重组抗CD52单克隆抗体亲和层析纯化工艺的优化
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摘要
目的优化重组抗CD52单克隆抗体亲和层析纯化工艺。方法采用试验设计(Design of Experiment,DOE)方法优化重组抗CD52单克隆抗体亲和层析纯化工艺,试验因子为中间清洗液Na Cl浓度(130~870 mmol/L)和洗脱液p H(3.50~4.20),试验响应变量为纯化后样品收率、纯度、残余宿主蛋白含量、外源DNA含量及蛋白A含量,通过DOE构建模型,预测中间清洗液Na Cl浓度和洗脱液p H范围。同时采用DOE方法验证预测工艺参数的稳定性。结果确定中间清洗液Na Cl浓度为400~700 mmol/L,洗脱液p H为3.55~3.75。试验因子在该参数范围内变动时,样品收率均大于80%,纯度均大于97%,外源DNA浓度均低于260 pg/mg,宿主蛋白含量均低于2 300 ng/mg,蛋白A含量均低于6.5 ng/mg。结论成功优化了重组抗CD52单克隆抗体的亲和纯化工艺,且具有较好的稳定性。
Objective To optimize the affinity chromatography process for purification of recombinant anti-CD52 monoclonal antibody(Mc Ab). Methods Affinity chromatography process for purification of recombinant anti-CD52 Mc Ab was optimized by Design of Experiment(DOE). The input factors were sodium chloride concentration(130 ~ 870 mmol/L)of intermediate wash solution and the p H value(3. 50 ~ 4. 20)of elution solution,while the responses were the recovery rate,purity,residual host cell protein content,exogenous DNA content and protein A content of purified samples,with which a data model was established by DOE. The ranges of sodium chloride concentration of intermediate wash solution and p H value of elution solution were predicted by the model,and the stability of process parameters were verified by DOE. Results The optimal sodium chloride concentration of intermediate wash solution was 400 ~ 700 mmol/L,while the optimal p H value of elution solution was 3. 55 ~ 3. 75. When the factors changed within the predicted ranges,all the recovery rates of samples were more than 80%,while the purities were more than 97%,the exogenous DNA contents were less than 206 pg/mg,the host cell protein contents were less than 2 300 ng/mg,and the protein A contents were less than 6. 5 ng/mg. Conclusion The affinity chromatography process for purification of recombinant anti-CD52 Mc Ab was successfully optimized,which showed high stability.
Objective To optimize the affinity chromatography process for purification of recombinant anti-CD52 monoclonal antibody(Mc Ab). Methods Affinity chromatography process for purification of recombinant anti-CD52 Mc Ab was optimized by Design of Experiment(DOE). The input factors were sodium chloride concentration(130 ~ 870 mmol/L)of intermediate wash solution and the p H value(3. 50 ~ 4. 20)of elution solution,while the responses were the recovery rate,purity,residual host cell protein content,exogenous DNA content and protein A content of purified samples,with which a data model was established by DOE. The ranges of sodium chloride concentration of intermediate wash solution and p H value of elution solution were predicted by the model,and the stability of process parameters were verified by DOE. Results The optimal sodium chloride concentration of intermediate wash solution was 400 ~ 700 mmol/L,while the optimal p H value of elution solution was 3. 55 ~ 3. 75. When the factors changed within the predicted ranges,all the recovery rates of samples were more than 80%,while the purities were more than 97%,the exogenous DNA contents were less than 206 pg/mg,the host cell protein contents were less than 2 300 ng/mg,and the protein A contents were less than 6. 5 ng/mg. Conclusion The affinity chromatography process for purification of recombinant anti-CD52 Mc Ab was successfully optimized,which showed high stability.
引文
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[4]RATHORE A S.Quality by design(Qb D)-based process development for purification of a biotherapeutic[J].Trends Biotechnol,2016,34(5):358-370.
[5]DAS A K,MANDAL V,MANDAL S C.Design of experiment approach for the process optimisation of microwave assisted extraction of lupeol from Ficus racemosa leaves using response surface methodology[J].Phytochem Anal,2013,24(3):230-247.
[6]GE Healthcare.UNICORNTM 6.1 method manual[M].uppsala,Sweden:GE Healthcare Bio-Sciences AB,2009:321-338.
[7]WANG L,LI Y H,RAO C M.Determination of residual host genomic DNA in recombinant products by real-time PCR[J].Chin J Pharm Anal,2009(10):1593-1596.(in Chinese)王兰,李永红,饶春明.实时定量PCR检测重组技术产品中宿主基因组DNA残留[J].药物分析杂志,2009(10):1593-1596.
[8]ZHANG F,DONG Y D,GUO S,et al.Comparison of three types of residual Chinese hamster ovary cell protein detection kits[J].Chin J Pharm Anal,2016(13):1107-1112.(in Chinese)张峰,董衍东,郭莎,等.3种中国仓鼠卵巢细胞蛋白残留量检测试剂盒的比较研究[J].中国药学杂志,2016(13):1107-1112.
[9]BI H,FAN W H,SHI X C,et al.Development of a method for determination of residual protein A of a kind of Fc fusion protein[J].Chin J Pharm Anal,2016(13):1107-1112.(in Chinese)毕华,范文红,史新昌,等.融合蛋白Protein A残留检测方法的建立[J].药物分析杂志,2014(2):297-300.
[10]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Republic of China(Vol III)[S].Beijing:China Med Sci Press,2015:363-366.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:363-366.
[11]CHON J H,ZARBIS-PAPASTOITSIS G.Advances in the production and downstream processing of antibodies[J].N Biotechnol,2011,28(5):458-463.
[12]URH M,SIMPSON D,ZHAO K.Affinity chromatography:general methods[J].Methods Enzymol,2009,463:417-438.
[13]CHOE W,DURGANNAVAR T A,CHUNG S J.Fc-binding ligands of immunoglobulin G:an overview of high affinity proteins and peptides[J].Materials(Basel),2016,9(12):994-1011.
[14]WEI J L,ZHANG B,LIU Y,et al.Development and optimization of purification process for humanized anti-human TNF monoclonal antibody[J].J Shenyang Pharmaceutical University,2017(1):79-83.(in Chinese)魏建玲,张波,刘颖,等.人源化抗人TNFα单克隆抗体纯化工艺的优化[J].沈阳药科大学学报,2017(1):79-83.