用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

doi:10.12034/j.issn.1009-606X.219166

过程工程学报 ›› 2020, Vol. 20 ›› Issue (1): 84-90.DOI: 10.12034/j.issn.1009-606X.219166

用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

葛佳^1,2，那向明^1,2，吴学星²，杨维兴³，郝冬霞^2*，马光辉^1,2*

1. 中国科学院大学化学工程学院，北京 100049 2. 中国科学院过程工程研究所生化工程国家重点实验室，北京 100190 3. 中科森辉微球技术(苏州)有限公司，江苏苏州 215000

收稿日期:2019-03-25 修回日期:2019-05-05 出版日期:2020-01-22 发布日期:2020-01-14
通讯作者: 郝冬霞 dxhao@ipe.ac.cn
基金资助:
国家自然科学基金项目;国家自然科学基金项目;北京市自然科学基金-北京科学技术研究院联合基金项目

Preparation of biomimetic peptide attached supermacroporous poly(glycidyl methacrylate) microspheres for monoclonal antibody purification and its performance

Jia GE^1,2, Xiangming NA^1,2, Xuexing WU², Weixing YANG³, Dongxia HAO^2*, Guanghui MA^1,2*

1. School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, China 2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China 3. Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd., Suzhou, Jiangsu 215000, China

Received:2019-03-25 Revised:2019-05-05 Online:2020-01-22 Published:2020-01-14

摘要/Abstract

摘要： 以仿生多肽配基FYEILHC为亲和配基、以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯[Dextran-poly(glycidyl methacrylate), Dextran-PGMA]超大孔微球为基质，制备用于单克隆抗体纯化的仿生多肽超大孔PGMA微球，在环氧氯丙烷中滴加2 mol/LNaOH使其表面衍生出环氧基，在表面修饰FYEILHC；用扫描电镜表征微球表面形貌，用AKTA蛋白纯化系统考察了Dextran-PGMA微球和琼脂糖微球对抗体的动态吸附量随线性流速的变化。结果表明，偶联FYEILHC后Dextran-PGMA微球仍能保持其大孔结构，在923 cm/h线性流速下，其对抗体的动态吸附量仅下降约8%，而琼脂糖微球的动态吸附量则迅速下降25%。表明在较高流速下，抗体在Dextran-PGMA微球上的传质性能较好。吸附?用0.1 mol/L NaOH原位清洗重复40次后，Dextran-PGMA微球对抗体的动态吸附量约为(21?1) mg/mL，表明微球具有良好的化学稳定性；血清中回收的抗体纯度为95.0%，表明仿生多肽亲和介质具有从复杂生物样品中纯化抗体的巨大潜力，可满足高流速、高通量抗体分离纯化需求。

关键词: 单克隆抗体, 纯化, 仿生多肽, 超大孔, 微球

Abstract: Recently, immunoglobulin G (IgG) has attracted great attentions in clinical medicine, biotechnology, stimulated the development of downstream purification technology of monoclonal antibodies. Now, the protein based affinity ligands during traditional chromatographic purification of IgG exist drawbacks including the high cost and low stability, which leads to the secondary contamination and biological toxicity to purified sample. Also the chromatographic agarose microspheres present shortcomings such as the gel compressibility and the mass transfer limitations. In this work, biomimetic peptide as the protein based ligands alternative, grafting on the supermacroporous dextran-poly(glycidyl methacrylate) microspheres (Dextran-PGMA) to overcome above drawbacks were prepared and to efficiently improve the IgG chromatographic separation. The hydroxy on dextran-grafted polymer chromatographic microspheres were converted to the epoxy groups in epichlorohydrin solution with 2 mol/L NaOH, and the epoxy groups were opened and coupled with biomimetic peptide, FYEILCH, to achieve the purpose of affinity modification. The SEM observed that the pore structure of macroporous was mostly maintained after coupling with the biomimetic peptide. The change of 10% dynamic binding capacity (DBC10%) of FYEILHC based on Dextran-PGMA microspheres and agarose microspheres under different flow rate (92~923 cm/h) was compared. The DBC10% of dextran-PGMA was declined only 8% under 923 cm/h, whereas the DBC10% of agarose was sharply declined about 25%. This phenomenon might be related to better mass transfer for IgG at higher flow rate on the Dextran-PGMA microspheres. The DBC10% of Dextran-PGMA microspheres maintained (21±1) mg/mL after cleaning-in-place (CIP) 40 cycles using 0.1 mol/L NaOH, indicating that the affinity medium had good chemical stability. The purity of recovered antibodies in plasma was 95.0%, demonstrating that this biomimetic peptide based affinity medium had great potential to purify IgG from complex biological samples. This biomimetic peptide grafted supermacroporous poly(glycidyl methacrylate) microspheres could meet the requirement for rapid and high-throughput separation of monoclonal antibody.

