CN111500671A - 一种注射用多西他赛胶束无菌检查方法 - Google Patents
一种注射用多西他赛胶束无菌检查方法 Download PDFInfo
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Abstract
本发明提供了一种注射用多西他赛胶束无菌检查方法,采用薄膜过滤法,培养物包括试验组和对照组,试验组的制备步骤包括:取注射用多西他赛胶束,用0.9%氯化钠溶液溶解后稀释作为供试液,按薄膜过滤法全量过滤至3个滤筒,同法制备6个滤筒,每个滤筒用pH7.0无菌氯化钠‑蛋白胨缓冲液冲洗,在最后一次冲洗液中加入试验菌液,在每个滤筒中加入培养基培养,逐日观察。本发明针对注射用多西他赛胶束抑菌特性,确定无菌检查过程注射液原液稀释、薄膜过滤选用器械、过滤后薄膜冲洗过程,阳性对照菌、培养基、缓冲液等,有利于多西他赛胶束无菌检查。
Description
技术领域
本发明属于医药检查领域,涉及化学品的检查方法,具体涉及一种注射用多西他赛胶束无菌检查方法。
背景技术
多西他赛属于M期的周期特异性药物,临床上主要用于晚期乳腺癌、非细胞肺癌和卵巢癌的治疗,具有促进小管聚合成微管,并抑制其降解的药理活性,主要通过减少小管数量以达到破坏微管网状结构发挥作用。胶束是表面活性剂溶液浓度超过临界浓度后,其分子以极性基团为外层、非极性基团为内核形成的有序组装体,具有包埋和输送药物的作用。胶束良好的生物相容性和可降解、结构易修饰、可改善抗肿瘤药物水溶性差的特性是其主要的优点。注射用多西他赛胶束卫生指标要求无菌,虽然采用无菌生产工艺生产,但依然可能存在部分休眠状态微生物。
无菌检查法是无菌药品无菌检查的方法,对产品工艺放行及上市后监管都非常重要,部分抗肿瘤药物,如柔红霉素、更生霉素、阿霉素等均具有不同程度的抑菌活性。同时,一些具有抗肿瘤活性的物质也表现出一定程度的抗菌活性,目前并无论文或专利文献公开过注射用多西他赛胶束无菌检查方法。注射用多西他赛胶束属于新型的强抗肿瘤活性药物,在建立无菌检查方法时关键在于对溶解稀释液种类、冲洗液种类、冲洗次数及冲洗量、阳性菌的选择的确定,冲洗量过大容易造成假阴性,冲洗量不足无法消除药物的抑菌活性也会造成假阴性。
发明内容
要解决的技术问题:本发明的目的是提供一种注射用多西他赛胶束无菌检查方法,针对多西他赛抑菌特性,调整无菌检查过程、培养基、缓冲液等要素,能够准确可靠的对多西他赛胶束注射液进行无菌检查。
技术方案:
一种注射用多西他赛胶束无菌检查方法,采用薄膜过滤法,培养物包括试验组和对照组,
所述试验组的制备步骤包括:取注射用多西他赛胶束15瓶,每瓶500mg,用0.9%氯化钠溶液溶解后稀释至500mL作为供试液,按薄膜过滤法全量过滤至3个滤筒,同法制备6个滤筒,每个滤筒用pH7.0无菌氯化钠-蛋白胨缓冲液冲洗3次,每次100mL,在最后一次冲洗液中加入试验菌液,在每个滤筒中加入培养基100mL,培养,逐日观察,作为试验组;
所述对照组的制备步骤包括:以等量0.9%无菌氯化钠溶液替代注射用多西他赛胶束供试液后采用相同的后续操作,作为对照组。
进一步的,所述薄膜过滤法采用的过滤器为三联NS01-AS3型全封闭无菌试验过滤培养器,孔径为0.22μm。
进一步的,所述培养基为硫乙醇酸盐流体培养基或胰酪大豆胨液体培养基。
进一步的,所述试验菌液为金黄色葡萄球菌菌液、大肠埃希菌菌液、枯草芽孢杆菌菌液、生孢梭菌菌液、白色念珠菌菌液、黑曲霉孢子悬液。
进一步的,所述硫乙醇酸盐流体培养基25℃的pH值为7.1±0.1;所述胰酪大豆胨液体培养基25℃的pH值为7.3±0.1。
进一步的,所述金黄色葡萄球菌菌液、大肠埃希菌菌液、枯草芽孢杆菌菌液的制备方法为:分别取金黄色葡萄球菌、大肠埃希菌、枯草芽孢杆菌的甘油冻存液1mL接种至胰酪大豆胨液体培养基中,32℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,分别制成每1mL含菌数<100cfu的试验菌液。
进一步的,所述生孢梭菌菌液的制备方法为:生孢梭菌的甘油冻存液1mL接种至硫乙醇酸盐流体培养基中,32℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含菌数<100cfu的试验菌液。
进一步的,所述白色念珠菌菌液的制备方法为:白色念珠菌的甘油冻存液1mL接种至沙氏葡萄糖液体培养基中,23℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含菌数<100cfu的试验菌液。
进一步的,所述黑曲霉菌液的制备方法为:黑曲霉孢子甘油冻存液恢复室温后直接稀释,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含孢子数<100cfu的试验菌液。
