CN112043685A - 重组人干扰素α1b突变体吸入溶液及其制备方法 - Google Patents
重组人干扰素α1b突变体吸入溶液及其制备方法 Download PDFInfo
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- human interferon
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Abstract
本发明提供一种重组人干扰素α1b突变体吸入溶液及其制备方法。该技术方案首先设计并表达了具有改进型氨基酸序列的重组人干扰素α1b突变体;不仅保持了既有功效,而且具有正确配对的二硫键,极大的提高了稳定性;同时,该突变体通过周质空间表达,有利于增强正确二硫键的形成、促进蛋白的正确折叠和减少杂蛋白水平,从而提高药物蛋白产量和稳定性。以该重组人干扰素α1b突变体为核心,本发明设计了雾化吸入溶液剂的配方及制备方法,填补了该剂型的空白,同时在稳定性、生物学活性等方面性能优异。本发明有效克服了重组人干扰素α1b水溶液的不稳定性,提高了药物靶向作用及患者用药的顺应性,并使临床雾化吸入给药具有了合法的给药剂型。
Description
技术领域
本发明涉及生物制品技术领域,具体涉及一种重组人干扰素α1b突变体吸入溶液及其制备方法。
背景技术
重组人干扰素α1b目前临床上多采用雾化吸入用于治疗呼吸道病毒感染,《重组人干扰素αlb在儿科的临床应用专家共识》也明确推荐用雾化吸入给药治疗儿童病毒性肺炎,但市场上重组人干扰素α1b产品现主要以注射剂为主,国内外均暂无雾化吸入给药的合法途径。
重组人干扰素α1b注射剂用于雾化吸入,其辅料成分无足够的临床资料证明其安全性,不同给药方式、给药剂量及治疗疗程,循证医学证据也仍不充足,尚需要进一步扩大临床研究的样本量,进行大规模多中心的临床研究,以获得更多的循证医学证据。此外,与注射给药相比,雾化吸入给药患者痛苦小、依从性高,尤其适用于呼吸***疾病的治疗。因此开发重组人干扰素α1b雾化吸入剂意义深远。
然而,常规重组人干扰素α1b的水溶液不稳定,这是目前制约重组人干扰素α1b雾化吸入剂开发的一个重要因素,在这种情况下,如何改善其水溶液的稳定性,成为开发雾化吸入剂剂型的重要技术节点。
发明内容
本发明旨在针对现有技术的技术缺陷,提供一种重组人干扰素α1b突变体吸入溶液及其制备方法,以解决目前,重组人干扰素α1b缺乏雾化吸入剂剂型的技术问题。
本发明要解决的另一技术问题是,如何改善重组人干扰素α1b水溶液的稳定性,从而为其雾化吸入剂剂型的开发奠定基础。
本发明要解决的再一技术问题是,采用注射液进行雾化吸入给药,其安全性和有效性不够可靠,且不符合法律法规。
为实现以上技术目的,本发明采用以下技术方案:
重组人干扰素α1b突变体吸入溶液,包括重组人干扰素α1b突变体,pH调节剂,保护剂;所述重组人干扰素α1b突变体的氨基酸序列如SEQ ID No.1、SEQ ID No.2或SEQ IDNo.3所示。
作为优选,还包括以下成分的其中一种或其中若干种:防腐剂,渗透压调节剂,吸收促进剂。
作为优选,所述pH调节剂为磷酸盐缓冲溶液或柠檬酸盐缓冲溶液;所述重组人干扰素α1b突变体吸入溶液的pH为5.5~8.0。
作为优选,所述保护剂选自以下成分的其中一种或其中若干种:人血白蛋白,甘露醇,海藻糖,明胶,吐温,EDTA-2Na,甘氨酸,精氨酸,葡萄糖,蔗糖,卵磷脂,氨丁三醇。
作为优选,所述防腐剂选自以下成分的其中一种或其中若干种:羟苯甲酯,羟苯乙酯,羟苯丙酯,苯甲醇,苯甲酸钠。
作为优选,所述渗透压调节剂为NaCl。
作为优选,所述吸收促进剂为月桂氮卓酮或二甲亚砜。
作为优选,在所述重组人干扰素α1b突变体吸入溶液中,所述重组人干扰素α1b突变体的含量为10万IU/2ml~600万IU/2ml。
在以上技术方案的基础上,本发明进一步提供了上述重组人干扰素α1b突变体吸入溶液的制备方法,包括以下步骤:
1)向配方量的pH调节剂中,加入配方量的保护剂、渗透压调节剂、防腐剂,搅拌至溶解;
2)超滤;
3)向其中加入配方量的重组人干扰素α1b突变体,搅拌至混合均匀;
4)过滤除菌。
作为优选,步骤4)完成后,还依次包括灌装步骤和外包装步骤;所述灌装步骤包括:对内包装材料进行清洗、干燥、灭菌处理,而后以定量体积进行灌装。
本发明提供了一种重组人干扰素α1b突变体吸入溶液及其制备方法。该技术方案针对常规重组人干扰素α1b水溶液稳定性较差的问题,首先设计并表达了具有改进型氨基酸序列的重组人干扰素α1b突变体;以此为基础制备了雾化吸入剂。
