CN115704821A - 一种cho细胞宿主蛋白残留量检测方法 - Google Patents
一种cho细胞宿主蛋白残留量检测方法 Download PDFInfo
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- CN115704821A CN115704821A CN202110900828.2A CN202110900828A CN115704821A CN 115704821 A CN115704821 A CN 115704821A CN 202110900828 A CN202110900828 A CN 202110900828A CN 115704821 A CN115704821 A CN 115704821A
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Abstract
本发明提供一种CHO细胞宿主蛋白残留量检测方法，所述方法包括配制含有表面活性剂的蛋白样品溶液，然后通过ELISA对蛋白样品溶液进行HCP检测，本发明提供了一种专属性强、准确度高、重复性好的CHO细胞宿主蛋白残留量检测方法，对蛋白的质量控制具有重要意义。
Description
技术领域
本发明涉及生物蛋白的质量控制领域，具体涉及一种CHO细胞宿主蛋白残留量检测样品处理及检测方法。
背景技术
许多抗体、疫苗或蛋白类药物的制备主要是通过生物体系合成，其中CHO细胞(中国仓鼠卵巢细胞)由于表达的蛋白最接近于天然蛋白分子、产物胞外分泌但很少分泌自身内源蛋白，对目标蛋白分离纯化工作十分有利，是目前被广泛使用的工程细胞具有表达遗传背景清楚、表达系统完善稳定、蛋白表达水平较高等优势。尽管采用多种方式进行纯化，但是CHO细胞仍然会有少量蛋白残留在生物制品的半成品和成品中，例如CHO细胞中残留的宿主蛋白(HCP)作为人类自身系统的外源蛋白，可能会在不同程度上引发机体的免疫应答，最终导致过敏反应或其他不良反应。因此需要建立合适的检测HCP的方法来监控最终生物制品或半成品的质量。
ELISA是目前应用最广泛的HCP检测方法。该方法相对简单，精良度好，方便设定控制范围和建立技术规范。目前已经有多种商品化的ELISA试剂盒可以用于HCP的检测，还可以通过自制多克隆抗体进行检测。虽然目前的检测方法给HCP的检测原理和方法提供了一种通行的指引，但是很少有人关注到检测样品处理对检测结果的影响，申请人发现如果检测样品处理不当，可能影响HCP的检测效果，如回收率、准确性、精密度等。
发明内容
本发明为了解决现有技术中HCP检测的缺陷，提供了一种回收率高、准确度和精密度好的CHO细胞宿主蛋白(HCP)残留量的ELISA检测方法，包括：
1)配制含有表面活性剂的待测蛋白样品溶液；
2)通过ELISA对步骤1)所述的样品溶液进行HCP残留量检测。
其中，所述表面活性剂选自吐温20或吐温80。在一些优选实施例中，所述表面活性剂的浓度为0.75-1.2wt％。在一些优选的实施例中，所述表面活性剂的浓度为0.83-1wt％。在一个具体的实施方案中，所述表面活性剂的浓度为0.83wt％。
其中所述蛋白为具有生物学会活性及临床应用价值的目标蛋白，所述蛋白可以为本领域已知的抗体、蛋白或多肽产品，也可以是本领域技术人员根据已知的生物学方法进行构建以及通过CHO细胞生产获得的蛋白。在一个具体的实施例中，本发明所述的蛋白为康柏西普，具有如SEQ ID NO:1所述的氨基酸序列。
在一些优选实施例中，本发明所述的蛋白样品溶液的浓度为0.92mg/ml-1.38mg/ml，优选1mg/ml。
