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A robust affinity chromatography system based on ceramic monoliths coated with poly(amino acid)‐based polymeric constructs
Polymer International ( IF 3.2 ) Pub Date : 2020-10-10 , DOI: 10.1002/pi.6142
Javier Sánchez Santiago 1 , Ramón L Cerro 1 , Carmen Scholz 2
Affiliation  

Traditional chromatographic separation systems are disadvantaged by low flow rates, a high pressure drop across the column, low capacity and poor reusability. Searching for more efficient separation systems we introduced the use of a ceramic monolith as robust support in bioseparations. A coating consisting of l‐asparagine as ligand, poly(l‐lysine) as spacer arm and a commercial poly(ethylene acrylic acid) film forming copolymer network (Michem 4983‐40R) was developed as a coating for these ceramic monoliths. Poly(l‐lysine) was synthesized by ring‐opening polymerization of ε‐trifluoroacetyl‐l‐lysine N‐carboxyanhydride and coupled to a commercial film‐forming poly(ethylene acrylic acid) network. This construct was then ‘decorated’ with l‐asparagine via the terminal amino functional groups of poly(L‐lysine) and coated onto the ceramic monolith to selectively bind l‐asparaginase. Adsorption/elution experiments showed reversible binding between l‐asparagine and l‐asparaginase, and the subsequent release of l‐asparaginase, and between 83% and 94% of the active enzyme was recovered by elution with d‐asparagine and NaCl solutions. The functional activity of the eluted l‐asparaginase was verified by a Nessler's assay. While traditional separation processes (adsorption and elution) using gel bead packings take many hours, the ceramic monolith system achieves the same of level of separation in about 1 h. This new system served as a proof of concept for its application in protein separation and purification. This work paves the way to a better understanding of the use of ceramic monoliths as stationary phase coated with a stable polymer construct for more robust and efficient supports in affinity chromatography. © 2020 Society of Industrial Chemistry

中文翻译:

强大的亲和色谱系统,基于涂覆有基于聚氨基酸的聚合物结构的陶瓷整体

传统色谱分离系统的缺点是流速低,色谱柱上的压降高,容量低和可重复使用性差。为了寻找更有效的分离系统,我们介绍了使用陶瓷整体材料作为生物分离中的可靠载体。开发了一种由l-天冬酰胺作为配体,聚l-赖氨酸作为间隔臂和商用聚(乙烯丙烯酸)成膜共聚物网络(Michem 4983-40R)的涂料,作为这些陶瓷整体涂料的涂料。聚(l-赖氨酸)是通过ε-三氟乙酰基-l-赖氨酸N的开环聚合反应合成的羧酸酐,并与市售的成膜聚(乙烯丙烯酸)网络偶联。然后,通过聚(L-赖氨酸)的末端氨基官能团用l-天冬酰胺“修饰”该构建体,并涂在陶瓷整体上以选择性结合l-天冬酰胺酶。吸附/洗脱实验表明可逆之间的结合天冬酰胺和-asparaginase,和随后释放-asparaginase,和83%,并且活性酶的94%之间,通过用洗脱回收d天冬酰胺和NaCl溶液。洗脱的的功能活性天冬酰胺酶已通过Nessler分析验证。尽管使用凝胶珠填料的传统分离过程(吸附和洗脱)需要花费数小时,但陶瓷整体系统在大约1小时内即可达到相同的分离水平。该新系统证明了其在蛋白质分离和纯化中的应用。这项工作为更好地理解使用陶瓷整料作为固定相涂覆了稳定的聚合物构造物,以在亲和色谱中提供更坚固和有效的载体铺平了道路。©2020工业化学学会
更新日期:2020-12-05
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