Void exclusion of antibodies by grafted-ligand porous particle anion exchangers

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• 作者：Rui Nian ; Cindy Chuah ; Jeremy Lee ; Hui Theng Gan ; Sarah Maria Abdul Latiff ; Wan Yee Lee ; Vincent Vagenende ; Yuan-Sheng Yang ; Pete Gagnon ; ^{pete_gagnon@bti.a-star.edu.sg} ; ^{pete@validated.com}
• 关键词：Anion exchange ; Ion exclusion ; Size exclusion ; Void exclusion ; Vntibody purification ; pH excursions
• 刊名：Journal of Chromatography A
• 出版年：2013
• 出版时间：22 March, 2013
• 年：2013
• 卷：1282
• 期：Complete
• 页码：127-132
• 全文大小：727 K

文摘

We describe a new variant of anion exchange chromatography in columns packed with porous particles that embody charged low-density polymer zones supported by a higher density polymer skeleton. IgG defies the norms of anion exchange and is excluded to the void volume at pH 3-10 and 0-4 M NaCl. Void exclusion also occurs with Fab, F(ab¡¯)₂, and IgM. Host cell protein contaminants mostly follow the usual norms of anion exchange and bind more strongly with increasing pH and decreasing conductivity. Sample buffer composition has no impact on partitioning so long as applied sample volume does not exceed the interparticle void volume of the column. Void-excluded antibody elutes in equilibration buffer. This seemingly conflicted collection of behaviors is reconciled by a variable size exclusion function mediated through the low-density polymer zones, the charge properties of the antibody species, and the pH and conductivity of the equilibration buffer. Current-generation porous particle anion exchangers that employ grafting techniques to achieve high charge density mediate void exclusion to varying degrees, with the best-suited achieving complete exclusion, and others as little as 65 % . Perfusive and non-grafted particle-based exchangers mediate as little as 50 % exclusion. Monoliths mediate no exclusion, due to their lack of an interparticle void volume. On qualified exchangers, the technique supports greater than 99 % reduction of host proteins, DNA, and endotoxin. Virus is reduced more than 99.9 % , and aggregates are reduced to less than 0.05 % . The method supports better process control than other anion exchange formats because pH excursions in conjunction with changes in salt concentration do not occur until after the antibody has eluted from the column.