在一个具体的实施方案中，本发明所述的检测方法，步骤1)中所述的样品溶液采用缓冲溶液进行配制，其中所述缓冲液型号为：CYGNUS I028-100。
在一个具体的实施方案中，本发明所述的检测方法，步骤2)中，将步骤1)所述的样品溶液加入碱性磷酸酶标记的HCP抗体中，形成抗原-抗体复合物，孵育后然后加入显色液进行显色，405nm(测定波长)/492nm(参比波长)读数，通过HCP标准曲线计算待测样品溶液中的HCP含量。
在一个具体的实施方案中，显色液为对-硝基苯磷酸酯。
在一个具体的实施方案中，本发明所述的检测方法包括：
1)利用CYGNUS I028-100缓冲溶液配制含0.83wt％吐温20或吐温80的蛋白样品溶液，其中蛋白浓度为1mg/ml；
2)将步骤1)所述的样品溶液加入碱性磷酸酶标记的HCP抗体中，形成抗原-抗体复合物，孵育后然后加入显色液进行显色，405nm(测定波长)/492nm(参比波长)读数，通过HCP标准曲线计算待测样品溶液中的HCP含量。
在步骤2)中，可通过常用的商购ELISA试剂盒，并按照试剂盒的操作步骤或进行适当调整后对样品溶液进行检测，常用的ELISA试剂盒型号例如：Cygnus F015 CHO HCP、Cygnus CM015 CHO HCP或Cygnus F550 CHO HCP。
本发明的技术效果在于通过大量的实验摸索提供了一种专属性强、准确度高、重复性好的CHO细胞宿主蛋白残留量检测方法，对蛋白的质量控制具有重要意义。
具体实施方式
本发明通过以下实施例对本发明的内容作进一步的详细说明，并不能用于限制本发明的保护范围。
本申请所用试剂及仪器：
CHO HCP ELISA kit(CYGNUS，Cat:F015)
样品稀释液(CYGNUS，Cat:I028)
二硫苏糖醇(GE，17-1318-01)
碘乙酰氨(Sigma，V900335-25G)
异丙醇(Fisher，Cat：A451-4)
氯化钠(成都市科龙化工试剂厂)
TritonX-100(Sigma，X100-100ml)
TCEP-HCl(Sigma，Cat：C4706-10G)
Tween-20(J.T.Baker，Cat:4116-04)
Tween-80(Sigma，Cat:P1754-25ml)
仪器设备
酶标仪(Spectraxmax i3X)
实施例1专属性实验
取康柏西普蛋白原液缓冲液(自制，不含康柏西普蛋白，辅料含有柠檬酸、蔗糖、精氨酸)，采用样品稀释液1(CYGNUS，Cat:I028)稀释3.3倍，按试剂盒(CYGNUS，Cat:F015)操作步骤进行CHO细胞蛋白含量检测，具体为：取200μl稀释后的蛋白原液缓冲液加入400μl碱性磷酸酶标记的CHO HCP抗体，形成捕获抗原-抗体复合物，混匀后，25℃静置2h；静置后，200μL/孔，25℃500rpm孵育2h；洗板后加入对-硝基苯磷酸酯(PNPP)显色基底200μL/孔，室温避光静置孵育90-110min；Tris缓冲液洗涤除去未反应的物质后，405nm(测定波长)/492nm(参比波长)读数，根据Protein A标准曲线计算Protein A含量。做3个平行样品，结果如表1。
HCP标准曲线制备：取HCP标准品，用试剂盒样品稀释液1(CYGNUS，Cat:I028)稀释至250ng/ml、75ng/ml、20ng/ml、4ng/ml、1ng/ml、0ng/ml；然后按照上述试剂盒操作步骤，测定不同浓度下HCP标准品在405nm(测定波长)/492nm(参比波长)读数，并制定HCP标准曲线。
表1
实验结果显示，康柏西普蛋白原液缓冲液3个平行样品的检测结果均为未检出，表明蛋白原液缓冲液对方法无明显干扰。
实施例2稀释缓冲液摸索
缓冲液配制
