WO2017114214A1 - 一种组合物及其应用、含有该组合物的稳定剂 - Google Patents

一种组合物及其应用、含有该组合物的稳定剂 Download PDF

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WO2017114214A1
WO2017114214A1 PCT/CN2016/110724 CN2016110724W WO2017114214A1 WO 2017114214 A1 WO2017114214 A1 WO 2017114214A1 CN 2016110724 W CN2016110724 W CN 2016110724W WO 2017114214 A1 WO2017114214 A1 WO 2017114214A1
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composition
stability
stabilizer
composition according
mannitol
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French (fr)
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韩宇东
周湘龙
黄宗文
麦有觉
董伍爱
韩克胜
许礼贵
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海口奇力制药股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03016—Phosphoprotein phosphatase (3.1.3.16), i.e. calcineurin

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  • the present invention relates to the field of biotechnology, and in particular to a composition and its use, a stabilizer containing the composition.
  • Protein is a macromolecular substance with a complex spatial three-dimensional structure, which is susceptible to denaturation due to external conditions. Protein stability is related to the amino acid species in the composition. The most common is cysteine. The protein is sensitive to external factors such as solution pH, oxygen in the air, etc. It is prone to structural transformation and affects activity.
  • Calcineurin is the only known protein phosphatase that relies on Ca 2+ /CaM (calmodulin) and consists of a 1:1 ratio of A and B subunits.
  • the A subunit (CNA) is a catalytic subunit and the B subunit (CNB) is a regulatory subunit.
  • CN plays an important role in the immune activation pathway, indicating that the key to T cell activation is not.
  • CNA protein phosphatase that relies on Ca 2+ /CaM (calmodulin) and consists of a 1:1 ratio of A and B subunits.
  • the A subunit (CNA) is a catalytic subunit
  • the B subunit (CNB) is a regulatory subunit.
  • CN plays an important role in the immune activation pathway, indicating that the key to T cell activation is not.
  • CN is easily inactivated and unstable, and CNB, as a regulatory subunit of the enzyme, can promote the activity of CNA, and has relatively small mo
  • CNB can stimulate T cell and NK cell proliferation, enhance NK cell killing activity, enhance macrophage phagocytosis and other immune functions, and recombinant human calcineurin B subunit (rhCNB) is innovative oncology drugs for genetic engineering in development.
  • rhCNB has obvious role in killing tumor cells.
  • Monomer is a protein containing two thiol structures. During production, placement, etc., it is susceptible to temperature, moisture, pH, light, oxidation and other factors to form dimers. Multimeric structures such as trimers, which cause the composition of each component to be unstable and cannot be quantitatively controlled.
  • the present invention provides a composition and use thereof, and a stabilizer containing the composition.
  • the invention solves the problem that the rhCNB components are stable and controllable, completes the stability investigation of the drug clinical declaration, satisfies the requirements of the drug declaration, and obtains the stabilizer composition of the invention through a large number of experiments, and effectively solves the rhCNB. Unstable problems in production and storage.
  • the present invention provides a stabilizer composition capable of stabilizing the proportion of rhCNB components, a composition consisting of inorganic salts, amino acids, sugars, polyols, and surfactants in a certain proportion, wherein the different components are respectively for the monomer of rhCNB.
  • the components such as dimers and multimers have their respective stabilizing effects, and can form synergistic and superimposed, so that the purity of rhCNB active components (including dimers and monomers) is over 99%, and the dimer stability is controlled. More than 98%, the impurity multimer content is less than 0.5%, reaching the international advanced level of this indicator of biopharmaceuticals.
  • the present invention provides the following technical solutions:
  • the present invention provides a composition comprising a mixture of one or more of a chloride, an amino acid, a saccharide or a surfactant.
  • the chloride is calcium chloride or zinc chloride.
  • the amino acid is tryptophan or lysine.
  • the saccharide is a mixture of one or more of trehalose, sucrose or mannitol.
  • the surfactant is a nonionic surfactant.
  • the nonionic surfactant is polysorbate-80.
  • the invention also provides the use of the composition in the preparation of a human calmodulin phosphatase B subunit stabilizer.
  • the present invention also provides a stabilizer for increasing the stability of a human calmodulin phosphatase B subunit component, including the composition and water.
  • the present invention also provides a method for preparing a stabilizer for improving the stability of a human calmodulin phosphatase B subunit component.
  • the method comprises the following steps:
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 Mix calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then add 50-150 parts of water for injection to obtain a mixed solution; then add activated carbon to stir and filter. Removing the carbon to prepare a first solution;
  • Step 3 taking a formula amount of polysorbate-80, adding water for injection, adding activated carbon, stirring, filtering and removing charcoal to prepare a second solution;
  • Step 4 Mix the first solution prepared in the step 2 and the second solution prepared in the step 3, and sterilize by filtration.
