and i / v. Treatment must be started by a physician. Further, with the permission of the doctor, the patient can inject himself a maintenance dose on his own (in cases where the drug is prescribed s / c or i / m).
Chronic hepatitis B: Altevir ® is administered subcutaneously or intramuscularly at a dose of 5-10 million IU 3 times a week for 16-24 weeks. Treatment is stopped after 3-4 months of use in the absence of positive dynamics (according to a study of the DNA of the hepatitis B virus).
Chronic hepatitis C: Altevir ® is administered subcutaneously or intramuscularly at a dose of 3 million ME 3 times a week for 24-48 weeks. In patients with a recurrent course of the disease and patients who have not previously received treatment with interferon alfa-2b, the effectiveness of treatment is increased with combination therapy with ribavirin. The duration of the combination therapy is at least 24 weeks. Altevir therapy should be carried out for 48 weeks in patients with chronic hepatitis C and genotype 1 of the virus with a high viral load, in whom by the end of the first 24 weeks of treatment, hepatitis C virus RNA is not detected in the serum
Laryngeal papillomatosis: Altevir ® is administered subcutaneously at a dose of 3 million IU / m 2 3 times a week. Treatment begins after surgical (or laser) removal of tumor tissue. The dose is selected taking into account the tolerability of the drug. Achieving a positive response may require treatment for 6 months.
Hairy cell leukemia: The recommended dose of Altevir for subcutaneous administration to patients after or without splenectomy is 2 million IU / m 2 3 times a week. In most cases, the normalization of one or more hematological parameters occurs after 1-2 months of treatment, it is possible to increase the duration of treatment to 6 months. This dosing regimen should be adhered to constantly, unless there is a rapid progression of the disease or the onset of symptoms of severe drug intolerance.
Chronic myeloid leukemia: The recommended dose of Altevir as monotherapy is 4-5 million IU / m 2 per day s / c daily. To maintain the leukocyte count, a dose of 0.5-10 million IU / m 2 may be required. If the treatment allows you to control the number of leukocytes, then to maintain hematological remission, the drug should be used in the maximum tolerated dose (4-10 million IU / m 2 daily). The drug should be discontinued after 8-12 weeks if therapy has not led to partial hematological remission or a clinically significant decrease in the number of leukocytes.
Non-Hodgkin's lymphoma: Altevir ® is used as adjuvant therapy in combination with standard chemotherapy regimens. The drug is administered subcutaneously at a dose of 5 million IU / m 2 3 times a week for 2-3 months. The dose must be adjusted depending on the tolerability of the drug.
Melanoma: Altevir ® is used as an adjuvant therapy for patients with a high risk of recurrence in adults after tumor removal. Altevir ® is administered intravenously at a dose of 15 million IU / m 2 5 times a week for 4 weeks, then subcutaneously at a dose of 10 million IU / m2 3 times a week for 48 weeks. The dose must be adjusted depending on the tolerability of the drug.
Multiple myeloma: Altevir ® is prescribed during the period of achieving stable remission at a dose of 3 million IU / m 2 3 times a week n / a.
Kaposi's sarcoma with AIDS: the optimal dose has not been established. The drug can be used in doses of 10-12 million IU / m2 / day s / c or i / m. In case of stabilization of the disease or response to treatment, therapy is continued until tumor regression occurs or drug withdrawal is required.
Kidney cancer:the optimal dose and regimen have not been established. It is recommended to use the drug subcutaneously in doses from 3 to 10 million IU / m 2 3 times a week.
Preparation of a solution for intravenous administration
The volume of the Altevir solution required for the preparation of the required dose is collected, added to 100 ml of sterile 0.9% sodium chloride solution and injected over 20 minutes.
Side effect
General reactions: very often - fever, weakness (they are dose-dependent and reversible reactions, disappear within 72 hours after a break in treatment or its termination), chills; less often, malaise.
From the side of the central nervous system: very often - headache; less often - asthenia, drowsiness, dizziness, irritability, insomnia, depression, suicidal thoughts and attempts; rarely - nervousness, anxiety.
From the musculoskeletal system: very often - myalgia; less often - arthralgia.
From the digestive system:very often - decreased appetite, nausea; less often - vomiting, diarrhea, dry mouth, change in taste; rarely - abdominal pain, dyspepsia; possibly a reversible increase in the activity of liver enzymes.
On the part of the cardiovascular system: often - a decrease in blood pressure; rarely - tachycardia.
Dermatological reactions: less often - alopecia, increased sweating; rarely - skin rash, itching.
From the hematopoietic system: possible reversible leukopenia, granulocytopenia, decreased hemoglobin levels, thrombocytopenia.
