CN107362352B - 一种蛋白或多肽组合物及其制备方法和用途 - Google Patents
一种蛋白或多肽组合物及其制备方法和用途 Download PDFInfo
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- CN107362352B CN107362352B CN201610315928.8A CN201610315928A CN107362352B CN 107362352 B CN107362352 B CN 107362352B CN 201610315928 A CN201610315928 A CN 201610315928A CN 107362352 B CN107362352 B CN 107362352B
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- hyaluronic acid
- polypeptide
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Classifications
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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- A—HUMAN NECESSITIES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
本发明公开了一种组合物及其制备方法和用途。所述组合物中含有蛋白或多肽和寡聚透明质酸(寡聚HA)或其盐;其液体形式时,蛋白或多肽0.1‑100μg/ml,寡聚透明质酸或其盐1‑20%。
Description
技术领域
本发明涉及生化领域,尤其涉及一种蛋白或多肽组合物及其制备方法和用途。
背景技术
1934年,Meyer等自牛眼玻璃体提取分离得到一种大分子多糖,命名为hyaluronicacid。透明质酸(hyaluronan,HA)是大分子直链糖胺多糖(glycosaminoglycan,GAG),由重复的二糖单元组成,即D-葡糖醛酸(β1-3)与N-乙酰基-D-葡萄糖胺(β1—4)。HA广泛存在于生物体内,具有多种生物学活性,作为许多组织例如皮肤、腱、肌肉和软骨的细胞的机械支撑,是细胞间基质的主要组成部分,在组织的湿润和润滑中起着重要作用。
HA及各自的盐已经用作药物,特别是治疗关节病中,用作天然器官和组织的辅助物和/或替代物,特别是在眼科和美容手术中,并用于化妆品制剂中作为优良的皮肤填充剂和护肤保湿剂。还开发了用于整形外科、风湿病和皮肤病的透明质酸产品。
根据分子量的不同,可以将透明质酸分为高分子量透明质酸(高2×106Da),中等分子量透明质酸(0.5-2×106Da),低分子量透明质酸(10k-500k Da)以及寡聚透明质酸(HA-Oligo,<10k Da)。不同分子量的透明质酸,其理化性质和应用领域有所不同。一般普通情况下不指明特定分子量时即指中等分子量透明质酸。体内组织损伤、炎症及肿瘤发生时,内源性的透明质酸酶可消化HA多聚体而产生低分子量透明质酸寡糖片段(hyaluronanoligosaccharides,o-HA)。这种寡聚透明质酸已可以在体外通过消化、降解过程而获得。
近年来,含生长因子创伤敷料(growth factors contained wound dressing,GFD)已经成为医用敷料领域发展的新亮点,生长因子的加入使敷料增加了促进创面愈合、提高创面愈合效果的功效。目前已有众多重组生长因子在加速慢性创面愈合或皮肤损伤修复过程中起到重要的作用,并在临床和化妆品领域进行应用,这些生长因子包括表皮细胞生长因子(EGF)、纤维细胞生长因子(FGF)、角质细胞生长因子(KGF-2)、血小板源性生长因子(PDGF)等。
