几种Protein A亲和层析填料纯化重组单克隆抗体效果的比较

doi:10.13523/j.cb.20151211

中国生物工程杂志

2015, Vol. 35

Issue (12): 72-77 DOI: 10.13523/j.cb.20151211

技术与方法

几种Protein A亲和层析填料纯化重组单克隆抗体效果的比较

何凌冰¹, 李乐², 邓义熹², 蒙国基², 于玉根²

1. 深圳国家高技术产业创新中心深圳 518057;
2. 深圳万乐药业有限公司深圳 518029

Screening of Protein A Resins for Purifying Combinant Antibody

HE Ling-bing¹, LI Le², DENG Yi-xi², MENG Guo-ji², YU Yu-gen²

1. State High-Tech Industrial Innovation Center, Shenzhen 518057, China;
2. Shenzhen Main Luck Pharmaceuticals Inc., Guangdong 518029, China

全文: PDF HTML

摘要：

随着表达技术的进步以及发酵技术的优化,细胞发酵上清液中抗体的滴度大幅度提高,必然对下游纯化工艺的处理能力有更高的要求。Protein A亲和层析是分离单克隆抗体的一种标准纯化方法。目前已经有很多种protein A亲和填料,其配体基本上都是大肠杆菌发酵的重组protein A。不同厂家生产的protein A填料有各自不同的基质或键合技术,从而造成填料对抗体亲和力以及对碱耐受性的差异。针对正在研发的WLB303单克隆抗体,比较了6种不同的protein A填料的动态载量,并从洗脱溶液种类和pH两个方面进行单克隆抗体纯化条件的筛选,分析纯化后样品SEC纯度和回收率。综合考虑载量、除杂效果、回收率三个因素,MabSelect填料是最适合本抗体纯化的,SEC纯度可以达到98%,回收率可以达到100%左右。基于这些数据,对MabSelect填料进行了使用寿命试验,200次循环之后载量仅衰减了5%。

关键词： 动态载量; 回收率; ProteinA; SEC纯度

Abstract:

With the significant increase in mammalian cell culture titers of monoclonal antibodies(mAbs) in the last decades, many large scale processes are facing a bottleneck in the protein A affinity chromatography, a standard for mAbs capture and purification from the cell culture. Six brands of protein A resin, with different base beads and ligands, were tested in three elution buffers. Concerning with the dynamic binding capacity(DBC), monomer purity of eluate and total recovery, MabSelect from GE Healthcare was selected for the ideal capturer. A further 200 cycles test showed a 5% decrease in the DBC of MabSelect.

Key words: ProteinA Dynamic Binding Capacity Recovery Monomer Purity

收稿日期: 2015-09-22 出版日期: 2015-12-22

ZTFLH:

Q819

通讯作者: 蒙国基 E-mail: mengguoji@wanle.com.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	何凌冰
	李乐
	邓义熹
	蒙国基
	于玉根

引用本文:

何凌冰, 李乐, 邓义熹, 蒙国基, 于玉根. 几种Protein A亲和层析填料纯化重组单克隆抗体效果的比较[J]. 中国生物工程杂志, 2015, 35(12): 72-77.

HE Ling-bing, LI Le, DENG Yi-xi, MENG Guo-ji, YU Yu-gen. Screening of Protein A Resins for Purifying Combinant Antibody. China Biotechnology, 2015, 35(12): 72-77.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151211 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/72

[1] Sjoquist J, Movitz J, Johansson I B,et al. Localization of protein A in the bacteria. Eur J Biochem,1972,30:190-194.
[2] Lindmark R, Thoren-Tolling K, Sjoquist J. Binding of immunoglobulin to protein A and immunoglobulin levels in mammalian sera. J Immunol Mothods,1983,62:1-13.
[3] Lofdahl S, Guss B, Uhlen M,et al. Gene for staphylococcal protein A. Proc Natl Acad Sci USA,1983,80:697-701.
[4] Guss B, Uhlen M, Nilsson B,et al. Region X,the cell-wall-attachment part of staphylococcal protein A. J Biochem,1984,138:413-420.
[5] Horstmann B J,Chase H A. Modeling the affinity adsorption of immunoglobulin-G to protein-A immobilized to agarose matrices. Chem Eng Res Des,1989,67:243-254.
[6] Hahn R, Schlegel R, Jungbauer A. Comparison of protein A affinity sorbents. Chromatography B,2003,790(1-2):35-51.
[7] McCue J T,Kemp G,Low D,et al. Evaluation of protein-A chromatography media. J Chromatography A,2003,989:139-153.
[8] Fahrner R L, Whitney D H, Vanderlaan M,et al. Performance comparison of protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol Appl Biochem,1999,30:121-128.
[9] Boschetti E, Jungbauer A. Handbook of Bioseparations. San Diego:Academic Press,2000.535.
[10] Gottschalk U. Bioseparation in antibody manufacturing:the good,the bad,and the ugly,Biotechnol Prog,2008,24:496-503.
[11] Kelley B. Industrialization of mAb production technology:the bioprocessing industry at crossroads. Mabs,2009,1:443-452.
[12] Liu H F,Ma J,Winter C,et al. Recovery and purification process development for monoclonal antibody production. Mabs,2010,2:1-20.
[13] Shukla A A, Gupta P, Han X. Protein aggregation kinetics during chromatography:case study for an Fc fusion protein. J Chromatography A,2007,1171:22-28.
[14] Gagnon P. Technology trends in antibody purification. J Chromatogr A,2012,1221:57-70.
[15] Vazquez-Rey M, Lang D A. Aggregates in monoclonal antibody manufacturing processes. Biotech Bioeng,2011,108:1494-1508.
[16] GE Healthcare Life Sciences.Regulatory Support File MabSelect,11-0028-20.

[1]	蒙国基, 邓义熹, 李乐, 罗豪晖, 于玉根. 变速上样在MabSelect填料上分离WLB303[J]. 中国生物工程杂志, 2016, 36(6): 65-75.
[2]	Yamuna Dasarathy, Mat s Ramberg, Mikael Andersson. 下游工艺开拓中如何系统地筛选离子交换层析介质[J]. 中国生物工程杂志, 1998, 18(2): 55-59.
[3]	RobertC.A.Yang, JohnLis, Raywu, 王雪松. 电泳后琼脂糖凝胶中DNA的洗脱[J]. 中国生物工程杂志, 1981, 1(4): 34-38.

Viewed

Full text

Abstract

Cited

Shared

Discussed