  • the temperature of the water for injection in step 2 is from 20 to 60 °C.
  • 50 to 150 parts of the water for injection is stirred at a rotation speed of 30 to 200 rpm as described in the step 2.
  • the activated carbon in the step 2 is activated activated carbon, and the activated carbon accounts for 0.05% to 0.5% by volume of the mixed liquid.
  • the step of adding the activated carbon for stirring in the step 2 is 10 to 30 minutes.
  • the number of rinses with water for injection is as described in step 3 from 2 to 4 times.
  • the activated carbon in the step 3 is activated activated carbon, and the activated carbon accounts for 0.05% to 0.5% by volume of the mixed liquid.
  • the agitation time in step 3 is from 10 to 30 minutes.
  • the filtration sterilization in the step 4 of the preparation method is sterilizing by filtration through a 0.2 ⁇ m filter.
  • the mass ratio of the rhCNB (in terms of protein amount) to the composition or stabilizer is 1:1.
  • composition or stabilizer is composed of calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80, and the like:
  • the present invention provides a composition comprising a mixture of one or more of a chloride, an amino acid, a saccharide or a surfactant.
  • the invention solves the problem that the rhCNB components are stable and controllable, completes the stability investigation of the drug clinical declaration, satisfies the requirements of the drug declaration, and obtains the stabilizer composition of the invention through a large number of experiments, and effectively solves the rhCNB. Unstable problems in production and storage.
  • the present invention provides a stabilizer composition capable of stabilizing the proportion of rhCNB components, a composition consisting of inorganic salts, amino acids, sugars, polyols, and surfactants in a certain proportion, wherein the different components are respectively for the monomer of rhCNB.
  • the dimer, multimer and other components play their respective stabilizing effects, and can form synergy and superposition, so that the purity of the active component of RhCNB is over 99%, wherein the dimer is stable at 98% or more, and the impurity polymer content is less than 0.5%, reaching the international advanced level of this indicator of biopharmaceuticals.
  • Figure 1 is a graph showing the results of the stability of the stability acceleration test of Example 1;
  • Figure 2 is a graph showing the results of the stability of the stability acceleration test of Example 1;
  • Figure 3 is a graph showing the results of the stability of the stability acceleration test of Example 1;
  • Figure 4 is a graph showing the results of the stability of the stability test of Example 1 for 6 months;
  • Figure 5 is a graph showing the results of the stability of the stability acceleration test of Example 2.
  • Figure 6 is a graph showing the results of the January 1st stability acceleration test of Example 2.
  • Figure 7 is a graph showing the results of the stability of the stability acceleration test of Example 2.
  • Figure 8 is a graph showing the results of the stability of the accelerated acceleration test of Example 2.
  • Figure 9 is a graph showing the results of the stability of the stability acceleration test of Example 3.
  • Figure 10 is a graph showing the results of the stability of the stability acceleration test of Example 3.
  • Figure 11 is a graph showing the results of the stability of the stability acceleration test of Example 3.
  • Figure 12 is a graph showing the results of the stability of the stability acceleration test of Example 3.
  • Figure 13 is a graph showing the results of the stability of the stability acceleration test of Example 4.
  • Figure 14 is a graph showing the results of the stability of the stability acceleration test of Example 4.
  • Figure 15 is a graph showing the results of the stability of the stability acceleration test of Example 4.
  • Figure 16 is a graph showing the results of the stability of the accelerated acceleration test of Example 4.
  • Figure 17 is a graph showing the results of the stability of the stability acceleration test of Example 5.
  • Figure 18 is a graph showing the results of the stability of the stability acceleration test of Example 5.
  • Figure 19 is a graph showing the results of the stability of the stability acceleration test of Example 5.
  • Figure 20 is a graph showing the results of the stability of the accelerated acceleration test of Example 5.
  • Figure 21 is a graph showing the results of the stability of the stability acceleration test of Example 6.
  • Figure 22 is a graph showing the results of the stability of the stability acceleration test of Example 6.
  • Figure 23 is a graph showing the results of the stability of the stability acceleration test of Example 6.
  • Figure 24 is a graph showing the results of the 6th month of the stability acceleration test of Example 6;
  • Figure 25 is a graph showing the results of the 0-month comparison of the proportional stability acceleration test
  • Figure 26 is a graph showing the results of the January stability of the proportional stability accelerated test.
  • the invention discloses a composition and an application thereof, and a stabilizer containing the composition, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the stabilizer according to the present invention is composed of calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol and polysorbate-80, and the composition thereof is as follows:
  • the preparation method of the stabilizer is:
  • compositions provided by the present invention, and the use thereof, and the materials and reagents used in the stabilizer containing the composition are commercially available.