Others: rarely - weight loss, autoimmune thyroiditis.
Contraindications to the use of the drug
- a history of severe cardiovascular disease (uncontrolled chronic heart failure, recent myocardial infarction, severe cardiac arrhythmias);
- severe renal and / or hepatic insufficiency (including those caused by the presence of metastases);
- epilepsy, as well as severe dysfunctions of the central nervous system, especially expressed by depression, suicidal thoughts and attempts (including a history);
- chronic hepatitis with decompensated cirrhosis of the liver and in patients receiving or recently receiving treatment with immunosuppressants (with the exception of a completed short-term course of GCS treatment);
- autoimmune hepatitis or otherwise autoimmune disease;
- treatment with immunosuppressants after transplantation;
- thyroid disease that cannot be controlled by conventional therapeutic methods;
- decompensated lung diseases (including COPD);
- decompensated diabetes mellitus;
- hypercoagulability (including thrombophlebitis, pulmonary embolism);
- severe myelodepression;
- pregnancy;
- lactation period (breastfeeding);
- hypersensitivity to drug components.
Use of the drug during pregnancy and breastfeeding
The drug is contraindicated during pregnancy and lactation (breastfeeding).
Application for violations of liver function
Application for impaired renal function
The drug is contraindicated in severe renal and / or liver failure (including those caused by the presence of metastases).
special instructions
Prior to treatment with Altevir for chronic viral hepatitis B and C, a liver biopsy is recommended to assess the degree of liver damage (signs of an active inflammatory process and / or fibrosis). The effectiveness of the treatment of chronic hepatitis C increases with combination therapy with Altevir and ribavirin. The use of Altevir is not effective in the development of decompensated liver cirrhosis or hepatic coma.
In case of side effects during treatment with Altevir, the dose of the drug should be reduced by 50% or the drug should be temporarily canceled until they disappear. If a side effects persist or reappear after dose reduction, or disease progression is observed, then treatment with Altevir should be discontinued.
If the platelet count falls below 50x10 9 / l or the granulocyte count is below 0.75x10 9 / l, it is recommended to reduce the dose of Altevir by 2 times with a blood test control after 1 week. If these changes persist, then the drug should be canceled.
If the platelet count falls below 25x10 9 / l or the granulocyte count is below 0.5x10 9 / l, it is recommended to discontinue the drug Altevir with a blood test control after 1 week.
In patients receiving interferon alpha-2b preparations, antibodies can be detected in the blood serum that neutralize its antiviral activity. In almost all cases, antibody titers are low, their appearance does not lead to a decrease in the effectiveness of treatment or the occurrence of other autoimmune disorders.
Overdose
Data on overdose of Altevir ® are not provided.
Drug interactions
The drug interaction between Altevir and other drugs has not been fully studied. Altevir should be used with caution simultaneously with sleeping pills and sedatives, narcotic analgesics and drugs that potentially have a myelodepressant effect.
With the simultaneous appointment of Altevir and theophylline, the concentration of the latter in the blood serum should be monitored and, if necessary, the dosage regimen should be changed.
When using Altevir in combination with chemotherapeutic drugs (cytarabine, cyclophosphamide, doxorubicin, teniposide), the risk of toxic effects increases.
Terms of dispensing from pharmacies
The drug is available by prescription.
Storage conditions and periods
The drug should be stored out of the reach of children, in accordance with SP 3.3.2-1248-03 at a temperature of 2 ° to 8 ° C; do not freeze. The shelf life is 18 months.
Transport at temperatures from 2 ° to 8 ° С; do not freeze.
"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 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 for producing 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 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 such as 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 allows 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 c.p f-crystals, 6 ill.
Drawings for RF patent 2242516
The invention relates to genetic engineering medicines, 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 encoding interferon synthesis.
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 leukocytes donated blood human induced by viruses and other inducers (SU1713591, RU 2066188, RU 2080873).
The main disadvantage of these methods of obtaining 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 strain SG 20050 with plasmid p280 / 21FN (Kravchenko V.V. et al. Bioorganic chemistry, 1987, v. 13, No. 9, pp. 1186-1193), strain E. coli SG 20050 with plasmid pINF14 (SU 1703691), strain E. coli SG 20050 with plasmid pINF16 (RU 2054041) and other. The disadvantage of technologies based on the use of these strains is their instability, as well as insufficient expression of interferon.
Along with the characteristics of the strains used, the efficiency 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 the 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 higher than -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 a 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 using 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 strain BL21 (DE3), in which the T7 RNA polymerase gene is located under the promoter of the lac operon 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 strain of E. coli producer 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 task 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 a recombinant strain of E. coli SX50 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;
Succession from 195 AD 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 AD includes a DNA fragment of 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. size 91 bp, containing the promoter of the gene β-lactomase (gene resistance to ampicillin - Amp R);
Sequence from 1230 AD until 2045 n. includes a DNA fragment of the plasmid pUC4K with 720 nt. until 1535 AD 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 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. The E. Coli SX50 strain is characterized by the following features.
