An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography.
Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.
Maeno
Katsuyuki
K
Shiseido Research Center, 2-2-1 Hayabuchi, Tsuzuki, Yokohama 224-8558, Japan. katsuyuki.maeno@to.shiseido.co.jp
Hirayama
Aya
A
Sakuma
Kenichi
K
Miyazawa
Kazuyuki
K
D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods*; D010767:Phosphorylcholine / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry
D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D013388:Succinimides; D010767:Phosphorylcholine; D012822:Silicon Dioxide; C001426:N-hydroxysuccinimide