  • Step 1 Weigh calcium chloride, tryptophan, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride, tryptophan, trehalose, sucrose, mannitol, and then adding 50-150 parts of 40 ° C water for injection, stirring at 30 rpm to prepare a mixed liquid; and adding the volumetric mass of the mixed liquid
  • the activated activated carbon with a score of 0.05% was stirred for 20 min, and the carbon was removed by filtration to prepare a first solution;
  • Step 3 Take the formula amount of polysorbate-80, rinse it twice with water for injection, add activated activated carbon with a volume percentage of 0.3%, stir for 10 min, filter and remove charcoal, and prepare a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 were mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then adding 50-150 parts of 60 ° C water for injection, stirring at 200 rpm to prepare a mixed solution; Adding 0.5% of activated activated carbon to the mixed liquor for 10 min, filtering and removing charcoal to prepare a first solution;
  • Step 3 Take the formula amount of polysorbate-80, rinse 4 times with water for injection, add 0.5% by volume of activated activated carbon, stir for 30 min, filter and remove charcoal to prepare a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 are mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then adding 50-150 parts of 20 ° C water for injection, stirring at 100 rpm to prepare a mixed solution; Adding activated carbon to the mixed volume of 0.2% by mass of activated carbon for 30 min, filtering and removing charcoal to prepare a first solution;
  • Step 3 Take the formula amount of polysorbate-80, rinse it with water for injection 3 times, add 0.05% by volume of activated activated carbon, stir for 20 min, filter and remove charcoal to prepare a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 are mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then adding 300-1500 parts of 20 ° C water for injection, stirring at 100 rpm to prepare a mixed solution; Adding 0.5% of activated activated carbon to the mixed solution for 20 min, filtering and removing charcoal to prepare a first solution;
  • Step 3 take the formula amount of polysorbate-80, rinse 4 times with water for injection, add 0.3% by volume of activated activated carbon, stir for 30 min, filter and remove charcoal to prepare a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 are mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then adding 300-1500 parts of 60 ° C water for injection, stirring at 200 rpm to prepare a mixed solution; Adding the activated carbon of the mixture to a volume fraction of 0.05% for 10 min, filtering and removing the carbon to obtain a first solution;
  • Step 3 take the formula amount of polysorbate-80, rinse twice with water for injection, add 0.05% by volume of activated activated carbon, stir for 10 min, filter and remove charcoal to prepare a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 are mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • Step 1 Weigh calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, polysorbate-80 according to the ratio;
  • Step 2 mixing calcium chloride or zinc chloride, tryptophan or lysine, trehalose, sucrose, mannitol, and then adding 300-1500 parts of 40 ° C water for injection, stirring at 30 rpm to prepare a mixed solution; Adding activated carbon to the mixed volume of 0.3% by volume of the activated carbon for 30 min, filtering and removing charcoal to prepare a first solution;
  • Step 3 taking a formula amount of polysorbate-80, rinsing with injection water for 3 times, adding 0.5% by volume of activated activated carbon, stirring for 20 min, filtering and removing charcoal to obtain a second solution;
  • Step 4 The first solution prepared in the step 2 and the second solution prepared in the step 3 are mixed, and sterilized by filtration through a 0.2 ⁇ m filter.
  • the stabilizers prepared in Examples 1 to 6 were mixed with 1 part of rhCNB, subjected to an accelerated test at 37 ° C, and sampled for biological activity and purity at 0, 1, 3, and 6 months (SEC-HPLC method).
  • FIGS. 1 to 24 The chromatograms of Examples 1 to 6 are shown in FIGS. 1 to 24, wherein FIG. 1 to FIG. 4 respectively show the results of the stability acceleration test of the first embodiment in January, January, March, and June; FIG. 5 to FIG. 8 shows the results of the stability acceleration test of Example 2 for 0 months, January, March, and June; and FIG. 9 to FIG. 12 respectively show the results of the stability acceleration test of Example 3 for 0 months, January, March, and June; 13 to FIG. 16 respectively show the results of the stability acceleration test of the embodiment 4 in January, January, March, and June; and FIGS. 17 to 20 respectively show the stability acceleration test of the embodiment 5 in January, January, and March. June results; 21 to 24 show the results of the stability acceleration test of Example 6 for 0 months, January, March, and June, respectively.
  • the specific spectrum data is shown in Tables 1 to 24.
  • Peak number keep time area height area% 1 10.367 4120 163 0.196 2 10.96 2064996 94266 98.479 3 12.363 27779 743 1.325 total 2096895 95171 100
  • Peak number keep time area height area% 1 10.188 4647 209 0.21 2 10.627 2191698 95669 98.812 3 11.846 21706 800 0.979 total 2218051 96677 100
  • Peak number keep time area height area% 1 10.179 13097 370 0.500 2 10.62 2248004 96228 98.262 3 11.829 26675 885 1.238 total 2287775 97484 100
  • Peak number keep time area height area% 1 10.246 4037 196 0.192 2 10.65 2065172 88437 98.272 3 11.938 32268 952 1.535 total 2101477 89585 100
  • Example 2 is the best, followed by Example 3, Example 5, and Example 6, Example 1
  • the worst case with 4 is the amount of mannitol added as the main support.