Key words: monoclonal antibody, purification, biomimetic peptide, supermacroporous, microsphere

葛佳那向明吴学星杨维兴郝冬霞马光辉. 用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能[J]. 过程工程学报, 2020, 20(1): 84-90.

Jia GE Xiangming NA Xuexing WU Weixing YANG Dongxia HAO Guanghui MA. Preparation of biomimetic peptide attached supermacroporous poly(glycidyl methacrylate) microspheres for monoclonal antibody purification and its performance[J]. Chin. J. Process Eng., 2020, 20(1): 84-90.

参考文献

[1]Salvalaglio M, Zamolo L, Busini V, Moscatelli D, Cavallotti C, .Molecular modeling of protein a affinity chromatography[J].Journal of Chromatography A, 2009, 1216(50):8678-8686
[2]Gahoual R, Heidenreich A K, Somsen G W, Bulau P, Reusch D, Wuhrer M, Haberger M.Detailed characterization of monoclonal antibody receptor interaction using affinity liquid chromatography hyphenated to native mass spectrometry[J].Analytical Chemistry, 2017, 89(10):5404-5412
[3]Zhao W W, Liu F F, Shi Q H, Sun Y, .Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands[J].Journal of Chromatography A, 2014, 1359:100-111
[4]Santos R, Rosa S A S L, Aires-Barros M R, Tover A, Azevedo A M.Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies:development and optimization of a washing step[J].Journal of Chromatography A, 2014, 1355:115-124
[5]Dutta A K, Tran T, Napadensky B, Teella A, Brookhart G, Ropp P A, Zhang A W, Tustian A D, Zydney A L.Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography[J].Journal of Biotechnology, 2015, 213:54-64
[6]Du K.Peptide immobilized monolith containing tentacle-type functionalized polymer chains for high-capacity binding of immunoglobulin G[J].Journal of Chromatography A, 2014, 1374:164-170
[7]Li R X, Dowd V, Stewart J D, Burton J S, Lowe R C.Design, Synthesis, and Application of a Protein A Mimetic[J].Nature Biotechnology, 1998, 6:190-195
[8]黄波.抗体亲和肽配基的理性设计和色谱表征，[J].天津大学, 2011, :99-104
[9]Sondermann P, Kaiser J, Jacob U,.Molecular basis for immune complexrecognition: a comparison of Fc-receptor structures[J].Journal of molecular biology, 2001, 309:737-749
[10]Sugita T，Katayama M，Okochi M, Kato R，Ichihara T， Honda H.Screening of peptide ligands that bind to the Fc region of IgG usingpeptide array and its application to affinity purification of antibody[J].Biochemical Engineering Journal, 2013, 79:33-40
[11]Wei Y P, Xu J D, Zhang L, Fu Y K, Xu X.Development of novel small peptide ligands for antibody purification[J].Rsc Advances, 2015, 5(82):67093-67101
[12]Yu M R, Li Y, Zhang S P, Li X N, Yang Y L, Chen Yi, Ma G H, Su Z G.Improving stability of virus-like particles by ion-exchangechromatographic supports with large pore size: Advantages ofgigaporous media beyond enhanced binding capacity[J].Journal of Chromatography A, 2014, 1331:69-79
[13]Zhou W Q, Gu T Y, Su Z G, Ma G H.Synthesis of macroporous poly(glycidyl methacrylate) microspheres by surfactant reverse micelles swelling method[J].European Polymer Journal, 2007, 43(10):4493-4502
[14]Zhang R Y, Li Q, Li J, Zhou W Q, Ye P L, Gao Y, Ma G H, Su Z G.Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation[J].Materials Science and Engineering: C, 2012, 32(8):2628-2633
[15]Wang X Y, Xia D H, Han H, Peng K, Zhu P J, Crommen J, Wang Q Q, Jiang Z J.Biomimetic small peptide functionalized affinity monoliths for monoclonal antibody purification[J].Analytica Chimica Acta, 2018, 1017:57-65
[16]Hahn R, Bauerhansl P, Shimahara K, Wizniewski C, Tscheliessnig A, Jungbauer A.Comparison of protein A affinity sorbents II. Mass transfer properties [J].Journal of Chromatography A, 2005, 1093:98-100
[17]Zhao L, Wei W, Huang Y D, Zhang R Y, Zhu K, Hao D X, Su Z G, Ma G H, .A novel rProtein A chromatographic media for enhancing cleaning-in-place performance[J].Journal of Immunological Methods, 2018, 460:45-50