具体来看,本发明通过基因工程手段将重组人干扰素α1b C86半胱氨酸突变成为丝氨酸、丙氨酸或天冬氨酸之一(氨基酸序列分别如SEQ ID No.1、SEQ ID No.2、SEQ IDNo.3所示;其编码基因的核苷酸序列分别如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6所示)。所得的重组人干扰素α1b突变体,不仅保持了既有功效,而且具有正确配对的二硫键,极大的提高了稳定性;同时,该重组人干扰素α1b突变体通过周质空间表达,有利于增强正确二硫键的形成、促进蛋白的正确折叠和减少杂蛋白水平,从而提高药物蛋白产量和稳定性。基于以上技术方案,使药物表达量、生物学活性和稳定性得到充分提高,同时保持了良好的安全性和有效性。以该重组人干扰素α1b突变体为核心,本发明设计了雾化吸入溶液剂的配方及制备方法,填补了该剂型的空白,同时在稳定性、生物学活性等方面性能优异。
本发明利用重组人干扰素α1b突变体,制备重组人干扰素α1b突变体吸入溶液,有效克服了重组人干扰素α1b水溶液的不稳定性,提高了药物靶向作用及患者用药的顺应性,并使临床雾化吸入给药具有了合法的给药剂型。
附图说明
图1是本发明具体实施方式中,发酵样品SDS聚丙烯酰胺凝胶电泳图谱。
图2是本发明具体实施方式中,原液SDS聚丙烯酰胺凝胶电泳图谱。
图3是本发明具体实施方式中,原液高效液相图谱。
具体实施方式
以下将对本发明的具体实施方式进行详细描述。为了避免过多不必要的细节,在以下实施例中对属于公知的结构或功能将不进行详细描述。以下实施例中所使用的近似性语言可用于定量表述,表明在不改变基本功能的情况下可允许数量有一定的变动。除有定义外,以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。
1、重组人干扰素α1b突变体C86S的制备
1.1重组人干扰素α1b突变体基因
采用基因合成技术已经十分成熟,可委托第三方生物技术服务公司进行合成,程序比较固定。可以根据指定的基因序列直接合成,实现定点突变的目的。
本实施例提供3种重组人干扰素α1b突变体(其氨基酸序列分别如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3所示;其中序列为SEQ ID No.1的重组人干扰素α1b突变体性能更优),这3种重组人干扰素α1b突变体是将重组人干扰素α1b(序列如SEQ ID No.7所示)第86位半胱氨酸分别突变成为丝氨酸、丙氨酸、天冬氨酸。
这3种重组人干扰素α1b突变体,其编码基因的核苷酸序列分别如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6所示。
1.2重组人干扰素α1b突变体C86S表达菌株构建、转化和鉴定。
本实施例中宿主菌选择的是大肠杆菌BL21(DE3)。
合成pelB-IFNa1b-86ser基因,Hind III和Nde I酶切回收目的片段,连接到pET-22b(+)的Hind III和Nde I位点,构建出重组人干扰素α1b突变体C86S表达质粒。通过化学转化导入大肠杆菌BL21(DE3)菌株中,氨苄青霉素筛选阳性克隆子,阳性克隆子子液体LB中过夜培养,提取质粒,酶切验证,得到重组人干扰素α1b突变体C86S表达菌株。
1.3重组人干扰素α1b突变体的发酵
将实施例1B中获取得到的重组大肠杆菌BL21(DE3)以适当的接种量接种至已灭菌的发酵培养基中,并加入适当的诱导剂进行诱导表达。该重组大肠杆菌BL21(DE3)所合成的重组人干扰素α1b突变体蛋白主要是以周质空间可溶性蛋白的方式存在。(发酵样品SDS聚丙烯酰胺凝胶电泳图谱如图1)
1.4重组人干扰素α1b突变体的纯化
将发酵所收集的菌体加入适当的蛋白抽提液,进行第一步初纯,再经过第二步阴离子交换层析以及第三步疏水层析得到纯度为大于95%且符合药典要求的原液。(原液SDS聚丙烯酰胺凝胶电泳图谱以及高效液相图谱如图2、图3)
2、重组人干扰素α1b突变体雾化吸入溶液的制备
重组人干扰素α1b突变体雾化吸入溶液处方组成见表1。
表1重组人干扰素α1b突变体雾化吸入溶液处方组成
制备方法:
(1)缓冲溶液的配制:称取处方量的pH调节剂,加入注射用水配制成缓冲盐溶液,并测定其pH值;
(2)初混:按处方量加入渗透压调节剂、保护剂、防腐剂、表面活性剂、金属离子螯合剂搅拌至溶解;
(3)超滤:将配好的缓冲液进行超滤;
(4)混合:加入重组人干扰素α1b突变体原液搅拌至混合均匀;
(5)过滤除菌:用0.22μm的微孔滤膜将配好的溶液进行过滤除菌;
(6)灌装:对内包装材料进行清洗、干燥、灭菌处理,调节好灌装体积(2ml)进行灌装;
