What Is Recombinant Interleukin?

Recombinant human interleukin-2 (rhIL-2) is a gene recombination product. It is a non-glycosylated protein with the same biological activity as natural IL-2. Its pharmacological effect lies in enhancing the immune response. Recombinant human IL-2. Approved by the FDA in the United States in 1992 under the trade name Proeukin. Since then, it has been marketed in Japan, Denmark and other countries. The approved clinical application indications are kidney cancer, angiosarcoma and melanoma. ^[1]

Chinese name: Recombinant human interleukin-2

Foreign name: Recombinant Human Interleukin-2
Relative molecular mass: 15500

Recombinant Human Interleukin 2 Pharmacopoeia Standard

Definition, composition and use of recombinant human interleukin-2

This strain is made from E. coli that highly expresses human interleukin-2 (referred to as human interleukin-2), and is a recombinant human interleukin-2 obtained after fermentation, isolation, and high purification. Suitable stabilizer, without preservatives and antibiotics. ^[2]

Basic requirements for recombinant human interleukin 2

The facilities, raw materials and auxiliary materials, water, utensils, animals, etc. used for production and inspection shall comply with the relevant requirements of "ordinance". ^[2]

Recombinant human interleukin 2 manufacturing

1 engineering strains ^[2]

1.1 Name and source ^[2]

The recombinant human interleukin-2 engineering strain is an E. coli strain transformed with a recombinant plasmid carrying the human interleukin-2 gene.

1.2 Establishment of seed batches ^[2]

It shall comply with the provisions of the Administrative Regulations on Bacterial Toxins for the Examination of Production of Biological Products.

1.3 Bacterial test ^[2]

The strains of the main seed batch and the working seed batch shall be subjected to the following comprehensive inspections.

1.3.1 Seeding LB agar plates

It should be in the form of a typical E. coli colony, without the growth of other bacteria.

1.3.2 Staining microscopy

Should be typical Gram-negative bacilli.

1.3.3 Resistance to antibiotics

Should be consistent with the original species.

1.3.4 Electron microscopy (work seed batches can be exempted)

It should be in the typical form of E. coli, free of mycoplasma, virus-like particles and other microorganisms.

1.3.5 Biochemical reactions

Should conform to the characteristics of E. coli biochemical reaction.

1.3.6 Human interleukin-2 expression

Culture in a shaker should be no less than the expression of the original strain.

1.3.7 Plasmid check

The restriction map of this plasmid should be consistent with the original recombinant plasmid.

1.3.8 Nucleotide sequence check of target gene (work seed batch can be exempted)

The nucleotide sequence of the gene of interest should be consistent with the approved sequence.

2 stock solution ^[2]

2.1 Seed liquid preparation

The certified working seed batch strains are inoculated in a suitable medium (which may contain an appropriate amount of antibiotics) for cultivation.

2.2 Fermentation medium

Use a suitable antibiotic-free medium.

2.3 Seed liquid inoculation and fermentation culture

2.2.3.1 Inoculate an appropriate amount of seed solution in the sterilized medium.

2.2.3.2 Fermentation at an appropriate temperature should be performed in accordance with an approved fermentation process and the corresponding fermentation conditions should be determined, such as temperature, pH, dissolved oxygen, feed, fermentation time, etc. The fermentation broth should be checked regularly for plasmid loss rate (Appendix G in Part III of the 2010 Pharmacopoeia).

2.4 Fermentation broth treatment

Collect and treat bacterial cells with appropriate methods.

2.5 Preliminary purification

Use an approved purification process for preliminary purification to achieve the required purity.

2.6 Highly purified

After preliminary purification, it is highly purified using an approved purification process to meet the 3.1 requirements, which is the recombinant human interleukin-2 stock solution. Add a suitable stabilizer, store it at a suitable temperature after sterilizing and filtering, and specify its validity period.

2.7 Stock solution test

Proceed according to 3.1.

3 semi-finished products ^[2]

3.1 Preparation and sterilization

3.1.1 Diluent preparation

Prepare dilutions according to approved formulas. It should be used for dilution immediately after preparation.

3.1.2 Dilution and sterilization

Dilute the qualified recombinant human interleukin-2 stock solution with the stabilizing agent to the required concentration with the diluent of item 2.3.1.1. After sterilization and filtration, it is a semi-finished product and stored at 2-8 ° C.

3.2 Testing of semi-finished products

Proceed according to 3.2.

4 finished products ^[2]

4.1 Batch

Should comply with the "Biological Product Batch Regulations".

4.2 Packing

Should comply with the "Biological product packaging and lyophilization regulations" and the 2010 edition of the third part of the Pharmacopoeia Appendix IA relevant provisions.

