CN115105628B - 一种高稳定性重组人表皮生长因子创面修护敷料及其制备方法 - Google Patents
一种高稳定性重组人表皮生长因子创面修护敷料及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种高稳定性重组人表皮生长因子创面修护敷料及其制备方法,属于生物技术制药技术领域。本发明通过生物酶技术将表皮生长因子和胶原蛋白反应产生分子内交联,起到保护表皮生长因子三维分子结构,使其活性中心构象在水溶液中稳定不易被降解,交联的表皮生长因子活性产品可常温保存,存放一年生长因子活性仍保持90%以上,储存条件宽松,产品有效期延长。
Description
技术领域
本发明涉及生物技术制药技术领域,尤其涉及一种高稳定性重组人表皮生长因子创面修护敷料及其制备方法。
背景技术
人表皮生长因子(hEGF)广泛存在于体液和多种腺体中,hEGF在体内的含量极微。随着基因工程技术的发展,生物学家通过基因重组技术,成功利用大肠杆菌为宿主生产hEGF,实现了工业化生产rhEGF。EGF可刺激多种动物细胞的增殖,常应用于促进人体表皮细胞、内皮细胞生长和修护,对于角膜损伤、烧烫伤及医美手术后等创面的修复和愈合有很好的疗效。
人表皮生长因子(hEGF)分子结构由53个氨基酸组成,分子量6201道尔顿,分子内有6个半胱氨酸组成的二硫键,形成3个分子内环型结构,组成生物活性所必须的受体结合区域,表皮生长因子是一种生物活性蛋白质,分子结构不稳定,在有水的环境中EGF分子结构易被破坏照成活力损失,通常表皮生长因子在水溶液中活性一般只能存活7-10天。
目前国内以表皮生长因子(EGF)为原料生产的表皮细胞修复制剂有凝胶、水剂、敷料、冻干粉等品种,为了保护表皮生长因子的活性,在生产产品一般需要添加适当保护剂和抗氧化剂以减缓EGF的失活,但也仅限于延缓EGF的活力下降,不能根本解决EGF的失活,如何维持产品中EGF分子结构稳定,是确保产品EGF活性及使用效果的关键。
发明内容
有鉴于此,本发明的目的是提供一种高稳定性重组人表皮生长因子创面修护敷料,该敷料具有保护表皮生长因子三维分子结构,使其活性中心构象在水溶液中稳定不易被降解,交联的表皮生长因子活性产品可常温保存,存放一年生长因子活性仍保持90%以上,储存条件宽松,产品有效期延长。
本发明的高稳定性重组人表皮生长因子创面修护敷料,由敷料基础液和表皮生长因子-水解胶原蛋白交联物(hEGF-HC)组成;
所述表皮生长因子-水解胶原蛋白交联物,包括以下重量百分数的原料:
重组人表皮生长因子 1.0-5.0%,
水解胶原蛋白 3.0-15.0%,
谷氨酰胺转氨酶 0.1%,
余量为纯化水。
优选的,所述表皮生长因子-水解胶原蛋白交联物占所述敷料的质量比例为0.2%。
优选的,所述重组人表皮生长因子的活性为7500万U/g。
优选的,所述水解胶原蛋白为猪水解胶原蛋白或牛水解胶原蛋白,分子量范围200KD-500KD。
优选的,所述谷氨酰胺转氨酶的活性为120U/g。
优选的,所述敷料基础液,包括以下重量百分数原料:
本申请另一个目的是提供一种高稳定性重组人表皮生长因子创面修护敷料的制备方法,包括以下步骤:
步骤S1、制备表皮生长因子-水解胶原蛋白交联物:
步骤S1-1、按比例称取表皮生长因子、水解胶原蛋白,用预冷20℃纯化水搅拌溶解;
步骤S1-2、加入谷氨酰胺转氨酶,在20℃恒温水浴条件下以60rpm的转速搅拌反应8-10小时,用0.22um微孔滤膜过滤,收集滤液,得到表皮生长因子-水解胶原蛋白交联物(hEGF-HC);放入冰箱冷藏备用;
步骤S2、制备敷料基础液::
步骤S2-1、按比例称量水、海藻糖、羟乙基纤维素、透明质酸钠,倒入加热搅拌锅,加热至90℃,搅拌溶解,保温30~35分钟;
