CN102178931B - Recombinant human interleukin-12 preparation for injection and preparation method thereof - Google Patents
Recombinant human interleukin-12 preparation for injection and preparation method thereof Download PDFInfo
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- CN102178931B CN102178931B CN2011101116406A CN201110111640A CN102178931B CN 102178931 B CN102178931 B CN 102178931B CN 2011101116406 A CN2011101116406 A CN 2011101116406A CN 201110111640 A CN201110111640 A CN 201110111640A CN 102178931 B CN102178931 B CN 102178931B
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Abstract
The invention discloses a recombinant human interleukin-12 preparation for injection and a preparation method thereof, and aims to provide a preparation which has the advantages of good stability and low moisture content. The invention has the technical point that each milliliter of preparation contains 2.5-10mu g of recombinant human interleukin-12, 50mg of filling agent, 5-20mg of freeze-dried protective agent, 2.30mg of disodium hydrogen phosphate, 0.52mg of sodium dihydrogen phosphate and 7.02mg of sodium chloride. The preparation method comprises the following steps of: (1) weighing disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride according to the formula proportions, adding water for injection, stirring for dissolving, regulating the pH value to 6.8-7.8 with sodium hydroxide or hydrochloric acid, and metering the volume to the formula proportion; (2) taking out the solution, adding the formula proportion of the filling agent, stirring for dissolving, then adding the formula proportions of the freeze-dried protective agent and recombinant human interleukin-12, stirring until the materials are uniform, carrying out sterile filtration, and respectively packaging in bottles of 1ml; and (3) freeze-drying. The invention belongs to the technical field of medicine preparation.
Description
Technical field
The present invention relates to a kind of preparation and preparation method, concrete saying of sending out relates to a kind of injection recombinant human interleukin--1's 2 preparations and preparation method, belongs to medical preparing technical field.
Background technology
Il-1 2 (IL-2) is important cytokine in the human body, can improve helper T lymphocyte quantity and active in to strengthen whole immunologic function, by regulating NK cell and macrophage-mediated nonspecific immunity and the specific immunity effect of Th1, CTL mediation, produce a series of biological effect.It is 30 multinomial that external recombinant human interleukin--1's 2 (rhIL-12) clinical trial protocol has reached, relate to multiple infectious disease (as hepatitis B, hepatitis C, acquired immune deficiency syndrome (AIDS)), kinds of tumors (as melanoma, renal carcinoma, rectal cancer, carcinoma of prostate, cervical cancer etc.), autoimmune disease and asthma etc., but approval listing not yet so far, il-1 2 is prepared into easy to use, is easy to the dosage form of absorption of human body, metabolism, apparent is particularly important.
Summary of the invention
At the problems referred to above, the present invention discloses a kind of injection recombinant human interleukin--1's 2 preparations and preparation method.
Previous purpose of the present invention is; a kind of injection recombinant human interleukin--1's 2 preparations are provided; every ml of formulation contains following compositions: the recombinant human interleukin--1 2 of 2.5～10 μ g; the filler of 50mg; the freeze drying protectant of 5～20mg; 2.30mg sodium hydrogen phosphate, the sodium dihydrogen phosphate of 0.52mg and the sodium chloride of 7.02mg.
Above-mentioned injection recombinant human interleukin--1's 2 preparations, described preparation contains the recombinant human interleukin--1 2 of 2.5 μ g or 5 μ g or 10 μ g.
Above-mentioned injection recombinant human interleukin--1's 2 preparations, described filler are glycine, mannitol, one of them of dextran or their compositions.
Further, above-mentioned injection recombinant human interleukin--1's 2 preparations, described filler is preferably mannitol.
Above-mentioned injection recombinant human interleukin--1's 2 preparations, wherein, described freeze drying protectant is one of them or their compositions in human albumin, sucrose, trehalose, lactose, the glucose; The preferred human albumin of filler.
A back purpose of the present invention is that the preparation method of the said preparation that provides comprises the steps: successively
(1) recombinant human interleukin--1's 2 raw materials are taken out from refrigerator below-10 ℃, place 4 ℃ of following freeze thawing, stand-by.
(2) take by weighing sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride by formula ratio, add water for injection and stir 10～15min, make dissolving fully, regulate pH value to 6.8～7.8 with sodium hydroxide or hydrochloric acid, be settled to formula ratio.
(3) take out above-mentioned solution, the filler that adds formula ratio stirs 10～15min, makes dissolving fully; the freeze drying protectant and the recombinant human interleukin--1 2 stirring 2min that add formula ratio again are even to material; aseptic filtration, aseptic subpackaged in 100 grades of workshops, every bottle of 1ml.