(7)外包。
高温稳定性试验:将样品存放在37℃±2℃恒温稳定性试验箱中,分别在0天、5天、15天、30天取样检测溶液的性状、pH值、渗透压和生物学活性,结果见表2。
表2 37℃高温稳定性试验结果
雾滴粒径分布检测:将2ml重组人干扰素α1b突变体吸入溶液倒入欧姆龙雾化器中雾化,采用马尔文sprytec激光粒度检测,检测结果如表3。
表3雾滴粒径分布检测结果
项目 | 处方1 | 处方2 | 处方3 | 处方4 |
D10(μm) | 1.921 | 1.652 | 1.543 | 1.642 |
D50(μm) | 3.890 | 3.643 | 3.514 | 3.647 |
D90(μm) | 6.865 | 6.348 | 6.248 | 6.579 |
微细粒子剂量检测:将2ml重组人干扰素α1b突变体吸入溶液倒入欧姆龙雾化器中雾化,按照2015版《中国药典》吸入制剂微细粒子空气动力学特性测定法(通则0951)装置3-NGI检测,检测结果如表4。
表4微细粒子剂量检测结果
项目 | 处方2 | 处方3 |
药物回收率(%) | 95 | 97 |
emitted dose(μg) | 21.70 | 22.4 |
FPD(μg) | 7.18 | 9.85 |
FPF(%) | 62 | 60 |
MMAD(μm) | 3.872 | 3.546 |
GSD | 2.211 | 2.687 |
递送速率与递送总量检测:将2ml重组人干扰素α1b突变体吸入溶液倒入欧姆龙雾化器中雾化,采用呼吸模拟器进行检测,检测结果如表5。
表5递送速率与递送总量检测结果
项目 | 处方2 | 处方3 |
递送速率(μg/min) | 1.5 | 1.2 |
递送总量(μg) | 6.6 | 7.2 |
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,并不用以限制本发明。凡在本发明的申请范围内所做的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 深圳科兴药业有限公司
<120> 重组人干扰素α1b突变体吸入溶液及其制备方法
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Claims (10)
1.重组人干扰素α1b突变体吸入溶液,其特征在于,包括重组人干扰素α1b突变体,pH调节剂,保护剂;所述重组人干扰素α1b突变体的氨基酸序列如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3所示。
2.根据权利要求1所述的重组人干扰素α1b突变体吸入溶液,其特征在于,还包括以下成分的其中一种或其中若干种:防腐剂,渗透压调节剂,吸收促进剂。
3.根据权利要求1所述的重组人干扰素α1b突变体吸入溶液,其特征在于,所述pH调节剂为磷酸盐缓冲溶液或柠檬酸盐缓冲溶液;所述重组人干扰素α1b突变体吸入溶液的pH为5.5~8.0。
4.根据权利要求1所述的重组人干扰素α1b突变体吸入溶液,其特征在于,所述保护剂选自以下成分的其中一种或其中若干种:人血白蛋白,甘露醇,海藻糖,明胶,吐温,EDTA-2Na,甘氨酸,精氨酸,葡萄糖,蔗糖,卵磷脂,氨丁三醇。
5.根据权利要求2所述的重组人干扰素α1b突变体吸入溶液,其特征在于,所述防腐剂选自以下成分的其中一种或其中若干种:羟苯甲酯,羟苯乙酯,羟苯丙酯,苯甲醇,苯甲酸钠。
6.根据权利要求2所述的重组人干扰素α1b突变体吸入溶液,其特征在于,所述渗透压调节剂为NaCl。
7.根据权利要求2所述的重组人干扰素α1b突变体吸入溶液,其特征在于,所述吸收促进剂为月桂氮卓酮或二甲亚砜。
8.根据权利要求1所述的重组人干扰素α1b突变体吸入溶液,其特征在于,在所述重组人干扰素α1b突变体吸入溶液中,所述重组人干扰素α1b突变体的含量为10万IU/2ml~600万IU/2ml。
9.权利要求2所述重组人干扰素α1b突变体吸入溶液的制备方法,其特征在于,包括以下步骤:
1)向配方量的pH调节剂中,加入配方量的保护剂、渗透压调节剂、防腐剂,搅拌至溶解;
2)超滤;
3)向其中加入配方量的重组人干扰素α1b突变体,搅拌至混合均匀;
4)过滤除菌。
10.根据权利要求9所述的制备方法,其特征在于,步骤4)完成后,还依次包括灌装步骤和外包装步骤;所述灌装步骤包括:对内包装材料进行清洗、干燥、灭菌处理,而后以定量体积进行灌装。
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