4.3 Specifications

Should be an approved specification.

4.4 Packaging

Should comply with the "Biological Product Packaging Regulations" and the relevant provisions of the 2010 edition of the three Pharmacopoeia Appendix IA.

Recombinant human interleukin 2 test

1 Stock solution test ^[2]

1.1 Biological activity

Determined according to law (Appendix XD in Part III of the 2010 Pharmacopoeia).

1.2 Protein content

Determination according to law (the second method of Appendix VIB in the third part of the Pharmacopoeia of the 2010 edition).

1.3 Specific activity

For the ratio of biological activity to protein content, each 1 mg of protein should not be less than 1.0 × 10IU.

1.4 Purity

1.4.1 Electrophoresis

Determined according to law (Appendix IVC in Part III of the 2010 Pharmacopoeia). By non-reducing SDS-polyacrylamide gel electrophoresis, the separation gel concentration is 15%, and the sample volume should be not less than 10 g (Coomassie Blue R250 staining method) or 5 g (silver staining method). Scanned by a scanner, the purity should be no less than 95.0%.

1.4.2 High Performance Liquid Chromatography

Determined according to law (Appendix IIIB in Part III of the 2010 Pharmacopoeia). Chromatographic columns are filled with gels suitable for chromatographic separation of proteins with a molecular mass of 5 to 60 kD; the mobile phase is 0.1 mol / L phosphate-0.1 mol / L sodium chloride buffer, pH 7.0 (with appropriate surface activity) The amount of sample should be no less than 20g, and the detection should be performed at a wavelength of 280nm. The theoretical plate count based on the peak of human interleukin-2 chromatogram should be no less than 1500. According to the area normalization method, the area of the main peak of human interleukin-2 should not be less than 95.0% of the total area.

1.5 molecular weight

Determined according to law (Appendix IV C, Part III of the Pharmacopoeia, 2010 edition). By reduction SDS-polyacrylamide gel electrophoresis, the separation gel concentration is 15%, the amount of sample should be not less than 1.0 g, and the molecular weight of the product should be 15.5kD ± 1.6kD.

1.6 Exogenous DNA residue

The dose per human should not be higher than 10ng (Appendix B in Part III of the 2010 Pharmacopoeia).

1.7 Host bacteria protein residue

It should not be higher than 0.10% of the total protein (Appendix C of Part III of the 2010 Pharmacopoeia).

1.8 Residual antibiotic activity

As determined by law (Appendix A of Part III of the 2010 Pharmacopoeia), there should be no residual ampicillin or other antibiotic activity. If the product contains SDS. The SDS concentration should be diluted to at least 0.01% before determination.

1.9 Bacterial endotoxin test

It should be less than 10 EU per 1 million IU (the third edition of the Pharmacopoeia of 2010 edition E gel limit test). If the product contains SDS, dilute the SDS concentration to at least 0.0025% before measuring.

1.10 isoelectric point

The main zone should be 6.5 to 7.5, and the isoelectric point spectrum of the test product should be the same as that of the reference product (Appendix IV D, Part III of the 2010 Pharmacopoeia).

1.11 UV Spectrum

Dilute the test sample to about 100-500 g / ml with water or physiological sodium chloride solution, and scan at 1 cm of the optical path and a wavelength of 230-360 nm. The maximum absorption peak wavelength should be 277 nm ± 3 nm (Appendix IIA in Part III of the Pharmacopoeia, 2010 Edition) .

1.12 Peptide Map

Determined according to law (Appendix E of the third part of the Pharmacopoeia of the 2010 edition), which should be consistent with the graphic of the reference substance.

1.13 N-terminal amino acid sequence (at least once a year)

Using an amino acid sequence analyzer, the N-terminal sequence should be:

(Met) -Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-Glu.

2 Semi-finished product inspection ^[2]

2.1 Bacterial endotoxin test

It should be less than 10 EU per 1 million IU (the third edition of the 2010 Pharmacopoeia Appendix E gel limit test). If the product contains SDS, dilute the SDS concentration to at least 0.0025% before measuring.

2.2 Sterility check

Inspection according to law (Appendix A of Part III of the 2010 Pharmacopoeia) shall comply with the regulations.

3 Finished product inspection ^[2]

3.1 Identification test

It should be positive according to the immunoblot method (Appendix III A in Part III of the 2010 Pharmacopoeia) or the immunospot method (Appendix III B in Part three of the Pharmacopoeia 2010).

3.2 Physical inspection

3.2.1 Appearance

It should be colorless or slightly yellow clear liquid.

3.2.2 Visible foreign bodies

Inspection according to law (Appendix VB of Part III of the 2010 Pharmacopoeia) shall comply with regulations.