缓冲液1：试剂盒样品稀释液(CYGNUS，Cat：I028)；
缓冲液2：5％TritonX-100，超纯水配制；
缓冲液3：1M NaCl，超纯水配制，加入10％异丙醇；
缓冲液4：50mM Tris缓冲液，250mM Arg-HCl，1M NaCl，pH 8.0，超纯水配制。
检测过程
分别取适量上述缓冲液，加入缓冲液1，接着加入康柏西普原液(自制，蛋白具有如序列1所述的氨基酸序列，浓度为10.9mg/ml，辅料含有柠檬酸、精氨酸、蔗糖，pH为7.5-8.3)或原液缓冲液，再加入HCP蛋白标准品后获得蛋白样品溶液(2.7mg/ml或0mg/ml)；按如实施例1中的试剂盒操作步骤进行HCP含量检测(根据标准曲线计算HCP含量)，并按下式计算HCP加标回收率：
其中：
原液加标检测值为康柏西普原液加入HCP标准品后经检测计算得到的HCP含量(浓度)；
原液检测值为康柏西普原液(未加入HCP标准品)经检测计算得到的HCP含量(浓度)；
理论加标量为加入的HCP标准品浓度。
结果如表2。
表2
以上数据可以看出，采用缓冲液1的缓冲体系，康柏西普蛋白中添加CHO细胞蛋白(Host Cell Protein，HCP)标准品加标回收率为61.2％，低于80％，但是不含康柏西普蛋白的HCP加标回收率在95.9％。推测可能是康柏西普蛋白与CHO细胞蛋白之间具有相互作用，导致CHO细胞蛋白加标回收率偏低。
实施例3稀释缓冲液摸索
缓冲液配制
缓冲液1：试剂盒样品稀释液CYGNUS，Cat：I028；
缓冲液5：5％TritonX-100，超纯水配制；
缓冲液6：1M NaCl，超纯水配制，10％异丙醇；
缓冲液7：50mM Tris，25mM NaCl，5mM EDTA，1M Arg-HCl，超纯水配制，pH 8.5。
检测过程
分别取适量上述缓冲液，加入缓冲液1，接着加入稀释康柏西普原液(10.9mg/ml)或原液缓冲液，再加入HCP标准品获得蛋白样品溶液(3.3mg/ml或0mg/ml)；其中一组样品，用缓冲液1稀释后，再添加25mM DTT室温静置10min，再添加25mM IAM室温静置10min；按如实施例1中的试剂盒操作步骤进行HCP含量检测(根据标准曲线计算HCP含量)，并考察CHO蛋白加标回收率。
结果如表3。
表3
首先，从实施例2和实施例3中蛋白浓度分别为2.7mg/ml和3.3mg/ml的结果(回收率分别为61.2％、31.3％)可以看出，蛋白浓度对检测结果产生影响。其次，添加了表面活性剂0.3％TritonX-100(不含康柏西普)的样品加标回收率为98.2％，说明该配方不影响试剂盒抗原抗体结合，且在康柏西普蛋白浓度3.3mg/ml时的加标回收率为60.3％，明显优于其他配方的缓冲液，表明表明活性剂可能可以通过调节抗原抗体反应而影响检测结果。
实施例4蛋白浓度摸索
分别采用缓冲液1(CYGNUS，Cat：I028)，稀释康柏西普蛋白原液(11.0mg/ml)，加入HCP标准品，使康柏西普蛋白浓度分别为2.75mg/ml、1.83mg/ml、1.38mg/ml、1.10mg/ml、0.92mg/ml、0.78mg/ml，再按如实施例1中的试剂盒操作步骤进行HCP含量检测。结果如表4所示。
表4
上述结果表明，采用缓冲液1将康柏西普原液稀释至10～12倍，即蛋白浓度0.92mg/ml～1.10mg/ml，HCP加标回收率较优。
实施例5
缓冲液配制
缓冲液8：5％TritonX-100，用缓冲液1(CYGNUS，Cat：I028)配制；
缓冲液9：5％TritonX-100，超纯水配置，其中含有30％异丙醇；
缓冲液10：5％Tween-20，用缓冲液1配制；
缓冲液11：6％Tween-80，用缓冲液1配制。