(4) open freeze dryer, arrange or access the lyophilizing program: pre-freeze is warmed up to-10 ℃ after 4 hours, keeps-10 ℃ 5 hours, be warmed up to 30 ℃ and kept 4 hours, end.
(5) quit a program after the EP (end of program), tamponade, Zha Gai are namely.
In the above-mentioned preparation method, the pre-freeze described in the step (4) is cooled to-40 ℃, needs 40 minutes, keeps 3 hours at-40 ℃ again, and cold-trap was prepared 20 minutes.
In the above-mentioned preparation method, be warmed up to-10 ℃ with 5 hours from described from-40 ℃ in the step (4), be warmed up to 30 ℃ with 6 hours from-10 ℃.
Compared with prior art, injection recombinant human interleukin--1's 2 preparations disclosed in this invention and preparation method, the product appearance of gained is good, moisture is low, can not reduce protein content and the biological activity of the rhIL-12 of production, good stability, preparation method is simple.
The specific embodiment
Below in conjunction with specific embodiment the present invention is described in further detail, but does not constitute any limitation of the invention.
Embodiment
Clinical research shows, recombinant human interleukin--1 2 (rhIL-12) clinical medicine dose is 1-500ng/kg, but the dosage level at 300-500ng/kg, groups of people show abnormal liver function, toxic and side effects such as oral ulcer, safer effective dosage is at 50ng-250ng/kg, and according to Chinese body weight about 50-60kg, the single bottle of specification of this product is decided to be: rhIL-122.5,5 or 10 μ g/ preparation units.
The preparation that pharmaceutical formulation forms is lyophilized injectable powder, serves as the screening foundation with lyophilized powder appearance character and rhIL-12 content and Determination of biological activity when filling a prescription screening.
RhIL-12 is the glycoprotein that is formed by disulfide-bonded by two subunits, and is soluble in water, and it is stable in the phosphate buffer of pH7.4, so adopted sodium hydrogen phosphate and sodium dihydrogen phosphate as the pH buffer agent.Every vial formulation contains sodium hydrogen phosphate 2.30mg and sodium dihydrogen phosphate 0.52mg, and the preparation pH value of making is about 7.4.Add sodium chloride simultaneously as osmotic pressure regulator, addition is every bottle of 7.02mg.
A kind of injection recombinant human interleukin--1 2 preparations of the present invention, prepared every ml of formulation contains the recombinant human interleukin--1 2 of 2.5 μ g or 5 μ g or 10 μ g, one of them of the glycine of 50mg, mannitol, dextran or their combinations, in 5～20mg human albumin, sucrose, trehalose, lactose, the glucose one of them or their combination, 2.30mg sodium hydrogen phosphate, 0.52mg sodium dihydrogen phosphate and 7.02mg sodium chloride.For selecting stabilized formulations of the present invention, the inventor has designed the embodiment of 13 different formulations, and every milliliter of included composition of every embodiment and experimental result are shown in Table 1.
The concrete preparation method of every embodiment in turn includes the following steps:
(1) recombinant human interleukin--1's 2 raw materials are taken out from refrigerator below-10 ℃, place 4 ℃ of following freeze thawing, stand-by;
(2) take by weighing sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride by formula ratio, add water for injection and stir 10～15min, make dissolving fully, regulate pH value to 6.8～7.8 with sodium hydroxide or hydrochloric acid, be settled to formula ratio;
(3) take out above-mentioned solution, the filler that adds formula ratio stirs 10～15min, makes dissolving fully, the freeze drying protectant and the recombinant human interleukin--1 2 stirring 2min that add formula ratio again are even to material, aseptic filtration, aseptic subpackaged in 100 grades of workshops, every bottle of 1ml;
(4) open freeze dryer, arrange or access the lyophilizing program: pre-freeze cools to-40 ℃, and temperature fall time shares 40 minutes ,-40 ℃ 3 hours, cold-trap was prepared 20 minutes, be warming up to-10 ℃ again, the heating-up time shares 5 hours, is warming up to-10 ℃ and keeps 5 hours, be warmed up to 30 ℃ again, heating-up time is 6 hours, is warming up to 30 ℃ and keeps 4 hours, finishes.
(5) quit a program after the EP (end of program), tamponade, Zha Gai namely get product.
The stability test example
The present inventor has carried out sufficient research to the stability of said preparation, comprise influence factor's test, accelerated test and Journal of Sex Research steady in a long-term, final certification said preparation comparatively stable under 4 ℃ of conditions, can make rhIL-12 keep long-term activity, show that said preparation has satisfied the stability requirement of medicine.