  • all the six examples can effectively stabilize and protect rhCNB, which proves the good protection effect of the combination stabilizer.
  • Step 1 Weigh trehalose, sucrose, mannitol according to the ratio
  • Step 2 mixing trehalose, sucrose, mannitol, and then adding 50-150 parts of 30-40 ° C water for injection, stirring at 30 rpm to prepare a mixed solution; and adding the volume fraction of the mixture to a activation of 0.3%.
  • the activated carbon was stirred for 30 min, filtered to remove charcoal, and then 1 part of rhCNB protein was added. After lyophilization, the accelerated test was carried out at 37 ° C. The biological activity and purity were sampled at 0, 1, 3, and 6 months (SEC-HPLC method). .
  • Fig. 25 shows the 0-month result map of the proportional stability acceleration test
  • Fig. 26 shows the January result map of the comparative stability acceleration test.
  • the specific spectrum data is shown in Tables 26-27.
  • the difference between the comparative example and the example stabilizer is that it does not contain amino acids and chlorides. These two materials are important components of the lyophilized stabilizer.
  • the multimer of the test results exceeded in January. 1%, the dimer is reduced to less than 98%, and the stabilizing effect is poor.
  • the multimer is less than 0.5% and the dimer is more than 98%, which is compared with the comparative example. The effect is obvious.

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Abstract

提供了一种组合物及其应用、含有该组合物的稳定剂。该组合物包括氯化物、氨基酸、糖类或表面活性剂中的一种或两者以上的混合物。还提供了该组合物在用于制备人钙调蛋白磷酸酶B亚基稳定剂中的应用，含有该组合物的稳定剂及其制备方法。

Description

一种组合物及其应用、含有该组合物的稳定剂
本申请要求于2015年12月30日提交中国专利局、申请号为201511024935.4、发明名称为“一种组合物及其应用、含有该组合物的稳定剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及一种组合物及其应用、含有该组合物的稳定剂。
背景技术
蛋白质是一种具有复杂的空间立体结构的大分子物质，易受外界条件的影响发生变性。蛋白质稳定性与组成中的氨基酸种类有关系，最常见的是半胱氨酸，蛋白质对溶液pH、空气中的氧等外界因素都较为敏感，容易发生结构的转变，影响活性。
钙调磷酸酶(Calcineurin,CN)是目前所知唯一一种依赖Ca2+/CaM(钙调素)的蛋白磷酸酶，由A，B亚基以1：1的比例组成。A亚基(CNA)是催化亚基，B亚基(CNB)是调节亚基。CN在免疫激活通路中发挥重要作用，示T细胞活化的关键没。CN作为大分子酶蛋白，易失活，不稳定，而CNB作为该酶的调节亚基，能促进CNA的活性，且相对分子质量小，性质稳定。由研究表明CNB能刺激T细胞和NK细胞增殖以及增强NK细胞的杀伤活性，增强巨噬细胞吞噬能力等免疫功能，且重组人钙调磷酸酶B亚基(Recombinant human Calcineurin B subunit,rhCNB)是开发中的基因工程创新肿瘤药物。
rhCNB具有明显的杀伤肿瘤细胞的作用，单体是一种含有两个巯基结构的蛋白，在生产、放置等过程中，易受温度、水分、pH值、光照、氧化等因素影响形成二聚体、三聚体等多聚体结构，造成各组分比例不稳定，无法定量控制的特点。
因此，提供一种能有效稳定重组人钙调蛋白磷酸酶B亚基组分比例的组合稳定剂具有重要的现实意义。
发明内容
有鉴于此，本发明提供一种组合物及其应用、含有该组合物的稳定剂。本发明为了解决rhCNB各组份比例稳定可控的难题，完成药物临床申报稳定性考察，满足药物申报的要求，通过大量的实验，得到了本发明中的稳定剂组合物，有效的解决了rhCNB生产、贮存过程中不稳定的问题。
本发明提供了一种能够稳定rhCNB组分比例的稳定剂组合物，由无机盐、氨基酸、糖类、多元醇、表面活性剂按一定比例组成的组合物，其中不同成分分别对rhCNB的单体、二聚体、多聚体等组分起各自的稳定作用，并能形成协同与叠加，使rhCNB有效成份(含二聚体和单体)纯度达99％以上，其中二聚体稳定控制在98％以上，杂质多聚体含量小于0.5％，达到生物药品本项指标国际先进水平。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种组合物，包括氯化物、氨基酸、糖类或表面活性剂中的一种或两者以上的混合物。
在本发明的一些具体实施方案中，以质量份计，包括如下组分：