步骤S2-2、待步骤S2-1中溶液的温度降至40℃以下,加入羟苯甲酯,搅拌溶解均匀,得到敷料基础液。
步骤S3:制备高稳定性重组人表皮生长因子创面修护敷料:按比例将表皮生长因子-水解胶原蛋白交联物加入到敷料基础液中,搅拌混合均匀,在洁净室环境的超净工作台灌装,封口,即得液体敷料产品。
本发明制备的敷料中添加了谷氨酰胺转氨酶,谷氨酰胺转氨酶是一种催化酰基转移反应的转移酶,它以生长因子肽链上的谷氨酰胺残基γ-酰胺基作为酰胺基供体,胶原多肽链中赖氨酸残基ε-氨基作为受体,形成蛋白质分子内ε-(γ-谷氨酰胺基)-赖氨酸异肽键使蛋白质分子发生交联,生成表皮生长因子-胶原蛋白复合物(hEGF-HC)。反应式如式I:
该交联产物性质稳定,谷氨酰胺转氨酶催化形成的共价键交联产物一般在无酶催化条件很难将其断裂。
谷氨酰胺转氨酶反应条件温和,PH适用范围广,尤其可在低温条件反应,交联过程避免表皮生长因子的活性损失,添加量小,交联反应完成后谷氨酰胺转氨酶残留很少,人体内本身也有谷氨酰胺转氨酶存在,不会对人体产生细胞毒性,对比其它使用化学交联剂安全性更高。
与现有技术相比,本发明具有以下有益效果:
本发明通过生物酶技术将表皮生长因子和胶原蛋白反应产生分子内交联,起到保护表皮生长因子三维分子结构,使其活性中心构象在水溶液中稳定不易被降解,交联的表皮生长因子活性产品可常温保存,存放一年生长因子活性仍保持90%以上,储存条件宽松,产品有效期延长。且本发明的制备方法操作简单,不需特殊设备和特殊反应条件,成本低廉,效果显著,反应得到的表皮生长因子-水解胶原蛋白交联物可应用于凝胶、水剂、敷贴类等表皮修复产品。
具体实施方式
下面结合实施例和对比例对本发明作进一步说明。
实施例1
一种高稳定性重组人表皮生长因子创面修护敷料,由敷料基础液和表皮生长因子-水解胶原蛋白交联物(hEGF-HC)组成;表皮生长因子-水解胶原蛋白交联物占所述敷料的质量比例为0.2%;
其中表皮生长因子-水解胶原蛋白交联物由以下重量百分数原料组成:
活性为7500万U/g的重组人表皮生长因子 1.0%,
分子量范围200KD-500KD的猪水解胶原蛋白 3.0%,
活性为120U/g的谷氨酰胺转氨酶 0.1%,
余量为纯化水;
所述猪水解胶原蛋白为猪皮提取的水解胶原蛋白。
敷料基础液由以下重量百分数的原料组成:
所述高稳定性重组人表皮生长因子创面修护敷料的制备方法,步骤如下:
步骤S1、制备表皮生长因子-水解胶原蛋白交联物:
步骤S1-1、按比例称取表皮生长因子、水解胶原蛋白,用预冷20℃纯化水搅拌溶解;
步骤S1-2、加入谷氨酰胺转氨酶,在20℃恒温水浴条件下以60rpm的转速搅拌反应10小时,用0.22um微孔滤膜过滤,收集滤液,得到表皮生长因子-水解胶原蛋白交联物(hEGF-HC);放入冰箱冷藏备用;
步骤S2、制备敷料基础液::
步骤S2-1、按比例称量水、海藻糖、羟乙基纤维素、透明质酸钠,倒入加热搅拌锅,加热至90℃,搅拌溶解,保温30分钟;
步骤S2-2、待步骤S2-1中溶液的温度降至40℃以下,加入羟苯甲酯,搅拌溶解均匀,得到敷料基础液。
步骤S3:制备高稳定性重组人表皮生长因子创面修护敷料:按比例将表皮生长因子-水解胶原蛋白交联物加入到敷料基础液中,搅拌混合均匀,在洁净室环境的超净工作台灌装,封口,即得液体敷料产品。
实施例2
一种高稳定性重组人表皮生长因子创面修护敷料,由敷料基础液和表皮生长因子-水解胶原蛋白交联物(hEGF-HC)组成;表皮生长因子-水解胶原蛋白交联物占所述敷料的质量比例为0.2%;
其中表皮生长因子-水解胶原蛋白交联物由以下重量百分数原料组成:
活性为7500万U/g的重组人表皮生长因子 3.0%,
分子量范围200KD-500KD的猪水解胶原蛋白 9.0%,
活性为120U/g的谷氨酰胺转氨酶 0.1%,
