title: High-throughput screening of packed-bed chromatography coupled with SELDI-TOF MS analysis: monoclonal antibodies versus host cell protein.
abstract: A feasibility study to couple high throughput screening of packed bed chromatography with mass spectrometric detection by SELDI-TOF MS is presented. As model system monoclonal antibodies (mAb) versus host cell protein (HCP) from an industrial cultivation was chosen. Packed bed chromatography was screened on a TECAN Evo Freedom 200 station using miniaturized chromatographic columns placed on a specially designed array carrier linked to a commercially available T-Stack module. Gradient elution of the bound proteins was performed by applying a multiple step strategy. When analyzing selected HCP peaks as well as the detected antibody peaks throughout the chromatographic runs a direct correlation between applied and detected components was established. The sensitivity of conventional protein A chromatography was found to be lower than SELDI-TOF MS analysis. During initial screening a shift in the elution pattern for one of the monoclonal antibodies detected with all four resins was identified to be a heterogeneity in the mAb glycosylation pattern. In addition, a detailed differentiation between various HCP fractions through out the chromatographic process using SELDI-TOF analysis let to the detection of HCP components possibly adhering to the mAbs during chromatographic separations.
authors.affiliation: Institut für Biotechnologie 2, Forschungszentrum Jülich, 52426 Jülich, Germany.
authors.forename: Pierre Schulze
authors.initials: PS
authors.lastname: Wierling
authors.forename: Ralf
authors.initials: R
authors.lastname: Bogumil
authors.forename: Esther
authors.initials: E
authors.lastname: Knieps-Grünhagen
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000302:isolation & purification*; D016466:CHO Cells / Q000378:metabolism; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002847:Chromatography, Agarose / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D011108:Polymers; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D018033:Antibodies, Bispecific; D011108:Polymers; C058606:Toyopearl 650