title: Utilization of a precolumn with size exclusion and reversed-phase modes for size-exclusion chromatographic analysis of polysorbate-containing protein aggregates.
abstract: Size-exclusion chromatography (SEC) is a useful method for quantification of protein aggregates because of its high throughput capacity and highly quantitative performance. One of the problems in this method concerns polysorbates, which are well-known additives for protein-containing products to prevent protein aggregation, but frequently interfere with the photometric detection of protein aggregates. We developed a new SEC method that can separate polysorbates from protein sample solutions in an on-line mode with a precolumn with size exclusion and reversed-phase mixed modes. The precolumn can effectively trap polysorbates in aqueous mobile phase, and the trapped polysorbates are easily eluted with acetonitrile-containing aqueous mobile phase to clean the precolumn. Small parts of protein aggregates may be also trapped on the precolumn depending on temperature and proteins. Setting appropriate column temperature can minimize such inconvenient trapping of aggregates.
authors.affiliation: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma 370-0013, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Fukuda
authors.affiliation: Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma 370-0013, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Iura
authors.affiliation: Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma 370-0013, Japan.
authors.forename: Shigehiro
authors.initials: S
authors.lastname: Yanagihara
authors.affiliation: Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: kano.kenji.5z@kyoto-u.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kano
mesh_terms: D002850:Chromatography, Gel / Q000295:instrumentation* / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation* / Q000379:methods; D011136:Polysorbates / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D013696:Temperature
chemical_list: D011136:Polysorbates; D011506:Proteins
keywords: MSpak GF-4A; Polysorbate; Protein aggregation; Size-exclusion chromatography; Temperature-dependent retention