余量为纯化水;
所述猪水解胶原蛋白为猪皮提取的水解胶原蛋白。
敷料基础液由以下重量百分数的原料组成:
所述高稳定性重组人表皮生长因子创面修护敷料的制备方法,步骤如下:
步骤S1、制备表皮生长因子-水解胶原蛋白交联物:
步骤S1-1、按比例称取表皮生长因子、水解胶原蛋白,用预冷20℃纯化水搅拌溶解;
步骤S1-2、加入谷氨酰胺转氨酶,在20℃恒温水浴条件下以60rpm的转速搅拌反应10小时,用0.22um微孔滤膜过滤,收集滤液,得到表皮生长因子-水解胶原蛋白交联物(hEGF-HC);放入冰箱冷藏备用;
步骤S2、制备敷料基础液::
步骤S2-1、按比例称量水、海藻糖、羟乙基纤维素、透明质酸钠,倒入加热搅拌锅,加热至90℃,搅拌溶解,保温30分钟;
步骤S2-2、待步骤S2-1中溶液的温度降至40℃以下,加入羟苯甲酯,搅拌溶解均匀,得到敷料基础液。
步骤S3:制备高稳定性重组人表皮生长因子创面修护敷料:按比例将表皮生长因子-水解胶原蛋白交联物加入到敷料基础液中,搅拌混合均匀,在洁净室环境的超净工作台灌装,封口,即得液体敷料产品。
实施例3
一种高稳定性重组人表皮生长因子创面修护敷料,由敷料基础液和表皮生长因子-水解胶原蛋白交联物(hEGF-HC)组成;表皮生长因子-水解胶原蛋白交联物占所述敷料的质量比例为0.2%;
其中表皮生长因子-水解胶原蛋白交联物由以下重量百分数原料组成:
活性为7500万U/g的重组人表皮生长因子 5.0%,
分子量范围200KD-500KD的猪水解胶原蛋白 15.0%,
活性为120U/g的谷氨酰胺转氨酶 0.1%,
余量为纯化水;
所述猪水解胶原蛋白为猪皮提取的水解胶原蛋白。
敷料基础液由以下重量百分数的原料组成:
所述高稳定性重组人表皮生长因子创面修护敷料的制备方法,步骤如下:
步骤S1、制备表皮生长因子-水解胶原蛋白交联物:
步骤S1-1、按比例称取表皮生长因子、水解胶原蛋白,用预冷20℃纯化水搅拌溶解;
步骤S1-2、加入谷氨酰胺转氨酶,在20℃恒温水浴条件下以60rpm的转速搅拌反应10小时,用0.22um微孔滤膜过滤,收集滤液,得到表皮生长因子-水解胶原蛋白交联物(hEGF-HC);放入冰箱冷藏备用;
步骤S2、制备敷料基础液::
步骤S2-1、按比例称量水、海藻糖、羟乙基纤维素、透明质酸钠,倒入加热搅拌锅,加热至90℃,搅拌溶解,保温30分钟;
步骤S2-2、待步骤S2-1中溶液的温度降至40℃以下,加入羟苯甲酯,搅拌溶解均匀,得到敷料基础液。
步骤S3:制备高稳定性重组人表皮生长因子创面修护敷料:按比例将表皮生长因子-水解胶原蛋白交联物加入到敷料基础液中,搅拌混合均匀,在洁净室环境的超净工作台灌装,封口,即得液体敷料产品。
对比例1
参照实施例2的制备方法以及原料比例,制备一种重组人表皮生长因子创面修护敷料。
区别在于,对比例1中添加的谷氨酰胺转氨酶预先90℃灭活,使表皮生长因子和水解胶原蛋白不发生交联反应。
一、表皮生长因子活性稳定性试验对比
对实施例1~3和对比例1中创面敷料中的表皮生长因子-水解胶原蛋白复合物活性进行检测,试验样品按规定温度条件保存,每隔一短时间取样检测,比较表皮生长因子活性,评价稳定性效果。表皮生长因子活性检测按照《中国药典》三部附录XH重组人表皮生长因子生物学活性测定方法。
(一)常温条件(0-30℃)保存,检测不同时间的生物学活性,检测结果如表1:
表1常温储存各样品活性下降对比
从表1可以看出,常温条件(0-30℃)下,表皮生长因子-水解胶原蛋白交联物活性稳定,12个月活力仅下降5%左右,而处于游离状态表皮生长因子产品12个月活性下降超过50%。
(二)破坏性试验(40℃存放),检测不同时间的生物学活性,检测结果如表2:
表2高温储存各样品活性下降对比
