High-throughput screening of packed-bed chromatography coupled with SELDI-TOF MS analysis: monoclonal antibodies versus host cell protein.
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.
Wierling
Pierre Schulze
PS
Institut für Biotechnologie 2, Forschungszentrum Jülich, 52426 Jülich, Germany.
Bogumil
Ralf
R
Knieps-Grünhagen
Esther
E
Hubbuch
Jürgen
J
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*
D018033:Antibodies, Bispecific; D011108:Polymers; C058606:Toyopearl 650