Figure PCTCN2016110724-appb-000001
在本发明的一些具体实施方案中，所述氯化物为氯化钙或氯化锌。
在本发明的一些具体实施方案中，所述氨基酸为色氨酸或赖氨酸。
在本发明的一些具体实施方案中，所述糖类为海藻糖、蔗糖或甘露醇中的一种或两者以上的混合物。
在本发明的一些具体实施方案中，所述表面活性剂为非离子型表面活性剂。
在本发明的一些具体实施方案中，所述非离子型表面活性剂为聚山梨酯-80。
在本发明的一些具体实施方案中，以质量份计，包括如下组分：
Figure PCTCN2016110724-appb-000002
Figure PCTCN2016110724-appb-000003
在本发明的另一些具体实施方案中，以质量份计，包括如下组分：
Figure PCTCN2016110724-appb-000004
在本发明的另一些具体实施方案中，以质量份计，包括如下组分：
Figure PCTCN2016110724-appb-000005
本发明还提供了所述组合物在用于制备人钙调蛋白磷酸酶B亚基稳定剂中的应用。
本发明还提供了一种提高人钙调蛋白磷酸酶B亚基组分稳定性的稳定剂，包括所述组合物和水。
本发明还提供了一种提高人钙调蛋白磷酸酶B亚基组分稳定性的稳定剂的制备方法，在本发明的一些具体实施方案中，包括如下步骤：
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入50～150份注射用水搅拌，制得混合液；再加入活性炭搅拌，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，加入注射用水，再加入活性炭，搅拌，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，过滤除菌。
在本发明的一些具体实施方案中，步骤2中注射用水的温度为20～60℃。
在本发明的一些具体实施方案中，步骤2中所述再加入50～150份注射用水搅拌的转速为30～200rpm。
在本发明的一些具体实施方案中，步骤2中所述活性炭为活化的活性炭，所述活性炭占所述混合液的体积质量分数为0.05％～0.5％。
在本发明的一些具体实施方案中，步骤2中所述再加入活性炭搅拌的时间为10～30min。
在本发明的一些具体实施方案中，步骤3中所述用注射用水冲洗的次数为2～4次。
在本发明的一些具体实施方案中，步骤3中所述活性炭为活化的活性炭，所述活性炭占所述混合液的体积质量分数为0.05％～0.5％。
在本发明的一些具体实施方案中，步骤3中所述搅拌的时间为10～30min。
在本发明的一些具体实施方案中，所述制备方法步骤4中所述过滤除菌为经0.2μm滤芯过滤除菌。
在本发明的一些具体实施方案中，所述rhCNB(以蛋白质量计)与所述组合物或稳定剂的质量比为1:1。
即：
组合物或稳定剂是由氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80共同组成，其配比如下：
①氯化钙0.02～0.1份、色氨酸0.05～0.25份、海藻糖0.2～2份、蔗糖0.2～2份、甘露醇1～10份、聚山梨酯-80 0.1～0.5份、rhCNB蛋白1份。
②氯化锌0.02～0.1份、赖氨酸0.05～0.25份、海藻糖0.2～2份、蔗糖0.2～2份、甘露醇1～10份、聚山梨酯-80 0.1～0.5份、rhCNB蛋白1份。
本发明提供了一种组合物，包括氯化物、氨基酸、糖类或表面活性剂中的一种或两者以上的混合物。本发明为了解决rhCNB各组份比例稳定可控的难题，完成药物临床申报稳定性考察，满足药物申报的要求，通过大量的实验，得到了本发明中的稳定剂组合物，有效的解决了rhCNB生产、贮存过程中不稳定的问题。
本发明提供了一种能够稳定rhCNB组分比例的稳定剂组合物，由无机盐、氨基酸、糖类、多元醇、表面活性剂按一定比例组成的组合物，其中不同成分分别对rhCNB的单体、二聚体、多聚体等组分起各自的稳定作用，并能形成协同与叠加，使RhCNB有效成分纯度达99％以上，其中二聚体稳定在98％以上，杂质多聚体含量小于0.5％，达到生物药品本项指标国际先进水平。
加入本发明提供的组合稳定剂，在37℃条件下储存6个月，rhCNB组分比例和生物活性没有明显变化，各项质量指标也无明显变化。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实施例1稳定性加速试验0月结果图谱；
图2示实施例1稳定性加速试验1月结果图谱；
图3示实施例1稳定性加速试验3月结果图谱；
图4示实施例1稳定性加速试验6月结果图谱；
图5示实施例2稳定性加速试验0月结果图谱；
图6示实施例2稳定性加速试验1月结果图谱；
图7示实施例2稳定性加速试验3月结果图谱；
图8示实施例2稳定性加速试验6月结果图谱；
图9示实施例3稳定性加速试验0月结果图谱；
图10示实施例3稳定性加速试验1月结果图谱；
图11示实施例3稳定性加速试验3月结果图谱；
图12示实施例3稳定性加速试验6月结果图谱；
图13示实施例4稳定性加速试验0月结果图谱；
图14示实施例4稳定性加速试验1月结果图谱；
图15示实施例4稳定性加速试验3月结果图谱；
图16示实施例4稳定性加速试验6月结果图谱；
图17示实施例5稳定性加速试验0月结果图谱；
图18示实施例5稳定性加速试验1月结果图谱；
图19示实施例5稳定性加速试验3月结果图谱；
图20示实施例5稳定性加速试验6月结果图谱；
图21示实施例6稳定性加速试验0月结果图谱；
图22示实施例6稳定性加速试验1月结果图谱；
图23示实施例6稳定性加速试验3月结果图谱；
图24示实施例6稳定性加速试验6月结果图谱；
图25示对比例稳定性加速试验0月结果图谱；
图26示对比例稳定性加速试验1月结果图谱。
具体实施方式
本发明公开了一种组合物及其应用、含有该组合物的稳定剂，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
1、本发明所涉及的稳定剂是由氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80共同组成，其配比如下：
①氯化钙0.02～0.1份、色氨酸0.05～0.25份、海藻糖0.2～2份、蔗糖0.2～2份、甘露醇1～10份、聚山梨酯-80 0.1～0.5份、rhCNB蛋白1份。
②氯化锌0.02～0.1份、赖氨酸0.05～0.25份、海藻糖0.2～2份、蔗糖0.2～2份、甘露醇1～10份、聚山梨酯-80 0.1～0.5份、rhCNB蛋白1份。
2、本稳定剂的配制方法为：