从表2可以看出,试验产品40℃存放条件,表皮生长因子-水解胶原蛋白交联物活性相对稳定,12个月活力下降30%左右,状态游离表皮生长因子产品6个月活性下降46.92%。12个月活性仅存12%。
二、应用效果对比
本实验采用Wister白鼠背部皮肤进行创伤模型的功效实验。
将创伤白鼠分为3组,分为空白对照组、实验组1和实验组2,每组10只。选用实施例2和对比例1制备的重组人表皮生长因子创面修护敷料,常温放置6个月后,作为白鼠创伤愈合试验敷料。
其中空白对照组不给药,实验组1采用实施例2制备的重组人表皮生长因子创面修护敷料涂抹创伤部位,每日一次;实验组2采用对比例1制备的重组人表皮生长因子创面修护敷料涂抹创伤部位,每日一次。
先用脱毛剂在白鼠背部脱毛,脱毛后继续喂养两天,然后按照白鼠体重的0.3%剂量注射水合氯醛溶液,白鼠麻醉后,脱毛处皮肤消毒,在背部中央距肩胛2cm处用手术剪剪去皮层,剪去皮肤直径1.6cm、面积2.0cm2,形成圆形创面。
每日常规观察白鼠活动、饮食等健康状态是否正常,并做好记录。
根据不同时间点对每组白鼠创伤面积进行观察并测量,创面部位凝块脱落且被新皮或结蹄组织覆盖判定为愈合,记录愈合25%、50%、75%和100%所需时间,结果如表3所示:
表3Wister白鼠创面愈合实验数据
从表3可以看出,与不用药对比,使用实施例2与对比例1敷料完全愈合时间提前4天和2天,使用含表皮生长因子的敷料比空白组试验明显愈合较快,实施例2为含表皮生长因子-水解胶原蛋白交联物,在样品保存半年后使用体现了(hEGF-HC)活性的较高稳定性。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种高稳定性重组人表皮生长因子创面修护敷料,其特征在于,所述敷料由敷料基础液和表皮生长因子-水解胶原蛋白交联物组成;
所述表皮生长因子-水解胶原蛋白交联物,包括以下重量百分数的原料:
重组人表皮生长因子 1.0-5.0%,
水解胶原蛋白 3.0-15.0%,
谷氨酰胺转氨酶 0.1%,
余量为纯化水;
敷料基础液,包括以下重量百分数原料:
纯化水 93.0~94.0%,
海藻糖 4.6~5.0%,
羟乙基纤维素 0.8~1%,
透明质酸 0.2%,
羟苯甲酯 0.1%;
所述水解胶原蛋白为猪水解胶原蛋白或牛水解胶原蛋白,分子量范围200KD-500KD;
所述谷氨酰胺转氨酶的活性为120U/g;
所述高稳定性重组人表皮生长因子创面修护敷料的制备方法,包括以下步骤:
步骤S1、制备表皮生长因子-水解胶原蛋白交联物:
步骤S1-1、按比例称取表皮生长因子、水解胶原蛋白,用预冷20℃纯化水搅拌溶解;
步骤S1-2、加入谷氨酰胺转氨酶,在20℃恒温水浴条件下以60rpm的转速搅拌反应8-10小时,用0.22um微孔滤膜过滤,收集滤液,得到表皮生长因子-水解胶原蛋白交联物;放入冰箱冷藏备用;
步骤S2、制备敷料基础液:
步骤S2-1、按比例称量水、海藻糖、羟乙基纤维素、透明质酸钠,倒入加热搅拌锅,加热至90℃,搅拌溶解,保温30~35分钟;
步骤S2-2、待步骤S2-1中溶液的温度降至40℃以下,加入羟苯甲酯,搅拌溶解均匀,得到敷料基础液;
步骤S3:制备高稳定性重组人表皮生长因子创面修护敷料:按比例将表皮生长因子-水解胶原蛋白交联物加入到敷料基础液中,搅拌混合均匀,在洁净室环境的超净工作台灌装,封口,即得液体敷料产品。
2.根据权利要求1所述高稳定性重组人表皮生长因子创面修护敷料,其特征在于,所述表皮生长因子-水解胶原蛋白占所述敷料的质量比例为0.2%。
3.根据权利要求1所述高稳定性重组人表皮生长因子创面修护敷料,其特征在于,所述重组人表皮生长因子的活性为7500万U/g。
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