但蛋白质或多肽的结构决定了其不稳定性。蛋白质分子内部的化学键的形成或断裂引起的水解、氧化、消旋,以及其高级结构的物理转变包括变性、聚集、沉淀、吸附等都会影响甚至致使蛋白生理活性丧失。所以,如何对蛋白质或含蛋白组合物的活性保护成为其实际应用中的重要问题。目前,对蛋白常用的保护方法有冻干固化、选择温和的蛋白保存或处理条件、选择添加适宜的蛋白质稳定剂,它们通常是一些多羟基化合物,如海藻糖、乳糖、蔗糖、葡萄糖、右旋糖酐、甘露醇和甘油等。
透明质酸是有等摩尔的氨基葡萄糖和葡萄糖醛酸双糖单位组成的直链多糖,透明质酸在水溶液中分子内的氢键令其形成螺旋结构,当浓度较高时可以形成网状结构,同时具有凝胶的弹性和液体的粘性这一双重特性,这种特性赋予其具有广泛的生理功能及应用前景。
Zhang等的研究显示,胰激肽原酶(PKase)在冻干时加入透明质酸或者透明质酸与海藻糖后,明显增加了PKase冻干品的储藏与复溶的稳定性。但寡聚透明质酸及其盐作为蛋白保护剂的研究和应用未见任何报道。
发明内容
本发明旨在提供一种蛋白或多肽的稳定的组合物。
在本发明的第一方面,提供了一种组合物,其中含有蛋白或多肽和寡聚透明质酸或其盐;其液体形式时,蛋白或多肽浓度为0.1-100μg/ml;以组合物的总体积计,寡聚透明质酸或其盐浓度为1-20%(w/v)。
在另一优选例中,所述寡聚透明质酸或其盐浓度为5-20%(w/v),更优选为5-15%(w/v)。
在另一优选例中,所述蛋白或多肽包括表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、角质细胞生长因子(KGF、KGF-2)、骨骼生长因子(SGF)和生长激素(GH)。
在另一优选例中,所述透明质酸盐选自透明质酸钠、透明质酸钾、透明质酸铵、透明质酸钙、透明质酸镁、透明质酸锌或透明质酸钴。
在另一优选例中,液体形式时磷酸盐缓冲液和/或生理盐水作为溶剂。
在本发明的第二方面,提供了一种如上所述的本发明提供的组合物的制备方法,所述方法包括步骤:
(1)将寡聚透明质酸或其盐与磷酸盐缓冲液和/或生理盐水混合,得到混合溶液1;
(2)将蛋白或多肽和混合溶液1混合,得到液体形式的如上所述的本发明提供的组合物。
在另一优选例中,将液体形式的组合物冷冻干燥得到固体形式的组合物。
在另一优选例中,步骤(1)中将寡聚透明质酸或其盐与磷酸盐缓冲液和/或生理盐水混合后振荡2-4小时;步骤(2)中将蛋白或多肽和混合溶液1混合后搅拌10-15分钟。
在另一优选例中,所述寡聚透明质酸或其盐、磷酸盐缓冲液、生理盐水是无菌的。
在本发明的第三方面,提供了一种寡聚透明质酸或其盐用作蛋白或多肽的稳定剂的用途。
在本发明的第四方面,提供了一种如上所述的本发明提供的组合物的用途,所述组合物用于制备药物组合物或化妆品。
在另一优选例中,所述化妆品是用于保湿的霜剂。
据此,本发明将寡聚透明质酸及其盐作为了蛋白保护剂。
具体实施方式
发明人经过广泛而深入的研究,惊奇地发现透明质酸或其盐是一种良好的蛋白质稳定剂。在此基础上,完成了本发明。
如本文所用,“寡聚透明质酸”是指分子量在10000以下的透明质酸或其盐;优选分子量为2000-8000,更优选为3000-5000。
一般寡聚透明质酸的一个聚合单位的分子量约为400,其聚合单位在25个以下。由于分子量相对较小在机体中穿透、吸收、生理行为相似,而与其它类的透明质酸有差别。寡聚透明质酸是不同聚合度分子的混合物,其分子量不是整齐划一的而是连续的,相同分子量或者说相同聚合度的寡聚透明质酸尚无法彼此严格纯化分离,分子量数值是取其正态分布峰值,其分布宽度则依厂家和质量而有所不同。再考虑分子量的测定误差,实践中并不再把不同分子量的寡聚透明质酸分别进行区别和研究。
如本文所用,“透明质酸(hyaluronic acid)”是指一种高分子的聚合物,由单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高级多糖。D-葡萄糖醛酸及N-乙酰葡糖胺之间由β-1,3-配糖键相连,双糖单位之间由β-1,4-配糖键相连。分子式:(C14H20NO11)n,结构式如下:
本发明提供的组合物可以是固体形式也可以是液体形式,以液体形式为例,所述组合物由以下含量的成份组成:蛋白或多肽、寡聚透明质酸或其盐、磷酸盐缓冲液和/或生理盐水;其中多肽或蛋白浓度为0.1-100μg/ml,优选为1-100μg/ml,更优选为5-50μg/ml;以组合物的总体积计,寡聚透明质酸或其盐浓度为1-20%(w/v),优选为5-20%,更优选为5-15%。
本发明提供的组合物的固体形式是其液体形式通过冷冻干燥或喷雾干燥而获得的干燥粉末状固体。
以本发明提供的组合物为液体形式为例,其制备方法由以下步骤构成:
(1)配置桶中加入适量纯水和氯化钠、磷酸氢二钠、磷酸二氢钠,封口后置于高压蒸汽灭菌锅中,121度灭菌20分钟;
(2)上述溶液冷却后,在百级洁净环境下加入适量寡聚透明质酸钠无菌干粉,封口后置于摇床中震荡溶解,条件为25℃2-4小时;
(3)取出上述溶液,在百级洁净环境下加入适量蛋白或多肽溶液,封口后继续震荡混匀2-3分钟。
本发明提到的上述特征,或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用,说明书中所揭示的各个特征,可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明,所揭示的特征仅为均等或相似特征的一般性例子。
本发明的主要优点在于:
1、本发明提供的组合物稳定,蛋白或多肽的生物活性得到良好保护。
2、本发明提供的组合物生物半衰期长。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。除非另外说明,否则所有的百分数、比率、比例、或份数按重量计。
本发明中的重量体积百分比中的单位是本领域技术人员所熟知的,例如是指在100毫升的溶液中溶质的重量。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
下述实施例中关于EGF的生物活性的测定方法:
Balb/c3T3细胞株用完全培养液于37℃、5%二氧化碳培养,控制细胞浓度为每1ml含1.0×105-5.0×106个细胞,传代后24—36小时用于生物学活性测定。弃去培养瓶中的培养液,消化和收集细胞用完全培养液配成每1ml含5.0×104-8.0×104个细胞的细胞悬液,接种于96孔板中,每孔100μl,于37℃、5%二氧化碳条件下培养。24小时后换成维持培养液。置37℃、5%二氧化碳养24小时。制备的细胞培养板弃去维持液,加入标准品溶液和供试品溶液,每孔100μl。置37℃、5%二氧化碳培养64—72小时。每孔加入MTT溶液20μl,于37℃、5%二氧化碳培养5小时。以上操作在无菌条件下进行。弃去培养板中的液体后,向每孔中加入DMSO 100μl,混匀后在本科标仪上,以630nm为参比波长,570nm为试验波长测定吸光度,记录测定结果。据此可以计算EGF的生物活性。
实施例1
表皮生长因子(EGF)溶液配制(20μg/ml)
实施例2
表皮生长因子(EGF)溶液配制(0.2μg/ml)
实施例3
碱性成纤维细胞生长因子(bFGF)溶液配制(70μg/ml)
实施例4
(1)将寡聚透明质酸钠5g与生理盐水90ml混合振荡2-4小时溶解,得到混合溶液1;
(2)将表皮生物因子EGF 10000μg与混合溶液1混合,加生理盐水至100ml,搅拌10-15分钟溶解,得到液体组合物100ml,含EGF 100μg/ml。
实施例5
(1)将寡聚透明质酸钠10g与生理盐水100ml混合振荡2-4小时溶解,得到混合溶液1;
(2)将表皮生物因子EGF 10000μg与混合溶液1混合,加生理盐水至200ml,搅拌10-15分钟溶解,得到液体组合物200ml,其中含EGF 50μg/ml,含寡聚HA 5%。
(3)将液体组合物进行真空冷冻干燥,得到固体组合物约12g。
实施例6
稳定性试验(生物活性)