①、按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
②、将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇投入配料罐，再投入适量20～60℃注射用水，启动搅拌，转速30～200rpm，搅拌溶解，再投入配液体积0.05％～0.5％的经活化的活性炭，继续搅拌10～30min，过滤除炭；
③、在另一配液罐中注入适量注射用水，投入已称量好的聚山梨酯-80，用已备好的注射用水冲洗2～4遍，洗尽残余，再投入溶液体积0.05％～0.5％的经活化的活性炭，继续搅拌10～30min，过滤除炭；
④、将②与③中的除热原溶液合并，混匀，0.2μm滤芯过滤除菌，待用。
3、加入上述组合稳定剂，在37℃条件下储存6个月，rhCNB组分比例和生物活性没有明显变化，各项质量指标也无明显变化。
本发明提供的组合物及其应用、含有该组合物的稳定剂中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1稳定剂的制备
Figure PCTCN2016110724-appb-000006
步骤1：按配比称量氯化钙、色氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙、色氨酸、海藻糖、蔗糖、甘露醇混合，再加入50～150份40℃注射用水搅拌，转速30rpm，制得混合液；再加入所述混合液的体积质量分数为0.05％的活化的活性炭搅拌20min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗2遍，再加入体积百分含量为0.3％的活化的活性炭，搅拌10min，过滤除炭，制得第二溶液；步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例2稳定剂的制备
Figure PCTCN2016110724-appb-000007
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入50～150份60℃注射用水搅拌，转速200rpm，制得混合液；再加入所述混合液的体积质量分数为0.5％的活化的活性炭搅拌10min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗4遍，再加入体积百分含量为0.5％的活化的活性炭，搅拌30min，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例3稳定剂的制备
Figure PCTCN2016110724-appb-000008
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入50～150份20℃注射用水搅拌，转速100rpm，制得混合液；再加入所述混合液的体积质量分数为0.2％的活化的活性炭搅拌30min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗3遍，再加入体积百分含量为0.05％的活化的活性炭，搅拌20min，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例4稳定剂的制备
Figure PCTCN2016110724-appb-000009
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入300～1500份20℃注射用水搅拌，转速100rpm，制得混合液；再加入所述混合液的体积质量分数为0.5％的活化的活性炭搅拌20min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗4遍，再加入体积百分含量为0.3％的活化的活性炭，搅拌30min，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例5稳定剂的制备
Figure PCTCN2016110724-appb-000010
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入300～1500份60℃注射用水搅拌，转速200rpm，制得混合液；再加入所述混合液的体积质量分数为0.05％的活化的活性炭搅拌10min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗2遍，再加入体积百分含量为0.05％的活化的活性炭，搅拌10min，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例6稳定剂的制备
Figure PCTCN2016110724-appb-000011
步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入300～1500份40℃注射用水搅拌，转速30rpm，制得混合液；再加入所述混合液的体积质量分数为0.3％的活化的活性炭搅拌30min，过滤除炭，制得第一溶液；
步骤3：取配方量的聚山梨酯-80，用注射用水冲洗3遍，再加入体积百分含量为0.5％的活化的活性炭，搅拌20min，过滤除炭，制得第二溶液；
步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，经0.2μm滤芯过滤除菌。
实施例7
实施例1～实施例6制备的稳定剂与1份rhCNB混合，37℃进行加速试验，第0、1、3、6月取样检测生物学活性和纯度(SEC-HPLC法)。
实施例1至实施例6的色谱图见图1至图24，其中，图1至图4分别示实施例1稳定性加速试验0月、1月、3月、6月结果；图5至图8分别示实施例2稳定性加速试验0月、1月、3月、6月结果；图9至图12分别示实施例3稳定性加速试验0月、1月、3月、6月结果；图13至图16分别示实施例4稳定性加速试验0月、1月、3月、6月结果；图17至图20分别示实施例5稳定性加速试验0月、1月、3月、6月结果； 图21至图24分别示实施例6稳定性加速试验0月、1月、3月、6月结果。具体谱图数据见表1～表24。
表1图1的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.017 7725 373 0.174
2 10.595 4360632 193361 98.395
3 11.788 63406 1913 1.431