Cultural and morphological characteristics
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 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.
Both mineral salts in ammonium and nitrate forms and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc. are used as a nitrogen source.
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 Identifier (1974) as a strain of the species Escherichia coli.
A method of industrial production of alpha-2b interferon
A feature of the proposed method is the development of technology that allows you to isolate interferon from the insoluble form that accumulates during fermentation, which allows to 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 strain SX50 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 and gel Sepharose Flow filtration resins such as Superdex 75.
The optimal conditions for the individual stages of obtaining interferon are as follows:
Fermentation is carried out with the continuous addition of substrates throughout the entire process, which causes high level 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;
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 CM Sepharose Fast Flow cation exchange resin, and gel filtration chromatography on 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 applying the described method is approximately 400-800 mg of interferon from 1 liter of culture medium. The quality of the resulting product 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:
The 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 an 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 pyrogen-free (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 conferring ampicillin resistance is replaced by the coding sequence of the kan gene and contains the transcription 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 with 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 ° С for 5 min, 35 PCR cycles (30 sec at 95 ° С, 30 sec at 56 ° С, 2 min at 72 ° С) and incubation for 10 min at 72 ° С. 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 seeded on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, clones are sifted out, plasmid DNA is isolated and a 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 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 seeded on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, clones are sifted out, plasmid DNA is isolated and restriction analysis is performed. As a result, plasmid pSX10 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 a synthetic DNA fragment of 20 bp encoding the SD sequence (Shine-Delgarno) and XbaI-BamHI a 501 bp DNA fragment encoding the human interferon alpha 2b gene.
To obtain Hind III - BamHI, a DNA fragment of the vector plasmid pSX3 (2529 bp) 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 template and primers TRP1 and PRP2, followed by processing of the amplified fragment with restriction enzymes Hindlll and EcoRI:
To obtain EcoRI-Xbal a synthetic 20 bp 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 enzymes 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 kanamycin. After incubation for 12 hours at 37 ° C, 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. Next, a 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 rare 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 carried out 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 the IFN3 and IFN4 primers:
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, 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 the IFN5 and IFN6 primers:
PCR is carried out under the following conditions: heating at 95 ° C for 5 min, 20 PCR cycles (30 sec 95 ° C, 30 sec 56 ° C, 10 min 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, 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 the IFN7 and IFN8 primers:
PCR is carried out under the following conditions: heating at 95 ° C for 5 min, 20 PCR cycles (30 sec 95 ° C, 30 sec 56 ° C, 10 min 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 seeded on LA medium containing 20 μg / ml kanamycin. After incubation for 12 hours at 37 ° C, 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 E. coli SX50 strain - interferon producer
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 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 revolutions 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 when the maximum optical density is reached (~ 25-30 pu) and interferon synthesis stops. 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. At the same time, 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. Insoluble 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 in the first stage is purified 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 to 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 50 mM Na (CH 3 COO) buffer, 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 for the isolation and purification of interferon makes it possible to obtain 4-8 g of highly purified interferon in one isolation cycle for 7-10 days from the biomass obtained from 10 l of the culture medium. The quality of the resulting interferon fully meets 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;
The 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 of 176 bp containing the tryptophan promoter (P trp), sequence from 177 to 194 bp. 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\u003e C), 39 (G\u003e T), 40 (A\u003e C), 42 (G\u003e T), 67 ( A\u003e C), 69 (G\u003e T), 70 (A\u003e C), 72 (A\u003e T), 96 (G\u003e A), 100 (A\u003e C), 102 (A\u003e T), 114 (A \u003e C), 120 (C\u003e G), 126 (G\u003e A), 129 (G\u003e A), 330 (C\u003e G), 339 (G\u003e A), 342 (G\u003e A), 487 (A\u003e C), 489 (A\u003e T), 495 (G\u003e 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 the plasmid pUC4K with 720 nt. until 1535 AD size 816 bp, 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 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 for producing 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 tryptophan content with the continuous addition of nutrient substrates, preferably glucose and yeast extract.
5. The method according to claim 3, in which before the dissolution of interferon, it is purified by removing soluble cellular components, including DNA, RNA, proteins, lipopolysaccharides, washing with buffer solutions containing detergents of the Triton XI 00 type, ureas.
6. The method according to claim 3, wherein after each chromatographic purification, sterilizing filtration is carried out through filters with pore sizes of 0.22 μm.