检测过程
分别取上述缓冲液50μl，加入缓冲液1，加入康柏西普原液(22.0mg/ml)，加入HCP标准品，使康柏西普蛋白终浓度为1.0mg/ml，样品溶液总体积300μl；再按如实施例1中的试剂盒操作步骤进行HCP含量检测。考察加标回收率，结果见表5。
表5
从表5实验结果可以看出，含有0.83％TritonX-100的样品溶液加标回收率超过130％，加入0.83％Tween-20和Tween-80表明活性剂的加标回收率分别为91.2％和106.6％。
实施例6重复性和准确度验证
采用缓冲液1(CYGNUS，Cat：I028)将康柏西普原液(22.0mg/ml)预稀释至3mg/ml，继续加入缓冲液1，加入Tween-80，加入不同浓度的HCP标准品，使康柏西普浓度为1.0mg/ml，含有1％Tween-80，样品溶液总体积300μl；再从中取200μl加入400μl碱性磷酸酶标记的HCP抗体，混匀后，25℃静置2h；静置后，200μL/孔，25℃500rpm孵育2h；洗版后加入PNPP显色基底200μL/孔，室温避光静置孵育100min；405nm(测定波长)/492nm(参比波长)读数。其中，PNPP显色液使用前，置于室温避光平衡1h，加标回收率及RSD结果见表6。
表6
实验结果显示，不同HCP加标浓度样品回收率在93％～123％范围内，各浓度3个平行样品RSD在1％～5％范围内。
序列表
<110> 成都康弘生物科技有限公司
<120> 一种CHO细胞宿主蛋白残留量检测方法
<130> KH20210611
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Claims (10)
1.一种CHO细胞宿主蛋白(HCP)残留量的ELISA检测方法，其特征在于所述检测方法包含步骤：
1)配制含有表面活性剂的蛋白样品溶液；
2)通过ELISA对步骤1)所述的蛋白样品溶液进行HCP检测。
2.根据权利要求1所述的检测方法，其特征在于所述表面活性剂选自吐温20或吐温80。
3.根据权利要求2所述的检测方法，其特征在于所述表面活性剂浓度为0.75wt％-1.2wt％。
4.根据权利要求3所述的检测方法，其特征在于所述表面活性剂浓度为0.83wt％-1wt％。
5.根据权利要求1所述的检测方法，其特征在于所述蛋白为康柏西普或阿柏西普。
6.根据权利要求5所述的检测方法，其特征在于所述蛋白样品溶液的浓度为0.92mg/ml-1.38mg/ml，优选1mg/ml。
7.根据权利要求1所述的检测方法，其特征在于步骤1)中所述的样品溶液采用缓冲溶液进行配制，其中所述缓冲液型号为：CYGNUS I028-100。
8.根据权利要求1所述的检测方法，其特征在于步骤2)中，将样品溶液加入碱性磷酸酶标记的HCP抗体中，形成抗原-抗体复合物，孵育后然后加入显色液进行显色，405nm(测定波长)/492nm(参比波长)读数，通过HCP标准曲线计算待测样品溶液中的HCP含量。
9.根据权利要求8所述的检测方法，其特征在于显色液为对-硝基苯磷酸酯(PNPP)。
10.根据权利要求1-9任一项所述的检测方法，其特征在于检测方法包括：
1)利用CYGNUS I028-100缓冲溶液配制含0.83wt％吐温20或吐温80的蛋白样品溶液，其中蛋白浓度为1mg/ml；
2)将样品溶液加入碱性磷酸酶标记的HCP抗体中，形成抗原-抗体复合物，孵育后然后加入显色液进行显色，405nm(测定波长)/492nm(参比波长)读数，通过HCP标准曲线计算待测样品溶液中的HCP含量。
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