总计   4431762 195648 100
表2图2的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.596 2058 233 0.097
2 10.917 2086892 94796 98.371
3 12.271 32506 848 1.532
总计   2121456 95877 100
表3图3的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.213 4387 232 0.198
2 10.627 2185666 94960 98.453
3 11.813 29960 894 1.35
总计   2220013 96086 100
表4图4的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.171 11565 386 0.492
2 10.618 2312881 97549 98.312
3 11.821 28157 947 1.197
总计   2352603 98882 100
表5图5的谱图数据
峰号 保留时间 面积 高度 面积％
1 9.992 3303 149 0.151
2 10.598 2157312 98598 98.904
3 11.788 20612 630 0.945
总计   2181227 99378 100
表6图6的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.238 6126 255 0.283
2 10.631 2134299 97707 98.501
3 11.913 26354 703 1.216
总计   2166779 98665 100
表7图7的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.217 7153 240 0.323
2 10.786 2179372 98451 98.515
3 12.088 25695 689 1.161
总计   2212220 99381 100
表8图8的谱图数据
峰号 保留时间 面积 高度 面积％
1 9.967 9974 297 0.467
2 10.622 2103952 95428 98.566
3 11.846 20643 641 0.967
总计   2134569 96366 100
表9图9的谱图数据
峰号 保留时间 面积 高度 面积％
1 9.992 7415 355 0.185
2 10.582 3924373 168502 98.136
3 11.721 67122 2100 1.679
总计   3998911 170957 100
表10图10的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.367 4120 163 0.196
2 10.96 2064996 94266 98.479
3 12.363 27779 743 1.325
总计   2096895 95171 100
表11图11的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.188 4647 209 0.21
2 10.627 2191698 95669 98.812
3 11.846 21706 800 0.979
总计   2218051 96677 100
表12图12的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.179 13097 370 0.500
2 10.62 2248004 96228 98.262
3 11.829 26675 885 1.238
总计   2287775 97484 100
表13图13的谱图数据
Figure PCTCN2016110724-appb-000012
Figure PCTCN2016110724-appb-000013
表14图14的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.563 4635 232 0.228
2 10.924 1999045 93766 98.304
3 12.204 29852 789 1.468
总计   2033533 94787 100
表15图15的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.354 4983 225 0.233
2 10.748 2103332 90760 98.248
3 11.988 32526 973 1.519
总计   2140841 91958 100
表16图16的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.179 6712 265 0.3
2 10.626 2206689 95615 98.536
3 11.913 26071 805 1.164
总计   2239473 96685 100
表17图17的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.271 3746 185 0.176
2 10.677 2093235 89104 98.289
3 11.938 32694 945 1.535
总计   2129674 90234 100
表18图18的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.033 9456 421 0.257
2 10.609 3628120 162204 98.604
3 11.904 41915 1364 1.139
总计   3679490 163989 100
表19图19的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.15 4968 214 0.233
2 10.675 2095273 89884 98.174
3 11.996 33998 914 1.593
总计   2134239 91012 100
表20图20的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.055 11819 485 0.327
2 10.61 3548256 153336 98.273
3 11.863 50534 1552 1.4