3.2.3 loading

Inspection according to law (Appendix I A in Part III of the 2010 Pharmacopoeia). It should not be lower than the indicated amount.

3.3 Chemical verification

3.3.1 pH

It should be 3.5 4.5 (Appendix V A in Part III of the 2010 Pharmacopoeia).

3.3.2 Osmolality

Determination according to law (Appendix V H in Part III of the 2010 Pharmacopoeia) shall meet the requirements for approval.

3.4 Biological activity

It should be 80% to 150% of the labeled amount (Appendix XD in Part III of the 2010 Pharmacopoeia).

3.5 Residual antibiotic activity

As determined by law (Appendix A of Part III of the 2010 Pharmacopoeia), there should be no residual ampicillin or other antibiotic activity. If the product contains SDS, dilute the SDS concentration to at least 0.01% before measuring.

3.6 Sterility check

Inspection according to law (Appendix A of Part III of the 2010 Pharmacopoeia) shall comply with the regulations.

3.7 Bacterial endotoxin test

Each one should be less than 10EU (2010 Appendix 3 Pharmacopoeia E Gel Limit Test). If the product contains SDS, dilute the SDS concentration to at least 0.0025% before measuring.

3.8 Abnormal toxicity test

Inspection according to the law (Appendix III of the 2010 Pharmacopoeia F mouse test method) should comply with the regulations.

3.9 Residual acetonitrile content

If acetonitrile is used in the process, perform gas chromatography (Appendix III C, Part III of the Pharmacopoeia, 2010 Edition). The chromatographic column is a quartz capillary column with a column temperature of 45 ° C, a gasification chamber temperature of 150 ° C, a detector temperature of 300 ° C, a carrier gas of nitrogen, and a flow rate of 4.0 ml per minute. Pick up 1.0ml of the above standard solution and 400l of sample solution for headspace injection, and determine the acetonitrile content of the test solution by comparing the peak areas of the standard solution and the test solution. The content of acetonitrile is not higher than 0.0004%.

Pharmacological effects of recombinant human interleukin 2

Interleukin-2 ^[3] (Interleukin-2, IL-2) is a cytokine produced by the activation of helper T lymphocytes (TH) [1-2]. IL-2 has the ability to dually regulate immune strain and can effectively induce cellular and humoral immune responses. Recombinant human interleukin-2 (rhIL-2) is a recombinant gene product, which contains 133 amino acid residues and a relative molecular mass of 15.5 k. Its pharmacological effect is to enhance the immune response.

The main physiological role of aldileukin ^[4] (IL-2) is to stimulate and maintain the differentiation and proliferation of T cells. The biological activities of IL-2 related tumors include: 1. Stimulate the proliferation and activation of natural killer (NK) cells and enhance their activity. 2. Induced cytotoxic lymphocytes and enhanced their cytolytic activity. 3. Induced lymphokine activated killer (LAK) cells. LAK cells have a broad spectrum of antitumor activity and can lyse a variety of tumor cells. IL-2 is not only an essential lymphokine that produces LAK cells, but also can maintain the proliferation and long-term growth of LAK cells in its presence. Therefore, IL-2 and LAK cells are often given at the same time in clinical antitumor treatment. It is the adoptive immunotherapy (AIT) of malignant tumors. 4. Stimulate the proliferation of tumor infiltrating lymphocytes (TIL) and enhance its activity. Animal experiments have proven that TIL has 50 to 100 times higher antitumor activity than LAK cells. 5. It can not only promote the proliferation of T cells, but also promote the proliferation and differentiation of cells. In animal models of tumor therapy, it has been proven that when T cell-mediated antitumor responses are strong, a very low amount of IL-2 is sufficient to stimulate antitumor effects; but when T cell-mediated antitumor responses are weak, it is necessary to High doses of IL-2. Low doses can significantly stimulate T cells, while activation of NK and LAK cells requires large doses. Because IL-2 cannot directly fight cancer, the purpose of medication is to maximize the excitement of the immune mechanism to strengthen the anti-cancer effect. The combination of IL-2 and LAK cells previously used in the treatment of malignant tumors is no longer recommended, and replaced with IL-2 and tumor infiltrating lymphocyte (TIL) combined therapy (adoptive therapy). 6. rhIL-11 is an active protein consisting of 177 amino acids. Its coding gene is located in the 13th arm of chromosome 19. Acting on the megakaryocyte level at a later stage of the megakaryocyte cell line, it promotes the proliferation and maturation of megakaryocytes and promotes the production of platelets. Although IL-11 does not have megakaryocyte colony-stimulating factor (Meg-CSF) activity, it has megakaryocyte proliferation factor activity and works in concert with GM-CSF and IL-3 to increase the amount of CFU-MegDNA.