按实施例1配置完整配方的溶液,以及不含寡聚HA的溶液,40℃放置10天后分别测定生物活性,同时配置以0.1%BSA(牛血清白蛋白)作为稳定剂的溶液作为对照。结果如下表所示。
溶液组成 | 生物活性(*10<sup>4</sup>IU/ml) |
EGF | 1.04 |
EGF+BSA | 1.89 |
EGF+寡聚HA | 2.21 |
BSA是经常使用的蛋白质保护剂。上述结果表明,寡聚透明质酸对EGF蛋白的生物活性具有较好的保护作用,且明显优于常用的蛋白保护剂BSA。
实施例7
EGF保护试验
按如上实施例配制完整配方的EGF多肽溶液,并加有不同浓度的寡聚HA进行保护,在40℃下放置,7天后测定EGF的生物活性数据。数据显示了不同寡聚HA对不同浓度的EGF多肽的保护能力。
实施例8
(1)将寡聚透明质酸钠10g与生理盐水100ml混合振荡2-4小时溶解,得到混合溶液1;
(2)将表皮生物因子EGF 10000μg与混合溶液1混合,加生理盐水至200ml,搅拌10-15分钟溶解,得到液体组合物400ml,其中含EGF 25μg/ml,含寡聚透明质酸钠2.5%,取样0.1ml。
(3)将液体组合物进行真空冷冻干燥,得到固体组合物约12g。
(4)精密称量(3)所得固体120mg,纯化水溶解定容至4ml。
(5)测定(2)取样和(4)所得溶液的EGF生物活性,分别为3.3×104IU/ml和3.1×104IU/ml。
(6)将(3)所得固体室温下(25℃)保存1个月,按(4)、(5)所述处理后测定溶液的EGF生物活性为3.15×104IU/ml。
结果表明,复溶后仍然很好地保持了EGF的活性。
以上所述仅为本发明的较佳实施例而已,并非用以限定本发明的实质技术内容范围,本发明的实质技术内容是广义地定义于申请的权利要求范围中,任何他人完成的技术实体或方法,若是与申请的权利要求范围所定义的完全相同,也或是一种等效的变更,均将被视为涵盖于该权利要求范围之中。
Claims (9)
1.一种组合物,其中含有蛋白或多肽和寡聚透明质酸或其盐;其液体形式时,蛋白或多肽浓度为0.1-100μg/ml;以组合物的总体积计,寡聚透明质酸或其盐浓度为5-20%(w/v);所述寡聚透明质酸或其盐是其中唯一的蛋白或多肽稳定剂;所述蛋白或多肽为表皮生长因子。
2.如权利要求1所述的组合物,其特征在于,所述寡聚透明质酸或其盐浓度为5-15%(w/v)。
3.如权利要求1所述的组合物,其特征在于,所述寡聚透明质酸盐选自寡聚透明质酸钠、寡聚透明质酸钾、寡聚透明质酸铵、寡聚透明质酸钙、寡聚透明质酸镁、寡聚透明质酸锌或寡聚透明质酸钴。
4.如权利要求1所述的组合物,其特征在于,液体形式时磷酸盐缓冲液和/或生理盐水作为溶剂。
5.一种如权利要求1-4任一项所述的组合物的制备方法,其特征在于,所述方法包括步骤:
(1)将寡聚透明质酸或其盐与磷酸盐缓冲液和/或生理盐水混合,得到混合溶液1;
(2)将蛋白或多肽和混合溶液1混合,得到液体形式的如权利要求1-4任一项所述的组合物;
所述组合物中蛋白或多肽浓度为0.1-100μg/ml;以组合物的总体积计,寡聚透明质酸或其盐浓度为5-20%(w/v)。
6.如权利要求5所述的制备方法,其特征在于,将液体形式的组合物冷冻干燥得到固体形式的组合物。
7.如权利要求5或6所述的制备方法,其特征在于,步骤(1)中将寡聚透明质酸或其盐与磷酸盐缓冲液和/或生理盐水混合后振荡2-4小时;步骤(2)中将蛋白或多肽和混合溶液1混合后搅拌10-15分钟。
8.一种寡聚透明质酸或其盐用作蛋白或多肽的唯一稳定剂的用途,所述寡聚透明质酸或其盐作为蛋白或多肽的唯一稳定剂的液体形式组合物中蛋白或多肽浓度为0.1-100μg/ml;以组合物的总体积计,寡聚透明质酸或其盐浓度为5-20%(w/v);所述蛋白或多肽为表皮生长因子。
9.一种如权利要求1-4任一所述的组合物的用途,其特征在于,所述组合物用于制备药物组合物或化妆品。
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