总计   3610608 155373 100
表21图21的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.042 3101 157 0.143
2 10.614 2134596 93152 98.495
3 11.896 29516 930 1.362
总计   2167214 94239 100
表22图22的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.246 4037 196 0.192
2 10.65 2065172 88437 98.272
3 11.938 32268 952 1.535
总计   2101477 89585 100
表23图23的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.017 6890 239 0.326
2 10.612 2073493 91392 98.096
3 11.896 33351 881 1.578
总计   2113734 92512 100
表24图24的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.421 9391 319 0.489
2 10.794 1879579 79778 98.056
3 12.079 29830 886 1.455
总计   1918799 80982 100
检测结果见表25。
表25 37℃加速6个月检测结果
Figure PCTCN2016110724-appb-000014
Figure PCTCN2016110724-appb-000015
试验结果表明，实施例1-6中组合稳定剂对rhCNB均具有明显的稳定和保护作用，加速试验0、1、3、6个月二聚体含量、生物学活性变化不明显，多聚体含量均≤0.5％，在质量控制目标范围内。在冻干成型性上面，实施例2最佳，其次是实施例3、实施例5、实施例6，实施例1 和4最差，主要原因是作为主要支撑物的甘露醇的加入量。总的来说，6个实施例均能对rhCNB起到有效的稳定和保护作用，证明了本组合稳定剂良好的保护效果。
对比例
海藻糖                   2份
蔗糖                     0.5份
甘露醇                   3份
步骤1：按配比称量海藻糖、蔗糖、甘露醇
步骤2：将海藻糖、蔗糖、甘露醇混合，再加入50～150份30～40℃注射用水搅拌，转速30rpm，制得混合液；再加入所述混合液的体积质量分数为0.3％的活化的活性炭搅拌30min，过滤除炭，再加入1份rhCNB蛋白，冻干后，在37℃下进行加速试验，第0、1、3、6月取样检测生物学活性和纯度(SEC-HPLC法)。
图25示对比例稳定性加速试验0月结果图谱；图26示对比例稳定性加速试验1月结果图谱。具体谱图数据见表26～27。
表26图25的谱图数据
峰号 保留时间 面积 高度 面积％
1 10.083 2912 153 0.117
2 10.613 2448004 108502 98.707
3 11.871 29144 1045 1.175
总计   2480060 109699 100
表27图26的谱图数据
峰号 保留时间 面积 高度 面积％
1 9.987 24832 552 1.264
2 10.673 1906044 82571 97.031
3 11.979 33482 849 1.704
总计   1964358 83972 100
检测结果见表28。
表28对比例37℃加速6个月检测结果
Figure PCTCN2016110724-appb-000016
对比例与实施例稳定剂的差别在于不含氨基酸和氯化物，此两种物料是组成本冻干稳定剂的重要成份，在对比例的加速试验中，1月检测结果多聚体已超过了1％，二聚体降低至98％以下，稳定效果较差，而本发明实施例加速试验6月结果中多聚体低于0.5％，二聚体在98％以上，与对比例稳定效果比较，作用明显。
以上对本发明所提供的组合物及其应用、含有该组合物的稳定剂进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (12)

  1. 一种组合物，其特征在于，包括氯化物、氨基酸、糖类或表面活性剂中的一种或两者以上的混合物。
  2. 根据权利要求1所述的组合物，其特征在于，以质量份计，包括如下组分：
    Figure PCTCN2016110724-appb-100001
  3. 根据权利要求1或2所述的组合物，其特征在于，所述氯化物为氯化钙或氯化锌。
  4. 根据权利要求1至3任一项所述的组合物，其特征在于，所述氨基酸为色氨酸或赖氨酸。
  5. 根据权利要求1至4任一项所述的组合物，其特征在于，所述糖类为海藻糖、蔗糖或甘露醇中的一种或两者以上的混合物。
  6. 根据权利要求1至5任一项所述的组合物，其特征在于，所述表面活性剂为非离子型表面活性剂。
  7. 根据权利要求1至6任一项所述的组合物，其特征在于，所述非离子型表面活性剂为聚山梨酯-80。
  8. 根据权利要求1至7任一项所述的组合物，其特征在于，以质量份计，包括如下组分：
    Figure PCTCN2016110724-appb-100002
  9. 根据权利要求1至8任一项所述的组合物在用于制备人钙调蛋白磷酸酶B亚基稳定剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述组合物与所述rhCNB(以蛋白量计)的质量比为1:1。
  11. 一种提高人钙调蛋白磷酸酶B亚基组分稳定性的稳定剂，其特征在于，包括如权利要求1至8任一项所述的组合物和水。
  12. 根据权利要求11所述的稳定剂的制备方法，其特征在于，包括如下步骤：
    步骤1：按配比称量氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇、聚山梨酯-80；
    步骤2：将氯化钙或氯化锌、色氨酸或赖氨酸、海藻糖、蔗糖、甘露醇混合，再加入注射用水，搅拌，制得混合液；再加入活性炭，搅拌，过滤除炭，制得第一溶液；
    步骤3：取配方量的聚山梨酯-80，加入注射用水，再加入活性炭，搅拌，过滤除炭，制得第二溶液；
    步骤4：将步骤2制得的第一溶液和步骤3制得的第二溶液混合，过滤除菌。
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