Pharmacokinetics of recombinant human interleukin-2 :

This product is mainly distributed in the kidney, liver, spleen and lungs. The kidney is the main metabolic organ, and cathepsin D of kidney tissue cells breaks down this product. ^{[5] The} distribution and elimination half-life (t1 / 2) in serum were 13 minutes and 85 minutes, respectively. ^[6]

Recombinant human interleukin 2 indications:

1. For the treatment of renal cell carcinoma, melanoma, breast cancer, bladder cancer, liver cancer, rectal cancer, lung cancer. It can be used for the control of cancerous pleural and ascites fluid, and can also be used for the culture of killer cells activated by lymphatic prisoners.

2. It is used to treat tumor patients after surgery, radiotherapy and chemotherapy, which can enhance the body's immune function.

3 For the treatment of congenital or acquired immunodeficiency. Improve patient's cellular immune function and anti-infective ability.

4 Treatment of various autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome and so on.

5. It has a certain therapeutic effect on certain viral, bacillary, and intracellular parasitic infectious diseases, such as hepatitis B, leprosy, tuberculosis, and Candida albicans infection. ^[5]

Dosage and Administration of Recombinant Human Interleukin 2

Dissolve it in sterile water for injection. The specific usage, dosage and course of treatment vary depending on the disease. Generally, the following methods are used (or as prescribed by your doctor).
1. Systemic medication:
(1) Subcutaneous (or intramuscular) injection: Recombinant human interleukin-2 (Ala) 600 ~ 1 million IU / time (1 or 2), dissolved by adding 2ml of water for injection, subcutaneous injection 3 times / week A course of treatment.
(2) Intravenous injection: 400 to 800,000 IU / m ₂ (1 to 2 sticks), 500 ml of normal saline, instillation time of not less than 4 hours, 3 times a day, 6 weeks as a course of treatment.
(3) Interventional arterial infusion: 500,000 to 1 million IU / time (1 to 2), once every 2 to 4 weeks, and 2 to 4 times as a course of treatment.
2. Regional and local administration:
(1) Intrathoracic injection: used for cancerous pleural and peritoneal effusion, recombinant human interleukin-2 (Ala) 1 to 2 million IU / m ₂ (1 to 2), as much as possible after the effusion is removed , 1 to 2 times a week, 2 to 4 weeks (or effusion disappeared) is a course of treatment.
(2) Local administration of tumor lesions: The dosage should be determined according to the size of the tumor. The dosage should be no less than 100,000 IU each time, once every other day, 4-6 times as a course of treatment. ^[5]

Recombinant human interleukin 2 adverse reactions

The most common adverse reactions are fever, chills, and muscle soreness, which are related to the dosage of the medication. Generally, they are transient fever (about 38 ° C), and there can also be high chills. The body temperature can be more than 3 to 4 hours after withdrawal. Return to normal. Individual patients may experience nausea, vomiting, rash, and cold-like symptoms. Subcutaneous injection may cause local swelling, induration, and pain, and all side effects can recover on their own. ^[7]

Recombinant human interleukin 2 precautions

This product must be used with caution under the control of an experienced oncologist. ^[7]

When using larger doses, this product may cause capillary leakage syndrome, which is manifested as hypotension, peripheral edema, temporary renal insufficiency, etc., and should be stopped immediately and actively treated symptomatically. It should be noted that the safe dose should be strictly controlled when using this product.

After the medicine bottle is opened, it should be used up at one time and not used repeatedly. Prefilled syringe packaging is for single use only and must not be reused. Use this product starting from a small dose and gradually increasing the dose. The safe dose should be strictly controlled. The low dose and long course of treatment can reduce toxicity and maintain antitumor activity.

Recombinant human interleukin-2 for pregnant and lactating women

Use with caution in pregnant and lactating women. Pediatric, elderly with severe cardio-cerebral-renal complications should be used with caution. ^[5]

Recombinant human interleukin 2 contraindications

1. Patients with a history of allergies to the ingredients of this product.

2. High fever, severe heart disease, hypotension, severe heart and kidney dysfunction, abnormal lung function or organ transplant.

3. Recombinant human interleukin-2 has had related toxic reactions in the history of medication: persistent ventricular tachycardia; uncontrolled arrhythmia; chest pain with ECG changes, angina pectoris or myocardium Infarction; palpitation of the heart; dialysis for renal failure> 72 hours; coma or toxic psychosis> 48 hours; refractory or refractory epilepsy; intestinal ischemia or perforation; gastrointestinal bleeding requires surgery surgery;