用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

Preparation of biomimetic peptide attached supermacroporous poly(glycidyl methacrylate) microspheres for monoclonal antibody purification and its performance

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	文康韦祎马光辉. 基于响应面法对制备高包埋率ROP-PLGA微球的影响因素分析[J]. 过程工程学报, 2021, 21(1): 83-91.
[2]	张敏韩林胡月月李健闫冰. 改性琼脂糖微球固定化甘油脱氢酶的研究[J]. 过程工程学报, 2020, 20(5): 591-598.
[3]	陶娇丽黄永东赵岚朱凯吴学星周丹妮苏志国马光辉刘红缨. 新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能[J]. 过程工程学报, 2019, 19(6): 1197-1203.
[4]	尤星力杨延丽苏志国张媛张松平. 鸡白痢沙门氏菌兔抗血清多抗IgG的纯化与稳定[J]. 过程工程学报, 2019, 19(6): 1204-1211.
[5]	韦巍黄永东赵岚吴学星朱天孝李冬雪靳海波张荣月苏志国马光辉. 新型耐碱蛋白A介质的制备与性能[J]. 过程工程学报, 2019, 19(3): 609-616.
[6]	赵春龙孙峙郑晓洪高文芳张延玲林晓. 碳酸锂的制备及其纯化过程的研究进展[J]. 过程工程学报, 2018, 18(1): 20-28.
[7]	赵培李艳王建梅王雪莹许敏王立秋. 利用新型膜乳化器制备粒径均一的PLGA微球[J]. 过程工程学报, 2017, 17(6): 1257-1264.
[8]	胡琳琳韦祎徐玉松居殿春马光辉. 两亲性刺突微球的制备及性能表征[J]. 过程工程学报, 2017, 17(4): 821-826.
[9]	钱西慧张冬海薛杨李晓飞李文辉陈运法. SiO2中空微球对高温硫化硅橡胶电气绝缘性能的影响[J]. 过程工程学报, 2016, 16(5): 781-787.
[10]	朱凯赵岚黄永东李强邱晗王启宝苏志国马光辉. 葡聚糖接枝型耐碱Protein A介质的层析性能提升研究[J]. 过程工程学报, 2016, 16(5): 856-861.
[11]	邹勇娟刘艳王月琦吴颉巩方玲马光辉. 壳聚糖微球及其与白油混合乳液对禽用新流二联疫苗佐剂的免疫效果[J]. 过程工程学报, 2016, 16(4): 639-646.
[12]	吴晶晶吴楠岳华吴兴兰中西秀树高晓冬马光辉. 壳聚糖季铵盐微球作为注射疫苗佐剂的安全性评价[J]. 过程工程学报, 2016, 16(4): 647-653.
[13]	陈涛史楠门吉英高保娇. 胰蛋白酶分子表面印迹材料的制备及其大分子识别特性[J]. 过程工程学报, 2016, 16(2): 272-278.
[14]	荆迎军高慧杨春燕. 表面修饰聚乙烯亚胺的壳聚糖微球对十二烷基苯磺酸钠的吸附特性[J]. 过程工程学报, 2015, 15(6): 1057-1062.
[15]	张博王瑛王启宝杨婷媛张贵峰王连艳. 快速膜乳化法制备尺寸均一的载硫酸亚铁明胶微球[J]. 过程工程学报, 2015, 15(5): 847-853.