With the prescription 1 in the table 1, prescription 12, the 13 prepared preparations of writing out a prescription, 4 ℃ of placements of simulation listing packing were respectively at 0,3,6,9,12,18 month sampling and measuring.The stability of investigating injection recombinant human interleukin-11 2 mainly detects 7 indexs investigations from character, protein content, biological activity, pH value, moisture, sterility test and the endotoxin of sample, the result presses the preparation of each formulation in biological activity and the comparison in 0 o'clock of different time points, have no significant change, show that injection rhIL-12 placed 18 months under plan listing condition of storage, its active nothing significantly descends, other every indexs all meet 2010 editions " Chinese pharmacopoeia regulations, illustrate that said preparation can long term storage under 4 ℃ of conditions, the shelf-life meets the medicine regulation.The result of study on the stability is detailed to be shown in Table 2.
Claims (4)
1. injection recombinant human interleukin--1 2 preparations; it is characterized in that; every ml of formulation contains following compositions: the recombinant human interleukin--1 2 of 2.5～10 μ g; the filler mannitol of 50mg; the freeze drying protectant human albumin of 5mg; 2.30mg sodium hydrogen phosphate and the sodium dihydrogen phosphate of 0.52mg as the pH value regulator, the osmotic pressure regulator sodium chloride of 7.02mg.
2. injection recombinant human interleukin--1's 2 preparations according to claim 1 is characterized in that, described preparation contains the recombinant human interleukin--1 2 of 2.5 μ g or 5 μ g or 10 μ g.
3. the preparation method of described injection recombinant human interleukin--1's 2 preparations of claim 1 is characterized in that this method comprises the steps: successively
(1) recombinant human interleukin--1's 2 raw materials are taken out from refrigerator below-10 ℃, place 4 ℃ of following freeze thawing, stand-by;
(2) take by weighing sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride by formula ratio, add water for injection and stir 10～15min, make dissolving fully, regulate pH value to 6.8～7.8 with sodium hydroxide or hydrochloric acid, be settled to formula ratio;
(3) take out above-mentioned solution, the filler that adds formula ratio stirs 10～15min, makes dissolving fully, the freeze drying protectant and the recombinant human interleukin--1 2 stirring 2min that add formula ratio again are even to material, aseptic filtration, aseptic subpackaged in 100 grades of workshops, every bottle of 1ml;
(4) open freeze dryer, arrange or access the lyophilizing program: pre-freeze is warmed up to-10 ℃ after 4 hours, keeps 5 hours at-10 ℃, is warmed up to 30 ℃ again and keeps 4 hours;
(5) quit a program after the EP (end of program), tamponade, Zha Gai are namely.
4. preparation method according to claim 3 is characterized in that, the pre-freeze described in the step (4) 4 hours was to be cooled to-40 ℃ of needs 40 minutes, keeps 3 hours at-40 ℃ again, and cold-trap was prepared 20 minutes.
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| CN111701012A (en) * | 2017-09-07 | 2020-09-25 | 康立泰药业有限公司 | Application of IL-12 in postoperative antitumor aspect |
| CN112485451B (en) * | 2020-12-01 | 2021-12-07 | 广州市进德生物科技有限公司 | Interleukin 6 freeze-drying calibrator, preparation method thereof and freeze-drying protective solution |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1471969A (en) * | 2002-07-29 | 2004-02-04 | 北京北医联合生物工程公司 | Medicinal use of recombined human interleukin-1 receptor antagonist |
| CN1927387A (en) * | 2005-09-05 | 2007-03-14 | 中国医学科学院基础医学研究所 | Application of interleukins-15 in preparation of medicine for tumor immunization therapy and immunotherapy drug |
| CN101002735A (en) * | 2007-01-24 | 2007-07-25 | 上海华新生物高技术有限公司 | Recombination interleukin-2 frozen-dried preparation, and its preparing method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1471969A (en) * | 2002-07-29 | 2004-02-04 | 北京北医联合生物工程公司 | Medicinal use of recombined human interleukin-1 receptor antagonist |
| CN1927387A (en) * | 2005-09-05 | 2007-03-14 | 中国医学科学院基础医学研究所 | Application of interleukins-15 in preparation of medicine for tumor immunization therapy and immunotherapy drug |
| CN101002735A (en) * | 2007-01-24 | 2007-07-25 | 上海华新生物高技术有限公司 | Recombination interleukin-2 frozen-dried preparation, and its preparing method |
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