进一步的,所述沙氏葡萄糖液体培养基25℃的pH值为5.6±0.1。
依据《中国药典》2015年版四部通则<1105>,本发明建立了2种注射用多西他赛胶束无菌检查方法,结果发现每个滤筒用pH7.0无菌氯化钠蛋白胨缓冲液冲洗3次,每次100mL时注射用多西他赛胶束的抑菌活性被基本消除,可采用金黄色葡萄球菌作为阳性菌进行无菌检查。
有益效果:
1. 本发明注射用多西他赛胶束均以金黄色葡萄球菌作为阳性对照菌,每个滤筒用pH7.0无菌氯化钠-蛋白胨缓冲液冲洗3次,每次100mL进行无菌检查可行。
2. 本发明针对多西他赛胶束抑菌特性,确定无菌检查过程注射液原液稀释、薄膜过滤选用器械、过滤后薄膜冲洗过程,阳性对照菌、培养基、缓冲液等,有利于多西他赛胶束无菌检查。
3. 本发明能够简单、有效、准确的对注射用多西他赛胶束进行检查,为产品质量控制以及合格率的检查提供依据。
具体实施方式
1. 实验仪器
LA2-4A1型生物安全柜购自新加坡Esco公司;KB240型培养箱购自德国Binder公司;Steritest TM Equinox集菌仪购自德国默克密理博公司;NS01-AS3型全封闭无菌试验过滤培养器购自北京牛牛基因技术有限公司;LASAI III尘埃粒子计数器购自美国PMS公司。
2. 试验菌株及处理方法
分别取金黄色葡萄球菌、大肠埃希菌、枯草芽孢杆菌的甘油冻存液1mL接种至胰酪大豆胨液体培养基中,32℃培养24h后待用;生孢梭菌的甘油冻存液1mL接种至硫乙醇酸盐流体培养基中,32℃培养24h后待用;白色念珠菌甘油冻存液1mL接种至沙氏葡萄糖液体培养基中,23℃培养24h后,待用;黑曲霉孢子甘油冻存液恢复室温后直接稀释后使用。按上述方式处理后,将培养的菌悬液(或解冻的孢子悬液)用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含菌数(或孢子数)小于100cfu的试验菌液。试验用菌株均购自中国食品药品检定研究院,编号及本实验中使用代数见表1。
表1 无菌检查方法验证用试验菌株
菌种名称 | 菌种编号 | 使用代数 |
金黄色葡萄球菌 | CMCC(B)26003 | 第3代培养物 |
枯草芽孢杆菌 | CMCC(B)63501 | 第3代培养物 |
大肠埃希菌 | CMCC(B)44102 | 第3代培养物 |
生孢梭菌 | CMCC(B)64941 | 第3代培养物 |
白色念珠菌 | CMCC(F)98001 | 第3代培养物 |
黑曲霉菌 | CMCC(F)98003 | 第2代培养物 |
3. 供试品操作过程
试验组:取本品15瓶,500mg/瓶,用0.9%氯化钠溶液溶解后稀释至500mL作为供试液。按薄膜过滤法全量过滤至3个滤筒,同法制备6个滤筒,每个滤筒用pH7.0无菌氯化钠-蛋白胨缓冲液冲洗3次,每次100mL,在最后一次冲洗液中分别加入<100cfu的6种试验菌液,并在每个滤筒中加入培养基100mL,培养,逐日观察。
对照组:以等量0.9%无菌氯化钠溶液替代注射用多西他赛胶束供试液后采用相同的后续操作,作为对照组。
4. 环境监测
本研究对进行无菌检查方法学验证环境的温湿度、换气次数、对外压差及尘埃粒子数(方法参照GB/T16292-2010)和沉降菌(方法参照GB/T16294-2010)等项目进行监测。
5. 适用性验证
按照建立的方法进了两家不同生产单位的注射用多西他赛胶束进行验证。注射用多西他赛胶束A来自无锡BD药物研究有限公司(规格500mg/瓶,批号DXTS20131123);注射用多西他赛胶束B来自苏州HTBA生物技术有限公司(规格500mg/瓶,批号:20141110)。采用三联NS01-AS3型全封闭无菌试验过滤培养器进行薄膜过滤后,每个滤筒经pH7.0无菌氯化钠蛋白胨缓冲液冲洗3次,每次100mL处理后药物抑菌活性基本消除,可进行无菌检查,结果可行。
5.1 环境监测结果
生物安全柜中≥5μm和≥0.5μm的尘埃粒子均为0个/m3,沉降菌检测结果为0cfu/皿,无菌室的对外压差为10Pa,环境温度23℃,相对湿度50%,换气次数41次/h,符合进行方法学验证要求。
5.2注射用多西他赛胶束无菌检查方法验证试验结果
研究发现按照每个滤筒冲洗3次,每次100mL处理后,最后一次冲洗液中加入菌液,再加入硫乙醇酸盐流体培养基培养,注射用多西他赛胶束A和注射用多西他赛胶束B中的6种验证菌株均在48h内生长良好,即待验证注射用多西他赛胶束在此处理条件下无抑菌活性或者抑菌活性可以忽略不计,见表2。
表2注射用多西他赛胶束无菌检查方法验证试验结果
部分抗肿瘤药物,如柔红霉素、更生霉素、阿霉素等均具有不同程度的抑菌活性。同时,一些具有抗肿瘤活性的物质也表现出一定程度的抗菌活性。在对春砂仁和益智仁中黄酮类物质的抑菌和抗肿瘤功能研究时证实二者均具有较强的抗菌和抗肿瘤活性。橘皮与橙皮精油对金黄色葡萄球菌、枯草芽孢杆菌和变形杆菌等3种指示菌均有抑制作用,其对肿瘤细胞凋亡或坏死的效果可能与其抗氧化性和抑菌性有一定的相关性。同时,Tubeufiaceae科真菌次生代谢产物均有较强的抑菌和抗肿瘤活性。本发明发现注射用多西他赛胶束经3次、每次100mL的pH7.0无菌氯化钠蛋白胨缓冲液冲洗后抑菌活性被基本消除,提示虽然抗肿瘤药物均有较强的抑菌性,但基于胶束剂型药物溶解性好、对滤膜的吸附性低的特性,在此处理条件下注射用多西他赛胶束的抑菌活性能够得到完全的消除,可作为无菌检查的方法。本发明未发现注射用多西他赛胶束对6种验证用菌株的某一种具有特别的抑菌活性,因此,可选择金黄色葡萄球菌作为阳性菌。
Claims (10)
1.一种注射用多西他赛胶束无菌检查方法,采用薄膜过滤法,培养物包括试验组和对照组,其特征在于:
所述试验组的制备步骤包括:取注射用多西他赛胶束15瓶,每瓶500mg,用0.9%氯化钠溶液溶解后稀释至500mL作为供试液,按薄膜过滤法全量过滤至3个滤筒,同法制备6个滤筒,每个滤筒用pH7.0无菌氯化钠-蛋白胨缓冲液冲洗3次,每次100mL,在最后一次冲洗液中加入试验菌液,在每个滤筒中加入培养基100mL,培养,逐日观察,作为试验组;
所述对照组的制备步骤包括:以等量0.9%无菌氯化钠溶液替代注射用多西他赛胶束供试液后采用相同的后续操作,作为对照组。
2.根据权利要求1所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述薄膜过滤法采用的过滤器为三联NS01-AS3型全封闭无菌试验过滤培养器,孔径为0.22μm。
3.根据权利要求1所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述培养基为硫乙醇酸盐流体培养基或胰酪大豆胨液体培养基。
4.根据权利要求1所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述试验菌液为金黄色葡萄球菌菌液、大肠埃希菌菌液、枯草芽孢杆菌菌液、生孢梭菌菌液、白色念珠菌菌液、黑曲霉孢子悬液。
5.根据权利要求3所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述硫乙醇酸盐流体培养基25℃的pH值为7.1±0.1;所述胰酪大豆胨液体培养基25℃的pH值为7.3±0.1。
6.根据权利要求4所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述金黄色葡萄球菌菌液、大肠埃希菌菌液、枯草芽孢杆菌菌液的制备方法为:分别取金黄色葡萄球菌、大肠埃希菌、枯草芽孢杆菌的甘油冻存液1mL接种至胰酪大豆胨液体培养基中,32℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,分别制成每1mL含菌数<100cfu的试验菌液。
7.根据权利要求4所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述生孢梭菌菌液的制备方法为:生孢梭菌的甘油冻存液1mL接种至硫乙醇酸盐流体培养基中,32℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含菌数<100cfu的试验菌液。
8.根据权利要求4所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述白色念珠菌菌液的制备方法为:白色念珠菌的甘油冻存液1mL接种至沙氏葡萄糖液体培养基中,23℃培养24h,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含菌数<100cfu的试验菌液。
9.根据权利要求4所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述黑曲霉菌液的制备方法为:黑曲霉孢子甘油冻存液恢复室温后直接稀释,用灭菌的0.9%氯化钠溶液进行10倍梯度稀释,最终制成每1mL含孢子数<100cfu的试验菌液。
10.根据权利要求8所述的一种注射用多西他赛胶束无菌检查方法,其特征在于:所述沙氏葡萄糖液体培养基25℃的pH值为5.6±0.1。
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CN113388663A (zh) * | 2021-06-09 | 2021-09-14 | 深圳善康医疗健康产业有限公司 | 一种难溶性植入剂无菌检查方法及无菌过滤装置 |
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